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TABLE OF CONTENTSBIOLOGY LABORATORY

PROTOCOL BOOK

SYLLABUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

HOW TO SUCCEED IN INTRODUCTORY BIOLOGY LABORATORY . . . . . . . . . . 7

MAKING AND KEEPING A LAB NOTEBOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

USE OF BINOCULARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

BIOMETRICS AND STATISTICAL ANALYSIS OF DATA . . . . . . . . . . . . . . . . . . . . . 19

GRAPH CONSTRUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

OBSERVATIONS VERSUS CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

ACCURACY AND PRECISION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

DETERMINING pH OF SOME COMMON SUBSTANCES . . . . . . . . . . . . . . . . . . . . 34

PERCENTAGE OF SUGAR IN SOFT DRINKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

PEPSIN DIGESTION OF PROTEINS AND EFFECTS OF ANTACIDS . . . . . . . . . . . 42

LIPIDS, EMULSIONS, AND EMULSIFYING AGENTS . . . . . . . . . . . . . . . . . . . . . . . 44

MICROSCOPE USE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

YEAST AND THE PRODUCTS OF ITS FERMENTATION PROCESS . . . . . . . . . . . 53

MAKING ROOT BEER AT HOME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

CELLS AND ORGANELLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

BUNSEN BURNER USE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

SAFELY HEATING TEST TUBES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

DIFFUSION AND OSMOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

LIGHT AND SPECTROPHOTOMETER USE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

PHOTOSYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

MITOSIS, MEIOSIS, AND GENETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

BLOOD TYPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

FRANKINCENSE AND MYRRH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

CLASSIFICATION OF ORGANISMS USED IN BIOLOGY 1081L AND 1082L LABS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

January 4, 2016 99

Grasshopper spermatogenesis slide,dissection

INSECTA/DICTYOPTERAGromphadorhina portentosa Madagascar Hissing Cockroach dissection

Chordata (Vertebrata) OSTEICHTHYESCoregonus clupeiformis Whitefish mitosis

AVES/GALLIFORMESPhasianidae

Gallus gallus Domestic Poultry egg: examine cells & inmayonnaise and waffles, tohatch

MAMMALIA/A RTIODACTYLABovidae

Bos taurus Domestic Cattle suet, milk for yogurt, dairyproducts, fat for soap

Capra domesticus Domestic Goat milk for yogurt & cheeseMAMMALIA/P RIMATES

HominidaeHomo sapiens Human vit. C usage, blood type,

blood pressure & pulse,visual reaction time

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Glycine max (Soja max) Soybean oil for soap, mayo, & salad,sprouts

Lens culinaris Lentil lentils & rice, sproutsCicer arietinum Garbanzo/Chickpea sproutsArachis hypogaea Peanut bird seed & peanut butterMedicago sativa Alfalfa sprouts, examine stem

structuresVigna sinensis Black-eyed Pea, Cowpea sproutsPhaseolus sp. “Bean” examine sprout structuresPhaseolus sp? Mung Bean sproutsTrifolium sp. Clover root nodule bacteria, sprouts

FagaceaeQuercus suber Cork Oak examine “cells”

CannabinaceaeHumulus lupulus Hops flavoring agent in brewing,

beer in wafflesLauraceae

Cinnamomum zelandicum Cinnamon soap additiveLaurus nobilis Bay seasoning in pizza sauceSassafras albidum Sassafras sassafras tea

UmbelliferaeDaucus carota Carrot veggies & mayonnaise,

salad, test vit. CPetroselinum crispum Parsley chlorophyll extraction

OleaceaeOlea europaea Olive oil for soap, mayonnaise, &

saladSolanaceae

Capsicum annuum Chili Pepper seasoning in mayonnaiseCapsicum frutescens Cayenne Pepper seasoning in mayonnaise

Green/Red Pepper veggies & mayonnaise, testvit. C

Lycopersicon esculentum Tomato examine cells, (salad), testvit. C, pizza sauce

Solanum tuberosum Potato examine cellsLabiatae

Ocimum basilicum Basil pizza sauceOriganum vulgare Oregano pizza sauceThymus vulgaris Thyme pizza sauce

RubiaceaeCoffea arabica Coffee beverage at waffle breakfast

CompositaeHelianthus annuus Sunflower seed butter, oil, sprouts, bird

seed, seeds in wafflesCarthamus tinctorius Safflower oil for soap & mayonnaise

PiperaceaePiper nigrum Black Pepper seasoning for salad

MyrtaceaeSyzygium aromaticum Clove soap additive

CucurbitaeCucumis sativus Cucumber veggies & mayonnaiseCucurbita pepo var. melopepo Zucchini veggies & mayonnaise

BurseraceaeBoswellia sacra (B. carterii) Frankincense demonstration of scentCommiphora myrrha Myrrh demonstration of scent

ANIMALIAANIMALIA NematodaAscaris sp. mitosis (& meiosis)

Annelida OLIGOCHAETALumbricus terrestris Earthworm dissection

Arthropoda CRUSTACEAPortunus sp. Blue Crab dissectionCambarus sp. Crayfish dissection

INSECTA/ORTHOPTERAAcrididae

January 5, 2017 3

SYLLABUSBIOLOGY LAB I, Spring 2017 Janet Stein Carter, Assoc. Prof., Em.-Adj., of Biologycourse #34-BIOL-1081L-004, MW 4:00-5:20 e-mail: Janet.Carter@uc.eduoffice hours: MW ~3:00 to ~4:00 (McD 215-L or the lab area)

COURSE DESCRIPTION:1 undergraduate credit. Biology I Laboratoryexpands upon concepts introduced in lecture,and is designed to develop a student’s abilityto think, work, and write like a scientist.Tools and techniques will include work withmicroscopes, biochemical tests, andexperimental design. Co-req.: Biol 1081.STUDENT LEARNING OUTCOMES OFTHE 1081L-1082L SEQUENCE:Student Learning Outcomes:1. Effectively use tools and commontechniques including microscopes,spectrophotometers, and dissection to makeobservations and gather data.2. Given a question or problem, generatea hypothesis and design an experiment,including suitable controls.3. Create and interpret graphicalrepresentations of experiments.4. Analyze results of experiments anddraw appropriate conclusions.5. Present results of experiments in oraland written form.6. Write a complete scientific report,including scholarly references.

REQUIRED EQUIPMENT:1. The lab protocols (your “textbook”) forthis course may be printed out from our Website. Go tohttp://biology.clc.uc.edu/courses/bio1081L/2. A bound 10 x 7� inch compositionnotebook with graph rulings (available in thebookstore). All notes taken during lab shouldbe entered DIRECTLY into this notebook.3. A pen, such as “Pilot Precise®” (ExtraFine tip), “Tombow Roll Pen, Jr.®” orRapidiograph, which writes with BLACKindelible (waterproof) ink. Water-solublemarkers, ballpoint pen (which is soluble inalcohol, etc.), and felt-tips which “bleed”through the page are NOT acceptable.4. Clear contact paper to mount handouts,specimens, etc. into lab notebook (seeprotocol).OPTIONAL RESOURCES ANDEQUIPMENT:1. While your primary drawings must bedone with your lab pen, you may wish to haveon hand a set of colored pencils with which toindicate color(s) of specimens. Magic markerswhich bleed through the page are not suitable.2. The following book may aid you inlearning how to write like a bIologist:Pechenik, Jan A. 1993. A Short Guide to

Writing about Biology. 2nd. Ed.HarperCollins College Publ., NY.

3. A variety of field guides to trees,

insects, wildflowers, fossils, birds, etc. isavailable in the bookstore, and similar booksare available from other local bookstores andthe Cincinnati Nature Center. Although notrequired, you should purchase those in whichyou have an interest. If there are field guidesyou do not wish to purchase, you may wish tomake arrangements to share, borrow, orotherwise have access to them for field use. 4. Other materials may be needed forcertain lab exercises, and are listed in theschedule and/or will be announced as needed.GRADES: Grades will be determined basedon the total of the points from the threequizzes (50 pt. each), two midterms (200 pt.each), final (200 pt.), and protocol/notebookgradings, (40 + 2 at 180 pt. each), plus pointsfrom any Abstracts and unannounced “pop”quizzes, thus a minimum of 1150 points willbe possible. A histogram (curve) of the totalscores will be constructed and analyzed usingstatistical methods. In general, the classmean will serve as the dividing line between“B” and “C” scores, and only those studentswhose scores are above the mean plus onestandard deviat ion uni t , therebydemonstrating superior mastery of thematerial covered, will receive an “A.” An“F” will be given when an individualrepeatedly scores at the bottom of the classand shows blatant disregard for good studyhabits and class attendance. Ten percent(10%) of the total possible points will bededucted per class period for late assignments(notebooks, papers, collections, etc.). Asstated in the Making and Keeping a LabNotebook protocol, “No books abandoned inmy mailbox will be considered as turned in,nor will they be graded – your portion of thegrade sheet must be filled out and turned inwith the notebook.” Any student who stopsattending class and does not go through theofficial withdrawal process will be given thegrade of “UW” – unofficial withdrawal – theequivalent of an “F.” Grades will be awardedbased on a straight A-B-C-D-F grading scale.

I realize that there are some medicalconditions which, legitimately, can precludea student from having an equal chance tolearn in this course. A very obvious examplewould be a student who had trouble hearingme speak, thus was at a great disadvantagebecause (s)he would miss what I was sayingin lecture. However, other, more subtle,conditions such as ADD and dyslexia can alsoadversely affect an equally-intelligentstudent’s opportunity to obtain informationand/or communicate to me that (s)he has

December 17, 2016 4

learned the needed material. It is not “unfair”to anyone to make arrangements tocompensate for such medical conditions, butrather, this can help insure that such peoplehave an equal chance at doing well in thiscourse. Obviously, however, such studentswould still have to demonstrate that, givenreasonable accommodations, they are capableof mastering the required material. Thus,students who need some type ofaccommodations in order to “level the playingfield” and put them on a par with the rest ofthe class should speak with me now, notafter grades have suffered.SCHEDULE: Note: Weather permitting, wewill be spending time outdoors studying localflora and fauna. Thus, the following scheduleis subject to change due to rain. Tests,

quizzes, and other due dates will be at thestated times unless an announcement to thecontrary is specifically made beforehand (withthe one exception that if it is raining when afield-hike quiz is scheduled, that quiz may bepostponed). Please wear/bring clothingsuitable for hiking through brambles and/ormud and/or poison ivy. Due to varioussafety regulations, long pants and sturdyboots/shoes are mandatory for field hikes.Inappropriately-dressed students will notbe permitted to participate in lab activitiesand will, thus, not have notes for any lab(s)missed. Since some of the hikes are mildlystrenuous, people with asthma, diabetes,and/or hypoglycemia should plan ahead andcome prepared to deal with any possiblereactions they might have.

MONDAYS WEDNESDAYS9-I — Intro to Lab, Lab Notebook How-To Scientific Paper

11-I — Biometrics (ht, wt, age, bp, pulse)

16-I — NO CLASS – MLK DAY

18-I — Accuracy & PrecisionPROTOCOL BOOK DUE

23-I — pH – BRING SUBSTANCES TOTEST

25-I — QUIZ I , Sugar in Soft Drinks –BRING POP TO TEST

30-I — Ret Quiz, DUE: Methods and MaterialsEffects of Conditions on Pepsin’s Function

1-II — Read Pepsin Results, Lipids –(BRING CLEAN JAR?)

6-II — Microscope Use 8-II — MIDTERM I

13-II — Return Test, (Snow-day Catch-up orPossible Hike)

15-II — Cells & OrganellesNOTEBOOKS DUE

20-II — Return NB,Diffusion & Osmosis

22-II — Spectrophotometer Use and Beer’sLaw (Riboflavin)

27-II — Hike 1-III — Fermentation – How Yeast HarvestEnergy

6-III —QUIZ II , Photosynthesis: Chromatography

8-III — Return Quiz, Photosynthesis: Spectraof PigmentsDUE: Abstract

13-17-X — NO CLASS – BREAK WEEK

20-III — Hike 22-III — MIDTERM II

27-III — Return Test, Mitosis and Meiosis Slides

29-III — Hike

3-IV — Genetics: Probability, Corn, PTCPaper — Bring coins

5-IV — QUIZ III , (incl. hike)

10-IV — Return Quiz, Blood Typing

12-IV — HikeNOTEBOOKS DUE

17-IV — Return NB, Bottling (BRING 2-LBOTTLES?), (Frankincense and Myrrh?)

19-IV — Hike

Week of 24-IV – TBA FINAL EXAM (incl Hike?)

TESTS AND QUIZZES: There will bethree quizzes worth 50 pts. each and twomidterms and a final worth 200 pts. each plusa number of 10-pt. “pop” quizzes to insure

you are reading the lab protocols before lab.Make-up tests will be given only in the eventof a valid excuse, and must be takenpromptly. Field-tests and pop quizzes cannot

December 17, 2016 97

Equisetum hyemale Scouring Rush examine structure

Pterophyta FILICINEAEPteris sp. or Pteridium sp. Fern examine structure

Gymnospermae CONIFEROPHYTAPinaceae

Pinus sp. Pine examine structuresGINKGOPHYTA

Ginkgo biloba Ginkgo examine leafAngiospermae MONOCOTYLEDONEAE

LiliaceaeAllium cepa Onion seasoning, examine cells, in

salad & stirfry, mitosisAllium sativum Garlic seasoning, salad & stirfryLilium sp. Lily examine ovary structure,

anther meiosisYucca sp. Yucca examine leaf structure

CommelinaceaeRhoeo spathacea Moses-in-the-Boat examine cells for Ca-oxalate

HydrocharitaceaeElodea sp. Waterweed examine cells, osmosis

GramineaeTriticum aestivum Wheat bread, pita, sprouts, waffles,

pizza crustHordeum vulgare Barley malt for brewing, sprouts,

beer in wafflesZea mays Maize, Corn sprouts, genetics, bird seed,

cornmeal in waffles, geneticcrosses, examine structures

Panicum miliaceum Millet bird seedOryza sativa Rice lentils & riceSaccharum officinarum Sugar Cane sugar for brewing, waffles,

osmosisDICOTYLEDONEAE

CruciferaeBrassica hirta (Sinapis alba) White Mustard seasoning in mayonnaiseBrassica oleracea Broccoli, Cauliflower,

Cabbage, Kaleexamine cells, veggies &mayonnaise, sprouts, salad,test vit. C, pizza ingredient

Raphanus sativus Radish sproutsChenopodiaceae

Beta saccharifera Sugar Beet sugar for brewing, etc.Spinacea oleracea Spinach chlorophyll, salad, test

vit. C, pizza ingredientRutaceae

Citrus limon Lemon in mayonnaise, salad, testvit. C

Citrus aurantium Orange test vit. C, juice at wafflebreakfast

GeraniaceaePelargonium hortorum Geranium examine cells, stomates,

chlorophyll extractionOxalidaceae

Oxalis sp. Shamrock chlorophyll extractionAceraceae

Acer saccharum Sugar Maple sap & maple syrupVitaceae

Vitis sp. Grape wine vinegar for saladRosaceae

Malus pumila Apple cider vinegar for mayonnaise& salad

Pyrus communis Pear examine leaf structureLeguminosae

January 8, 2016 96

CLASSIFICATION OF ORGANISMS USED IN BIOLOGY 1081LAND 1082L LABS

Copyright © 1990 J. L. Stein Carter

KINGDOMKINGDOM Phylum or Division CLASS/ORDERFamily

Genus species Common Name Use(s) in LabsMONERAMONERA

Lactobacillus acidophilus Lactobacillus yogurt, Gram+ bacteriaLactobacillus bulgaricus making yogurtStreptococcus thermophilus making yogurtSpirulena sp. Spirulena chlorophyll, examine cellsEscherichia coli colon-dwelling Gram�Rhizobium sp. legume root-nodule bacteria

PROTISTPROTISTAA RhizopodaAmoeba sp. Amoeba examine structure

Apicomplexa or SporozoaPlasmodium vivax Malaria organism examine structure

ZoomastiginaTrypanosoma gambiense African Sleeping Sick. examine structure

CiliophoraParamecium sp. Paramecium examine structure

DinoflagellataPhytoplankton, Plankton examine structure

BacillariophytaDiatoms examine structure

EuglenophytaEuglena sp. examine structure

ChlorophytaChlamydomonas sp. examine structureChlorella sp. Chlorella examine structureVolvox sp. examine structureUlothrix sp. examine structureOedigonium sp. examine structureUlva sp. Sea Lettuce examine structureClosterium sp. Desmids examine structureSpirogyra sp. examine structure

PhaeophytaChondrus crispus Irish Moss, Carrageen examine structure, tasteGelidium spp. Agar-agar gelling mediumPorphyra tenera Nori taste

FucaceaeFucus sp. or Laminaria sp. Kelp examine structure, taste,

popcorn seasoningRhodophyta

Dulse examine structure, tasteMyxomycota

Physarum sp. Plasmodial Slime Mold examine structureFUNGIFUNGI ASCOMYCETES

Penicillium roqueforti Roquefort Mold blue cheese in mayonnaiseSaccharomyces cerevisiae Bakers’/Brewers’ Yeast fermenting sugar, brewing,

bakingBASIDIOMYCETES

Agaricus sp. Commercial Mushroom pizza ingredientPLANTPLANTAEAE Bryophyta HEPATICA

Marchantia sp. Liverwort examine structureMUSCI

Moss examine structureTracheophyta/Psilophyta

Psilotum sp. examine structureLycophyta

Lycopodium sp. Wolf's Claw examine structureSphenophyta

Equisetum arvense Horsetail examine structure

January 8, 2016 5

be made up. Tests will cover material fromeach lab session that is included, and gradeswill not be adjusted for any labs that you miss– “I wasn’t there” is not a valid excuse.Students who miss a test should makearrangements to make it up BEFORE the nextclass period. Requests to make up tests afterthe tests have been returned and discussedwill be denied unless a student has a validexcuse (like a doctor’s note). Optionally, amore difficult make-up test may be written(but graded on the same curve as everyoneelse). Only one test may be made up late, and

then only with a valid excuse. If more thanone test is missed, subsequent tests willreceive a “zero.” This means that if you skipone test because you “don’t feel like it,” thenmiss a second test due to illness, you haveused up your one chance and will receive a“zero” on the second test. It has been myexperience that students who don’t take a teston time because they think they need moretime to study end up doing no better (if notworse) when they do finally take the test.There will be a 10% per class period penaltyfor a late test.

July 1, 2013 6

VARIOUS ACADEMIC POLICIES: (Supplement to Course Syllabi)Janet Stein Carter, Associate Professor of Biology

1081L, 1082L , LAB COURSEATTENDANCE AND MAKE-UPPOLICY : Attendance at all lab sessions isrequired. Because of the heavily-scheduleduse of the lab room, it will be nearlyimpossible to arrange make-up time if youmiss a lab. Students will, however, beresponsible for all material covered in all labsessions, and it is your responsibility to knowwhat happened in a lab you missed. For yourown health and safety and in consideration ofthe health and safety of others around you, nosmoking will be permitted during the labperiod (including while on hikes). Also,because of possible contamination by toxicchemicals or pathogenic bacteria, food and/orbeverages should not be consumed in the labrooms unless called for as part of anexperiment. Please wear/bring clothingsuitable for hiking through brambles and/ormud and/or poison ivy. Due to varioussafety regulations, long pants and sturdyboots/shoes are mandatory for field hikes.Inappropriately-dressed students will notbe permitted to participate lab activitiesand will, thus, not have notes for any lab(s)missed. Some of the hikes will be mildlystrenuous, so if you have diabetes,hypoglycemia, or allergies, make sure youcarry appropriate medication with you. Whilehiking, drinking water in non-breakablecontainers is permissible and is especiallyencouraged in very hot weather. Smoking andlittering are prohibited.

POLICY ON CELLULAR PHONES ANDBEEPERS: ALL cellular phones andbeepers will please be TURNED OFF duringclass. Ringing cellular phones or beepersand/or people answering calls and talking onthe phone during class are extremely rude andinconsiderate of your classmates who aretrying to hear what the instructor has to sayand very disruptive of the class. Please givethe number of the college switchboard(732-5200) to any family members who mayneed to reach you, and in an emergency, thecollege security guard or receptionist willnotify you. Lab students who own a cellphone may, optionally, wish to bring that cellphone (turned off, please) along on hikes incase of emergency.

WITHDRAWAL POLICY : The Universityhas set up time limits within which you maywithdraw at first without the instructor’ssignature, and later, only with the instructor’ssignature. The registration office can supplyyou with the actual dates for each quarter. If

you need to drop a course, it is yourresponsibility to do so by the appropriatedeadline. Be advised that if you decide towithdraw and choose to leave yourwithdrawal slip in my mailbox the last day,you run the risk that I will not get it until afterthe deadline. If you wish to withdraw, see mein person to get my signature. Any studentwho stops attending class and does not gothrough the official withdrawal process willbe given the grade of “UW” – unofficialwithdrawal – the equivalent of an “F”.

UNIVERSITY OF CINCINNATIACADEMIC DISHONESTY POLICY :The following prohibitions against cheatingand plagiarism are from the University ofCincinnati Student Code of Conduct. Allstudents are expected to adhere to thesepolicies. Failure to do so will result inappropriate disciplinary action.

CHEATING is defined as any dishonesty ordeception in fulfilling an academicrequirement such as:1. Using unauthorized material during anexamination such as tape cassettes, notes,tests,2. Obtaining assistance with or answers toexamination questions from another personwith or without that person’s knowledge,3. Furnishing answers to examinationsquestions to another person,4. Possessing, using, distributing orselling unauthorized copies of anexamination,5. Representing as one’s own anexamination taken by another person, or6. Taking as one’s own an examination inplace of another person.

PLAGIARISM is defined as:1. Submitting another’s published orunpublished work, in whole, in part, or in aparaphrase, as one’s own without fully andproperly crediting the author with footnotes,citations or bibliographical reference, or2. Submitting as one’s own, originalwork, materials that have been producesthrough unacknowledged collaboration withothers.THIS INCLUDES PUTTING SOMEONEELSE’S CUMULATIVE LISTS IN YOURLAB NTOEBOOK WITHOUT CITINGTHAT PERSON AS THE AUTHOR, ASWELL AS SUBMITTING SOMEONEELSE’S PLANT COLLECTION WITHFAKE LABELS REPRESENTING IT ASYOUR OWN WORK!!!

July 1, 2013 95

FRANKINCENSE AND MYRRHCopyright © 1993 J. L. Stein Carter

I. OBJECTIVE:To learn what these plant resins look and smell like and how they have been used by

humans.

II. BACKGROUND:This time of year, many people in

this country are beginning to sing songs whichmention frankincense and myrrh, yet mostpeople know very little about these two plantresins. Frankincense comes from the plantsBoswellia sacra or B. carterii, and myrrhcomes from Commiphora myrrha or C.molmol. To the Magi (in ancient Persianthese were a priestly caste who wereastrologers and thought to have occultpowers), gold symbolized royalty/kingship,the frankincense, because of its use inreligious rituals, symbolized divinity, and themyrrh, commonly used as an embalmingagent, was suppose to have foretold death.

Boswellia and Commiphora spp. areall scrubby trees in the family Burseraceae(bursa = a hide or purse), and grow in aridareas near the southern coast of the Arabianpeninsula. Ideal conditions are hot tropicalsun, limestone soil for frankincense andbasaltic soil for myrrh, and salty dew blownin from the Arabian Sea. It is thought that2000 years ago, this area exported about 3000tons of frankincense annually to just Greeceand Rome, but today only a few tons areproduced each year, yet even the sands of thebeaches near the ancient ports still smell offrankincense. The bark of these trees ischipped away so that the milky sap oozes out,and after several weeks is hardened and readyto be collected. Frankincense hardens intotranslucent yellowish lumps, while myrrh is areddish-brown color.

Frankincense has been used inreligious rites in many of the Middle-Easternreligions from Egypt, Greece, Rome, India,and beyond. It has also been used inperfumes and other cosmetics – both

frankincense and myrrh were ingredients inthe sacred anointing oil of the Israelites. TheRomans in Nero’s time used it to mask theodor in their cremation rites. Historically, ithas also had a number of other medicinaluses. In modern Arabia it is used as aninfusion for upset stomach, burned as incensewhile the men are sitting around talking afterdinner, and chewed because it is thought to begood for the teeth and gums. Considering theblended aroma of sewage, garbage, dung,dead animals, etc. baking in the noontime sunin ancient cities, the burning of frankincenseand other incenses probably was a necessityfor other reasons.

The name “myrrh” is derived fromthe old Hebrew and Arabic word mur ,meaning bitter (myr also means ointment orperfume). Traditionally, demand for myrrhwas less and the cost higher thanfrankincense, although, today it is much morewidely used medicinally (both for humans andin veterinary medicine). Myrrh has been usedas an anointing oil, a fumigant and incense, incooking and embalming, and medicinally. Itpreserved and perfumed the royal mummiesof Egypt. Due to its antiseptic properties,myrrh is still used as a mouthwash and garglefor inflammations of the mouth and pharynxand used in some tooth-powders. It is alsoused externally on wounds. Burning myrrh issaid to repel mosquitoes. Myrrh containsconstituents that stimulate gastric secretionsand promote peristalsis, thus some peoplebelieve it is an aid to digestion. Historically,it has been used for cancer, leprosy, syphilis,and as an aid in chest and menstrualproblems, but there is inconclusive evidencethat it really works for these conditions.

III. MATERIALS NEEDED:frankincense and myrrh resins heat source

IV. PROCEDURE:1. Examine the resins, taking notes ontheir appearance, smell (and taste?).2. Your instructor will demonstrate“burning” the resins to release their odors.

Note that they are not actually set on fire, butrather, heated until they smolder – in normaluse, pieces are placed on smoldering charcoal.

V. DATA:Take notes on your observations of these resins (appearance, smell, etc.).

VI. CONCLUSIONS:Which of the smells do you like better?

January 4, 2016 94

Figure 68. Stirred, Agglutination in Rh8. Watch to see if any of the samples

show agglutination. This would appear asgrainy clumps of red blood cells (RBCs)suspended in a clear solution (as opposed to agenerally red color). Rh is slower toagglutinate, so don’t give up too soon.Reaction with a particular antiserum (=antibodies) indicates that you have that

antigen on your RBCs – you have that bloodtype. If no agglutination appears in any of thesamples, you have O� blood.

9. Note any agglutination in your labnotebook. Place the slide in the designateddishpan of 10% Clorox for decontamination.Properly dispose of your cotton ball/Kimwipeand your 3 toothpicks in the designatedcontainer.

10. MAKE SURE ALL BLOOD-STAINED ITEMS ARE DISPOSED OFPROPERLY. Nothing should go in theregular trash, nothing should be left on thetable tops, and nothing should be left on thefloor.

VI. DATA:Take notes on procedure, problems, etc. Record your blood type in your notebook and

in the computer as directed. Copies of all information will then be provided so that you cancalculate what percentages of the class are each of the blood types.

VI. DISCUSSION:1. Now that you know your blood type,

to whom (what blood type) can you give bloodand from whom may you receive blood?

2. Do you know the blood types of anyother members of your family? People whohave been in the military have had their bloodtyped, as well as anyone who has ever givento a blood bank. If you can get enoughinformation, try to figure out the Punnettsquare for your parents having you and yoursiblings and/or the Punnett square for you andyour spouse/fiancé having children. ABOwould be one gene and Rh would be another,

thus each alone would be a monohybrid cross,but the two together would be a dihybridcross.

3. For your blood type, figure out whatpercentage of the general population of theU. S. has the same ABO/Rh type as you.How do each of the percentages for thevarious blood types for the class compare tothe percentages of those types for the generalpopulation? In one previous lab sections,everyone in the class had either type O+ or O�,a rather atypical distribution.

January 4, 2016 7

HOW TO SUCCEED IN INTRODUCTORY BIOLOGY LABORATORYCopyright © 1998 J. L. Stein Carter

Welcome to this first semester ofthe science majors’ biology lab sequence. Weknow that students like to do well in theircourses. In order to help you do well in thiscourse, the following guidelines have beenprepared. Students who adhere to theseguidelines will be much more likely tosucceed in this course.

There are prerequisites for thiscourse. These prerequisites are not there tokeep students out of the course, but rather totell you what background you need to have inorder to understand the material presented inthis course. Students who lack theappropriate background knowledge will findthe material presented in this course to bedifficult, if not impossible, to understand.However, the University of Cincinnatirecently implemented a new computer systemto keep track of courses, registration, etc., andonly built into that system the ability to listother UC courses as prerequisites. Thus,while these prerequisites still stand, the newsystem allows no way to notify you of these inany of the University’s official publications.It is assumed that students in this science-majors’ course have successfully completedhigh-school biology, chemistry, and algebra.The purpose of this course is not to re-introduce the basic concepts learned in thosecourses, but to build on that foundation.There is not time in this course to re-explainterminology, concepts, and mathematicalcalculations which students should knowbefore enrolling.

If you lack these prerequisites, it isin your own best interest to not attempt totake this course at this time. In the past,students who had thought this didn’t apply tothem and that they were exceptions and could“handle it” have ended up either withdrawingor failing. You will do much better if youfirst take the time to prepare yourself properlyto take this course. If you cannot read at acollege level, you won’t be able to understandthe text book for the accompanying lecturecourse or the lab protocols for this course, soany needed developmental reading and/or

English courses should be completed first.Any needed developmental math coursesshould also be completed before taking thiscourse: you will be expected to performcollege-level mathematical calculations in thiscourse. If you have not had high-schoolbiology and/or chemistry, you should take atleast one quarter of General Biology and/orGeneral Chemistry, as needed, beforeattempting this course. If you have had nohigh-school nor junior-high science courses(and are taking other developmental courses),you may need to take the DevelopmentalScience course before attempting GeneralBiology or General Chemistry. See anacademic advisor (Dr. Peggy Hager is theacademic advisor for science majors) or theLearning Center staff for further guidance andinformation.

In order to do well in this course,you will need to spend an appropriate amountof time doing homework. As a national, if notinternational “given,” college students areexpected to spend at least two hours out ofclass on preparation and homework for everyhour spent in class, thus a student carrying 12credit hours should plan to spend a minimumof an additional 24 hr/week doing homework.Since this is a one credit hour course thatmeets for three hours/week, that means youshould plan on devoting anywhere from twoto six hours per week to homework for thiscourse – yes, YOU. If you have a job and/orfamily responsibilities that prevent you fromspending an amount of time whichcorresponds to the course load for which youare registered, you should reduce your courseload to reflect the amount of homework timeyou have available. Past students who hadthought they were different and could get bywith less (or no) study time have ended upwithdrawing or failing the course. Almost toa person, those who have needed to withdrawdue to lack of sufficient study time havecommented they thought they were different,thought they could “handle it,” but found outthe “hard way” that to do well requiresadequate time. Consider the following:

Reality Check – Where Does Your Time Go?In one week there are 7 × 24 = 168 hr/wkIf taking 12 cr hr if + biol lab @ 1 cr = 3 lab hr if also + chem lab @ 1 cr = 3 lab hr

= 12 hr/wk = 14 hr/wk = 16 hr/wkIt is a national, if not international “given” that college students need to plan on spending 2 hrout of class studying and doing homework for each hour in class, thus...

12 × 2 = 24 14 × 2 = 28 16 ×2 = 32so, added on, that’s

12 + 24 = 36 hr/wk 14 + 28 = 42 hr/wk 16 + 32 = 48 hr/wkIf that person is also working a full-time job (40 hr/wk), then added on, that’s....

= 76 hr/wk = 82 hr/wk = 88 hr/wk

January 4, 2016 8

But the person also has to get there. Some of you live closer and some live farther away. Thus,as a rough estimate, let’s say the person spends a total of 2 hr/da driving × 7 da/wk = 14 hr/wk.

= 90 hr/wk = 96 hr/wk = 102 hr/wkThe person also needs to eat some food to keep going. Let’s figure ½ hr each for breakfast andlunch and 1 hr for dinner, including cooking time. That’s 2 hr/da × 7 da/wk = 14 hr/wk, so

= 104 hr/wk = 110 hr/wk = 116 hr/wkLet’s give our person some time for religious observance (church, synagogue, mosque, etc.) ofabout 2 hr/wk, so that’s...

= 106 hr/wk = 112 hr/wk = 118 hr/wk

If our person sleeps the recommended 8 hr/night × 7 = 56 hr/wk, that’s= 162 hr/wk = 168 hr/wk = 174 hr/wk

Thus, at this point, the amount of time “left over” is6 hr/wk (< 1 hr/da) 0 hr/wk ��6 hr/wk

If, instead, the person gets inadequate sleep (which negatively affects mental functioning, thusstudying ability, grades, driving safety, etc.) of only 6 hr/night × 7 = 42 hr/wk, that’s

148 hr/wk 154 hr/wk 160 hr/wkThen, the “left-over” time becomes

20 hr/wk (= 2.8 hr/da) 14 hr/wk (= 2 hr/da) 8 hr/wk (~ 1 hr/da)

Out of that “left-over” time must come the following (and you can probably think of more):getting dressed in the morning, extra time at school in between classes, socializing (dates,texting, phone chats, etc.), shopping (groceries, etc.), house cleaning, caring for children, workingon the car, participation in sports, etc., doctors’ and dentist’s visits, watching TV, readingnewspapers and “extra” books, surfing online (U-Tube, Facebook, Twitter, etc.), etc., etc., etc.

A portion of the grade in this courseis based on demonstrated good record-keepingand documentation skills – necessary, butsometimes time-consuming tasks in anybiological or medical field. This is not a“fluff” course. This is not a course to take ifall you’re looking for in college is easy “A”s.If you don’t have the time, the interest, or theself-discipline right now to devote to suchtasks, you would be better off, gradewise, towait until you can devote the needed timeand/or energy to this course. If you arementally at a point in your life where you arefeeling burned out on school, would rather bedoing other things than studying for classes,or are unsure of where you want to go fromhere, that’s OK, but then you should considerpursuing those activities now. You canalways come back and take classes later whenyou are feeling more motivated to be here. Todo well in this, or many of your other classes,you need to be mentally, psychologicallyready to tackle the workload. Remember,your transcripts are “forever.”

You will enjoy this course muchmore if you give up any preconceived notionsyou may have about what you think thiscourse should not cover. Yes, we know someof you are Pre-Med or Pre-Pharm, but thatdoes not include everyone in the class. Thiscourse was designed to provide anintroduction to a broad spectrum of biologicaltopics. Yes, we’ll be covering somebiochemistry and anatomy-type topics, but wewill also be covering field biology andlearning about the plants and animals thatsurround us. All of these topics are valid

parts of the study of biology. Even doctorsand pharmacists don’t spend all their timeworking, and sometime when you’re at a parkwith your family, you (or they) just mightwish you knew the name of “that flower overthere.” Additionally, much currentpharmaceutical research is focusing onpotential drugs found in plants, for which aknowledge of the plants, themselves, isneeded. Allow yourself to enjoy this courseby experiencing the delight, the wonder, the“ah-ha!” of discovering something you didn’tknow before.

Many of you have, no doubt, heardthat places like College of Pharmacy or theMed School are very selective. Please beaware that the purpose of this course is NOTto give everyone “A”s so you can all end upthere. Realize that if we would give you all“A”s, the Med School and Pharmacy woulddeem those grades to be meaningless andrefuse to accept anyone from Clermont.College of Pharmacy and Med School don’twant “A”s just for the sake of the gradesthemselves, but rather, use those grades assigns that students have successfully masteredand know the material covered in this course.Your job, then, while you are here, is NOT toget an “A” at any cost, but to work yourhardest to absorb/learn/understand everythingyou can, because you’ll need it as afoundation for the topics covered in yourclasses down there. Cheating won’t allowyou to learn the background material youneed, and even if you aren’t caught here, thatwill catch up to you in future classes forwhich you are not prepared. Besides, would

January 4, 2016 93

Figure 65. Wax Circles

Figure 66. Antisera Added

Figure 67. Blood Added

cause someone else’s blood to enter your body(like stick yourself with a used lancet) noranything that would risk exposing anyone elseto your blood. To avoid exposure to anyblood-borne pathogens, each person shouldlance his/her own finger. If, for some reason,it becomes necessary to help someone else,wear gloves. Also due to Federalhealth/safety regulations, NO BLOOD ORBLOOD-STAINED ITEMS SHOULD BEPUT INTO THE TRASH CAN OR DOWNTHE DRAIN – this could endanger peopleand/or cause legal problems for the school. Aspecial container has been designated fordisposal of lancets (and other “sharps”).Toothpicks, cotton, etc. should be placed inthe specially designated container to bedecontaminated. Do not leave these itemslying around the lab – YOU MUST CLEANUP YOUR OWN MATERIALS!!! Yes, youknow your blood is safe, but the lab staff andmaintenance people that clean up the labwon’t know if it came from you or someoneelse.

Before performing this lab exercise,review safety issues and proper procedure byviewing and becoming familiar with theinformation presented on the Web page.http://biology.clc.uc.edu/courses/bio112/blood.htm.

1. Obtain a microscope slide. If it isnot clean, clean it. The slide must be veryclean so it does not interfere with thereaction.

2. With a wax pencil, draw two lineson one surface of the slide to divide thesurface into thirds (do NOT peel the waxpencils just to play with them). In each third,draw a large circle that fills the space. Labelthe circles “A,” “B,” and “D” or “Rh” in thecorner OUTSIDE the circles (Figure 65).Also, put your initials on a corner of yourslide.

3. Obtain three toothpicks (one foreach circle) and place near the circle forwhich each will be used. Obtain anAutolet®, load a lancet into it, and place anorange finger platform on it.

4. Place one drop of the appropriateantiserum (at room temperature) near theedge, but within each of the circles. Be verycareful – be sure you do NOT crosscontaminate the sera! Do NOT let them touchor mix (the wax pencil rings should help tocontain them). Do NOT let the droppers getmixed up.

5. Choose a little-used finger (usuallyleft ring finger). Clean this fingertip withalcohol on a cotton ball or Kimwipe, and let itair dry. Keep the cotton ball/Kimwipe nearbyand clean – you’ll need it again. Dangle thehand down to increase the amount of blood inthe fingers (don’t touch anything with theclean finger). Press on the “bottom” of thefingertip with the thumb of the same hand (tohelp hold blood in the fingertip) and with aquick, deep jab (press the orange platformtightly against your finger), lance the “fleshy”part of the side of the fingertip on the “little-finger side” of the tip. Note that the lancet issterile when you remove its cap, so do nottouch the tip with anything before using it.

6. Working quickly, let one drop ofblood drop into each circle but not touchingthe antisera yet. Do not get any antiserum onyour finger. Keep your arm below your heartand your hand below your elbow, andmassage the hand and/or finger towards thetip to get more blood if needed. When youhave your three drops of blood, you may thenapply gentle pressure to the wound with yourcotton ball/Kimwipe (use a clean one if yoususpect that your previous one may havebecome contaminated with someone else’sblood) to stop the bleeding. Note that theblood will flow better if the hand is helddown (below the heart) rather than up.REMEMBER TO PROPERLY DISPOSEOF THE LANCET BEFORE SOMEONEELSE GETS STUCK WITH IT. The cap doesnot need to go into the “sharps” container –only the lancet.

7. Using one toothpick for each circle,stir the blood and antiserum untilhomogeneous, but do not overmix. Do thisfor each of the three. Place on the Rhviewbox which will also warm the slide. Thewax pencil circles will help to keep thesamples isolated and contained.

July 1, 2013 92

(no B or AB) or where 40% of the peoplehave type B (with a mixture of the othertypes).

There are other blood factors too,such as the MN group in which a person canbe type M, type N, or type MN. Otherantisera are available to test for this bloodgroup. Note also that Rh factor is actually

also a multiple allele blood group like theABO blood group. The most common allelefor Rh factor is the D allele, and this is theonly one which is commonly tested. Althoughcomparatively rare, there are other alleles anda person who tests negative to the D allelecould possibly be Rh+ blood for one of themore rare alleles.

III. SAFETY CONSIDERATIONS:In this lab exercise, we will be

working with human blood, thus a number ofsafety precautions must be observed toprevent the spread of blood-borne pathogens.THINK before you act, use common sense,and do not do anything that could bring youinto direct contact with someone else’s bloodor risk exposing anyone else to your blood.Students who are behaving in an unsafemanner will be asked to leave the lab, and ifneeded, the security guard will be asked toescort such persons out of the area. Youabsolutely, positively MUST!!! CLEAN UPafter yourself, down to the last toothpick! Allsoiled items must be properly disposed of inthe designated locations. Make sure youobserve the following precautions:

1. ABSOLUTELY NO FOOD ORDRINKS SHOULD BE CONSUMED WHILETHIS LAB IS BEING PERFORMEDAND/OR UNTIL THE LAB ROOM ISTOTALLY FREE OF ANY BLOOD-STAINED ITEMS AND THE TABLE TOPSHAVE BEEN DECONTAMINATED.

2. Keep your hands out of your eyesand mouth.

3. Wear gloves if you need to helpsomeone else.

4. Use only a sterile lancet (if it hasbeen sitting out on the desk top – which itshouldn’t be – it’s no longer sterile, andmight be contaminated with someone else’sblood) on your finger. Do not set a lancetwithout its cap, loose, on the desk top – it willbecome contaminated with air-borne bacteriaor someone else’s blood. Also, do not attemptto recap a used lancet. Use only cleantoothpicks and Kimwipes or cotton balls.

5. When you are finished with the

lancet, immediately put it in the hard-walledsharps container. THIS IS THE ONLYPLACE WHERE USED LANCETS SHOULDBE PLACED, and ONLY “sharps” should beplaced in the sharps container.

6. Any blood-stained or potentiallyblood-stained disposables such as lancet caps,toothpicks, Kimwipes, and/or cotton ballsmust immediately be placed in the designatedlocation so that they may be disinfected by labpersonnel. Do NOT let any toothpicks dropon the floor – you will be using three, so youshould be disposing of three (if needed, countthem as you dispose of them). NONE OFTHESE ITEMS SHOULD BE PLACED INTHE REGULAR TRASH!

7. Blood-stained, glass microscopeslides should NOT be cleaned off at the sink.They should be placed “as is” into a 10%solution of Clorox in the designated dishpan.Gently place the slides into the solution sothat no splashing occurs (goggles to preventeye damage are strongly recommended). Donot let your fingers come into contact with thesolution. 10% Clorox is supposed to beeffective at disinfecting blood products, whichis why it is being used. When all the slidesare collected, lab personnel will see thatfurther steps are taken to insure properdecontamination.

8. If any blood drops anywhere, wipeup the spill with a Kimwipe or paper toweldipped in 10% Clorox-water to disinfect thearea, then place the paper in with the waste tobe disinfected. If the spill is large,immediately notify your instructor and ask forhelp, because the area must be treated with10% Clorox-water to disinfect it.

IV. MATERIALS NEEDED:clean microscope slidewax pencillancetanti-A, anti-B, and anti-D (Rh) sera70% alcohol (EtOH) and cotton or Kimwipe3 toothpicksRh viewbox

V. PROCEDURE:First, a note of caution: For many,

many years countless technicians have safelyperformed blood typing on patients. With therecent epidemics of the AIDS and hepatitis

viruses, however, someone might question thesafety of doing this lab. If you exercisecommon sense and caution, this will not be aproblem. Do not do anything that would

July 1, 2013 9

you want that kind of a pharmacist or doctorcaring for you and your family? Anotherpiece of this reality is that, despite everyone’sglorious plans for their futures, not all of youwill end up in Med School or College ofPharmacy or wherever. While I wish you allthe best of luck, the reality is that all of you,each and every one, yes YOU, need to startplanning now for “Plan B” just in case.Relax, enjoy the course, learn as much as youcan, earn the best grade you are honestlycapable of earning, and don’t make yourselfand everyone around you miserable byfocusing only on grades.

I can tell you that, over the years,many past students, while enrolled in thesecourses, have said (complained?) that thework needed to maintain their lab notebookshas not been “hard” work, but has required a

significant amount of time. However, to aperson, every graduate/alumnus who hasreturned for a visit has told us stories of howmuch better prepared they were, how muchfarther ahead of their classmates in upper-division courses, because of the discipline ofhaving to keep lab notebooks for Clermontbiology courses.

Please weigh your options and yourlevel of commitment carefully. If you havethe proper background knowledge for thiscourse, if you are willing to put the necessarytime into preparation and homework, if you’recurious, inquisitive, and interested in learningmore about the living world around you, andif you’re willing to work hard and try yourbest to learn the most you can and have fundoing it, your chances of succeeding in thiscourse are greatly increased.

July 1, 2013 10

MAKING AND KEEPING A LAB NOTEBOOKProtocol Copyright © 1982 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To learn how to organize and record scientific data.2. To furnish a record of experiments conducted, data gathered, and facts

learned for future reference.

II. BACKGROUND:We humans are good at forgetting

things, or perhaps only half rememberingthem, or perhaps remembering them wrong.Thus, over several thousand years, we havedeveloped written communication as a way ofrecording present findings and knowledge andpassing that information on to others.

This is especially important inscientific investigation. In science, as in otherfields, current knowledge is based on pastknowledge and future knowledge will bebased on what we discover now. Oftenscientists gather data over a period of severalyears before analyzing and drawingconclusions from them. These conclusionsand the new facts discovered are the buildingblocks for the next discovery. If someoneneglects to write down some data that aresignificant or records them in such a mannerthat they are unintelligible, they do theinvestigator no good, and may even lead to afalse conclusion. Scientists keep these data in

lab notebooks, and increasingly, computersare being used to analyze data.

However, what if you are notplanning to work in a biology lab? Of whatuse to you is all this record-keeping?Obviously, nurses, lab technicians, doctors,pharmacists, and social workers need to keeprecords on their clients, but a few minutes’reflection should reveal that there is some sortof record-keeping in any profession. Thegood record-keeping habits that you shoulddevelop as you create your biology labnotebook should aid you in maintainingdetailed records in whatever profession youhave chosen. Additionally, you may, in thefuture, find yourself in a position where ayoung person, perhaps your own child, needsideas for a science fair project or something todo on a rainy afternoon, or there may be someother reason you would want to rememberhow you did that experiment so long ago.

III. MATERIALS NEEDED:A. A printed copy of the lab protocols forthis class, available from our Web site at: http://biology.clc.uc.edu/courses/bio1081L/.B. A bound 10 × 7� in. compositionnotebook with graph ruling. All notes takenduring lab should be entered DIRECTLYinto this notebook.

C. A permanent writing utensil. Pencil,water-soluble marker, ballpoint pen (alcoholsoluble), and/or “Sharpie” markers (“bleed”through page) are not acceptable. BLACKcolor should be used. The best type of utensilto use would be a technical pen which writeswith India/drawing ink. Besides writing,these pens are also better for drawingillustrations and xerox better than ballpoint ifneeded. Such pens include theTombo Roll Pen, Jr.®, Pilot Precise®, and

Rapidiograph® as well as an old-fashionedcrow-quill pen and drawing ink (not reallyhandy for field hikes).D. Clear contact paper. This should havea smooth surface, not patterned, and shouldbe transparent enough to see the details ofspecimens mounted under it. Other factors touse when comparing various brands includeability to be lifted up to reposition something,cost, ability to be written upon, tendency ofthe adhesive to ooze out beyond the edges,and resistance to yellowing and shrinking.

E. Optional colored pencils to indicatecolor in illustrations. All illustrations shouldalways be outlined with your lab pen first, butoptionally may be shaded with appropriatecolors after that.

IV. PROCEDURE:INITIAL NOTEBOOK SET-UP:

A. Place your name, lab section, and seatnumber on the front cover of your notebook.B. With your book closed, mark acrossthe edges of all pages at the bottom edge ofthe first square, then count down two squaresfrom there, and draw a line across the edgesof all pages (again, following the printed

line). Count down six more lines, and drawanother line across the edges of the pages.These lines will guide you as you placehandouts and data on the notebook pages.C. Starting with the first sheet of paper,number the right-hand pages with oddnumbers (1, 3, 5, etc.) placed in the upper

July 1, 2013 91

BLOOD TYPINGProtocol and additional information Copyright © J. C. TanProtocol Copyright © 1980 D. B. FankhauserBackground and additional information Copyright © 1989 J. L. Stein Carter

I. OBJECTIVES:1. To study blood types as related to human genetics.2. To learn how blood types are determined.

II. BACKGROUND:Our blood cells have a variety of

chemicals on their surfaces called antigens(derived from antibody generating). Bloodcells from different people have differentantigens (anti = against, opposite) on them,and which ones a person has depend on theperson’s genes. A person cannot developantibodies against his/her own antigens – onejob of the immune system is to be able to tell“me” from an “invader.” Our immunesystems make proteins called antibodies thatfight against specific antigens. This is usefulin fighting off invading cells, but means thata person could develop an allergic-typereaction if exposed to blood cells fromanother person with a different blood type.

If you ever need to receive blood, itis important that you receive the proper typeof blood. The wrong type could trigger animmune response and cause agglutination(agglutin = glued together), or clumping, ofthe red blood cells (RBCs). Blood can betyped by mixing it with various antisera thatmimic this clumping behavior, indicating thepresence of the antigen being tested.Agglutination with an antiserum thusindicates that you have that blood type whilelack of agglutination indicates a lack of thatparticular antigen.

If a person with type A blood comesinto contact with type B blood, that personwill develop antibodies to type B and wouldhave a severe reaction to any further attemptsto give a type B transfusion. The opposite istrue for type B blood. People with type ABblood will not develop antibodies to either Aor B antigens and thus could receive any kindof blood (ABO blood group) as a transfusion.Type O blood can develop antibodies to bothA and B antigens and so can receive only typeO blood. However, type O blood will notcause types A or B blood to form antibodiesbecause it has no antigens. Thus, in anemergency, type O can be used as the“universal donor” (using the same type is stillpreferred and safest) while type AB isreferred to as the “universal recipient.”

Another “problem” involving bloodtypes is that of Rh or Rhesus factor, sonamed because first discovered in Rhesusmonkeys. The problem arises when an Rh�

mother has an Rh+ baby. The first baby isusually not affected, but if, during birth, anyof the baby’s blood comes into contact withthe mother’s, she could develop anti-Rh+

antibodies which could “attack” the blood ofsubsequent Rh+ babies. Note that Rh+

indicates the presence of the Rh antigen andRh� indicates a lack of this particular antigen.Because of potential Rh incompatibility, it issuggested to all first-time expectant parentsthat they have their blood typed. Postpartumshots (Rho-Gam) are now available whichtrick the new mother’s immune system into“thinking” that she already is producing anti-Rh+ antibodies so production of theseantibodies never starts. Also, an Rh� womanwho experiences a miscarriage should have aRho-Gam shot when she goes to the hospital.Note that an abortion, especially of the type inwhich the baby is pulled to pieces, would bemore likely to cause the mother’s blood to beexposed to that of her baby with possiblesubsequent Rh incompatibility than a regularbirth, especially since the instruments used toremove the baby also frequently cause smallto large lacerations on the woman’s cervixand/or uterus. Since Rh+ is the presence ofthe Rh factor and Rh� is the absence of thatfactor, Rh� blood can develop anti-Rh+

antibodies, but Rh+ blood which has the factorcannot develop antibodies against Rh or itwould destroy itself. Thus, Rh+ can acceptRh� blood, but Rh� cannot accept Rh+.

The human ABO blood group is anexample of multiple alleles for one gene.With eye color in humans, a person can beBB, Bb, or bb as brown or blue are the onlytwo choices for this gene. For the ABO bloodgroup gene, however, there are more choices.There are three possible alleles at this genesite, namely IA, IB, and i. Thus, a personcould be IAIA or IAi and have type A blood, IBIB

or IBi and have type B blood, IAIB and havetype AB blood, or ii and have type O. Oneallele is inherited from each parent, thus, ababy’s blood type is used as court evidence inpaternity suits. About 45% of the populationof the U. S. is type O, 42% type A, 10% typeB, and 3% type AB. About 85% of thepopulation is Rh+ and 15% Rh�. Thus, aperson’s chances of being, for example, O�

would be 45% × 15% or 6.75%. The rarestblood type in the U. S. would be AB� whichwould be only about 0.45% of the population.These various alleles are not evenlydistributed among all humans. In somecultures, 100% of the people have type Oblood, while other cultures exist where 75%of the people have type A and 25 have type O

July 1, 2013 90

Figure 63. Sample Pedigree

d. Two Different Coins, Two of Each

Coin 1 Coin 2 Individ. Tot. ÷6= Expected

HH HH

HH HT

HH TT

HT HH

HT HT

HT TT

TT HH

TT HT

TT TT

Totals 96 16 16

Record your data in the computer as requested so class data can be printed fordistribution. Do all calculations as indicated, and fill in your personal final results on the abovecharts. Include any other observations/notes you wish to take and try to answer all questions.Record data on corn and any other notes, observations, and answers to questions. Include allPunnett squares indicated.

VIII. DISCUSSION:1. How closely did your coin tosses come to the expected values? Do your individual data

or the lab data come closer to the expected values? Why?

2. Using square to represent males andcircles to represent females and using a whitecircle/square to represent blue eyes and a

black circle/square to represent brown eyes,draw a pedigree for as much of your family asyou are able (grandparents, parents, aunts,uncles, siblings, cousins, etc.). Can you tellwhich of the brown-eyed people areheterozygous? What is your genotype? Canyou predict what color eyes your childrenmight be likely to have? Note: gray eyes areconsidered to be a variation on blue, whilegreen, to further complicate things, is a totallyseparate gene. Apparently, a person can bedominant or recessive for the blue/green geneseparate from the blue/brown gene, and eyecolor is actually influenced by both.

From this pedigree, it becomes obvious thatthe people marked with an asterisk (*) areheterozygous. Can you figure out why/how?

July 1, 2013 11

Figure 2. Sample Notebook Page

right hand corner.D. Pages C-3 should be reserved for yourTable of Contents. Today, using the marksyou made on the edges of the pages asguidelines, in large capital letters, two lineshigh at the top of the page, label these pagesas “Table of Contents, p.1”, etc.

E. The last several pages of your notebookwill serve as cumulative lists of organisms

that were seen on field hikes. Starting fromthe last page and working forward, title apage for each of these topics:

WOODY PLANTSHERBACEOUS PLANTSBIRDSINSECTS, SPIDERS, AND OTHER

ARTHROPODSOTHER ANIMALSGREEK AND LATIN WORDSTEMS

NOTEBOOK PAGE FORMAT:

Pages 4 and following are for your specimens,data, any additional handouts, etc. Here aregeneral guidelines for page layout:A. You must take ALL NOTES ANDDATA DIRECTLY INTO THENOTEBOOK (more information is included,below). That means you should begin takingtoday’s notes on or after page 4, not on aseparate sheet of paper. The one exception tothis rule is that, for certain of the labs, a datatable is included in the protocol, and in thatcase, you may take your data on the protocol.

B. Title each page in bold CAPITALletters, two lines high (on lines 2 and 3,following the marks you made on the edgesof the pages). The title should tellspecifically what is on that page.C. Date each page on the upper leftcorner. Pages bearing handouts (such asprintouts of class data) should be dated withthe date the handout was placed in the book.If an experiment continues over several days,these dates may be added to the left-hand sideof the page as data are recorded.

D. Skip six more lines before starting towrite on line 9 of that page. These six lines

are to be used for cross references. Often,there is not enough room on one page for allthe data you may collect from a given lab, orperhaps you may be given computer printoutsof class data at a later date. If you don’t“guess” ahead of time how many pages areneeded for each lab and “run out of room,”you may continue your data in another placeIF you provide appropriate cross references,such as “computer printouts, p. 45,” “datacontinue on p. 32,”and/or “data continuedfrom p. 15.” Make sure that you referenceboth TO and FROM pages, and be specific.Do not just say “cross reference, p. 67,”which is not very useful.E. Use a new page for each topic, eachexperiment or hike. Do not put data from twounrelated topics on the same page, even ifthey were done on the same day. If datacontinue elsewhere in your book, use the sixlines under the title to cross reference to orfrom the appropriate pages (for example:“graph on page 35”), being specific in yourreferences. To get you started today, it issuggested that you begin taking “first day”notes on page 4.

OBTAINING AND DEALING WITH LAB PROTOCOLS:A. You must go to the Biology Web site athttp://biology.clc.uc.edu/courses/bio1981L/ assoon as possible to print a copy of the labprotocols for this course.B. You should, then, cut the pages in half

(forming 5½ × 8½ in. pages) and insure thatthey are in the correct order. Fill in therequested information on the cover page.C. Then, take the whole stack to a localoffice supply store to have them permanently

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bound with a black plastic cover on the backand a clear plastic cover on the front. This is

less expensive than what the bookstore wouldprobably charge.

CUTTING HANDOUTS AND USE OF CONTACT PAPER:A. Use clear Contact Paper to mount allspecimens and any additional handouts.Include at least one “souvenir” specimen fromeach hike (identified and the page dated).Any writing on the page should be done firstif possible, since it is impossible to write onContact Paper with these waterproof pens.B. For many of the labs we will be doing,you will be asked to submit your data onlineon our Web site. Then, once everyone hashad time to submit their data, you areresponsible for returning to the Web site toprint out a copy of the class data. Thatprintout must be included in your labnotebook. In order to fit into your labnotebook, it is suggested that these handoutsbe trimmed so you end up with pages whichare 5½ × 8½ inches.

C. It is suggested that (before removingthe wrapper?) you cut the 18-in. wide roll ofcontact paper in half down the center to maketwo 9-in. “mini-rolls.” Then, cut off pieces6½ in. wide. These should suffice for most ofthe handouts, giving a ½ in. sticky border all

around the page. If you just randomly chopup the roll, you will waste a lot of contactpaper to get what you need.D. Remove the backing from the contactpaper, then place the contact paper on a table(desk, etc.) STICKY SIDE UP. Position yourpaper or specimen on the contact paperRIGHT-SIDE DOWN avoiding bubbles bypressing from the center out to the edges. Forpapers, it helps if you hold them curved so thecenter of the page touches the center of thecontact paper first, then gradually “roll” thepage down onto the contact paper. Be verycareful when positioning handouts--if you tryto reposition them, the “ink” will stick to thecontact paper and smudge.

E. After the handout or specimen is on thecontact paper, turn the whole thing over andposition it in your lab notebook. Press fromthe center out to make it adhere. If you wishto be able to see both sides of a specimen ora field quiz with notes on the back, you maycut out a hole in the page before mounting thespecimen and put contact paper on both sides.

ENTRY OF DATA:A. All data and observations must beentered DIRECTLY into the lab notebook.In science, thoroughness and accurate recordsof first impressions are of greater importancethan “prettiness,” so DO NOT RECOPYnotes from another page into your labnotebook — if you wish to include somethingon a separate page, use contact paper to putthat page directly into your lab notebook.Both notes from field hikes and data from labexperiments should be included.B. Anything that can be illustrated shouldbe – do as many illustrations as possible.This both enables you to learn to concentrateon really looking at the item in question andhelps you to remember what you saw.Illustrate all new equipment as it is used andlabel significant parts. Illustrations should beLARGE enough to clearly show the detailsyou observed. All illustrations and graphsshould be clearly labeled, both as to whatthey are (a “title”) and also the significantstructures, axes of graphs, coordinates,magnification, etc.

C. For each hike we take, your notebookshould include three things: a) your notes onwhat we saw, b) at least one identifiedspecimen (the more species you get, the easierit is to remember what you saw) of somethingyou saw on the hike (but take ONLY whatyou need – do not rip out huge handfuls ofplants only to throw them away – have respect

for the life around you), and c) a map of ourpath with significant features (includingwhich way is north ) and locations noted forall new species seen. Specimens should bepressed and dried (in a phone book?) beforemounting under contact paper, or they will getmoldy. Mounted specimens should be labeledwith their identity and location collected, andthe page they’re on should bear the date andname of the hike on which they werecollected. Data on new species seen shouldalso be transferred to your cumulative lists inthe back of the notebook.D. While things should be reasonablyorganized and intelligible, you are not beinggraded on prettiness of the notebook. If youmake a mistake, just draw one, single line(NOT an ink blob) through it to indicate thatit was an error and start over. Do not tear outpages or use white-out!!! If you need to putsomething on a separate sheet of paper, donot recopy the information into the notebook,but rather, contact-paper in the loose sheet.

E. If excessive bleed-through of ink is aproblem in your notebook (the paper and pensvary somewhat), you may use the right-handpages for notes and illustrations and left forhandouts and specimens (reverse if you areleft-handed). However, if you choose to dothis, the data on the right-hand pages shouldgo with the handouts on the left-hand pages.

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VII. DATA: PART BEnter your numbers into your lab notebook and into the computer as requested.

Tabulations of class data will be provided via computer printouts.a. Single Coin

Side Individ. Tot. % Expected

H

T

Tot. 100 100 100

b. Two of Same Coin

Side Individ. Tot. % Expected

HH

HT

TT

Tot. 100 100 100

c. Two Different Coins

Coin 1 Coin 2 Individ. Tot. % Expected

H H

H T

T H

T T

Totals 100 100 100

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should be deaf and the owner would know notto use him for further breeding. DrawPunnett squares to illustrate these twopossible crosses. Also, using Punnettsquares, show how two hearing dogs couldproduce deaf offspring.

b. Incomplete Dominance: In four-o-clock flowers (as well as roses, hibiscus,snapdragons, etc.), if a red-flowered plant iscrossed with a white-flowered plant, theoffspring all have pink flowers. If two ofthese pink flowers are crossed, some of theoffspring are red, some are pink, and someare white. Draw the Punnett squares toillustrate the first and second filial (F1 and F2)crosses and predict how many of the F2offspring should be each of the three colors(red, pink, white). Hint: this is one of theextremely few cases where it may be helpfulto use different letters (R for red and W forwhite), although “r” is often used, even in thiscase, for white flowers.

[A bit of explanation: for many ofthe genes we’ve discussed so far, thedominant allele codes for “make xxx”, andthe corresponding recessive allele codes for“we don’t know how to make xxx” resultingin whatever is the “default” phenotype.However, in this case, the “R” allele is codingfor “make red” and the “r” or “W” allele iscoding for “make white” (not just absence ofred), thus an individual who is Rr (RW)would have two sets of instructions – “makered” plus “make white” – resulting in pink.]

c. Trihybrid Cross (tri = three): InGuinea pigs, black hair (B) is dominant overwhite (b), rough coat texture (R) is dominantover smooth (r), and short hair (S) isdominant over long hair (s). Determine thepossible gametes, then diagram the Punnettsquare for a cross between a rough, black,short-haired Guinea pig (homozygous) and asmooth, white, long-haired one (assumingthese three genes are on separatechromosomes). What would the phenotype ofthe offspring be? If two of the offspring werecrossed, draw the Punnett square for the F2generation. Hint: each time, make a list ofthe possible gametes first, making sure eachhas exactly one copy of each of the genes.What would the genotype and phenotyperatios be for the F2 cross?

d. Sex-linked Genes: Sex-linked genesare usually X-linked genes because the Xchromosome is longer than the Y, thus X-linked genes are often indicated by X� orsomething similar. There is no correspondinggene on the Y chromosome (but women whohave two X chromosomes could have the“opposite” – dominant or recessive – allele ontheir other X chromosome). Red-green

colorblindness (and hemophilia) is an X-linked recessive gene in humans. If a manand woman, both with normal vision, marryand have a colorblind son, draw the Punnettsquare that illustrates this. If the man diesand the woman remarries to a colorblind man,draw a Punnett square showing the types ofchildren that could be expected from thismarriage. How many of each could beexpected? Also, as mentioned above,androgen insensitivity syndrome (AIS) is anX-linked recessive allele which causes theperson’s body to be insensitive to the effectsof testosterone. Draw the Punnett square fora cross between a woman who is a carrier forthe AIS allele and a normal man. Whatwould the phenotype ratio of their childrenbe?

e. Y-linked Genes: An extra finger(total = 6) on each hand is a dominant traitcarried on the Y chromosome. If a man withthis trait marries a normal woman, draw thePunnett square for this marriage. Whatproportion of the offspring could be expectedto have this trait? Does the sex of theoffspring influence the presence of the extrafinger? Could one of their daughters pass thistrait on to her sons? Why would all Y-linkedtraits be dominant traits?

f. Sex-influenced Traits: Baldness inhumans is a dominant, sex-influenced trait.This gene is on the autosomes, not the sexchromosomes. A man who is BB or Bb willbe bald and will be normal only if he is bb. Awoman will only be bald if she is BB andnormal if she is Bb or bb. It’s almost asthough “B” was dominant in men and “b” inwomen. Another sex-influenced trait iscomparative length of index and ring fingers– most men have the ring finger longer andmost women have the index finger longer.

1. If two parents are heterozygousfor baldness, what are the chances of theirchildren being bald? Use a Punnett square toillustrate this. Note: you need to set this upas a dihybrid cross to include the sex of thechildren because XXBb will have a differentphenotype than XYBb.

2. A non-bald man marries a non-bald woman. They have a son and a daughter.If the son becomes bald, what are the chancesthat his sister will, too? Use a Punnett squareto show this cross.

3. A woman’s mother is bald buther father is not. Her brother is rapidly goingbald. She is an acrobat who hangs by herhair. Should she change her profession beforeshe goes bald (and maybe become ageneticist?)? Use a Punnett square to showthis.

VI. DATA: PART ATake notes, answer questions, draw and label everything for the slides you examine.

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F. As soon as possible after an experimentis completed, while it is still fresh in yourmind, analyze data and draw conclusions. Bydoing these things as you go along, yournotebook will be more organized, and also,there will be less work to do the night beforethe notebooks are due. All tests, quizzes,grade sheets, etc. should also be placed intothe notebook. When these items are returnedto you, you should take notes on correctanswers to questions, items to be improved ordone differently, etc.

G. If you are absent and need to get notesfrom a classmate, you must cite the source ofthat information--otherwise it’s plagiarism.Do your classmates the courtesy ofacknowledging their hard work.

H. If you run out of space and need to starta second book, you should give that book itsown table of contents and start, again withpage 1, etc. Do not continue with page 166,etc. – that’s too confusing.

FORMAT FOR TABLE OF CONTENTS:A. When your notebook is turned in, youwill need a computer-generated Table ofContents, mounted with contact paper. It issuggested that you set up a computer file atthe beginning of the semester, and add newpages after each lab session.B. Again, if you get that word processor orspreadsheet file set up now, and keep up withentering page information, then all you’llhave to do the night before notebooks are dueis to print the file and mount it in your bookwith contact paper.

C. Optionally, if it is helpful to you, asyou enter data in your notebook, you maycreate a temporary table of contents bywriting the required information on the pages

reserved for the Table of Contents, and thenmount the typed version over the written one.D. To create your table of contents, listEACH INDIVIDUAL PAGE NUMBER downthe left side of the page, the correspondingtitle of the specific experiment or subjectmatter on that page in the center, and the dateon the right. List EACH page in order one-by-one. If a page is blank, enter its number,but leave the title and date columns blank.Later, if you put something on that page, youcan type up a small strip of information tocontact-paper over the original blank spot.

E. To receive “excellent” for this categoryon your notebook grade, the table of contentsshould be done using a computer.

Page Topic Datecover Table of Contents

1 Table of Contents2 Table of Contents3 Table of Contents4 Notes on How to Keep a Notebook 10-VI5 Notes on Lab Safety 10-VI6 I Fell in the Mud Hike Notes 20-VI7 etc., etc. 30-VI

CUMULATIVE LISTS:A. As previously mentioned, on the lastseveral pages of your notebook, keepseparate, cumulative lists of organisms seenon field hikes, including separate pages for:

1. WOODY PLANTS2. HERBACEOUS PLANTS3. BIRDS4. INSECTS, SPIDERS, AND OTHER

ARTHROPODS5. OTHER ANIMALS6. GREEK AND LATIN WORDSTEMS

B. List each species that we saw, once, inchronological order by the first time it wasseen. For each species, list 1) common name,2) scientific name (look up in a field guide--insects may be more difficult), 3) the family

and/or order to which the organism belongs,4) the date we first saw it on a hike, and 5)the page number(s) in your notebook wherefurther information about this species can befound (where it was seen, what it looked like,etc.--your hike notes).C. For the wordstems, list each wordstemand its translation. Include page references tothe protocol or class notes where thewordstem and its definition were given.

D. To receive “excellent” for this categoryon your notebook grade, these cumulative listsshould be done with a word processor. Notethat you, personally, will be held responsiblefor the correctness and completeness of the

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information (including spellings) that hasbeen included.E. In the past, students have occasionally“borrowed” cumulative lists from one, hard-working student in the class. If you choose touse someone else’s work in your cumulativelist (not suggested), you must give credit byciting the author of that work--otherwise it’s

plagiarism. You must also include your ownpage references, etc. You will be heldpersonally responsible for any errors in thecumulative lists in your notebook, and sayingthat someone else did it is not an acceptableexcuse. In general, kind-hearted, well-meaning students are discouraged from doingall the work for the whole class.

# Common Name Scientific Name Family Date Pg1 Dandelion Taraxacum officinale Compositae 20-VI 332 Queen Anne’s LaceDaucus carota Umbelliferae 25-VIII 65

NOTEBOOK ILLUSTRATIONS:Notebook illustrations are intended to recordvisual data, including shape, unique traits,relative size, relationship to other features,etc. They require you to LOOKCLOSELY at the object or specimen,reinforce its structure in your mind, and allowreview of these data in the future. Do not tryto rush through drawing illustrations, but takethe time to really look closely at the itemyou’re trying to draw, think about all itsstructures, and consider how you can bestrepresent those in a drawing. The followingguidelines should make your illustrationsuseful for this purpose.

A. Illustrate a single (large) illustrationper page unless it is a secondary illustrationto expand on the primary subject of the page.Multiple pieces of equipment with related usemay be drawn on the same page.B. The name of the illustration should bethe title of that page (don’t forget the date).C. Below the title, give the crossreference(s) to the location(s) of the portion ofthe protocol which you followed and any textor resource (protocol or your notes) whichmight give additional information on thedrawing and its significance.D. For microscopic views, first scan theentire specimen to find a characteristic viewwith all features mentioned in the protocol.Adjust the microscope (lighting and focus) foroptimum resolution.E. Draw a LARGE line drawing of theobject or specimen with a black lab pen.Make it fill most of the page. Eachmicroscopic specimen examined should haveits own page, although 100× and 400× viewsmay be placed on the same page. It is notnecessary to enclose your drawing in a circle

– just illustrate the specimen. If desired, youmay subsequently color in your illustration torepresent actual colors seen. Do not useunrealistic colors just to use color. Do not usecolored pencils to make your initial drawingbecause they are too faint and indistinct andbecause they can run if they get wet.F. Label all features listed in the protocolor mentioned in lab. Take care to spell themcorrectly (refer to the protocol if in doubt).G. Briefly describe the function orsignificance of each feature.H. In the legend below the illustration,especially for microscope slides, give thesource of the specimen (if known), thepreparatory treatment (staining, etc.) to whichit was subjected, and the stain’s specialsignificance (if any) to the features observed.I. Give the power of magnification to thelower right of your illustrations for allmicroscopic views.J. On the page where the protocol ismounted, cross reference to the page wherethe illustration may be found. Also, a noteplaced in the margin by the correspondingcommentary could be helpful.K. Use any excuse for an illustration--illustrate every new piece of equipment orapparatus, labeling all functional features(name and function). Draw at a scale largeenough to clearly show the necessary details.Color may be used to show the actual color ofthings seen. This often makes the illustrationmore valuable as a record of what you saw.Title your graphs, labeling coordinates, notingsignificant points/phases, especially accordingto time, and giving conditions under whichthe experiment was performed.

ANALYSIS OF DATA:A. For each lab exercise, at least one pageshould be set aside for conclusions andanalysis of data. This page should containthree sections, labeled in capital letters alongthe left edge of the page with indented textunder each heading: ANSWERS TO

QUESTIONS (most protocols contain thoughtquestions to be answered), CONCLUSIONS(in your own words, in detail, whatconclusions can you draw from the datagathered and how can unexpected data beexplained; what did you learn from this lab?),

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Record your numbers in the chart for Step Cand in the computer.

d. To simulate possible offspring of adihybrid cross produced by random union ofan egg and sperm from two parents eachheterozygous for two traits obtain four coinsof two types (2 pennies + 2 nickels) and place

together in a container. Shake thoroughly andtoss out onto the tabletop. Tally the resultsfor 96 such tosses on the chart for Step D.Rather than figuring out percentages, divideall your numbers by 6 to obtain how many outof 16 were of that type. Enter your numbersonline.

� � H T � � B bH HH HT B BB BbT HT TT b Bb bb

Figure 63. Punnett Squares

3. Geneticists use Punnett squares toshow what can be expected from a crossbetween two parent organisms.

First, the possible gametes fromeach parent are determined (like Steps A or Cabove). The possible gametes from the maleparent are usually written across the top of thePunnett square and those from the femaleparent are usually written down the left sideof the square. From this, the possiblegenotypes of the offspring are calculated byfilling in the small boxes. Figure 62illustrates Steps A and B above. “B”represents the brown-eyed allele, “b”represents the blue-eyed allele, and HH, HT,and TT have been converted to theappropriate genotypes. The Punnett squarethus obtained is 2 × 2 or 4 boxes in size. One

of the gametes (technically, which alleleended up in that sperm) from the father hasbeen written above each column and one ofthe gametes (technically, which allele endedup in that egg) from the mother has beenwritten in front of each row. Geneticists listthe dominant allele first. The male gameteshave been copied into the boxes under eachand the female gametes into the boxes next toeach, placing the dominant allele first. Thisgives four possible genotypes for theoffspring, which correspond to the results ofStep B above. From this, figure out theexpected frequency/percentage of eachgenotype and phenotype of offspring.

4. From Steps C and D above, let coin 1 represent some gene, A, where H=A and T=a. Letcoin 2 represent some gene, B, where H=B and T=b.

Draw a Punnett square that is 4boxes on a side (total of 16 boxes). “Translate” the gametes from Step C into A,a, B, and/or b and write in along the sides ofthe square (you should have four gameteswith one of each gene for each parent – AB,Ab, aB, ab). Remember: gametes should haveONE ALLELE FOR EACH GENE! Fill in

the boxes and the results should match withwhat you got in Step D. Note that whendoing a dihybrid cross, the “A” alleles shouldbe written next to each other and the “B”alleles should be kept together. Determinethe genotype and phenotype ratios that youwould expect from this cross.

5. Individually, examine the monohybrid (mono = one) and dihybrid crosses illustrated bycorn. Count at least three rows of kernels, but please do not mark on the corn. Ears of cornrepresenting other genetic combinations may also be available.

What genes are illustrated by thesecrosses and what are the two alleles for eachof these genes? Which is dominant and whichis recessive – how can you tell? DrawPunnett squares for the first filial (F1) andsecond (F2) crosses (fili = son or daughter).

For each Punnett square, what genotype andphenotype ratios would be predicted? Countthe kernels of corn. How closely to thepredicted phenotype ratios do these samplescome?

6. Do these sample problems to illustrate various principles of genetics (if you run out of timein lab, these may be done on your own). Optionally, these may be done over the Web, but if youdo that, do not neglect to take notes in your lab notebook.

a. Test Backcross or Testcross: Anorganism which has two of the same allelesfor a trait is said to be homozygous (homo =like, alike). When the two alleles aredifferent (1 dominant and 1 recessive), theorganism is said to be heterozygous. In dogs,there is an hereditary deafness caused by arecessive allele, “d.” A kennel-owner has amale dog that she wants to use for breeding

purposes if possible. The dog can hear, so theowner knows his genotype is either DD orDd. If the dog’s genotype is Dd, the ownerdoes not wish to use him for breeding so thatthe deafness allele will not be passed on.This can be tested by breeding the dog to adeaf female (dd). If the male’s genotype isDD, all of the puppies will be able to hear(Dd), but if his genotype is Dd, half of them

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including those found in cabbage varieties,including broccoli, brussels sprouts, andcauliflower, and it has been noticed thatoften, people who are TT dislike the taste ofthose (and other strongly-flavored) foods,while, in general, non-tasters tend to be morewilling to eat a more varied diet, includingmore dark-green, leafy vegetables.

Thiourea is another chemical thattastes very bitter to some people (dominanttrait) and is tasteless to others (recessive).

A third chemical that tastesdifferent to different people is sodiumbenzoate, a controversial food preservative.Typically, a solution of 0.1% is used for foodpreservation, and researchers have shown thata 0.1% solution was tasted by about 25% ofthe people on whom it was tested.Interestingly, however, for people who aretasters, the taste varies. Some people think ittastes sweet, some salty, some sour, and somebitter.

d. Tongue Rolling: People who canroll their tongues into a tube have at least oneR allele (R�) and those who can’t are rr.

e. Widow’s Peak: If your hairlineforms a “V” on your forehead, you are W�and if your hairline is straight, you are ww.

f. Ear Lobes: If your earlobes aredetached, you have at least one E (genotypeE�), but if they are attached, you are ee.

g. Little Finger: Hold your hand outwith fingers together. If the end joint of yourlittle finger bends in, you are F�. If it is

straight, you are ff.h. Hitchhiker’s Thumb: Hold out your

hand like you’re hitchhiking. If your thumbbends back at quite an angle, you are H�. Ifyour thumb is fairly/nearly/almost straight,you are hh.

i. Mid-digital Hair: Look closely atthe middle segments (not knuckles or joints)of all of your fingers to see if any of themhave hair growing on them (If it’s a finger youuse a lot, the hairs may be worn down tostubble, so look closely.). If there is any hairon any of them, you are D�. If all yourmiddle segments are totally bald, you are dd.

Considering just these 9 traits, andassuming all are simple one-gene-with-two-alleles situations would give 29 or 512different possibilities, and the lab room onlyholds 20 students. Note that, in humans with23 pairs of chromosomes, a gamete wouldhave 223 = 8,388,604 possible combinations ofchromosomes (each bearing numerous genes)from that parent. Any couple could have 223

× 223 = 70,368,744,177,644 (70 trillion)different possible children, based just on thenumber of chromosomes, not actual genes.Thus, based on the number of chromosomes,the chance of 2 siblings (other than identicaltwins) being exactly identical is 1/70 trillion.To make things even more complex,crossingover, or exchange of segmentsbetween homologous chromosomes duringsynapsis, adds further variation.

2. Work individually on the following coin tosses, recording your data on the following charts(in the Data section). When you have all your numbers, record the specified data on thecorresponding Web page. After everyone has entered their data, you may print out the class data.

a. Most organisms have two sets ofchromosomes, and only one from each pair ispassed on, at random, to the offspring throughthe process of meiosis by which eggs andsperm are formed. Thus, since thechromosomes contain or are made up ofgenes, the eggs and sperm get only one allele(alternate forms for genes; allelo = oneanother, parallel) for each gene. Obtain acoin. Let us assume that heads represents,say, brown eyes and tails represents blueeyes, and that this “parent” has one allele forbrown eyes and one for blue (a heterozygote– hetero = other, different, zygo =a yoke). Toillustrate the chances of having one or theother of these alleles in any particular gamete(egg or sperm – gamet = a wife or husband),toss the coin on the tabletop 100 times. Using“chicken scratches” record the number ofheads and the number of tails on the chart forStep A.

b. To illustrate the outcomes when thepossible alleles from two heterozygousparents unite in an offsprin g, obtain twocoins of the same type (two pennies?). Tossthese, together, 100 times. Tally and record

on the chart for Step B the numbers of a) twoheads, b) one head and one tail, and c) twotails. As in the previous part, let us assumethat these represent eye color in humans. Inhumans, brown eye color is dominant overblue: that is, if an individual has one allelefor brown and one for blue, that individualwill have brown eyes. Thus, in your cointosses, HH or HT would be brown-eyed,while TT would be blue-eyed. What ratio ofbrown to blue eyes did you get (i. e. whatratio of HH and HT combined versus TT)?

c. In a dihybrid (di = two) cross,geneticists look at two traits (such as eyecolor and tongue-rolling ability) passed onfrom parents to offspring. Assuming that thetwo genes involved are not on the samechromosome, they are inherited independentlyof each other. To show the possible gametes(eggs/sperm) produced by a parentheterozygous for both traits, obtain twodifferent coins (penny and nickel) and tossthem together 100 times. Record how manyof each of the combinations H1+H2, H1+T2,T1+H2, and/or T1+T2 are obtained. Whatratio/percentage of each were obtained?

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SUGGESTIONS (where did you haveproblems, what can be improved, and mostimportantly, what further experimentation is

suggested by the results of this experiment).A suggested page format is:

B. After each experiment is completed,analyze your data as indicated, draw anyconclusions that you can, and answer anythought questions in the handout in fullsentences (since you must do this on aseparate page and don’t have the questionsthere to refer to them when you are studyingfor a test). Also, make note of anYthing thatcould/should be checked into in more depth infurther experiments, questions which thisexperiment raises for you, and/or suggestionsfor improvements, changes, or other related

experiments. Note any difficulties youencountered in doing each experiment.

C. You are not to just do the experiment,but also to spend time thinking about it.Why are you here? What did someone hopeyou would learn by taking this lab? Why didwe bother to do this experiment? What didyou learn from it? What do your resultsmean? Thus, part of your notebook grade willbe based on how much thought you put intoyour conclusions, discussion, and suggestions.

WHEN THE NOTEBOOK IS TURNED IN:*** WARNING ***

My policy is that assignments will have 10% per class perioddeducted if turned in late. Since notebooks are worth 200 points, thatmeans that for every lab period a notebook is late, 20 points will bededucted from your score. No books abandoned in my mailbox willbe considered as turned in, nor will they be graded – your portion ofthe grade sheet must be filled out and turned in with the notebook.

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111 LAB 1997 1st GRADE NAME: SEAT: SECT:LAB handout not/data conclus HIKE map list specim

Syllabus (3) 24-IX Intro HikeNotebook How-To (6) 1-X Ordovician FossilsAbstract How-To (2) 15-X Quiz HikeGraphing (2) 29-X ?Collection (8) LAB handout not/data conclus

Binoculars (2) Sug. in Pop Data (5)Observ. & Concl. (2) Lipids (5)Acc. & Prec. (4 ) Spectrophotometer (6)Acc. & Prec. Data (4) Spectro. Computer (3)Acc/ Pre Pop Quiz (1) First Hike Quiz (1)pH (4) Microscope (5)pH Computer Data (6) Beer (5)Sugar in Pop (4) PB & J Sand. (1)ILLUSTRATIONS? (list page numbers)Lab Map w/ Shower 100 mL Vol. Flask Mayo EquipmentNotebook Page Set-up Balance/Weights SpectrophotometerObs/Concl Organism Interpolation CuvetteOpp & Alt. Leaves dH2O w/ Siphon 1.0 & 5.0 mL PipetsSimple/Cmpd Leaves pH Meter Beer’s Law GraphMeniscus pH Electrode MicroscopePasteur Pipet/Bulb Sample pH Paper Antlion Pit250 mL Beaker Stove (“Big Bertha”) Other (Specify):100 mL Grad Cylinder ThermometerSUGGESTIONS? (list page numbers)

OVERALL excellnt20

good15

adequat10

poor5

needsimprvmnt

01. Table of contents with page titles & dates, word

processing, odd pages numbered, each pagelisted

2. Waterproof ink, pages titled in large capitalletters, dated

3. All handouts present & mounted with contactpaper

4. New page for each experiment/hike, crossreferences, organization

5. Detailed notes from each experiment directlyinto notebook, graphs. &/or illustrations asneeded

6. Specimen(s), map, & notes on species seen foreach hike

7. Quality of discussion/conclusion sections(answers, summary)

8. Suggestions for further experimentation &/orimprovements (part of conclusions)

9. Cumulative lists of species seen with allinformation included word processing

10. Handed in on timeTOTAL:

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enable them to swim.Reportedly, 2n = 8 for this

organism. Remembering that grasshoppersare an example of XO inheritance, technicallythe 2n number should be 7 (because there isno Y to pair with the X chromosome), andthus, in anaphase I of spermatogenesis, onedaughter nucleus should get 3 autosomes andan X chromosome while the other should getonly the 3 autosomes. (Female grasshopperswould have two X chromosomes, thus, inoogenesis, all daughter cells would beexpected to receive 4 chromosomes.)

5. Animal Meiosis/Mitosis: Ascarismegalocephala is a type of large roundwormthat parasitizes horses. This slide is a cross-section (x.s.) of a female, and at the point inher body from where these slides were cut,most of what’s visible is the uterus. Theprocess of egg development in Ascaris issimilar to humans: before the “egg” hascompleted meiosis, first the sperm nucleus

enters. That triggers completion of meiosis inthe “egg,” including formation of polarbodies, then the sperm and egg nuclei unite toform a zygote, then the zygote begins todivide, forming a 2-celled, then a 4-celled,etc., embryo. This whole process is usuallynot visible all on one slide, so depending onwhich slide you are viewing, you will seeportions of this process. Depending on whichslide you are viewing, the uterus containsnumbers of either “eggs” in the process ofbeing fertilized and undergoing meiosis, ornewly-fertilized and rapidly dividing zygotesand young embryos. Note that if you areviewing a slide with zygotes/embryos on it,you may see both 2-celled and 4-celledembryos, in various stages of mitosis.Observe cells in each stage of mitosis. Whenare asters present? Find a cell in which thechromosomes are well spread and try to countthem – what is the chromosome number ofthis organism?

V. PROCEDURE: PART B1. As a class, examine various human traits which are each thought to be controlled by onlyone gene. Note: many of these, previously thought to be one-gene traits, are turning out to becontrolled by several genes. Traits which may be examined include:

a. Sex: We all have at least one Xchromosome. Usually, a person with two Xchromosomes (genotype XX) is female, and aperson with a Y chromosome (genotype XY)is male. However, it is important toremember that sex is a phenotype, not agenotype. Sex is not about how many X or Ychromosomes a person has, but rather, howthe genes on those (and other) chromosomesare expressed.

In humans, sex is a phenotypewhich is determined by the influence or lackof influence of the genes located on the Ychromosome, whereas in fruit flies, sex isdetermined by the effects of the ratio of thenumber of X and Y chromosomes (if anabnormal number). Thus in humans, XO andXXX would typically also be female whileXXY would typically be male. In humans, acombination such as XXXY would produce amale phenotype because of the effects of thegenes on the Y chromosome, but in fruit flieswould produce a female phenotype because ofthe relatively larger number of Xchromosomes. However, to “complicate” thesituation, in humans, there is an X-linkedrecessive mutation for androgen insensitivitysyndrome (AIS) which causes all cells in theperson’s body to be insensitive to the effectsof testosterone. Thus, even if such a personwould have a Y chromosome, most of itsalleles would have no effect and that personwould be female.

b. Eye color: We typically talk aboutthis in terms of B� coding for brown eyes,and bb coding for blue eyes. Again this is anover-simplification. According to the OMIM

Web site, there are at least two genes whichcontribute to eye color. One of these geneshas an allele that codes for “make brownpigment” and another allele for “we don’tknow how to make brown” = default bluecolor. The other gene has an allele for “makegreen pigment” and an allele for “we don’tknow how to make green”. While, startingout, it may be easier to understand howgenetics works if we only look at the brown-blue gene, in reality, eye color phenotype is acombination of the effects of the alleles forboth of these genes.

c. P T C - T a s t e r : P T C(phenylthiocarbamide) is a chemical thattastes bitter to some people while otherscannot taste it at all. Here, too, while we tendto talk about this in terms of tasters (T�) andnon-tasters (tt), the reality is a slightly morecomplicated situation. This gene actually hasmultiple alleles, one for “tasting” and at leasttwo different forms of “non-tasting.” Peoplewho are TT often find the test paperextremely bitter, while people who are Tt mayfind it bitter, but not extremely so. There isalso variability (controlled by other genes) inthe concentration needed to trigger a tastereaction, so some people might not notice abitter taste at a lower concentration, butwould react negatively to a higherconcentration. It is reported that, in the“average” US population, about 70% ofpeople are tasters and 30% non-tasters.

The PTC molecule contains aN�C=S group that is thought to be related toits bitter taste. This group is also found inseveral other bitter-tasting chemicals,

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meiosis. The testes themselves are 2n, whilethe sperm formed within them are 1n. Eachtestis is organized into a series of side-by-sidetesticular lobes, all of which open at theirproximal (basal, closest) end into the vasdeferens. Within each lobe are a number oftesticular cysts, each surrounded by aseptum made of connective tissue. In eachcyst are a number of cells that are undergoingmeiosis, and typically, all the cells in a givencyst are in the same stage of meiosis.Typically, the cysts in the distal (apical,farthest from the vas deferens) region contain2n, primary germ cells (cells which give riseto sperm) called spermatogonia or cells invery early prophase I. As the cells within thecysts undergo meiosis, the cysts travel fromthe distal to the proximal end of the lobe, sothat cysts near the proximal end contain“finished,” 1n spermatozoa.

The slide to be examined is alongitudinal section (l.s.) of a grasshoppertestis, and should contain one or more oblonglobes, sliced “lengthwise” so a number ofcysts and a variety of stages in meiosis shouldvisible in each lobe. At the rounded, distalend, notice the 2n spermatogonia (goni =seed). These cells undergo meiosis orspermatogenesis (genesis = origin, birth) toform the 1n sperm. As the cells undergomeiosis I, they are first called primar yspermatocytes (cyto = cell), then during

meiosis II, secondary spermatocytes, then(when meiosis is complete) spermatids. Therounded spermatids are transformed, via thematuration process called spermiogenesis,into mature spermatozoa (zoa = animal) asthey develop flagella.

While many of the primaryspermatocytes are in prophase I, try to findprimary spermatocytes in other stages ofmeiosis I. In metaphase I, the tetrads moveto the center of the cell, line up there, and areattached by their centromeres to the spindlefibers. with each of the homologouschromosomes in a pair attaching to a spindlefiber from the “opposite” centriole/pole. Inanaphase I, the whole chromosomes (not justthe chromatids as in mitosis) are pulled to thepoles, with one homologous chromosomefrom each pair being pulled to each pole.This is the reduction division that ischaracteristic of and central to the process ofmeiosis. Meiosis I ends with telophase I, inwhich the secondary spermatocytes areformed. The secondary spermatocytes may bedistinguished from the primary spermatocytesby their smaller size. They undergo meiosisII, a process that’s similar to mitosis in thatthe sister chromatids are separated (and thus,become chromosomes). The resulting 1n cellsare the spermatids. The spermatids undergoa maturation process called spermiogenesiswhich involves changing from a rounded to apointed shape and the growth of flagella to

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Figure 5. Light Path through Binoculars

USE OF BINOCULARSProtocol Copyright © 1982 D. B. FankhauserBackground and additional information Copyright © 1993 J. L. Stein Carter

I. OBJECTIVE:To learn how to use binoculars

II. BACKGROUND:Binoculars (bi = two; ocul = eye)

are often used in biology to view animals,especially birds, in the field when a close-upview is not possible. Having the right kind ofbinoculars and knowing how to care for anduse them can, thus, be an asset to you, bothnow as you take this course and later as youperhaps go on a walk in the woods with yourfamily. If you ever purchase a pair ofbinoculars for yourself or your family, youwill need to be aware of “what to look for” soyou end up with a usable pair of binoculars.

Binoculars come in a variety ofpowers of magnification and diameter of theobjective lenses. The binoculars here in theBiology Lab have an overall magnification of7× life size. The diameter of the objectivelenses is 35 mm. To admit enough light tosee clearly, binoculars should have objectivelenses which are at least 5 times greater insize than the power of magnification (7 × 5 =35 mm). In dim light, the binoculars must letin more light in order to see clearly, thus, theobjective lenses should be even larger (for

example, 50 mm for 7× magnification).Another important characteristic of binocularsis the field of view, which is expressed aseither an angle measurement or as the numberof feet you are able to see (side-to-side) at1000 yards distance. For these binoculars,this is 6.5° or 341 ft. (compare with 7.0° or367 ft. for 7 × 50 binoculars).

While telescopes (tele = far; scope= see, watch, look) are also used for birding,an advantage of binoculars over a telescope isthat seeing with both eyes allows for depthperception. Telescopes are more frequentlyused in astronomy (astro, aster = star; nomos= law, system of laws) where depthperception is not as important, but having agreater power of magnification and widerobjective lenses to let in more light areimportant considerations.

One important word of caution –NEVER look directly at the sun throughbinoculars (or a telescope). Doing so couldcause permanent eye damage or blindness!

III. MATERIALS NEEDED:binoculars corresponding to your seat number – please leave lens caps in the drawer

IV. PROCEDURE:A. Parts of the binoculars:

Obtain the binoculars thatcorrespond to your assigned seat number,leaving the lens caps in the drawer. Note thatthe neck strap is (hopefully) coiled neatlyaround the binoculars. You are expected tomake sure the strap is neatly coiled and thelens caps replaced each time you put thebinoculars away after use. Become familiar with your binoculars andtheir parts. In your lab notebook, draw apicture of the binoculars you are using and

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label the following, underlined parts.1. The barrels are the main body of thebinoculars and contain prisms that reflect thelight from the objective lenses to the ocularlenses. Note that the barrels are hingedtogether at the center.

2. The objective lenses are on the “front”of the binoculars – note that these are 35 mmacross. These are achromatic lenses (a- =not, without; chromo = color) which aremade of several layers of glass bondedtogether to prevent “rainbows” of color whilelooking through the binoculars. They are alsocoated with a substance to help transmit lightbetter.3. The ocular (ocul = an eye) lenses

(eyepieces) are mounted on the ends of thebarrels closest to your eyes. The oculars canbe moved closer together or farther apart toadjust to the width of your eyes by bendingthe hinged center of the binoculars. Practiceflexing this hinge.4. Note the interpupillary distance scaleon the central joint of the binoculars. Thisindicates the distance between your eyes (70to 80 mm).

5. On the side of the outer barrel of theright ocular there is a white scale, the diopterscale, – you will use this to customize thefocus to your eyes.6. The center wheel is used to focus thebinoculars.

B. Use of the binoculars:1. Put the strap around your neck and,while on field trips, also hold the binocularsso they don’t swing. Avoid dropping orbanging the binoculars! Please keep yourfingers off the lenses. If there are fingerprintson the lenses, clean with your breath and lenspaper, being careful to not scratch lenses withgritty dirt.

2. Adjust the interpupillary distance tosuit your eyes by bending the center hinge ofthe binoculars – note the interpupillarydistance scale reading and record in yournotebook (which setting lines up with thesmall white dot?).3. Find some object upon which to focusand use the center wheel (which focuses bothoculars together) to adjust the focus so that itis correct for your left eye.

4. Note the white dot on the righteyepiece. Line up the “0" mark (of thediopter scale) with this dot, then rotate theright ocular to adjust the diopter setting untilthe right eye is in focus, too. Note/drawwhere on the +/� scale the dot falls. Recordin your notebook for future reference.

5. If not already done, record numbersfrom scales so that next time you use thebinoculars, you can quickly adjust them toyour needs by setting to the appropriatenumbers.6. Now that the binoculars are set for youreyes, use only the center wheel to focus onwhatever you wish to see.

7. Practice focusing on several targets(first stationary then moving) such as treebranches, birds, etc. to familiarize yourselfwith use of the binoculars.

C. Storage of the binoculars:1. Wind the neck strap NEATLY andsecurely around the center of the binocularsso that it holds itself in place.2. Flatten the binoculars and/or use thecenter wheel to move the oculars all the waydown if needed so that the binoculars fit intothe drawer.

3. Place the lens caps on the lenses of thebinoculars. Double check to make sure youhave done all of these things before puttingaway the binoculars.

4. Carefully put away the binoculars.

V. DATA:Record all observations and data. Draw pictures and/or take notes where needed.

Especially, draw and label your own picture of the binoculars – you see more when you have todraw it yourself.

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slides of lily (Lilium sp.) anthers showing meiosis to form pollen (B-680, -682, -682b, -683,-684, -685)

slide of grasshopper testis showing spermatogenesis (including meiosis) (G-145)PART Bmono- and dihybrid cross genetic corn earsPTC, thiourea, and sodium benzoate taste test paperscoins (total of 4 of 2 kinds)

IV. PROCEDURE: PART AWorking individually, obtain and

examine under the microscope each of theslides discussed below. Try to find cells ineach of the stages of mitosis (mito = a thread;-sis = the act of) or meiosis (meio = less):interphase (inter = between, among),prophase (pro = before, in front of),metaphase (meta = between, with, after),anaphase (ana = up, throughout, again), andtelophase (telo = end, complete). Draw whateach of these cells you observe looks like.Label such things as nucleus, nucleoli,nuclear envelope, chromosomes (chromo =color; soma = body), spindle fibers, asters(aster = star), etc. if/when present. Forfurther information, you may also wish torefer to the chapters on mitosis and meiosis inyour lecture text.

1. Plant Mitosis: An onion root tip isa rapidly-growing (meristematic) portion ofthe onion, thus many cells in various stages ofmitosis may be seen (Figure 61). Ininterphase, note that the chromosomes arelong and entangled and are not individuallyvisible, thus the nucleus of such a cell wouldbe fairly-evenly colored and may also containone or more nucleoli. During prophase, thechromosomes contract and become distinct,thus the nucleus of a cell in prophasefrequently has a “grainy” appearance. Bymetaphase, the chromosomes are contracted,distinct, and lined up on the metaphase plate.Try to find and draw a cell that clearly showschromosomes with sister chromatids andcentromere (this doubled structure should bevisible by late prophase or metaphase). Tryto find a cell in which the chromosomes arefairly well spaced and count them. What isthe diploid chromosome number of onion?Are there asters present in any stage(s)? Inanaphase, note the disjunction of the sisterchromatids. In telophase, note the formationof the new cell plate. How manychromosomes should be included in each ofthe daughter nuclei?

2. Animal Mitosis: In whitefish, as inhumans, the blastula (blasto = bud, sprout; -ula = little) is the hollow ball stage inembryonic development, and thus ischaracterized by rapid growth. In prophase,note the chromatin within the nucleus (thenuclear envelope is intact in early prophase,although not visible). Are there any asters atthis stage? In metaphase, note that thechromosomes are lined up alone the equator

of the cell. Asters are readily visible andpolar fibers are attached to the centromeres ofeach chromosome. In anaphase, note thedisjunction of the chromosomes. In telophase,note the constriction type of cytokinesis.

3. Plant Meiosis: A lily plant is 2n,like humans. In the anthers, the pollen isformed, but since it is only 1n (technically,pollen is a 1n male “gametophyte” because itmakes the sperm – the gametes – while theparent plant is referred to as a “sporophyte”),meiosis must occur here. Examine the seriesof slides showing various stages in meiosis:B-680 (30-4532): Lily Anther, general

structure – These anthers just have theprecursor 2n germ cells in them.

B-682 (30-4550): Lily Anther, earlyprophase I – Chromosomes have pairedand have begun meiosis.

B-682b (30-4556): Lily Anther, lateprophase I – Chromosometetrads/bivalents should be visible.

B-683 (30-4562): Lily Anther, first meioticdivision – Chromosomes separate,resulting in 2 daughter cells that are1n.

B-684 (30-4568): Lily Anther, second meioticdivision – Sister chromatids areseparated, resulting in a total of 4daughter cells that are 1n.

B-685 (30-4574): Lily Anther, pollen tetrads– Because of the way plants reproduce,these would be the precursors to themicrospores which will subsequentlygerminate and grow into the malegametophytes, also known as pollen.Within the pollen, two sperm nucleiwill be produced. Pollen and spermare not the same thing, but rather,pollen is a plant generation that makesthe sperm.

B-686 (30-4586): Lily Anther, mature pollengrains – These are the mature malegametophytes (pollen) with their twosperm nuclei visible in each.

Note that the end result of meiosis is fourdaughter cells. Remember that in prophase Iand metaphase I of meiosis, the homologouschromosomes are paired (synapsis) intobivalents or tetrads. How many of thesebivalents do you see? What is the diploidchromosome number of lily?

4. Animal Meiosis: Similarly, ingrasshopper testes (note: “testis” is singular,“testes” is plural), sperm are formed by

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Figure 61. Comparison of Mitosis in Onion Root Tip and Whitefish Blastula

MITOSIS, MEIOSIS, AND GENETICSCopyright © 1989 J. L. Stein CarterMitosis protocol Copyright © 1989 D. B. Fankhauser

I. OBJECTIVES:1. To observe mitosis and meiosis in plant and animal cells.2. To explore probability as it applies to genetics.3. To learn how to make and use Punnett squares.

II. BACKGROUND:In his studies on inheritance in peas,

Gregor Mendel showed that an offspringformed by the fertilization of an egg by asperm gets half its alleles from each parent.Exactly which alleles for each trait theoffspring receives from the parents followsthe laws of probability. This can beillustrated by coin tosses. If a single coin istossed, it will land heads or tails with a 50:50chance of each. If two coins are tossed, eachhas a 50:50 chance of being heads or tails.Thus, the probability of obtaining, forexample, two heads should be ½ × ½ = ¼.The genotype of an organism is its actualgenetic make-up; which two alleles for a trait(gene) it actually has. Its phenotype (pheno= show, seem, appear) is how those genes areexpressed; what it “looks like”. For example,a person with one allele for brown eyes andone for blue will have the genotype Bb, but

that person’s phenotype will be brown eyesbecause brown is dominant over blue (likegetting one head plus one tail when two coinsare tossed together).

The processes of mitosis andmeiosis were discovered in the 1870s and1890s, respectively. It was later concludedthat the movement of the chromosomes inmeiosis was responsible for the behavior ofthe alleles during reproduction, as Mendelnoted. Mitosis (mito = a thread) is theprocess of replication and division of thechromosomes as a cell divides to make twocells. Meiosis (meio = less) is a special typeof division in which the chromosome numberis reduced by half, resulting in gametes (sexcells: eggs and sperm) with only onechromosome from each of the pairs that arepresent in our somatic cells (general bodycells – soma = body).

III. MATERIALS NEEDED:PART Amicroscopeslide of onion (Allium cepa) root tip mitosis (B-552)slide of whitefish (Coregonus clupeiformis) blastula mitosis (E-1025)slide of Ascaris megalocephala (a roundworm) uterine section showing meiosis or mitosis in

eggs, zygotes, or embryos (E-325, -335, -354, -355)

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BIOMETRICS AND STATISTICAL ANALYSIS OF DATACopyright © 2000 J. L. Stein Carter (based on an idea from Bloom and Krekeler, 1963)

I. OBJECTIVES:1. To learn more about metric system measurements.2. To learn more about reading equipment and interpolation of digits.3. To learn how to calculate averages and standard deviations to analyze data.4. To study an example of Darwin’s concept of intraspecific variation as

illustrated in a population of humans (Homo sapiens).

II. BACKGROUND:In biology, gathering numerical data

to test one’s hypothesis and subsequentlyperforming a statistical analysis on those dataare of utmost importance when interpretingthe data and drawing conclusions from them.Due to a variety of factors, despite the mostcareful observations, there will always bevariation in the data collected, necessitatinga statistical analysis of those data.Biometrics is the application of statisticalmethodology to analyze biological data.

In collecting data, it is important toknow how to correctly read the equipmentbeing used, which frequently involvesinterpolation to obtain the last digit of thedata. Interpolation is “reading between thelines” — for example, if you’re looking at aclock that only has 5-min. markings on it andyou read a time of 8:53, you are interpolatingthe “3” by estimating how far between the“0” and the “5” the minute hand of the clockis. Also, in biology, as in other sciences, themetric system is used. Thus, we measure anorganism’s weight in grams or kilograms andits height in centimeters or meters.

To evaluate these numbers, it isnecessary to employ several statisticalconcepts. The mean or average (X�) of a setof data is a measure of “central tendency” ofa group of numbers, such that the total of thedeviations of the numbers above the mean isequal to the total of the deviations of thenumbers below the mean. For example, forthe numbers 1, 3, 5, 7, and 9, the mean is 5,so the deviations of each of the numbers fromthat mean are �4, �2, 0, 2, and 4,respectively. Note that the absolute values of(�4) + (�2) and 2 + 4 are equal. Further,note that the sum of the deviations around amean should always be 0. The mean is thetotal of the values divided by the number ofdata points. This is expressed mathematicallyas: X�� = (��xi)/n. �� means sum, xi means allthe individual values, and n means thenumber of items. The closer the mean of agroup of numbers is to the true value, themore accurate that group of numbers is.

Another concept that is sometimesused is that of the median, which is the datapoint above and below which one-half of thedata points lie. That means that if there is anodd number of data points, the median is thenumber that’s in the “middle” of the list. If

there is an even number of data points, themedian is the average of the middle two. Forexample, for the numbers 2, 6, 7, 14, and 56,the median is 7. For the numbers 2, 6, 7, 9,14, and 56, the median is (7 + 9)/2 = 8.

The mean is preferred over themedian as a measure of central tendency in agroup of data, but there might be somesituations where the median would be a betterindicator. If a distribution is symmetrical, themean and median should be about the same,but if a distribution is skewed, then themedian might be a better measure to use thanmean. For example, if a statistician waslooking at family income in an area wherefour families had incomes of under $20,000while one family had an income of over$1,000,000, then median would be a betterindicator of “typical” family income in thatcommunity. The median is less sensitive toextremes in the data than the mean. Forexample, as pointed out above, the mean ofthe numbers 1, 3, 5, 7, and 9 is 5, and so isthe median. However, for the numbers 1, 3,5, 7, and 34, the median is still 5, but themean is 10.

One other concept that is only usedoccasionally is that of mode. The mode is thenumber that occurs with the greatestfrequency. For example, if 2 students get ascore of 50 on a test, 3 students get 80, and 1student gets a 90, then the mode is 80 — themost students got that score (by the way,since the middle score would be one of the80s, that is also the median, and the mean ofthose numbers would be 71.67). However, ifyou are collecting data on some experimentwhich requires that you weigh somethingthree times, and you get three entirelydifferent weights, the concept of mode reallydoesn’t mean much.

When analyzing data, it is alsouseful to determine how spread-out, howdispersed, those data are. One indication ofthis is the range of the data, which is equal tothe highest number (the maximum) � thelowest number (the minimum ). This can beexpressed as range = xmax �� xmin.

The standard deviation, s, is one ofthe most commonly-used measures of thecentral tendency or dispersion of the data, inother words, a measure of how far from themean the data are scattered. Thus, the

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smaller the standard deviation is, the moreprecise, the closer to agreement with eachother, the data are. In many cases, if thestandard deviation is as large as or greaterthan the mean, that would indicate that theexperimenter needs to re-examine his/herexperimental technique! If the means of twogroups of data are not farther apart from eachother than the standard deviation of eachgroup, then one cannot draw the conclusionthat there is a statistically-significantdifference between the two groups (to reallybe sure, one should do a “t-test” on the data).Standard deviation is expressed

mathematically as .

These calculations can easily bedone on a calculator or computer. Since somany people use the mean and standarddeviation to analyze data, most calculatorsand spreadsheet software have built-infunctions to do those calculations.

To more easily visualize statisticaldata, often a histogram is constructed. Ahistogram is a bar graph in which the X-axisrepresents the range of possible valuesdivided into discrete categories, and the Y-axis represents the number of individuals who“fit into” each category (frequency ofindividuals observed at each value). Intheory, given a large-enough sample size, the

histograms for weight and height for adulthumans should look like “bell” curves.

The pressure-sensitive instrumentused to measure blood pressure is called asphygmomanometer (sphygmo = pulse;mano = wide, roomy, rare, thin; meter = tomeasure). When the pressure is pumped upto around 140-150 mm Hg for a normalperson, this will cut off the flow of blood inthe brachial artery (brachi = arm) of the armbeing tested. As the pressure is slowlyreleased, when the cuff pressure equals thesystolic pressure (systol = a contraction) inthe artery, a sound can be heard with eachpulse as the blood surges under the cuff. Asthe pressure decreases further, at some point(the diastolic pressure: diastol = standingapart) the sound will disappear as the bloodflows freely under the cuff. “Average” bloodpressure for younger adults is considered tobe 120/80 mm Hg.

A person’s pulse is another measureof cardiovascular (cardio = heart; vascul = alittle vessel) fitness, especially how quicklythe pulse returns to a “resting” rate followingexercise. As the left ventricle of the heartcontracts, blood is forced into the aorta and oninto the arteries. The resulting wave ofdistension that passes over the arterial systemis called the pulse. The pulse travels morerapidly than the actual blood flow.

III. MATERIALS NEEDED:medical balancestopwatch

stethoscopesphygmomanometer

III. PROCEDURE:AGE:

1. For this lab, you are asked to work ingroups of 4 to 5 students. Exchange your datawith the other members of your group, anduse your group numbers.2. In your lab notebook, make a list of allthe people in your group. Record eachperson’s sex and age to the nearest 0.5 yr.

3. Use the medical balance to determinethe height and weight (in metric units) ofeveryone in your group (see below).4. While your group is waiting to use themedical balance, determine each person’spulse and blood pressure (see below).

HEIGHT AND WEIGHT:1. With the help of your group members,use the medical balance to determine yourheight in centimeters (to the nearest 0.1 cm)and your weight in kilograms (to the nearest0.01 kg). You may wish to remove your

shoes to obtain a more accurate heightmeasurement. Make sure you obtain readingswith the correct number of decimal places.Record height and weight data for all groupmembers directly into your lab notebook.

PULSE:1. Within your group, work in pairs

with one person being the technician and theother the “victim.”

2. Use your fingers to locate the pulsein the person’s wrist area.

3. The pulse rate can be determined bycounting the number of beats in 30 sec. andmultiplying by two to get beats per minute.

Take three readings and average them todetermine the person’s average pulse rate.Round to one decimal place.

4. Trade roles to obtain the otherperson’s pulse rate.

5. Make sure you record the averagepulse rates for everyone in your group.

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would be worst? Why?

6. As may be observed in televisedunderwater photography, under/in the oceaneverything looks blue because the waterfilters out (absorbs) all other colors of light.Mostly blue light is reflected/transmitted, andthus available for use by seaweeds (orcameras). Thus, it would be suspected thatseaweed should contain (a) pigment(s)capable of absorbing blue light. Whatcolor(s) would you expect that/those pigment

to be? Do any of the pigments we’vediscussed fit that description?

7. It is thought that seaweed-typeorganisms (Kingdom Protista) evolved first,and land plants (Kingdom Plantae) evolvedfrom them. How would your answer to theprevious question on ocean-dwellingseaweeds explain the color(s) of light used byterrestrial plants for photosynthesis? There isplenty of green and yellow light given off bythe sun, so why don’t plants use those colorsfor photosynthesis?

OPTIONAL ADDITIONAL EXPERIMENT(S):A. The anthocyanins in leaves such asthose of red cabbage are soluble in water, andmay be extracted by putting red cabbage in ablender with water, then straining off thepulp. Adding acid changes the color of thecabbage “juice” to a bright, cherry red, addinga base turns it a dark, forest green, and addingtap water sometimes changes it to blue.Anthocyanins are also soluble in methanoland ethanol, and so may be extracted usingone of those, but at least with ethanol, withina few hours the solution fades to clear. Oncespotted onto chromatography paper (eitherfrom a methanol or ethanol extract or using apenny to apply pigment directly), it appearsthat the anthocyanins will not move using thesolvent systems typically used forchromatography of plant pigments.Optionally, a spectrum could be obtained forfreshly-extracted pigment from red cabbageleaves. A comparison of spectra for redcabbage juice in acidic and basic solutionswould also be interesting. Experimentationwith various chromatography solvents couldbe done to try to find a system that wouldallow anthocyanins to move.B. Blue-green algae (bacteria-relatives inKingdom Monera) like Spirulena also containa bluish pigment, phycocyanin, which is

water-soluble and is not used inphotosynthesis. Because Spirulena is amicroscopic organism, using a penny to “roll”pigments onto chromatography paperwouldn’t work. The pigments in Spirulenawould have to be extracted in some solvent(like was done for the spinach/dried parsleyin Part B), then spotted onto chromatographypaper if so desired. Phycocyanin appears tobe insoluble in EtOH (or only slightly).Optionally, a spectrum of water-extractedSpirulena could be taken (use water as theblank) and/or experimentation undertaken totry to find a chromatography solvent withwhich phycocyanin could be separated fromthe various photosynthetic pigments alsopresent in Spirulena.C. It might be interesting to try to extractpigments from carrots or other vegetableshigh in �-carotene, then performing paperchromatography on the extract and obtainingabsorption spectra for the pigments thusisolated.

D. Health-food stores often sell“chlorophyll extract”. It might be interestingto obtain some of that to use forchromatography and spectral analysis.

July 1, 2013 80

be set to 350 nm and the zero and blankrechecked. Absorbance readings should beobtained for all specimens the group istesting. Then the spectrophotometer shouldbe set at 375 nm, the zero and blankreadjusted, and another set of readingsobtained. Readings of absorbance should betaken at 25 nm intervals from 350 to 800 nm(350, 375, 400, etc.). Each time the

wavelength is changed, it is necessary torecheck both the zero and the blank to getcorrect readings. Readings should beobtained for each of the bands being testedbefore changing wavelength. Readingsshould be recorded in students’ lab notebooksin chart form with columns for wavelengthand for each of the samples. Also, datashould be entered online.

VI. DATA:PART A:1. The resulting bands on thechromatography paper should be drawn (thencolored with colored pencils?) and described(color, location with respect to the solventfront and/or original spot). A tentative

identification should be assigned to each ofthe pigments based on the list of pigmentcolors mentioned in the Background.Remember to draw any new equipment used.

PART B1. All spectrophotometer readings shouldbe recorded in group members’ labnotebooks.2. Data for the absorption spectra of allsolutions/bands tested should also be enteredonline (once per group – per set of data, notmultiple entries of the same data). When alldata have been entered, you may then returnto the Web site to print out the class data.3. For each sample the group tested, agraph of wavelength (on the X- or horizontalaxis) versus absorbance (on the Y- or verticalaxis) should be constructed. The graphingprotocol should be used as a reference onproper graphing techniques. Absorptionmaxima (peaks) and minima for each of the

solutions tested should be noted. Again,because the concentrations of the solutionswere not standardized, the heights of thepeaks (which, you should recall, are merelyconcentration-dependent) are not significant(differences in concentrations of solutions arenot being examined in this experiment), butrather, the locations of the peaks relative tothe wavelengths tested are important data.Because this graph represents data which donot exhibit a proportional correlation,sequential points should be connected in “dot-to-dot” fashion, and the graph will not be astraight line graph.4. Any other significant notes,observations, and data should be included.

VII. DISCUSSION:In your discussion, include the following:

1. From the colors of the individualbands on the chromatogram, which pigmentdoes each of these bands appear to represent(see Background section)? Which is thesmallest or fastest-moving molecule? Whichis the slowest? Theoretically, it has beenreported that the carotenes (bright yellow-orange) move the farthest followed byxanthophylls (slightly greenish yellow), thenchlorophyll A (bluish green), and finallychlorophyll B (pea green). Did yourchromatogram come out that way?

2. At what wavelength(s) did each ofthe isolated pigments absorb the most/leastlight? Do the observed absorption maximaand minima correspond to those reported inthe literature for each of those pigments?Were the tentative identifications of the bandscorrect – do the absorption data support theiden t i f i ca t i ons mad e b a s ed oncolor/appearance? To what colors do thesewavelengths correspond?

3. The absorption spectrum of themixed pigments tested should be comparedwith the spectra from the various “known”pigments. By matching the peaks, which of

the individual pigments does the mixedpigment solution contain? Also, at whichwavelengths did the mixed plant pigmentsabsorb the most light – where were theabsorbance peaks? To what colors do thesewavelengths correspond? At whichwavelength did they absorb the least light –where was the absorbance closest to zero? Towhat color does this correspond?

4. If methylene blue, food coloring, orany other pigments were also tested, the sameanalysis should be done for each pigmenttested. The absorption maxima and minimashould be determined for each of the colorstested. What wavelength(s) of light is/areeach of the colors absorbing (thereforeunavailable to a plant), and whatwavelength(s) is/are each color not absorbing(therefore reflecting or transmitting andavailable to a plant). If a plant was placedinto a solution containing this/thesepigment(s), what wavelength(s)/color(s) oflight would be available to the plant to use?

5. Based on the class results, if coloredlights were shined onto plants, which color(s)would be best for photosynthesis? Which

July 1, 2013 21

BLOOD PRESSURE:1. Again, work in pairs within your

group. Often, but not always, the non-writingarm is used and should be resting on the table– rearrange the chair as needed. It is helpfulto work in a quiet room so you can hear thebeating. Please keep conversation to aminimum so others can hear.

2. Make sure the stethoscope (stetho= chest, breast; scope = see, watch, look) andsphygmomanometer are working properly andclean the ear pieces of the stethoscope withalcohol to prevent transfer of ear infections.

3. Wrap the sphygmomanometer cuffaround the arm above the elbow. Close thevalve nut securely, BUT NOT SO TIGHTLYTHAT IT GETS STUCK SHUT (test it first).Place the diaphragm of the stethoscope overthe artery under the bottom of the cuff (overthe bend of the elbow) and insert ear piecesfacing forward in your ears.

4. Pump up the cuff until no beatingsound is heard (approximately 140-150 mmHg – usually not necessary to go over 160unless the person knows he/she has highblood pressure).

5. Very slightly loosen the valve nut so

that the pressure drops slowly. Watch thepressure gauge and listen for sounds.

6. When the systolic pressure isreached, the needle on the gauge will pulsateslightly and you will begin to hear beatingsounds. Remember this number.

7. As the pressure drops, the soundswill first become louder, then softer, thendisappear. Remember the number at whichthe sounds become muffled. This is thediastolic pressure.

8. OPEN THE VALVE ALL THEWAY AND LET THE CUFF TOTALLYDEFLATE even if you need to try again!!!You MUST allow restoration of circulation inbetween readings and it is VERY importantthat you not keep an inflated cuff on a persontoo long.

9. Record systolic/diastolic pressuresfor each of three trials. Average the threesystolic values and the three diastolic valuesto determine the person’s average bloodpressure (average systolic/average diastolic).

10. Switch places and test your partner.11. Make sure to write in your lab

notebook the data for everyone in your group.

DATA ANALYSIS:1. Submit your initials, sex, age (to thenearest 0.5 yr), height in centimeters (to thenearest 0.1 cm), weight in kilograms (to thenearest 0.01 kg), average pulse (rounded tothe nearest 0.1 BPM), and average bloodpressure (rounded to the nearest 0.1 mm Hg)online as requested. When everyone hasentered his/her data, you may re-visit that

Web page to print out the overall data.2. As described below, perform astatistical analysis on the data for the peoplein your group. Determine the mean, median,maximum, minimum, range, and standarddeviation for age, height, weight, averagepulse, and average blood pressure.

IV. DATA:1. As requested above, record the age,height, weight, average pulse, and averageblood pressure of all group members into yourlab notebook and enter your data online.2. Using the data from the people in yourgroup, calculate the mean, median, range, andstandard deviation for age, height, weight,pulse, and blood pressure (systolic anddiastolic pressures will need to be consideredseparately). Make sure to enter all your workdirectly into your lab notebook. Whatpercentages of your group are males andfemales?

3. Make histograms of the group’s data asfollows. (Obviously, with only 4 or 5 datapoints, this will probably end up lookingrather “empty,” but hopefully, you’ll get theidea of how it is supposed to work.

a. Looking at age, with only four orfive people, if we would group those into 1-yrcategories, everyone might be in a separatecategory, yet, if we’d group by 5-yrcategories, everyone might all end up in thesame category. Thus, let’s try 2.5-yr age

categories. For the categories under-15,15.00-17.4, 17.5-19.9, 20.0-22.4, 22.5-24.9,25.0-27.4, 27.5-29.9, 30.0-32.4, 32.5-34.9,35.0-37.4, 37.5-39.9, and 40-and-over, recordhow many of your group members fall intoeach of those categories.

b. Make a similar list of height by 5-cm categories (for example: 150.0-154.9,155.0-159.9, etc.) from the minimum to themaximum of those data, and make a list ofweight by 10-kg categories (for example:50.00-59.99, 60.00-69.99, etc.) from theminimum to the maximum of those data. List average pulse in 5 BPM categories (forexample, 75.0-79.9 BPM), and averagesystolic and diastolic blood pressures (listseparately) in 5 mm Hg categories. Determinehow many of your group members fall intoeach of those categories.

c. Set up a histogram (graph) for eachcomparison (i.e. age, weight. . .). Each“block” on the X-axis represents one category(for example, on the height histogram, one ofthe units on the X-axis would represent the150.0 - 154.9 cm group). The Y-axis

July 1, 2013 22

represents the number of people in eachcategory. For each category on the X-axis,draw a bar the appropriate height to representthe number of people in that category. Makesure to properly title your graph and label theaxes (see the Graphing protocol).

d. On the X-axis of each graph,indicate where the mean (calculated above)would be located. Indicate the position of themean + one standard deviation unit and theposition of the mean � one standard deviationunit (for example, if X� = 5.00 and s = 0.20,those two points would be at 5.20 and 4.80,respectively), as well as the mean ± twostandard deviation units (which would be5.40 and 4.60 for the above example).

e. You should end up with separate

histograms for distributions of age, height,weight, pulse, systolic BP, and diastolic BP.Also, create a histogram for the sex of yourgroup members (a total of 7 graphs).4. As a group and/or individually,complete the practice problems in thefollowing statistical analysis worksheet.Show all your work in your lab notebook.

5. After you print out the class data,compare your histograms with those for theclass data. How are they similar? Different?Are the means in the same places? Howsimilar or dissimilar are the standarddeviations? Do any of the histograms form abell curve?

V. CONCLUSIONS:1. Based on your statistical analysis ofyour group’s data and/or the analysis of theclass data, do there appear to anystatistically-significant differences in eitherage, height, weight, pulse, systolic, ordiastolic blood pressure when comparingmales vs. females? Upon what actualnumbers (cite them) do you base yourconclusion?2. Based on your statistical analysis ofyour group’s data and/or the analysis of theclass data, do there appear to anystatistically-significant differences in eitherheight, weight, pulse, or systolic or diastolicblood pressure when comparing people who

are under 25 with those who are 25 and over?Upon what actual numbers (cite them) do youbase your conclusion? What percentage ofeach of those groups is male and whatpercentage is female? Based on your firstconclusion (RE sexual differences), if the sexdistribution in these two age groups is notsimilar, what effect would that have on thevalidity of your statistics and conclusions forthe age group comparison?3. Before looking at the data, whichwould you have expected to be more differentbetween the under/over 25 groups — weight,height, pulse, or blood pressure? Why? Doyour data support this hypothesis?

STATISTICAL ANALYSIS PRACTICE PROBLEMS

Two groups of Biology 1081 students achieved the following scores on the third test:Group 1: 94, 94, 91, 88, 85, 85, 81, 79, 79, 77, 75, 70, 65, 56, 56, 53Group 2: 67, 64, 63, 62, 60, 54, 54, 49, 47, 36, 33, 32, 30, 26, 12

From these scores, the range, the differencebetween maximum and minimum scores, andthe mean, the average, can be calculated(Range = Xmax � Xmin, and Mean = X� =(�Xi)/n) [note that �� means sum--add themup], so

Group 1 Group 2Range 94 � 53 = 41 67 � 12 = 55Median 79 49�Xi 1228 689n 16 15X� 1228÷16 = 76.75 689÷15 = 45.93

Two means and ranges might be closetogether, causing one to assume the two

samples were similar. However, in onesample, the scores could be more closelygrouped with only a few at the extremes, andin another sample, the scores could be morewidely spread. Thus, it is useful to calculatethe variance, and from that, the standarddeviation as indicators of how spread out orclustered the data are (dispersion). If you areinterested in the variance of only thoseindividuals in your sample, s2 = �(Xi � X�)2/nfor this finite group of data. However, if thisgroup is a representative sampling of a largerpopulation, then it has been found that�2 = �(Xi � X�)2/(n�1) is a better estimate ofthe population variance. Thus, for our twogroups of students:

July 1, 2013 79

stopped by removing the paper from the flaskand replacing the stopper. Thechromatogram should be observed and drawn,especially noting the colors of the variousbands that are visible. The paper should behandled carefully, and no marks should bemade on it.7. As a class, the identical bands will beput together and the pigments re-dissolved.One labeled 10 × 130 test tube will besupplied for each band Using (CLEAN )scissors, the various bands should be cut apartfrom each other (remember which is which).Each should then be placed as far into thebottom of the designated 10 × 130 test tube aspossible. Everyone’s identical bands (i. e. allouter yellow bands) should go into the sametube to make the solutions as concentrated aspossible. After everyone’s bands have beencollected, the instructor (or an appointed class

member) should place about 5 mL of 100%ethanol into each tube. Each tube should belabeled (if not done previously) and coveredwith Parafilm®. Each label should includethe order and color of the band that tubecontains (for example, “outer yellow”). Thecovered tubes should be placed in thedesignated rack for storage until the next labperiod.8. All tubes being saved must be properlylabeled and covered, then placed into a rackand stored in an appropriate location untilnext period. “Clean” chromatography solventshould be returned to the reagent bottle forreuse. All glassware should be washed andplaced in the racks to dry. All scraps ofchromatography paper and spinach should bedisposed of properly and any other generalclean-up should be done.

PART B: SPECTRA OF PIGMENTS1. For this part of the experiment,students will be working in groups, based onthe number of spectrophotometers availableand the number of students in the lab section.(Optionally, as a class, a drop of each color offood coloring may be diluted with 100%EtOH so these may also be tested.) Someonein the class may grind a piece of spinach leafor some dried parsley with a mortar andpestle, then add 100% EtOH to extract theplant pigments. A small test tube, rack, glassfunnel, and a piece of circular filter papershould be obtained. The test tube should beplaced into the rack and the funnel into thetest tube. The paper should be folded in half,then in quarters (half of half) as demonstratedby the instructor, then inserted into thefunnel. The newly-extracted pigment solutionshould be poured through the filter paper toremove any particles. If this solution is verydark green, it may need to be diluted withmore ethanol (in step 4).

2. The tubes containing the isolated bandsfrom the chromatograms (and thosecontaining the diluted food coloring) will bedistributed among the groups of students,.thus each group should have some mixedspinach or parsley pigments and at least oneof the isolated bands and/or color of foodcoloring to test.3. One (CLEAN – without methyleneblue stains) cuvette should be obtained foreach solution the group will be testing plusone for plain EtOH, making sure to matchglass colors (types/brands of cuvettes). If it isnecessary to label the cuvettes, ONLYPENCIL SHOULD BE USED, lightlywriting in the white area provided. DO NOTUSE WAX MARKER OR LAB PEN!However, since it is so difficult to remove

markings from cuvettes, it is better to just linethem up in a test tube rack in a pre-determined order corresponding to the labeledtest tubes from the pigments being tested.Each cuvette should be tested for the presenceof unwanted, left-over methylene blue byplacing a small amount of 100% EtOH in it,swirling, and holding the tube against a whitesurface. Cuvettes should not be cleaned outwith water because water could interfere withthe readings. For this experiment only EtOHshould be used to clean out cuvettes. Thecuvette that will serve as the blank shouldhave about 4 or 5 mL of 100% ethanol addedto it. Later, each pigment solution to betested will be poured directly from its testtube into its own cuvette.

4. While the individual bands areprobably dilute enough, the concentration ofthe mixed parsley or spinach pigment solutionmay need to be adjusted so the readings for itare not off the scale. Remember, Beer’s Lawsays that, by diluting a sample, its absorbance(at all wavelengths) will decrease.5. The isolated pigment bands are diluteenough that they are probably OK as is and donot need to be diluted. Each should bedecanted into a separate, clean (check first formethylene blue) cuvette, taking care to notinclude any of the paper pieces.

6. Absorbance readings will be obtainedfor all pigments at 25 nm intervals, and theprocess of obtaining readings will be quickerif all samples for which the group isresponsible are tested at a given wavelengthbefore changing to the next wavelength (alltested at 350 nm, then all at 375 nm, etc.). Itis much more time-consuming to test onesample at all wavelengths, then test a secondsample, etc. Initially, the wavelength should

July 1, 2013 78

IV. MATERIALS NEEDED:PART A:

Fresh spinach (or other leaves of one’s choice – Optionally, if available, leaves may bepicked from outdoors. Brightly-colored autumn leaves could be interesting to test.)

Freshly-made solution of 90% petroleum ether + 10% acetoneWhatman #1 chromatography paperPenny (or other coin)250-mL Erlenmeyer flask with rubber stopper (#8) to fit it, T-pinForceps and scissors95 or 100% EtOH13×100 test tubes (one per pigment per lab section)Parafilm®

PART B:Tubes of pigments from Part B95 or 100% EtOH(Optional red, yellow, blue, and green food color)Additional 13×100 test tubesSpectrophotometer, cuvettes in plastic rack, lens paper

V. PROCEDURE:PART A: PAPER CHROMATOGRAPHY1. A 250-mL Erlenmeyer flask, #8stopper, and T-pin should be obtained. A fewmillimeters (depth) of the 90% petroleumether + 10% acetone solution should bepoured into the bottom of the flask and thestopper placed on the flask so that the“fumes” can start to accumulate in the flaskwhile the next few steps are performed. Theair in the flask must become saturated withfumes from the solvent or the chromatographywon’t run properly.

2. A piece of chromatography paperslightly longer than what will fit into the flaskshould be obtained. It is important to touch itas little as possible, preferably only at theedges. The bottom edge of the paper shouldbe cut as straight as possible. The papershould be long enough that when a T-pin isused to attach it to the underside of thestopper, the tip of the paper will reach towithin a few millimeters of the bottom of theErlenmeyer flask. This paper must be kept asclean as possible, handled only by the edges,and only set on clean surfaces. If it isnecessary to mark on the chromatographypaper, only pencil should be used, not pen(ballpoint ink is alcohol-soluble).

3. A spinach (or other) leaf should beobtained. The chromatography paper shouldbe laid on a piece of clean paper and the leaflaid over the chromatography paper. Theedge of a penny (or other coin) may be used toroll (smash) a stripe of color across the paperabout 1.5 to 2 cm above the end. The leafshould be moved so a new portion of the leafis over the stripe, and re-rolled with the pennyover/onto the same place on the paper todarken the stripe. This process should berepeated several times as needed to obtain adark stripe. The stripe should be allowed to

dry before proceeding.

4. The chromatography paper should beheld up next to the flask to judge the exactlength of paper needed such that when thepaper is pinned to the bottom side of thestopper, the bottom end of the paper will bejust below the surface of the solution. Ifneeded, the top end of the strip should befolded over at the right place.5. The flask shouldnot be left open whilepinning the paper to thestopper. With the flaskplaced in its “permanent”location, the stoppershould be quickly flippedupside-down on top ofthe flask so the flaskremains sealed. Theflask should be kept openthe minimum amount oftime possible. The T-pinshould be used to attach the top (non-pigmentend) of the paper to the center bottom of therubber stopper. When the paper is securelyattached, the stopper should quickly beflipped right-side-up and inserted into theflask in such a way that the paper does nottouch the sides. The bottom of the papershould be barely in the solvent so that thesolvent will be soaked up. It is imperativethat you not move, jostle, or slosh the flaskonce the paper is soaking!

6. As the solution is absorbed into thepaper by capillary action, it will carry thevarious pigments up from the “center”. Whenthe farthest band is about 1.0 to 0.5 cm awayfrom the top of the paper or close to touchingthe T-pin, the chromatography may be

July 1, 2013 23

GROUP 1 GROUP 2Xi Xi � X� (Xi � X�)2 Xi Xi � X� (Xi � X�)2

94 17.25 297.5625 67 21.07 443.8094 17.25 297.5625 64 18.07 326.4091 14.25 203.0625 63 17.07 291.2788 11.25 126.5625 62 16.07 258.1485 8.25 68.0625 60 14.07 197.8785 8.25 68.0625 54 8.07 65.0781 4.25 18.0625 54 8.07 65.0779 2.25 5.0625 49 3.07 9.4079 2.25 5.0625 47 1.07 1.1477 0.25 0.0625 36 �9.93 98.6775 �1.75 3.0625 33 �12.93 167.2770 �6.75 45.5625 32 �13.93 194.1465 �11.75 138.0625 30 �15.93 253.8756 �20.75 430.5625 26 �19.93 397.3456 �20.75 430.5625 12 �33.93 1151.4753 �23.75 564.0625

n = 16 � = 2701 n = 15 � = 3920.92X� = 76.75 s2 = 2701÷16 = 168.81 X� = 45.93 s2 = 3920.92÷15 = 261.39

s = = 12.99 s = = 16.17OR: �2 = 2701÷15 = 180.07 �2 = 3920.92÷14 = 280.06

� = = 13.42 � = = 16.74

In a “normal” distribution, a graphof scores on the x-axis versus number ofpeople who got that score on the y-axis shouldbe a bell curve with the mean in the center.68% of the people should be within one std.

dev. unit to either side of the mean and 95%should be within two units of the mean.

Luckily, most scientific calculatorsand PCs will perform these calculations fromentered data and a few simple commands.

PROBLEMS:1. A group of ecology students was studying and comparing two different woodland areasby taking 20 sample plots in each area. In each woods the combined number of sugar plus redmaple trees in each of the 20 plots was found to be:Woods A: 1, 0, 0, 0, 0, 0, 1, 0, 1, 0, 0, 1, 0, 1, 2, 0, 1, 0, 0, 0Woods B: 0, 1, 0, 2, 0, 2, 0, 4, 0, 3, 0, 1, 0, 0, 0, 8, 0, 1, 0, 4Calculate the range, median, mode, mean, s, and � for the number of maples per plot for eachof these two areas.2. A student was trying to determine the amount of vitamin C in an orange. For fivetrials, the student obtained the values of 21.82, 96.38, 15.28, 107.94, 13.33, and 12.57 mg ofvitamin C per 100 g of orange. Calculate the range, mean, and standard deviation for these data.What does the statistical analysis reveal about this student’s data?3. Another student put 100 mL of distilled water into a graduated cylinder and weighedit. This student then put 100 mL of distilled water into a volumetric flask and weighted that.For 100 mL of water in the graduated cylinder, the student obtained weights of 99.87, 99.65, and99.76 g. For 100 mL of water in the volumetric flask, the student obtained weights of 99.76,99.74, and 99.78 g. Which of these pieces of glassware is more precise? Why (based on whatdata)?4. In the above example of student test scores, if in each case, the professor assigned the classmean as the dividing line between the Bs and Cs, the mean +1 standard deviation unit as thedividing line between the As and Bs, and the mean �1 standard deviation unit as the dividingline between the Cs and Ds, construct a histogram for those categories for each test (notice thatthe actual point numbers would be different between the two tests) – in other words, how manystudents got what grades on each of the tests?Suppose the 16th student took the second test late and got a score of 79. How would that changethe statistics and assigned grades for that test?

July 1, 2013 24

GRAPH CONSTRUCTIONProtocol Copyright © 1989 D. B. FankhauserBackground and additional information Copyright © 1992 J. L. Stein Carter

I. OBJECTIVE:To be able to correctly graph scientific data.

II. BACKGROUND:One of the ways that scientists

present data is in the form of a graph.Probably the most frequently used type ofgraph is the XY graph. In this type of graph,a quantity, such as time, that is independentof the experiment, is plotted along thehorizontal, or X- axis, and a second variable,the values of which depend on the first, isplotted along the vertical, or Y- axis. Thus,

for each experimental point along the X-axis,there is a corresponding measured value alongthe Y-axis, and the intersection of these twodistances/values describes the location of onedatum point on the graph.

To be meaningful, a graph must becreated in an orderly, logical fashion, which isthe subject of this protocol.

III. MATERIALS NEEDED:graph paper (your lab notebook)a straightedge such as a rulera pen with permanent, black ink (your lab pen)

IV. PROCEDURE:

A. DETERMINE THE LIMITS OF THEX-AXIS AND THE Y-AXIS: Examine the data set for the x-axis and notethe minimum and maximum values. Repeatfor the y-axis.B. ASSIGN VALUES TO AXES TOINCLUDE LIMITS:The values assigned to the coordinates mustmeet the following requirements:

1. They should include the limitsdetermined in step 1

2. They should make an adequatelylarge graph (fill the page)

3. They should not exceed theavailable space on the graph.Count the number of squares available alongeach axis, and fit the limits of the data to

meet these division values.The quantity zero should oftenbe the left and/or bottom mostspace. A division on an axisshould equal some convenientfactor of 1, 2, 5 or 10 (Othervalues will make plotting thedata difficult.). For example,divisions of 0.02 units wouldbe acceptable, divisions of0.25 or 0.33 units would not.C. CONSTRUCT AXES,MARK WITH REGULARVALUES:Draw the x-axis below andthe y-axis to the left of theselected open area on thegraph paper. Label each axiswith a title that tells what theaxis represents and what unitsare being used. Mark off theselected regular values with a

small line at the regular intervals selected instep 2. Label each small line with itscorresponding value. Be certain to maintainlinearity (note that if units are 0.02, 0.04,0.06, 0.08, 0.10, the next division is 0.12, not0.20!). Use regular spacing as determined bythe pre-printed lines on the page – that’s whyyou are using graph-ruled pages.

D. ENTER DATA POINTS:For the first point, locate the appropriatevalue along the x-axis and then move upalong that line until the appropriate value ofy is reached (interpolate as needed). Doublecheck that you have not shifted from thedesired location, and make a dot at the point.Draw a small circle around the point, making

July 1, 2013 77

PHOTOSYNTHESISPortions of protocol Copyright © 1986 D. B. FankhauserPortions of protocol, background and additional information Copyright © 1989 J. L. Stein Carter

I. OBJECTIVES:1. To discover what pigments are present in plant leaves and to separate/isolate these

pigments from each other2. To determine absorption spectra for each of the pigments found, noting maximum and

minimum absorbances

II. BACKGROUND:Chlorophyll A (chloro = green,

phyll = leaf) is the pigment used by plants toconvert energy from the sun into chemicalenergy useful to the plant, but other pigmentspresent in leaves also help to “harvest” lightenergy. This energy is stored by convertingcarbon dioxide and water to sugar. Thechemical reaction for this is 6 CO2 + 12 H2O �� C6H12O6 + 6 O2 + 6 H2O.This sugar is stored by the plant as starch(thus the occurrence of photosynthesis couldbe demonstrated using the iodine test forstarch). Benedict’s solution could be used totest for the presence of sugar (usually foundin the leaf veins, indicating transfer of sugarfrom one part of the plant to another).

Besides chlorophylls A and B,various other pigments, including carotenes(carot = carrot), xanthophylls (xantho =yellow), and anthocyanins (antho = a flower,cyano = blue, dark blue), are often found inplant leaves. The chemical structures of thesemolecules are illustrated in many organicchemistry and cell physiology books. Becauseof their different colors, many of the carotenesand xanthophylls are capable of “capturing”solar energy that the chlorophyll cannot andtransferring that energy to the chlorophyllenabling photosynthesis to occur.Anthocyanins are not involved inphotosynthesis.

Once a mixture of these pigmentshas been extracted from a leaf together,because each of these pigments, including thechlorophylls, has a different chemicalstructure and formula, the mixed pigmentscan be separated from each other be a processknown as paper chromatography (chromo= color; graph = to write). In this process,the mixed pigments are dissolved in a mixtureof two (or more) solvents and allowed to soakinto a piece of paper by capillary action.Typically, one of the solvents used is more

covalent while the other is more polar orionic, and their molecular weights differconsiderably. Various of the leaf pigments,thus, are more, or less, soluble in the differentsolvents, so as the solvent system wets thepaper, the various pigments move into/acrossthe paper at various rates depending on theirsizes (molecular weight), relative number ofcovalent or ionic bonds in the molecule, andother factors based on their chemicalstructures: normally, the smallest move fastestand farthest.

Once the pigments are separated, atentative identification of each may be made(to be confirmed by obtaining an absorptionspectrum of each.). Chlorophyll A appears asa blue-green band while chlorophyll B is ayellow-green band. Carotenes are brightyellow to orange while xanthophylls are aslightly greenish yellow. Anthocyanins arereddish, violet, or blue, and are not soluble inorganic solvents, thus typically do not moveup the chromatogram at all. The fact thateach of these pigments appears as a differentcolor is an indication that each is absorbingdifferent wavelengths of light. Remember thecolor(s) that we see is whatever the plant hasNOT absorbed (For example, chlorophyll Alooks green because it is not absorbing andusing green light). According to theliterature, chlorophyll A has two absorptionpeaks (absorbs the most light) at around428 nm (blue-violet range) and at around 660to 700 nm (red range), while chlorophyll Babsorbs best at around 453 and 643 (to650) nm. Beta-carotene, the most commoncarotene (and precursor of vitamin A), has anabsorption peak at a wavelength of 451 nm (atthe blue-violet end of the spectrum). Each ofthese pigments or the mixture as extractedfrom the leaf can be examined with aspectrophotometer to determine its absorptionspectrum, thus confirming its identity.

III. SAFETY CONSIDERATIONS:The chemicals in the solvent system

being used in Part A and the ethanol used inPart B are flammable, thus should be keptaway from any open flames. Pouring ofsolvent system to/from the Erlenmeyer flask

should take place in a fume hood, and thereagent bottle and your flask shouldimmediately be capped. It probably is also agood idea to not breathe too much of it.

January 4, 2016 76

this may not be needed. Repeat steps 6through 8 for the rest of your solutions.

9. In your cuvette, obtain ~4 mL of the“unknown” and measure its absorbance.

10. Empty the cuvette andrinse thoroughly with dH2O, blot the lip andreplace upside-down in the designated plasticrack to drain – do not mix cuvettes andregular test tubes.E. Compare the absorbances of yoursamples. If that of the second dilution is notclose to 2× that of the first dilution, the third2× that of the second, the fifth 10× that of thefirst, etc., you did not pipet carefully enough.As time allows, you may discard the “bad”solutions and start over again. Repeat untilyou get satisfactory results (Yes, all of thedata go into your notebook). High-qualityresults come from careful pipetting. Now isthe time to develop proper technique.

F. Submit the data from your best resultson the Beer’s Law data-submission Webpage. When data are all entered, you shouldgo back to the Web site and print out the class

data.G. Clean all test tubes and place in properplace. Do not use a brush to clean cuvettes!Do not mix cuvettes and regular test tubestogether. Place cuvettes in designated plasticrack to dry such that they are not touchingeach other (so they don’t scratch each other).Place used pipets in the designated receptacle(located by the sink), not back with the cleanones. Only if you are the last person in thelast lab section for the day to use thespectrophotometer should it be turned off. Ifanother lab is to follow, leave thespectrophotometers on.

H. Referring to the “Graphing” protocoland accompanying Web page for instructionson proper technique, make a graph of yourdata as explained below. Then, use yourgraph to determine the concentration of the“unknown,” based on its absorbance reading.Depending on your results and how muchtime is left, you may wish to repeat theexperiment to see if you can get better data.Make sure all data are recorded online.

V. DATA:Take notes on all procedures,

supplemented with illustrations wherehelpful. Record all absorbance measurements(with corresponding milliliters of riboflavinadded) both in your notebook and thecomputer. For your five tubes, make a graphof absorbance (on the Y-axis) versusmilliliters of riboflavin (on the X-axis). Agraph that includes data from the whole classwill be generated by the Web server. On your

graph, let every two lines across the page inyour notebook equal 0.1 mL (from 0 to 1.0) ofriboflavin added, and every line up the pageequal 0.020 absorbance units (from 0 to0.800). Label (title) the axes of yourgraph(s). Make sure you use equal-sizedunits on your axes. For example, if you’reusing 0.02, 0.04, etc., then 0.12 follows 0.10– NOT 0.20!

VI. DISCUSSION:1. How does your graph compare to thegraph for the whole class? Is either of them astraight line? Which is closest? How is thestraightness of the line affected by yourpipetting technique?2. Using your graph and the class graph,what is the concentration of the unknownsolution?3. Design a procedure using thespectrophotometer to find out what theabsorption spectrum of riboflavin looks like –how much light does a given, “constant”solution absorb at various wavelengths and atwhat wavelength(s) does it absorb the most

and/or the least light?4. People who take B-vitaminsupplements know that any excess riboflavin(B2), beyond what a person needs, is excretedin the urine, giving the urine the characteristicyellow color of riboflavin. Design aprocedure to determine what percentage of agiven dose of riboflavin is retained/used bythe body vs. what percentage is excreted inthe urine.5. Include any other comments, ideas,suggestions regarding pipetting and/orspectrophotometer use.

January 4, 2016 25

it easier to see, and preserving the integrity ofthe point. Repeat until all data have beenentered.E. CONNECT THE CIRCLES:There are two main types of graphs we willbe constructing

1. In one type, the function beinggraphed is supposed to be linear (but youractual data may not be). In this case, use aruler to determine the best fit straight linethat comes closest to going through the points.Some points will be above the line and somewill be below. Try to have about the samenumber of points about the same distancesabove and below the line. Draw a straightline with the ruler, but do not violate theinterior of any circles through which the linegoes.

2. In the other type of graph, thefunction is suppose to be non-linear – some

kind of a curve. In this case, it is appropriateto connect the circles (in order) toapproximate the curve. In this case, the rulercan help you connect consecutive pairs ofcircles, but some smoothing may be desirableat noticeable curves to obtain the best fitcurve. Again, do not violate the circles orobscure the points therein.

3. Occasionally a third type, a bargraph, may be employed. In this case, the x-axis is generally a list if items which havevalues corresponding to the y-axis. In mostcases, the bars should be of uniform width.F. TITLE THE GRAPH:Write the title in CAPITAL LETTERS abovethe graph, below indicate where the datacame from. Provide titles for both axesindicating what each scale represents,including units.

January 4, 2016 26

OBSERVATIONS VERSUS CONCLUSIONSCopyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To learn to distinguish between making observations and drawing

conclusions.2. To develop one’s power of observation; to learn to see details.

II. BACKGROUND:So often in scientific investigation,

the small, seemingly insignificant details endup being the most important key to theproblem at hand. Yet, because of the culturein which we live and/or because of ourunfamiliarity with a field, we do not noticethese things. Often, too, we misinterpretwhat we see, mistaking conclusions forobservations, and thus, come to a wrongconclusion overall.

For example, if I observe an antcarrying a weed seed, it is just that. Unless Iactually see that ant or its nestmates eatingthat seed, I cannot say that the ant is carryinga piece of food. Perhaps that seed is merelyin the way and being removed, perhaps it willserve as a substrate upon which the ants willgrow fungus to eat, or perhaps it will servesome other function. Thus, if I make the“observation” that the ant is carrying a piece

of food, it might lead me to a false conclusionlater on that, for example, the anthill is beinginvaded by an unwanted fungus. As anotherexample, “The ants are under the appleslice,” is an observation, but “The ants arehiding under the apple slice,” or “The ants arelooking for food under the apple slice” areboth making assumptions or drawingconclusions about the ants’ behavior or theirrelationship to the apple.

Also, it is amazing how frequentlyor how long we can look at something andnever see that which is perfectly obviousabout it. This is the basis of many of ouroptical illusions. As biologists in a three-dimensional world of living organisms, wehave the opportunity to use our senses oftouch, hearing, and chemoreception to aid oursense of sight, yet we frequently use onlywhat we see.

III. PROCEDURE:We will be going on a short hike

outdoors (weather permitting) to makegeneral observations as to what sorts of plantsand animals surround us. Pick one of thesemany organisms, (ant colony, tree, beetle,wildflower, bird, etc.) and list in your labnotebook at least twenty (20) things youobserve about it. Record descriptive thingslike its smell, its sound, how does it feel(texture), what does it look like – shape,color, etc., etc. (be specific on size – howmany centimeters?). Don’t worry if you don’tknow what this organism is called – actually,knowing what it is might bias you and temptyou to make conclusions instead – but dothink about what traits/characteristics of thatorganism might be distinguishing features you

could potentially use to identify it (How manylegs does it have? How are its leavesarranged?). Be careful you do not include anyconclusions among your observations. Youmay find that the first two or three are “easy”and then it gets harder to think of things –that’s normal. Just sit there and think awhile, and often, a number of newobservations will suddenly come to you.Drawing a picture of your organism wouldprobably help you to “see” it better, providingyou really look at it to see how to draw it andnot just think, “I can’t draw,” and make onlya quick sketch. Especially with plants,remember to observe the whole organism notjust a portion of it.

IV. DATA:1. List your observations in your labnotebook. Be as precise and descriptive aspossible. You should draw a detailed pictureof your organism and label the features youobserved.2. From your list of observations, write aparagraph describing the organism you chose– do NOT attempt to identify it.

3. Trade notebooks with someone elsefrom the class and, without knowing whatyour partner’s organism is, read his/herdescription of that organism. From the

description ONLY draw a picture of theorganism. Don’t “read between the lines”based on your knowledge, but rather attemptto draw what is described. As you aredrawing, and especially once you havefinished, comment in that person’s notebookon the ease/difficulty with which you wereable to draw the picture from his/herdescription (It may have said the flowers wereyellow, but did it say where on the plant theywere?). Note good points and bad/missinginformation in the description. Sign yourname.

January 4, 2016 75

Tube # mL dH2O mL Riboflavin1 3.9 0.12 3.8 0.23 3.6 0.44 3.3 0.75 3.0 1.0

reading the numbers.5. During transfer, the pipet should be

held horizontally to prevent dripping, but itshould be held vertically when delivering thesolution. Never hold the pipet upside-down.C. Set up five test tubes according to thefollowing chart. These are the dilutionswhose absorbance you will be measuring withthe spectrophotometer. Be sure that yourtubes are labeled so you know which iswhich. Add the appropriate amount of dH2Oand the riboflavin. Make sure the tubes areclean – sometimes they get put away dirty,and anything in your solution will change thereadings you get. (Hint: Leave them cleanfor the next students, which could be you.)

Notice that the total amount of liquid in eachtube is 4.0 mL so they should all look likethey are about equal, remembering the tubesvary slightly in shape.

Mix each solution using a vortex.Solutions should not be mixed by invertingthe tube with your thumb on top becausechemicals from your thumb could dissolve inthe solution and change your readings. Also,some chemicals could damage your thumb(riboflavin won’t hurt you, though).D. Read the absorbance at 450 nm foreach of your samples as follows:See separate instructions, online, for the“new” Spectronic-200 spectrophotometers.Use your tubes of plain water (= 0 mL ofriboflavin), 0.2 mL, 0.7 mL, and 1.0 mL toplot the graph. Use your 0.1 mL and 0.4 mLsamples as “unknowns.”

1. Set the wavelength to 450 nm withthe spectrophotometer’s upper right-handknob.

2. Without any sample in thespectrophotometer, adjust the machine to readinfinite (�) absorbance (= 0% T) by rotatingthe zero-adjust (left-hand) knob. This tellsthe machine that, when it’s totally dark

inside, all of the light is being absorbed. Ifyou are looking directly at the needle, youwill not be able to see its reflection in themirror behind it (remember parallax error?).

3. Obtain two of the special cuvettes,making sure they are the same color ifexamined from the top (some appear moregreenish than others) and making sure theyare clean. Handle these only by the top edgeand place only in a plastic rack. Fill one ofthe special cuvettes with about 4 mL of dH2O(~1.5 in). Remember to PLACE IT IN APLASTIC TEST TUBE RACK ONLY.Gently polish off fingerprints with lens paperonly – anything else (including Kimwipes)would be too rough and scratch the cuvette,interfering with your readings – just beforeeach time you take a reading. Hold the tubeby the top edge only – fingerprints can changeyour readings. Insert into spectrophotometerwith marks lined up and close lid.

4. Use the lower right knob on thespectrophotometer to adjust the absorbance toread 0.000 absorbance (= 100% T). Thiscorrects for any light absorbed by the glassand water (yes, water does absorb light – thatis why underwater pictures all look blue – theother colors have all been absorbed). Bydoing this, we can compensate for the lightabsorbed by the glass and water and read justwhat light the sample absorbs. Note that ifanother solvent than water is used, thatsolvent must be used to “blank” the machine.

5. Remove the “blank” from themachine. Pour the solution from tube #1 intoa second cuvette, polish with lens paper,place into machine with proper orientation,and close the lid.

6. Read the absorbance (not %T) tothree decimal places – interpolate the thirdplace. Any absorbances greater than 0.7 canbe read to two decimal places. Note that thescale goes RIGHT TO LEFT and is alogarithmic scale – the scale intervals getsmaller as the scale goes farther left.

7. Return the solution to its test tube.Blot – do not rub or wipe – the solution fromthe RIM ONLY with a Kimwipe, but do notrinse. When measuring a number of solutionsof increasing concentration, you will diluteeach successive one less if any droplets left inthe cuvette are from the last solution than ifthey are of water. Do not use paper towel onthe cuvette, do not use Kimwipes except toblot the rim, do not use test tube brush or anyother “scratchy” item in or on the cuvettesbecause scratches can change thespectrophotometer readings.

8. Place the next solution into thecuvette, polish the tube, measure theabsorbance, and record in your lab notebook.Optionally, your readings may be better if youdouble check the machine with the plainwater blank in between readings, although

July 1, 2013 74

Figure 55. Scale on Spectronic 20

III. MATERIALS NEEDED:spectrophotometer1 mL and 5 mL pipets and pipet filler5 13×100 mm (small) test tubestest tube rack2 cuvettes for spectrophotometer in PLASTIC – not metal – racklens paper (and Kimwipe)2 × 10–4 M riboflavin solution (0.0753 g/L)vortex

IV. PROCEDURE:You should work individually on

this lab. Each person MUST learn how to usea pipet and practice using it. We will bedoing labs this and next semester where youwill be working individually and will need toknow how to pipet!!!A. Turn on the spectrophotometer and letit warm up for at least 30 min. before use.

B. Using the following procedure, usepipets to deliver the specified amounts ofliquids into your test tubes.

1. Note that you have the proper sizeof pipet. Notice the markings on the pipet:

a. The total amount that thepipet can contain. If the pipet has a frostedband around the top end, it is a serologicalpipet, and the last drop of liquid must beblown out to correctly deliver the desiredamount (if you are emptying the whole pipet).

b. The subdivisions whichare marked on the pipet – it would not bepossible to accurately deliver 1.05 mL ofliquid with a pipet that was calibrated in0.1 mL increments.

c. Remember to account forwhether you are reading the numbers goingup or down. If you deliver from 0.0 to 0.7 mLmarkings, you have delivered 0.7 mL ofliquid, but if you deliver from the 0.7 mL

marking to the bottom you have onlydelivered 0.3 mL (in a 1 mL pipet).

2 To suck up fluid, immerse the tipabout 0.5 cm below the surface and bracegently against the lip of the container. Watchyou don’t press too hard and tip it over.

3. Use a pipet filler to suck up liquidto a level slightly higher than the amount youneed. NOW is the time to learn how to use apipet filler – EACH AND EVERY PERSON– and not in subsequent labs when accuracyand speed are more important. Don’t rushthrough this lab, but take the time to reallylearn how to use the pipet. Do not suckliquids up into the pipet filler – it can ruin thefiller and/or contaminate future pipettings andyour solution. If solution gets into the filler,you can assume that both the filler andsolution within the pipet are contaminatedand need to be dumped, the filler given to thelab staff to repair, and you need to start overagain. Your instructor will demonstrateproper technique and use of a pipet filler.

4. Use the pipet filler to adjust thevolume. The bottom of the meniscus shouldjust touch the calibration line. If needed,touch, do not wipe, off the excess fluid fromthe tip, being careful not to pull any of thesolution out of the pipet. Remember toaccount for whichever direction you are

July 1, 2013 27

V. CONCLUSIONS/DISCUSSION:Include not only you conclusions

about the organism you have been observing,but also your conclusions about the exerciseas a whole – what did you learn from this lab?

Look critically at your list of“observations.”1. In double-checking your list, are thereany conclusions listed among yourobservations? Ask yourself, “How did I knowthat?” If your answer is, “Because I saw it,”it’s probably an observation, but if youranswer is, “That’s kind of what looked likewas going on,” it might very well be aconclusion.

2. If you find any conclusions, can yourephrase them as observations – what did youreally observe/see/hear?3. Were there any conclusions youthought of putting down as observations butcaught yourself in time? How might thesehave changed your view of that organism?

4. What is the most interesting/excitingthing you discovered about your organism thatyou never knew before? Why did you pickthat organism to begin with?5. Based on your observations, are thereany conclusions that you can make about yourorganism?

ASSIGNMENT TO BE TURNED IN AT THE BEGINNING OF THE NEXT CLASS:(If assigned — 5 pt. just for doing it and turning it in)

In scientific investigation, anotherkey to success is being able to logically thinkthrough the procedure to be followed to gatherthe desired data, then to successfullycommunicate this to someone else in anintelligible manner. For the next class, write(in paragraph form on a separate piece ofpaper) a description of the procedure to befollowed in making a peanut-butter-and-jelly

sandwich. Assume your audience is acomputer (maybe an android?) that has noidea how to go about this, but will exactlyfollow whatever directions you give it. Whenthis paper is returned, you will be asked toplace it in your notebook, so you should writeon only one side of the page (and you maywish to limit page size to about 5½ × 8 in.).

July 1, 2013 28

ACCURACY AND PRECISIONProtocol Copyright © 1980 D. B. FankhauserBackground and additional information Copyright © 1989 J. L. Stein Carter

I. OBJECTIVES:1. To learn how to use a balance to weigh objects.2. To determine the accuracy of various lab glassware as well as the precision

obtainable when using each of these.II. BACKGROUND:

If a person goes into a restaurantand orders a cup of coffee, how much coffeewill be given to the person? Will the coffeearrive in a large, earthenware mug or adelicate, china cup? Do all coffee cups holda cup of coffee? If, on the other hand, anotherperson is following a recipe that called for acup of coffee, how much coffee will thatperson use? Will there be a difference in howthe coffee is measured in these twosituations?

Recording numerical data is animportant part of scientific research. Thereliability of these data can influence theconclusions drawn from the experiment.Although “accuracy” and “precision” are usedinterchangeably in common speech, inscientific language, they mean two differentthings. The “true value” of any number is aphilosophical idea which we take as agiven/known thing; for example, scientists saythat exactly 100.0000 mL of water weighexactly 100.0000 g at 4° C (theoretically99.823 g at 20°C – room temperature). An“error ” in data is the numerical differencebetween the measured value and the truevalue. An “accurate” result is one thatagrees closely with the true value (has lesserror); for 100 mL of water, a weight of100.001 g is more accurate than 100.009 g,and that is more accurate than 100.01 g.“Precision,” on the other hand, refers toagreement among a group of data, but saysnothing about their relationship to the truevalue. Three measurements of 100.009,100.008, and 100.007 g might be moreprecise than three measurements of 100.009,100.002, and 99.995 g, and yet may not bemore accurate.

In the above example, which ofthese methods of measuring coffee is the mostaccurate? If a measuring cup is used, willthat always measure exactly one cup ofcoffee? Why or why not? What factor(s)could be sources of error in the user’smeasurement? Which of these methods ofmeasuring coffee would be the most precise?Why?

There is a variety of glassware herein the Biology Lab – beakers, graduatedcylinders, Erlenmeyer flasks, volumetricflasks – that could be used for a lab exercisein which students would be required tomeasure 100 mL of distilled water (dH2O).Because these various types of lab glassware

are designed for different purposes, theiraccuracy and precision vary. Certain types ofglassware are manufactured with greaterprecision than other types and/or yield moreaccurate measurement of volume. Knowledgeof the relative accuracy and/or precision of thevarious types of glassware can aid indetermining the appropriateness of a piece ofglassware for a desired use. For example, ifa student needs several identical 100 mLsamples, which measuring utensil should bechosen? Why?

When a scientist comes up with ananswer to a question like the preceding onethat might be right yet needs to be tested tosee if it is true, this is called a hypothesis(hypo = under, beneath; thesis = anarranging). Any testable answer to theprevious question such as, “I think that the___ glassware is more precise (or moreaccurate),” is a hypotheses.

Once a scientist has formed anhypothesis, it is then necessary to figure outhow that hypothesis can be tested. Thescientist would need to decide what to do(procedure/methods) and what data areappropriate to gather to uphold or disprovethe hypothesis. At times, scientists may endup gathering “negative” data that actuallydisprove their hypotheses. For this glassware,what could be done – what steps could befollowed – to find out if the ___ glasswarereally is the most precise/accurate? Is itenough to use one piece of glassware orshould several kinds/styles be tried? Is itenough to take one reading on each piece ofglassware or should several tests/trials beperformed on each piece?

If a person places a desired amountof water into a piece of glassware, how willthat person know if the container is correctlyfilled? How will (s)he know the container isfilled the same amount every time? Whenviewed from the side, the surface of the waterin a transparent glass container is acharacteristic shape that is a clue to solvingthis dilemma.

Because of water’s affinity for glass(glass is hydrophilic, hydro = water, philio= brotherly love), the edges of the water’ssurface will creep up the walls of thecontainer slightly. Especially in small-diameter glassware, the surface of the wateris, thus, noticeably curved. This curvedsurface of the water is called a meniscus

July 1, 2013 73

approx. color seenwhen transmitted

� in m� or reflected400-435 violet435-480 blue480-490 green-blue490-500 blue-green500-560 green560-580 yellow-green580-595 yellow595-610 orange610-750 red

Figure 54. Colors of Visible Light

bodies, so must be consumed frequently. Anyexcess in a person’s diet is excreted by theurinary system, often turning the person’surine a bright yellow color. Riboflavin isused in the Krebs cycle, and is important inmaintenance of healthy mucus membranesand skin.

Visible light, that which can be seenby the human eye, is only a small portion of alarger spectrum known as the electromagneticspectrum, and can be further subdivided bywhat we call color (Figure 54). Note that ifwhite light is passed through a solution thatabsorbs certain wavelengths while others aretransmitted, we see only the wavelengths thatare transmitted and thus, hit our eyes, notthose that are absorbed. For example, sinceriboflavin appears yellow, we would expect itto absorb light in the blue-violet range. Ablack piece of paper absorbs all colors that hitit and reflects back none, thus appearingblack because of an absence of color(s)reflected.

Bouguer, in 1729, and Lambert, in1760, said that for a solution of a light-absorbing chemical such as riboflavin (Figure53) or chlorophyll, the thickness (amount) ofsolution through which the light must passaffects how much light it absorbs. Beer, in1852, said the concentration of the solutionalso affects how much light is absorbed. Forexample, a 2 cm thick “layer” of solution willabsorb more light than a 1 cm thick “layer” ofsolution and a 6 M solution will absorb morelight than a 4 M solution. If we let “P” standfor the initial amount or power of the lightwhich is shining on a sample, then we cancall Pi the initial amount of light before itgoes through the sample and Pf the finalamount of light left after it goes through thesample. Since the sample, we assume,

absorbs some of the light, then Pf is less thanPi. We can, then, talk about the amount oflight that is transmitted. This is called thetransmittance (T), so T = Pf /Pi. Somechemists use the term “percent transmittance”(%T) such that %T = 100 × Pf /Pi. Chemistsalso use the term “absorbance” symbolized by“A,” which is equal to the logarithm of 1/T[A = log(1/T)].

As previously mentioned,absorbance is related to the length of the paththe light must travel through the absorbingmedium and the concentration of the solution.It has been found that this is a directrelationship, so that if the length issymbolized by “b” and the concentration issymbolized by “C” (not to be confused withthe speed of light, symbolized by “c”), thiscan be expressed mathematically as A = bCKwhere “K” is a constant value for each kind ofchemical. This is called Beer’s Law. Notethat for several concentrations of the samesolution, if you make a graph of A versus c,you should have pretty close to a straight linebecause b and K stay the same.

It is possible to make use of aninstrument called a spectrophotometer(spectro = a sight, the spectrum; photo =light; meter = measure) to studyconcentrations of various solutions and evenpredict the concentration of an “unknown”solution using the amount of light the solutionabsorbs. A spectrophotometer has a lightsource, usually a special light bulb. The lightpasses through a narrow slit or lens to focus itinto a small beam and then through some kindof device – a prism or diffraction grating –which separates the light into a spectrum.The spectrophotometer has another fine slit(or diffraction grating) to let only a narrowband of the colored light go through. Thecolor is chosen/adjusted by a knob whichmoves the prism (or diffraction grating), thusfocusing a different portion of the spectrumon/through the slit. The light then passesthrough the sample to a detector (aphotoelectric cell) which is electricallyconnected to the meter on the machine. Manyspectrophotometers have both A and %T ontheir scales – we will use only the absorbance(A) scale. The maximum and minimum Acan be set to compensate for factors withinthe machine and for any light absorbed by thewater (or other solvent) used to make thesolution.

July 1, 2013 72

Figure 53. Riboflavin

LIGHT AND SPECTROPHOTOMETER USEProtocol Copyright © 1979, 1982, and 1983 D. B. FankhauserBackground and additional information Copyright © 1989, 1992, and 1993 J. L. Stein Carter

I. OBJECTIVES:1. To learn how to use a serological pipet

and pipet bulb.2. To learn how to use a

spectrophotometer.

3. To become familiar with some basicproperties of light.

4. To investigate the principle known asBeer’s law.

II. BACKGROUND:Two instruments frequently used by

biologists are serological pipets andspectrophotometers. There are a number ofexercises and experiments in this and the nexttwo quarters of Biology Lab in which youeither need to accurately measure a smallamount of liquid (the purpose of a pipet) ordetermine how much light is absorbed by asolution (the purpose of a spectrophotometer).Additionally, knowing how to properlyconstruct a graph of one’s data is also animportant lab skill. In this experiment, theprinciple known as Beer’s Law (named aftera person, not the beverage) will be used todevelop and perfect students’ lab skills: if thepipet is used correct ly, i f thespectrophotometer is read correctly, and if thegraph is constructed correctly, each person’sdata should form a straight line graph. Errorsin reading the pipet, delivering the correctamount of liquid, and/or reading thespectrophotometer will lead to a graph whichis not a straight line, while errors in graphconstruction can make good data appear to be“wrong.” The challenge, then, is to do itright, thereby getting a nearly-straight line onone’s graph.

One of the primary goals in this labis to learn to use a pipet correctly. The pipetsin the Biology Lab are serological (sero =serum, whey) pipets, which have a slightlydifferent design than the pipets some of youmay have used in the Chemistry Lab.Serological pipets are calibrated such that thelast drop of liquid should be blown out, andthe markings go all the way to the tip. Notethe various markings, bands, and color-codingon the various sizes of pipets. We will beusing Beer’s Law to test the accuracy of yourpipetting. You will be making solutions ofvarying concentrations of riboflavin (vitaminB2), using pipets to measure the specifiedvolumes of water and riboflavin. In theory, ifyou have pipetted accurately, Beer’s Law saysthat a graph of concentration (amount ofriboflavin added) versus absorbance should bea straight line. The less accurately youmeasure the water and/or riboflavin, thefarther from a straight line your data pointswill be. How close can you get?

When diluting chemicals, there aresome important terms that are frequentlyused, and thus with which you will need to be

familiar, including:Aliquot: a measured sub-volume of sample.Diluent: material with which the sample is

dilutedDilution factor: ratio of final volume

(aliquot plus diluent volume) dividedby the aliquot volume (Vf/Vi) — forexample, (0.1 mL aliquot of sample +9.9 mL of diluent)/0.1 mL of sample =10 mL/0.1 mL = 1 to 100, 1:100 or 102

more diluteConcentration factor: ratio of aliquot

volume divided by the final volume(Vi/Vf) — for example, 0.1 mL ofsample/(0.1 mL of sample + 9.9 mL ofdiluent) = 0.1 mL/10 mL = 0.01 or 10�2

more concentrated.Thus, to prepare a desired volume of solutionof a given dilution:

1. Calculate the volume of the aliquot:aliquot volume = concentrationfactor × final volume

2. Calculate the volume of the diluent:volume of diluent = (final volume -sample aliquot volume)

3. Measure out the correct volume ofdiluent, add the correct volume ofaliquot to it, mix.

Riboflavin (vitamin B2) is a brightyellow color, and is what gives vitamin Bcomplex pills their yellow color. It is a water-soluble vitamin, and thus is not stored in our

July 1, 2013 29

Figure 7. Meniscus

(Figure 7) (menisc = a crescent). Glasswareis designed such that the correct way tomeasure an amount of water is to line up theBOTTOM of the meniscus with the top ofthe line on the glassware, or to determine howmuch is there, by looking at where the bottomof the meniscus falls in comparison to thelines on the glassware. By lining up thebottom of the meniscus with the top of theline in question, a person can come closer tofilling the container with the same amountevery time. Unlike the beakers we will beusing in this lab, the buret pictured in Figure7 is read from the top, down. Note that thebottom of the meniscus in Figure 7 is at27.53 mL, (NOT at 27.4, etc.).

However, if what looks like 100 mLof dH2O is put in a container, how will theexperimenter know if it is really 100 mL orhow close to 100 mL it really is? By whatmeans can one determine how much water isactually in the container? How mightknowing the true value of the weight of wateraid in this determination?

It is possible to make use of achemical property of substances calleddensity to determine the accuracy of thesevolumes. At a given temperature, a givenvolume of a substance weighs a given amount– density is weight per volume. For water at25° C (77° F), 1.00 mL should weigh0.99707 g or 100 mL should weigh 99.707 g. Thus, determining the weight of the“100 mL” of water can show how close thevolume really is.

What about the weight of thecontainer? How can one obtain the weight of

just the water? Do all containers of the sametype weigh the same amount? How wouldany differences in container weights affect theprocedure used in this experiment?

In science, it’s not enough to do theexperiment, look at the data, and decide if thenumbers are close enough to each other to beconsidered the “same.” Rather, some kind ofstatistical analysis must be done. If a givencontainer is filled only once, and the weightwas, say, 98.87 gm, how will theexperimenter know if the container was filledwrong or if it was manufactured wrong? It is,thus, important to obtain at least threemeasurements for each item being tested.

To help evaluate these numbers, itis necessary to need to employ a couple ofstatistical concepts. The mean or average (X�)of a set of data is the total of the values ofthose data divided by the number of datapoints. This is expressed mathematically as:X� = (�xi)/n. �� means sum, xi means all theindividual values, and n means the number ofitems. In the examples above, (100.009 +100.008 + 100.007) ÷ 3 = 100.008 g average,and (100.009 + 100.002 + 99.995) ÷ 3 =100.002 g average. Thus, the second set ofdata is more accurate – closer to thetheoretical value of 99.707. The closer themean of a group of numbers is to the truevalue, the more accurate that group ofnumbers is.

The standard deviation, s, is ameasure of the central tendency or dispersionof the data, in other words, a measure of howfar from the mean the data are scattered. Thisis expressed mathemat ica l l y as

July 1, 2013 30

.

In the above examples, (100.009 - 100.008)2 = 0.0012 = 1 × 10�6

(100.008 - 100.008)2 = 0.0002 = 0 × 10�6

(100.007 - 100.008)2 = -0.0012 = 1 × 10�6

� = 2 × 10�6

(100.009 - 100.002)2 = 0.0072 = 4.9 × 10�5

(100.002 - 100.002)2 = 0.0002 = 0 × 10�6

( 99.995 - 100.002)2 = -0.0072 = 4.9 × 10�5

� = 9.8 × 10�5

Since the standard deviation of 0.000816 g issmaller than that of 0.00572 g, this indicatesthat the data in the first example are closer toeach other – they are more precise.

For a student’s individual data,these calculations can easily be done on acalculator. For the large numbers of datacollected from the whole class, the computercan be used to calculate the mean andstandard deviation. Thus, all data will beentered into the computer for analysis anddistribution to the whole class.

III. SAFETY CONSIDERATIONS:One of the pieces of equipment you

will be using today is a small, plastic pipet.This is similar to a “medicine dropper” in thatit is a tube with a bulb at one end. If a pipetis held “upside down,” even slightly, theliquid in it most likely will run down into thebulb. If you were using a glass pipet with arubber bulb, this might cause the bulb todeteriorate more quickly. In any event, youmay assume that past students were not verycareful and the insides of the rubber bulbs arecontaminated with something. Thus, if youare using a rubber bulb and any of yoursolution gets into the bulb, you can assumethat your whole pipet full of solution iscontaminated. To prevent this, pipets shouldalways be held such that the open end is atleast a little lower than the bulb end, with analmost horizontal position (bulb end slightlyhigher) being useful while transferring a fullpipet from one container to another.

Another piece of equipment we willbe using is a graduated cylinder. This is aglass cylinder supported by a plastic base tokeep it upright. Note that the graduatedcylinders also have a plastic collar around

them. This collar should always remain nearthe top of the cylinder, as it serves a veryimportant function there. Contrary to whatmany students guess, the collar is NOT usedto indicate water level – that’s what thepainted lines on the cylinder and your eyesare for. Rather, since these cylinders tend tobe “top heavy,” the purpose of a properly-placed collar is to help prevent breakage ofthe lip of the cylinder if accidentally tippedover. The collar should be above thecalibration lines, high enough up so that if thecylinder gets knocked over on its side, it willland on the collar, not the end of the cylinder.Obviously, if the cylinder is heading for thefloor, you’re out of luck.

In this and all subsequent labs, onevery important safety procedure you need tofollow is to clean up after yourself. If youleave “mystery” containers abandonedsomewhere, the lab staff have no way ofknowing it’s “only” water. Each person isresponsible for cleaning up his/her own areaand equipment. Remember, your motherdoesn’t work here – you have to clean upyour own mess!

IV. METHODS AND MATERIALS:Note: due to the recent decrease in lab time,you will probably not have time to test allthree pieces of glassware as explained here.Try to get at least two of the three done in theavailable time.A. The balance that corresponds to eachtable number (even seat number ÷ 2) will beobtained. If it was properly stored, theweights are (or should be) slid over to theright side so that the balance does not swingwhen carried. Balances should be supportedby the base when carried – not “grabbedaround the neck.” Each pair of lab partnerswill share a balance, but each person will dohis/her own samples. Be aware that, cometest time, each person will be expected toknow how to use the balance. Now is the

time to learn.B. The balance may not be “in balance” atzero but for today, this will not be a problembecause the difference in weight between afull and empty beaker will be calculated tosee how much the water weighs alone, andthe weight of the beaker will be off by thesame amount no matter if it is full or if it isempty.C. The object to be weighed will be placeon the left pan of the balance, and the weightsmoved over until they balance the weight ofthe object. The proper way to read a balanceis to see if the needle swings the same amountto both sides of the center line when observedfrom STRAIGHT in front of it. When thebalance stops swinging, it might stick due to

July 1, 2013 71

VI. DISCUSSION:1. What does the presence of chloride ionin the water in the cylinders in “Osmosis”indicate was happening – where did thechloride ion come from?2. What do you think caused theBrownian motion you observed? Couldsomething (what?) have been bombarding andjostling the particles to cause them to move?From this, can you figure out why you hearsound even in a quiet room?

3. In “Diffusion,” does the rate ofdiffusion just after adding methylene bluechange by 15 to 30 minutes later?4. In “Hypotonic/Hypertonic Solutions,”when you first examined the Elodea leaf,could you see the plasma membrane? Explainwhat caused the change when you addedsaltwater. What happened to the plasmamembrane when the saltwater was added?What is responsible for keeping themembrane in its normal position? What

happened to the plasma membrane whendistilled water was added? Do you think itwould be possible that this cell couldexplode? Why/why not?5. Plant cells have cell walls and animalcells do not. Predict what you might expect tosee if you added 15% salt to a solution ofblood cells. What if you added distilled waterto them?

6. If the milk-food coloring demonstrationwas done, this is actually a rather complicatedsystem. Milk is an emulsion, and detergent isan emulsifying agent. However, detergent isalso a surfactant, a substance which reducesthe surface tension of water and/or similarlyaffects the tension at the boundary of waterwith another liquid. Chemicals like foodcoloring tend to be “big” organic molecules ofsome sort. Based on your knowledge ofbiology and chemistry, suggest possibleexplanations for what might be going on here.

July 1, 2013 70

Another similar demonstration thatmay be done if some milk is available, is toplace some milk in a saucer. Gently add onedrop each of red, yellow, green, and blue foodcoloring, each in a different quadrant of themilk. Then, gently add one or two drops ofdish detergent to the center of the milk.Observe what happens.

HYPERTONIC, ISOTONIC, AND HYPOTONIC SOLUTIONS — (if time allows)

(Plasmolysis in Plant Cells)1. Make a wet mount of an Elodea leaf asbefore (using tap water). Examine and drawa typical cell as seen under the microscope.2. Put a drop at a time of 15% saltsolution at one edge of the coverslip andobserve what happens to the leaf. DO NOTGET THIS ON THE MICROSCOPE!!! Ifsome does get on the microscope, wipe it offimmediately and thoroughly! Record howmany drops of salt solution were added tocause a change in the cells. Draw anddescribe this change.3. Now, add distilled water a drop at atime. DO NOT GET THIS ON THEMICROSCOPE!!! If some does get on the

microscope, wipe it off immediately andthoroughly! If the slide gets too wet, absorbsome of the water with a paper towel orKimwipe. Observe what happens and recordhow many drops of water were needed tocause a change in the cells. Draw anddescribe this change.

WHEN YOU ARE DONE: makesure your microscope is CLEAN AND DRYbefore putting it away. Make sure there is nosalt solution spilled on it – especially checkaround the hole where the condenser comesup through the stage. Remember to follow allthe steps for proper storage of the microscope(4× lens down, rheostat down, light off, cordcoiled neatly, stage down, cover on).

V. DATA:Record all observations, draw

pictures wherever possible, indicate colors ofobjects observed, and label all recognizable

cell organelles. For “Diffusion” a chart oftime and observations may be helpful. Enter“Osmosis” data into the computer.

July 1, 2013 31

Figure 8. Balance Scale

friction and not stop in the right place.Failure to look straight at the needle results inparallax error , that is, the apparent readingwill look like more or less than the trueamount if not observed from straight ahead.The weight is obtained by adding together allthe weights, reading the smallest one to thenearest 0.01 g. This is called interpolating(Figure 8) and involves thinking of animaginary scale with 10 divisions in betweenwhatever are the smallest divisions that aremarked (on these balances, the 0.1 g divisionsare the smallest divisions shown). On thisimaginary scale, how far over does the pointergo – one mark? four marks? nine marks? –those represent the 0.01 gram divisions. Thisnumber should be recorded in your notebook.D. An empty, dry 250 mL beaker will beweighed (by each person) to the nearest0.01 g. and this number (= weight ofcontainer) recorded. The second decimalplace will be interpolated – the balance willbe read to TWO DECIMAL PLACES .E. The beaker will be filled with dH2O tothe 100 mL mark. The BOTTOM of themeniscus should just touch the top of thecalibration line when looking STRAIGHT atit. The volume will be carefully adjustedwith a Pasteur pipet (medicine dropper) andrubber bulb. Any extra drops of water will beremoved with a Kimwipe (every extra drop ofwater adds weight) and any trapped airbubbles removed by “rotating” the containerto dislodge them (they subtract weightbecause air weighs less than water, and thosebubbles are taking up space that should befilled with water).F. The filled container will be weighed,again to the nearest 0.01 g (= weight ofcontainer + water) and this weight recordedjust above the last number. When the weightof the empty container is subtracted from theweight of the filled container, this willdetermine the weight of the water. Excesswater on the outside of the beaker or thebalance will throw off the readings.G. Some of the water will be poured intoa spare container (the one in which water wasobtained), then the weighed beaker carefullyrefilled to the line as before (to simulate usingthree different samples of water withoutwasting water), and weighed again. Afterrecording this weight, the weight of the emptybeaker will be subtracted to obtain the actualweight of the water. This whole process willbe repeated three times so three weights forthe water (as though weighing three differentwater samples) are obtained.

H. The three values for the weight of thewater will be added together and divided bythree to get the mean weight of water. Fromthis, the standard deviation will be calculated:

1. The difference between each,individual value and the mean forthat piece of glassware will befound.

2. Each of these differences will besquared.

3. The squares for each piece ofglassware will be summed.

4. The sums will be divided by thetotal number of trials (3).

5. The standard deviation for thatpiece of glassware is the squareroot of the answer to step 4.

I. Steps C through H will be repeated fora 100 mL graduated cylinder, then for a100 mL volumetric flask. The volumetricflasks are numbered, thus students will obtainthe flasks corresponding to their seatnumbers. The same dH2O will be re-used inthese trials. One note: the plastic collar onthe graduated cylinder should remain aroundthe top of the cylinder, It is there to preventthe neck of the cylinder from breaking iftipped over, and if needed, it may be slid up,above the graduations so the graduations canbe clearly seen.J. CLEAN UP!!! “Used” dH2O shouldbe poured into the designated container. Wetglassware should be placed in one of the dishracks by the sinks to dry – students should notplace wet glassware back on the shelves. Thebalances should be clean and dry and theweights slid over to the right before replacing.Any spills will be cleaned up by theresponsible party. Remember, your motherdoesn’t work here – you’ve got to clean upyour own mess.K. Your data should be entered online.When all have entered their data, you mayprint out a copy of the class data for inclusionin your lab notebook.

IV. DATA:All procedural notes, numbers,

calculations, and other data should berecorded as requested (lab notebook andcomputer). As mentioned above, a suggested

format to make doing the calculations easierwould be:

July 1, 2013 32

XXX Piece of GlasswareTrial # I II IIIwt. of glassware + H2O:wt. of empty glassware: ___ ___ ___

wt. of H2O:Very carefully-drawn, labeled

pictures of all new equipment (balance withlabeled weights, balance scale andinterpolation, proper balance swing, avoidingparallax error, various glassware withappropriate markings, meniscus, etc.) usedshould be made in students’ lab notebooks.Drawings of the various glassware should belarge enough (0.25 to 0.50 page) to clearlydraw and see the lines on the glassware, and

should include representations of thegraduations exactly as they appear on theglassware. A vaguely beaker-shaped blobwith a bunch of lines on it is unacceptable,and attention should be paid to thelength:height ratio of the glassware.Drawings should be made to show the naturalproportions of each piece of glassware.Students should develop their observationalskills by noticing where the lines are reallylocated on the pieces of glassware and whatunits of volume are represented by each line.Students should also examine glassware andnote any other significant markings or labels.

V. CONCLUSIONS:Assuming each container was

supposed to hold/measure exactly 100.00 mLof dH2O at room temperature,1. For which container were individualmeasurements most precise (closesttogether)? In other words, which type ofcontainer is most likely to be filled to thesame amount each time? For which type wereindividual measurements least precise –where was “human error” the biggestproblem?2. For which type of container were theclass measurements most precise? In otherwords, which type was manufactured mostprecisely – which has the least variationamong the different pieces of glassware ofthat type? Which was worst – which showedthe most difference – which wasmanufactured the least carefully?3. Which type of glassware was mostaccurate? If they were designed to holdexactly 100.00 mL of H2O and that is supposeto weigh exactly 99.71 to 99.82 g, dependingon temperature, which glassware came the

closest (for individual trials and for the wholeclass)? Which was the least accurate?4. If we have the technology tomanufacture the more accurate, more preciseglassware, of what use is the less accurate,less precise glassware? Why bother tomanufacture it? When would it beappropriate to make use of each of the threestyles of glassware? By the way, in terms ofbeing careful in the lab, which labware is themost expensive to replace if it gets broken?5. It should be noted that the beaker andthe graduated cylinder have their markingspainted on them while the volumetric flaskhas its marking etched into the neck. If two250 mL beakers are placed side by side, the(painted) markings are all at the same level.If, however, two (or more) volumetric flasksare compared, the etched markings are all atdifferent levels on the necks. What does thissay about the accuracy and/or precision ofthese types of glassware?6. Why would it be important to use dH2Orather than tap water?

July 1, 2013 69

should not start to dry out.F. Using the electronic balance, weigh thebag to the nearest 0.01 gm and record theweight in your lab notebook and thecomputer. It will be quicker to record eachweight in the computer as the bags areweighed rather than waiting to record all theweights at the end.G. Gently submerge the bag into yourcylinder of water.H. Every 15 min. for the next 60 min.,remove the bag and gently dry it as before,then weigh it. Record the weight in your labnotebook and in the computer. Note anychange in the way the bag feels – does itincrease or decrease noticeably in turgidity?Is there any visible evidence of the passage ofmolecules from the bag to the external water(color change)?I. After you take your last reading, withthe bag still out of the water, add a coupledrops of silver nitrate (AgNO3) to the water inthe cylinder and observe what happens. Ifchloride ion (Cl�) is present, it will react withthe silver ions (Ag+) to form silver chloride(AgCl) which is not water-soluble and, thus,shows up as a white precipitate by thefollowing chemical reaction: AgNO3 + Cl� �

AgCl� + NO3�. Is there chloride ion present

in your cylinder? CAUTION: SILVERNITRATE STAINS SKIN BLACK UNTILIT WEARS OFF – DO NOT GET THISON YOUR SKIN OR SPILL ANY!!!Optionally, a flame test could be conducted totest for the presence of Na+ and/or Benedict’sSolution could be used to test for glucose inthe cylinder water.J. Do the following calculations for eachtime for each bag of solution:

1. For each time subtract the initialweight of the bag from the weight at the endof that time to determine the change in weightof that bag, or

wtfin �� wtini = ��wt.2. For each of these,

��wt/wt ini × 100 = %��wt (percent change in weight).

3. In your notebook, make a graph oftime (minutes from the start) on the X-axis,versus percent weight change on the Y-axisfor each of your five bags (five lines).K. Enter your data into the computer asyou weigh each bag each time. While thebags are soaking, complete the rest of the labexercise. Empty the tubes after testing.

BROWNIAN MOTIONMake a wet mount of carmine

particle (a red dye) suspension and examineunder the microscope. Describe what yousee. Are any of the particles, especially thesmaller ones, moving? This type of motion iscalled Brownian motion after Robert Brownwho first described it.

DIFFUSIONAs a class, put some tap water in a

beaker. Set the beaker on a table and let it situntil calm. Gently add a couple drops ofmethylene blue (or another dye) with thedropper near the surface to disturb the wateras little as possible. Observe what happensover time. Do not bump or move the beakeronce the dye is added.

July 1, 2013 68

DIFFUSION AND OSMOSISCopyright © 1988 J. L. Stein Carter

I. OBJECTIVE:To observe Brownian motion, diffusion, and osmosis.

II. BACKGROUND:The cytoplasm of cells is 70 to 95%

water. Dissolved or dispersed in that waterare various salts, sugars, proteins, etc. whichmake up a complex mixture of molecules.

Molecules in liquids and gases arein constant motion due to their kinetic energy.Substances dissolve in water and dispersethroughout a solution because they areconstantly in motion. Diffusion is thetendency for molecules of any substance tospread out randomly into the available space.Substances will diffuse from moreconcentrated to less concentrated areas orsolutions. Passive transport is diffusionacross a biological membrane. Sometimes,though, solute molecules are too large to gothrough a semipermeable membrane.

Osmosis (osmo = to push; -sis = the act of),then is a special case of passive transport inwhich water diffuses across a selectivelypermeable membrane from less to greaterconcentration to try to equalize theconcentrations of the solutes. If a cell and itswatery environment have the sameconcentrations of solutes, they are said to beisotonic (iso = equal; tono = tone, tension,stretched). If the environment has a greaterconcentration of solutes, it is hypertonic(hyper = over, above), and the cell willshrink as it loses water. If the environmenthas a lesser concentration of solutes, it ishypotonic (hypo = under, beneath) and thecell will swell or, in the case of animals, evenburst as it gains water.

III. MATERIALS NEEDED:microscope, slide, coverslipsuspension of carmine particlestest tubes and rackmethylene blue (or another dye)15% salt solution, dH2OElodeasilver nitrate (AgNO3) – caution: do not get on skindialysis tubingbalance100 mL graduated cylinder(s)25% solutions of salt (NaCl), glucose, sucrose, and albumin

IV. PROCEDURE:OSMOSIS

The class should divide into groupsof four or five people. Each group should testthe following substances. All data gatheredshould be entered into the computer. Youwill, then, receive a copy of all the class data.

For each group:

A. Obtain five pieces of dialysis tubingapproximately nine inches (20 cm) long. Ifnot already cut and soaking, cut with scissorsand soak each under cool, running tap water,rolling the end between your fingers until itopens up. Run tap water through it until it iscompletely open.B. Tie a knot in one end of each tube toseal it, as close to the end as possible. Fillthe tube with tap water, and while pinchingthe open end to hold it shut, gently squeezethe tube to check for leaks. If you find a leak,get a new piece of tubing.

C. For your group, obtain five 100 mLgraduated cylinders and fill eachapproximately � to ¾ full with dH2O. Labelone for each of the solutions to be used.D. Fill each tubing “bag” with one of thefollowing solutions – each of the groupsshould test all five of these solutions.

1. tap water2. 25% salt (NaCl) in dH2O3. 25% glucose (C6H12O6) in dH2O4. 25% sucrose (C12H22O11) in dH2O5. 25% egg albumin in saline solution

When filling the bags, leave them a littleflaccid (flacc = flabby) or limp because lateron they may absorb water and become turgid(turg = swell, swollen) or rigid, even to thepoint of bursting if too full. E. Tie a knot in the open end of the “bag”to seal it. Avoid trapping air bubbles inside.When the tube is sealed, rinse it under tapwater to remove any spills, then dry by gentlyrolling on a paper towel. Do not let it getoverly dry – the surface of the bag should bedry enough to not mess up the balance, but

July 1, 2013 33

Density of Water at Various Temperatures (from Chemistry handbooks)temp °C temp °F density

0 32.0 0.999871 33.8 0.999932 35.6 0.999973 37.4 0.999994 39.2 1.00000

. . .18 64.4 0.9986219 66.2 0.9984320 68.0 0.9982321 69.8 0.9980222 71.6 0.9978023 73.4 0.9975624 75.2 0.9973225 77.0 0.9970726 78.8 0.9968127 80.6 0.99654

. . .35 95.0 0.9940636 96.8 0.9937137 98.6 0.9933638 100.4 0.9929939 102.2 0.99262

July 1, 2013 34

DETERMINING pH OF SOME COMMON SUBSTANCESProtocol Copyright © 1980 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To learn what the pH values are for some commonly-used substances.2. To become familiar with various methods for determining pH.3. To learn how to interpolate, understand, and evaluate the validity of meter readings.

II. BACKGROUND:“pH” is the negative logarithm of

the hydrogen ion concentration in asolution. This can be expressedmathematically as pH = ��log[H +], and isexplained, below.

Many biological processes aredependent on the pH or hydrogen ion, H+,concentration of the surrounding solution.Acid foods like sauerkraut and pickles do notspoil easily because many pathogenic (pathos= disease, suffering; gen = bear, produce)bacteria do not grow well in acidic condItions.Lemons taste sour and soap tastes bitter to usbecause of their respective acidity or basicity(alkalinity). Our digestive tract – mouth,stomach, intestines, etc. – changes the pH ofour food from acid to base and back severaltimes as it is digested.

Various solutions have varyingconcentrations of hydrogen ion, H+, andhydroxide ion, OH�. Even plain water, H2O,dissociates a little bit to form H+ and OH�(hydrogen ions – technically, hydronium ions– and hydroxide ions). In any solution, if you

multiply the concentration of H+ (expressed inmolarity) times the concentration of OH�, theproduct always comes out to 1 × 10�14. Thus,if a solution is neutral, neither acid nor base,we would expect the concentrations of H+ andOH� to be equal, each at 1 × 10�7 M. If youincrease the concentration of H+ in a solution,the concentration of OH� automaticallydecreases so the product is still 1 × 10�14. Ifa solution is an acid and contains more H+,then the concentration of the H+ would begreater than 1 × 10�7 M and that of OH�would be less than 1 × 10�7 M. If a solutionis a base (alkali) and contains more OH�, thenwe would expect the opposite situation.

When working mathematical problemslike 0.0000001 × 0.0000001, it is easier towrite it as (1 × 10�7 ) × (1 × 10�7 ) and add theexponents to figure out the product, i. e., (�7)+ (�7) = �14, so (1 × 10�7 ) × (1 × 10�7 ) =1 × 10�14. You may recall from high schoolmath that logarithms can be used to findanswers to multiplication problems, andessentially, this is what we’ve just done, i. e.,

if (1 × 10�7 ) × (1 × 10�7 ) = 1 × 10�14

then log(1 × 10�7 ) + log(1 × 10�7 ) = log(1 × 10�14 )or (�7) + (�7) = (�14)

This, then, is the basis of pH units.“pH” is defined as the negative logarithm ofthe hydrogen ion concentration which canbe expressed mathematically as pH =��log[H+]. For example, if the H+concentration is 1 × 10�7 M, then the log[H+]= �7, and the negative logarithm or pH = 7.Chemists have defined pH as the negativelogarithm because most of the H+

concentrations with which they deal are lessthan one (for example, 0.1 M, 0.01 Metc.).and it’s easier if all the numbers. arepositive (whereas the logarithms all arenegative). This means that if a solution is anacid, the pH is less than 7 and if it’s a base,the pH will be greater than 7. Note thatbecause this is a logarithmic scale, a changeof one pH unit represents a 10× change in H+

concentration. Thus, going from pH 1 to pH2 is going from an H+ concentration of 0.1 Mto 0.01 M, and this could have a profoundeffect in an organism’s body.

There are different ways ofmeasuring the pH of a solution. The first iswith pH paper. These paper strips have beenimpregnated with special dyes that change

color as the pH changes. Different dyes aresensitive to (change color at) different pHranges. Most indicator papers have a mixtureof dyes so that they are sensitive to a widerpH range. Note that pH paper is not the sameas litmus paper – litmus paper only changespink-purple to indicate acids vs. bases.

A pH meter has an extremely thin(and fragile!) glass electrode which ispermeable only to H+ (technically H3O

+,hydronium) ions. Hydrogen ions from the

July 1, 2013 67

inner blue cone is the hottest part of theflame, and the object to be heated should bepositioned just above it. CAUTION: theflame of a properly-adjusted Bunsen burnerwill be nearly invisible in a well-lit room –don’t forget you have it lit and burn yourself!

9. When finished, turn off the gas at thedesk stopcock by turning the handle at rightangles to the hose. CAUTION: the upperportion of the burner will be hot, handle withcare. Let cool before putting away.

SAFELY HEATING TEST TUBESCopyright © 1996 J. L. Stein Carter

Always wear goggles. Adjust the burner to a low flame (about 1to 1.5 inches tall).

Never hold a tube straight up above a flame.It might suddenly start to boil causing thecontents to explode out.

Hold the tube at an angle, but keep itmoving so one spot doesn’t suddenlyheat and explode the contents out of thetube.

Keep the tube moving so it doesn’texplode and remove it from theflame before it starts boiling.

July 1, 2013 66

BUNSEN BURNER USEProtocol Copyright © 1992 D. B. FankhauserBackground and additional information Copyright © 1992 J. L. Stein Carter

Learn these parts before you start:desk stopcockmouth of barrelneedle valveflint striker

barrelburner nippleair intake holes

PROCEDURE:

1. Take proper personal safetyprecautions:

a. If your hair is long enough to pullback in a rubberband, then it’s longenough that you must do so – burnthair stinks!

b. Tuck in, fasten down, roll up, or insome other way secure any looseclothing. The only accident we’veever had here was when a studentcaught a baggy sleeve on fire.

c. Be EXTREMELY ALERT andaware of exactly where every litburner in the room is. The color ofa properly-adjusted flame is nearlyinvisible, thus very dangerous.

2. Attach rubber hose to bench-topstopcock, and the other end to the burnernipple (if not already attached).3. Confirm that the needle valve at thebase is gently closed (screw it in, clockwise).4. Practice using the flint striker: Lookinto the cup, press the flint against the fileand squeeze to strike. If you don’t getsignificant sparking, press the flint harder. Ifyou still get no spark, check to see that theflint is not worn out. (Don’t waste the flintby needlessly striking over and over...) Onceyou get good sparks, move to the next step.Suggestion: try it left-handed and press upand in with your thumb.5. Open the bench stopcock by turning thehandle so that it is in line with the hose (it isoff when it is at right angles with the hose.)6. a: With flint striker handy, open the

needle valve slightly while listening

at the mouth of the burner. Whenyou hear a slight hissing sound:

b: hold the striker just over the mouthof the burner, and strike it severaltimes until it lights.

c: If it doesn’t light, confirm that theburner is hissing, and try it again.If you fail again, turn off the gas atthe bench stopcock, and practicegetting good spark from the striker.Repeat steps 6a and 6b until youget it lit.

7. Adjust the air mix by rotating thebarrel: screwing it down reduces the air,screwing it up increases the air. The flamewill indicate a proper mix: If it is yellow and“soft,” you need more air. If it is roaring andpopping, you need less air. Too much airmay make it pop and go out. Many studentsare tempted to try to alleviate a yellow flameby turning down the gas, when what is reallyneeded is more air. If the air is properlyadjusted, the flame should be nearly invisible,no matter what size it is.8. Adjust the gas with the needle valveagain until you have a flame of suitable heightand intensity. Having an “invisible” flamedoes NOT mean turning the gas so low thatthere is a danger of the flame going out – itmeans having a suitably-large flame with theair adjusted properly. Make any necessaryfinal corrections in air so that the flame formsa nearly invisible blue cone. The tip of the

July 1, 2013 35

solution can cross this glass membrane, thuscausing a very small electrical current whichis measured by the machine. At different H+

concentrations, different amounts of H+ ionscross the glass membrane, thus the machinecan “read” different H+ concentrations, andthe face of the meter shows this in terms ofpH units. Please note that these glasselectrodes are very thin and fragile and quiteexpensive. Thus, you must exercise greatcare in using the pH meter – your instructorwill demonstrate correct use of a pH meter.

A further note on pH electrodes: Asyou may know, it takes two electrodes forelectricity to flow, and pH meters are noexception. Older pH meters were equippedwith two separate electrodes, both of whichwere lowered into the solution to be tested.Newer meters have a “combination”electrode, one which combines both

electrodes into one glass housing. For thisreason, our meters may appear to have onlyone electrode attached, but if you look at theother end of the wire lead, you can see thatthere are, indeed, two wires which attach tothe meter. A combination electrode will havea small whitish spot on one side just abovethe glass electrode. This is the otherelectrode (the calomel electrode) – it isimportant that this spot be under the surfaceof your solution for the machine to correctlymeasure the pH of the solution.

Also, it is very important to notethat if you turn a pH meter to “READ”without the electrodes fully immersed in asolution, it will try to send electrical currentthrough them, but won’t be able to do socorrectly. The result is that the electrodeswill cease to function properly, and you willget meaningless numbers from the pH meter.

III. SAFETY CONSIDERATIONS:A. Disposal of Waste1. Federal law says that all acids andbases must be neutralized to pH 7 beforedisposing of them down the drain, and theymust be washed down the drain with lots ofcold water. With your instructor’s approval,you may mix some of your substances togetherto neutralize them: for example, pop, vinegar,or lemon juice could be mixed with detergent.UNDER NO CI RCUMSTANCESSHOULD BLEACH (CLOROX) BEMIXED WITH AMMONIA !!! Thiscombination will release toxic chlorine gas.

Test your mixture with pH paper to make sureit is pH 7 before disposing of it.2. “Clean” window cleaner, etc. obtainedfrom here in the lab can be placed back intothe original containers for future use.

3. Please take left-overs of any chemicalsyou bring in to test back home with you.Because we must inventory and properlydispose of any chemicals here at the College,having “extra” chemicals lying around causesa lot of extra work for the lab staff.

B. Personal Safety1. For this lab, “play it safe” and do notuse concentrated, strong acids or bases suchas toilet cleaner or drain cleaner. The acidityof vinegar and the alkalinity of soap aredifferent enough to get the point across.

If you are dealing with concentratedacids or bases and/or are working in asituation where some acid or base couldsplash, you should be wearing goggles. Wehave goggles available in the lab area, so ifyou don’t see them around, check with yourinstructor or the lab manager if you need orwant to wear them. Goggles may not be“fashionable,” but they sure beat going blind(or having to endure the eye-wash).

2. If you get a concentrated acid or baseon your skin, immediately rinse the area offwith lots of cold water and notify yourinstructor. Strong bases can be neutralizedwith vinegar, and strong acids with bakingsoda. For large spills, notify the lab staff sothey can assist with proper clean-up.3. Our MSDS books are available in thelab area, and you are welcome to use these atany time to find information on health andsafety precautions when working with variouschemicals. If time allows, you might trylooking up some of the chemicals you’retesting to see if they’re in the books, and if so,what the sheet has to say about it/them.

IV. MATERIALS NEEDED:You may bring samples to be tested (note:some samples may need to be dissolved inwater first – see protocol) such as yogurt,buttermilk, milk, ice cream, sauerkraut,distilled water, well water, mineral water, tapwater, baking soda, baking powder, cream oftartar, vinegar, salt, sugar, various fruit juices,soft drinks (compare “flat” to fresh?), coffee,tea, herb tea, vitamin C, Tums, Rolaids, etc.

(maybe try “before” and “after” readingswhen added to 0.1 M HCl or other acid), Milkof Magnesia, aspirin, Tylenol, etc., dilutesolution(s) of HCl or NaOH (lye), urine(compare fresh to stale?), saliva, soilsample(s), ammonia, laundry or dishdetergent, Windex, Ivory or other soap,Clorox, other cleaning supplies, etc., etc.Please note that some household chemicals

July 1, 2013 36

including toilet bowl cleaners (Vanish) anddrain cleaners (Drano) are extremelyconcentrated, strong acids (Vanish) or bases(Drano), to the point that they really aren’tsafe to handle without goggles, gloves, labaprons, and special training. Also, unlikeyour home, Clermont College falls undercertain federal regulations which prohibitdrain disposal of these concentrated acids andbases without first going to a lot of trouble to

neutralize them. Thus we would ask that youplease NOT bring samples of them to test.pH meterStandard buffer solutions (pH 7.00 plus either

4.00 or 10.00)pH indicator paperdH2O in wash bottleKimwipes®glassware as needed

V. PROCEDURE:A. Preparation of Sample(s)1. Select a substance to be tested.Substances must be water-soluble, not oil-soluble (hand lotion, butter, furniture polish),and must not coat the electrode (hair spray,white-out, mascara, concentrated liquiddetergent). Solvent-based substances(fingernail polish remover, perfume) shouldalso be avoided. Some of the above areavailable in the lab. You may wish to bringsomething in from home to test. Withinreason, use your imagination.2. Liquids may be used “as is” or may bediluted (record how much substance and howmuch water were mixed). Thick liquids likedish detergent or shampoo must be diluted.3. Items like fruit need to be squeezed orblended (and put through a strainer if needed)to extract the juice.4. Thick liquids (like yogurt or Milk ofMagnesia) need to be diluted with distilledwater until “thin” enough to easily rinse offthe pH meter electrode. If you do need todilute something, record this in your notebookbecause this does, of course, change the H+

concentration.5. Solid substances need to be dissolvedin dH2O (note that the pH meter will workonly with water-soluble solutions, not things

like oil). Weigh a 100 mL beaker. Add about1 gm of sample and re-weigh, subtracting theweight of the beaker to determine the exactweight of your sample (two decimal places).Multiply the weight of your sample by 20 andadd that many milliliters of water to get a 5%solution. Make note of this because, again,dilution changes the pH.6. For any liquids or solutions, you maywish to test further the effect of dilution onpH. Measure 5 mL of your solution, place intoa graduated cylinder, and dilute to 50 mL fora 1/10 dilution (which would be expected tochange the pH by one unit). Pour into a clean100 mL beaker to determine the pH.7. Substances like Tums and Rolaids arebuffers, that is they minimized the change inpH from a pre-determined value. Thus, if youadd acid or base to a solution of one of them,you would not expect the pH to change verymuch – an interesting experiment to try.They “neutralize” stomach acid because theyare at a nearly-neutral pH and do not allowmuch variation from that point.8. Make sure you label all beakers ofsamples as you make them up and dispose ofthem properly. That way, someone won’taccidentally stick their fingers in something.

B. Use of pH PaperTear no more than 5 cm (2 in) of

paper off the roll – be conservative and don’ttake more than what you need – trying not tolet the end go inside the case. Dip about 2 cm(½ to ¾ in) into the fluid to be tested.Compare the color with the chart on the roll.Check both sides of the color chart becausethere may be some slight variation in your test

sample. Be careful not to get sample on yourfingers, especially if it’s something caustic.Record the pH in your notebook. Optionally,when dry, your pH paper strips may befastened into your notebook as well.Otherwise, dispose of them in the trash, anddo not leave them on the lab table or the floor.

C. Use of pH Meter1. Your instructor will demonstrateproper use and care of the pH meters. Pleaseobserve carefully and take good notes in yourlab notebook so you know how to operate themachine (and get a reliable reading) withoutbreaking it.2. The electrode(s) have been soaking inpH 7.00 buffer and the machine has beenwarming up for at least 30 min. Notice thatthe beaker in which the electrode(s) is/aresoaking is labeled as containing the buffer.

Locate the beaker which should be nearbythat is labeled “WASTE”, the squirt-bottlelabeled “dH2O”, and a box of Kimwipes.Note that while the buffer is not extremelydangerous, it would be a good idea to avoidgetting it on your hands and to rinse it off ifyou do.3. Observe and draw the various knobsand the toggle switch on the machine. Payclose attention to the labels for(“Temperature,” “Standardize,” “Slope”) and

July 1, 2013 65

Figure 40. Air Bubbles Look Like This!

stored in the central vacuole. Draw and labelwhat it looks like.J. Bird’s EggBirds’ eggs are among the largest cells.While the shell, the membrane just within it,and the albumen are non-cellular secretions ofthe hen’s reproductive tract, the yolk (and infertile eggs, the initial zygote – unfertilizedeggs only have the female half of thechromosomes) is a single cell. In fertile eggs,the embryo develops into a multicellular

chick. The demonstration chicken egg hasbeen heated to coagulate the albumen andyolk. Examine, draw, and label this cut-openegg (NOT for microscopic examination –there’s not much to see that way).K. Other CellsOther materials and/or prepared slides may beavailable, and if so, examine and draw. Ifavailable, examine a drop of pond water tosee what “lives” there (draw).

V. DATA:Draw and label all cell types you observe. Take notes on what you see, too.

VI. DISCUSSION:Of the cells we examined, which

was the smallest and which the largest (basedon how many it took to fill the field of view)?In addition to general comments, make a chart

of the various organelles, etc. that youobserved which contains the followinginformation:

Organelle/Structure Organism(s) Where Found General Description/Notes Function

Also (letters correspond to aboveletters):

A. Cork: Did you see anything beside cellwalls? Why/why not? What do you thinkhappened to the cytoplasm of these cells?B. Elodea: Did you see cyclosis?Describe what you saw.C. Potato: What effect did the iodinehave? Relate this to the lab we did on sugarsand starch.D. Oral mucosa/buccal smear: How is theshape of a cell influenced by being part of aclump versus being solitary – are the edgescurved or are there straight edges withcorners? Did you see many bacteria? Whatdid they look like?E. Onion epidermis: What is the generalsize and shape of these cells? were thenucleoli in the nuclei visible? If so, how didthey appear?F. Tomato: What did you see under themicroscope that could account for the redcolor of a tomato?G. Broccoli or geranium leaf: Doepidermal cells have chloroplasts? Do guardcells have chloroplasts? Did you see any leaftissue that did contain significant numbers ofchloroplasts, thus where photosynthesiswould be occurring? Can you figure out whatfunction the stomates might serve?

J. Did you have time to examine anythingelse? What sorts of interesting “creatures”lived in the pond water you examined? Didthey appear to be single or multicellular? Doyou have any other observations to discuss orconclusions you can make?

July 1, 2013 64

Figure 36. Tomato Nucleus and Cytoplasm,400×, Methylene Blue stain

Figure 37. Broccoli Epidermis, 400×

Figure 38. Rheo Flower Petal with OxalateCrystals, 100×

Figure 39. Oxalate Crystals, 400×

the red “pulp” or tissue beneath the skin(NOT the skin itself). Gently spread it out abit on your slide and make a wet mount. Youshould not have a big, red blob on the slide,rather, the smear should be thin enough thatyou can just barely see it. Examine under lowpower and draw, then under high power anddraw. Note whether cells that are stillattached to each other and cells that havecome free from the rest are the same shape –does being in contact with other cellsinfluence the shape of a cell? Locate the cellwalls. Notice the small, rust-coloredchromoplasts (chromo = color) – which givethe tomato its color – located in thecytoplasm. The majority of the cell is acentral vacuole and the cytoplasm will appearas thin streaks of grayish or speckled matter.Optionally, stain with methylene blue byputting a drop at the edge of the coverslip anddrawing it through by touching the other sidewith a Kimwipe (see end of #3 for moredetailed directions for this method). DON’TGET IT ON THE MICROSCOPE!!!

H. Broccoli or Kale EpidermisTear a broccoli, kale, or geranium leaf“sideways” so that a portion of the lower(clear) epidermis (epi = upon, over; derm =skin) is exposed. Cut this off with a razorblade or scalpel and make a wet mount of it.Observe and draw. Label epidermal cells(clear and irregular), their cell walls, and the

smaller, oval areas that resemble cat-eyeswhich consist of a pair of crescent-shapedguard cells surrounding a small opening, thestomate (stoma = mouth). Notice the greenchloroplasts in the guard cells. On your slide,you will probably also see larger, rounded,green (due to chlorophyll) mesophyll cells(meso = middle; phyll = leaf).

I. Moses-in-the-Boat Flower PetalIf enough are available, make a wet mount ofa petal of a Moses-in-the-Boat (Rhoeo) flowerpetal (a few of the other flower parts alsowork). If not enough are available foreveryone to make mounts, one will be set upas a class demonstration. These cells containlong, slender crystals of calcium oxalate (oxa= sharp, acute, acid), similar to the oxalic

acid that givest h e p l a n tOxalis its tastea n d n a m e .T h e s e a r eprimarily an“ e xc r e t i o n ”product of theplant and are

July 1, 2013 37

any divisions/markings on the knobs.4. Use a thermometer to determinecurrent room temperature. Set the“Temperature” knob to that temperature.Record the room temperature in your labnotebook.5. Note that on a combination electrode(everything in one so only one electrodeneeded rather than two) there is a smallwhitish spot called the “calomel” on the sidejust above the glass electrode – this must bebelow the surface for the machine to functionproperly. With the electrode submerged farenough to include the calomel, set the pHmeter on “Read.” Since the electrode is inpH 7.00 buffer, the indicator needle shouldread exactly 7.00 (If you are looking exactlyat the needle, you will not see its reflection inthe mirror behind it – remember parallaxerror?). You may need to wait a little whilefor the needle to stabilize. If the reading isnot 7.00, slowly turn the “Standardize” knobuntil it is, then put the meter back on“Standby.” If the calomel is not submerged,the machine will behave erratically. Alwaysremember to turn the right knob back to“Standby” before removing it from asolution. Failure to do so can mess up theelectrode so it doesn’t give meaningfulreadings.6. After double checking to make sure themachine is on “Standby,” gently raise theelectrode out of the buffer (note: if you dothis with the machine on “Read” it will messup the electrode – make sure it is on“Standby”). Use the bottle of dH2O toTHOROUGHLY rinse off the electrode,catching the drips in the “waste” beaker.Gently TOUCH off the excess water (do notrub or wipe) with a Kimwipe.7. CAREFULLY lower the electrode intoa beaker of either pH 4.00 or pH 10.00 buffer(record in your lab notebook which you use)far enough to include the calomel. Set themeter to “Read” and use the “Slope” knob

(note: do NOT use the “Standardize” knob forthis – make sure you use the “Slope” knob) toadjust the meter to the corresponding pH(4.00 or 10.00). Then, remember to put themeter back on “Standby,” and once again, liftup the electrode and thoroughly rinse it andpat it dry.8. CAREFULLY lower the electrode intoyour sample far enough to include thecalomel. Set the machine to “Read” and,when stable, take your reading, rememberingto interpolate the last decimal place (read totwo decimal places). Do not let the electrodehit bottom or it will break. Set the machineback to “Standby” when you are done, andremember to record your reading in your labnotebook. After setting the machine to“Standby,” raise the electrode(s) remove yoursample, and THOROUGHLY rinse all samplematerial off the electrode(s) into the “waste”beaker.9. If you have another/more sample(s) totest, repeat step 8 for any subsequentsample(s). After you are finished with allyour samples, thoroughly rinse the electrodeto make sure it is clean, pat it dry, then lowerthe CLEAN electrode carefully into the pH7.00 buffer for storage. Under NOcircumstances should the electrode be leftin “mid-air” where it will dry out. Pleasemake sure before you leave a station that theelectrode is back in the pH 7 buffer solution.10. Clean up all spills. Leave the area neatand dry. Claim and clean up your beaker(s)of sample. Please do not leave your beakersof solutions lying around for someone else toclean up and please remember to dispose ofwaste properly. Make sure the waste beakerand beaker of buffer as well as bottle of dH2Oand box of Kimwipes are still there for thenext person. Thoroughly clean all yourglassware and place in the appropriate rack(s)to dry. Double-check to make sure theelectrode is clean and has been placed backinto the beaker of pH 7.00 buffer.

VI. DATA:Record all data as indicated in the

Procedure, plus any other notes andobservations you might have. Illustrate a pHmeter, labeling the various knobs. Payspecial attention to getting the actual face ofthe meter drawn correctly, including theactual markings, the needle, and the mirror.Also, remember to draw any other equipment

used that is new to you. Samples of pH paperused to test substances may also be includedin your notebook. Enter the requested data onthe appropriate Web page. Printouts of classdata will be made available, and should beincluded in your notebook when you receivea copy.

VII. CONCLUSIONS:1. You should construct some kind ofchart or graph to indicate the pH values of thevarious substances tested by people in yourlab section. Indicate which are acids andwhich are bases.2. Since pH is a logarithmic number, howwould you expect the 1:10 dilution to affect

the pH of that solution? How did thiscompare with the actual readings obtained?Of what reasons can you think as to why theremight be a difference between expected andactual values?3. Do you have any further comments oninteresting or unusual observations?

July 1, 2013 38

PERCENTAGE OF SUGAR IN SOFT DRINKSProtocol Copyright © 1982 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To learn about specific gravity and density and how these may be used to

gather information about a solution or substance, especially its concentrationin percentage by weight.

2. To explore the sugar content of various types of soft drinks.

II. BACKGROUND:Sugars, especially glucose (gluco =

sweet, -ose = carbohydrate ending), are amajor source of energy for all living things.Plants produce glucose by photosynthesis andconvert that and other monosaccharides intovarious disaccharides such as sucrose (tablesugar – sucro = sugar) or convert it intostarch to store it more easily. Animals whicheat these plants can make use of this energysource and also are attracted to the sweet tasteand smell.

We humans have gone a stepfarther. We frequently add sugar to foods thatnormally and naturally do not have it (or haveit only in small quantities) just because wecrave the taste of it for its own sake. We havefought whole wars because of sugar – thereare sources that suggest that the Boston TeaParty was caused not by the British tearegulations, but because of their molassesregulations. As our sugar consumption hasrisen in western nations, so have our rates ofthe “stress” diseases: diabetes (dia = across,through; bainein, badis = to go, walk, step)and hypoglycemia (hypo = under, beneath;glyco = sweet), heart and circulatoryproblems, dental caries, malnutrition,decreased resistance to infections, etc. whichare not nearly as prevalent (if at all present)in the Third World nations. An increasingnumber of nutritionists and other medicalpeople are now in agreement that refinedsucrose (or any sugar) is a mind-altering,addicting drug (if you don’t think so, trydoing without, and you will probablyexperience the same withdrawal symptoms asany drug addict).

As of 1986, when Laurel Robertson,et. al. revised their book, Laurel’s Kitchen,Americans were averaging � lb. of sugar perperson (including children) per day, whichcomes to about 127 lb. per person per year.As of 1982, when Francis Moore Lappérevised Diet for a Small Planet, ¼ of theaverage American’s intake of cane and beetsugar came from soft drinks (pp. 126-127).According to Laurel’s Kitchen, (p.421) softdrink consumption in the U. S. rose from 1.6drinks per person per year in 1850 to 620

drinks per person per year in 1981.According to the July 1998 issue of BetterNutrition, the average American sugarconsumption has risen to 148 lb. per personper year, which is over 1/3 lb. or 600 KCalper day! In this experiment, we will analyzea number of types of soft drinks to see howmuch sugar they contain.

Hopefully you recall from theAccuracy and Precision Lab that density isdefined as grams of solution per milliliter ofsolution (gm/mL), and that by definition, thedensity of water is 0.997 or 0.998 gm/mL at20 to 25°C – room temperature. If anysolutes are dissolved in the water, the weightof a given volume of the solution increases –the solution becomes more dense. Peoplewho have done a lot of swimming probablyhave heard that a person floats higher inocean water and especially in very salty waterlike the Dead Sea or Great Salt Lake. This isbecause relative to the density of our bodies,these salty waters are more dense (and weless dense). When sugar is added to water, italso makes the solution more dense, thus theweight of a known volume of solution can becorrelated to the amount of sugar in it.Specific gravity is defined as the density of asubstance divided by the density of water atthe same temperature, and thus is a unitlessquantity. In the metric system, for ourpurposes, density and specific gravity areequal (but in the English system, this is nottrue) thus, by weighing equal volumes ofwater and soft drink, we can determine thespecific gravity of the soft drink (wt. of popper 100 mL ÷ wt. of dH2O per 100 mL = wt.of pop ÷ wt. of dH2O because the 100 mL/100mL cancels out). We can, then, look up thepercentage of sucrose (sucro = sugar; -ose =a carbohydrate ending) from a chart or graphof specific gravity versus percent sucrose.From this, it is possible to calculate howmuch sugar is in a can of soft drink. Notethat we are assuming that the other solutes insoft drinks do not occur in large enoughamounts to affect the density of the solutionvery much.

III. MATERIALS NEEDED:1 can/bottle soft drink of your choice100 mL volumetric flask w/ your seat no.

balancehot water bath (heat source)

July 1, 2013 63

Figure 32. Purple Onion Epidermis, 100× Figure 33. Onion Epidermis Cytoplasm andNucleus, 400×

Figure 35. Tomato Pulp, 100×, MethyleneBlue stain

the nucleus, which should have stained darkblue and which may contain one or more evendarker blue nucleoli (nucleolus), the centralvacuole which, again, takes up most of thecell (this vacuole contains watery “sap” and isseparated from the rest of the cell by amembrane that cannot be seen without specialstain), and the cytoplasm which may be found

especially near the edges and corners of thecell as well as occasional streaks across thecell – you may be able to see cyclosis (cyclo= circle, wheel; -sis = the act of). You mayhave to adjust the iris diaphragm or light levelto get optimal contrast. Note how many cellsit takes to fill the field of view lengthwise andwidthwise.

As time and specimens allow, also examine these:F. Yeast CellsYeast cells should be fairly oval in shape. Do you see any cells with reproductive budsattached? Can you see any of the organelleswithin the cells? Yeast cells do have a thincell wall and clear cytoplasm. The nucleuscannot be seen unless special stainingtechniques are used. After viewing the slide“as is” at each power, remove the slide fromthe microscope and add a small drop ofmethylene blue by touching the tip of thedropper near the side of the coverslip. The

dye will run under the coverslip, and the yeastwill absorb the dye and turn blue. Whatdifference(s) does the methylene blue make inthe “visibility” of the yeast cells or theirorganelles? Note any other observations (forexample – have all of the cells taken up thedye equally?).

G. Tomato PulpFrom a tomato (or red pepper), take a bit of

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Figure 1. Potato nucleus, 100×, Iodine stain

Figure 2. Buccal Smear, 400×, MethyleneBlue stain

them are not visible – containing starchgrains. Since some cells were cut open, therewill be loose starch grains.

Generally, the nucleus is obscured by thestarch grains, but if you are very lucky, youmay see one, especially if you stain the cell.Examine single starch grains and note theconcentric layers (the light has to be justright). Remove your slide from themicroscope and put a tiny drop of iodine toone side of the coverslip. If necessary, use aKimwipe or paper towel touched to theopposite side of the coverslip to pull theiodine underneath the coverslip. Examineand draw you potato slice now. Note in yourdrawing either by words or colored pencilswhat colors things are now (especially makenote of what color the starch grains are now).Optionally, try a potato slice with methyleneblue to attempt to see the nucleus.

D. Buccal SmearBy carefully following directions, make abuccal smear slide as follows. Use yourfingernail to scrape the lining of your cheek(the oral mucosa). Spread this material over0.5 cm2 in the center of a clean slide – makea buccal smear (bucca = cheek). Allow it toair dry. When it is dry, pass it through theflame of a Bunsen burner three times – it isnot necessary to bake, scorch, brown, or cookthe specimen, just pass it through the flame,right-side up (it should NOT be too hot tohold). Note: this is not designed to dry out awet specimen, either, merely help it to stickto the slide better (like three-day old spaghettidried onto a plate). This warming will fix thecells to the slide so the stain won’t wash themoff (if you are careful). Place the slide,smear-side up, on a paper towel and put dropsof methylene blue onto the smear to cover it.Let the stain sit on the slide for exactly oneminute (time it), then tip the slide and allowthe excess stain to run off. Rinse GENTLY intap water from one of the SQUIRT BOTTLESso labeled (NOT under the faucet), shake offthe excess water, and allow the slide to airdry right-side up (you may use a Kimwipe todry the bottom and edges of the slide, but donot attempt to blot the area where the smearis or you will wipe it off). You do not need acoverslip with this slide. Examine and draw.Look for small, usually somewhat oval orround cells alone or in small groups. Ifgrouped, note how this affects their shape.Focus up and down with the fine adjustmentto see if you can observe any thickness to thecells. The nucleus should show up as adarker blue oval or round region near thecenter of each cell. Tiny, darkly-stainedobjects which adhere to the cell membranesare bacteria which are commonly found in themouth. The cytoplasm will be a pale blue.Optionally, if someone has a lot of interestingbacteria, your instructor may set up amicroscope to view them with the oilimmersion lens at 1000×. Note how flat orrounded these cheek cells appear to be – canyou relate this to their function as a lininglayer of cells? What is the ratio of thediameter of the nucleus to the overalldiameter of these cells? Again, how manydoes it take to span the field of view?E. Onion EpidermisPeel a small piece of the transparentepidermis from a layer of an onion (NOTE:YOU DO NOT WANT the whole, thick,fleshy part, just the transparent “skin” layer).Place it on a slide with a drop of water. Adda small drop of methylene blue, then put thecoverslip on. Examine and draw. Pickseveral cells to examine in more detail anddraw. Focus up and down with the fineadjustment to see the third dimension of thecells. Label the thin cell walls between cells,

July 1, 2013 39

SAMPLECALCULATIONS:

wt. of flask + H2O: 161.55

wt. of flask 61.83wt. of H2O 99.72

wt. of flask + pop: 166.17wt. of flask 61.83wt. of pop 104.34

wt. of pop ÷ wt. of H2O:104.34/99.72 = 1.0463(specific gravity)

from the chart, 1.0463corresponds to 11.5% sugar

11.5% = 0.115

0.115 × 104.34 g (wt. of pop) =12.00 g of sugar per 100 mL

x = 3.55 × 12.00 = 42.60 gsugar/can, and then,42.60 g/can × 0.22433 tsp./g =9.56 tsp of sugar/can of pop

250 mL beakerglass stirring rodthermometer (use glass – be careful)

Pasteur pipet & bulbice bath

IV. PROCEDURE:A. Pour about 125 mLdH2O (at room temperature)into a clean 250 mL beakerand insert the thermometer(thermo = heat; meter = tom e a s u r e ) t o c h e c ktemperature. Please bec a r e f u l w i t h g l a s sthermometers – they breakeasily. Obtain the balance foryour table number.B. While the thermometeris equilibrating, weigh aclean, dry 100 mL volumetricflask (the one with your seatnumber on it) to the nearest0.01 gm. Record its weight inyour lab notebook. You mustuse the same one each time –each flask weighs a differentamount. THIS IS THE ONLYCHANCE YOU WILL GETTO OBTAIN A DRYWEIGHT FOR YOURFLASK.

C. Read the temperatureof the dH2O. If it is not righton 20° C, use the ice bath toadjust it. Set the beaker inthe ice bath, and whileconstantly moving it, also stir the water witha glass stirring rod (temporarily remove thethermometer so it doesn’t break). Monitorthe temperature frequently because it maychange rapidly. Remove the beaker from theice bath when the temperature is just above20° C, and keep stirring it. As the coldbeaker absorbs heat from the water, the waterwill continue to cool. Note that, if it is closeto 20° to begin with, it may only take a fewseconds in the ice bath to adjust thetemperature.D. When the water is at exactly 20° C,pour 100 mL into the volumetric flask. Toavoid getting air bubbles trapped in the flask,pour the water gently down the side of theflask. Any air bubbles in the flask or excessH2O will change the weight. Remember thatthe bottom of the meniscus should be evenwith the calibration line on the neck of theflask. Use a Pasteur pipet (medicine dropper)to adjust the volume and then, dry off anyexcess water droplets.

E. Using the same balance as before (andwithout any further “adjustments” to it),weigh the flask plus water. Record theweight in your lab notebook. Subtract the

weight of the flask alone todetermine the weight of thedH2O.F. Pour used dH2O intothe designated container for“recycling”. Invert the flaskto drip dry, supported in a testtube rack. in a location whereit will not get knocked over.

G. Pour about 125 mL ofyour favorite soft drink intothe 250 mL beaker. Ifpossible, recap the left-overso f t d r i nk to avoidcontamination.H. “De-gas” the soft drinkby heating it to about 80°C (Itmay not get quite that hot).Place your beaker in the hotwater bath and stir itoccasionally with the glassstirring rod. Periodically,monitor the temperature withthe thermometer. Do not boilyour sample or water willevaporate and change theconcentration of the solution –you only want to get rid of thecarbonation. Stirring gentlywill speed things up. Heat

and stir until there is no more “fizz” left –until the pop is totally flat. Lack of “fizz” ismore critical than temperature reached.

I. Place the beaker of sample into the icebath. As before, gently swirl the beaker andstir the pop with the glass rod. Periodicallymonitor the temperature as before(CAUTION: thermometers are fragile).When the temperature is a few degrees above20° C, remove the beaker from the ice bath.Continue swirling and/or stirring until thetemperature reaches 20°.J. Place 100 mL of de-gassed soft drink inthe SAME volumetric flask you used beforeand adjust the level as before. Remember towatch for bubbles and excess droplets.

K. Using the same balance as before,weigh the flask plus soft drink. Record thisweight in your lab notebook and subtract tofind the weight of the soft drink.L. Determine the specific gravity of thesoft drink by dividing the weight of 100 mL ofit (from step K) by the weight of 100 mL ofdH2O (from step E).

M. Use the provided chart to determine the

July 1, 2013 40

percent sucrose by weight in your soft drink.This chart was excerpted from a much largerversion in one of the chemistry handbooks.What this number means is that your popcontains that percentage of sugar, or for 100 gof pop, that number of grams out of the 100would be sugar (however, your sampleprobably did not weigh exactly 100 g).N. Using the decimal form of your percentsucrose (for example, 12% = 0.12), multiplythat times the weight of the sample (from stepK) to calculate the weight (number of grams)

of sucrose in your sample. For example, ifyour sample weighed 104.34 g and was11.5% sugar, the sample would contain12.00 g of sugar.O. However, a can of soft drink containsmore than the 100 mL in your sample. If youlook at the side of the can, somewhere it willsay that it contains 12 oz, which is equivalentto 355 mL. Thus, the number of grams ofsugar in a can of soft drink can be calculatedusing a ratio:

which can be simplified to:grams in 100 mL × 3.55 = grams/can.

P. However, many people don’t reallyhave a visual idea of how big a gram is, so amore useful figure might be the number ofteaspoons of sugar per can. To allow us tocalculate that number, knowing that one cup(1 C) equals 48 tsp., a cup of sugar wasweighed. The weight of that cup of sugar,those 48 tsp, was found to be 213.97 g. Thus,each gram of sugar is equivalent to0.22433 tsp. Thus, to determine the numberof teaspoons of sugar in your can of soft drink,multiply the number of grams you calculatedper can (from step O) by 0.22433.Q. Look at the side of the can/bottle tofind out how much sugar the manufacturerclaims is in “one serving.” Beware! If yoursoft drink is in a can, the manufacturer willtell you a “serving” is 12 oz, but for the samesoft drink in a 2 L bottle, suddenly, a

“serving” is only 8 oz. So that we can easilycompare the amount of sugar in various softdrinks, we want to convert “everything” to theequivalent of a 12-oz serving. Thus, if abottle of soft drink claims to have 32 g ofsugar per 8 oz “serving”, that’s equivalent to32 × 12 / 8 = 32 × 1.5 = 48 g of sugar per“normal” 12-oz serving.

R. Record data in the computer asindicated.S. CLEAN UP AFTER YOURSELF!!! Thoroughly rinse all pop off thermometersand stirring rods. Thoroughly rinse out yourvolumetric flask by gently inserting the watersupply tubing up into the flask so clean waterwill “push” the sticky pop out. Thoroughlyrinse all pop out of the beaker. Check youwork area and clean up any spilled pop. Anypop that gets left on glassware or table topsmakes a sticky mess when it dries.

V. DATA:In your lab notebook, record all data

and observations as indicated in theprocedure. Take any other notes you feel areimportant. Draw anything new or that willhelp you to remember. When everyone hasentered data into the computer, the computer

will analyze the class data and calculateaverages for each brand/flavor of pop used.These data will be xeroxed and distributed toall class members, so save space in yournotebook for these data.

VI. CONCLUSIONS:In your discussion, include:1. Do you drink soft drinks frequently?You may wish to figure out how much sugaryou get from soft drinks per day or week ormonth. For example:

# of cans/day × g/can (from step O) = g/dayg/day × 7 days/week = g/weekg/week × 1 lb/453 g = lb/weeklb/week × 52 weeks/year = lb/year

lb/year ÷ 5 = # of 5-lb sacks/year

Would you put that much sugar in a cup of teaor coffee?2. As you compare the class data, whichsoft drinks had the most sugar? You maywish to comment on the implications,healthwise, for someone who drinks a lot ofsoft drinks.

July 1, 2013 61

Figure 3. Elodea Leaf, 400×

Figure 4. Potato, 40×

trapped. Rather, lower the coverslip, tilted atan angle, until the lower edge touches theslide at the edge of the water drop.

Then, slowly lower the upper edge ofthe coverslip. The drop should spread outunder it without air bubbles being trapped.Wipe off any excess water from the bottom,edges, etc. with a Kimwipe so the microscopedoesn’t get wet.

Examine your slide under 40, 100, and400× (ALWAYS START AT 40× =4× objective). Draw what you see at eachpower. Remember to make your drawingslarge enough. Carefully focus up and downwith the fine adjustment to observe the factthat these cells are three-dimensional(adjustment of the iris diaphragm and rheostatmay help you to see this better. How muchsize variation can you see? If you see a roundobject (or several) with a broad, black ringaround the outside, it’s an air bubble.

As you examine the followingmaterials, take notes on and make drawingsof what you are viewing. Refer toillustrations in the handout and label all partsindicated on these illustrations and your owndrawings Each drawing should be ¼ to ½page. Do not draw circles around everything,but do watch relative space and proportion.Use your lab pen to outline drawings and fillin with color later, if desired. Label withpower of magnification to lower right of eachpicture. Wash and dry slide and coverslipbetween each specimen and when you aredone. Remember to return slides toappropriate places when you are finished withthem.A. Cork BarkWhen Robert Hooke first saw and namedcells, he was examining cork bark. From apiece of cork, shave off a VERY THIN sliceand make a dry mount of it by just placing itunder a coverslip. Your slice should be thinenough that you can almost see through it andthe coverslip does not rock back and forth ontop of it. Draw and label what you see. Areall the “cells” you see the same size andshape or not?

B. Elodea LeafPick an Elodea leaf. Put it in the middle of aslide with a drop of tap water, and put acoverslip on it. Note the cells that make upthe midrib of the leaf and notice that the leafis “3-D.” Locate one cell (usually ones nearthe edge work well) to examine more closely.Draw cells as they appear under the variouspowers of magnification. Label the cell wall,cytoplasm (cyto = cell), chloroplasts (chloro= green; plasti = formed, molded) – greenovals within the cell, if visible the larger,oval, transparent, nucleus (if you find it –usually difficult to see) within the cytoplasm,

and the very large central vacuole (vacu =empty) which takes up almost the whole cell,or so it seems. Using the fine adjustment,focus up and down to observe the centralvacuole and the small surrounding layer ofcytoplasm. Look for a region in that cell oranother where the chloroplasts are moving,indicating that cyclosis (cyclo = a circle,wheel; -sis = the act of), or cytoplasmicstreaming, is occurring as it often does in leafcells. You do not need to draw the wholefield of view – draw a representative areawith a few cells. Note the general size andshape of the cells – how many of them does ittake to fill the field of view (length? width?)under both low and high powers?

C. Potato PulpFrom a potato, take a small, very THIN slice(you should be able to see through it). Makea wet mount of your slice. Examine anddraw. Note the cell walls and the leucoplasts(leuco = white) – membranes delineating

July 1, 2013 60

Figure 26. Cork Bark, 400×

Figure 27. Side view of coverslip application

CELLS AND ORGANELLESBuccal Smear Protocol Copyright © 1980 D. B. FankhauserCopyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To study various cells and the organelles of which they are composed.2. To learn how to make a buccal smear.

II. BACKGROUND:All living things are made up of

cells. Some organisms, like yeast, are onlysingle-celled, while others, like humans,contain many cells. Cells are bounded by aplasma membrane which is so thin it is ofteninvisible even with a light microscope. Cellsof organisms such as plants have a cell walloutside the plasma membrane. The mostimportant organelle (-elle = small) withineukaryotic cells is the nucleus. Rememberthat,unlike yeast, humans, and other sucheukaryotes (eu- = good, well, true), bacteria,which are prokaryotes, (pro = before, in frontof; karyon = nut, kernel, nucleus) do not havetheir DNA organized into a nucleus. When acell is “stained,” the nucleus often takes dyewell, especially in the region(s) of thenucleolus (nucleoli). The region between thenucleus and the plasma membrane is calledthe cytoplasm (cyto = cell), which contains anumber of other kinds of organelles. Some ofthese are visible only with an electronmicroscope and/or special stainingtechniques, while others are easily visiblewith a light microscope. Various vacuoles(vacu = empty) are usually visible. Manyplant cells have a large central vacuole whichoften takes up more space than the cytoplasm.In plants, chloroplasts (chloro = green; plasti= formed, molded) are easily seen, as well asvarious other plastids including leucoplasts(leuco = white) and chromoplasts (chromo =color). If present, cilia (cilium = eyelid,eyelash, small hair) and flagella (flagellum =a whip) can sometimes be seen by theirshadow (or with a special stain), although not

in great detail. There are special stainsavailable to enable us to see certain of theother organelles.

The smallest cells we know of aresome bacteria, the largest are bird eggs, theyolk portion of which is a single cell. Thealbumen (alb = white; album = the white ofan egg), outer membrane, and shell are non-cellular products of the hen’s reproductivetract. The longest cells we know of are nervecells. To reach from a human’s spinal cord totoes, a nerve cell has to be about three to fourfeet long – imagine a giraffe’s nerve cells.

III. MATERIALS NEEDED:slide and coversliprazor blade and/or scalpeltoothpicksKimwipes and/or paper towelmethylene blueiodine solutionmicroscopeBunsen burner* opt. colored pencils

corkElodeapotatoonion* tomato or red pepper* broccoli, kale, or geranium leaf* Moses-in-the-Boat flowers (Rhoeo)* hard-boiled egg, cut in half* pond water or other optional materials

IV. PROCEDURE:Obtain a blank slide and coverslip and

check to make sure that they are clean. Placeyour specimen and one drop of water on thecenter of the slide.

Do not just drop the coverslip onto theslide because large air bubbles will be

July 1, 2013 41

Specific Gravity of Sucrose Solutions at 20°/20° Csp. gr. % sucrose sp. gr. % sucrose sp. gr. % sucrose sp. gr. % sucrose

1.00000 0.0 1.01490 3.8 1.03010 7.6 1.04582 11.41.00039 0.1 1.01530 3.9 1.03050 7.7 1.04625 11.51.00078 0.2 1.01570 4.0 1.03090 7.8 1.04668 11.61.00117 0.3 1.01610 4.1 1.03130 7.9 1.04711 11.71.00156 0.4 1.01650 4.2 1.03170 8.0 1.04754 11.81.00195 0.5 1.01690 4.3 1.03211 8.1 1.04797 11.91.00234 0.6 1.01730 4.4 1.03252 8.2 1.04840 12.01.00273 0.7 1.01770 4.5 1.03293 8.3 1.04882 12.11.00312 0.8 1.01810 4.6 1.03334 8.4 1.04924 12.21.00351 0.9 1.01850 4.7 1.03375 8.5 1.04966 12.31.00390 1.0 1.01890 4.8 1.03416 8.6 1.05008 12.41.00429 1.1 1.01930 4.9 1.03457 8.7 1.05050 12.51.00468 1.2 1.01970 5.0 1.03498 8.8 1.05092 12.61.00507 1.3 1.02010 5.1 1.03539 8.9 1.05134 12.71.00546 1.4 1.02050 5.2 1.03580 9.0 1.05176 12.81.00585 1.5 1.02090 5.3 1.03621 9.1 1.05218 12.91.00624 1.6 1.02130 5.4 1.03662 9.2 1.05260 13.01.00663 1.7 1.02170 5.5 1.03703 9.3 1.05302 13.11.00702 1.8 1.02210 5.6 1.03744 9.4 1.05344 13.21.00741 1.9 1.02250 5.7 1.03785 9.5 1.05386 13.31.00780 2.0 1.02290 5.8 1.03826 9.6 1.05428 13.41.00819 2.1 1.02330 5.9 1.03867 9.7 1.05470 13.51.00858 2.2 1.02370 6.0 1.03908 9.8 1.05512 13.61.00897 2.3 1.02410 6.1 1.03949 9.9 1.05554 13.71.00936 2.4 1.02450 6.2 1.03990 10.0 1.05596 13.81.00975 2.5 1.02490 6.3 1.04032 10.1 1.05638 13.91.01014 2.6 1.02530 6.4 1.04074 10.2 1.05680 14.01.01053 2.7 1.02570 6.5 1.04116 10.3 1.05723 14.11.01092 2.8 1.02610 6.6 1.04158 10.4 1.05766 14.21.01131 2.9 1.02650 6.7 1.04200 10.5 1.05809 14.31.01170 3.0 1.02690 6.8 1.04242 10.6 1.05852 14.41.01210 3.1 1.02730 6.9 1.04284 10.7 1.05895 14.51.01250 3.2 1.02770 7.0 1.04326 10.8 1.05938 14.61.01290 3.3 1.02810 7.1 1.04368 10.9 1.05981 14.71.01330 3.4 1.02850 7.2 1.04410 11.0 1.06024 14.81.01370 3.5 1.02890 7.3 1.04453 11.1 1.06067 14.91.01410 3.6 1.02930 7.4 1.04496 11.2 1.06110 15.01.01450 3.7 1.02970 7.5 1.04539 11.3 1.06153 15.1

July 1, 2013 42

PEPSIN DIGESTION OF PROTEINS AND EFFECTS OFANTACIDS

Protocol and background information Copyright © 1995 J. L. Stein Carter

I. OBJECTIVES:1. To study how pepsin digests proteins in our diet.2. To examine factors which can denature enzymes, causing them to cease

functioning.3. To study the effects of antacids on pepsin’s activity.

II. BACKGROUND:In the human digestive system,

there are many enzymes to help digest ourfood. Enzymes are a special subclass ofproteins that serve as biological catalysts(substances which enable a chemical reactionto happen, but are not used up in thatreaction). Just like any other proteins,enzymes can be denatured, but in the case ofenzymes, denaturing them typically causesthem to lose their ability to function.Changes in temperature, pH, or saltconcentration, or a change from a hydrophilicto hydrophobic solvent can all denatureproteins.

In the stomach, the enzyme pepsinfunctions to break proteins into smallerpolypeptides. The pancreas secretes anumber of digestive enzymes includingmaltase (which breaks maltose into twoglucose molecules), sucrase (which breakssucrose into glucose and fructose), and lactase(which breaks lactose into glucose andgalactose), trypsin and chymotrypsin (whichfurther digest protein), and pancreatic lipase(which breaks fats into glycerol and fattyacids) into the small intestine where mostdigestion and absorption takes place.

Enzymes like pepsin and trypsin aresecreted in inactive forms (pepsinogen andtrypsinogen) and require certain conditions

for conversion to their active forms.Pepsinogen, for example, needs HCl forconversion to pepsin and a pH range of 1 to 3.This pH range is also necessary for properfunctioning of the pepsin. Too great of achange in pH can denature proteins such asthese enzymes, thus causing them to ceasefunctioning.

The pH of the stomach environmentis generally around 1.5 to 3.5, which asmentioned, is necessary for the activation andoptimal activity of pepsin. Most over-the-counter antacid medications react with normalstomach acid (HCl) to produce a more nearlyneutral pH (closer to a pH of 7) solution, andas the pH rises above 4, pepsin activitydecreases or stops. Additionally, it has beenobserved that because our bodies are designedto maintain a normal, constant internalenvironmental balance – homeostasis –typically, consumption of antacids results inthe stomach actually secreting morehydrochloric acid to try to compensate for theimbalance caused by the antacids. Thus,while consumption of antacids may be helpfulwhen prescribed by a doctor to soothe gastriculcer, casual use is probably not a good idea.TV advertisers are only interested in yourmoney, not your heath.

III. MATERIALS NEEDED:16 × 150 test tubes with capswax pencil1% pepsin solution (1 g pepsin in 100 mL or

10 g/L)boiled 1% pepsin solution0.5% NaHCO3 (sodium bicarbonate, baking

soda) (= 5 g/L)0.1N HCl (= 31.15 mL of conc. HCl/ gal or

8.24 mL/L of solution)

hard-boiled egg whiteknife and cutting boardantacid(s) of your choice (Students are invited

to bring samples from home. Recordwhich you use)

250 mL beaker for each antacid samplemortar and pestle (optional)incubatorrefrigerator (optional)pH meter, buffer solutions, and thermometer

IV. PROCEDURE:1. Obtain (at least) eight (or more)

16 × 150 test tubes and number them. Obtainan additional test tube for each antacid you

will be testing.2. In these tubes place the following

solutions:

July 1, 2013 59

MAKING ROOT BEER AT HOMEProtocol Copyright © 1992 D. B. FankhauserBackground and additional information Copyright © 1994 J. L. Stein Carter

I. BACKGROUND:Fermentation has been used by

mankind for thousands of years for raisingbread, brewing beer and for fermenting wine.The products of the fermentation of sugar bySaccharomyces cerevisiae are ethyl alcoholand carbon dioxide. Carbon dioxide is whatgives effervescent drinks their bubbles, andthe action of yeast on sugar can be used tocarbonate beverages (this is also what adds

the bubbles to champagne). We will set up afermentation in a closed system, and capturethe carbon dioxide produced, using it tocarbonate the resulting root beer. You may,of course, adjust the quantities of sugar and/orextract to taste. (Zatarain’s or Hire’s are bothavailable at Kroger. Dr. Fankhauser prefersZatarain’s.)

II. MATERIALS NEEDED:clean 2 liter plastic soft drink bottle with caproot beer extract1 cup sugar¼ tsp powdered baker’s yeast

III. PROCEDURE:1. Thoroughly rinse out a 2 liter plastic soft

drink bottle. With a funnel, add:1 level cup of sugar (swirl in bottom

to make concave to catch extract)1 Tbsp of root beer extract

2. Half fill the bottle with fresh cool tapwater (hopefully without an excess ofchlorine), and swirl to dissolve.

3. Add: ¼ teaspoon powdered baker’s yeast

(Fleischmann’s or other brand)Swirl to dissolve.

4. Q.s. to the neck of the bottle with freshcool water, leaving about an inch of head

space. Securely screw the cap down toseal the bottle.

5. Place at RT (room temperature) for fourdays. Store in cool place. Move torefrigerator ON (overnight) beforeopening.

Note: Bottles left at room temperature toolong will explode! Storing them in therefrigerator after the initial fermentationperiod until consumption is highlyrecommended. Also, if it looks like a bottle isbeginning to stretch, carefully loosen the capto relieve the pressure. Then, tighten the cap,again, and refrigerate until use.

IV. TO SERVE:Use caution when opening the bottle

as it will be under high pressure. Loosen thelid a little, and let some of the built-up CO2escape before removing the lid all the way.Note that there will be a sediment (the matterthat settles at the bottom of a liquid; sedi =sit, sitting, sink down) of yeast at the bottom

of the bottle, so that the last bit of root beerwill be turbid (cloudy or muddy inappearance; turbid = disturbed, confused).Decant (to pour off the liquid from acontainer without disturbing or removing thesediment on the bottom) carefully if you wishto avoid this sediment.

July 1, 2013 58

In this popcorn, the brewer’s yeast provides anumber of the B vitamins. Kelp providesiodine, needed by your thyroid gland. Thekelp, brewer’s yeast, and popcorn arecomplementary protein sources, thus form acomplete protein when combined. By mixing

the butter 50:50 with vegetable oil, it ispossible to have the good buttery taste, yetreduce the amount of cholesterol (cholesterolis found only in animal products, thus wouldbe in the butter, but not vegetable oil).

July 1, 2013 43

#1 10 mL dH2O#2 5 mL dH2O + 5 mL 1% pepsin solution#3 5 mL dH2O + 5 mL 0.5% NaHCO3#4 5 mL dH2O + 5 mL 0.1 N HCl#5 5 mL 0.5% NaHCO3 + 5 mL 1% pepsin#6 5 mL 0.1 N HCl + 5 mL 1% pepsin#7 5 mL 0.1 N HCl + 5 mL BOILED 1% pepsin solution#8 5 mL 0.1 N HCl + 5 mL 1% pepsin solution

3. Mix each solution with a vortex.4. Use a pH meter to determine the pH

of each test tube. Record the pH values inyour lab notebook.

5. For each antacid you wish to test, ina 250 mL beaker, obtain 100 mL of 0.1 NHCl. Use a pH meter to determine the pH ofthis solution and record in your lab notebook.This should be in the range of how much HClmight be present in your stomach.

6. To this solution, add one dose of theantacid you are testing (if more than oneantacid is being tested, each needs a separatebeaker of solution). This would be analogousto the effect on your stomach acid caused bytaking a dose of that antacid. Stir tocompletely dissolve and mix (note: tabletsmay need to be ground, first, with a mortarand pestle). Determine the pH, again, andrecord in your lab notebook. How much didthat dose of antacid change the pH of your“stomach?”

7. Obtain a test tube for each antacidyou are testing, label (#9, #10?, etc.), and intoit (them), place 5 mL of the corresponding

solution(s) you just mixed + 5 mL of 1%pepsin solution. Check the pH again.Remember to record tube number(s),contents, and pH in your lab notebook.

8. Obtain 5 mm-sized cubes of eggwhite and add one piece to each test tube.

9. Cap the tubes and incubate allEXCEPT #8 at 37°C for 48 hours. Store #8 atroom temperature in the designated location.Optionally, prepare another tube with thesame contents as #8 and place it in therefrigerator.

10. The next class period, obtain yourtubes and record what happened in each. Isthere egg white still present or not? Is it stillwhite or has it turned translucent? Is it stillin one piece or broken up? Is there any smellor any other change? Note, since what we’relooking for, here, is whether or not, or howmuch the pepsin functioned, concentrate onthat, not whether mold formed on top.

11. After recording your data in yournotebook, clean up all your test tubes. Enteryour data online. Once everyone has enteredtheir data, go back and print the class data.

V. DATA:Create a table in your notebook similar to the following:

tube # contents pH reaction?

The following rating system can be used to indicate the results of this experiment:(3) +++ no egg white observed, completely digested(2) ++ some egg white present but decreased mass and very transparent(1) + egg white slightly transparent and only slightly changed in size(0) � no change in the condition of the egg white

VI. CONCLUSIONS:1. What effect(s) does pH have on

pepsin’s ability to digest egg white?2. What effect(s) does the addition of

antacid have on stomach pH and on pepsin’sability to digest egg white? Of whatsignificance would this be in “everyday life?”

July 1, 2013 44

Figure 10. Triglyceride

Figure 11. Chemical Reaction for Saponification

LIPIDS, EMULSIONS, AND EMULSIFYING AGENTSSoap Protocol Copyright © 1979 and Mayonnaise Protocol Copyright © 1978 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To study properties of common

lipids.2. To investigate lecithin in egg yolk

as an example of an emulsifyingagent useful for making theemulsion known as mayonnaise.

II. BACKGROUND:

Lipids (lipo =fat) are ag r o u p o fo r g a n i cc omp oundswhich are allhydrophobic( h y d r o =water, phobia= fear), thatis, they do notm i x w i t h

water. Most lipids with which we arefamiliar are oils or fats--tri glycerides (tri =three)--with the general chemical formulashown in Figure 10, where R1, R2, and R3 arehydrocarbon chains (radical). The length ofthese chains and the number of C=C doublebonds determine if the lipid will be an oil, fat,or wax.

When such a compound is mixedwith lye the chemical reaction shown inFigure 11 takes place. This process is calledsaponification. Because the soap formed(sodium salt of a fatty acid) has an ionic end(“head”) it is soluble in water, and because ithas a long hydrocarbon “tail” it is soluble inlipids. Thus, soap is an emulsifying agent,something which is soluble in both oil andwater, thus enabling the two to mix.

Another emulsifying agent, lecithin(lecithos = egg yolk), is a phospholipid

(phos = light; phoro = bear, carry--these referto phosphorus, which “glows in the dark”)present in egg yolk and is reputed to be usefulin removing cholesterol from one's arteries.Another rich source of lecithin is soybeans.

Phospholipids have the same basicstructure as a triglyceride. The difference,however, is that phospholipids have one ofthe carbons of the glycerol (glycer = sweet; -ol = alcohol ending) joined to a phosphate (-ate = to treat, to make, to form, characterizedby having) group which, in turn, is joined tosome other hydrophilic (philia = brotherlylove) organic molecule (the glycerol is joinedto only two hydrocarbon “tails”). Thus,having a hydrophilic “head” and twohydrophobic “tails” makes phospholipids,such as the lecithin in egg yolk, goodemulsifying agents enabling us to enjoy theemulsion known as mayonnaise. Figure 12shows the full structure of lecithin with all thehydrocarbon “tails” illustrated rather thanusing the abbreviation “R.”

Mayonnaise, which is thought tohave originated in the Mediterranean port cityof Mahon, Minorca (hence the name), isprimarily a mixture of oil with lemon juice orvinegar, emulsified by the lecithin in the egg.

Other examples of biologicalemulsions include such things as blood andmilk in mammals as well as various milkyplant saps.

July 1, 2013 57

frothy/foamy.C. Add the oil and 2 C of flour. Beatapprox. 1000 strokes by hand or for a whilewith the electric mixer to develop the gluten,a protein that makes the structure of the breadthat holds in the CO2.

D. Gradually, add approx. another 4 C offlour. The dough should stick together butnot be too stiff. Optionally, you may addraisins, sunflower seeds, chopped nuts,cinnamon, or chopped onion and variousherbs such as basil and sage, or whateversounds like a good bread ingredient. Youmay also substitute soy, rye, oat, or other flouror meal for 1 to 2 C of the whole wheat flour.E. Knead the dough about 10 min until itbecomes elastic.

F. Coat lightly with oil and place in anoiled bowl. Cover with a clean, damp cloth orkitchen towel. Allow to rise in a warm place(a gas oven that’s turned off is good) for aboutone hour until doubled in size.G. If not already done, coat the loafpans/cookie sheet with oil and dust with flour.

H. “Punch down” the dough (deflate itsomewhat), and form into two loaves. Placeinto the loaf pans or form round loaves on thecookie sheet.I. Cover lightly with the towel and letrise until double again (maybe another houralthough it shouldn’t take as long this time).

J. Bake at 350° F for about one half houror until done (check it after ½ to ¾ hr). Signsof doneness are a) if the sides have shrunkenaway from the pan and the crust is brownedand b) when you turn it out of the pan and tapon the bottom, it sounds “hollow.” If not, putit back in the pan and back into the oven fora while. Time may vary depending on anumber of factors.

K. Turn out onto a wire rack to cool. Tokeep for more than a day or two, store inrefrigerator in a plastic bag. May be frozen.

Variations: You may add from 1 to2 C up to not more than half the total of adifferent kind(s) of flour. Unbleached whiteflour will make a lighter-colored, higher-rising bread but lower in vitamins and fiber.Rye bread can be made by adding up to halfrye flour and some caraway seeds. A coupletablespoons of unsweetened cocoa and instantcoffee will make it dark like pumpernickel(using molasses in place of honey will helpdarken it, too). A cup of soy flour plus a cupof wheat germ will increase the proteincontent of the bread. Instead of loaves, youcould braid the bread by forming three strandsand braiding them (bake on cookie sheet).Dinner rolls could be made by forming intosmall balls and baking on cookie sheet or inmuffin tins (will not take as long to bake).

Note: this bread contains wheat and would notbe OK for anyone with a gluten sensitivity.

V. DATATake notes on what you tried.

VI. CONCLUSIONS:Make any comments and/or suggestions you can think of. In the past, a Biology lab

tradition has been lab brew accompanied by popcorn seasoned with Fankhauser popcornseasoning. Hopefully, we can still sample the popcorn.

FANKHAUSER POPCORN SEASONING:4 T brewer’s yeast2 T powdered kelp1 T saltNote: Larger batches may be made by using the same proportions

of ingredients (like 4 C + 2 C + 1 C, etc.).Mix thoroughly (in blender if yeast is in flakes). Store in tightly-

sealed jar.

TO MAKE A BATCH OF POPCORN:1. Place about 2 T (walnut-sized piece) ofcoconut oil into a heavy pot and place the poton the largest burner on the stove. Turn theburner on high.2. When the oil is melted and hot (a cornkernel will sizzle), add 400 mL corn kernelsand cover. Shake until all the corn is popped.

3. Pour the popped corn into a grocerysack (with top turned over twice to stiffen it).

4. While shaking the bag to stir and mix,slowly drizzle on 30 to 40 mL of a 1:1butter/oil mixture (provides butter flavor withless cholesterol). Then, dust with 30 to40 mL of seasoning mixture.

July 1, 2013 56

in the bottles. If the last bottle is just a tinybit low, you may q. s. with water.N. Cap the bottles and check for leaks.Store in a 60 to 70° F location for a minimumof 10 to 15 days to allow residual yeast toferment the added sugar. This lastfermentation adds carbonation to the finalproduct. Note that if you bottle the producttoo soon and/or add too much sugar and/or gettoo much of the yeast sediment into thebottles, too much CO2 will be formed and thebottle(s) may/will explode.

O. Swirl the sediment in the jug to loosenand dispose of it. Immediately andthoroughly wash out the jug, making sure toscrub off any yeast stuck to it, and place in arack to dry. The yeast mixture is difficult toimpossible to remove once it has dried.P. While current University policy doesnot allow us to let you take any of this labbrew home, if you later decide to make someon your own, after the beer, wine, or mead

has aged at least two weeks, to taste,refrigerate upright for at least 12 hours. Donot shake or disturb. When you open it,decant it into a pitcher (or into glasses, butyou must pour it all out and not stop midway),avoiding the yeast sediment at the bottom.Again, rinse out the bottle as soon as possible,before the left-over yeast dries. The flavorimproves with age, but mead in particular,should be consumed within a year. Do not letthe bottles freeze or get too warm or they mayexplode. It is a good idea to store the bottlesin a location that will not be damaged if onedoes explode – it does occasionally happen.

Note that the left-over yeast,especially the strain used by commercialbreweries, is processed to remove some of thebitter flavor, killed, and purified, then driedand sold as brewer’s yeast in the health-foodstores or used as a supplement in livestockfeed. It is an excellent source of the Bvitamins and other nutrients.

V. DATA:Take notes on any verbal

information provided by your instructor andon what your class did. Record anydeviations from the written protocol (did youuse a different type of sugar?). Make sure toinclude all observations and data. Draw

pictures, especially of any new equipment(airlock on bottle), and/or take notes whereneeded. Optionally, a “souvenir” yeast packetor piece of malt label (only if the can isempty and the label no longer needed) may beincluded.

VI. CONCLUSIONS:In your discussion, you should

include any of the following that apply.1. As time and interest allow, look up andread about how breweries make beer.Summarize your findings and cite source(s).

2. As time and interest allow, look up andread about how wine is made. Summarizeyour findings and cite your source(s).3. As time and interest allow, visit one ofthe local breweries or wineries for a tour.Tell about your visit and what you learned.

4. As time and interest allow, study theformation of CO2 by yeast by baking a batchof bread at home. How easy or hard was it tomake? How did it smell while baking? Whatmakes it smell that way – what chemical isbeing evaporated/released? How did it taste?How well did the yeast do its job?5. In step #1, why do you think it isimportant that the water used to dissolve theyeast be warm but not too hot?

OPTIONAL AT-HOME EXPERIMENT: BREADIII. MATERIALS NEEDED:

½ C honey2 T or 2 pkg yeast2 C warm (not hot) H2Owhole wheat flour (approx. 6 C or so)¼ C oil (safflower is highest in vitamin E, olive has a good taste,

others will work, too)big mixing bowlmeasuring cup(s) and spoonswooden spoon (and/or electric mixer)2 loaf pans or cookie sheet (coated with oil and dusted with whole wheat flour)

IV. PROCEDURE:A. In a large bowl, mix the honey andwarm water. If the water is too hot, it willkill the yeast. Warm tap water is OK.

B. Sprinkle in the yeast and gently stir todissolve. Wait about 10 min to make sure theyeast is alive: the mixture should start to get

July 1, 2013 45

Figure 12. Lecithin

III. MATERIALS NEEDED:bring a clean container to take your

mayonnaise homeblenderrubber scrapermeasuring cup and spoonsjuicercutting board and knifeegg

oil (safflower, corn, soy, olive, peanut)lemon juice and/or vinegardry mustard(opt. other spices such as red pepper)apples, carrots, blue cheese, and garlicopt. other veggieshot, soapy water

IV. SAFETY CONSIDERATIONS:A. Your cooperation is requested inkeeping the oil in the designated space and inusing only designated glassware to measureit--if spilled on the floor, it is VERY slipperyand dangerous.

B. Do not insert the scraper into theblender while the blender motor is running.The blender could propel a scraper out at highspeed possibly injuring you (and/or paintingthe ceiling with mayonnaise).

V. PROCEDURE:Your cooperation is requested in

keeping the oil in the designated space in thelab room and in using only designatedglassware to measure it--if spilled on thefloor, it is VERY slippery and dangerous.This recipe makes about 1½ C of mayonnaise,and you should bring a clean container inwhich to take it home.A. Each person should place in theblender (take turns using the blender):

1 egg2 T lemon juice or vinegar (or a

mixture of them)1 t dry mustard

optional ingredients such as red orblack pepper, salt, or whatevertastes good to you

B. Briefly mix on high speed to blend.C. TURN OFF BLENDER, then, ifnecessary, scrape down sides with rubberscraper. CAUTION: DO NOT INSERTSCRAPER WITH BLENDER MOTORRUNNING!!!D. With blender motor running (high),slowly and carefully drizzle in 1 C of salad oil(safflower is highest in vitamin E, or usecorn, olive, peanut, soy, etc.) through the holein the lid – put lid on but remove the center

July 1, 2013 46

first. Occasionally turn off the blender toscrape the sides. You may need tooccasionally stop and “burp” the blender if anair bubble gets trapped under the mayonnaiseas it thickens.E. Transfer your finished mayonnaise to aclean container with lid and store inrefrigerator. If you are not the last person inyour group to use the blender, you can justscrape your mayonnaise out with a spatula.After the last person, the blender will need tobe cleaned with soapy water.F. After everyone is done, all utensilsshould be thoroughly cleaned with hot soapywater. Oil is difficult to remove fromlabware, yet it is very important to do so. Becareful around sharp blender blades. All

spilled oil on the floor, countertops, etc.should be thoroughly cleaned up--spilled oilon the floor can be very slippery anddangerous. Carefully clean all spills off yourlab bench so it's clean for the next classcoming in. If yours is the last lab section forthe day, a thorough cleaning is necessary.G. If blue cheese, garlic, and veggies areavailable, your instructor will make bluecheese and/or garlic dip. Then you areencouraged to try some with the veggies.H. Optional at home: Grated blue cheese,onion, and/or various other herbs or spicesmay be stirred into mayonnaise to give it avariety of flavors. Try your mayo on asandwich or whatever and record how ittasted or any other observations.

VI. DATA:TAKE NOTES – Record all

observations and notes. What kind of oil didyou use to make your mayonnaise? Did youuse vinegar or lemon juice? What otheringredients did you add? Did you try any“variations” from the original recipe? What

did your mayonnaise look like when it wasdone? How did it taste? If soap was made,you should save space for notes on the finalstep in the soap-making process. You maywish to monitor the pH of the soap as it cures.Draw any equipment, etc. as needed.

VII. CONCLUSIONS:Include the following:1. How did your mayonnaise turn out?Do you have any suggestions for ingredientsthat you would like to try next time you makemayonnaise?2. When cleaning up after themayonnaise, compare the ease of cleaning theblender jar with the emulsion in it versuscleaning the measuring cup used to measurethe oil. Which was easier to clean? Why?3. Commercial soap has the glycerolremoved to make it harder and last longer.Homemade soap, with the glycerol in is lessprone to cracking and is supposedly more

soothing to the skin. How do you feel oursoap compares to your favorite brand of soap?Which is more drying, which lasts longer,which is a more neutral pH, which lathersbetter, etc?4. How did your additive(s) affect yourbar of soap? Was it the color you expected?Did it smell the way you expected: Does itclean any differently?5. Devise/Suggest an experiment either totest the ability of various emulsifying agentsto emulsify a given lipid or of various lipidsto be emulsified by a given emulsifying agent.

July 1, 2013 55

B. For all recipes, start by adding 1 tspyeast and ¼ tsp sugar to about ¼ C warmwater. Let the mixture sit for about 15 min.to “proof.” It should become bubbly or frothy.If nothing happens – it’s dead – try again.

For Recipe A1 — “Lab Brew”C. In a pot, place a few inches of waterand the can containing the malt extract(opened and with label removed). Heat thecan of malt until it can be poured.D. When the malt is softened and can bepoured/measured, in a separate pot, measureand mix the � C hopped malt, the 1¼ Csucrose, and about 5 to 5½ C water.

E. Slowly heat to boiling but do not let itboil over. Stir slowly to prevent burning.Have hot pads ready to remove the pot fromthe heat if/when needed.F. Put a little under ½ gal cold water intothe jug, then add the hot sugar solution witha funnel. Avoid running the hot solutiondown the sides of the jug or it may crack fromthe heat. Pour some down a funnel directlyinto the cold water, then swirl to warm thebottle. Repeat the pour-and-swirl until all thehot liquid has been transferred.

G. Fill the jug to the “hip” with coldwater. In pharmaceutical jargon, one wouldsay, “Q. s. to the hip,” where “q. s.” standsfor “quantum sufficit” in Latin which meansas much as suffices, as much as is necessary.This “final” mixture is called the “wort.”For Recipe A2 — “Lab Brew”C-G. Follow recipe A1, except, in place ofthe malt extract and sugar, use � C (= ¾ C +2 T) of just malt (OK to round to ~1 C).

For Recipe B — WineC. In a blender, grind a “bunch” of grapes(weigh first?) with about 5 to 5½ C of water.D. Add about 1� to 1½ C sugar and blendto mix it all together.

E. Note: this mixture will not be cookedbecause the natural yeasts on the grape skinsaid the fermentation process and would bekilled by the heat.F-G. Pour the mixture into the jug and q. s.to the hip of the jug.

For Recipe C — MeadC. In a pot, mix 3 C honey and 5 to 5½ Cwater. Slowly heat to boiling but do not let itboil over. Stir slowly to prevent burning.Have hot pads ready to remove the pot fromthe heat if/when needed.D. Add 1 oz (~3-4 in long piece) chopped,fresh gingerroot, 2 tsp whole cloves and either2 tsp ground nutmeg or 1 whole nutmeg thathas been smashed with a hammer.

E. Boil another 5 min, making sure itdoesn’t boil over.F. Put a little under ½ gal cold water intothe jug.

G. Then add the hot sugar solution with afunnel. Avoid running the hot solution downthe sides of the jug or it may crack. Pour itdown a funnel directly into the cold water.Q. s. to the hip with cold water.Then, for all recipesH. If the yeast solution is bubbly, add it tothe jug. In beer brewing, this is called“pitching the wort,” with “pitching”referring to setting up something (likepitching a tent), not throwing something (likepitching a ball) – the wort is being set up toferment. If it’s not bubbly, mix up some newas per step #1. It is very important to add allthe cold water first – if the solution is too hotwhen the yeast is added it will kill the yeast.

I. Place an airlock on top of the bottle andfill with water to the correct level. This willallow CO2 from the fermentation process toescape without permitting entrance of outsideair that contains O2 needed by bacteria thatcould cause the wort to spoil or turn tovinegar. Label the bottle with the lab section,date, and contents (type of malt, sugar, honey,grapes, and/or yeast).J. After 1 to 2 days (next lab period),the beer should have a good head on it (calledthe “high kreusenen” stage). Wine or meadshould be actively bubbling. Check to makesure the level of the water airlock is OK.

K. After about 4 days, if you are makingbeer, the foam should subside, or if you aremaking wine or mead, the fermentationshould have slowed a bit, at which point, fillthe jug up to the bottom of the neck withwater. In pharmaceutical jargon, one wouldsay, “Q. s. to the neck,” where “q. s.” standsfor “quantum sufficit” in Latin which meansas much as suffices, as much as is necessary.Replace the airlock. Note: you may be ableto do this step as soon as the next lab period.L. After about 8 to 10 days or so (longerif the room is cooler), bubbling should havedecreased to a very slow rate. Periodicallycheck to insure there’s water in the airlock.

M. About two to three weeks after thebrew was made, it will be ready for bottling.In each 2-L bottle, add 2 tsp. sugar using afunnel. Carefully decant (carefully pour offthe liquid or supernatant [super = over,above; natant = swimming], leaving thesediment on the bottom) or siphon the beer,wine, or mead into a large, clean beaker orbucket, avoiding the sediment on the bottom.From that container, pour the liquid into thebottles, leaving about 1½ inch of head-room

July 1, 2013 54

Figure 23. Airlock

always twines in a clockwise direction.“Humulus” is a Medieval latinization of anAnglo-Saxon word, “humule.” In the U. S.today, most hops are grown in Washington,Oregon, California, and Idaho.

Like a certain other member of theCannabinaceae, the part of the hops plant thatis used is the female strobilus (a part of thefemale plant surrounding the flowers). Thishas also been used medicinally for centuries.The primary constituents responsible for themedicinal properties and bitter flavor are twochemicals called humulone and lupulone.These are unstable in the presence of lightand air, thus dried hops rapidly lose theirflavor and medicinal effectiveness (and mustbe used for brewing within a relatively shorttime after harvest). Hops is a well-known

sedative and has long been used as a sleepaid. Animal and human research has shownthat, indeed, it is a CNS depressant. Hops isalso used herbally as a diuretic and antibiotic.

Within the yeast cells, the actualchemical reactions that turn sugar into alcoholare catalyzed by a number of enzymes (en =in; zym = yeast) – biological catalysts thathelp other chemicals to react. Althoughnumerous enzymes are found in livingorganisms, each one with its own specializedfunction, the very first such chemicals to bestudied were those involved in the process offermentation. Since they were found in yeast,they were called “enzymes.” We now knowof many more enzymes, most of which are notfound in yeast, yet the name is still used.

III. MATERIALS NEEDED FOR A ONE GALLON BATCH: one 1-gal. jug (left-over from apple cider?)airlock (Figure 23) made from rubber stoppers and glass tubinghot pads, cooking pot(s), stove, wooden spoonmeasuring cups and spoonscan opener or blender if needed for the recipe you choose2 CLEAN 2-liter soft-drink bottles with their plastic lids – if you have

some, please bring them in when we are ready to bottle

1 tsp live yeast, preferably brewer’s, ale, or wine yeast, but regular bakingyeast will work with a slightly different final product

¼ tsp table sugar (sucrose)¼ C warm waterFor Recipe A1 — “Lab Brew”� C hopped malt extract1¼ C table sugar (sucrose)5½ C watera little under ½ gal of cold H2Omore cold water

For Recipe A2 — “Lab Brew”� C (= ¾ C + 2 T) of malt extract (OK to round to ~ 1 C)5½ C watera little under ½ gal of cold H2Omore cold water

For Recipe B — Winea “bunch” of grapes~5 C water1� to 1½ C table sugar (sucrose)more cold water

For Recipe C — Mead~5 C water3 C honey1 oz (~3-4 in long piece) fresh gingerroot, chopped2 tsp whole cloves2 tsp ground nutmeg or 1 whole nutmeg, smashed with a hammera little under ½ gal of cold H2Omore cold water

IV. PROCEDURE:A. Your instructor, perhaps with yourinput, will decide which of the followingrecipes your class will make as a whole-classproject. Various people from the class are

encouraged to volunteer to do different partsof the procedure so everyone can be involvedin some way. Everyone should watch what’sgoing on and take notes.

July 1, 2013 47

MICROSCOPE USEProtocol Copyright © 1986, Photographs Copyright © 2002-2004 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To become familiar with the parts of a microscope.2. To learn how to use a microscope.3. To use the microscope to view yeast cells.

II. BACKGROUND:Back in the 1600s, a number of

scientists were working on the invention andimprovement of the microscope. In 1663,Robert Hooke used this new tool to view asection of the bark of the cork oak tree(Quercus suber), and he saw many smallcompartments which he called “cells” (cell =a small room). Shortly thereafter, Antonievan Leeuwenhoek made use of the microscope(micro = small; scope = see, watch, look) toview many tiny organisms and cells for thefirst time. Today, the field of microscopy hasimproved considerably, and not only do we

have light microscopes like those we will beusing in this lab, but also electronmicroscopes which use a beam of electronsrather than light to “see” a specimen.

Because microscopes are expensiveand because of the number of other studentsin other lab sections/classes who also usethese microscopes, it is important to follow afew simple rules that will insure that goodcare is taken of the microscopes. These rulesand requirements will be covered in theprocedure to follow.

III. MATERIALS NEEDED:microscope – will be assigned based on

where you are sitting. You shouldalways use the same one,

corresponding to your seat number.prepared slide of the letter “e”slides and coverslips

IV. PROCEDURE:First, your instructor shouldA. show the class the microscope-use videos on the Biology Web site, andB. point out to you all the microscope parts listed here on an actual microscope and draw andlabel an illustration of the microscope on the board as you do the same in your lab notebook.

CARRYING AND STORAGE OF THEMICROSCOPE:

To lift/carry a microscope, grasp thearm firmly in one hand and lift/support the

base with your other hand. Microscopes arelocated in the cupboard beneath your desk.When removing/replacing your microscopefrom/into the cabinet, be very careful not tobump it, especially the lenses, or you maydamage it. Obtain the microscope thatcorresponds to your assigned seat number, setit on the desk top, and return the plasticdustcover to the cupboard. Because space inthe cupboard is at a minimum, to avoidaccidents, it is imperative that you properlycoil the cord around the microscope.Hopefully, the last person stored it away thatway, so observe the (hopefully) neatly-coiledcord on your microscope. Practice (yes, thismay seem silly, but DO IT) wrapping andunwrapping the cord a couple times. Thecord should be coiled in a clockwisedirection. Make sure the cord is not twistedor kinked and that the end is securely tuckedinto the rest for storage. You are expected tomake sure the cord is neatly coiled each timeyou put it away after use (this is a majorsource of rivalry among the various labsections) so that when your lab partner getsout his/her microscope, yours won’t fall onthe floor. If your microscope is not properly

July 1, 2013 48

stored, rest assured that students in the otherlab sections will complain to their instructorand blame you. When you are getting readyto use the microscope, as you are plugging inthe cord, lay the excess across the tabletop –NOT dangling down in front of you and the

cabinet door. If the cord is dangling down,the risk is great that you will accidentally tripover it, catch it as you attempt to open thedoor, etc., and pull the microscope off thetable.

THE MICROSCOPE AND ITS PARTS:Become familiar with your

microscope and its parts. As your instructorgoes through the names and locations of themicroscope parts and draws a right-side viewon the board, you also should draw a full-page-sized picture (right-side view) of themicroscope in your lab notebook and label thefollowing, underlined parts, noting theirfunctions.

A. Note the path traveled by the light.Start your notebook illustration by indicatingthe light path as a (lower) vertical lineconnected to a (upper) line that angles to theupper left. This will help you to align thevarious parts as you draw them.B. The base is the main support for themicroscope.

C. The arm supports the stage and theoptical head. Again, note that the proper wayto carry a microscope is to grasp the arm withone hand and support the base with the otherhand.D. An electric light is mounted on the baseof the microscope, and is controlled by bothan on-off switch and a rheostat (rheo = flow,current) which adjusts the brightness. Pleasekeep the light off when not in use to avoidheat build-up. If you finish looking at oneslide and need to clean it off and/or go getanother, turn off the light in between. Also,

turn off the light before unplugging andstoring the microscope. To extend the life ofthe bulb, when turning the light off, alwayslower the rheostat to the lowest light levelbefore switching on or off the light.

E. The stage is the flat area upon whichthe specimen is placed. It has a hole in thecenter through which light may pass.

F. The iris diaphragm and condenser arelocated under the stage. The condenserfocuses the light going through the specimenand the iris diaphragm is used to regulate the

July 1, 2013 53

Figure 21. Yeast

Figure 22. Yeastand Acetobacter

YEAST AND THE PRODUCTS OF ITS FERMENTATIONPROCESS

Protocol Copyright © 1982 D. B. FankhauserBackground and additional information Copyright © 1988 J. L. Stein Carter

I. OBJECTIVES:1. To study about the process of fermentation as a source of energy for cells.2. To relate this to “everyday life” by making “lab brew.”3. To illustrate one of the classic principles in the field of microbiology.

II. BACKGROUND:Many microorganisms (micro =

small), notably yeasts and bacteria, extractenergy from their food (glucose) byfermentation. The best-known method offermentation is alcohol fermentation in whichthe overall chemical reaction is: (sugar)C6H12O6 � 2CO2 + 2CH3CH2OH (ethylalcohol). Humans have known about andutilized this fact for many thousands of years.CO2 liberated by yeast helps our bread to rise,while the liberated alcohol gives it itswonderful smell. The Egyptians and manysubsequent civilizations have fermentedgrains such as barley to break the starch downto malt (maltose), then glucose, and finallyalcohol. For at least that long, people havealso known that various fruits, especiallygrapes, could also be fermented to producealcoholic beverages. In this lab, we will bestudying the process of fermentation asperformed by the yeast, Saccharomycescerevisiae (sacchar = sugar; myces = fungus;Ceres = goddess of grain; vis = to see; -ia =state of, condition of, disease).

Louis Pasteurwas a famous Frenchmicrobiologist who livedin the 1800s. Peopleinvolved in the wineindustry of that dayasked him to researchwhy some bottles of winewere OK while others

spoiled and turned into vinegar (vin = wine;aigre = sour). Pasteur discovered that thewhitish bloom on the skins of grapescontained a number of small, oval cells whichhe identified as yeast (Figure 21). These arethe yeast that turned the smashed grapes intowine.

In wine thathad turned to vinegar,Pasteur also found small,rod-shaped bacteria(Figure 22) (this shapeis called bacillus) as a“contamination” or“infection” in the wine.These were found tob e l on g t o g e n u s

Acetobacter (aceto = vinegar; bacter = rod).Pasteur discovered that if the wine was heatedto 63°C and held at that temperature for 30

minutes, the Acetobacter would be killed.This process is named in his honor:pasteurization. Some people object totreating wine in this way, claiming that theflavor is changed. Today, by law, all winesold in the United States must have sulfitesadded to kill anything living in it (and somehighly allergic humans).

There is, however, another way toinhibit growth of Acetobacter. It has beendiscovered that while yeast do not need thepresence of oxygen (O2) to do fermentation,Acetobacter do need O2 to turn alcohol intovinegar, more specifically acetic acid(CH3COOH). Thus, if O2 can be eliminated,the Acetobacter cannot grow. However, sincethe process of fermentation is evolving CO2,the fermentation vessel cannot be sealed or itwill explode. This necessitates the use of anairlock (Figure 23) which allows the CO2produced to bubble out through a waterbarrier which simultaneously prohibits O2from entering. We will, therefore, be makinguse of Pasteur’s discoveries to keep our brewfrom turning into malt vinegar.

For centuries, beer has beenflavored by the addition of various bitterherbs, for example, meadowsweet (Spiraealatifolia), alehoof (which is another name forGill-over-the-Ground, Glecoma hederacea),and/or alecost or costmary (Chrysanthemumbalsamita). In the eleventh century,Bavarians started adding hops (Humuluslupulus) to the brew to act as a preservative(it helps extend the “shelf-life” of the beer)and to flavor the beer, as a replacement forthe bitter herbs previously used. Thispractice was borrowed by the British in thesixteenth century. Modern breweries startwith a cooked “mash” of sprouted barleywhich is fermented by a special strain ofyeast. Added to this is a water-extract or“tea” from hops. For home-brewing, cans ofbarley malt with hops extract added can bepurchased to add to sugar water to make beer.This is what we will use in this experiment.

Hops (Humulus lupulus) is in thefamily Cannabinaceae. Its native habitatincludes damp areas where it can be foundtwining tightly around willow (and other)trees. Because of this, the Greek philosopherPliny called it lupus salictarius which means“willow wolf.” Interestingly, apparently it

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Proper Storage of Microscope_____1. Turn rheostat down to #1 and turn off

light._____2. Thoroughly clean off any immersion oil

and spills._____3. Rotate 4× objective until clicked into

place._____4. Mechanical stage not sticking out to the

side, moved all the way back to the arm._____5. Return diopter adjustment on left ocular

to 0 (in the middle)._____6. Wind cord neatly in the right direction

and tuck end to secure._____7. Tighten loose screws under mechanical

stage & opt. head screw, secure bluefilter.

_____8. Lower the stage.

_____9. Replace dust cover in correctconfiguration.

_____10. Place in cupboard with arm facing towardyou.

there are a couple things to try. Assumingyou’ve already tried the fine adjustment, thenext thing to check is the position of the irisdiaphragm lever. If the iris diaphragm is allthe way open, you won’t be able to get muchin focus — try closing the iris diaphragmmost of the way to see if that helps. Trymoving the condenser height adjustmentknob, because changing that can help.If you’ve tried making those adjustments, andthat hasn’t helped, and your image is stillreally blurry, there is a chance that “somestudent” in “one of those other classes” gotimmersion oil on the 40× objective (which

it’s not designed for) and then didn’t clean itoff (which “they” are supposed to do). Get apiece of lens paper out of your drawer and usethat to clean the 40× objective (gentlybreathing on the paper can help). Whileyou’re at it, check the 100× oil-immersionlens too – chances are, if the 40× is dirty, the100× may be dripping oil that was nevercleaned off. While you don’t want to breakthe lenses in the process, you also don’t wantto be so “gentle” with them that you don’t doany good. The tips of the lenses are springloaded, so they will “give” with sufficient,gentle pressure.

STORAGE OF THE MICROSCOPE:A. Turn the rheostat down to the lowestnumber, then make sure the light is off. Windthe cord NEATLY and securely in the properdirection around the bottom of the arm andthe front of the lamp, tucking in the end, thenfully lower the stage to help hold it in place.The 4× lens should be in place below theoptical head and the mechanical stage shouldnot be sticking out to either side (use thecontrol knobs to move it to the center).B. Clean up all spills on the microscope.Carefully check through everything else onthis list and make sure it’s all OK.

C. Place the dust cover over themicroscope, making sure that the numbers on

the cover and microscope match (Do you havethe right cover?). Double check to make sureyou have done all of these things beforeputting away the microscope.D. Carefully put away the microscope withthe arm of the microscope toward the door ofthe cabinet (so the arm can conveniently begrasped) and clean up anything else in yourarea. Make sure all slides have been returnedto the proper place and all trash is removed.The drawer and microscope cupboard areNOT trash receptacles!

E. Report any problem with thei n s t r u me n t t o you r i n s t r u c t o rIMMEDIATELY.

V. DATA:Record all observations and data.

Draw pictures and/or take notes whereneeded. Especially, to help your powers ofobservation, draw and label your own pictureof the microscope – you see more when youhave to draw it yourself. Also include

drawings of the letter “e” as it looks to thenaked eye and under the various powers(label these drawings as to which is which), agroup of yeast cells under 40 and 100×, anddetail of one good representative yeast cellunder 400×.

VI. CONCLUSIONS:In your discussion, you should include: 1. In general, what can you say about theimage you see in a microscope as compared to

the direction you specimen is really facingand the direction you move the specimen?

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Figure 17.Letter “e”

amount of light passing through the specimen(which also, as in a camera, influenced thedepth of field). Locate the iris diaphragmlever and the condenser height adjustmentknob. Often, if you cannot see a specimenclearly, it is because the amount of lightpassing through needs to be greater or less.G. Mounted on the stage is a mechanicalstage which includes a specimen holder. Thisis designed to hold the slide in place so itdoesn’t move while you’re looking at it and tomove the slide around from place to place.Note the vernier scales that indicate theposition of the slide (side-to side and front-to-back) – jotting down these numbers makes itpossible to return to an exact location on aslide. The mechanical stage is controlled bythe low drive coaxial stage controls located onthe lower right-hand side of the stage.

H. A pair of large knobs, called the coarseadjustment, which permit rapid raising orlowering of the stage, are located on the sidesof the arm. Memorize the direction to rotatethis knob to lower or raise the stage.I. A pair of smaller knobs, called the fineadjustment, which permit smaller adjustmentsin the stage height are located “inside” thecoarse adjustment knobs.

J. The optical head, the body of themicroscope which contains the lenses, is bentat an angle on these microscopes. There is anoptical head retaining screw which holds theoptical head onto the microscope. DO NOTLOOSEN THIS SCREW! If it is left loose,the optical head will fall off (and land on thefloor and break).K. The nosepiece is a revolving plate onthe lower side of the optical head which holdsthe objective lenses. Note that as it is rotated,

each lens CLICKS into place. A frequentcause of students seeing “strange” images isan improperly seated objective lens.L. On the nosepiece, a 4× scanningobjective or lens (red band), a 10× low-powerobjective (yellow), a 40× high-powerobjective (blue), and a 100× oil immersionlens (white) may be found. DO NOT USET H E O I L I M M E R S I O N L E N SUNTIL/UNLESS YOU HAVE RECEIVEDSPECIFIC INSTRUCTIONS TO DO SOAND HOW TO USE IT. Note that theselenses are color-coded, and remember whichcolor is which for easy identification. ONLYlens paper (located in the drawer in front ofyou – let your instructor know if your supplyis getting low so we can replenish it) shouldever be used on the lenses, and only ifabsolutely necessary. Never use paper towel,Kleenex, or even Kimwipes on the lenses –ever!!! (NOTE: the drawer in front of you isNOT a trash receptacle – dispose of trashproperly. Also, there are A&P slides and abottle of immersion oil in the drawer – theseare off limits to General and IntroductoryBiology students!)

M. The oculars (ocul = an eye) lenses fitinto the top of the tube and are 10×. Becareful they don’t fall out – they are notattached. The oculars can be moved closertogether or farther apart to adjust to the widthof your eyes. As you move them in and out,note the interpupillary distance scale whichindicates how far apart they are spread. Noteon the left, outer barrel of the left ocular thereis a white line running up the side and someother markings, the diopter adjustment scale– you will use this to adjust the focus to youreyes.

USE OF THE MICROSCOPE:A. Place the microscope on the table infront of you with the arm away from you.Plug in the cord and drape the excess acrossthe tabletop – NOT HANGING DOWN!!!

B. You may need to adjust the diaphragm(now or later) so that the proper amount oflight passes through your specimen.C. If the microscope was properly stored,the 4× objective should be in position in thelight path (under the optical head). If not,swing the nosepiece around until it clicks intoposition.

D. Obtain a prepared slide ofthe letter “e.” Observe (anddraw) which way the “e” ismounted on the slide. Handlethese slides only by the edges orthe labeled end and use lenspaper or a Kimwipe to clean if

needed – don’t put your thumbprint in thecenter of the specimen! Place the slide ontothe stage, specimen-side up. By looking fromthe side and the front, use the mechanicalstage controls to try to place the “e” as closeto the center of view (optic center) as youcan.

E. Carefully, LOOKING AT WHATYOU’RE DOING FROM THE SIDE, use thecoarse adjustment to raise the stage as far asit will go without hitting the lenses – DONOT LET THE LENS TOUCH THE SLIDE.NEVER use the coarse adjustment to raise thestage while looking through the microscope oryou could smash the lens into the slide.Again, check from the side and front to alignthe “e” as closely as possible.F. If you were successful at getting the “e”directly under the objective, you should see a

July 1, 2013 50

faint haze when looking through the RIGHTocular. Slowly, move the stage DOWN usingthe coarse adjustment (make sure you’regoing down, not up) until your specimencomes into view. If the “e” is not visiblewhen you first look through the ocular, movethe slide a little one way or another whilelooking through the RIGHT ocular watchingfor a haze or dark spot to pass by. Back up tothat spot, then proceed to focus.

G. Make fine adjustments in the focuswith the fine adjustment knob if needed sothat what you see in the RIGHT ocular is infocus. On the base of the left (outer) side ofthe LEFT ocular is a white line. Slightlyfarther up on the LEFT ocular, there are someshorter lines, a “0", a “+”, and a “�”. Noticethat, as you turn the diopter adjustment ringon the left ocular, various of the shorter linesline up with the longer line. With the “0" linematched up with the longer line, look throughthe LEFT ocular and turn it until it, too, is infocus (do NOT adjust the main focus knobs –you are adjusting now for differences betweenyour two eyes). Hopefully, each eye shouldbe in focus now. Next, you need to adjust thespread of the oculars to match yourinterpupillary distance by moving the ocularsin or out until you can comfortably see theimage through both eyes at the same time. Inyour lab notebook, draw the scales for boththe diopter adjustment ring and theinterpupillary distance scale, indicating theexact numbers/position that are correct forYOUR eyes. The next time you use yourmicroscope, you can, then, adjust themicroscope to these numbers and it should becorrect for your eyes without having to gothrough this whole process again.

H. While, in most cases, having the

condenser height as high as it will go will bebest, you may need to adjust this formaximum ease of viewing. The knob for thisis on the left side, under the stage. Whilelooking through the microscope, raise andlower the condenser, and observe whathappens to the light. The rheostat, located onthe lower right side of the microscope, can beused to adjust the amount of light put out bythe lamp, and will need to be adjusted to acomfortable brightness depending on whichobjective lens you are using. Also, practiceadjusting the iris diaphragm while lookingthrough the microscope, and observe whathappens to the light. Just like in a camera, asmaller aperture of the iris diaphragm willgive greater depth of field (more of a 3Dview), but let less light through, thusnecessitating increasing the voltage to thelamp to compensate.I. Note that the oculars are 10×, so if youare using a 4× objective, everything ismagnified 40×, if you are using a 10×objective, everything is magnified 100×, ifyou are using the 40× objective, it’smagnified 400× and if you are using the 100×oil immersion lens, it’s magnified 1000×.

J. Use the mechanical stage controls tomove the slide slightly to the right, left,toward, and away from you. Record in yournotebook your observations of what happensto the image as you move the slide. Draw apicture of what the “e” looks like to theunaided eye and what it looks like under themicroscope – is it right-side up? Do not makeyour drawings so small that you can’t tellwhat they are when you go back to study forthe final. It is suggested that drawings be atleast ¼ to ½ page in size so that you can

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easily draw and see the details of what youobserved. You don’t have to worry aboutdrawing a circle, but do indicate to the lowerright of your drawing what magnification itrepresents. If you do draw a circle to give anidea of scale, be conscious of how much of ityour specimen fills. For example, if it takesthree of a certain kind of cell to span thecircle, don’t draw 20 of them. Refer to theNotebook Protocol section on how to drawillustrations.K. Now, WITHOUT MOVING THEFOCUS, carefully swing the 10× objectivearound until it clicks into place whilewatching from the side. Watch you don’t hitthe stage or the slide with the lenses. Do becareful that you really do have enoughclearance – watch from the side! Mostmodern microscopes, including these in thelab, are paracentral and parfocal meaningthat a specimen which is in focus and in theCENTER of the field of view at 40×, will bemore or less in focus and within the field ofview at 100×, and if in the center and in focus

at 100×, it will be more or less in focus and inthe field of view at 400×. (On some antiquemicroscopes, however, it may not work tofocus in this way.) Use the fine adjustment tocorrect the focus as needed, then view anddraw the letter “e” at 100×. Switch to the40× objective to observe the “e” at 400×. Donot use the coarse adjustment with the 40×objective in place – only fine adjustmentshould be necessary and is the ONLY one youshould use or you could smash a lens. Youmay need to readjust the diaphragm and/orrheostat. Again, draw your sample as it looksat 400× and label. Practice using the controlsto follow/trace the “e” at 400×. Note theindividual silver grains.L. When you are done, return to the 4×objective and lower the stage somewhat, thenmake sure your slide is free of fingerprintsand return it to the slide box. Avoid parallaxerror – make sure you get the slide straight ina slot and facing the same direction as the restof the slides.

TROUBLESHOOTING — SOME “EASY” MICROSCOPE REPAIRS:A. If the light doesn’t turn on, the firstthing to try is to push the red “reset” buttonon the outlet into which the cord is plugged.Also, check to make sure the “problem” isn’tthat the light is really on, but the rheostat isset on #1, so it just “looks like” it’s not on. Ifyou’ve tried both of those, and that doesn’thelp, then notify your instructor or the labstaff (so they can check the fuse and bulb).

B. If you can’t see your specimen becausethe view is too dark, try turning up therheostat and/or opening the iris diaphragmmore.C. Unless it has been lost, there may be ablue filter under the iris diaphragm, held inplace by a black, plastic ring. Since that’sonly held in place by spring(s), occasionally,if it gets bumped (like when you’re trying towrap the cord?), it pops out. If that happens,just carefully pop it back into place.

D. If the mechanical stage is loose andwobbly and slides are slipping under the stageclip rather than it holding them securely, itsscrews are loose. The mechanical stage isheld in place by two “big,” silver-coloredscrews under the stage, along the “right”edge. If those screws are loose and need to betightened, do so!E. Although you were told, above, to notloosen the optical head retaining screw,occasionally some other student in “one ofthose other classes” will do so. Thus, if youfind that someone else has left this screwloose, tighten it! If someone left the opticalhead crooked, you may loosen the screw just

a tiny bit to straighten out the optical head,then make sure to tighten it, again.

F. If you’re looking through themicroscope and you can see your specimen inhalf the view, but the other half the view isdark, that probably means the lens isn’ttotally clicked into place. Rotate thenosepiece until the lens is clicked into place.G. If you had your specimen in view andfocused using the 4× objective and the 10×objective, but then “lost it” when you went tothe 40× objective, there are two things youneed to check. Go all the way back to the 4×objective, and re-find and re-focus yourspecimen. Make sure the area you wish toexamine is exactly in the center – use themechanical stage controls to center it. Then,turn the nosepiece until the 10× objective isin place and re-focus there. Again, insure thatthe area you wish to view is exactly in thecenter. When you go to the 40× objective,watch from the side to make sure it’s notgoing to hit, but don’t move the stage down!!!The lens will come very close to the coverslip,but it should just barely clear it, and if youmove the stage way down because you“think” it’s going to hit, you’ll never find andbe able to focus on your specimen. If, indeed,the lens does come so close that it won’t clearthe coverslip, that means that you won’t beable to get that specimen in focus with the40× objective, and moving the stage downwon’t help.

H. If your specimen was in focus at 40×and 100×, but when you go to 400×, you cansee the specimen but can’t get it into focus,