biology 1 lab manual 201405

Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Table of Contents:

FHSB 1214 Biology I

FHSC 1214 Cell Biology

Experiment Description Page

Introduction Exercise 1: Writing of Lab Reports Exercise 2: Notes on Biological Drawings

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Practical 1 Cell Biology Studies I

Practical 1 Biological molecules I

Identification of Biochemical in Their Pure Form

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Practical 2 Cell Biology Studies II

Practical 2 Biological molecules II

Investigation of Action of Saliva and HCl in Two Carbohydrate Solutions

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Practical 3 Cell Biology Studies III

Practical 3 Enzyme studies I (Experiment 1) Optional: Practical 3 Enzyme studies I (Experiment 2)

Investigation of the Effects of Catalase Concentration on Hydrogen Peroxide Synthesis of Starch Using an Enzyme Extracted from Potato Tuber

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Practical 4 Cell Biology Studies IV

Practical 4 Enzyme studies II

Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide

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Practical 7 Cell Biology Studies VII

Practical 5 Cell studies I

Exercise 1: The Microscope and Its Uses Exercise 2: On-site Assessment Exercise 3: Preparation of Wet Mount Exercise 4: Preparation of Microscopic Slides Exercise 5: Measurement with a Microscope Exercise 6: Observation of Starch Grains (Additional practice tasks if time permits) Exercise 7: Observation of Hair (Additional practice tasks if time permits)

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- Practical 6 Cell studies II

Extraction of Cell Organelles by Differential Centrifugation

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Practical 5 Cell Biology Studies V

Practical 7 Cell studies III

Determination of Solute Potential of Potato Cell Sap

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Practical 6 Cell Biology Studies VI

Practical 8 Cell studies IV

Effects of Various Treatments on Pieces of Stained Potato Cells

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Practical 8 Cell Biology Studies VIII

Practical 9 Energetics I

Respiration of Germinating Beans

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Practical 9 Cell Biology Studies IX

- Exercise 1: Microscopic Examination of Cells at Various Stages of Plant Mitosis Exercise 2: Reproductive Tissues in Plants (Histology of Plant - Lily Reproductive Structures) [Meiosis]

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FHSB 1214 Biology I

FHSC 1214 Cell Biology

Experiment Description Page

Practical 10 Cell Biology Studies X

- Exercise 1: Mitosis and Meiosis Modelling Exercise 2: DNA Replication Modelling

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- Practical 10 Energetics II

Respiration of Yeast

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Appendix : Case study


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E-lab manuals: towards saving the earth More and more people all over the world and in Malaysia are getting involved in saving the planet electric cars, wind and solar energy, and paper-less projects. What can YOU do?

Beginning January 2011, CFS PJ Biology has piloted test e-lab manuals in an attempt to join global conservation efforts to save the planet by saving trees. Less paper means more trees will be left standing to absorb carbon dioxide and reduce the greenhouse effect. Dont you & your future loved ones deserve to enjoy a cooler planet? Another advantage is that full-colour biology pictures will be accessible to students for the first time. Important rules on tests and lab assignments Summary:

No MCs or any valid reasons accepted for late/missed assignments and tests.

It is the students responsibility to submit another assignment in-lieu or sit for a replacement test when announced (on different topic).

The lecturer will NOT remind students to submit late/missed assignments nor attend replacement tests. (Treating you as adults.)

Details:

Unacceptable: MCs, valid reasons (chicken pox, met with an accident, menstrual cramps, stomach ache, dog died etc, non-valid reasons (forgetfulness; lateness; server/ IT problems). This is to be fair to everyone as fake MCs and liars are present in Malaysia.

If a student fails to submit an assignment or misses a test, the lecturer will NOT remind you to submit a new assignment nor to sit for a replacement test. The replacement test will be announced to everyone in general and not to individual absentees. Those who are supposed to attend must turn up and will not be reminded. It will be conducted at the end of the semester on a different topic (usually more difficult) when all students are so busy with tests and assignments.

It is the responsibility of the student to choose one other assignment to be submitted later in the semester when all students are so busy with tests and assignments.

If a submission is done online, a minimum of 7 days are given to submit your assignment. As such, no excuses will be entertained if theres a server/ IT failure or technical problems with your UTAR account. Hence, you have an option to submit your assignment on day 1 to be safe, or on day 7 to be stupid. If youre late, a link for Late submissions is available if you dont mind getting a 50% discount, or you may choose to submit another assignment which the lecturer will not bother to remind you about.

I acknowledge reading the above & agree to be bound by terms therein. Your signature:


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How YOU can do well in BIOLOGY

Follow the 4As and you can expect As. ttitude

Attend ALL lectures, tutorials and practicals on time without fail.

Be attentive in class and revise your notes after class while the topic is still fresh in your mind. Why waste time re-reading 2-3 months later?

Do your assignments faithfully as they carry marks for the finals.

Come prepared for lessons (i.e. read up beforehand).

Read up beforehand before attending lectures so that you wont be lost and wasted hours of your life week after week.

Why stress yourself out if you can avoid it? Do NOT count on last minute revision for tests and examinations, as it will be too late to catch up and seek help in areas where you may find confusing or unclear of.

Why panic before exams because you cant find this or that? Keep separate files for lecture, tutorial and practical. File up the respective notes systematically so that you do not lose them along the semester.

Do you expect the lecturer/ tutor to be available all the time to answer your questions? It is YOUR responsibility to take the initiative to clear your doubts or satisfy your curiosity to understand certain scientific phenomena by reading up on the relevant topics.

ttendance for lectures, tutorials and practicals

Lectures, tutorials and practicals carry marks that count towards your finals.

You are expected to be present at ALL lectures, tutorials and practicals.

Absence from any lesson must be accompanied by a photocopy of your medical certificate presented to your lecturer/ tutor at your next meeting.

If you know in advance that you will not be able to attend the practical for a particular week, you are expected to inform your tutor latest by the Friday before the affected week.

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Based on a true story A professor at the National University of Singapore recounts how on one occasion a student consulted him days before the exam. Student: Prof, could you explain this page to me please? Professor: What dont you understand about this page? Student: EVERYTHING. Professor: But I already went through this during lecture. Student: Oh, I didnt attend most of the lectures actually. As for the next page, could you explain this page to me please? ... and this page too and that too Prof: Im sorry, I cant help you. Student: (Hmmmph, HES so selfish. Hey, I paid to study here!) What do YOU think?

If the student failed, whose fault was it?

Was this student clever in skipping lectures?

Was it fair for the student to make demands on the lecturers precious time to answer his questions?

How would the student have benefited himself if he looked up books and other sources of information for himself first?


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ssignments

Use proper A4 foolscap for all handwritten assignments.

Write neatly and legibly in blue or black ink. Your tutor reserves the absolute right to reject your assignment and ask you to re-do the assignment should he/she consider it to be below the expected quality.

Submit your assignment on time. Late submissions may entail mark deduction or not be graded at all.

ssessments

ALL academic tests and examinations help prepare you better for the finals.

As such, to sit for them all is not only compulsory, but beneficial. After sitting for one, youll just want to sit for another, and another, and another

Absence from tests and examinations MUST be covered by a medical certificate, or will be considered to have failed the tests.

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Introduction Exercise 1 Writing of Lab Reports

hy should I bother writing lab reports in the correct way? The Foundation Programme is designed to prepare you for undergraduate studies at UTAR which will require the writing of lab reports all years generally. At the end of your third

year, you may have an opportunity to work on scientific projects which will culminate in an official scientific report. Depending on the quality of your report, the golden chance remains of publishing your report in a scientific journal. Such recognition may open doors of opportunity (e.g., strengthen application for scholarships and further studies etc.). Science professors are evaluated in most parts of the world by the papers they write. Format of a lab report Your lab report should be preceded by a cover page which contains the following:

Name

Partners name Group

Date

Program

Unit code

Unit description

Year and semester of study

Title of lab report

Lecturers name

W


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Example:

Your lab report should contain the following sections:

Title

Objective

Apparatus, materials and methods (if your assignment is submitted online, this step may be omitted)

Observations and/or results with discussion

Conclusion The following guidelines on report writing are those required by the actual internationally-recognized scientific community. The text in quotation marks in the following section is taken from Warren D. Dolphin of Iowa State University. Credit has been given to the author by citing the source. This is good practice as opposed to plagiarism, in which copied material is claimed as the possession of the copyist. 1 Apparatus, materials and methods As the name implies, the materials and methods used in the experiments should be reported in this section. The difficulty in writing this section is to provide enough detail for the reader to understand the experiment without overwhelming him or her. When procedures from a lab book or another report are followed exactly, simply cite the work, noting that details can be found in that particular source. However, it is still necessary to describe special pieces of equipment and the general theory of the assays used. This can usually be done in a short paragraph, possibly along with a drawing of the


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experimental apparatus. Generally, this section attempts to answer the following questions:

1. What materials were used? 2. How were they used? 3. Where and when was the work done? (This question is most important in field

studies.)

2 Observations and/or results with discussion Results The results section should summarize the data from the experiments without discussing their implications. The data should be organized into tables, figures, graphs, photographs, and so on. But data included in a table should not be duplicated in a figure or graph. All figures and tables should have descriptive titles and should include a legend explaining any symbols, abbreviations, or special methods used. Figures and tables should be numbered separately and should be referred to in the text by number, for example:

Figure 1 shows that the activity decreased after five minutes.

The activity decreased after five minutes (fig. 1). Figures and tables should be self-explanatory; that is, the reader should be able to understand them without referring to the text. All columns and rows in tables and axes in figures should be labelled. This section of your report should concentrate on general trends and differences and not on trivial details. Many authors organize and write the results section before the rest of the report. 2.1 Recording Qualitative Data Qualitative experiments include those that require observations of non-quantifiable data such as observations of colour, slides and whole specimens. Below are guidelines on reporting a segment of qualitative experiments. Liquid in container: Be careful to distinguish accurately among solution, suspension, emulsion etc. Often, mixture is a safe descriptive term to employ. It is your responsibility to look up the definitions as studied in secondary school.

KI solution was added to the starch suspension

emulsion of lipid droplets in water Amount of light penetrating solution Be careful to distinguish accurately among transparent, translucent and opaque. It is your responsibility to look up the definitions as studied in secondary school.


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Colour Some descriptions of colour are unacceptable as they are ambiguous.

Light/pale brown, instead of beige

Murky/ cloudy white, instead of milky If theres a change in colouration, you may choose to report as follows.

The initial blue colouration of the mixture turns green, then yellow and may finally appear brick red.

If the transition cannot be easily seen, at least state the initial and final colours. If there is no change, one must state the colour (e.g., it remained blue). It is incomplete to only report there was no colour change without at least recording the initial colour. Precipitate One should comment on the precipitate colour and relative quantity. To do so, the mixture must be left to settle.

Colour of precipitate - green, yellow, brick red precipitate

Amount of precipitate - a little, moderate amount, abundant Example: When describing observations involving Benedicts test, one should report that when one shakes the test tube containing Benedicts solution and precipitate, the entire mixture will take the colour of the precipitate. This colour upon shaking is recorded and also the amount of light penetrating solution (transparent/ translucent/ opaque).

Moderate amount of brick red precipitate suspended in solution, which bore a tinge of blue. Solution was opaque.

Note: Particles cannot be regarded as precipitate. (e.g. groundnut particles in water.) 2.2 Recording Quantitative Data Quantitative experiments include those that require observations of quantifiable data such as time, quantity, weight, etc. Tabulation and graphing

There are two categories of data normally used in reporting quantitative results raw data and processed data. Raw data refers to the readings obtained from measurements (e.g., length, weight, height, quantity, etc.).

The table must be accompanied by the following features:

Informative table title

Gridlines

Columns/ rows with appropriate headings and units (units and calculations should not be in the table body)


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All processed data related to and required for plotting graph must be shown in the table. E.g. Averages, rate of yeast respiration in terms of no. of bubbles formed per minute.

Precision and decimal places: One must express data according to the precision afforded by the instrument. E.g., if the instrument can weigh an item as light as 0.1 g, then do not record it as 0.10 g, so as to correctly reflect the precision of the instrument. Note that the decimal places in the table must be the same for the same unit of measurement, and reflect the precision of the instrument. If a measurement unit is converted to percentage or any other unit, one is not bound by the precision of the instrument. However, the recording should maintain a consistent and reasonable use of the number of decimals (e.g., avoid too many decimals 88.8888888 %). Note that the table and graph below feature such consistency of decimal places.

Precision of processed data can be presented in the following manner:

Averages calculated need not follow the decimal places of the raw data.

Processed data involving summation and/ or subtraction should follow decimal places of the raw data.

Decimals arising from processed data involving multiplication and/ or division should be reasonable (e.g., not unnecessarily long).

Sample table: Title: Mass of precipitate of standards at various concentrations of glucose solutions.

Precipitate mass (g)

Glucose concentration (%)

Reading 1 Reading 2 Reading 3 Average

4 0.1 18.6 18.4 18.7

2 8.2 9.3 9.0 8.8

1 5.2 4.5 4.8 4.8

0.5 2.3 1.8 2.1 2.1

0.1 0.4 0.3 0.4 0.4

Graph

Plot a graph that will show the trend of the investigation. Include the following in the plotting of graph:

Informative title

x-axis : labelled, including units (independent variables)

y-axis : labelled, including units (dependent variables)

appropriate scale used

points plotted


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Shape of graph can only be drawn using pencil, blue and black ink pen

points plotted according to table of data

best fit line/ curve Sample graph:

Note: The line of the plot does not go beyond the concentrations used (no extrapolation

of points). Hence, one should not extrapolate otherwise it is a claim that a certain y value is predicted for a certain concentration.

Avoid clashing headings with clashing units (e.g., headings with two different units but both have gram in their units gram eggs vs. gram nutrients per gram plain feed)

Mass of eggs laid in a week (g)

Amount of nutrients (g/ g plain feed)

0.30

0.25 0.20 0.15 0.10 0.00

Mean 78.0 74.0 69.3 62.7 59. 7 58.0

2.3 What if I dont obtain desired results? For the purpose of your UTAR lab report, if you dont obtain the desired results, just record them as they are. By right, you should repeat it however, you may be constrained by a limited amount of supplied solutions in the UTAR lab and time. Hence, if your repeats involve consuming more solutions, please ask your tutor first. You may put a footnote concerning the expected results. In your discussion, be sure to explain the possible reasons for the anomaly.

Average mass of precipitate of standards at various

concentrations of glucose solutions

0

2

4

6

8

10

12

14

16

18

20

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Concentration of glucose solution (%)

Ave. precipitate mass (g)


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3 Discussion This section should not just be a restatement of the results but should emphasize interpretation of the data, relating them to existing theory and knowledge. Speculation is appropriate, if it is so identified. Suggestions for the improvement of techniques or experimental design may also be included here. In writing this section, you should explain the logic that allows you to accept or reject your original hypotheses. You should also be able to suggest future experiments that might clarify areas of doubt in your results. 3.1 General Comments on Style 1. All scientific names (genus and species) must be italicized. Underlining indicates

italics in a typed paper. 2. Use the metric system of measurements. Abbreviations of units are used without a

following period. 3. Be aware that the word data is plural while datum is singular. This affects the

choice of a correct verb. The word species is used both as a singular and as a plural.

4. Numbers should be written as numerals when they are greater than ten or when

they are associated with measurements

6 mm or 2 g

two explanations of six factors.

When one list includes numbers over and under ten, all numbers in the list may be expressed as numerals; for example,

17 sunfish, 13 bass, and 2 trout.

Never start a sentence with numerals. Spell all numbers beginning sentences.

5. Be sure to divide paragraphs correctly and to use starting and ending sentences that indicate the purpose of the paragraph. A report or a section of a report should not be one long paragraph.

6. Every sentence must have a subject and a verb.

7. Avoid using the first person, I or we, in writing. Keep your writing impersonal, in the

third person. Instead of saying, "We weighed the frogs and put them in a glass jar," write, "The frogs were weighed and put in a glass jar."

8. Avoid the use of slang and the overuse of contractions.


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9. Be consistent in the use of tense throughout a paragraph--do not switch between past and present. It is best to use past tense.

10. Be sure that pronouns refer to antecedents. For example, in the statement,

"Sometimes cecropia caterpillars are in cherry trees but they are hard to find." Does "they" refer to caterpillars or trees?

After writing a report, read it over, watching especially for lack of precision and for ambiguity. Each sentence should present a clear message. The following examples illustrate lack of precision:

"The sample was incubated in mixture A minus B plus C."

Does the mixture lack both B and C or lack B and contain C?

"Protection against Carcinogenesis by Antioxidants"

The title leaves the reader wondering whether antioxidants protect from or cause cancer.

The only way to prevent such errors is to read and think about what you write. Learn to reread and edit your work.

Identify trends/ patterns by in words the trend shown in the graph. Remember to make reference to the values shown on the graph. Explain all the observations or trend obtained during the investigation.

As temperature increases from 25 oC to 50OC, rate of yeast respiration/ mean number of bubbles formed per 3 mins. increases proportionately/ linearly from 7 to 28.

In summary, the discussion should be correctly applying the theoretical concept involved in the experiment.

4 Conclusion

State the general trend obtained through the investigation and provides a concise conclusion about the investigation. 5 Literature Cited This section lists all articles or books cited in your report. It is not the same as a bibliography, which simply lists references regardless of whether they were cited in the paper. The listing should be alphabetized by the last names of the authors. Different journals require different formats for citing literature. For articles: Fox, J.W. 1988. Nest-building behavior of the catbird, Dumetella carolinensis. Journal of Ecology 47: 113-17.


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For Books: Bird, W.Z. 1990. Ecological aspects of fox reproduction. Berlin: Guttenberg Press. For chapters in books: Smith, C.J. 1989. Basal cell carcinomas. In Histological aspects of cancer, ed. C.D. Wilfred, pp. 278-91. Boston: Medical Press. When citing references in the text, do not use footnotes; instead, refer to articles by the author's name and the date the paper was published.

Fox in 1988 investigated the hormones on the nest-building behavior of catbirds.

Hormones are known to influence the nest-building behavior of catbirds (Fox, 1988).

When citing papers that have two authors, both names must be listed. When three or more authors are involved, the Latin et al. (et alia) meaning "and others" may be used. A paper by Smith, Lynch, Merrill, and Beam published in 1989 would be cited in the text as:

Smith et al. (1989) have shown that... This short form is for text use only. In the Literature Cited, all names would be listed, usually last name preceding initials.


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Introduction Exercise 2 Notes on Biological Drawings ______________________________________________________________________

Drawings are an aid to precise observations and for this reason they are an important part of laboratory work. In the practical examination, the examiner will have only your written recordings and drawings to assess you. Therefore full recordings and neatly labelled drawings are of great importance. Keep the following points in mind when making drawings: 1. Use a sharp, pointed HB/2B pencil. 2. Drawings should be as large as possible and made to fit into the space available. 3. Attention must be given to the general shape and proportion of the specimen.

First consider what you want to show. Then plan your drawing so that various parts are in proportion and fit on the page. Small marks indicating the length and breadth of the drawing are of great help in planning, and a faint outline can be rapidly drawn to show the relative positions of the parts.

4. The final outline should be drawn with clean firm lines (not sketchy broken lines). Details should be put in clearly with a sharp pencil. If important details are too small to be shown in proportion, they can be shown in an enlarged drawing on the side.

5. Drawings should be accurate records of your observations. The biologist is not expected to be an artist, but to become, in some degree, a draughtsman. Clear and accurate line drawings are needed.

6. Shading and colouring should be avoided. It should be possible to make the drawing perfectly clear by the judicious use of thick and thin pencil lines and careful cross-hatching. Get into the habit of making your drawings large and clear.

7. As important as the drawing is the labelling. This should be done neatly in pencil and the letters printed. Each label should be connected to the appropriate part of the drawing by a clear guideline without arrowheads. Do not label too close to the drawings, and never write on the drawing itself. Always make sure that each drawing is fully labelled before you leave it. Guidelines to the labels must be drawn with pencil and ruler and never crossing one another.

8. Each drawing must always have a title. The title should specify whether it is a transverse section, s longitudinal section, whole mount, etc.

9. Magnification of drawing can be stated if necessary. Calculate using the formula, Magnification = size of drawing___

Actual size of specimen 10. Plan diagrams of microscopic sections should not include any cell structure.

They are outline drawings showing relative amounts and distribution of various tissues.

11. In making high-power detailed drawings, repeated features need not all be drawn but only a small representative portion showing a few large accurate cells (3 or 4 adjacent cells) of each type must be indicated.

12. It is sometimes appropriate, particularly when drawing live specimens, to make succinct notes to the labels. These are called annotated drawings, which are particularly valuable as they combine a record of structure with functional observations. Annotations must be beneath the labels.


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Example of an annotated drawing Title: Detailed drawing of a Hydrilla leaf cell Magnification power: 10x. 40x

Note: The distinction between a plan diagram and a drawing: A drawing is an exact and accurate representation of an object, unlike a diagram which is a simplified outline. Warning: Memorized textbook drawings or diagrams, bearing little likeness to the specimens or observations will not be awarded marks.

Cell wall (made of cellulose, stained yellow)

Chloroplast (moves in cytoplasm, site of photosynthesis; stained brownish with iodine solution)

Cytoplasm (granulated; found at periphery of cell; stained light yellow with iodine solution)

Cell vacuole (contains salt and sugar solution; bound by membrane tonoplast; colourless)

Nucleus (doubled membrane organelle embedded in the cytoplasm; control center of the cell; stained orange brown with iodine solution.)


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A tissue map or plan diagram refers to a generalized outline of the tissue regions of a specimen. If such is requested, no detailed drawings of individual cells are required. The illustration below is a cross section of a leaf. The items in the square box are detailed drawings of cells whereas those of the vascular bundle reflect a tissue map or plan diagram.

Is the picture below a drawing or diagram?


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General Marking Scheme It is your responsibility to read the guidelines in the introduction to this lab manual. In the past many clever students lost marks simply because they failed to look up instructions already provided. Would you like to be as clever as them? This is an example of what a marking scheme may look like for: General instructions for students:

One to two slides may be drawn [please consult your lecturer]. If two drawings are to be done, each student is allowed only 30 mins per slide for drawings to be done in the 1st half.

The second half of the practical will be used to assess students microscope and / or identification skills on one type of slide. Students are required to identify at least any three structures which they choose.

For online submissions, students are required to upload pictures of their drawings (more instructions on WBLE).

In order not to lose marks unnecessarily, please ensure that you comply with the instructions on writing lab reports at the beginning of this manual, including your particulars (e.g., name, group, etc.) as stated in the instructions.


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Checklist for biological drawings This section may comprise 10 marks out of 20 marks. Any mistake will result in subtraction of 1 mark.

Please read full version for your personal success

Items Remarks from Lab Introduction

1. Appropriate & comprehensive (detailed and complete) title

The title should specify whether it is a transverse section, s longitudinal section, whole mount, etc.

2. Written indication of objective used (multiplied by __x ocular lens)

3. Requirement for detailed drawing or plan diagram instruction adhered to

A tissue map or plan diagram refers to a generalized outline of the tissue regions of a specimen. If such is requested, no detailed drawings of individual cells are required.

4. Correctly labelled items

5. Annotations if requested annotated drawings, which are particularly valuable as they combine a record of structure with functional observations.

6. Drawing is as what is seen under microscope (i.e., not textbook-perfect picture)

Memorized textbook drawings or diagrams, bearing little likeness to the specimens or observations will not be awarded marks.

7. Magnification if requested

Magnification = ___size of drawing Actual size of specimen 8. Method of calculation if

requested

9. Early submission up to tutor to delete marks according to lateness

10. Overall impression of drawings (e.g., neatness)

a. Satisfactory b. Quite good c. Good d. Very good e. Excellent


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General guidelines for biological photo(s)

please read full version for your personal success

Items Remarks from lab introduction

1. Title - Appropriate & comprehensive (detailed and complete)

Specify whether it is a transverse section/cross section, longitudinal section, whole mount, etc.

2. Magnification - Written indication of objective used (multiplied by __x ocular lens)

10x. 4x or 40x / 10x. 10x or 100x

3. Label - Correctly labelled items

Label box should not overlap the photo. Label line should point to the specific area. Label line should not overlap each other.

4. Annotations if requested Include the structure and function Annotation should be beneath the label.

5. Images and tidiness Photos taken must be clear.

6. Late submission - up to tutor to delete marks according to lateness

General instructions for lecturers: Two areas of assessment:

1) Biological drawings 1st hour allocated for drawing Kindly instruct students to draw slides according to availability (as supplied). The manual provides options to lecturers to request 1 detailed drawing or 2 plan diagrams. Note: if students are to submit drawing on the spot, 1 drawing should suffice. Recommended time allocation: Briefing (5-10 mins) + Slide familiarisation & reading (30 mins) + drawing (30 min if 1 detailed drawing; longer if 2 plan diagrams required)

2) On-site assessment 2nd hour allocated for on-site assessment. On-the-spot identification of 3 structures (10 marks); 2-3 students assessed within 3 minutes. Drawing (10 marks) Recommended time allocation: On-site assessment (50 min).

General instructions for lab reports:

Prepare your answers on your own A4 sheets of paper.

You are not required to re-write questions.


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Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Identification of Biochemical in Their Pure Form ______________________________________________________________________ Objective: To identify the components of the solution in its pure form with various food tests and state the justifications. Important notice: Any heating that has to be done in the following tests should be carried out in a water bath at 95oC. Direct heating of test-tubes should not be taken place. Apparatus & Equipments: Test tubes Test tube rack Water bath, 95oC Spatula Test tube holder Materials:

Iodine

1 M hydrochloric acid

Sudan III

Starch solution

Corn oil

Egg albumin

1% copper sulphate solution

DCPIP (dichlorophenolindophenol) solution

Ascorbic acid (or vitamin C tablet, or lemon juice)

1% sucrose solution (Analar sucrose must be used to avoid contamination with a reducing sugar Benedicts reagent

1 M sodium hydroxide (or potassium hydroxide or sodium hydrogen carbonate)

Millons reagent 1% glucose solution

Absolute ethanol

Introduction The nutrients in the food you eat supply your body with energy for growth and repair. These principle substances include carbohydrates, proteins, fats, minerals and vitamins. We can test for the presence of these important compounds in food by using chemical reagents that react in predictable ways in the presence of these nutrients. Please refer to the notes given above on:

How to record qualitative data. (Marks will be awarded based on proper recording.)

What to do if you dont obtain the desired results.


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Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the days experiment. Procedures: The following tests are to be done in pairs unless otherwise specified. Part 1: Identification of Carbohydrates Test for reducing sugars The reducing sugars include all monosaccharide, such as glucose and fructose, and some disaccharides, such as maltose and lactose, use 0.1 1% sugar solutions. Common tests for reducing sugars include Benedicts test (described below) and Fehlings test (not done here). See basis of test below for explanation of the following reaction:

Benedicts test for reducing sugars:

Procedure* Basis of test Observation

Reducing sugar test

Add 2 cm3 of any one solution of the reducing sugar provided to test-tube. Add an equal volume of Benedicts solution. Using a test-tube holder, shake and heat at a high temperature for one minute (a water bath is provided), shaking continuously to minimize spitting.

Benedicts solution contains copper sulphate. Reducing sugars reduce soluble alkaline blue copper sulphate containing copper (II) ions, Cu2+ to insoluble red-brown copper oxide containing copper (I). The latter is seen as a precipitate.

[Note: report after shaking and after contents settle down; see introduction pg. 9]

*: Please do NOT remove measuring cylinder or any other item from the stations provided. Observe and report characteristics of tube contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate. Test for non-reducing sugars The most common non-reducing sugar is sucrose, a disaccharide. If reducing sugars have been shown to be absent (negative result in above test), a brick-red precipitate in


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the test below indicates the presence of a non-reducing sugar. If reducing sugars have been shown to be present, a heavier precipitate will be observed in the following test than with the reducing test if non-reducing sugar is also present. The proper procedure for testing for an unknown carbohydrate sample for non-reducing sugars involves:

First test for reducing sugars: Benedicts test on the unknown fresh sample Why is this step necessary? What results will one get which will cause this step to be called a negative test?

Second test for reducing sugars: Benedicts test on the acid-hydrolysed unknown sample What results will one get which will cause this step to be called a positive test?


Non-reducing sugar test

Add 2 cm3 of fresh sucrose solution to a test tube. Add 1 cm3 O.1 M hydrochloric acid. Using a test-tube holder, heat at a high temperature for one minute. Carefully neutralize with equal volume (1 cm3) of 1 M sodium hydroxide or sodium hydrogen carbonate or potassium hydroxide. (Care is required because effervescence occurs.) Finally, add an equal volume (4 cm3 ) of Benedicts solution to the acid-hydrolysed sugar solution. Using a test-tube holder, shake continuously to minimize spitting when heating at a high temperature for one minute (a water bath is provided).

A polysaccharide or disaccharide can be hydrolyzed to smaller component constituents by boiling with O.1 M hydrochloric acid. Sucrose is hydrolyzed to glucose and fructose, both of which are reducing sugars and give the reducing sugar result with the Benedicts test.


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Additional Information The mixture is likely to bump violently during heating and extra care should therefore be taken. The test is semi-quantitative, that is, a rough estimation of the amount of reducing sugar present will be possible. The final precipitate will appear green to yellow to orange to red-brown with increasing amounts to reducing sugar. The initial yellow colour blends with the blue of the copper sulphate solution to give the green colouration. Is the precipitate that of reducing sugar or copper oxide?

*: Please do NOT remove measuring cylinder or any other item from the stations provided. Observe and report characteristics of tube contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate. Test for starch Starch is only slightly soluble in water, in which it forms a colloidal suspension. It can be tested as a mainly solid in suspension.


Iodine test

***Note: The starch prepared for you is already cooked starch. Add a few drops of 1% cooked starch solution on a white tile. Add a few drops of I2/KI solution (iodine). Be sure to mix them together on the tile with an object such as your pen cover.

A polyiodide complex is formed with starch.

*: Please do NOT remove measuring cylinder or any other item from the stations provided.


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Part 2: Identification of Lipids Lipids include oils (such as corn oil and olive oil), fats and waxes.


Sudan III

Sudan lll is a red dye. Add 2 cm3 of oil to 2 cm3 of distilled water in a test-tube. Add a few drops of Sudan III and shake.

Fat globules are stained red and are less dense than water.

Emulsion test

Add 2 cm3 fat or oil to a Test-tube containing 2 cm3 of absolute ethanol. Dissolve the lipid by shaking vigorously. Add an 4 cm3 volume of cold (or tap) water.

Lipids are immiscible with water. Adding water to a solution of the lipid in alcohol results in an emulsion if tiny lipid droplets in the water which reflect light and give a white, translucent appearance.

[***Note: report after shaking and after contents settle down]



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Part 3: Identification of Proteins A suitable protein for these tests is egg albumen.


Millons Test

Add 2 cm3 protein (albumin) solution or suspension to a test-tube. Add 1 cm3 Millons reagent. Using a test-tube holder, heat at a high temperature for one minute (a water bath is provided). Millons reagent is poisonous: be extremely careful!

Millons reagent contains mercury acidified with nitric acid, giving mercury (II) nitrate and nitrite. The amino acid tyrosine contains a phenol group which reacts to give a red mercury (II) complex. This is a reaction given by all phenolics and is not specific for proteins. Protein usually coagulates on boiling. Thus appearing solid. The only common protein lacking tyrosine likely to be used is gelatin.

Biuret Test

Add 2 cm3 (albumin) protein solution to a test tube. Add an equal volume of 5% sodium hydroxide (or potassium hydroxide) solution and mix. Add 2 drops of 1% copper sulphate solution and mix. No heating is required.

A test for peptide bonds. In the presence of dilute copper sulphate in alkaline solution, nitrogen atoms in the peptide chain form a purple complex with copper (II) ions, Cu2+. Biuret is a compound derived from urea which also contains the CONH group and gives a positive result.



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Part 4: Identification of Vitamin C (ascorbic acid) ***Note: If more than 5 drops of ascorbic acid are required to turn DCPIP to colourless, please dilute the latter significantly. This test can be conducted on a quantitative basis if required, in which case the volumes given below must be measured accurately. A suitable source of vitamin C is a 50/50 mix of fresh orange or lemon juice with distilled water. Vitamin C tablets may also be purchased.


DCPIP test

Using 0.1% ascorbic acid solution as a standard. Add 1 cm3 of DCPIP solution to a test-tube. ***Add the 0.1% ascorbic acid to the DCPIP drop by drop until it becomes approximately colourless (or by stirring gently if youre provided with a syringe needle/ glass rod). Note the no. of drop(s) of ascorbic acid solution used.

DCPIP is a blue dye which is reduced to a colourless compound by ascorbic acid, a strong reducing agent.

Additional Information Shaking the solution would result in oxidation of the ascorbic acid by oxygen in the air. The effects of shaking and of boiling could be investigated.



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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills Based Assessment: Tabulation of qualitative data

1. Tabulate your observations above for each biochemical food test executed, according to the guidelines provide in the introduction on writing lab reports. Note: The table in the lab manual for this task is not presented correctly.

2. Wrong results are alright for this experiment. 3. No need to write procedure, basis of test, discussion or conclusion. 4. You may choose to construct one or more tables. 5. For tests involving carbohydrates, observe and report characteristics of tube

contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate as above.

o Liquid mixture, solution, suspension, emulsion? transparent, translucent, opaque?

o Colour state initial and final colours?

o Precipitate (if any) colour of precipitate? amount of precipitate?

Better understanding of terms:

Mixture

Solution

Suspension Option 3: Skills Based Assessment: Critical thinking/ Discussion 1. How could you determine the concentration of ascorbic acid in an unknown sample?

2. You are provided with three sugar solutions. First one contains glucose, second one

is a mixture of glucose and sucrose, and lastly is sucrose solution. (a) How could you identify each solution? (b) Supposing that the apparatus were available, and time permitted, briefly discuss

any further experiments you could perform to confirm your results. 3. After carrying out Benedicts test, a student concludes that the obtained positive results prove that glucose is present. True or false? Provide a reason. 4. After carrying out Benedicts test, a student identifies the coloured precipitate as reducing sugar. True or false? Provide a reason. 5. A student pours Benedicts solution into a tube containing a carbohydrate. No colour change is obtained. The student concludes that the carbohydrate is not a reducing sugar. True or false? Provide a reason.


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6. A student adds acid to a solution of sucrose followed by neutralization and, finally, Benedicts test. A negative (instead of positive) result is obtained. Explain why. 7. Why does sucrose yield positive results after carrying out the non-reducing sugar test? What are the components of sucrose? For those who have done Sem 3 3. How would you make 100 cm3 of a 10% glucose solution? 4. Starting with stock solutions of 10% glucose and 2% sucrose how would you make

100 cm3 of a mixture of final concentration 1% sucrose and 1% glucose?


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Practical 2 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two Carbohydrate Solutions ______________________________________________________________________ Objective: Students are expected to state the objective of this experiment. Apparatus & Equipments: Boiling tubes Metal test tube racks Pipette filler Graduated glass pipette, 10ml Water bath, 37-40oC Water bath, ~90-95oC Beaker Pasteur pipette Materials: Carbohydrate solution A Carbohydrate solution B Benedicts solution 3 M Hydrochloric acid 3 M Sodium hydroxide (or potassium hydroxide) Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the days experiment. Procedures: This experiment is to be done in pairs. To avoid congestion, each pair should collect the following before beginning the experiment:

8 ml NaOH

16 ml Benedicts Solution 2ml Solution A

42ml Solution B

2ml HCl

1 pipette and 1 rubber teat (to be washed with distilled water each time before reuse)

5 ml measuring cylinder (to be washed with distilled water each time before reuse)

Metal test tube racks (not wooden) Overview Please see tables 1 & 2 on the next page to get a rough idea of what is required in the experiment. Can you identify in the instructions that follow, how the tubes are to be placed under various temperatures and time periods? Carry out your investigation as follows. 1. Prepare two test tubes containing 2 ml solution A and 2 ml solution B respectively.

Add 2 ml Benedicts solution to each test tube. Heat both tubes together in the hotter (~90-95oC) water bath for two minutes. Record the results in table 1.

2. Pipette 10 ml solution B into each of four test-tubes and, label the tubes 1, 2, 3 and

4 respectively with labelling paper (or masking tape) near mouth of tube. Write the initials of your group name or individuals.


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3. Place tubes 1 and 2 in a water bath of ~37o, and tubes 3 and 4 in a water bath of ~95oC (It doesnt matter how long you put it in at this stage as no saliva or HCl have been added yet).

4. Salivate into a separate test-tube till it reaches a height of about 1 cm - 1.5 cm.

Dilute the saliva with an approximately equal volume of distilled water.

5. Ensure that the following two steps (5 and 6) adding of saliva or HCL into the respective tubes (mentioned in the next sentence and below) is done approximately at the same time. (Why is this necessary?)

Note: for the following, please ensure that the respective tube into which saliva is going to be dropped does NOT leave the water bath (especially 95 oC) for more than 30 seconds! (Why is this necessary?) 6. Use a 5 ml measuring cylinder to measure out 2 ml of the diluted saliva prepared in

(3) and pipette 1 ml each into tubes 1 and 4. Shake the contents of the tubes well to ensure thorough mixing.

7. Use a measuring cylinder to measure out 2 ml HCl and pipette 1 ml each into tubes

2 (already in water bath of ~37oC) and 3. Place tubes 3 and 4 in a water bath set at 95 oC. Let tubes 1, 2 (already in water bath of ~37oC), 3 & 4 (recently in water bath of ~95oC) incubate at their respective temperatures (see Table 2) for 35 minutes from this moment.

8. Label 4 more new tubes (either test tubes or boiling tubes) as follows: 1, 2, 3 and

4. After 5 minutes of incubation of tubes labelled 1 to 4 prepared previously, pour out about one-third of the total volume of the contents from all these tubes into the respective newly labelled test tubes (e.g., 1 into 1, 2 into 2 etc.). Ensure that the volume in each of the tubes 1-4 is approximately the same (why is this important?). Straightaway, place back the original tubes (labelled 1-4) back into the respective temperatures of incubation.

9. Neutralize the acid in each of tube labelled 2 and 3 with 2ml of sodium hydroxide (or potassium hydroxide) (each). Shake each tube (2 and 3) to ensure uniform mixing.

10. Remove 1ml of the solution from each tube (1 to 4) into new tubes and label

appropriately as you wish as long as you dont get confused. To carry out Benedicts test, add an equal volume of Benedicts solution (1 ml) for each tube. Using a test-tube holder, shake and heat at a high temperature for one minute (use the hotter water bath provided), shaking continuously to minimize spitting. Record your observations in table 2.

11. Wash the test tubes 1 to 4. After 35 minutes of incubating tubes 1 to 4, pour out

about one-third of the total volume from test samples from all the tubes into the respective tubes labelled 1- 4.

12. Neutralize the acid in each test tube labelled 2 and 3 with 1ml of sodium

hydroxide (or potassium hydroxide). (Why is neutralization necessary?) Remove 1ml of solution from each tube 1 to 4 and carry out Benedicts test with an equal


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volume of Benedicts solution (1 ml) for each tube. Remember to heat your sample (please see previous. Record your observations in table 2.

13. Add a few drops of fresh solution A and B separately spaced on a white tile. On

each solution, add 1-2 drops of I2/KI solution (iodine). Be sure to mix them together on the tile with an object such as your pen cover. Record your observations in the table 1.

Note: no penalization for unexpected results. Please refer to Practical 1 Exercise 1 Writing lab report. Table 1: (title)

Observations Conclusions

Solution A

Benedicts test: Iodine test:

Solution B

Benedicts test: Iodine test:

Table 2: (title)

Tube Contents Temp (C)

Benedicts TestColour Observation

After 5th min (from tubes 1 4

into 1 4)

After 35th min (from tubes 1 4 into

1 4) 1 10 ml solution B

1 ml saliva 37

2 10 ml solution B 1 ml 3 M HCl

37

3 10 ml solution B 1 ml 3 M HCl

95

4 10 ml solution B 1 ml saliva

95


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Guidelines Observations For Benedicts test and Iodine tests, please follow lab manual guidelines for students on writing lab report on the following:

o Liquid mixture, solution, suspension, emulsion? transparent, translucent, opaque?

o Colour state initial and final colours?

o Precipitate colour of precipitate? amount of precipitate?

Conclusions Absence/presence of what type of carbohydrate? Results and Discussions: 1. The results and discussion sections of your report should not exceed 2 pages. 2. Ensure that the guidelines for constructing tables and recording results for this

experiment are adhered to (see introduction). 3. If youre required to write a discussion straight-to-the-point, follow the numbering

below (please check with your lab tutor). If your report is full-length, write your discussion in prose form (please check with your lab tutor). Theory to apply: Refer to relevant information from lecture topics on biological molecules and enzymes.

Discussion should contain:

1) Name of enzyme involved 2) Specific action(s) of enzyme involved 3) 4) Effect of HCl on substances (e.g., Solution B) 5) Effect of temperature on substances (e.g., Solution B, saliva content) 6) Product:

a. Identification (make suggestion(s)/ educated guesses) b. Structure (e.g., chemical classification etc.)

7) Basis of test() used 8) Which carbohydrate is more complex, A or B? Give a reason. 9) Conclusion: Summary of results


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Practical 3 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of the effects of catalase concentration on hydrogen peroxide

Objective: To investigate the effects of different catalase concentration on the decomposition of hydrogen peroxide. Apparatus and Materials: 5 test or boiling tubes Scalpel/ pen knife 1 beaker (500cm3) White tile 1 beaker (250cm3) Mortar and Pestle 1 Retort Stand (optional) Weighing boat 1 rubber bung with delivery tube 4 filter funnel and filter paper (optional) 4 test tubes or plastic vials (if provided) Potato 1% hydrogen peroxide solution Hand-held pipette **Caution: Hydrogen peroxide is formed continuously as a by-product of chemical reactions in living cells; it is a very toxic (poisonous) substance. Note to lecturer: This experiment may be done together with Experiment 2 if the lab session is 3 h long. Introductory instructions:

You may perform this experiment in groups of 3-5. Introduction: Enzymes are proteinaceous molecules that speed up chemical reactions within living systems. In this experiment, the effect of catalase on hydrogen peroxide is investigated. Catalase is an enzyme present in the cells of plants, animals and aerobic (oxygen requiring) bacteria. It promotes the conversion of hydrogen peroxide, a powerful and potentially harmful oxidizing agent, to water and molecular oxygen.

2H2O2 + catalase 2H2O + O2

Warning: H2O2 is corrosive. For the person handling, please wear gloves. Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the days experiment. Procedures: 1. Optional: Set up an electric water bath at 37oC. (If this is not provided, its ok.)

2. Depending on the size of the rubber bung holding the delivery tube, select either one

boiling or test tube and label it as tube A.

3. From the potato sample given, cut (with a pen knife/ knife/ scalpel) and weigh 5g of potato using a weighing boat so as not to dirty the weighing balance.


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4. Cut the potato samples into smaller pieces (the smaller the easier for you to mash) and mash the potato sample using the mortar and pestle. Note: dont spend too much time on this it doesnt have to be KFC mashed potato quality! Add 6 cm3 of distilled water to the potato samples after the mashing process.

5. You can do two things: (i) separate the solid mashed potato from the liquid in any

way you choose and pouring the liquid into a test tube; or (ii) by filtering the mashed potato sample (with filter paper and funnel) and collect the filtrate in a test tube or plastic vial (if provided) [using filter paper and funnel is more time-consuming].

6. Fill an empty test tube with tap water (see picture below).

7. Add 5cm3 of hydrogen peroxide into Tube A using the hand-held pipette provided.

8. Draw 1cm3 of the filtrate from the mashed potato samples and add to Tube A.

Immediately close the test tubes with the rubber bung. Seal the end of the delivery tube furthest away from the bung with parafilm.

9. Set up the apparatus as shown below (if retort stand is provided; if not just use each

others hands). Note: You need neither the water bath nor retort stand.

10. Remove the parafilm and immediately immerse the tube in the water bath quickly

(use a beaker for this and pour into it water from an electric water bath) and start your watch. Count the number of gas bubbles produced for 2 minutes and record it. After you finish, return the water you took back to the electric water bath. [Note: water can maintain the heat in it for quite some time.]

11. Optional, depends on time available: To get a 2nd measurement, dispose the contents of tube A. Repeat step 7 to 10. After you finish, return the water you took back to the electric water bath.

12. Repeat Step 2 to 11 but with 10g of potato, then 15g and finally 20g (optional,

depends on time available).


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13. Record the data in table 1. Your class is to record their data on the whiteboard. Calculate the averages in order to plot graphs.

Table 1: (title)

(What heading should you write here?)

5g 10g 15g *20g

Number of Attempt

1st 2nd *3rd 1st 2nd *3rd 1st 2nd *3rd *1st *2nd *3rd

Number of gas bubbles produced

*Optional, depends on time available. After the experiment Please dispose of potato pieces, masking tape, parafilm etc. into the dustbins provided. Please clean the sink, removing any potato pieces, masking tape, parafilm etc. Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data (Table 1) Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel) of the average number of bubbles produced against potato samples used. Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table, write a discussion or conclusion. Option 4A: Skills-Based Assessment: Discussion Data provided to students to discuss Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. Font Arial, size 11. Margins: 1 inch from top, bottom, left and right (no need to change if youre using the standard/ default size when MS Word opens). Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. *Option 4B: Skills-Based Assessment: Discussion Students use own data to discuss From the data you have collected in the practical, account fully for the results which you have obtained. Discuss any anomalous data/ results that you might have. Explain the trend or pattern of the graph.


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Practical 3 Experiment 2 (FHSC 1214 Cell Biology ONLY) Synthesis of Starch Using an Enzyme Extracted from a Potato Tuber ______________________________________________________________________ Objective: To find out which compounds the enzyme in the potato can act on to produce starch (investigate three possible substrates: glucose, maltose and glucose-1-phosphate). Apparatus and Materials: Centrifuge and centrifuge tubes Glucose-1-phosphate (2%) Test tube rack Glucose solution (2%) Pestle and mortar Maltose solution (2%) Knife Iodine solution Labelling paper (or masking tape) Potato tuber Test tubes Pipette White tile Procedures: Introductory instructions:

Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment.

You may perform this experiment in pairs.

Take 5 ml iodine only when ready to begin the reaction.

Groups may have to take turns to centrifuge, depending on the number of groups and holders in the centrifuge.

NOTE: After carrying out steps 1 to 2, proceed to Experiment 2. Return to Experiment 1 only during the waiting periods of Experiment 2.

A. Extracting the enzyme from potato tissue 1. Peel a medium-sized half potato. Cut half of it into small cubes on a white tile (the

smaller the easier for you to grind). Grind a few pieces of potato cubes in a pestle and mortar with 20cm3 of water.

2. Separate the aqueous part of the extract from the solid as best as possible. You can

do this by pouring it out while restraining the solids with your fingers or an appropriate instrument. Divide the aqueous part of the extract into two equal portions and pour them into two centrifuge tubes. As far as possible, avoid letting sand and solid matter to get into the tubes.

3. Spin the extracts in a centrifuge for ten minutes at 5000 rpm so that the starch, cell

walls and other solid matter will settle at the bottom of the centrifuge tubes. The starch-free liquid above the deposit, or supernatant, should contain the enzyme.

4. Using a teat pipette, carefully, without disturbing the deposit beneath, withdraw as

much the clear enzyme solution as possible from the centrifuge tube. 5. To check whether this enzyme solution is starch-free, transfer a few drops of it into a

test tube and add 2 drops of iodine solution onto it. If a blue colour appears, then the potato extract would need to be centrifuged again.


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B. Attempting starch synthesis 6. Label three clean test tubes G, M and G1P, respectively. Use a separate teat pipette

and measuring cylinder in each case to place 3 cm3 of glucose solution in the G tube, 3 cm3 of maltose solution in the M tube, and 3cm3 of glucose-1-phosphate solution in the G1P tube.

7. To synthesise starch, pour 2 cm3 of the enzyme solution (the liquid or supernatant

you obtained after centrifuging above) into the substrate tube (G, M and G1P), mix well and note the time.

8. For each substrate, place 15 discrete drops of iodine solutions on clearly labelled

piece of white tile. 9. After one minute of the reaction use a teat pipette to place one drop of enzyme-

substrate solution onto one existing drop of iodine solution on the white tile. Stir with a suitable object (e.g. woodsplint or tooth pick) and record the colour produced. Repeat at intervals of 1 minute over 15 minutes, all the three tubes simultaneously.

Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Discussion Discuss the following questions: 1. Draw the structural formula of the substrates. What features of the starch-

synthesizing substrate molecule might have been recognized by the starch-synthesizing enzyme?

2. The synthesis of polymers such as starch requires metabolic energy. What was the

energy source in the successful reaction? 3. The enzyme isolated from potatoes is known as starch phosphorylase. In the intact

potato tuber it is also used to break down starch. How did conditions in the test tube favor starch synthesis? In what circumstances does the enzyme bring about starch synthesis in a potato?

4. In plant leaves, starch accumulates in chloroplasts. The synthesis of starch requires

ATP. Where do you think this ATP comes from?


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Practical 4 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide Solution

Objective: To investigates the enzymatic effect of various materials in the hydrogen peroxide solution. Apparatus & Equipment: Beaker Test tubes Either: water bath (95oC) or Bunsen burner Materials: Fresh Liver Potato cubes Manganese dioxide Hydrogen peroxide** Wood splints **Caution: Hydrogen peroxide is formed continuously as a by-product of chemical reactions in living cells; it is a very toxic (poisonous) substance. Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the days experiment. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. Wear gloves when handling liver tissue, so as not to be contaminated by any pathogen associated with the liver tissue used. Please stick to using one pair of gloves per person to prevent wastage. [Note: using boiling tubes may provide better results.] 1. Label six fresh empty test or boiling tubes 1, 2, 3, 4, 5, 6 and stand them in a rack. 2. Using a razor blade, cut the provided liver into several pieces of roughly 0.8 cm x 0.8

cm x 0.5 cm. 3. Place one piece of liver into tube 1. 4. Boil 100 cm3 of water in a beaker. (If youre using a water bath set at 95oC, this step

is not necessary). 5. Place the second piece of liver into the bottom of tube 2. Using a wooden splint,

gently spread the liver, without mashing it, over as wide an area as possible of the bottom of the test or boiling tube. Place tube 2 in the boiling or water bath (95oC) for about five minutes.

6. Using the weighing balance, measure out two 0.5 g portions of manganese dioxide

powder each onto a weighing boat. Pour each portion into tube 5 and tube 6.


40

7. Put tube 6 in the beaker of boiling water or water bath (95oC) for five minutes. 8. After five minutes let cool tube 2 and 6. 9. *Now put the third piece of liver into test or boiling tube 3. With the wooden stick

provided, mash it gently into a pulp.

10. Now put the third piece of liver onto a white tile. With a mortar and pestle, mash it gently into a pulp. Scoop the pulp into tube 3.

11. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4. 12. Prepare another six fresh empty test or boiling tubes and stand them in a rack. Put 5

cm3 of hydrogen peroxide into each of them. 13. Next, quickly add hydrogen peroxide into the test or boiling tubes 1, 2, 3, 4, 5, and 6.

If needed, you may push down some materials with one end of the wood splints provided.

**Step 12 and 13 are to be done quickly. 14. Using the parafilm provided, stretch it quickly seal the mouth of the test or boiling

tubes by stretching the film over it. In order to prevent the parafilm from being displaced if a lot of gas is produced, secure the parafilm covering the side of the test or boiling tube with masking tape.

15. Leave for 20 minutes or till when you see quite a lot of gas being produced in some test or boiling tubes as evidenced by the bulging of parafilm from the test or boiling tube mouths.

16. Once enough gas has accumulated in some test or boiling tubes, insert a glowing

splint (flame extinguished but glow remains) into each tube one at a time by just penetrating the parafilm with it. You may use the same splint.

Why is it important to test each test or boiling tube at least without too much difference in the duration of sealing among the tubes?

17. Record all your observations in the table. Record your observations on each tube

immediately after the reaction has started. [Note: be sure to use the following terms correctly: glowing splint glowed brighter, flame rekindled, effervescence (bubbles) observed, reference to sound, etc.]


41

Table 1: (title)

Test Tube

Contents with 5 cm3

hydrogen peroxide Observations before and after using wood splint

1

Fresh liver

2

Boiled liver (cooled)

3

Pulped liver

4 Potato cubes

5

Manganese dioxide (untreated)

6

Boiled manganese dioxide (cooled after heating)

Washing up Thoroughly wash and scrubbed all apparatus containing liver pieces with detergent or Dettol solution provided to rid it of unpleasant odours.


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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of qualitative data Tabulate the results you obtained (unexpected results accepted). The results table should not exceed 1 page. Option 3: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. Font Arial, size 11. Margins: 1 inch from top, bottom, left and right (no need to change if youre using the standard/ default size when MS Word opens). Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. Please be sure to address the following: 1. What is the equation of the reaction observed? 2. What plant or animal organelle is involved? 3. What effect does pulping the liver have upon the reaction? Account for this. 4. What effect does boiling the liver have upon the reaction? Account for this (include

reference to enzyme structure (bonds, molecular motion, shape, active site). 5. What were the differences between the reactions with fresh liver and with fresh

potato cubes? Account for these differences (include reference to enzyme structure (bonds, molecular motion, shape, active site)

6. What were the differences between the effects on the reaction of boiling the liver and

heating the manganese dioxide? Account for these differences (include reference to susceptibility (sensitivity) to heat, enzyme shape, bonds etc).


43

Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 1 The Microscope and Its Uses

Microscope usage (revision) Note to lecturer: Before any microscope work (viewing of histological slides) commences, please ensure students have gone through this introductory session. Objective: To study the uses of microscope and its maintenances. To learn microscopic techniques such as focus the object with correct illumination under different power of magnifications. Introduction: The microscope is a basic tool of the biologist. It is a valuable precision optical instrument easily damaged by careless usage. It is very important for the student to become familiar with the parts of the microscope and the procedures in the handling of it. Treat your microscope well and it will serve you well. Apparatus and Materials: Binocular Microscope Cover slips Microscope slide Newspaper (1 page) Plastic millimeter ruler Wash bottle Setting up the Microscope: The microscope when not in use is usually kept in a case. Remove it by grasping the handle arm while placing one hand under the base. Set it down gently on the laboratory table and at a reasonable distance from the table edge. Always keep the microscope upright in the vertical position and never touch any of the lens surfaces with the fingers since it will deposit a thin film of oil on the glass.


44

Parts of the Microscope:

Component Function

Arm For lifting and carrying the microscope.

Base To provide stability.

Body tube To house the lenses.

Eyepiece or ocular lenses

This is a set of lenses that rests loosely at the top end of the body tube. It is obvious that if the microscope is tilted while being carried, the lens may fall out and be ruined. The magnification of the eyepiece (given as 10X) is printed on the metal part of the ocular.

Revolving nosepiece

Located at the lower end of the body tube, it carries 3 objectives of different lengths. Rotating this part changes the magnification of the objectives.

Objective lenses

They are of different magnifications with the following visible properties:

Objectives Magnification Length Lens opening

Scanning lens 4x Shortest Widest

Low power lens 10x short wide

High power lens 60x longest narrowest


45

Component Function

Focusing adjustments

These comprise two knobs located on either side of the microscope which are used to change the distance between the object being viewed and the objective lens. Changing the distance determines the focus. For the object to be viewed in focus under high magnification, the lens must be much closer to the object than when it is under low magnification.

Coarse adjustment Made by the large knob beside the body tube for focusing under low power magnification.

Fine adjustment Made by the small knob, which is for focusing under high power magnification and accurate focusing.

Precautions when using the focusing adjustments: Turn both adjustment knobs at the same time. Do not overturn the adjustment knobs (i.e. do not force them to

go beyond their limits) Do not use the coarse adjustment knobs when focussing under

the 60x objective lens.

Stage This is the platform for slides and specimens to be viewed under the microscope.

Mechanical stage

This movable portion of the stage is attached to the specimen holder and allows the slide to be moved in different directions to facilitate viewing.

Specimen holder

This holds the glass slide in place.

Vertical feed knob

Rotating this moves the glass slide in the vertical direction.

Horizontal feed knob

This moves the glass slide in the horizontal direction.

Condenser Located just beneath the stage of the microscope, it incorporates a lens which collects light on the stage to bear on the object.

Built-in light source

This is situated below the iris-diaphragm to provide light for illuminating the object. It can be switched on or off.

Brightness adjustment knob

This provides adjustment to the illumination brightness.

Main switch This ensures that power is turned on or off.


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Preliminaries before Use: 1. Use the coarse adjustment to raise the body tube so that the objective can clear

the stage when the revolving nosepiece is turned. 2. Turn the nosepiece until the scanning objective is in-line with the eyepiece. You

should hear a soft click or else feel a distinct falling into place as the objective moves into position. If not, the field of view is totally dark or an illuminated crescent instead of a complete circle.

3. Turn the diaphragm to its largest opening. 4. Look into the eyepiece and make a final adjustment to the light adjustment knob

so that the field of view (i.e., the lit circle which you see) is evenly illuminated. Any glare should be removed by adjusting the diaphragm.

5. Should either of the lenses appear dirty, wipe it gently with a piece of special

lens paper. Use a circular motion with very light finger pressure. You should never use any other type of paper or cloth. Discard the lens paper after use.

6. The microscope is now ready for use. 7. If youre using a binocular compound light microscope like the diagram above,

position it so that the stage faces you. 8. Connect the microscope to the power supply and turn on the built-in light. 9. Ensure that the microscope stage is at its lowest position. This will prevent breaking

of slides and lenses by mistake when adjusting the objectives by moving the stage with the coarse adjustment knob.


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Using a higher power objective 1. Great care must be taken when using higher power objectives. DO NOT focus the

high power objectives with the coarse adjustment knob. 2. Most microscopes have parfocal objectives. This means that if one switches from

viewing a specimen in sharp focus under a lower power objective to a higher one, the object should automatically come approximately into focus. Only some slight further focussing with the fine adjustment knob is required to see the specimen clearly. Therefore, if youre using the higher power objectives, do not use the coarse knob to refine focus or youll risk breaking the slide and lenses.

If the objectives are not parfocal, adjust the stage such that it is about 1cm from the low-power objective. Change to the high-power objective and then adjust the stage with the coarse adjustment knob until it is about 1mm away from the objective. This is determined by looking from the side of the microscope. Using the fine adjustment knob and looking through the eyepiece now, slowly bring the object into focus. Repeat the procedure carefully if the first attempt at finding an object under high-power magnification is unsuccessful.

3. When changing from one objective to another, you will hear a click when the

objective is set in position. 4. You are now ready to switch from the scanning objective to a higher power objective

after obtaining a sharp focus of the object. 5. If required, adjust the fine adjustment knob to see the specimen clearly. 6. If youre going to switch to the next higher power objective, look from the side of the

microscope and move the revolving nosepiece slowly till that higher power objective clicks into position. Be careful that it does not touch the slide (normally it shouldnt unless the specimen is too thick and also covered by a thin cover slip).

7. Take care that the lower end of the high power objective does not touch the cover

slip. If this happens, you must repeat the whole procedure focusing again, starting with the scanning objective.

Trouble-shooting Below are some common problems associated with not being able to find and focus on an object under high-power magnification.

Is the objective lens in position?

Is the cover slip on the slide facing upwards?

Is the object in the centre of the stage?

Are the lenses clean and free from dirt and moisture?

Is the condenser adjusted and focused?


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Oil Immersion: If your microscope comes with a 100 x objective, please do NOT use it. Used the improper way, it will break. If you require a particularly high magnification, immersion oil may be used. Fluid with the same refractive index as the objective lens is placed between a special objective lens and the cover slip so that it touches both. The fluid permits a larger cone of light rays to enter the objective from the specimen, and this increases the resolving power obtainable. Microscope Care: Like all laboratory instruments, the microscope needs proper care for best service. Observe the following: 1. Turn the resolving nosepiece until the scanning objective is in position. 2. Adjust the boy tube so that the lower end of the objective is about 1 cm above the

stage. 3. Ensure that the stage surface is clean and dry. 4. Return the microscope in an upright position to its storage case. Activity: Manipulation Skill practice task Note to lecturer: this activity may be graded. Any mistake will result in subtraction of 1 mark.

Microscope manipulation checklist Observed

Yes No

Skill: Manipulation

1. Position compound light microscope so that the stage faces you.

2. Ensure that the microscope stage is at its lowest position.

3. Position the specimen holder such that it is roughly in the middle of the stage and not at either left or right extremes.

4. Secure the slide in position correctly with the specimen holder

5. Ensure that the scanning objective is first employed.

6. Ensure that the field of view is a complete circle and not totally dark or an illuminated crescent.

7. Both eyes open and used to look through the eyepieces.

8. Adjust the brightness adjustment knob to give the right amount of light for viewing the object details clearly (i.e., instead of either too dark or too bright, obscuring the objects finer details).

9. Focus on image accurately and sharply by using the coarse and fine adjustment knobs.

10. When using the next higher power objective, look from the side of the microscope to ensure that it does not touch the slide.

11. When using higher power objectives (e.g., 40 X onwards), only the fine adjustment knob is used (i.e., not the coarse adjustment knob).


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 2 On-site Assessment

Each student will be assessed on-the-spot identification of 3 structures within certain minutes (10 marks) (The duration will be decided by the tutor). This section may comprise 10 marks out of 20 marks. Any mistake will result in subtraction of 1 mark. Checklist for on-site slide structure identification

Observed

Yes No

Skill: Manipulation

12. Position compound light microscope so that the stage faces you and

ensure that the microscope stage is at its lowest position.

13. Position the specimen holder such that it is roughly in the middle of

the stage and not at either left or right extremes.

14. Ensure that the scanning objective is first employed.

15. Ensure that the field of view is a complete circle and not totally dark

or an illuminated crescent.

16. Both eyes open and used to look through the eyepieces.

17. Adjust the brightness adjustment knob to give the right amount of

light for viewing the object details clearly (i.e., instead of either too

dark or too bright, obscuring the objects finer details).

18. When using the next higher power objective, look from the side of

the microscope to ensure that it does not touch the slide.

19. When using higher power objectives (e.g., 40 X onwards), only the

fine adjustment knob is used (i.e., not the coarse adjustment knob).

20. Focus on image accurately and sharply by using the coarse and fine

adjustment knobs.

Skill: Identification

21. Able to name the specimen from the slide or identify two - three

structures from the slide.

Total marks


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 3 Preparation of Wet Mount

Materials for microscopic examination are usually placed on the glass slide of standard size, the microscope slide. In most cases, the materials are then covered by small thin piece of glass, the cover slip. Both microscope slide and cover slip should be very clean before use. Cleaning microscope slides Hold the microscope slide by the edges between the index flinger and the thumb and dip in water. Then wipe dry using a soft tiss

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