lab notes for bio 2420 - austin community · pdf filelab notes for bio 2420 _____ these are...

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Look here for explanations, reactions, photos etc. http://www.austincc.edu/microbugz

Lab Notes for Bio 2420

______________ These are the Rob Lewis lab notes for Micro 2420.

The ACC Microbugz site (http://www.austincc.edu/microbugz/) provides explanations of tests and their

reactions, including photos of positive and negative reactions etc. for most of the lab processes and

procedures]

Because the content of each lab is not set in concrete, the contents/dates can shift, depending on

scheduling and availability… so check the syllabus/schedule and refer to the appropriate pages here.

_________________

http://www.austincc.edu/microbugz/

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Contents Introduction to Lab ............................................................................................................................5

Lab 1 – Safety & Ubiquity .......................................................................................................................... 5

Lab 2 - Ubiquity (cont.), Morphology, Intro to Microscope, Begin Aseptic Technique ............................ 6

Lab 3 – Aseptic Technique (cont.), Morphology (cont.), Isolation Streak, Unknown 1 ............................ 7

Lab 4 – Morphology (cont.), Staining Techniques, Intro to Oil Lens ......................................................... 9

Lab 5 - Special Stains: Negative/ Capsule, Gram’s .................................................................................. 10

Gram’s Stain .................................................................................................................................................... 10

Combination Capsule/Negative Stain ......................................................................................................... 11

Lab 6 - Special Stains: Acid Fast, Endospore, Flagella ............................................................................. 12

Acid Fast Stain (Kinyoun Method) ............................................................................................................. 12

Endospore Stain ............................................................................................................................................. 13

Flagella Stain .................................................................................................................................................. 13

Begin Labs on Staphylococcus sp. and Streptococcus sp. ................................................................... 14

Lab 7 – Begin Gram Positives (Staphs & Streps) ..................................................................................... 15

Phenylethyl Alcohol Agar (PEA): ................................................................................................................ 15

Blood Agar: ...................................................................................................................................................... 15

Unknown #2 (Staphs & Streps): ................................................................................................................ 15

Lab 8/9 – Staphs & Streps (cont)- MSA, Coagulase, Catalase ................................................................. 16

Mannitol Salt Agar (MSA) ........................................................................................................................... 16

Coagulase test ................................................................................................................................................ 16

Catalase test ................................................................................................................................................... 17

Lab 10 – Staphs & Streps (cont)- BEA, Bacitracin, Optochin ................................................................... 18

BEA (Bile Esculin Agar) slants .................................................................................................................... 18

Bacitracin Susceptibility disk ..................................................................................................................... 18

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Optochin Susceptibility disk ....................................................................................................................... 18

Begin Enteric Bacteria (Enterobacteriaceae) ..................................................................................... 19

Lab 11/12: Media for Enterics – EMB, HE, Mac ..................................................................................... 20

EMB (Eosin Methylene Blue) Media ........................................................................................................... 20

Mac (McConkey) Media ................................................................................................................................. 20

HE (Hektoen Enteric) Media ....................................................................................................................... 21

Phenol Red Sugar Broths ............................................................................................................................. 22

MR/VP Media .................................................................................................................................................. 22

SIM (Sulfur Indole Motility) Media.......................................................................................................... 22

Lab 14: Media for Enterics (cont.) – TSI, Citrate .................................................................................... 23

TSI (Triple Sugar Iron Agar) ..................................................................................................................... 23

Simmons Citrate Agar .................................................................................................................................. 23

Lab 15: Media for Enterics (cont.) – Urease, Ornithine Decarboxylase ................................................. 24

Urease Broth .................................................................................................................................................. 24

Ornithine Decarboxylase Media ................................................................................................................. 24

End of Enteric Medias .................................................................................................................................. 24

How Environmental Conditions Affect Growth: ................................................................................. 25

How does Presence or Absence of Oxygen Affect Growth? ................................................................... 25

What are the Affects of Osmotic Pressure on Bacterial Growth ............................................................ 26

What are the Affects of Temperature on Growth .................................................................................. 27

What are the Affects of UV Irradiation on Growth: ................................................................................ 28

Eukaryotic Parasitology .................................................................................................................... 29

Organisms for Lab Exam 4 – Parasitology ............................................................................................... 29

Demonstration Experiments ............................................................................................................. 34

The Standard Plate Count to Determine Bacterial Concentration ......................................................... 34

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Antibiotic Sensitivity Plate ...................................................................................................................... 35

Ouchterloney Double Diffusion Ig Plate ................................................................................................. 36

Rapid Bacterial ID Systems ...................................................................................................................... 37

Streptococcus Antibody Typing .............................................................................................................. 38

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Introduction to Lab

Lab 1 – Safety & Ubiquity

Objectives:

Safety – lab arrangement, rules, intro, safety video, emergency number (223.7999)

Media (broth, agar, plates)

Labeling, incubation and disposal

Ubiquity – handle plates, swabs, tubes, label, sample, disposal, incubate

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Lab 2 - Ubiquity (cont.), Morphology, Intro to Microscope, Begin Aseptic

Technique

Objectives:

Observe ubiquity plate for morphology, discuss factors affecting growth, normal flora

Learn to set up and use microscope (inter-pupillary distance, individual occur adjust, light),

observe and critical focus

Perform sterile transfers to broth, slant. Perform streak for isolation on plate

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Lab 3 – Aseptic Technique (cont.), Morphology (cont.), Isolation Streak,

Unknown 1

Objectives:

Observe streak plate for isolated colonies, troubleshoot

Observe slants, plates, broths for morphology (3 different orgs for each table)

Perform isolation streaks to prepare organism standards (Zoo) for morphology unknowns,

incubate 37C:

Staph. aureus Staph. epidermidis Strep. pyogenes

Mycobacterium

smegmatis

Bacillus subtilis Klebsiella

pneumonia

Escherichia coli Proteus mirabilis or

vulgaris

Pseudomonas

aruginosa

Choose and streak unknown #1 for isolation, label, write unknown number in notebook,

incubate in 37C.

Preserve original unknown #1 tube (labeled with name) in 4C fridge.

Perform example (practice/demo) streak for isolation using mixed culture (E.coli + Serratia

marcessans -- has a temperature sensitive enzyme that produces pink/orange pigment at 30C,

NOT at 37C). Place this mixed streak plate in 30C incubator.

Example Streaking Patterns:

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Lab 4 – Morphology (cont.), Staining Techniques, Intro to Oil Lens

Objectives:

Observe the plates prepared for Unknown #1 standards for morphology.

Gather the plates from all three tables for comparison. As a group, select the best

representative plate from the three choices to be preserved as the standard for that organism.

Observe Unknown plate for morphology, re-streak if not separated into colonies

Observe mixed practice streak plate, troubleshoot

Learn how to prepare smear and heat fix for staining

Perform a simple stain on unknown #1

Learn to use oil lense to look at your unknown

Discuss Gram’s stain, perform initial Gram’s stain on unknown.

Staph. aureus

(G+ coccus)

Staph. epidermidis

(G+ coccus)

Strep. pyogenes

(G+ coccus)

Mycobacterium

smegmatis

(G+ bacillus/rod)

Bacillus subtilis

(G+ bacillus/rod)

Klebsiella

pneumonia

(G- bacillus/rod)

Escherichia coli

(G- bacillus/rod)

Proteus vulgaris or

P. mirabilis

(G- bacillus/rod)

Pseudomonas

aruginosa

(G- bacillus/rod)

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Lab 5 - Special Stains: Negative/ Capsule, Gram’s

Objectives:

Understand and perform Gram’s stain

Understand how to report Gram stain results

Understand and perform combination capsule/negative stain

Gram’s Stain

Understand and perform Gram’s stain on Unknown (G+ = purple, G- = pink):

1. Smear Heat fix

2. Stain

i. Crystal Violet (#1) 30 – 45 secs, rinse H20

ii. Gram’s Iodine (mordant) (#2) ~1 min, rinse H20

iii. Gram’s Decolorizer Acid Alcohol (#3) ~15 secs, rinse H20

iv. Counter Stain Safranin (#4), rinse H20

3. Blot

4. View

Notes:

o Determine and report gram morphology of unknown, eliminate organisms from

potentials list based on gram morphology.

o Know the gram reactions of the nine morphological stand organisms.

o Understand how to report Gram stain results (gram rxn + shape)

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Combination Capsule/Negative Stain

Understand and perform combination capsule/negative stain:

1. Use a loop to mix a drop of water + a drop of congo red, or india ink, or Nigrosin

+ bacteria at end of slide

2. Use another slide to spread the smear like a blood smear

3. Air dry. DO NOT heat fix! (melts glycocalyx/ sugar coat capsule). OK to use slide

dryer. NO Rinse

4. Flood smear with crystal violet, 1 minute

5. GENTLE rinse H20, blot, dry, observe

Know which organism(s) yielded a positive capsule stain. Know significance of capsule.

Notes: Capsules present as clear halos against a dark filed with a stained bacteria in the

center.

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Lab 6 - Special Stains: Acid Fast, Endospore, Flagella

Objectives:

Understand and perform Acid Fast stain w/ Kinyoun method

Understand and perform Endospore stain

Recognize a flagella stain. Understand function and significance of flagella

Acid Fast Stain (Kinyoun Method)

Understand and perform Acid Fast stain w/ Kinyoun method

(looks for mycolic acid in cell wall):

1. Smear and heat fix

2. Carbolfuchsin 5 mins, rinse H20

3. Acid Alcohol decolorize, 2 mins, rinse H20

4. Brilliant Green (or Methylene Blue) counter stain, 1 min, rinse H20

5. Dry and observe

Notes: Acid fast cells stain red. Non-acid fast cells stain blue/purple

Know which organisms yielded at positive acid fast stain. What the acid fast stain tests for and its

significance.

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Endospore Stain

Understand and perform Endospore stain:

1. Smear and Heat fix

2. Place slide over steam bath

(do not allow bath to run dry! (take some initiative…)

3. Place a small piece of bibulous paper or paper towel over the smear

4. Saturate the paper with malachite green, 5mins

5. Remove bibulous paper with forceps and throw in trash!

6. H20 rinse

7. Counterstain with safranin, 2 minutes, H20 rinse

Notes: Endospores stain green. Parent cells stain red.

Know which organisms yielded at positive endospore stain. What the endospore stain tests for

and its significance.

Flagella Stain

(Demo and discussion only) Recognize a flagella stain. Understand function and significance of flagella.

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Begin Labs on Staphylococcus sp. and Streptococcus sp.

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Lab 7 – Begin Gram Positives (Staphs & Streps)

Objectives:

Demonstrate & understand Phenylethyl Alcohol Agar (PEA)

Demonstrate & understand Blood Agar:

Phenylethyl Alcohol Agar (PEA):

1. Streak example organisms for isolation on PEA

2. Incubate 37C

Notes:

o Inhibits G- (why?)

o Used to select for G+

Blood Agar:

1. Streak example organisms for isolation on blood agar

2. Incubate 37C.

Notes:

o Complex, enriched media, everything grows!

o Hemolytic patterns: alpha (), beta(), gamma() o Understand that many organisms can hemolyze blood, but hemolysis is used mainly to

differentiate the Streps.

Unknown #2 (Staphs & Streps):

1. Choose Unknown tube

2. Write your name on tube (replace in 4C when finished)

3. Write number in notebook and on plates, etc. (Record keeping!)

4. Streak Unknown for isolation on to Blood and TSA

5. Perform Gram stain

6. Incubate 37C

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Lab 8/9 – Staphs & Streps (cont)- MSA, Coagulase, Catalase

Objectives:

Demonstrate and understand the use of Mannitol Salt Agar (MSA)

Demonstrate and understand use of Coagulase test

Demonstrate and understand use of Catalase test

Mannitol Salt Agar (MSA)

1. Streak example organisms for isolation on MSA

2. Incubate

Notes:

o Salt inhibits (low to no growth) Streps, Staphs and Entercoccus faecalis tolerate

(grow)

o Has acid indicator. If mannitol (a sugar) is fermented, acid results. Turns media

from red to yellow (acid is formed).

o Read the media for two things: growth? Fermentation of mannitol.

(Note that Entercoccus faecalis is not a Staph but can tolerate the salt and grow,

but is Catalase negative)

o Know what the test is used for. What organisms give a positive result? What

organisms does it differentiate?

Coagulase test

1. Inoculate tubes of serum with test organisms

2. Incubate 37C, observe

Notes:

o This is an ancillary test, not required if MSA is employed, provides similar results.

o Tests for the enzyme Coagulase found in Staph. Aureus, NOT Staph. epidemidis

o Enzyme coagulates serum and makes it become a gel. To observe, tip tube sideways

and observe for presence or absence of serum flow.



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Catalase test

1. Place organism on clean slide

2. Add Hydrogen peroxide

3. Observe for bubbles

Notes:

o Tests for Catalase enzyme (breaks hydrogen peroxide into Oxygen and Water)

o Differentiates Staph from Strep.



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Lab 10 – Staphs & Streps (cont)- BEA, Bacitracin, Optochin

Objectives:

Demonstrate and observe use of BEA (Bile Esculin Agar) slants

Demonstrate and observe use of Bacitracin Susceptibility disk

Demonstrate and observe use of Optochin Susceptibility disk

BEA (Bile Esculin Agar) slants

1. Inoculate slant, incubate

2. Observe dark brown rxn is positive

Notes:

o Used to differentiate enterococci and Group D Streptococcus from non-group D

Streptococcus.

(Group D Streps are usually alpha or gamma hemolytic)

o Based on ability to hydrolyze esculin in the presence of bile

o Enterococcus faecalis and Strep. Bovis are positive.

Bacitracin Susceptibility disk

1. Streak ½ blood plate for confluence with each organism

2. Place a Bacitracin disk in the center of the area

3. Incubate and look for inhibition of growth

Notes:

o Differentiates Strep pyogenes (inhibited) from Strep agalactia (no inhibition)

Optochin Susceptibility disk

1. Streak ½ blood plate for confluence with each organism

2. Place a Optochin disk in the center of the area

3. Incubate and look for inhibition of growth

Notes:

o Differentiates Strep pneumoniae (inhibited) from Strep sanguis (no inhibition)

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Begin Enteric Bacteria (Enterobacteriaceae)

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Lab 11/12: Media for Enterics – EMB, HE, Mac

Objectives:

Demonstrate and observe use of EMB (Eosin Methylene Blue) media

Demonstrate and observe use of Mac (McConkey) media

Demonstrate and observe use of HE (Hektoen Enteric) media

EMB (Eosin Methylene Blue) Media

1. Streak for isolation on EMB

2. Incubate and observe

Notes:

o Pink colonies are Lac +

o Green sheen metal-flake coloration is diagnostic for fecal coliforms (E.coli)

o The Methylene Blue Inhibits G+

Mac (McConkey) Media

1. Streak for isolation on Mac


Notes:

o Pink colonies are Lac +

o Mac inhibits G+

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HE (Hektoen Enteric) Media

1. Streak for isolation on HE


Notes:

Technicolor (Red, Yellow, Orange colonies = NOT Salmonella or Shigella

Green/blue colonies w/o black centers is potential Shigella or Salmonella

Green/blue colonies w/ black centers is potential Salmonella, NOT Shigella,

The black colony centers in HE are the result of Hydrogen Sulfide generation. The

Reaction is gaseous Hydrogen Sulfide (H2S) combines with Fe2+ in the media to

make FeS (insoluble black ppt)

Slightly inhibits G+

General Notes for these media:

Useful in selecting G- over G+

Enterics are first separated into Lactose positive (Lac+) (fermentation) and Lactose negative

(Lac-) (fermentation) groups.

These media enable determining Lactose fermentation via color changes relating to pH and acid

formation.

Essential to help divide the organism into correct group to perform appropriate tests.

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Lab 13: Media for Enterics (cont)- PR Sugars, MR/VP, SIM

Objectives:

Demonstrate and observe use of Phenol Red sugar broths

Demonstrate and observe use of MR/VP media

Demonstrate and observe use of SIM (Sulfur Indole Motility) media

Phenol Red Sugar Broths

1. Inoculate PR sugars


o Yellow = + sugar use

MR/VP Media

(http://www.austincc.edu/microbugz/mrvp_test.php)

1. Inoculate 2 tubes MR/VP media per organism

2. Incubate, add reagents:

In one tube , add 15 drops MR+ = red

30 drops VP-a + 10 drops VP-b VP+ = red ring after 30 mins

SIM (Sulfur Indole Motility) Media

1. This media is a “deep”, (no slant in tube). Inoculate by stabbing down into media

2. Incubate, observe:

a. H2S production, black or no black, black indicates H2S (Sulfur), if black, cannot read

motility

b. If not black, read motility. Non-motile grows only in stab line

c. Layer 5 drops of Kovac’s reagent onto media – red = + for Indole production

Notes:

IMViC is an acronym for a specific series of tests for Lac+ organisms:

Indole Methyl-Red Voges- Proskauer Citrate

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Lab 14: Media for Enterics (cont.) – TSI, Citrate

Objectives:

Demonstrate and observe use of TSI (Triple Sugar Iron Agar)

Demonstrate and observe use of Simmons Citrate Agar

TSI (Triple Sugar Iron Agar)

(see microbugz for explanation)

1. Inoculate TSI with stab AND streak


Notes:

o Results are reported as slant rxn/butt rxn H2S rxn

Red = alkaline = K ; Yellow = acid = A

(example: R/Y + H2S, OR K/A +H2S)

o Y/Y w/o H2S means either contaminated (multiple organsims) or a lac + organism is

on the media…

Simmons Citrate Agar

(part of IMViC results)

1. Inoculate Simmons Citrate with streak on slant

2. Incubate and observe, blue = Citrate +

Notes: Ensure you use the correct tests for the appropriate type of organism. Spurious results will lead

you to crazy, incorrect identifications…

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Lab 15: Media for Enterics (cont.) – Urease, Ornithine Decarboxylase

Objectives:

Demonstrate and observe use of Urease broth

Demonstrate and observe use of Ornithine Decarboxylase Media

Urease Broth


1. Inoculate Urease broth

2. Incubate and observe.

Notes: bright pink= urease +

Ornithine Decarboxylase Media


1. Inoculate Ornithine Decarboxylase broth, overlay with mineral oil (NOT immersion oil!!)

2. Incubate and observe.

Notes:

o yellow reaction is Ornithine Decarboxylase negative(-)

o (more) purple reaction is Ornithine Decarboxylase positive(+)

End of Enteric Medias

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How Environmental Conditions Affect Growth:

How does Presence or Absence of Oxygen Affect Growth?

Prepare demonstration of different environments Oxygen concentrations) to compare growth of various

organisms:

1. Inoculate plates for the incubator, brewer jar and candle jar.

2. Inoculate thioglycolate tubes (close screwcap completely, then loosen ¼ turn).

3. Place all in 37C incubator.

4. Observe for relative growth.

Notes:

The environments are:

o ~[20%] O2 = Our regular incubator

o Low O2 (20%> O2 >0%) = Candle Jar

o ~[0%] O2 = Brewer Anaerobic Jar (catalyst removes Oxygen through molecular

binding)

o Multiple zones = Thioglycolate tube. Contains indicator that turns green in

presence of O2. . Bottom of tube is anaerobic.

Understand the ideas of strict anaerobe, strict aerobe, facultative anaerobe. Which of the test

organisms exhibit each trait?

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What are the Affects of Osmotic Pressure on Bacterial Growth

1. (per table) Inoculate a set of Tryptic Soy NaCl broth tubes (1, 3, 5, 7, 9, 11% NaCl)

2. Incubate, observe for growth or no growth. Compare the organisms.

Notes:

Physiologic [NaCL]= ~0.90%

Discuss osmotic pressure, osmosis, selective membranes, gradients

Think about why salt is used to cure foods etc.

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What are the Affects of Temperature on Growth

1. (per table) Inoculate a set of organisms on Tryptic Soy Agar plates

2. Label carefully for the temperatures 4F (fridge), 23F (room temp), 37F (incubator), 50F

(back incubator)

3. Incubate each set of plates in appropriate temperature location. Observe for relative

growth. Compare the organisms.

o psychrophiles vs. mesophiles vs thermophiles

o Growth rates and temperature, enzyme activity vs. temperature

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What are the Affects of UV Irradiation on Growth:

1. (per table) Inoculate a set of organisms on Tryptic Soy Agar plates

2. Label carefully for the exposure times:

0 secs,

30 secs,

1 min (60 secs),

2min (120 secs),

5 min (300 secs),

10 min ( 600 secs) .

3. Ensure UV lights are on, remove plate lids, expose for the appropriate time. Replace the

covers. Turn the lights off when completed.

4. Incubate each set of plates in 37F incubator. Observe for relative growth. Compare the

organisms.

Discuss radiation intensity, effects of distance and wave length

Discuss what damage UV creates, what about other forms of irradiation?

What part do spores play?

How is irradiation used in commercial applications?

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Eukaryotic Parasitology Additional study documents on blackboard and the website.

Organisms for Lab Exam 4 – Parasitology

Things to know:

Scientific Name and Common Name (if any)

How it is categorized (for example: Zygomycete or Cestode…)

Disease it causes (if any)

How is the organism contracted/passed (if applicable)

Where is this disease found in the world

Characteristic appearance

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Fungi and Molds

Things to know:

Fungal groups:

Ascomycetes

Basidiomycetes

Zygomycetes

Deuteromycetes (fungi imperfecti)

Basic fungal anatomy:

rhizoid (hyphae)

sporangiophore

sporangium

spore

mycelium (stolon)

Ascomycetes

Saccharomyces cervisiae

Candida albicans

Aspergillus niger

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Zygomycetes

Rhizopus stolonifera (zygomycete)

Basidiomycetes

Mushrooms, puffballs, etc.

Deuteromycetes

Penicillium sp. (deutero – looks like an ascomycete)

Protozoan

Amoeboid

Entamoeba histolytica (dysentery)

Ciliate

Balantidium coli (balantidiasis)

Flagellated

Giardia lamblia

Trichomonas vaginalis

Trypanosoma brucei

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Sporozoan

Plasmodium Toxoplasma gondii

Helminth

Trematode

Clonorchis sinensis (oriental liver fluke)

Paragonium westermani (lung)

Schistosoma mansoni

Cestode

Dipylidium caninum (dogs and cats... intestinal distress)

Echinococcus granulosus (carnivore, hydatid cyst)

Hymenolepsis nana (dwarf tapeworm)

Taenia saginata (beef)

Taenia solium (pork)

Nematode

Ascaris lumbricoides (intestinal round worm)

Enterobius vermicularis (pinworm)

Ancyclostoma duodenale (hook)

Necator americanus (hook)

Strongyloides stercoralis (skin lungs)

Wuchereria bancrofti (filiariasis)

Movie Stars

Organisms from the movie Eaten Alive (The Body Snatchers):

Acanthamoeba keratinitis (amoeba corneal infection)

Ascaris lumbricoides (intestinal round worm)

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Dermatobia hominis (Bot fly)

Entamoeba gingivalis (tooth amoeba)

Enterobius vermicularis (pinworm)

Hirudinia sp. (leech)

Pediculus humanus sp. (Human louse)

Taenia saginatta (beef tapeworm)

Taenia solium (pork tapeworm)

Vandellia cirrhosa (Candiru)

Wuchereria bancrofti

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Demonstration Experiments

The Standard Plate Count to Determine Bacterial Concentration

Prepare standard plate count to determine number of bacteria in a solution.

1. Prepare serial dilutions of bacteria. Use pipetter (9 ml blanks, 1ml solute)

2. Plate the dilutions (0.1ml into plate, massage in with glass hockey-stick) – DO NOT set

alcohol on fire!

3. Incubate

4. After incubation, count colonies and determine which plate has between 20-250

colonies. Ignore the rest.

5. Determine bacterial concentration of original sample.

Objectives

Understand:

serial dilutions (solute, solvent),

A= Solute, B= Solvent and dilution equals:

use of and importance of determining bacterial concentration.

speed of bacterial growth and the concept of logarithmic growth.

A

A+B

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Antibiotic Sensitivity Plate

Prepare and observe use of antibiotic sensitivity plate to test antibiotic sensitivity/resistance in bacteria.

1. Streak media plate for confluence, 1 bacterial genus per plate

2. Place antibiotic disks on media, ensure good contact to prevent loss of disks when inverting

plates.

3. Incubate plates and observe for relative zones of inhibition. Compare the organisms for

sensitivities/resistances.

Notes: In real life, you would likely be required to measure the zones to determine which is

most effective because various antibiotics travel different distances within the agar due to

molecular size and charge differences…

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Ouchterloney Double Diffusion Ig Plate

Prepare and observe Ouchterloney Double Diffusion Immunoglobulin Precipitation Plate. (look here for

more info: http://amrita.vlab.co.in/?sub=3&brch=70&sim=689&cnt=1)

1. Obtain plate (w/wells) and reagents.

2. Fill wells with reagents as directed (do not overfill or contaminate pipettes- it destroys the

reagents and ruins your results)

3. Incubate - right-side up!

4. Observe - look for precipitation bands caused by homologous antigen-antibody interaction

http://amrita.vlab.co.in/?sub=3&brch=70&sim=689&cnt=1

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Rapid Bacterial ID Systems

Prepare and observe one of the types of bacterial rapid tests.

This is one kind of API rapid test:

Here is the Enterotube II:

Some reactions, side by side:

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Streptococcus Antibody Typing

This demonstration uses specific monoclonal antibodies against surface antigens specific to each of the

recognized Streptococcus groups (A-G) to determine the antibody grouping of each Streptococcus group.

The test hinges on two principles:

Matching homologous antibody and antigen bind specifically and result in agglutination.

The commercial monoclonal antibody is bound via the constant region (FC) of the antibody to a

blue latex bead. This leaves the variable region of the antibody (Fab) available for binding with

the Streptococcus surface antigens. The blue beads enable agglutination to be more easily

detected.

The demonstration will show the reactions of Streptococcus pyogenes and Streptococcus agalactiae

against Anti Strep Group A and Anti Strep Group B reagents.

Materials

Toothpicks for mixing

Micropipette set to 100 microliters w tips.

Reaction card(s)

Live cultures of Streptococcus pyogenes and Streptococcus agalactiae

Typing Reagents, A & B

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Procedure

Use the following guide for the organism and reagent placement:

Streptococcus pyogenes + Anti A

Streptococcus pyogenes + Anti B

Streptococcus agalactiae + Anti A

Streptococcus agalactiae + Anti B

1. Place 100 microliters of the cultures in the appropriate well on each card. Use a new pipette tip

for each organism. Do not cross-contaminate your tests.

2. Gently shake the antibody reagents.

3. Add 1 drop of the appropriate reagent to the appropriate well on each card. Do not touch the

tip to the card. Allow the drop to fall from the bottle tip.

4. Mix each well with a new toothpick (do not contaminate any well with a “used” toothpick, it will

ruin the results.)

5. Observe the wells, watching for agglutination.

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Resources & Facts

Here is a link to a textbook discussion of human relevant ones if you have interest:

http://www.microbiologybook.org/fox/streptococci.htm

Quick Facts

(The names of ones we worked with in lab are highlighted.)

Know what organisms and issues Streptococcus groups A & B (the ones we typed in the lab) include:

Group A - Streptococcus pyogenes

https://www.cdc.gov/groupastrep/diseases-public/index.html

strep throat, impetigo, Rheumatic fever, Acute glomerulonephritis, Scarlet fever, bacteremia, toxic-

shock syndrome and necrotizing fasciitis

Group B - Streptococcus agalactiae

https://www.cdc.gov/groupbstrep/index.html

pneumonia and meningitis (neonates, elderly), occasional systemic bacteremia.

In pregnancy, colonize intestines and female reproductive tract, increasing risk of premature membrane

rupture during pregnancy, causing transmission to the fetus

Group C - a bunch of streps you don't know

Streptococcus equisimilis (animals)

Streptococcus equi (animals)

Streptococcus zooepidemicus (animals)

Streptococcus dysgalactiae (human, β-haemolytic streptococci that can cause pharyngitis and other

pyogenic infections similar to group A streptococci )

Group D - non-hemolytic or weakly beta. Rarely cause illness.

Streptococcus bovis

Streptococcus equinus

Group E - Enterococci, including:

Enterococcus faecalis

life-threatening infections in humans, endocarditis, septicemia, urinary tract infections, meningitis,

other infections- especially in nosocomial environment.

Frequently found in root canal-treated teeth (30% to 90% of cases) Root canal-treated teeth are ~9x

more likely to harbor E. faecalis than cases of primary infections.

Enterococcus faecium

Enterococcus durans

http://www.microbiologybook.org/fox/streptococci.htm

https://www.cdc.gov/groupastrep/diseases-public/index.html

https://www.cdc.gov/groupbstrep/index.html

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What to learn:

Importance of Streptococcus typing

How the test works (homologous ag-ab, agglutination, latex beads amplify visual)

What the results show and what diseases groups A & B are associated with.

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