Download Lab Notes for Bio 2420

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.
_________________
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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
2|Page
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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
4|Page
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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
5|Page
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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
6|Page
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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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8|Page
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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)
10 | P a g e
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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.
12 | P a g e
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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.
13 | P a g e
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Begin Labs on Staphylococcus sp. and Streptococcus sp.
14 | P a g e
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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
o
o
Complex, enriched media, everything grows!
Hemolytic patterns: alpha (), beta(), gamma()
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
15 | P a g e
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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
o
o
o
Salt inhibits (low to no growth) Streps, Staphs and Entercoccus faecalis tolerate
(grow)
Has acid indicator. If mannitol (a sugar) is fermented, acid results. Turns media
from red to yellow (acid is formed).
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)
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
o
o
o
This is an ancillary test, not required if MSA is employed, provides similar results.
Tests for the enzyme Coagulase found in Staph. Aureus, NOT Staph. epidemidis
Enzyme coagulates serum and makes it become a gel. To observe, tip tube sideways
and observe for presence or absence of serum flow.
Know what the test is used for. What organisms give a positive result? What
organisms does it differentiate?
16 | P a g e
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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.
o
Know what the test is used for. What organisms give a positive result? What
organisms does it differentiate?
17 | P a g e
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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)
18 | P a g e
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Begin Enteric Bacteria (Enterobacteriaceae)
19 | P a g e
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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
2. Incubate and observe
Notes:
o
Pink colonies are Lac +
o
Mac inhibits G+
20 | P a g e
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HE (Hektoen Enteric) Media
1. Streak for isolation on HE
2. Incubate and observe
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.
21 | P a g e
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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
2. Incubate and observe
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
22 | P a g e
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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
2. Incubate and observe
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…
23 | P a g e
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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
(see microbugz for explanation)
1. Inoculate Urease broth
2. Incubate and observe.
Notes: bright pink= urease +
Ornithine Decarboxylase Media
(see microbugz for explanation)
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
24 | P a g e
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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?
25 | P a g e
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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.
26 | P a g e
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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
27 | P a g e
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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?
28 | P a g e
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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
29 | P a g e
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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
30 | P a g e
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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
31 | P a g e
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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
A
Understand:

serial dilutions (solute, solvent),
A+B
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.
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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
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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, toxicshock 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
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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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