1. Biochemical Characterization
Supplemental instruction
Fall 2011
Chattahoochee Technical College
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Selective media
:allows the growth of certain types of organisms, while inhibiting the growth of other organisms.
-MSA(Manitol Salt Agar)
-EMB(Eosin Methylene Blue)
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Differential media
:allows one to distinguish between or among different microorganisms based on a difference in colony appearance (color, shape, or growth pattern) on the medium.
-MR-VP
-MSA(Manitol Salt Agar)
-SIM
-Phenol red broths
-EMB(Eosin Methylene Blue)
-Blood agar
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Is TSA (Trypticase soy agar ) differential agar or selective agar?
Differential
Neither!!! (sorry!)
TSA and NA media are not differential or selective growth medium.
Both Gram-positive and Gram-negative bacteria grow on this medium. Hence,
the purpose of this medium is to select bacteria based upon differences in colony morphology.
I said wrongfully on Thur.(11/3/11) …Sorry!!
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Mannitol Salt Agar (MSA)
-selective and differential
-ability to tolerate 7% salt concentration and ferment mannitol.
-Why Selective?
inhibit the growth of Gram negative bacteria (selective for Gram positive)
-Why differential?
differentiates the salt tolerant organisms on their ability to ferment mannitol.
Growth with no mannitol fermentation.
Growth with mannitol fermentation.
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Mannitol Salt Agar (MSA)
-selective and differential A: B: C:
Positive(mannitol fermentation)
Inhibit growth
Negative
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EMB(Eosin Methylene Blue)
-selective and differential
-Why Selective?
inhibit the growth of Gram positive bacteria (selective for Gram negative)
-Why differential?
differentiates coliforms (Lactose fermenters)
Fermenters ~ Dark purple(or black) or Green sheen
Non-fermeners ~ pink or colorless
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EMB(Eosin Methylene Blue) A C B A: B: C:
Positive
Negative
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Hemolytic reaction test
-differential test: test for hemolytic reactions
 differential for the ability of hemolysis.
-used Blood agar
-“alpha”, “beta”, “gamma” hemolysis Don’t say “positive” or “negative.”
Alpha hemolysis ~ partially break down hemoglobin; a greenish discoloration that surrounds a bacterial colony
Beta hemolysis ~ complete lysis of red blood cells and hemoglobin. Clearing of the blood around the colonies.
Gamma hemolysis ~ no hemolysis
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Hemolytic reaction test A B C A: B: C:
Alpha hemolysis
Gamma hemolysis
Beta hemolysis
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Extracellular Amylase test
-Differential test
 differential for the ability to produce extra-cellular amylase to digest starch.
-used starch agar
-test with gram-iodine (used mordant in Gram stainning)
 Iodine is used to test the presence of starch.
Black/Blue ~; presence of starch; absence of enzyme (Negative)
Light brown ~ absence of starch; presence of enzyme (Positive)
Negative
Positive
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SIM Test
-Differential test (media)
-Semisolid
Hydrogen Sulfide production (from sodium thiosulfate)
 Reduce sulfur?
Black ~ reduced sulfur or H2S produced (positive).
Yellow ~ Not H2S produced (negative)
Indole test
Indole production from the amino acid, tryptophan
use Kovac’s agent
 Indole produced?
Motility
Does the bacterium have one or more flagella that propel the cell?
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SIM Test
Kovac’s reagent + +
reduced sulfur(H2S produced)
produce indole
Motile? No
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SIM Test
Which one contains bacteria that have flagella?
Right SIM tube
Left SIM tube (sorry!)
What type of test?
What is the purpose of this test?
Which one is positive?
Indole test
Determine if indole is produced.
Left SIM tube
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Catalase Test -Differential test
-used to identify organisms that produce the catalase(enzyme).
Catalase ~ detoxifies hydrogen peroxide by breaking it down into water and oxygen gas (bubble!).
Bubble! +
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Oxidase Test -Differential test
-used to identify to determine if a bacterium produces certain cytochrome c oxidases.
 can therefore utilize oxygen for energy production with an electron transfer chain.
-Aerobes, facultative aerobes, microphilles
-Turn blue-purple
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Phenol red(PR) broth
Differential test
Contain pH indicator phenol red.
-Detect fermentation of carbohydrate depending on the type of carbohydrates.
PR glucose broth, PR mannitol broth, PR lactose broth, PR sucrose broth or other carbohydrates.
A/G
Fermentation with acid and gas production
A/-
Fermentation with acid but no gas production
-/-
No Fementation
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Phenol red(PR) broth a) b) c) d)
Red ~ No Fementation
Pink ~ No Fementation but degradation of peptone(protein)
Yellow/No gas
~ Fementation and no gas production
Yellow/ Gas
~ Fementation and gas production
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Methyl Red/Voges Proskauer (MR/VP)
-used to determine which fermentation pathway is used to utilize glucose.
-MR-VP broth +
MR test
-tests for mixed acid end products from glucose fermentation.
-use methyl red.
Red ~ mixed-acid produced (positive)
Yellow ~ no acid produced (negative)
VP test
-test for acetoin production from glucose fermentation.
- use reagent A & reagent B
- Red ~ acetoin produced (positive)
Yellow to brown ~ No acetoin produced (negative)
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Methyl Red/Voges Proskauer (MR/VP)
Red
Positive!
MR(+)
A mixed-acid fermentation
3 drops of methyl red.
Not red
negative
MR(-)
15 drops of VP reagent A
3 drops of VP reagent B
Red
Positive!
VP(+)
acetoin production
After 10-60min.
Not red
negative
VP(-)
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