biochemical characterization supplemental instruction designed by pyeongsug kim ©2010...
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Biochemical CharacterizationBiochemical CharacterizationSupplemental instruction
Designed by Pyeongsug Kim ©2010 [email protected]
Fall 2011Chattahoochee Technical College
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Selective mediaSelective media
-MSAMSA(Manitol Salt Agar)
:allows the growth of certain types of organisms, while inhibiting the growth of other organisms.
-EMBEMB(Eosin Methylene Blue)
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Differential mediaDifferential 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.
-MSAMSA(Manitol Salt Agar)
-EMBEMB(Eosin Methylene Blue)
-SIMSIM -MR-VPMR-VP
-Phenol Phenol red red brothsbroths
-BloodBlood agaragar
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Is TSA (Trypticase soy agar ) differential agar or selective agar?
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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!!
Differential Neither!!!Neither!!! (sorry!)
Mannitol Salt Agar (Mannitol Salt Agar (MSAMSA))-selective and differential-ability to tolerate 7% salt concentration and ferment mannitol. -Why SelectiveSelective? inhibit the growth of Gram negative bacteria (selective for Gram positive)-Why differentialdifferential? 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 (Mannitol Salt Agar (MSAMSA))
A:B:C:
Positive(mannitol fermentation)Inhibit growthNegative
-selective and differential
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EMBEMB(Eosin Methylene Blue)
-selective and differential-Why SelectiveSelective? inhibit the growth of Gram positive bacteria (selective for Gram negative)-Why differentialdifferential? differentiates coliforms (Lactose fermenters) Fermenters ~ Dark purple(or black) or Green Green sheensheen Non-fermeners ~ pinkpink or colorless
EMBEMB(Eosin Methylene Blue)
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AAA CB
A:B:C:
PositiveNegativePositive
Hemolytic reaction testHemolytic reaction test
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-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.”•AlphaAlpha hemolysis ~ partially break down hemoglobin; a greenish discoloration that surrounds a bacterial colony• BetaBeta hemolysis ~ complete lysiscomplete lysis of red blood cells and hemoglobin. Clearing of the blood around the colonies. •Gamma Gamma hemolysis ~ nono hemolysis
http://www.midlandstech.edu/science/kelleherk/225/labmaterials/sel_diff_media.html
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A B C
A:B:C:
Alpha hemolysisGamma hemolysisBeta hemolysis
Hemolytic reaction testHemolytic reaction test
Extracellular Amylase testExtracellular Amylase test
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-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/Black/BlueBlue ~; presence of starch; absence of enzyme (Negative) Light brown ~ absence of starch; presence of enzyme (Positive)
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Negative
Positive
•Indole test Indole production from the amino acid, tryptophan use Kovac’s agent Indole produced?
SIM TestSIM Test
•Hydrogen SSulfide production (from sodium thiosulfate)
Reduce sulfur? BlackBlack ~ reduced sulfur or H2S produced (positive). YellowYellow ~ Not H2S produced (negative)
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•Motility Does the bacterium have one or more flagella that propel the cell?
-Differential test (media)-Semisolid
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reduced sulfur(H2S produced) produce indole
Motile? No
++
Kovac’s reagent
SIM TestSIM Test
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SIM TestSIM Test
Which one contains bacteria that have flagella?
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
Right SIM tubeLeft SIM tube (sorry!)
Catalase TestCatalase 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 TestOxidase Test-Differential test-used to identify to determine if a bacterium produces certain cytochrome c oxidases. can therefore utilize oxygen for energyutilize oxygen for energy production with an electron transfer chain. -Aerobes, facultative aerobes, microphilles-Turn blue-purpleblue-purple
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-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.
Phenol red(PR) broth Phenol red(PR) broth
A/GFermentation with acid and gas production
-/-No Fementation
A/-Fermentation with acid but no gas production
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a)
b)
c)
d)
Red ~ No Fementation
Pink ~ No Fementation but degradation of peptone(protein)
~ Fementation and no gas production
~ Fementation and gas production
Yellow/No gas
Yellow/ Gas
Phenol red(PR) broth Phenol red(PR) broth
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Methyl Red/Voges Proskauer (MR/VP)Methyl Red/Voges Proskauer (MR/VP)
•MR testMR test-tests for mixed acid end products from glucose fermentation.-use methyl red. RedRed ~ mixed-acid produced (positive)YellowYellow ~ no acid produced (negative)
+
-used to determine which fermentation pathway is used to utilize glucose. -MR-VP broth
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•VP testVP test -test for acetoin production from glucose fermentation. - use reagent A & reagent B - Red Red ~ acetoin produced (positive) YellowYellow to brownbrown ~ No acetoin produced (negative)
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RedPositive!MR(+)
RedPositive!VP(+)
Methyl Red/Voges Proskauer (MR/VP)Methyl Red/Voges Proskauer (MR/VP)
3 drops of methyl re
d.
A mixed-acid fermentation
Not rednegativeMR(-)15 drops of VP reagent A
3 drops of VP reagent Bacetoin production
After 10-60min. Not rednegativeVP(-)