Biochemical Tests

Objectives
Tests to know
       

Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Mannitol fermentation Lactose fermentation

Indole Production Test

The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole, pyruvic acid and ammonia as end products. Tryptophanase differentiates indole-positive indoleenterics, such as E. coli and P.vulgaris from indole-negative indoleenterics, such as S. marcescens.  Media and Reagents: SIM with tryptophan and Kovacs reagent.  Method: Inoculate medium and incubate at 37C for 24-48 37 24hours. After incubation, add five drops of Kovacs reagent to the surface. Do not stir or shake the tube.  Expected Results:

 

Positive test : Kovacs reagent combines with indole and turns the surface red. Negative test: No red color development

Results for indole test

Methyl Red Test



 

Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive. If they use methyl-redbutylene glycol pathway and produce neutral end products, then they are called methyl-red-negative. methyl-redMedia and reagents: MR-VP medium and methyl red indicator MRMethod: Inoculate broth and incubate at 37C for 2-5 days. After 37 2incubation, transfer 2.5 ml of inoculate to another tube and add five drops of methyl red. Roll between the palms of hands to disperse methyl red. Expected results:
 

Positive test: acids + methyl red = red solution Negative test: neutral end products + methyl red = yellow color

Results for Methyl Red Test

Negative

Positive

Voges Proskauer Test

It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics that produce neutral endend-products, such as acetoin are detected by VP test.  Media and Reagent: MR-VP medium and Barritts Reagent MRA (contains alpha-naphthol) and Barritts Reagent B alpha(contains KOH).  Method: Inoculate medium and incubate at 37C for 48 37 hours. After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritts Reagent A and two drops of Barritts Reagent B. Gently mix and let it sit for 10-15 minutes to allow time for color development. 10 Expected results:

 

Positive test: acetoin + alpha-naphthol + KOH = red color alphaNegative test: alpha-naphthol +KOH = copper color alpha-

Results for VP test

Citrate Utilization
Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli  Media and Reagent: Simmons Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue  Method: Inoculate the slant and incubate at 37C for 24-48 37 24hours.  Expected results:

 

Positive test: Growth and color changes to blue Negative test: No growth and color remains green

Results for Citrate Test

Positive

Negative

H2S Production


  

Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product. endMedia and Reagent: SIM with cysteine and ferrous sulfate (detects H2S) Method: Inoculate the media and incubate at 37C 37 for 24-48 hours. 24Expected Results:
 

Positive Test: H2S production = Black Negative Test: No H2S production = No blackening of medium

Results of H2S production

Negative

Positive

Urea Utilization


  

Some bacteria produce urease, an enzyme capable of breaking down urea and produce alkaline end products. This distinguishes Proteus from other bacteria Media and Reagent: Urea Broth with phenol red Method: Inoculate the media with a loop and incubate at 37C for 24 hours. 37 Expected Results:
 

Positive test: production of alkaline end products = pinkish red color Negative test: No color change

Results for Urea Test

Positive

Negative

Motility Test


  

This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella. Media and reagent: Deep agar Method: Inoculate deep with a needle and incubate at 37C for 24-48 hours. 37 24Expected results:


Positive test: Growth spread away from the line of inoculation = motile Negative test: Growth only occurred at the line of inoculation = Non-motile Non-

Results for Motility Test

Mannitol Fermentation
Mannitol Salt Agar contains 7.5% NaCl, which is inhibitory to most bacteria. Bacteria that can grow on this agar can be differentiated based on mannitol fermentation. Fermentation of mannitol results in acidic products which turn phenol red pH indicator from red to yellow.  Media and reagent: MSA and phenol red indicator  Method: Streak MSA plate and incubate at 37C for 2 days. 37  Expected results:

 

Positive test: Mannitol fermentation occurred = growth and color changed to yellow Negative test: No mannitol fermentation = may or may not grow and no color change

Results for mannitol fermentation

No Growth

Negative

Positive

Lactose Fermentation
MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects GramGramGramnegative bacteria, such as E. Coli. It also differentiates lactoselactose-fermenting bacteria, such as E. Coli from nonnonlactose fermenting bacteria.  Media and Reagent: MacConkey Agar and neutral red dye  Method: Streak MAC plate and incubate at 37C for 2 37 days.  Expected results:

 

Positive test: Lactose fermentation = Growth and color change to pink Negative test: No lactose fermentation = May or may not grow and no color change

Results of Lactose Fermentation

Yeah!