Biochemical Tests

Objectives

Tests to know Lactose fermentation

Indole

Motility

H2S production in SIM

Methyl Red/Voges Proskauer

Citrate

Oxidase test

Lactose Fermentation

MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as E. Coli. It also differentiates lactose-fermenting bacteria, such as E. Coli from non-lactose fermenting bacteria.

Media and Reagent: MacConkey Agar and neutral red dye

Method: Streak MAC plate and incubate at 37°C for 2 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

Enteric Gram-negative rods

Lactose-fermenters (LFs) Escherichia coli

Klebsiella pneumoniae

Non-lactose fermenters (NLFs) Salmonella enterica

Shigella spp.

Proteus mirabilis

Pseudomonas aeruginosa

Pink on Macconkey Agar

Pale on Macconkey Agar

Results of 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 enterics, such as E. coli and P.vulgaris from indole-negative enterics, such as S. marcescens.

Media and Reagents: SIM with tryptophan and Kovac’s reagent.

Method: Inoculate medium and incubate at 37°C for 24-48 hours. After incubation, add five drops of Kovac’s reagent to the surface. Do not stir or shake the tube.

Expected Results: Positive test : Kovac’s reagent combines with indole and turns the

surface red.

Negative test: No red color development

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 37°C for 24-48 hours.

Expected results: Positive test: Growth spread away from the line of

inoculation = motile

Negative test: Growth only occurred at the line of inoculation = Non-motile

Results for Motility Test

SIM Medium (Sulfur Reduction Test, Indole Production, Motility)

Indole Production Purpose: The indole test

is used to identify bacteria capable of producing indole using the enzyme

tryptophanase.

Kovac’s Reagent

Results of H2S production

Positive Negative

Sulfur Reduction Purpose: This test is used

to identify those bacteria

capable of reducing

sulfur.

Motile Positive for H2S production

. SIM Tube Results

A. Indole positive and

hydrogen sulfide

positive

B. Hydrogen sulfide

positive

C. Indole positive and

motility positive (note

fuzzy growth away

from stab line)

D. Negative Control

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 butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative.

Media and reagents: MR-VP medium and methyl red indicator

Method: Inoculate broth and incubate at 37°C for 2-5 days. After

incubation, 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

Positive Negative

Methyl Red (MR) Test

Purpose: Used

to identify

bacteria that

produce stable

acid end

products by

means of mixed

acid

fermentation of

glucose

Voges Proskauer Test It is used to identify enteric bacteria based on their pattern

of glucose metabolism. The enterics that produce neutral end-products, such as acetoin are detected by VP test.

Media and Reagent: MR-VP medium and Barritt’s Reagent A (contains alpha-naphthol) and Barritt’s Reagent B (contains KOH).

Method: Inoculate medium and incubate at 37°C for 48 hours. After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritt’s Reagent A and two drops of Barritt’s Reagent B. Gently mix and let it sit for 10-15 minutes to allow time for color development.

Expected results:

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

Negative test: alpha-naphthol +KOH = copper color

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: Simmon’s Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue

Method: Inoculate the slant and incubate at 37°C for 24-48 hours.

Expected results: Positive test: Growth and color changes to blue

Negative test: No growth and color remains green

SIMMONS CITRATE

Purpose: The

citrate utilization

test is used to

determine the

ability of an

organism, using

the enzyme

citrase, to use

citrate as its sole

carbon source

تعتبر نتيجة االختبار موجبة عند تحول لون البيئة من

االخضر الى االزرق بعد انتهاء فترة التحضين

Escherichia coli دائما سالبة الختبار استغالل السترات

Enterobacter aerogenes دائما موجبة الختبار استغالل

السترات

IMViC Reactions

I M Vi C

Escherichia coli + + – –

Proteus vulgaris + + – –

Klebsiella pneumoniae – – + +

Enterobacter spp. – – + +

Serratia marcescens – – + +

Citrobacter freundii – + – +

Citrobacter koseri + + – +

وسط كليغلر

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae. Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. In the picture below the organism on the right (Pseudomonas aeruginosa) is oxidase positive. TOP

Oxidase Test

Oxidase test

Oxidase positive Oxidase negative

Campylobacter spp. Bordatella parapertussis

Brucella spp.

Kingella kingae Acinetobacter spp.

Moraxella spp. Enterobacteriaceae

Neisseria spp.

Pseudomonas spp.