Biochemical tests

To identify bacteria, we must rely heavily on biochemical testing. The types of

biochemical reactions each organism undergoes act as a "thumbprint" for its
identification.

Purposes of biochemical tests

1. Test for metabolism of carbohydrates and related products.

 Sugar fermentation test
 O/F test
2. Test for specific break down products
 Indole test
 MR and VP tests
3. Test to show ability to utilize a specific substance
 Citrate (Simon citrate medium)
4. Test for enzymes
Catalase, oxidase, urease ….
5. Test for metabolism of protein and amino acids

Fermentation of carbohydrates

A wide variety of carbohydrates may be fermented by various bacteria in order

to obtain energy and the types of carbohydrates which are fermented by a
specific organism can serve as a diagnostic tool for the identification of that
organism.

Procedure: incubate tubes of media containing:

1. A single carbohydrate (such as lactose or glucose)
2. pH indicator (such as phenol red)
3. Durham tube (a small inverted tube to detect gas production).

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 If the particular carbohydrate is fermented by the bacterium, acid end
products will be produced which lowers the pH, causing the pH indicator to
change color (phenol red turns yellow) .If gas is produced along with the acid,
it collects in the Durham tube as a gas bubble.

Oxidation Fermentation Test

Principle

To determine the oxidative or fermentative metabolism of a carbohydrate

or its non-utilization.

Fermentation is an anaerobic process and bacterial fermenters of

carbohydrates are usually facultative anaerobes.

Oxidation is an aerobic process and bacterial oxidizers’ are usually strict

aerobes.

Hugh and Leifson medium is a semi-solid medium in tubes containing

glucose and a pH indicator bromothymol blue which turn to yellow in acids
production and turn to blue in alkalinity.

Procedure

Two tubes are inoculated by stabbing and one is immediately sealed with
oil to produce anaerobic conditions

1. Organisms that cannot break down the carbohydrate aerobically or

anaerobically eg Alcaligenes feacalis produce an alkaline reaction in
the open tube and no change in the covered tube
2. Oxidizing organisms eg Pseudomonas spp produce an acid reaction in
the open tube only
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 3. Fermenting organisms eg Enterobacteriaceae produce an acid reaction
throughout the medium in both tubes

IMViC tests
IMViC: it’s a group of tests used mainly to identify Enterobacteriaceae
members which include:

1. Indole test
2. Methyl red test
3. Voges-Proskauer test
4. Citrate test

1. Indole test

Indole is a component of the amino acid tryptophan. Some bacteria have the
ability to break down tryptophan for nutritional needs using the enzyme
tryptophanase. When tryptophan is broken down, the presence of indole can
be detected through the use of Kovacs' reagent. Kovac's reagent, which is
yellow, reacts with indole and produces a red color on the surface of the test
tube.

Results: Indole-Positive reaction: red color ex. E.coli; Negative reaction: yellow
color ex. Klebsiella

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MR-VP test
MR test:

Principle to test the ability of the organism to produce acid end product from
glucose fermentation, this is a qualitative test for acid production.

VP test:

To determine the ability of the organisms to produce neutral end product

(acetoin) from glucose fermentation.

procedure
1. Inoculate the tested organism into 2 tubes of MR-VP broth
2. Incubate the tubes at 37°C for 24 hours
3. AFTER INCUBATION: Run the MR test in the tube 1, and the VP test
in tube 2.
– For methyl red: Add 6-8 drops of methyl red reagent.
– For Voges-Proskauer: Add 12 drops of Barritt's A (-naphthol), mix,
4 drops of Barritt's B (40% KOH), mix
– Let sit, for at least 1hour

Results
MR results:

Red: Positive MR (E. coli); Yellow: Negative MR (Klebsiella)

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Voges-Proskauer results

Pink: Positive VP (Klebsiella), yellow: Negative VP (E. coli)

Citrate Utilization Test:

Simmons Citrate agar is a defined medium containing sodium citrate as the

sole carbon source. The pH indicator, bromthymol blue, will turn from green
at neutral pH (6.9) to blue when a pH higher than 7.6 is reached (alkaline). If
the citrate is utilized, the resulting growth will produce alkaline products
changing the color of the medium from green to blue. (Blue color= positive
reaction eg; Klebsiella) ;( green color=negative reaction eg; E.coli)

Hydrogen sulfide (H2S) test:

SIM medium: it’s a medium used to detect three tests

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S for H2S production, I for indole and M for motility

In SIM medium some bacteria are capable of breaking down amino acids
containing sulfur (cystine, methionine) to produce hydrogen sulfide (H2S).

Positive for H2S production: Proteus and Salmonella.

To test for hydrogen sulfide production, a medium with a sulfur source and
iron salts, inoculated and incubated, if the sulfur is reduced and hydrogen
sulfide is produced, it will combine with the iron salt to form a visible black
ferric sulfide (FeS) in the tube.

Types of Culture Media

• Simple (basal, ordinary) Culture Media: media that contain the basic
nutrients (growth factors) that support the growth of bacteria without
special nutrients, and they are used as basis of enriched media. E.g.
Nutrient broth, nutrient agar, peptone water. They are for the growth of
non-fastidious organisms like E.coli.
• Enriched Culture Media: media that are enriched with: Whole blood e.g.
blood agar. Lysed blood (heated to 80C) e.g. Chocolate agar.

• Selective Media: it is a media, which contains inhibitor substances that

prevent the growth of microorganisms other than the bacteria for which
the media is prepared for. For example EMB (Eosin Methylene blue):
enteric isolation media.
• Differential Media (indicators): Contains indicators, dyes, etc, to
differentiate microorganisms. E.g. MacConkey agar, which contains neutral
red (pH indicator) and is used to differentiate lactose fermenter (E. coli) and
non-lactose fermenter (Salmonella).

MacConkey agar

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MacConkey agar is both selective and differential medium. It contains bile
salts and the dye crystal violet, which inhibit the growth of gram-positive
bacteria and select for gram-negative bacteria. It also contains the
carbohydrate lactose, which allows differentiation of gram-negative
bacteria based on their ability to ferment lactose.

Organisms which ferment lactose produce acid end-products which react

with the pH indicator neutral red, and produce a pink color. Non-lactose
fermenting bacteria will be pale colorless colonies.

Eosin Methylene Blue

Eosin Methylene Blue is a slightly selective stain for Gram-negative bacteria

provides a color indicator distinguishing between organisms that ferment
lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella),and
inhibits the growth of Gram-positive bacteria. It is contain of two stains,
eosin Y and methylene common application of this stain.

Lactose fermentation produces acids, which lower the pH. This encourages
dye absorption by the colonies, which are now colored purple-black.

On EMB if E. coli is grown it will give a distinctive metallic green sheen

Lactose non-fermenters may increase the pH by deamination of proteins.

This ensures that the dye is not absorbed. The colonies will be colorless.

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