Biochemical tests for characterization of bacteria

(I) Indole test: this test is performed on bacterial species to determine the ability of the organism
to convert tryptophan into the indole. This division is performed by a chain of a number of
different intracellular enzymes, a system generally referred to as "tryptophanase."
Indole is generated by reductive deamination from tryptophan via the intermediate molecule
indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-
NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole,
pyruvic acid, ammonium and energy. Pyridoxal phosphate is required as a coenzyme.

Method: Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24–48
hours before performing the test. Following incubation, add 5 drops of Kovac’s reagent (isoamyl
alcohol, para-Dimethylaminobenzaldehyde, concentrated HCl) to the culture broth.
The para-Dimethylaminobenzaldehyde reacts with indole present in the medium to form a red
rosindole dye. The isoamyl alcohol forms a complex with rosindole dye, which causes it
to precipitate. The remaining alcohol and the precipitate then rise to the surface of the medium.
A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer
of the broth. A negative result appears yellow.

Indole-Positive Bacteria: Bacillus alvei, Escherichia coli, Flavobacterium sp., Haemophilus

influenzae, Klebsiella oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Enterococcus
faecalis, and Vibrio sp.
Indole-Negative Bacteria: most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp.,
most Haemophilus sp., most Klebsiella sp., Neisseria
sp., Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp.

(II) Methyl-red test (MR test): MR test is used to identify bacteria producing stable acids by
mechanisms of mixed acid fermentation of glucose. MR test is used to identify enteric bacteria
based on their pattern of glucose metabolism. All enterics initially produce pyruvic acid from
glucose metabolism. Some enterics subsequently use the mixed acid pathway to metabolize
pyruvic acid to other acids, such as lactic, acetic, and formic acids. These bacteria are called
methyl-red positive and include Escherichia coli and Proteus vulgaris. Other enterics
subsequently use the butylene glycolpathway to metabolize pyruvic acid to neutral end products.
These bacteria are called methyl-red-negative and include Serratia
marcescens and Enterobacter.
Method: MR-broth containing glucose is prepared. An isolate is inoculated into a tube with a
sterile transfer loop. The tube is incubated at 35 °C for 2–5 days. After incubation, 2.5 ml of the
medium are transferred to another tube. Five drops of the pH indicator methyl red is added to this
tube. The tube is gently rolled between the palms to disperse the methyl red.

Enterics that subsequently metabolize pyruvic acid to other acids lower the pH of the medium to
4.2. At this pH, methyl red turns red, a positive test. Enterics that subsequently metabolize
pyruvic acid to neutral end products lower the pH of the medium to only 6.0. At this pH, methyl
red is yellow, a negative test.

(III) Voges-Proskauer Test (VP test): The VP test shows if the bacterium has butanediol
fermentation and can split glucose to acetoin via pyruvat and further to 2,3-butanediol according
to:

2 pyruvate + NADH --> 2CO2 + 2,3-butanediol.

If KOH (potassium hydroxide) is added, acetoin will be converted to diacetyl (= 2,3-

butanedione), which reacts with alpha-naphtol and forms a pink complex.

Method: Suspend one colony from the pure culture, which is to be investigated, in VP/MR
medium (containing glucose). Incubate at 30-37ºC during 24-48 h.

Add 0.2 ml of 40% KOH and then 0.6 ml of alpha-naphtol solution.

Positive test result: color change to pink. Negative test result: no color change.

Klebsiella spp. and Enterobacter spp. are VP positive

Escherichia coli, Salmonella spp. and Shigella spp are VP negative.

(IV) Citrate Utilization test: Some bacteria can utilize citrate as the only carbon source and the
citrate test is positive if the bacterium has this capacity to grow in such medium. It is often used
to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing
citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic
acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it
reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The
alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result
(the tube on the right is citrate positive). Klebsiella pneumonia andProteus mirabilis are
examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate
negative.
Method: Inoculate a tube containing citrate medium with a small amount of bacteria. It is
also possible to streak or perform a deep inoculation into Simmons citrate tube. Simmons
citrate medium contains sodium citrate and an indicator. Incubate at 30-37ºC during 24-48 h.

Positive test result: growth in citrate medium or growth with colour change to blue in
Simmon's citrate tube.

Negative test result: no growth in citrate medium or growth growth but no colour change
(still green colour) in Simmon's citrate tube.