Biochemical test

Biochemical test

IMVIC test:

It refers to indole, methyl red, Voges-Proskauer and citrate utilization tests. IMVIC are the 4 biochemical reactions which are used to differentiate the number of Enterobacteriaceae family. The pattern of these biochemical reactions is the different organism and hence they can be distinguished from each other.

• Indole test:

The basic principle of this test is to determine the ability of an organism to split the amino acid “tryptophan” into indole.This test is one of the important biochemical tests to differentiate between general and even between species of the bacteria, particularly of the Enterobacteriaceae group. Tryptophan is an amino acid that can be oxidized by certain bacteria to form three major indolic metabolites; indole,skatole, and indoleacetic acid.Various intracellular enzymes involved are collectively called “tryptophanase”that mediate the production of indole.

The major intermediate in tryptophan degradation is indole pyruvic acid from which indole can be formed through deamination and skatole through decarboxylation of indoleacetic acid. The enzyme “Tryptophanase” catalyse the deamination reaction,attacking the tryptophan molecule only in its side chain and leaving the aromatic ring intact in the form of indole. The degradation of tryptophan releases indole,pyruvic acid, ammonia, and energy. The pyruvic acid can be further metabolized either by way of the glycolytic cycle or can enter the krab’s cycle to release CO₂,H₂O and a large yield of energy. The NH₃ can be used to synthesize new amino acids by utilizing the energy present for the anabolic reaction.

The indole,split from the tryptophan molecule,can be detected by a regent which involves a chemical combination producing a distinct colour. The presence or absence of indole formation is used for bacteria identification.Indole, if present,combines with the aldehyde present in either Kovacs reagent to give a red colour in the alcohol layer. The color reaction is based on the presence of the pyrrole structure present in indole. The alcoholic layer extracts and concentrates the red colour complex. The characteristics of some of the bacteria towards indole test are:

E.coli(usually +),Klebsiella-Enterobacter(usually-)Bacillus alvei(+),Proteus mirabilis(-)

• Methyl red

This test is used to detect the production of sufficient acid (PH below 4.2) by certain microorganisms. Bacteria which carry out mixed acid fermentation produce sufficient acid by fermentation of glucose and changes the colour of methyl red indicator to the red.At the host,test organism is inoculated into the MR-VP broth or glucose-phosphate broth and incubated for 24 hours. First of all, bacteria utilize glucose and produce pyruvic acid. However,this slightly acidity of pyruvic acid is neutralized bu phosphate buffer. When culture is incubated further, the carry organisms carries out mixed acid fermentation produce sufficient acid bringing the ph of the solution below 4.2. When methyl red indicator is added in such culture, c a colour of methyl red changes to red. It is positive MR test. In the case of negative test colour of solution does not change after addition of methyl red indicator.

• Voges-Proskauer test

The basic principle of this test is to determine the ability of some organisms to produce a neutral end product, acetylmethylcarbinol (acetoin CH₃.CO.CHOH.CH₃) from glucose fermentation. Glucose is metabolized to pyruvic acid which is the key intermediate in glycolysis.From pyruvic acid, an organism may follow many pathways. The production of acetone is one pathway for glucose degradation in bacteria. The Voges-Proskauer test for acetone is used primarily to differentiate E.coli from the Klebsiella-Enterobacter groups although other members of the Enterobacteriaceae are capable of producing a positive VP. The bacteria of Enterobacteriaceae are characteristically classified as mixed acid or formic acid fermenters indicating that their end products of glucose fermentation are acidic: formic acid,acetic acid, succinic acid, ethanol,hydrogen, and carbon dioxide. These mixed acid fermenters can be further divided into two groups. The major end product of pyruvate utilization by the Klebsiella-Enterobacter groups,Serratia,Bacillus and many other microorganisms, is 1,2-butanediol.However, the VP test is based on the detection of acetoin, a precursor to 2,3-btaneidol production.One molecule of acetoin is formed by the decarboxylation of two molecules of pyruvic acid. Acetoin is an intermediate stage in the conversion to 2,3-butanediol. Both acetoin and 2,3-butanediole are neutral products from glucose fermentation.

2Pyruvate→ acetoin +2CO₂

In the presence of atmospheric oxygen and alkali,the neutral end products”acetoin and 2,3-butanediol”are oxidized to diacetyl, which is the reactant for the colour produced in the VP test. Therefore, diacetyl is an oxidation product of acetoin.

The VP test is based on the reaction between diacetyl and the quinidine nucleus present in peptone under alkaline condition. The first reagent added to an incubated aliquot is the catalyst(alpha)-naphthol.An alcoholic solution of alpha-naphthol in that it acts as a colour intensifier. The alpha-naphthol combines with the product of the reaction of diacetyl and the quinidine type compound arginine present in peptone early in the reaction.The second reagent is 40% KOH which when agent to hasten the oxidation of acetoin to diacetyl. Diacetyl is the essential reactant that gives a colour reaction with KOH and the peptone.

• Citrate Utilization test

The basic principle of citrate utilization test is to determine the ability of an organism to utilize citrate as the sole source of carbon for metabolism with resulting alkalinity. Normally, citrate metabolism involves a condensation of acetyl, with coenzyme and oxaloacetate to enter the Krebs's cycle. Citrate metabolism by most bacteria is rapid via the tricarboxylic acid cycle or the citrate fermentation pathway. In bacteria, the cleavage of citrate involves an enzyme system without the intervention of the coenzyme A. This enzyme is called citrate or citrate demolase.The enzyme requires a divalent cation for its activity which is supplied by magnesium or many sense. Thus, the utilization of citrate depends on the presence of enzyme produced by organisms that break down the citrate to oxaloacetate acid and acetic acid as an intermediate product. These products are later converted to pyruvic acid and carbon dioxide. The medium used in this experiment is Simmon's citrate agar which contains sodium citrate as the only source of carbon and energy. The indicator used in it is bromothymol blue. When the citrate is metabolized by the organism, the CO₂ generated combines with sodium and water to form sodium carbonate, which is an alkaline product. The carbonate so produces changes the colour of the indicator from green to blue which is a positive test.

• Triple sugar iron (TSI) agar test

TSI agar contains lactose(1%), sucrose(1%), glucose(1%), FeSO₄, sodium thiosulphate and phenol red as indicator. It is used to differentiate organisms belonging to family Enterobacteriaceae. It is based on the basis of the difference in carbohydrate fermentation and H₂S gas production. So this test is also known as H₂S production test. The colour of phenol is yellow in acidic pH and red in alkaline.

Procedure:At first organism is inoculated into the medium and incubated for the 24 hours.During incubation,initially all organism ferment glucose both in slant and butt and produce acid. Therefore, both slant and butt become yellow due to the colour change of the indicator. But after some time, glucose become depleted in the media. Now it organism can ferment lactose or sucrose the continue to produce acid in both slant and butt and hence both remain yellow up to 24 hours. However,if an organism cannot ferment lactose or sucrose, it utilizes peptone of media and produces ammonia,utilization of peptone occurs first in aerobic condition ie. On slant. Therefore slant become red due to alkaline pH caused by amino and butt remain yellow ( ie.alkali / acid, red/yellow)

References

Debey, RC and D K Maheshwari. A text book of Microbiology. India: s.chand and company Ltd., 1999.

MacFaddin, H W. Biochemical test for Identification of medical bacteria. the Williams and Wilkins company, n.d.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.