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Differentiation of Escherichia coli from coliforms

Jvo Siegrist

AnalytiX Volume 8 Article 5

BIOCHEMICAL TESTS AS HYGIENE INDICATORS

Escherichia coli and coliforms are important indicator organisms for hygiene status.

In August 2008, the discovery of E. coli-contaminated beef in the United States prompted a nationwide recall of beef. The source turned out to be one supplier that had a history of contaminated beef products. The common sources of E. coli in beef are feces-contaminated animal carcasses, water supply, and/or other hygiene problems. Even in Switzerland, where drinking water is unusually pure, there are rare cases of fecal contamination by liquid manure. Detection of such pathogens is critical in maintaining hygiene.

Detection and Identification of E. COLI

E. coli is an aerobe, rod-shaped, motile, Gram-negative intestinal bacterium that ferments lactose and other carbohydrates (Table 1). Detection is possible because the bacterium ferments dextrose (D-glucose) by producing mixed acids (e.g., lactic, acetic, and formic acids) that is made visible with the addition of methyl red indicator. There are many other methods to detect the presence of E. coli. For example, Voges and Proskauer found a test to detect acetoin and 2,3-butanediol produced when Klebsiella and Enterobacter ferment glucose. Under alkaline conditions, these two compounds oxidize into diacetyl. Diacetyl then reacts with creatine (a guanidine derivative) and appears as a pinkish-red compound, or it reacts with a-naphthol and appears cherry-red in color.

We offer a broad range of biochemical tests available for the differentiation and identification of these microorganisms.

Table 1: Carbohydrates Differentiation Discs (available in single packs of 25 disks; or package size of 10 x 25 disks). Key: [+] = positive reaction, yellow color; [-] = negative reaction is listed below.

Note:
Carbohydrates 1 denotes Adonitol, Arabinose, Cellobiose, Dextrose, and Dulcitol
Carbohydrates 2 denotes Fructose, Galactose, Inositol, Lactose, and Maltose
Carbohydrates 3 denotes Mannitol, Mannose, Melibiose, Raffinose, and Rhamnose
Carbohydrates 4 denotes Salicin, Sorbitol, Sucrose, Trehalose, and Xylose

Characteristics of E. coli bacterium

Some other characteristic enzymes can also be detected by their interactions. Tryptophanase cleaves Tryptophan into pyruvate, indol, and ammonia; by using reagents (Kovac’s and DMCA), researchers can detect indole production (Figure 1). ß-Galactosidase is detected with ONPG (2-Nitrophenyl ß-D-galactopyranoside), a chromogenic substrate that turns yellow after cleavage. Further, the ability to reduce nitrate to nitrite can be detected with the addition of sulphanilic acid and a-naphthylamine, which results in a red precipitate (prontosil). Finally, lysine is degraded by E. coli to cadaverine by the lysine decarboxylase. As a result of an alkaline reaction, the indicator (bromocresol purple) will change colour from yellow to purple.

Kovac’s indole reaction (from left to right: blank, negative, positive)

Figure 1.Kovac’s indole reaction (from left to right: blank, negative, positive)

TSI Agar: From the left,we see the mediumwithout organisms, followed by an extreme reaction in the butt of the tube and on the slant surface; the second tube fromleft shows the typical reaction when E. coli organisms are present.

Figure 2.TSI Agar: From the left, medium without organisms, followed by an extreme reaction at the bottom of the tube and on the slant surface; the second tube from left shows the reaction typical to E. coli organisms.

Interesting differentiation results are obtained with the inoculation of TSI Agar slants. Due to the formation of an acid during fermentation of lactose, sucrose and glucose, the pH level usually drops. However, in the case of oxidative decarboxylation of peptone alkaline products, the pH rises. The increase in pH is indicated by phenol red, which changes colour from red orange to yellow in an acidic environment and upon alkalinization, it turns deep red. E. coli shows an acidic reaction (yellow) and gas formation at the bottom of the test tube and an acid reaction (yellow) on the slant surface.

An overview of the important biochemical reactions of E. coli is given in Table 2.

Table 2.Biochemical reactions of E.coli Key: AG/A acid (yellow) and gas formation in butt of tube and acid (yellow) on slant surface
Test products for differentiation and identification
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Reagents for differentiation and identification
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