Differentiation of Escherichia coli from coliforms

By: Jvo Siegrist, AnalytiX Volume 8 Article 5

Escherichia coli and coliforms are important indicator organisms for hygiene status. A broad range of biochemical tests is available for differentiation and identification of these organisms

Product Manager Microbiology ivo.siegrist@sial.com

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 contamination of its beef products. The usual sources of E. coli in beef are faeces-contaminated animal carcasses, water supply, and/or other hygiene problems. Even in Switzerland, where drinking water is unusually pure, there are rare cases of faecal contamination by liquid manure. Detection is critical to maintaining hygiene.

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

Organisms (ATCC) Adonitol Arabinose Cellobiose Dextrose Dulcitol Fructose Galactose
  (Fluka 55876) (Fluka 80372) (Fluka 56481) (Fluka 63367) (Fluka 73044) (Fluka 53901) (Fluka 89608)
  Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas
Citrobacter freundii (8090) - - + + + - + + - -     + +
Enterobacteraerogenes(13048) + + + + + + + + - - + + + +
Escherichiacoli(25922) - - + + - - + + - - + + + +
Klebsiellapneumoniae(13883) + + + + + + + + - - + + + +
  Inositol Lactose Maltose Mannitol Mannose Melibiose Raffinose
  (Fluka 89614) (Fluka 28816) (Fluka 77653) (Fluka 94438) (Fluka 94445) (Fluka 93196) (Fluka 94226)
  Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas
Citrobacter freundii (8090) - - + + + + + + + + - - - -
Enterobacteraerogenes(13048) + + + + + + + + + + + + + +
Escherichiacoli(25922) - - + + + + + + + + + + - -
Klebsiellapneumoniae(13883) + + + + + + + + + + + + + +
  Rhamnose Salicin Sorbitol Sucrose Trehalose Xylose
  (Fluka 93999) (Fluka 92971) (Fluka 93998) (Fluka 94309) (Fluka 92961) (Fluka 07411)
  Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas Acid Gas
Citrobacter freundii (8090) + + - - + + + + + + + +
Enterobacteraerogenes(13048) + + + + + + + + + + + +
Escherichiacoli(25922) + + - - + + - - + + + +
Klebsiellapneumoniae(13883) + + + + + + + + + + + +
 
Table 3 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


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 (see Figure 1). β-Galactosidase is detected with ONPG (2-Nitrophenyl β-D-galactopyranoside), a chromogenic substrate that turns yellow after cleavage has occurred. Further, the ability to reduce nitrate to nitrite can be detected with the addition of sulphanilic acid and α-naphthylamine, which results in a red precipitate (prontosil). Finally, lysine is degraded by E. coli to cadaverine by the lysine decarboxylase. Because this is an alkaline reaction, the indicator (bromocresol purple) will change colour from yellow to purple.

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

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

Figure 2 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,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.

Interesting differentiation results are obtained with the inoculation of TSI Agar slants. Due to the formation of 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. This increase is indicated by phenol red, which changes colour in acidic surroundings from red-orange to yellow; upon alkalinisation, it turns deep red. E. coli shows an acid reaction (yellow) and gas formation in the butt of the test tube and an acid reaction (yellow) on the slant surface.

An overview of the important biochemical reactions of E. coli is included in Table 1. Sigma-Aldrich products available for differentiation are listed in Tables 2 and 3.

Biochemical test Reaction
Catalase +
Citrate utilisation (Simmon’s citrate Agar, Fluka 85463) -
TSI Agar (Fluka 44940) AG/A
Gelatin liquefaction (Nutrient Gelatin, Fluka 70151) -
Indole Production +
Nitrate Reduction +
Urease (Urea Broth, Fluka 51463; or Christensen’s Urea Agar, Fluka 27048) -
Voges-Proskaur -
Methyl Red +
Presumptive test (Lauryl sulphate Broth, Fluka 17349) +
Phenylalanine deaminase (Phenylalanine Agar, Fluka 78052) -
Motility (SIM Medium, Fluka 85438; or Tryptone Agar, Fluka 93655) +
Lysine (LD Broth, Fluka 66304) +
ONPG (β-galactosidase) +
Oxidase -
 
Table 1 Biochemical reactions of E.coli Key: AG/A acid (yellow) and gas formation in butt of tube and acid (yellow) on slant surface


Cat. no. Name Description (Engl) Package size
75554 Aminopeptidase Test For the detection of L-alanine-aminopeptidase in microorganisms. It is found almost exclusively in Gram-negative croorganisms. 50 ea
29333 Barritt’s Reagent A These reagents are used in the Voges-Proskauer test for detection of acetoin production 100 mL
39442 Barritt’s Reagent B by bacterial cultures. 100 mL
88597 Catalase Test A reagent to detect the enzymes catalase and peroxidase. 100 mL
05686 DMACA Indole Disks Detection of tryptophanase activity. 50 ea
49825 DMACA Reagent   50 mL
96343 HybriScan®D E. coli NEW Genetic based detection and identification of Escherichia coli in water and food samples. 96 tests
60983 Kovac’s Reagent for indoles E. coli is able to split tryptophan into indole and alpha-aminopropionic acid. The reagents listed enable the detection of indole. luka 67309 contains isoamylic alcohol as solvent, while Fluka 60983 contains n-Butanol as solvent. Both formulations are more stable than the old formulation with amyl alcohol. 100 mL
67309 Kovac’s Reagent for indoles   100 mL
78719 Kovac’s Reagent Strips   25 ea
08714 Methyl Red Solution Differentiates between bacteria based on level of acid production from glucose(high/low/none). 100 mL
38497 Nitrate Reagent A Reagents, disks and kits for the detection of nitrate reduction by bacteria. 100 mL
39441 Nitrate Reagent B   100 mL
51138 Nitrate Reagent Disks Kit   50 ea
73426 Nitrate Reduction Test   1 ea
07689 O’Meara’s Reagent Used in the Voges-Proskauer test for the detection of acetoin production by bacterial cultures. 100 mL
49940 ONPG Disks Testing for β-galactosidase. 50 ea
07345 Oxidase Reagent acc. Gaby-Hadley A Reagents, disks and strips for the detection of cytochrome oxidase activity of microorganisms, an important differentiation step for Gram-negative bacteria. 100 mL
07817 Oxidase Reagent acc. Gaby-Hadley B   100 mL
18502 Oxidase Reagent acc. Gordon-McLeod   100 mL
40560 Oxidase Strips   100 ea
70439 Oxidase Test   50 ea
 
Table 2 Tests and reagents for differentiation and identification

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