**3. Isolation of** *E. coli* **and quality control**

### **3.1. Isolation of** *E. coli*

Different options are available for the isolation of *E*. *coli*. The choice depends on target strain and objective of isolation. The ability to ferment lactose gives an option to use MacConkey agar to discriminate *E*. *coli* from other nonlactose fermenting coliforms from fecal, stool, food, water, and soil samples. Sample suspension (for solid samples) is made at any concentration, for example, 5% in normal saline or phosphate buffer solution and inoculated onto MacConkey agar followed by 18–24 h incubation at 37°C. Pink, round medium-sized colonies are picked as *E*. *coli* suspect colonies. All *E*. *coli* strains can be captured on MacConkey agar, and this approach gives a wide spectrum of strains to work on. Incubation of inoculated culture media at 45°C selects for thermophilic *E*. *coli* strains.

plates at 37°C overnight. The media plates should have no microbial growth after incubation. This will ensure that the isolates obtained after inoculation come from the samples and not due to contamination. Moreover, uninoculated media plate should be incubated simultaneously with inoculated media plates. Use of reference positive controls strains, e.g. *E*. *coli* ATCC

Isolation and Characterization of *Escherichia coli* from Animals, Humans, and Environment

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For water samples, quality control measures may involve the use of blank and sample replicates. The true samples and the blanks are simultaneously incubated. The blank sample will tell that the sampling equipment has not been contaminated. The replicate results will assess

Confirmation of *E*. *coli* isolates can be done by biochemical, enzymatic, or molecular methods. The choice of the method depends on many factors including availability of resources. The confirmation methods include biochemical methods, such as IMViC and Analytical Profile Index 20E (API 20E) systems, enzymatic methods, for example, use of brilliance *E*. *coli* agar or

*E*. *coli* isolates can be confirmed biochemically by the use of a traditional method called IMViC tests. This is a set of four tests that are used to differentiate members of the family Enterobacteriaceae. IMViC is an abbreviation that stands for the Indole, Methyl red, Voges-Proskauer, and Citrate utilization tests. In Indole test, the bacteria are tested for their ability to

The indole reacts with the aldehyde in the Kovac's reagent to give a red or a pink ring at the top of the tube. Peptone water in a tube, which contains tryptophan, is inoculated with bacteria isolate to be tested. The mixture is incubated overnight at 37°C. Then, a few drops of Kovac's reagent are added to the mixture and formation of a red or a pink colored ring at the

Methyl red test detects the ability of a bacterium to produce acid from glucose fermentation. Methyl red, a pH indicator, remains red in color at a pH less or equal to 4.4. The bacterium to be tested is inoculated into glucose phosphate (MRVP) broth, which contains glucose and a phosphate buffer and incubated at 37°C for 48 h. Three to five drops of MR reagent are added to the tube. Red color development is a positive reaction that occurs when the bacteria have produced enough acid to neutralize the phosphate buffer. Yellow discoloration occurs to

Voges-Proskauer test is used to detect the presence of acetoin in the bacteria-containing media. Acetoin is oxidized to diacetyl in the presence of air and sodium hydroxide. Diacetyl, in the presence of alpha-naphthol, reacts with guanidine to produce red color. In order to perform VP test, the test bacterium is inoculated into glucose phosphate (MRVP) broth in a

Petrifilm Select *E*. *coli* count plate, and molecular techniques such as MALD-TOF.

produce indole from tryptophan (amino acid) using the enzyme tryptophanase.

top is a positive reaction. *E*. *coli* are indole-positive bacteria.

MR-negative bacteria. *E*. *coli* are MR-positive bacteria.

tube and incubated for 72 h.

25922, will also help to ensure the isolates are the targeted bacteria.

the presence of variation for which explanations should be sorted out.

**4. Confirmation of** *E. coli* **isolates**

**4.1. IMViC tests**

The concentration of sample suspension may be set at different levels such as 1 g of solid sample in 19 ml of normal saline or phosphate buffer solution (5%), 1 g in 9 ml (10%) or 1 g in 4 ml of diluent (20%). However, the concentration of sample suspension will affect the number of colonies on the culture plate. This is well evidenced in bacteria count procedures whereby higher dilution, like 10<sup>5</sup> , will give lower number of bacteria than low dilutions, for example, 101 . This is because the bacteria growth rate depends on initial cell density in the sample [8].

Sample suspension can be enriched by 24 h incubation at 37°C in nondifferential broth such as Muller-Hinton or nutrient broth. This procedure will allow multiplication of *E*. *coli* and hence increase the chance of *E*. *coli* isolation especially when infrequent strains, such as pathogens, are the target. The generation (doubling) time for *E*. *coli* at 37°C incubation is 17–18 min [8], therefore, in 18–24 h incubation there will be 60–80 *E*. *coli* cell generations. However, clonal variability will decrease when samples are enriched because same bacteria increase in number. Therefore, this procedure is suitable when the research aims at a mere presence of a single specific strain and not its variants.

The weight of the sample and the volume of diluent used in making the sample suspension may affect the probability of bacteria recovery. Large sample weight normally increases the sensitivity of the isolation procedure. For example, in *E*. *coli* studies to isolate nonsorbitol-fermenting Shiga toxin-producing *E*. *coli* (NSF STEC) whereby *E*. *coli* broth was used to enrich fecal samples, different prevalence measure was obtained. When 10 g of sample was suspended in 90 ml of *E*. *coli* broth, the prevalence of Shiga toxin-producing *E*. *coli* (STEC) obtained was 1.3% [9], while the suspension of 20 g in 180 ml of same diluent resulted into a prevalence 11.1% NSF STEC [10].

Purification of *E*. *coli* colonies can be done in nondifferential media such as blood or nutrient agars. Depending on the degree of colony density, a series of inoculations can be desired until pure, single, or solitary colonies are obtained.

### **3.2. Quality control**

These are procedures undertaken to validate the accuracy of the bacteria isolates. Among the measures of quality control in isolation of *E*. *coli* include incubation of uninoculated media plates at 37°C overnight. The media plates should have no microbial growth after incubation. This will ensure that the isolates obtained after inoculation come from the samples and not due to contamination. Moreover, uninoculated media plate should be incubated simultaneously with inoculated media plates. Use of reference positive controls strains, e.g. *E*. *coli* ATCC 25922, will also help to ensure the isolates are the targeted bacteria.

For water samples, quality control measures may involve the use of blank and sample replicates. The true samples and the blanks are simultaneously incubated. The blank sample will tell that the sampling equipment has not been contaminated. The replicate results will assess the presence of variation for which explanations should be sorted out.
