Eosin methylene blue agar

Fresh faeces were collected aseptically from the different groups of rats prior to their euthanasia and the enumeration of bacteria from freshly voided, serially diluted faecal pellets was carried out on different selective media, according to the pour plate technique described by Harrigan and MacCance [25 ]. Culture media were obtained from Oxoid Ltd, Basingstoke, United Kingdom and included MacConkey agar (coliforms), Eosin methylene Blue agar (Escherichia coli), Slanetz and Bartley medium (Enterococcus spp.) and Mannitol salt agar (Staphylococcus spp.), De Man Rogosa and Sharpe (MRS) agar (Lactobacilli) and Centrimide agar (Pseudomonas aeruginosa). Bacteria were enumerated as colony forming units per gram of faeces (CFU/g).

Approximately 90 mL of buffered peptone water (Merck Biolab, Modderfontein, South Africa) and 10 g of faecal matter was aseptically added into a stomacher bag and homogenised for 2 min using a stomacher device. The sample was then incubated at 37 °C for 12–14 h. This resuscitation step allows for better recovery of bacterial cells due to frozen storage [48 (link)].
The pour plate technique was used, using a violet red bile dextrose agar (VRBDA) (Merck Biolab, Modderfontein, South Africa) and 10⁻⁴ and 10⁻⁵ dilutions of the original sample. These dilutions were found to produce single colonies in the range of 25 to 250 colonies per plate, thus allowing single colonies to be easily obtained. The plates were inverted and incubated overnight at 37 °C.
Characteristic colonies from the VRBDA plates were streaked onto an eosin methylene blue agar (Oxoid, Hampshire, UK). The plates were inverted and incubated overnight at 37 °C.
Five presumptive E. coli colonies were selected per animal species, and their identities were confirmed using Gram’s stain and the citrate utilisation test using Simmons citrate agar (Oxoid, Hampshire, UK). Glycerol stock cultures were made of the confirmed E. coli isolates.

For isolation of E. coli, swabs from the obtained specimens were inoculated in McConkey's broth (Oxoid, Hampshire, UK), followed by incubation for 24 h at 37 °C. A loopful of broth-culture was streaked onto MacConkey's agar, and eosin methylene blue agar (Oxoid, Hampshire, UK). The suspected colonies were identified according to their colonial characters, hemolytic activity, microscopical examination using Gram's staining, motility test, hemolytic activity on blood agar, and biochemical reactions (oxidase, catalase, indole, lactose fermentation, methyl-red, citrate-utilization, H₂S, Voges-Proskauer, and urease tests) as described by Quinn¹⁹ .
For isolation of other bacterial pathogens, swabs from the processed specimens were inoculated on nutrient agar, blood agar, mannitol salt agar, cetrimide agar, and MacConkey's agar (Oxoid, Hampshire, UK), then the inoculated plates were incubated for 24–48 h at 37 °C. The obtained pure colonies were identified according to their colonial characters, morphological characters, and biochemically as described by Quinn¹⁹ .

A sample of Sui Wu’u (25 g) was collected aseptically and placed in a sterile tube containing 45 mL of 0.1% sterile Butterfield’s phosphate-buffered dilution water (Merck). Serial decimal dilutions were prepared in sterile 0.1% (wt/vol) peptone water for microbiological testing. Total aerobic mesophilic microflora was enumerated in Potato Count Agar (Oxoid, UK) after 48 h of incubation at 35°C according to Maturin and Peeler [26 ]; total E. coli in Eosin Methylene Blue Agar (Oxoid) after 48 h of incubation at 35°C according to Nuraida et al. [27 ]; total Salmonella in Xylose Lysine Deoxycholate Agar (Oxoid) after 48 h of incubation at 35°C according to Brichta-Harhay et al. [28 (link)]; and total S. aureus according to ISO 6888-1:1999 Amd 2 [29 ] using Baird-Parker agar (BPA) specific media (Oxoid) plus egg yolk with the spread plate method after incubation at 37°C for 48 h. Typical S. aureus colonies on BPA media are round and black with a clear zone around the colony. After incubation, plates with 30–300 colonies were counted. Microbiological data are presented in colony-forming units (CFU)/g.

The acidity of fermented milk samples was ascertained using a pH indicator paper (Whatman®). Both the raw and fermented milk samples were diluted tenfolds using sterile distilled water before inoculating 1 ml onto MacConkey agar (Oxoid, UK) and subcultured on Eosin Methylene Blue agar (Oxoid, UK) using spread plating technique. The plates were incubated at 37°C for 24 hours and bacterial colonies that are circular, moist, smooth, and pinkish on MacConkey agar but have green metallic sheen on Eosin Methylene Blue agar were presumed to be Escherichia coli. All presumptive colonies were subjected to a panel of conventional biochemical tests (IMViC) and isolates that were positive for indole and methyl red tests but negative for Voges Proskauer and Citrate utilization tests we identified as E. coli. The identified E. coli isolates were further subcultured onto sorbitol MacConkey agar (Oxoid, UK) and incubated at 37°C overnight. Nonsorbitol fermenters that appear as smooth, circular, and colourless colonies were tentatively identified as Escherichia coli O157:H7 as earlier described [13 ].