Salmonella shigella agar

Bacteria were isolated according to the process defined by Harrigan and McCance (1976 ). Firstly, 9 mL of sterile distilled water was dispensed into seven test tubes. Then, 1 mL of the effluent was dispensed into the first test tube and serially diluted at a ratio of 1:10. Water samples from the receiving surface water body were diluted serially at a ratio of 1:7. About 1 mL of each of the effluent and water samples was inoculated on aseptically prepared MacConkey agar (MA) (HiMedia Laboratories Pvt. Ltd, Mumbai, India), Eosine Methylene Blue (HiMedia India) and Salmonella Shigella agar (HiMedia India) using the pour plate technique. Plates were incubated at 35 °C ± 2 °C for 18 –24 hours.
The total heterotrophic and coliform counts were determined on nutrient agar (NA) and MA plates. The maximum limit of heterotrophic count for drinking water is 1.0 × 10² cfu/mL (Environmental Protection Agency [EPA] 2002 ), whereas the maximum limit for wastewater is 400 cfu/mL. Morphologically distinct colonies were sub-cultured twice on corresponding media. Pure colonies obtained were stored both on NA slants and nutrient broth containing 15% glycerol (volume/volume [v/v]), for further investigation.

Stool specimens received at respective laboratories were cultured for enteric pathogens, including Shigella spp. by standard methods [17 ]. Sample was streaked onto MacConkey agar and a selective medium (either deoxycholate citrate agar or xylose lysine deoxycholate agar or Salmonella-Shigella agar) (all media by HiMedia, India) by the quadrant isolation technique [18 ]. The plates were incubated at 37 °C for 24 h. Isolates were identified by Gram’s staining, colony characteristics, and biochemical tests.
After identification of isolates, they were subcultured onto nutrient agar slants in screw capped tube, incubated overnight and then transported to NPHL in a cold box. All reported isolates of Shigella were re-confirmed by serotyping using commercially available antisera from Denka Seiken, Japan, and were preserved in tryptic soy broth with 20 % glycerol at −75 °C for further use. Antibiotic susceptibility test was performed for all the identified Shigella isolates by Standard Kirby Bauer’s disc diffusion technique. The antibiotics used for analysis were ampicillin (Amp-10 mcg), cotrimoxazole (Sxt-25 mcg), nalidixic acid (NA-30 mcg), ciprofloxacin (Cip-5 mcg), mecillinam (Mec), and ceftriaxone (CRO-5 mcg).

ISO standards were used to determine the microbiological load of the following bacteria during 28 days: total mesophyll aerobic flora (FAMT), Salmonella sp., Staphylococcus sp., Vibrio sp., and Escherichia sp. A 25 g of slices of the preserved fish sausage was ground under aseptic conditions in a mortar with a sterile pestle and homogenized for 2 min on a vortex. Samples were serially diluted and plated in triplicate on nutrient agar (plate count agar Hi-Media) and selective agars such as thiosulfate citrate bile salts sucrose (TCBS) agar (Hi-Media), Salmonella-Shigella agar (SSA Hi-Media), mannitol salt agar (MSA Hi-Media), Man, Rogosa and Sharpe (MRS; Hi-Media), and Pseudomonas selective agar (Drigalski agar) (Hi-Media) and incubated at 37°C for 24 and 48 h for FMAT.
The total number of colonies present in the sample unit is given by the formula as follows:
$\begin{array}{c}\text{N}=\frac{\mathrm{\Sigma }c}{\left(n1+0.1n2\right)\times d}\times \frac{1}{V}\times \frac{\text{Vsm}}{\text{Vpr}},\end{array}$ where n1 is the number of plates counted at the lowest retained dilution, n2 is the number of plates counted at the second retained dilution, d is the dilution rate at which the first counts are made: lowest dilution, ∑C is the total number of colonies on the retained plates, V is the volume of the inoculated test sample in ml, Vsm is the volume of the stock suspension in ml, and Vpr is the mass of the product (g) that made up the stock suspension.

Three clinical isolates (Salmonella Typhi, Salmonella Enteritidis and Salmonella Typhimurium from Pasteur Center, Yaoundé-Cameroon) and one bacterial strain (Salmonella Typhi ATCC6539 from American Type Culture Collection) were used for antimicrobial evaluation. The culture media used were Salmonella-Shigella Agar (SSA from HiMedia Laboratories, India) and Mueller Hinton Broth (MHB from HiMedia Laboratories, India).

Samples (25 g) of the raw and processed products were enriched with 225 mL nutrient broth containing 5 g peptone, 5 g sodium chloride, 1.5 g beef extract, and 1.5 g yeast extract per 1000 mL of water, pH 7.4 (HiMedia M002) and incubated at 35 °C for 24 h. Selective enrichment was then performed by transferring 25 mL of the enriched homogenate to 225 mL of tetrathionate broth (HiMedia M032) and the culture incubated at 37 °C for 24 h. A loopful of the Tetrathionate broth culture was streaked on Salmonella-Shigella Agar (HiMedia, M108) and plates incubated at 37 °C for 24 h. Typical Salmonella colonies (colourless colonies with black centres) were further identified using the Triple Sugar Iron (HiMedia, M021I) test. For this test, a sterilized inoculation needle was used to touch the top of a well isolated colony and then inoculated into the Triple Sugar Iron agar by first stabbing through the centre of the media to the bottom of the tube, followed by streaking on the surface of the agar slant. The slants were incubated at 35 °C for 24 h. Salmonella forms a red slope (alkaline) and yellow (acid) butt indicating fermentation of glucose, with or without gas (cracks and bubbles in the medium), and with or without H₂S production (blackening of the medium).