Sub-culturing of presumptive Salmonella

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3.6.2 Selective enrichment

From the non-selective enrichment sample mixture, 0.1 ml was transferred to 10 ml of Rappaport-Vassiliadis RV medium, and another 1 ml mixture to 10 ml of tetrathionate TT broth. The tube mixture compositions were then shaken vigorously by Vortex. RV medium were incubated at 42 ± 0.2°C for 24 ± 2 hours in circulating, thermostatically-controlled water bath. TT broths were incubated at 43 ± 0.2°C for the same time frame.

3.6.3 Plating on selective solid media

The RV and TT broth mixtures after the period of incubation were then shakenmixed vortex and streaked onto Bismuth sulfite BS agar, xylose lysine desoxycholate XLD agar, and Hektoen enteric HE agar with 3 mm loopfuls 10 µl. The streaked plates then were incubated at 35 °C for 24 ± 2 hours. Note: BS plates were prepared a day before streaking, and stored in dark at room temperature until streaked. Typical salmonella colonies from the plating would be as follows: 1 Hektoen Enteric HE agar indication of Salmonella being present be denoted by blue-green to blue colonies with or without black centers. Many cultures of Salmonella produce large, glossy black centers or appear as almost completely black colonies; 2 Xylose lysine desoxycholate XLD agar would be denoted by pink colonies with or without black centres; 3 Bismuth sulfite BS agar Brown, gray or black colonies sometimes with metallic sheen would be an indication of Salmonella. The surrounding usually is brown at first, but may turn black in time with increased incubation, producing the so-called halo effect. The atypical Salmonella colony morphology would be as follows: 1 HE and XLD agars would produce yellow colonies with or without black centers; 2 BS agar some of the strains would show green colonies with little or no darkening of the surrounding medium.

3.6.4 Sub-culturing of presumptive Salmonella

Typical Salmonella colonies from BS agar, HE agar, and XLD were then transferred into triple sugar iron agar slants TSI and lysine iron agar slants LIA using sterile streak and butt stab needle. The streaked and butt stabbed TSI and LIA slants 39 were done aseptically by heat fixing and working on clean surfaces. Incubation of TSI and LIA slants was at 35°C for 24 ± 2 hours; tubes capped loosely to maintain aerobic conditions while incubating slants to prevent excessive H 2 S production. Salmonella in culture would typically produce alkaline red slant and acid yellow butt, with or without production of H 2 S blackening of agar in TSI. In LIA, Salmonella typically would produce alkaline purple reaction in butt of tube. Distinct yellow in butt of tube would indicate that it is an acidic negative reaction. However, elimination must not be solely based on this discoloration in the butt of tube. Generally most Salmonella cultures would produce H 2 S in LIA. Some non- Salmonella cultures produce a brick-red reaction in LIA slants. After incubation period, the results of reactions were noted and recorded, and those giving typical Salmonella sp. characteristics were transferred into biochemical reaction medias: MRVP- broth Methyl Red-Vogers Prausker, Urea broth, Simmons citrate agar and Lysine decarboxylase broth. The TSI and LIA cultures that gave typical Salmonella species reactions yellow butt for TSI cultures; blackening due to H 2 S gas production and LIA slants had purple butt; blackening due to H 2 S gas were then transferred into tubes containing nutrient agar slants for further determination of establishing the species by serology test for those slants after passing for Salmonella species biochemical reactions using urease test, Simmons citrate agar and MR-VP test. For presumptive subculturing control stock from Dr Ratih s Salmonella species were used. Using a sterile inoculating needle the center of the colonies were lightly touched, then inoculation of TSI slant was done by streaking the slant and butt stubbing them. Without flaming, LIA slant were inoculated by stabbing butt twice and then the slant streaked. Since lysine decarboxylation reaction is strictly anaerobic, therefore, the LIA slants must have deep butt 4 cm. Storing of this picked selective agar plates was at 5-8°C.

3.6.5 Biochemical reaction confirmation