Biochemical reaction confirmation Isolating and identifying Salmonella species

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

For Biochemical confirmation, growth from the TSI slants showing typical Salmonella growth were used. The TSI slants mixed with HE agar, BS agar and XLD agar showing typical Salmonella growth were shown by yellow butt for TSI and H 2 S gas production showed by blackening of the media. Typical Salmonella colonies from TSI 40 mixed cultures were then transferred into MR-VP broths, into Urea broth, and into Simmons citrate agar slants for biochemical reaction confirmation. Urease test . With sterile needle, inoculate growth from each presumed-positive TSI slant cultures were put into tubes of urea broth. Since occasional, uninoculated tubes of urea broth turn purple-red positive test on standing, include uninoculated tube of this broth as control. Incubation was done for 24 ± 2 h at 35°C. Negative urease test is an indication of Salmonella species and is given by no change in reaction or no color change orange whilst positive urease reaction pink color is an indication of non Salmonella species. MR-VP test. Tubes containing MR-VP broth were inoculated with TSI tubes containing typical Salmonella characteristics and incubation done at 35 °C for 48 hours. Into 5 mls of the broth, 5 6 drops of methyl red were added, the reaction read after a few minutes: pink-red an indication of Salmonella species whilst no color change an indication of non Salmonella species. Into 1 ml of MR-VP broth, 0.6 mls of α -naphthol solution and 0.2 mls of potassium hydroxide 40 KOH. After 4 hours the reactions are observed and results if positive Salmonella species gave negative reaction no color change yellowishcloudy whilst non Salmonella species gave positive reaction color change to maroon-red. Simmons Citrate agar test. Agar slants of Simmons citrate were streaked and butt stubbed with TSI cultures of typical Salmonella reactions and incubated at 35 °C for 96 hours. The original color of the prepared slants was green and after incubation at the correct conditions, positive Salmonella sp. was indicated by blue colored slants whereas ones remaining greenish were considered as negativenon Salmonella sp.

3.6.6 Serological somatic O tests for Salmonella sp.