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Figure 6. Diagram of the procedure for detection of Salmonella sp.
3.6.1 Non-selective enrichment resuscitation
Swabs were placed into 225 ml lactose broths in Erlenmeyer flasks with lids; the contents were swirled to loosen the bacterial cells from the cotton swabs of the flask and
let stand for 60 ± 5 minutes at room temperature. The lactose broth and swab sample mixtures were then incubated at 35°C for 24 ± 2 hours. Swab samples were for tea
towels food preparation tables and hands of food handlers. For the utensils washing water 25g of the samples were weighed out and added to 225 ml of lactose in sterile
capped Erlenmeyer flasks 500 ml, followed by incubation at 35°C for 24 ± 2 hours.
25g of each swab isolate samples; 25g of water sample 225ml of lactose broth; mix for 2 minutes
Let stand for 60±2minutes Incubate: 24±2 hours at 35°C.
0.1ml of culture into 10ml RV medium Incubate: 42 ± 0.2°C for 24 ± 2 hours
1ml of culture into 10ml TT broth Incubate: 43 ± 0.2°C for 24 ± 2 hours
XLD, HE, BS agar media 35 °C for 24 ± 2 hours.
Inspect plates for the presence of characteristic colonies and any primary bochemical reactions 1 Confirmation of suspected colonies
2 Purify suspected colonies in Nutrient Agar; incubation at 35°C for 24 ± 2 hours Biochemical confirmation
Serology using antisera.
- Agglutination reaction.
Read reactions
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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
Kategori