This article details a method for isolating Salmonella from mixed bacterial cultures using selective media. The process involves the use of selenite cystine medium and Xylose lysine deoxycholate agar to differentiate Salmonella from non-Salmonella species based on hydrogen sulfide production.
Inoculate a culture containing both Salmonella and non-Salmonella species into a selenite cystine medium, and incubate.
Selenite inhibits the growth of selenium-sensitive bacteria by disrupting their metabolic processes.
However, selenium-tolerant bacteria, including Salmonella, can metabolize selenite and grow.
Streak the culture onto Xylose lysine deoxycholate or XLD agar, and incubate.
Salmonella reduces sodium thiosulfate in XLD agar to hydrogen sulfide, which reacts with ferric ions in the medium to form black iron sulfide, producing colonies with black centers.
In contrast, non-Salmonella species that do not produce hydrogen sulfide form colonies without black centers, allowing visual differentiation from Salmonella.
Select a black-centered colony and streak it onto Salmonella chromogenic agar for enhanced specificity.
Chromogenic agar contains Salmonella-specific enzyme substrates linked to chromophores.
Salmonella enzymes cleave these substrates, releasing the chromophores and forming pigmented colonies.
Select these colonies for further analysis.
Add 100 microliters of the pre-enrichment culture to five milliliters of selenite crystal medium. Then, incubate the culture at 36 degrees Celsius for 18 to 24 hours. Next, collect one loop of the culture and spread it onto a plate.
Then, incubate the plate at 36 degrees Celsius for 18 to 24 hours. After incubation, select suspicious colonies and transfer them to a plate. Incubate the plate at 36 degrees Celsius for 18 to 24 hours.
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