Lab Report: Isolation and Identification of Salmonella

Introduction

Salmonella, a genus of rod-shaped gram-negative bacteria within the Enterobacteriaceae family, is responsible for the second most common intestinal infection, known as "Salmonellosis." Salmonella outbreaks are frequently linked to contaminated eggs, meat, poultry, fruits, and vegetables. The two most common Salmonella serotypes are typhimurium and enterica, causing non-typhoidal and typhoidal diseases, respectively. Symptoms of Salmonella infection include diarrhea, abdominal cramps, and fever, with potential complications like focal infection and reactive arthritis. In most cases, Salmonella infections last for 3-7 days, with an infectious dose ranging from 10^3 to 10^9 bacterial cells per gram of food.

Week 1: Isolation of Salmonella

Step 1 - Pre-enrichment

During this experiment, a group of three students, under the guidance of our lab instructor, conducted the following steps:

1. 11 grams of ground turkey were mixed with 100ml of water and placed in a stomacher for 2 minutes.
2. 1 ml of the mixture (excluding the precipitate at the bottom) was diluted with 9 ml of water. This dilution was then added to Selenite cysteine broth and incubated for 24 hours at 35°C, as Salmonella can thrive in this medium due to the presence of sulfur in cysteine.
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3. Streaking was performed on three different selective media: Bismuth sulfate (BS), Brilliant Green Sulfa (BGS), and Mackoney (MAC) for purification.
4. Mackoney agar was chosen as the purification medium, and a full loop of cultured Selenite cysteine media was streaked on the three selective media in all four quadrants. These plates were incubated for 24-48 hours at 35°C.

Week 2: Observation of Colonies

Observations of colonies on the cultured selective media were as follows:

• Bismuth Sulfate (BS): Black shiny metallic sheen-like colonies were observed on this media, which appeared dull green in color.
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• Brilliant Green Sulfa (BGS): Fade pink-like colonies were observed, and the media exhibited a bright green color.
• Mackoney (MAC): Transparent/colorless colonies were observed on this media, and the media itself was a faded pink color. Mackoney agar is a non-lactose fermenter.

Step 5 - Biochemical Testing

The selected colonies were marked on the plates and isolated for biochemical testing. Isolated colonies from three plates were taken, added to a sterile solution, and mixed thoroughly. The sample was then pipetted into three Triple Sugar Iron (TSI) test tubes corresponding to MAC, BS, and BGS, respectively. A sterilized loop was used to inoculate the test tubes, and they were incubated for 24 hours at 35°C.

For API 20E testing, the sample from the prepared solution was added to each well of the API sheet and incubated for 24 hours at 35°C. Reagents were added to specific wells post-incubation, and reactions were observed and recorded using the API interpretation table.

Results

Based on the testing, the results were as follows:

• TSI Testing: Alkaline red over the slant and a bed of acidic yellow color (K/A with H2S) were observed on the TSI slant.
• API 20E Testing:

Conclusion

Unacceptable profiles can result from impurities or mixed purities on the cultured plates, which can be influenced by physical, chemical, or external factors. In the case of BS, an unacceptable profile was observed. However, excellent identification of Proteus mirabilis was achieved for BGS and MAC. No Salmonella was detected in any of the three samples, indicating that the tested food sample does not contain any Salmonella species.

References

1. Best, L. (2019). Foodborne Diseases (Lab Manual). Public Health, Department of Public Health, Cape Breton University, Sydney NS.
2. Salmonella Food Poisoning. (n.d.). Foodborne Illness. Retrieved from https://foodborneillness.com/salmonella_food_poisoning/
3. Infectious Dose of Salmonella. (n.d.). Medscape.com. Retrieved from https://www.medscape.com/answers/228174-77482/what-is-the-infectious-dose-of-salmonella
4. Salmonella Food Poisoning. (n.d.). Salmonella.com. Retrieved from https://about-salmonella.com/