Preparation of Salmonella typhimurium Culture

Take a culture plate containing Salmonella typhimurium, a pathogenic bacterium.

Using a sterile bacterial loop, pick a single, well-isolated colony to ensure culture purity and inoculate it into brain heart infusion broth.

Incubate with shaking for proper aeration and uniform nutrient distribution, promoting bacterial proliferation.

Dilute the overnight culture into fresh broth to prevent nutrient depletion and maintain exponential growth.

Continue incubating with shaking.

Use a spectrophotometer to measure the optical density (OD) to track bacterial growth.

Allow the culture to reach an OD of approximately one. This indicates the exponential growth phase, during which the bacteria actively utilize nutrients to undergo rapid proliferation.

Transfer the culture to a tube and centrifuge to separate the bacteria.

Discard the supernatant and resuspend the bacteria in a buffer to preserve bacterial integrity.

Centrifuge and remove residual media.

Resuspend the bacteria in fresh buffer to obtain a uniform, contamination-free suspension.

To start Salmonella typhimurium culture, use a bacteria loop to inoculate a single bacterial colony into 5 milliliters of brain heart infusion broth. Then incubate the suspension in a 37 degrees Celsius incubator overnight while shaking. Transfer one milliliter of this bacterial suspension into 19 milliliters BHI broth in a conical flask on the following day.

Incubate again in 37 degrees Celsius incubator while shaking. When the OD₆₀₀ reaches one, remove the flask from the incubator and transfer the culture into a 50 milliliter tube. Then centrifuge the tube at 5,400 g at 4 degrees Celsius for 15 minutes.

After removing the supernatant, resuspend the bacterial pellet in 10 milliliters sterile PBS. After repeating the centrifugation once, resuspend the pellet in 4.9 milliliters of PBS.