This article outlines the process of culturing bacteria from a frozen stock to prepare a pure culture for further experiments. It details the steps involved in streaking, incubating, and maintaining bacterial growth in a liquid medium.
Begin with a frozen stock of bacteria.
Use a sterile inoculation loop to streak the frozen bacteria onto a nutrient agar plate. Incubate the inverted plate to prevent water condensation onto the agar surface.
The bacteria utilize the nutrients, grow on the agar surface, and form distinct visible colonies.
Select a single colony and inoculate it into a liquid medium containing nutrients to initiate a pure culture.
Incubate the tube under vigorous shaking conditions.
Shaking maintains aeration in the tube and keeps the cells suspended, preventing them from settling at the bottom.
It also ensures an even distribution of nutrients throughout the medium, promoting exponential bacterial growth.
This results in a dense, metabolically active bacterial suspension.
The bacterial culture in the liquid medium is now ready for further experiments.
To use the stock, with a sterile inoculating loop, remove a small amount of frozen stock and streak the bacteria to obtain single colonies on LB agar plates.
Incubate the plates lid-side down for 12-16 hours at 37 degrees Celsius. Obtain previously prepared plate. Inoculate 4 milliliters of liquid LB broth in a sterile 10 milliliter culture tube with a single colony by touching the surface of the single colony with a sterilized loop and transferring the colony into the liquid broth. Then, incubate the culture with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius.
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