Streaking for Single Colonies

Learn why streaking for single colonies is essential when starting liquid cultures from glycerol stocks or bacterial stabs. This prevents potential mutants in the mixed population from affecting downstream experiments.

Collect bacterial glycerol stock or stab, LB agar plate with appropriate antibiotic, sterile toothpicks or sterile loop, Bunsen burner, dry incubator, and ensure good sterile technique is ready.

Inspect the LB agar plate for contamination and ensure it is dry. If condensation is present, place the plate next to a flame with the lid open for about 10 minutes to dry it out.

Take a sterile toothpick and insert it into the stabbed culture, dabbing at regions with visible bacterial growth. For glycerol stocks, immerse the sterile toothpick into the semi-frozen solution. Use only a small amount of cells.

Gently streak the first toothpick across roughly 1/3 of the plate. Dispose of it, use a new sterile toothpick to streak from where the first ended across another 1/3 of the plate. Repeat with a third sterile toothpick to complete the pattern.

Close the plate and place it upside down to prevent condensation from dripping onto the colonies and disturbing them. This orientation protects the newly streaked colonies.

Place the plate in a dry incubator at 30 or 37 degrees Celsius overnight or for at least 12 hours to allow individual colonies to form.