Preparing a bacterial culture involves inoculating a sterile growth medium with bacteria and allowing them to multiply under controlled conditions. Here's a step-by-step guide:
Steps to Prepare a Bacterial Culture
-
Select a Single Colony: Using a sterile technique, choose a well-isolated single colony from a bacterial growth plate (e.g., an LB agar plate). This ensures that your culture starts from a genetically uniform population. You can use a sterile pipette tip, inoculation loop, or toothpick for this.
-
Inoculate the Liquid Medium: Transfer the selected colony into a sterile liquid growth medium. Luria-Bertani broth (LB broth) is a common choice. If your bacteria requires antibiotic selection (e.g., if they contain a plasmid with an antibiotic resistance gene), add the appropriate antibiotic to the LB broth.
- For example, if your bacteria are resistant to ampicillin, add ampicillin to the LB broth to a final concentration of 100 μg/mL.
-
Mixing: Gently swirl the pipette tip or toothpick in the liquid medium to dislodge the bacteria and disperse them evenly. This ensures that the bacteria are well-distributed throughout the broth.
-
Incubation: Cover the culture loosely with sterile aluminum foil or a cap that is not airtight. This allows for gas exchange while preventing contamination. Incubate the culture at the optimal temperature for the bacteria (typically 37°C for E. coli) in a shaking incubator. Shaking provides aeration, which promotes bacterial growth. Incubate for 12-18 hours or until the culture reaches the desired density.
-
Aeration (Shaking): Place the culture in a shaking incubator. This provides aeration, which is crucial for optimal bacterial growth. A typical shaking speed is 200-250 rpm.
Key Considerations
- Sterility: Maintaining sterility is crucial to prevent contamination by unwanted microorganisms. Use sterile materials (pipette tips, tubes, growth media) and work in a clean environment (e.g., near a Bunsen burner).
- Growth Medium: Choose a suitable growth medium based on the nutritional requirements of the bacteria. LB broth is a general-purpose medium, but other media may be more appropriate for specific bacterial species.
- Temperature: Incubate the culture at the optimal temperature for the bacteria. Most common lab strains of E. coli grow well at 37°C.
- Aeration: Ensure adequate aeration during incubation. This is typically achieved by shaking the culture.
- Antibiotics: If your bacteria contain an antibiotic resistance gene, include the appropriate antibiotic in the growth medium to maintain selective pressure.
- Culture Density: Monitor the culture density during incubation. The optimal density depends on the downstream application. A common target is an OD600 (optical density at 600 nm) of 0.6-0.8, which usually means the culture is in log phase.
Example Table: Culture Parameters
| Parameter | Recommendation |
|---|---|
| Growth Medium | LB broth (or appropriate medium for bacteria) |
| Temperature | 37°C (or optimal for bacteria) |
| Shaking Speed | 200-250 rpm |
| Incubation Time | 12-18 hours (or until desired density) |
| Antibiotic (if req) | Appropriate antibiotic for selection |
By following these steps, you can successfully prepare a bacterial culture for various downstream applications, such as protein expression, DNA isolation, and antimicrobial susceptibility testing.
