To grow bacteria in liquid culture, you'll typically inoculate a sterile broth medium with a bacterial sample and incubate it under appropriate conditions to allow for bacterial growth. Here's a step-by-step guide:
Steps for Growing Bacteria in Liquid Culture
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Prepare the Growth Medium: The most common medium is Luria-Bertani broth (LB). If your bacteria require a specific antibiotic resistance, add the appropriate antibiotic. For example:
- Add the antibiotic (e.g., kanamycin) to the LB broth to achieve the desired final concentration (e.g., 1X concentration from a 1000X stock). Swirl or mix thoroughly to ensure even distribution of the antibiotic.
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Sterilize the Medium: Sterilize the LB broth in a suitable container (e.g., a glass flask or test tube) using an autoclave. This ensures that no contaminating microorganisms are present. Typically, autoclaving occurs at 121°C for 15-20 minutes. Let the medium cool to a temperature safe for handling before inoculation.
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Inoculate the Medium: Using sterile technique is critical.
- If using a bacterial colony from a plate: Gently touch a single, well-isolated colony on the agar plate with a sterile pipette tip or inoculating loop.
- If using a frozen stock: Thaw the stock on ice. Use a sterile pipette to transfer a small volume to the broth.
- Touch the tip or loop to the liquid medium and gently swirl. Be sure that at least one cell gets dislodged into the broth.
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Incubate the Culture:
- Place the inoculated tube or flask in an incubator set to the optimal growth temperature for your bacteria (e.g., 37°C for E. coli).
- Shake the culture: Most bacterial cultures grow best with aeration, so use a shaker incubator. A shaking speed of 200-250 rpm is often suitable. If a shaker is not available, loosely cap the container to allow for gas exchange.
- Incubation Time: Grow the culture for the appropriate amount of time, usually overnight (12-16 hours). However, the optimal incubation time may vary depending on the bacterial species and desired cell density. Monitor the culture's turbidity (cloudiness) to assess growth.
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Monitor Growth: As the bacteria grow, the liquid medium will become more turbid (cloudy). You can visually assess the growth or use a spectrophotometer to measure the optical density (OD) at a specific wavelength (usually 600 nm or OD600).
Example: Growing E. coli in LB broth with Kanamycin
Let's say you want to grow E. coli with kanamycin resistance in LB broth.
| Step | Description |
|---|---|
| 1. Medium Preparation | Prepare LB broth according to the manufacturer's instructions. |
| 2. Add Kanamycin | Add kanamycin to the LB broth to a final concentration of 50 μg/mL (if using a 1000X stock, add 1 mL of kanamycin stock per liter of LB). |
| 3. Sterilization | Autoclave the LB broth with kanamycin. |
| 4. Inoculation | Pick a single E. coli colony from a plate and transfer it to the sterile LB broth. |
| 5. Incubation | Incubate at 37°C with shaking (200 rpm) overnight. |
| 6. Growth Observation | Observe turbidity in the culture. Measure OD600 to quantify growth. |
Important Considerations:
- Sterility: Maintaining sterility throughout the process is essential to prevent contamination. Use sterile equipment and work in a clean environment (e.g., a biosafety cabinet).
- Bacterial Species: Different bacterial species have different growth requirements. Ensure you are using the appropriate medium, temperature, and incubation conditions for the specific bacteria you are culturing.
- Aeration: Adequate aeration is important for most bacterial cultures.
- Antibiotics: If using antibiotics, ensure they are added after the medium has cooled to prevent degradation.
