Making E. coli agar plates involves creating a nutrient-rich solid medium that supports the growth of E. coli bacteria. Here’s a step-by-step guide:
1. Prepare the Agar Medium:
- Gather materials: You'll need agar powder, distilled water, a suitable growth medium (e.g., LB broth or minimal medium), any desired supplements (e.g., glucose, amino acids), and antibiotics (if selecting for specific strains).
- Mix the ingredients: Typically, for LB agar, you would mix approximately 15 grams of agar powder per 800 ml of distilled water. Add any desired growth medium concentrate according to the manufacturer’s instructions, along with your chosen carbon source (e.g., glucose). The final volume should be 1 liter. Exact amounts will vary depending on the medium being used; consult the medium's instructions.
2. Autoclave the Medium:
- Sterilize the mixture: Autoclave the agar mixture at 121°C (250°F) for 15 minutes to sterilize it. This eliminates any contaminating microorganisms. Allow the autoclave to fully depressurize before opening.
3. Cool and Supplement the Medium:
- Cool down: Let the autoclaved agar cool down to approximately 50°C (122°F). This is important because adding heat-sensitive supplements at higher temperatures can degrade them.
- Add supplements and antibiotics (if required): At 50°C, aseptically add any desired supplements or antibiotics to the cooled agar. Use sterile technique to prevent contamination. Antibiotics should be filter-sterilized before addition to the medium.
4. Pour the Agar Plates:
- Pour the plates: In a sterile environment (e.g., near a Bunsen burner or in a laminar flow hood), pour approximately 32-40 ml of the cooled agar mixture into each sterile Petri dish. This volume generally yields about 25-30 plates per liter of medium.
- Swirl gently: Immediately after pouring, gently swirl the plate to ensure even distribution of the agar.
5. Allow to Solidify:
- Solidification: Allow the agar to solidify completely at room temperature. This usually takes about 30-60 minutes.
6. Storage:
- Storage: Once solidified, store the agar plates upside down in sealed plastic bags in the refrigerator (4°C or 39°F) to prevent condensation and dehydration. They can typically be stored for several weeks.
Important Considerations:
- Sterility: Maintain strict sterile techniques throughout the entire process to prevent contamination.
- Antibiotics: When using antibiotics, ensure they are properly stored and used at the correct concentration for effective selection.
- Medium Type: The specific agar medium used (e.g., LB agar, minimal agar) will depend on the experimental needs.
- Quality Control: Regularly check plates for contamination before use.
By following these steps, you can create E. coli agar plates suitable for a variety of microbiological applications.
