Preparing an agar plate is a straightforward process involving several key steps to create a solid growth medium for microorganisms. The following guide details the process using a recipe and sterile techniques, as described in the references:
Steps for Preparing an Agar Plate
1. Prepare the Medium
- Use a Recipe: Start by making the medium according to the specific recipe for the microorganisms you intend to culture. This usually involves combining nutrient broths with distilled water.
- Add Agar: Mix in the desired amount of agar powder, usually around 1% w/v (weight per volume) which will solidify the medium. Stir until evenly distributed.
2. Sterilize the Medium
- Autoclave: Sterilize the mixture by autoclaving it for 25 minutes at 121°C (250°F) under 15 PSI of pressure. This step is crucial to kill any existing microorganisms that might contaminate the agar plate.
3. Cool It Down
- Cool: Allow the autoclaved medium to cool down slightly to approximately 50°C (122°F). The agar solidifies around 45°C (113°F), and pouring when too hot could cause condensation and can damage the plastic Petri dishes.
4. Add Supplements (Optional)
- Supplement: Add any heat-sensitive supplements (like antibiotics) after the medium has cooled to prevent their degradation. These are added to provide specific growth conditions for the desired microorganisms. Mix gently to distribute them evenly.
5. Pour the Plates
- Pour: Working in a sterile environment (e.g., under a laminar flow hood or near a Bunsen burner flame), pour the molten agar medium into sterile Petri dishes. Fill each dish to a consistent depth, generally about 3-4 mm.
6. Allow to Set
- Set: Allow the poured agar to solidify and cool completely. This will take about 20-30 minutes. Avoid moving or disturbing the dishes during the setting process.
7. Dry the Plates
- Dry: Remove any condensation on the lid and invert plates. Leave the plates with their lids slightly ajar to reduce condensation overnight in a low temperature incubator or a sterile environment. This drying process reduces excess moisture which could interfere with the growth of microorganisms.
8. Use or Store
- Use or Store: Your agar plates are now ready for use. If you're not using them immediately, store them in a sealed bag or container at 4-8°C to prevent them from drying out. They can be stored for up to a few weeks under these conditions.
Summary of Steps in Table Form
| Step | Action | Notes |
|---|---|---|
| 1 | Prepare Medium | Mix recipe and agar |
| 2 | Autoclave | Sterilize for 25 mins |
| 3 | Cool It | Allow to cool to around 50°C |
| 4 | Supplement (Optional) | Add heat-sensitive substances |
| 5 | Pour Plates | Pour into sterile Petri dishes |
| 6 | Set | Allow to solidify |
| 7 | Dry | Remove condensation |
| 8 | Use or Store | Use or store in a sealed container at 4-8°C |
