1. Solution Preparation: Begin by adding a portion of the required water to the container. Weigh all the necessary ingredients according to the recipe, and add them one by one to dissolve. Once all materials are fully dissolved, top up with water to reach the desired volume. For substances like peptone or meat extract, it's essential to heat the mixture to ensure complete dissolution. Any water lost during heating should be replenished once all components are dissolved.
When preparing a solid medium, take the previously prepared liquid medium, bring it to a boil, and then add the measured agar. Continue heating until the agar is completely melted. Stir constantly to prevent the agar from sticking to the bottom and burning.
2. pH Adjustment: Use a pH test strip or a pH meter to check the pH of the medium. If the pH is not within the required range, adjust it using 10% HCl or 10% NaOH. Ensure the final pH matches the specifications for your culture.
3. Filtration: Filter the prepared medium using filter paper, gauze, or cotton. When using gauze, fold it into six layers for better filtration. With filter paper, fold it into a cone shape and place it in a funnel for efficient filtering.
4. Packaging: After filtration, pour the medium into appropriate containers. For slant media, transfer the medium into test tubes. For plate, liquid, or semi-solid media, use Erlenmeyer flasks. While pouring, hold the spring clip to control the flow and carefully fill each container. Avoid letting the medium stick to the neck or mouth of the container to prevent contamination from moisture on the cotton plug.
The amount of medium in each test tube depends on its size and purpose. For example, a 15×150 mm test tube used for slants should contain about 3–4 ml (1/4 to 1/3 of the tube’s height). For deep cultures, a 20×220 mm tube should be filled with 12–15 ml. In conical flasks, fill approximately halfway.
5. Adding the Cotton Plug: After filling, insert a cotton plug into the mouth of the container. The main purpose of the plug is to allow air to pass while preventing microbial contamination. Use clean, dry cotton—avoid absorbent cotton as it may become unusable. To make the plug, press the cotton between your thumb and index finger, forming a long, cylindrical shape. Insert it tightly into the container without over-tightening. Ensure that about two-thirds of the plug is inside the container, leaving a small part exposed for easy removal. Cover the containers with thick paper and tie them securely for sterilization.
6. Preparing Slant and Plate Media: After sterilization, you can create slant or plate media. The medium should remain in a liquid state before being poured. For slant media, place the test tubes on a wooden block so that the medium naturally slopes as it cools. For plate media, after pouring the medium into Petri dishes, let it solidify for about 15 minutes. Then, stack the plates, invert them, and incubate. After 24 hours, check for contamination. If no growth is observed, the medium is ready for use. This method is also ideal for storing cDNA libraries created using plasmid vectors.
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