1. Solution Preparation: Begin by adding a portion of the required water to a vessel. Weigh all the necessary ingredients according to the recipe and add them one by one to dissolve. Once all components are fully dissolved, top up the volume with water. For substances like peptone or meat extract, heating is necessary to ensure complete dissolution. Any water lost during heating should be replenished after all materials are dissolved.
When preparing a solid medium, take the previously prepared liquid medium, bring it to a boil, then add the measured agar. Continue heating until the agar is completely melted. Stir continuously to prevent the agar from sticking to the bottom and burning.
2. pH Adjustment: Test the pH of the medium using a pH test strip or a pH meter. If the pH is not within the desired range, adjust it using 10% HCl or 10% NaOH until the correct value is achieved.
3. Filtration: Filter the prepared medium using filter paper, gauze, or cotton wool. When using gauze, fold it into six layers for better filtration. For filter paper, fold it into a cone shape and place it in a funnel for efficient filtering.
4. Sterilization and Packaging: After filtration, transfer the medium into appropriate containers. For slant media, fill test tubes; for plates or liquid media, use Erlenmeyer flasks. While dispensing, hold the spring clip with one hand to control the flow, and carefully pour the medium into each container. Avoid letting the medium stick to the nozzle or bottle mouth to prevent contamination from moisture soaking the cotton plug.
The amount of medium per test tube depends on its size. For example, a 15×150 mm test tube should be filled with about 3–4 ml (1/4–1/3 of the height) for slant media. A 20×220 mm tube can hold 12–15 ml for deep cultures. In flasks, fill approximately half of their volume.
5. Cotton Plug Insertion: After filling, insert a cotton plug into the opening of the test tube or flask. The purpose of the plug is to filter air and prevent microbial contamination. Use clean, dry cotton—avoid absorbent cotton that may become unusable. To make the plug, shape the cotton to fit the container, forming a long, firm rod. Insert it so that about two-thirds remain inside the container, with a small part exposed for easy removal. Cover the containers with thick paper and tie them securely before sterilization.
6. Preparing Slant and Plate Media: After sterilization, if you need to create slant or plate media, ensure the medium is still liquid. For slant media, place the test tubes on a wooden board to allow the medium to cool and solidify at an angle. For plate media, work near the edge of the lab bench. Light an alcohol lamp, open the flask slightly, and quickly pour the medium into Petri dishes. Fill each dish with about 10 ml to cover the bottom. Allow the medium to solidify for 15 minutes, then stack the plates, invert them, and incubate for 24 hours. If no contamination occurs, the medium is ready for use. This method is also suitable for storing cDNA libraries created on plasmid vectors.
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