Introduction:

Glycerol stocks are essential for long-term storage of microorganisms such as bacteria and yeast. They enable researchers to maintain the viability of these cultures while minimizing genetic mutations that may occur during repeated subculturing. In this article, we’ll walk you through the process of preparing glycerol stock in 9 simple steps.

Step 1: Prepare Materials and Reagents

Begin by gathering the necessary materials and reagents:

– Microbial culture

– Sterile cryogenic tubes or vials

– Sterile inoculating loop or pipette

– Sterile 50% glycerol solution

– Incubator (temperature depends on the microorganism being cultured)

– Freezer capable of maintaining -80°C

Step 2: Grow Microbial Culture

Start by culturing your microorganism in suitable growth medium, under required conditions, and incubate until it reaches the log phase or stationary phase, depending on the specific organism.

Step 3: Prepare Glycerol Solution

If not already available, prepare a sterile 50% glycerol solution by combining equal volumes of glycerol and sterile distilled water. Mix well to ensure uniformity.

Step 4: Chill Glycerol Solution

Place the sterile glycerol solution in a refrigerator or on ice for at least 15 minutes prior to use. This helps to reduce heat shock when mixing with the microbial culture.

Step 5: Label Cryogenic Tubes

Label your cryogenic tubes or vials with relevant information such as date, strain, and sample identification.

Step 6: Transfer Microbial Culture

Using a sterile inoculating loop or pipette, transfer a small amount (e.g.,1 mL) of microbial culture into each labeled cryogenic tube.

Step 7: Add Glycerol Solution

Add an equal volume (e.g.,1 mL) of chilled sterile 50% glycerol solution to the microbial culture in each tube and mix gently by pipetting or vortexing. This should result in a final concentration of approximately 25% glycerol in the tube.

Step 8: Store Tubes

Place the cryogenic tubes or vials in a -80°C freezer for long-term storage. Ensure that they are tightly sealed and stored upright.

Step 9: Maintain Records

Document the addition of each new glycerol stock in your lab notebook or other record-keeping system. This should include details such as strain, date, freezer location, and any relevant notes about the culture’s growth.

Conclusion:

By following these 9 steps, you can successfully prepare glycerol stocks for long-term storage of your microorganisms. This practice is essential for maintaining culture viability and consistency during research applications.