Glycerol stock is used in laboratories to store cultures for long periods of time.When liquid cultures are added to a solution of 50% glycerol, they become stable and can be stored safely.If you mix your samples, freeze them at 80 C to make sure they stay viable for as long as possible.
Step 1: You want to store thebacteria in a liquid culture.
In order for a stock to be effective, it must be combined with a liquid culture.You need an Erlenmeyer flask filled with lysogeny and the correct concentration of antibiotic to make a liquid culture.If you have prepared a liquid culture, you can put thebacteria into storage or isolated the plasmid DNA.LB liquid, also known as lysogeny broth, is a type of fluid used to growbacteria.It can be purchased from a store that sells laboratory equipment.There are a lot of different variables involved in the process of incubating.It is best performed by a trained technician in a controlled laboratory setting.
Step 2: A 50% glycerol solution can be created by liquefying pure glycerol in distilled water.
Measure out 10 mL of both liquids using a sterile pipette and combine them in a single flask.Shake the flask thoroughly to evenly mix the liquids.Diluting the glycerol will prevent it from damaging the cells.Scientists prefer to use a solution that is less than 40% in order to avoid compromising the culture.maximum longevity in storage can be provided by a 50% mixture.
Step 3: 50 microliters of 50% glycerol solution are transferred to a microfuge tube.
Carefully move the glycerol to avoid spills.You can place the sample into cold storage if you use this same container.If possible, use a tube with a screw top.During mixing and storage, snap top tubes can come open accidentally.
Step 4: You can add 50 microliters of your culture to the solution.
Use a fresh pipette to measure out an equal quantity of the liquid culture and pour it directly into the glycerol solution in the tube.The final proportions of your glycerol stock should be 50%.Reducing the amount of time it can survive in cold storage could be achieved by using disproportionate amounts of liquid culture.If you use a lower concentration of glycerol, you will need to adjust the amount of the culture.You can get more information from the literature relating to the strain.
Step 5: The tube has a lid.
If you use a snap top tube, push down the lid until you hear a soft click.Before moving on, confirm that the lid is secure.
Step 6: Shake the tube to mix the cultures.
When both liquids are evenly distributed, whisk the tube back and forth a few times.The cell will begin to absorb the solution at this point, which will protect it from damage.Shake the microfuge tube to make sure it doesn't come loose and keep your thumb pressed against the lid.
Step 7: The sample should be labeled.
Write the name of the culture on a small test tube label or strip of masking tape and stick it to the microfuge tube where it will be obvious.You can write the classification info on the lid with a felt-tipped marker.The exact strain of the sample or its origin can be useful during analysis if you record any other information.You can keep track of your sample containers contents as they are shuffled around in and out of storage if you label them.
Step 8: The sample should be frozen at 80 C.
The sample should be placed in a laboratory freezer with a constant temperature of 80 C.The culture will stay viable for a long time under these conditions.The temperature of the freezer needs to be fixed at 80 C.The preservedbacteria may die if it is set any warmer.
Step 9: A small amount of frozenbacteria is needed to prepare it for analysis.
When you need to revive your sample, open the lid of the freezer and remove the microfuge tube.Use a sterile inoculation loop, toothpick, or pipette tip to collect a small amount of frozen material from the top of the sample and streak thebacteria onto an LB agar plate for examination or testing.Don't let thebacteria thaw.It is possible to keep the stock tube on dry ice.When absolutely necessary, pull your sample out of the freezer.Its lifespan will be reduced considerably by repeated thaw and refreezing.