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Storing Bacterial Strains

Introduction

If you are using a bacterial strain for a science project, it is a good idea to store the strain, at least temporarily, until after you are done with your experiment. After all, you may find that your experiment fails on the first try, or that the results of your experiment raise more exciting questions that you want to address with additional experiments. To ensure that you don’t “run out of” bacteria, here are two possible storage methods:

Short-term Storage of Bacterial Strains

Materials and Equipment

  • Permanent marker
  • Agar petri dish (1); same type as you are using for your experiment. If your experiment does not specify a type of media, chose nutrient agar. Nutrient agar is very rich and will support the growth of a wide range of bacteria.
  • Bacterial culture
  • Mixing tool; use one of the following
    • Sterile inoculating loop (1)
    • Sterile cotton swab (4); from a new package counts as sterile
    • Sterile toothpick (4); autoclaved
  • Bunsen burner (if using sterile inoculating loop)
  • 37°C incubator (optional)
  • Parafilm® or a resealable plastic bag
  • Refrigerator

Procedure

  1. Using a permanent marker, write the date and name of the bacterial strain on the bottom of the agar petri dish.
  2. Dip the sterile inoculating loop, cotton swab, or toothpick in the bacterial culture and follow the standard lab bacterial streaking protocol.
  3. Incubate the streaked agar plate either overnight in a 37°C incubator, or at room temperature, for 48 hours. Make sure the plate is resting on its lid to prevent water condensation from falling onto the agar.
  4. After incubation, you should see that the bacteria have grown on the agar plate.
    1. There should be regions of heavy bacterial growth where you first started streaking, and individual colonies where you finished the streaking procedure.
    2. Examine the colonies carefully. All colonies should have similar morphologies (textures, colors, and border shapes). If there are colonies with different morphologies, then you have a contaminant.
      • If you have a contaminant, you will need to streak out each type of colony on a separate plate to get a pure culture and use other information about the bacteria you are working with (for example colony morphology, media preferences, and antibiotic susceptibility/resistance) to determine which bacteria is the one you want.
  5. To prevent the agar plate from dehydrating, which will eventually kill the bacteria, either wrap the plate in Parafilm, or place it in a resealable plastic bag.
  6. Bacterial plates should be stored in a refrigerator. They will be good for up to one month under these conditions.
    Caution: It is not recommended to store bacteria in a refrigerator that is used to store food. If you absolutely must store your bacteria in the same refrigerator as food, place the bacteria plate inside two layers of Parafilm or resealable bags to minimize the risk of contaminating the food.

     

Long-term Storage of Bacterial Strains

Materials and Equipment

  • Permanent marker
  • Fresh liquid bacterial culture; for most bacteria, a culture grown overnight, or for approximately 8–12 hours, works best for freezing
  • Micropipette (P1000) (1) and sterile tips (2)
  • Sterile microcentrifuge or screw-cap tube (1)
  • Sterile glycerol (autoclave to sterilize)
  • Liquid nitrogen (optional)
  • Resealable plastic bag
  • Freezer

Procedure

  1. Using a permanent marker, label a sterile microcentrifuge or screw-cap tube with the date and the name of the bacteria.
  2. Using a micropipette, add 150 µl of sterile glycerol to the tube.
  3. With a new tip, use the micropipette to transfer 850 µl of the bacterial culture to the same tube.
  4. Cap the tube and invert it several times to thoroughly mix the glycerol and bacteria.
  5. If you are going to store the bacteria in a special -80°C freezer, you should first snap-freeze the bacterial stock by dropping it in a container of liquid nitrogen. If you are storing the bacteria in a regular -20°C freezer, the bacterial stock can be placed there with no further treatment.
    Caution: It is not recommended to store bacteria in a freezer that is used to store food. If you absolutely must store your bacteria in the same freezer as food, place the bacterial stock inside two layers of freezer-tolerant resealable bags to minimize the risk of contaminating the food.

     

Credits

Parafilm® is a registered trademark of American National Can Company.

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