Storage of Bacterial Cultures at 4°C

Storing bacteria in a refrigerator is practical for short periods of time. Bacteria are best preserved by freezing at ultralow to cryogenic temperatures, or by freeze drying. However, some bacterial cultures may be used daily, thus having a readily available active culture can be practical. Cultures stored at 4°C are sensitive to more rapid die off than frozen or freeze dried bacteria. Often researchers store cultures on plates which are very susceptible to fungal contamination and drying. Consequently, refrigerated bacterial stocks are best stored in small vials containing agar media. Many protocols call for the use of half dram vials, which is simply a small volume glass vial with a screw on cap (a dram is a volume measurement of 1/8 oz.). Small 1-2 ml cryogenic vials, that are pre-sterilized, can also be used for storing stock cultures. Cryogenic vials are more readily available and less expensive.

Preparation of the Vials

  1. Prepare enough culture media, e.g., tryptic soy agar, so that each vial will hold 1-2 ml. Sterilize the media in an autoclave for 15 min. at 121°C. Remove media from the autoclave and allow it to cool down in a water bath to between 55 and 65°C.
  2. In a biosafety hood, prepare a rack for the cryovials that is tilted about 45° on its side. A small stack of paper towels often works well for supporting a tilted rack. When media is pipetted into the cryovials and placed in the rack, an agar slant will form in the vial as it cools.
  3. All work in the hood requires aseptic technique. Place cryovials, pipettes (either a serological or repeating pipette will work), in the hood.
  4. Transfer the liquid agar to the hood. Work efficiently so that the agar does not solidify. Fill each vial to half full, loosely cap, and place in the rack. The agar should form a short slant when in the rack.
  5. Allow the agar to cool. Tighten the caps to seal the vials and store at 4°C.

Preparing Bacterial Stocks

  1. Obtain a prepared vial and allow it to equilibrate to room temperature. Using an inoculating needle/loop and aseptic technique, open the vial and streak the surface of the agar with a sample of bacteria.
  2. Organisms that are facultative anaerobes can also be stabbed into the solid agar using the needle. This does not work well if using a loop.
  3. Label the tube with the organism and date.
  4. Culture the organism in an incubator until growth is visible, then seal and store at 4°C.

Using Refrigerated Bacterial Stocks

  1. Bacteria stored on slants in small vials may only be viable for a couple of months up to a year. Do not depend on them to be viable for years.
  2. To use, aseptically open the vial and remove a sample from the slant or agar stab. Streak on to a suitable medium.
  3. Cap the vial and return to the refrigerator.