How to Freeze-Dry a Bacterial Culture (Lyophilization)

Lyophilization, or freeze drying, is a short laboratory procedure

Bacteria cultures in red agar gel in petri dishes
Credit: Phillip Hayson / Getty Images

Freeze-drying, also called lyophilization or cryodesiccation, is the process of removing water from a product after it's frozen and placing it in a vacuum. This allows ice to change from a solid to a vapor, without going through a liquid phase.

Ice (or other frozen solvents) are removed from a product through the process of sublimation and bound water molecules are removed through the process of desorption.

The Basics of Lyophilization

One of the best ways to store a bacterial, fungal, yeast or other microorganism culture for long periods of time is to use the process of freeze-drying. This short laboratory procedure can be undertaken with any commercially available freeze-dryer that will preserve your culture collection. 

Because lyophilization is the most complex and expensive form of drying, the process is usually restricted to delicate, heat-sensitive materials of high value. Substances that are not damaged by freezing can usually be lyophilized so that refrigerated storage is unnecessary.

This process can take as little as three hours, or as long as 24 hours (not including culture growth time).

Products You'll Need

  • Freeze-dryer
  • Autoclave
  • Nutrient or other appropriate agar plates
  • Incubator to grow the culture
  • Glass rod
  • Lyophilization buffer
  • Crimp-top vials with rubber stoppers (and a crimper to apply the caps)
  • Freezer

The Step-by-Step Process of Lyophilization

  1. Grow your overnight culture, or lawn, of the microorganism on Luria broth or other appropriate nutrient agar plates.
  2. Prepare sterile crimp-cap vials by autoclaving (method of sterilizing using steam, pressure and heat) ahead of time, with the caps (rubber stoppers) placed loosely on top. Place paper labels printed with the culture's identification inside the tubes prior to autoclaving. Alternatively, use tubes with caps designed for sterility.
  3. Add 4 milliliters of lyophilization buffer to the plate. If necessary, the cells can be suspended using a sterile glass rod.
  4. Quickly transfer the culture suspension to the sterilized vials. Add approximately 1.5 milliliters per vial. Seal with the rubber cap.
  5. Freeze the culture suspension inside the vials by placing the vials in a freezer set at minus 20 degrees Celsius.
  6. Once the cultures are frozen, prepare the freeze-dryer by turning it on and allowing time for the appropriate temperature and vacuum conditions to stabilize. Do this according to the manufacturer's instructions for the particular brand of freeze-dryer you're using.
  7. Carefully, and aseptically, place the vial caps loosely on top of the vials so that moisture can escape during the freeze-drying process. Place the vials into a freeze-dryer chamber and apply the vacuum to the chamber according to the manufacturer's instructions.
  8. Allow the culture time to completely lyophilize (dry out). This may range from a few hours to overnight depending on the volume of each sample and how many samples you have.
  9. Remove the samples from the freeze-dryer chamber according to the manufacturer's instructions and immediately seal the vials with the rubber cap and crimp the tops.
  10. Store the lyophilized culture collection at room temperature.