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 then placed under a vacuum) allowing 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, and as long as 24 hours, not including culture growth time.

The Step-by-Step Process of Lyophilization

  1. First, grow an overnight culture, or lawn, of the microorganism on LB or other appropriate nutrient agar plate.

  2. Next, prepare sterile crimp-cap vials by autoclaving 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.

  1. Add 4 mL lyophilization buffer to the plate. If necessary, the cells can be suspended using a sterile glass rod.

  2. Quickly transfer the culture suspension to the sterilized vials. Add approximately 1.5 mL per vial. Seal with the rubber cap.

  3. Freeze the culture suspension inside the vials by placing the vials in a -20 degrees Celcius freezer.

  1. 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.

  2. Carefully, and aseptically, place the vial caps loosely on top of the vials, so that moisture can escape during the freeze-drying process, and place the vials into a freeze-dryer chamber. Apply the vacuum to the chamber according to the manufacturer's instructions.

  3. Allow the culture time to completely lyophilize (dry out). This may take from a few hours to overnight depending on the volume of each sample and how many samples you have.

  4. Remove the samples from the freeze-dryer chamber according to the manufacturer's instructions and immediately seal the vials with the rubber caps, then the crimp tops.

  5. Store the lyophilized culture collection at room temperature.

Products You'll Need

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

Reference:

SP Scientific. Basic Principles of Freeze Drying. http://www.spscientific.com/freeze-drying-lyophilization-basics/