The basics of lyophilization

By Carol Horton

Lyophilization, or the process of freeze-drying, is a valuable method for preserving materials for longer-term storage and stability. Similar to cryopreservation, when we talk about lyophilization we focus on the management of water during the preservation process. However, during lyophilization, water is removed through a process called sublimation to yield a stable, dehydrated material with a relatively indefinite shelf-life.

Multiple glass vialsTo lyophilize a microbial culture, there are three main steps that are performed. Initially, the material is frozen using a cryoprotectant, typically 20% skim milk, which differs from the freezing process used in cryopreservation. After the strain is frozen, the majority of the water is removed during a primary drying stage, which is achieved by applying both heat and pressure to vaporize the material. Here, a vacuum pump removes the non-condensable gasses from the system, while a condenser collects the water molecules before they enter the vacuum pump. After the initial drying stage, bound water must be desorbed under low pressure and low condenser temperatures.

There is a delicate balance between the heat and pressure requirements for lyophilization, as well as the rate at which condensable and non-condensable gases are collected or removed—this applies to both the primary and secondary drying stages. For this reason, it’s best to source a commercial freeze-dryer if you are thinking about this form of preservation.

Most freeze-dried cultures are stable at 2-8°C; however, lower temperatures will yield a longer shelf life. Further, since freeze-dried materials are hygroscopic, it’s imperative to store them sealed and away from moisture. Likewise, both heat and oxygen can impact the storage of a lyophilized culture. In fact, oxygen behaves like a free radical when it comes into contact with the dried material, and heat accelerates this process.

When recovering freeze-dried cultures from ATCC, a small amount of medium can be added to the material to rehydrate. Then, the entire re-suspended mixture can be transferred to a growth medium that has either been recommended by ATCC, or that was used prior to preservation. ATCC always recommends rehydrating the entire freeze-dried pellet during recovery; attempting to remove and culture only a portion of the pellet may yield poor to non-existent growth. Further, storage of opened materials may cause a rapid decline in viability due to exposure to oxygen and atmospheric moisture.