Lyophilization, or freeze-drying of bacterial cultures, stabilizes the cultures for long-term storage while minimizing the damage that may be caused by strictly drying the sample. Many microorganisms survive well when lyophilized and can be easily rehydrated and grown in culture media, after prolonged periods of time in storage.

Lyophilization is also used in the biotechnology and biomedical industries to preserve vaccines, blood samples, purified proteins, and other biological material.

This short laboratory procedure can be used with any commercially available freeze dryer to preserve your culture collection.

Working Principle of Lyophilization

Lyophilization is based on a simple principle of physics called “SUBLIMATION”. Sublimation is the process of transition of a substance from solid to the vapor state without passing through an intermediate liquid phase. The process of lyophilization consists of:

• Freezing of the product to convert the water in the product to ice form,
• Sublimation of ice directly into water vapor under vacuum.
• Drawing off the water vapor
• Once the ice has been sublimated, the products are freeze-dried and can be removed from machine.

The principle advantages of lyophilization as a drying process are:

• Minimum damage and loss of activity in delicate heat-liable materials
• Speed and completeness of rehydration
• Possibility of accurate, clean dosing into final product containers
• Porous, friable structure

The principle disadvantages of lyophilization are:

• High capital cost of equipment (about three times more than other methods)
• High energy costs (2-3 times more than other methods)
• Long process time (typically 24 hour drying cycle)

The lyophilization process

The technical explanation of lyophilization

Lyophilization involves the removal of water or other solvents from a given product by a process called sublimation. This occurs when the ice of a frozen product converts directly to the gaseous state without passing through the liquid phase. This enables the preparation of a stable product that is easy to use and store at ambient temperatures.

A low pressure environment is pre-requisite to allow this process to take place. In order to start the removal of water, the pressure inside the freeze dryer must be below the “triple point value” for the product, whilst also maintaining the temperature of the sample below its freeze point in the lyophilization process.

Pre-freezing – first stage of the lyophilization process

The sample is frozen, which means the water in the product is converted to ice, thereby the phase has changed from liquid to solid.
Slow pre-freezing will produce lager ice crystals, which are easier to lyophilize, whilst fast pre-freezing results in smaller crystals.

Primary drying – Second stage of the lyophilization process

In the second stage of lyophilization the sublimation process starts. The ice formed in the pre-freeze step is removed from the sample by the direct transition of the “solid” ice to a vapour without passing through a liquid phase. The resultant vapour is collected by the condenser, which has a lower temperature and pressure than the product. The vapour is thus converted back to ice on the condenser surface.
The “energy” required for this process to occur is provided by a gentle heating of the sample, which will start the sublimation process and eventually the sample will dry.

If too much energy (heat) is applied to the sample during this stage the condenser of the lyophilizer may not be able to condense the volume of vapours fast enough, the ice condenser temperature will subsequently rise along with its vapor pressure, thus increasing the risk of the sample melting.

Secondary drying – Third stage of the lyophilization process

Finally, any residual water present, which is strongly bound to the molecules of the sample, is converted to vapour and removed from the sample.

This water has invariably a vapour pressure lower than that of water in its “free” form.
Removal of the water in this final stage of lyophilization will be performed at higher product temperatures, consequently, any biological activity of the sample will not be impaired or affected. This usually involves increasing the temperature and lowering the pressure to provide enough energy to break down the molecular bonding. A process called desorption.