LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.13
FREEZE-DRYING OF FUNGI INTRODUCTION During freeze-drying organisms are dehydrated in the cooling phase preceding the actual drying. Because large, septate and/or thick-walled
FREEZE-DRYING OF FUNGI
During freeze-drying organisms are dehydrated in the cooling phase preceding the actual drying. Because large, septate and/or thick-walledpropagules dehydrate slowly, they have to be cooled at -1°C/min. despite the adverse effects (protein denaturation) of slow cooling. Since thin-walled propagules £ 5mm dehydrate much faster, they are preferably cooled instantaneously to below -75°C (see also cryopreservation).
During drying the lyoprotectant is converted into a glass. A glass is a liquid in which viscosity is so high that the molecules are immobilized. It is an ideal formulation to store dehydrated organisms because due to the immobility of the molecules there is neither enzyme- nor chemical activity. In the cooling phase preceding drying the bulk of extra-cellular water crystallizes and the remaining non-crystallizable part of the lyoprotectant becomes a glass. During primary drying the ice-crystals are evaporated, leaving a glass interwoven with channels. During secondary drying water evaporates through these channels from the glass, making it even more viscose. Because the viscosity increases, the temperature at which the glass is stable increases. By slowly raising the temperature, evaporation of water from the glass is enhanced. The batch is dried when the glass is stable at the storage temperature.
The temperature-regime, that must be applied to prevent melting (= collapse) of the glass during freeze-drying must be established with a freeze-drying microscope or a Differential Scanning Calorimeter (DSC) for each lyoprotectant/protocol.
For each lyoprotectant/protocol the glass transition temperature (Tg) of the dried product must be estimated by DSC. Tg is the temperature at which a glass melts during warming. The dried product must be stored at least 10 degrees below the Tg.
The temperature in the freeze-drying device must be raised at1°C/min to reach the secondary drying phase to avoid collapse.
Admission of oxygen during processing should be avoided as oxygen stimulates production of free radicals in dried products. Therefore vials should be closed either under vacuum or filled with inert gas.
When vials are closed with rubber stoppers, constrictable vials should be used which must be flame-sealed within three months as the stoppers produce toxic gasses and tend to become leaky.
For each batch vacuum, product-, shelf- and condenser temperature has to be registrated. Independent measurement of formerly mentioned parameters must be possible. When registration of formerly mentioned parameters is not possible, RMC must be established regularly.
The freeze-drying device must be sterilizable or vials have to contain bacterial filters.
Residual moisture content (RMC) of the dried products has to be checked for each protocol/lyoprotectant.
The vacuum should be checked for each sealed tube (if not sealed under inert gas).
Replacements should be made according to the maintenance plan, depending on Tg and RMC in relation to the storage temperature.
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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