LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.13.2
COMMENTS: FREEZE-DRYING OF FUNGI
1. Ampoules should be sealable; rubber stoppers are to be avoided or only used for a period of less than three months, because (a) they produce toxic gasses, and (b) they become leaky.
2. Choice of freeze dryer. The more critical the process, the more demands on the freezedryer. There are three basic types:
a..Manifold with or without external cooling. Depends on the capacity of the vacuum pump. The process can hardly be influenced. External radiation may strongly influence the temperature. Only suited for non-critical organisms, better to be avoided.
b. Spinfreezer. The cooling rate is by definition fast. Micro-organisms with large cells or thick walls have little chance of survival. The process is usually reproducible. The process is by necessity two-stepped (aeration between the steps necessary).
c. Shelf freezers. The most flexible and controllable method. A shelf freeze-dryer should:
a. Control the processes by independent registration of vacuum, product temperature, shelf temperature and condenser temperature (not by calculation of vacuum by registering the temperature or vice versa, because especially in small dryers this can be erroneous).
b. Be able to perform a pressure rise test to estimate the end of primary drying. If not, vials with a bacterial filter should be used, especially when pathogenic organisms are concerned. During freeze-drying with open vials microorganisms can cause cross-contamination; when ampoules are not sealed under vacuum, but under inert gas, the chances are even greater.
c.Be regularly be maintained and checked.
All processes should be reproducable and controlled.
1. For small, single-celled microorganisms (e.g. bacteria) the cooling rate is generally not critical, as they have a favourable surface/volume ratio and a thin cell wall. For large celled organisms (e.g. many fungi) a programmable freezer is necessary.
2.. Entry of oxygen during the process has to be avoided. When two-step freeze-drying is necessary, an inert gas (nitrogen) should be applied between the two steps. Vials should be closed either under vacuum or filled with inert gas.
3. It is important that the residual moisture content of the product reaches a certain minimum (below 2.5%). Independent measurement of vacuum and various temperatures enable a controlled process. If this is failing, each batch should be tested by the Karl Fischer method, to ensure quality (shelf life).
4. It is important to know the glass transition temperature of the Iyoprotectant, as the shelf temp has to be below the glass transition temperature of the product. This has to be established only once.
5. It is important to know the glass transition temperature of the dried product, as the storage temp has to be at least 10 degrees below the glass transition temperature of the dried product. This has to be established only once.
6. After freeze-drying the following tests are necessary:
a. Viability and quality of the strain for every batch of each strain (results should belogged or stored in database)
b. Vacuum check for all tubes (if not sealed under inert gas)
c. Karl Fischer test for residual moisture content, if controls are insufficient.
7. File registration form for freeze drying process per batch (vacuum, product temperature, shelf temperature, condenser temperature, time).
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998, updated August 1999
© The CABRI Consortium 1999 -