Method of freeze-drying organisms

Details Method of freeze-drying organisms Method of freeze-drying organisms

1999-11-04

2001-11-27

Redding, David A. (Department: 1744)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S001300, C435S040500, C435S284100, C435S288400, C435S305200, C435S307100

Reexamination Certificate

active

06322994

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the freeze-drying of multiple samples of microorganisms such as bacteria in a multiwell format that may be stored and used at room temperature.
BACKGROUND TO THE INVENTION
Freeze-drying has been used in various applications for several years, such as in the food industry and pharmaceutical industry. Freeze-drying has also been used to prepare microorganisms such as bacteria for long term storage. In particular microorganism depositories such as the American Type Culture Collection often freeze-dry cultures for distribution since properly freeze-dried microorganisms remain viable at room temperature and can be transported without the need for solid carbon dioxide.
However, freeze-drying equipment is too expensive for day-to-day use by laboratory scientists who tend to keep microorganisms either as glycerol stocks at −80° C. or in liquid nitrogen. Both forms of storage are relatively expensive and also carry the risk of loss of sample viability if the equipment fails. Nonetheless, a significant amount of storage space in −80° C. freezers is given over to the storage of bacterial strains as glycerol stocks, for example to store bacterial colonies selected during cloning procedures. There is therefore a need to provide laboratory scientists with a means of storing large numbers of different microorganisms in an easily accessible format that does not require expensive freezer space.
SUMMARY OF THE INVENTION
We have now found that it is feasible to freeze-dry multiple samples of different microorganisms in microtiter plates, without cross contamination, resulting in microtiter plate “libraries” of microorganisms that may be stored at room temperature at high density. The resulting saving in high cost freezer space is likely to compensate for any initial outlay of freeze-drying equipment. Furthermore, the use of a room temperature microtiter plate format is more compatible with recent advances in automated machinery for screening bacterial libraries picked by machine.
Accordingly the present invention provides a method for freeze-drying multiple samples of viable microorganisms which method comprises:
(i) providing a container comprising multiple wells;
(ii) dispensing multiple liquid samples, each sample comprising a viable microorganism, into separate wells of the container;
(iii) placing the container in a freeze-drying apparatus; and
(iv) freeze-drying the samples present in the wells under conditions that substantially maintain the viability of the microorganisms.
Preferably the container is a microtiter plate.
In a preferred embodiment, the viable microorganisms have been grown prior to freeze-drying in a culture medium comprising an excipient and the microorganisms are freeze-dried in said culture medium.
Preferably the wells of the container are provide with sterile sealing means. More preferably, the sealing means permit removal of a portion of a freeze-dried microorganism sample from a well whilst maintaining a sterile seal.
The present invention further provides a container comprising multiple wells, each well comprising a viable freeze-dried sample of a microorganism. Preferably, the container is a microtiter plate.
In a preferred embodiment, the container further comprises sterile sealing means for each well. Preferably the sterile sealing means allow the removal of a portion of a freeze-dried microorganism sample from a well whilst maintaining a sterile seal.
The present invention also provides a container of the invention obtained by the method of the invention.


REFERENCES:
patent: 4879239 (1989-11-01), Daggett et al.
patent: 5858770 (1999-01-01), Perlman
patent: 5925511 (1999-07-01), Fuhr et al.
Franks; “Effective Freeze-Drying: A Combination of Physics, Chemistry, Engineering and Economics”; The Institute of Marine Engineers; 1994-1995; pp. 32-39.
A Guide to Freeze Drying for the Laboratory; An Industry Service Publication; 1998; pp. 3-11.
LESLIE et al., “Trehalose And Sucrose Protect Both Membranes and Proteins in Intact Bacteria During Drying”,Applied And Environmental Microbiology, vol. 61(10):3592-3597, (1995).
BARBAREE et al., “Problems In Freeze-Drying: II. Cross-Contamination During Lyophilization”,Developments in Industrial Microbiology, vol. 26:407-409, (1984).

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