Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Preserving or maintaining micro-organism
Reexamination Certificate
1998-09-24
2003-08-26
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Preserving or maintaining micro-organism
C424S093400, C424S282100, C435S252100, C435S252330, C435S822000, C435S849000, C435S879000, C435S909000
Reexamination Certificate
active
06610531
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method of preserving bacteria for long-term storage without causing changes to the characteristics of the organism. More particularly, this invention relates to a method of preserving the bacteria by drying the bacteria in the presence of a preservative.
DESCRIPTION OF THE PRIOR ART
The maintenance of live bacteria in a stable form is critical for the conduct of basic microbiological studies and the development and production of diagnostic assays and vaccines. Furthermore, transport of bacterial strains between research or production facilities often requires the organisms to be in a preserved state to prevent damage or genetic alteration. Several methods exist for the preservation of bacterial cells. The two most widely used methods are freeze-drying (Crowe and Crowe 1991; Crowe et al., 1992) and desiccation (Carpenter et al., 1987 and 1988; Oliver et al., 1995; Roser, 1991; Shier, 1988). Methods for the freeze-drying of prokaryotic organisms, e.g. bacteria, has been relatively successful. Because successfully freeze-dried cells can be stored in the absense of freezing conditions, these procedures are more convenient and less expensive compared to super-cooled systems such as with liquid nitrogen. Cryoprotectants are included when cells are frozen in order to prevent the damaging effects of water crystals. Similarly, desiccation requires anhydroprotectants to prevent destruction of bio-molecules as the water is removed during the preservation process.
Some anhydrobiotic organisms are actually able to survive in a nearly completely dry or desiccated state without freezing. Members of the tardigrades, for example, are capable of surviving under these conditions (Crowe and Cooper, 1971). These organisms are highly complex, with heads, limbs and internal body parts similar to those of insects. Anhydrobiosis is made possible, in large part, through the elaboration and distribution of a sugar, trehalose, which supports cellular membrane structure against collapse by substituting for water at the polar head groups of the lipids (Crowe and Crowe, 1991, Crowe et al., 1992; Leslie et al., 1995; Mansure et al., 1994). It has been suggested that trehalose has cryoprotective properties (Crowe et al., 1992; Israeli et al., 1993; Leslie et al., 1994). U.S. Pat. No. 5,059,518 and 5,409,826 describe the use of trehalose as a stabilizer for preserving human cells by lyophilization.
Chemically, trehalose is a non-reducing disaccharide consisting of two linked glucose molecules and has approximately half the sweetness of sucrose. Empirical evidence indicates that high concentrations of trehalose in the tissues of certain insects and desert plants allows them to survive in a state of suspended animation under conditions of water deficiency (Hirsh, 1987). It has also been suggested that trehalose is an important factor in the survival of frogs during the frozen winter months (Lee et al., 1992).
U.S. Pat No. 5,149,653 describes the use of trehalose in the preservation of viruses. Unlike mammalian cells, however, live virus vaccines can not be easily frozen without loosing their immunogenic effect. Therefore, they must either be kept in aqueous media under cool sterile conditions such as in a refrigerator or stored at room temperature in the presence of preserving agent such as trehalose.
Depending on the species, bacteria can be stored either at room temperature, refrigerated as slant cultures on nutrient agar, or frozen. Storage of bacteria on agar, for those species that will tolerate it, is relatively convenient. However, it has been shown that organisms can genetically alter over time, especially the genetic material which are carried on plasmids. Additionally, not all bacteria can be stored for long periods on nutrient agar. Although most bacteria can be frozen, like viruses and eukaryotic cells, some alterations to the bacteria can occur upon thawing. Furthermore, recovery rates of bacteria are variable among species and among freezing conditions. Also, the process for freezing of cells is often relatively complex, requiring either super-cooled systems such as liquid nitrogen or mechanical freezers. In some circumstances, especially field conditions or operations studies conducted in developing countries, the availability and maintenance of super-cooled systems or mechanical freezers, or even refrigerators, is often problematic.
The prior art identified addresses the use of trehalose in the preservation of mammalian cells by lyophilization or, in the case of viral viruses by evaporation at ambient temperatures. The use of trehalose for the preservation of bacteria, which have distinct membrane structure compared to mammalian cells or viruses is not previously described. Although many strains of bacteria can withstand freezing and drying in the absence of a special preserving agent, because of the presence of a rigid outer wall, the efficiency of recovery is often poor. Furthermore, many, more fragile bacteria, are incapable of being preserved without cryoprotective additives. The prior art does not lead to a simple, effective and easy to conduct method of bacterial preservation applicable for a large range of bacterial species not requiring equipment such as freezers or the availability of liquid nitrogen. Additionally, the use of divalent cations, in conjunction with trehalose, as stabilizing agents is not previously described.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is to provide a new, effective and economical process for the preservation of bacterial species without requiring special equipment beyond what is typically found in microbiology laboratories.
An additional object is that the bacteria can be easily reconstituted with a high rate of survival.
A further object is that the bacteria retain characteristics and are not detectably altered after freezing and reconstitution.
These and additional objects of the invention are accomplished by drying bacteria in the presence of the cryopreservative trehalose and more preferably in the presence of certain divalent cations. The method of preservation involves drying bacteria in the presence of specific cryopreservation materials such that the organisms can be reconstituted in a viable form with little or no genetic damage. This process is capable of being used on a number of different bacterial genera and species, beyond those immediately described here. Following preservation and reconstitution the cells retain their genotypic and phenotypic characteristics.
REFERENCES:
patent: 4875921 (1989-10-01), Paau
patent: 5695541 (1997-12-01), Kosanke et al.
patent: 5800978 (1998-09-01), Goodrich, Jr. et al.
patent: 0120111 (1984-03-01), None
Mateczun Alfred J.
Peruski, Jr. Leonard F.
Harris Charles H.
Hemby, Jr. Joseph K.
Naff David M.
The United States of America as represented by the Secretary of
Ware Deborah K.
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