Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-03-03
2002-05-21
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S182000
Reexamination Certificate
active
06391576
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for isolating a microorganism, and more particularly to a method for isolating a microbe which would otherwise be difficult to isolate using conventional plate culture methods.
2. Discussion of the Background
Conventionally, isolation of a microbe has been conducted by a plate culture method. This method includes spreading a sample solution containing various species of microbes and placing the seeded plates in nutritional and environmental conditions which enable the microbes to grow and form colonies on the plates. Subsequently, the formed colonies are isolated from the plate. However, the plate culture method is not useful for isolating a microbe that is unable to form colonies on the plate under the conventional nutritional and environmental conditions of the plate culture method. To find suitable conditions, plating is usually repeated many times under various nutritional and environmental conditions until such time that the microbe will form a colony. However, it is an impractability to formulate and prepare suitable nutritional and environmental conditions which allow unknown microbe species to grow on a plate for subsequent isolation.
In view of the above, only limited microbe species can be isolated from a sample containing various microbe species by the plate culture method. For example, Torsvik et al (Appl. Environ. Microbiol. 56, 782-787 (1990)) report that the proportion of microorganisms capable of forming a colony was 0.3% of all microorganisms existing in soil. Wagner et al (Appl. Environ. Microbiol. 59, 1520-1525 (1993)) report that the proportion of microorganisms capable of forming a colony was only 1 to 15% of all bacteria existing in active sludge. Staley et al (Annu. Rev. Microbiol. 39, 66-68 (1985)) report that the proportion of microorganisms capable of forming a colony was 0.1 to 1% of all microorganisms existing in neutral lake water. Ferguson et al (Appl. Environ. Microbiol. 42, 49-55 (1984)) and Kogure et al (Can. J. Microbiol. 25, 415-420 (1979) and Can. J. Microbiol. 26, 318-323 (1980) examined microbes in the sea and report that the proportion of microbes capable of forming a colony was 0.001 to 0.1% of all microorganisms in the sea. Jones (Freshwater Biol. 7, 67-91 (1997)) examined lake water and sediments and reports that the proportion of microbes capable of forming a colony was 0.25% of all microbes in both cases.
Although a considerably large number of microbe species fail to be isolated by the plate culture method as described above, isolation of such microbe species is intensely required in various fields. Examples of such fields includes the food industry, the medical and pharmaceutical industries, and environmental protection from pollution such as water protection. Furthermore, it is necessary to isolate microbe species of which isolation is difficult by the plate culture method for finding and utilizing novel functions of such microbes or for suppressing harmful activities thereof in the natural environment. Examples of such microbes are those that may be found in extreme environments such as the deep sea and from organisms such as humans, insects, soil protozoa, and sea organisms. Thus, a significant need exists for the isolation of microbe species that could not normally be isolated by prior methods of microbe isolation, e.g., plate culture methods.
SUMMARY OF THE INVENTION
Thus, the object of the present invention is to provide a method for isolating a microbe species from a sample containing various microbe species whereby isolation by the known plate culture method would otherwise be difficult.
This object is achieved by the present method comprising the steps of:
encapsulating microbe cells in agarose gel particulates, wherein some of the particulates contain a single cell, whereas the other particulates contain more than one cell;
subsequently incubating the particulates in nutritional and environmental conditions that enable the microbe contained in the sample solution that can grow on a plate of a plate culture method to grow in the agarose gel particulate; and
after incubation, isolating the particulates having single cells from the group of the particulates having more than one cell.
REFERENCES:
patent: 4647536 (1987-03-01), Mosbach et al.
patent: 4659655 (1987-04-01), Rose
patent: 4695548 (1987-09-01), Cantor et al.
patent: 4927761 (1990-05-01), Reading et al.
patent: 5643569 (1997-07-01), Jain et al.
patent: 07252157 (1995-10-01), None
Kurane Ryuichiro
Manome Akira
Tsuchida Takayasu
Gitomer Ralph
Japan Bioindustry Association
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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