Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Patent
1996-10-17
1998-05-26
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
435 72, 435 21, 435 34, 435 39, 435196, 4352536, C12Q 106, C12Q 142, C12Q 166, C12N 120
Patent
active
057563039
DESCRIPTION:
BRIEF SUMMARY
FIELD OF TECHNOLOGY
The present invention relates to a culture medium and a microbiological test method employing said culture medium, more specifically, to a culture medium employed in a microbiological test by ATP-luciferase method wherein the culture medium causes no disturbance in measurement due to the emission derived from a substance other than a microorganism and to a microbiological test method which employs said culture medium and is simple, accurate and highly reliable.
The present invention can be utilized effectively in a microbiological test of beer, more typically in a microbiological test of a bottled draft beer, which contains a small count of microorganisms.
BACKGROUND TECHNOLOGY
Adenosine triphosphate (ATP) is present specifically and locally in a viable cell. ATP-luciferase method wherein ATP is subjected as a coenzyme to luciferin-luciferase reaction and then a slight amount of the light emitted in proportion with the content of ATP is detected by a highly sensitive detector whereby confirming the presence of the microorganisms is now regarded as an interesting rapid test method to examine microorganisms.
In such a microbiological test by ATP-luciferase method, a conventional medium has not been able to be employed in a test for a small count of the microorganisms as a test for the microorganisms in a bottled draft beer, even using membrane filtration, since the medium itself emits a light.
In an attempt to solve this problem, a method of removing free ATP by washing a membrane prior to luciferase treatment was proposed (Laid-open Japanese Patent Application, Unexamined No. H2-163098).
However, this method required complicated processes which was not suited to a large scale treatment of samples.
As another method of removing free ATP, a method of treating medium components with apyrase (ATP lytic enzyme) is known (Neth. Milk Dairy J. 43, 347, 1989).
However, apyrase has an ATP lytic ability varying depending on the medium components, and can not decompose sufficiently in some cases. Accordingly, it is not effective in all medium components.
Therefore, a medium has been desired which suppresses the emission due to free ATP sufficiently, has a low background emission level and has no emissions which may cause false identification of microorganisms.
An objective of the present invention is to provide a culture medium which eliminate the problems experienced conventionally as mentioned above, and which is employed in a microbiological test by ATP-luciferase method without causing disturbance in measurement due to the emission of a substance other than a microorganism, as well as a simple, accurate and highly reliable method for microbiological test employing said culture medium.
Accordingly, another objective of the present invention is to provide a microbiological test method by which free ATP which disturbs the measurement is eliminated effectively, the background emission level is suppressed at a low level, cells can be counted even in the order of several cells in a test sample, the signals due to the microorganisms are distinguishable from those of background, for example, in a test by ATP-luciferase method for microorganisms trapped on a membrane filter, and thus the reliability of the test is ensured.
DISCLOSURE OF THE INVENTION
Thus, the present invention provides as the first aspect a culture medium for microbiological tests prepared by treating medium components containing adenosine triphosphate (ATP) with an acidic phosphatase.
The present invention also provides as the second aspect a microbiological test method characterized by using the culture medium for microbiological tests which is the first aspect of the present invention as a culture medium for microbiological test when the microbiological test is conducted by ATP-luciferase method.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a graph indicating the amount of the emission of each yeast extract after enzymatic treatment in Example 1.
FIG. 2 shows the amount of the emission of each culture medium in Example 3 .
REFERENCES:
patent: 3745090 (1973-07-01), Chappelle et al.
patent: 3971703 (1976-07-01), Picciolo et al.
APS Abstract Satou et al Japan 08-224096 (Sep. 3, 1996).
Derwent Abstract 90-235350/31 Japan Organo KK J02163098 (Jun. 22 1990).
Nakakita Yasukazu
Sato Toshihiro
Lilling Herbert J.
Sapporo Breweries Limited
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