Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-05-05
1999-10-05
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 34, 435962, 435963, 435 4, 4352831, 422 50, 422 681, 422255, 422256, 252349, C12Q 142, C12Q 104, C12Q 100, G01N 3353
Patent
active
059622474
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to an assay for microorganisms and to a device for use in such an assay.
BACKGROUND OF THE INVENTION
Rapid microbial testing by ATP bioluminescence demands assays that will work in a variety of sample matrices. The microorganisms can be determined by lysing them and measuring the light emitted in the presence of luciferase, as a function of the ATP contained in the microorganisms. However, before detection can take place, it is often necessary to separate the organisms from the remainder of the sample, which may contain contaminating amounts of ATP.
Separation of the microorganisms from their environment poses particular problems in aqueous samples also containing fatty material, e.g. milk. It has been proposed to centrifuge such samples with a clearing agent in a centrifugation tube, to give a pellet comprising the intact microorganisms, supernatant liquid and, above that, a fatty layer. The fatty layer and the liquid can be removed by aspiration, but the removal of fatty material is inefficient and time-consuming. It is also unsuited to ready use by unskilled personnel, e.g. for simple testing of samples of milk at a large dairy or distribution depot. If the sample is cream, which may contain 40% fat or more, the problems are greatly magnified and the technique of aspiration is not considered suitable.
Centrifugation tubes are known, which additionally comprise, as a sliding fit therein, a less deep inner tube whose base is a filter material. Sample is introduced into the tubes when fitted together, and centrifugation enhances filtration, leaving solids on the filter.
SUMMARY OF THE INVENTION
It has now been appreciated that, by slight adaptation of a centrifugation tube of the type described above, such that the base of the inner tube has one or more apertures sufficiently large to allow the passage of particles, the inner tube can be used to remove efficiently, not solids but rather a fatty deposit lying above an aqueous supernatant layer. Such a tube can therefore be used to solve the problems associated with the separation of microorganisms from cream. Aspiration is unnecessary. After the fatty material has been removed, supernatant liquid can be removed simply by decantation, although aspiration may be used if desired.
DESCRIPTION OF THE INVENTION
The term "centrifugation tube" is used herein merely to define the outer of the two mating tubes in a device of the invention. It may conveniently be provided with a tapering base in which a solid pellet is formed, on centrifugation. The inner tube is less deep, only in order to define a volume, beneath its base, in the centrifugation tube. This volume will be determined only by the need to ensure that, in a sample of a given amount, the fatty content will lie above its base, after separation of the sample, on centrifugation.
Unlike two-part centrifugation tubes of the type already known, the base of the inner tube according to the invention is adapted not to retain solids, and indeed to allow the passage of all components in the sample in either direction. It is thus used, not to cause separation during centrifugation, but only after centrifugation has caused separation into a solid pellet, and supernatant liquid and fatty layers.
The fatty layer is simply removed by taking out the inner tube, leaving the centrifugation tube containing solid material (including intact microorganisms) and supernatant liquid.
On removal of the inner tube, supernatant (aqueous) liquid drains through the aperture(s) in the inner tube. The layer of fatty material is sufficiently firm that it is retained by the inner tube, without immediately draining out, and can be disposed of separately.
The liquid can now be poured off. In order to remove liquid completely, aspiration may be used, but an equally effective way of ensuring that any liquid remaining after decantation does not affect the result is to add an ATPase. Microorganisms in the solid pellet can now be assayed by conventional means, e.g. by bioluminescence.
ATPase
REFERENCES:
patent: 1469221 (1923-10-01), Kristofek et al.
patent: 4683058 (1987-07-01), Lyman et al.
Foote Nicholas Peter Martin
Nelson David
Celsis International plc
Leary Louise N.
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