Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1996-09-16
1998-08-25
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 18, 435 24, 435 29, 435 34, 435 6, 435968, 435183, 435874, 435876, 435 4, 25230116, 436 13, 436 71, 436800, 437 16, 422 50, 422 55, 426 34, 426330, 426 33, 426601, 426417, C12Q 137, C12Q 102, C12Q 104, G01N 3353, A23C 912
Patent
active
057982210
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel method for conditioning a liquid sample, such as a milk sample, for determining the amount of bacteria therein by fluorescence determination, in particular by flow cytometry, as well as a method for performing the determination.
It is known to determine the number of bacteria in milk by staining with a fluorochrome, irradiating with light in a waveband in which the fluorochrome absorbs, and assessing the resulting fluorescence.
Thus, EP-A 8826, published on 19 Mar. 1980, discloses a method for counting bacteria in a bacteria-containing liquid sample, such as milk. Prior to the counting procedure, the milk is subjected to lysis of somatic cells by means of EDTA and centrifugation to isolate the bacteria from the other components of the sample (that is, fat globules and somatic cell walls), and the suspension of bacteria so isolated is treated with a proteolytic enzyme such as subtilisin, and stained with a combination of a fluorochrome, in particular acridine orange, EDTA, and a detergent. After this staining of the isolated bacteria, the bacterial suspension is applied as a thin film on the outer rim of a rotating disc, the film is subjected to irradiation, and the light signals emitted from the stained bacteria are used as the basis for determining the number of bacteria in the sample.
In recent years, attempts have been made to perform determination of bacteria in milk using simpler and faster techniques, in particular flow cytometry.
Thus, FI published patent specification No. 68654, published on 9 Sep. 1984, discloses a method in which the amount of somatic cells and bacteria in natural suspensions such as milk is measured by homogenizing at 45.degree.-55.degree. C., adding a phosphate buffer, pH 7.4, containing 20-100 .mu.g of ethidium bromide per ml buffer, and subjecting the resulting suspension to fluorescence determination in a flow cytometer.
A more recent strategy, suggested in EP-A 342501, published on 9 May 1988, comprises treating the milk with a clarification reagent essentially consisting of a non-ionic detergent, a C.sub.1-8 alcohol and a buffer of pH more than 10, and a colour reagent such as ethidium bromide, and excitation and fluorescence determination in a flow cytometer. The clarification will selectively break down the non-cellular particles contained in the milk without affecting any microbial or somatic cells which may be present.
Thus, the strategies suggested in the art comprise measurement of both somatic cells and bacteria, the most recent strategy involving removal of fat globules prior to such measurement.
In connection with comprehensive research leading to the present invention, it has been found that to obtain signal to noise ratios in flow cytometry fluorescence assessment which will ensure reliable results which are in agreement with accepted standard techniques (Standard Plate Count), it is necessary to degrade and solubilize the casein micelles/particles which are inherently present in milk, as the presence of casein has a tendency to lead to precipitation of protein and formation of protein coatings in the apparatus employed. It has also been found that the means available for the degradation of casein, i.e. proteolytic enzymes, as well as those available for staining of the bacteria, i.e. bacteriologically specific fluorochromes, will give rise to a certain degradation of the somatic cells, resulting in cell debris which will impair the signal to noise ratio, which means that the somatic cells must be lysed, degraded and solubilized, contrary to the teaching of the two above-mentioned patent applications relating to flow cytometry. On the other hand, it has also been found that contrary to the most recent of the above-mentioned patent applications and contrary to the teaching of EP-A 8826, it is possible to obtain a satisfactory signal to noise ratio in the fluorescence assessment without removing the fat globules, thereby very considerably simplifying the sample conditioning and the instrumentation used.
The present inventio
REFERENCES:
patent: 5256532 (1993-10-01), Melnicoff et al.
patent: 5375606 (1994-12-01), Slezak et al.
Foss Electric A/S
Leary Louise N.
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