Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Patent
1997-07-25
1999-11-02
Wallenhorst, Maureen M.
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
436 63, 436164, 436177, 435 29, 435 34, 435 39, 435808, 4352887, 422 8205, 422 8208, 422101, G01N 2176
Patent
active
059768924
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention consists in a method and installation for counting cells and micro-organisms, in particular in food products or biological fluids.
SUMMARY OF THE INVENTION
There are various prior art methods of determining the presence of micro-organisms and cells, in particular cells, yeasts, mold, parasites or viruses in a food product, and of measuring the concentration of said micro-organisms and cells, for example for the purposes of quality control.
A first method entails culturing a sample of the product to be tested and, after a few days of incubation, observing the culture dish under a microscope in order to count the micro-organisms and cells.
This counting method necessitates a time-delay between taking the sample and obtaining the result, and is therefore not suited to real time testing. Moreover, this method leads to an assessment rather than to a precise count.
Another prior art proposal is to mark the micro-organisms and cells by means of chemical coloring agents in order to render them fluorescent, which makes the identification and counting of the micro-organisms and cells easier and introduces the possibility of automating the process by acquisition of a microscope image followed by signal processing on a computer. The effect of the signal processing is to recognize the marked micro-organisms and cells by discriminating them from the background and counting the number of objects recognized.
To speed up counting it has been proposed to combine this method of marking and automatic recognition of the marked objects with a filtration system increasing the concentration of the micro-organisms and cells to be counted.
European patent EP-A-0 333 560 describes a method of this kind in which the sample is first placed in contact with a fluorescent probe or antibody, after which the marked micro-organisms and cells are counted individually.
The system can also process cells such as polynuclear leukocytes in milk or human blood and urine. In this case the system is substituted for a flow cytometer.
The above prior art document suggests carrying out the count by microscope image analysis.
The applicant has developed an installation for automatic counting of marked cells and micro-organisms and cells sold under the tradename "COBRA" which counts micro-organisms and cells by scanning a plurality of focal planes of a membrane and carrying out image processing for each focal plane in order to recognize and to count the events looked for.
A device of this kind offers very high performance in the context of laboratory analysis and has made it possible to propose routine test approved by official bodies for testing food products in particular. Installations of this type are costly, however, since they utilize high-performance image acquisition means and sophisticated image processing systems. They are also unsuitable for use outside the laboratory, in the field, and are beyond the resources of smaller laboratories.
They also have two major drawbacks. Firstly, the prior art counting method is effected by sampling the total surface of the membrane, each image corresponding to a particular field of observation from several hundred or several thousand possible fields of observation. For the error rate of the count to be acceptable, the number of observation fields within which automatic counting is carried out must be increased, the number of fields to be analyzed being determined statistically and according to the time that can reasonably be allocated to carrying out an analysis. If the number of micro-organisms and cells, and therefore the density of events, is very low, the error rate becomes high. Moreover, the samples are no longer uniformly distributed and there are edge effects in particular.
Prior art systems have attempted to reduce the error rate by scanning the membrane in a manner that varies the position of the field of observation and the depth of the field of observation. This increases the number of image analysis sequences and therefore increases the overal
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Biocom S.A.
Wallenhorst Maureen M.
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