Process for quantitative determination of micro organismes.

The present invention relates to a quantitative research process in a medium in liquid, semi-liquid, gel, paste, microorganisms, even present in extremely small quantities.

For extremely small amount is meant in the sense of the present invention, a number of microorganisms that can be equal to one.

Qualitative analysis and quantitative fast to the presence of microorganisms such as bacteria, yeasts, molds and parasites in food products, hygiene products or fluids and monitoring a fermentation process during its realization, are very useful and important in the various industries concerned.

usually is used as the detection technique, culturing a sample of the product to be tested. This detection method has the drawback of being long to implement the micro-organisms present developing within 2 to 30 days. In addition, the count of microorganisms by this method is not absolutely precise and allows only a rough estimate of the number of microorganisms at the start; the identification of microorganisms, by this method, remains part.

Also known is another technique for detecting, by image analysis, but this, as implemented so far, is expensive and difficult to implement; Indeed, to arrive at a correct count or sensitive detection of microorganisms present in the sample, it is necessary to analyze all the sample fields, which makes this very long search.

It is also known to mark cells, especially microorganisms by chemical or organic dyes, in particular dyes of nucleic acids, which mark mostly dead cells and non-living cells or fluorochromes such as derivatives fluorescein and phycoerythrin, that are used for many years, through their coupling to antibodies.

The use of polyspecific or monospecific made fluorescent antibodies have been described in the prior art, in particular by the Applicant in FR-A-2,598,513 which aims polyspecific anti-yeast and anti-mold.

However, these fluorescent antibody make all fluorescent cells, whether living or dead, which is a major drawback, ie to selectively determine the presence of a contaminant living in a food or hygiene, or to determine evolution or the smooth running of a fermentation process.

Other dyes have also been described in the literature, in particular carboxyfluorescein diacetate (CFDA) and fluorescein diacetate (FDA), the latter being used for the detection of bacteria as described in a number of articles (TH CHRZANOWSKI et al, "Applicability of the fluorescein diacetate method of Detecting active bacteria in fresh water," which appeared in 1984 in Microbial Ecology. (10: 179-185), B. Lundgren "fluorescein diacetate as a stain of metabolically active bacteria in soil ", published in OIKOS, 1981 (36: 17-22), Jarnagin et al." The use of fluorescein diacetate and ethidium bromide as a stain for Evaluating viability of mycobacteria "appeared in Stain Technol., 1980 (55, 4, 253-258), J. et al Schnürer "Fluorescein diactetate hydrolysis as a measure of total microbial activity in soil and litter," appeared in Applied Environm Microbiol, 1982 (43, 6, 1256 -... 1261), which SONTAG describe these dyes and their mechanism of action, however, is usually of estimation methods, or when the method is quantitative, it uses a chemical dye in combination nucleic acids. these are few methods appropriate to the serial analysis.

detection techniques according to the prior art do not enable the selective detection of live microorganisms, especially present in very small quantities.

WO-A-8605206 describes for example a method for assessing the concentration of microorganisms present in a sample relative to a normal population: involving the induction of fluorescence upon cleavage with an enzyme specific to microorganisms, a non-fluorescent dye previously added and measurement of the total fluorescence emitted by the emsemble microorganisms, this method does not detect concentrations of less than 10² cells / ml.

Accordingly, the invention has set itself the aim of providing a research method for detecting and counting microorganisms, very fast and easy to implement, sensitive, specific and inexpensive, allowing the counting the nearest unit said microorganisms.

The present invention relates to a quantitative method for determining microorganisms normally present or may be contained as contaminants in a complex medium in the liquid, semi-liquid, gelled or pasty, in particular food products, products of hygiene or biological fluids, which method comprises steps of obtaining sample and of adding a positive viability marker capable of marking living microorganisms, selected from non-fluorescent substances or fluorescent bit, entering the microorganism, the said substances then being cleaved or modified so as to provide a fluorescent product, said product being trapped for the most part or completely inside of said microorganism, which method is characterized in that it comprises:<ul><li>A. for the specific and selective marking of the microorganisms:<ul><li>a) separating said microorganisms by filtration and / or centrifuging of a sample suspension, obtained directly when the starting sample is liquid, or obtained by contacting the sample with a pH adjusting buffer of the medium to an alkaline pH, where the starting sample is semi-liquid or pasty;</li><li>b) intracellular marking of the said microorganisms by incubating with at least one positive viability marker for 5 to 10 min at 37 ° C,</li><li>c) treating the sample obtained in b) with ultrasound for 10 s, to individualize the microorganisms, and</li></ul></li><li>B. for the enumeration of said microorganisms made fluorescent:<ul><li>d) passage of the sample obtained in c) in a flow cytometer, and</li><li>e) counting by means of said flow cytometer of each microorganism marked by the measuring / intracellular fluorescence emitted by each of said microorganisms.</li></ul></li></ul>

by microorganisms is meant in the sense of the present invention, including bacteria, yeasts, molds and parasites.

The term marker positive viability, a substance which does not color the living cells and not the dead cells and cells whose color intensity is proportional to their metabolic activity.

Vital dyes in the sense of the present invention are dyes of nucleic acids and are combined with the genetic material of cells both living and dead.

These include, by way of example of vital dyes, ethidium bromide, iodide propydium of the mythramycine A, DAPI, the dye marketed under the name Hoechst 33258 dye marketed under the name Hoechst 33342.

The flow cytometry, known per se is described in numerous documents in particular in EP-A-177718.

The combination of means according to the invention has the advantage of allowing the realization in series of assays, quick to implement, reliable and counts to the nearest unit. This is not a concentration assessment method compared to a standard population, so it is not necessary to know the species or type of microorganism prior to analysis.

According to an advantageous mode of carrying out said process, the pH adjusting buffer is used as dissolution medium.

According to yet another mode of advantageous implementation of said method, the pretreatment further comprises the action of appropriate enzymes.

Treatment (before and after labeling) allows the individualization of the cells, forming the origin of the colonies, and thus increases the sensitivity of detection.

The marker positive viability is selected from non-fluorescent substances or low fluorescent, which penetrate into the microorganism, the said substances then being cleaved or modified so as to provide a fluorescent product, said product being trapped for the most part or totally within said microorganism.

viability markers, in accordance with the invention are selected from the group which comprises the FDA, 6-CFDA, 5-CFDA, the fluorescein dilaurate ester, 5,6-CFDA-N-hydroxy succinimide fluorescein dipropionate, di-beta-D-galactoside fluorescein, phosphate, 3-O-méthylfluorescéine, pentaacétoxy the ester of 2 ', 7'-bis (carboxyethyl) -5 (6) -carboxyfluorescein (BCECF / AM) , diacetate azidofluorescéine, acetate of 4-methylumbelliferyl, 4-methylumbelliferyl beta-D-galactoside, 4-methylumbelliferyl alpha-D-mannopyranoside, 4-nonanoate méthyllumbelliferyl phosphate, 4-methylumbelliferyl, the alanine-7-amino-4-methylcoumarin, glycine-7-amino-4-methylcoumarin, proline-7-amino-4-methylcoumarin, valine-7-amino-4-methylcoumarin, glycyl-L-proline -7-amino-4-methylcoumarin, 1,4-diacetoxy-2,3-dicyanobenzene (ABD), hydroethidine, resorufin acetate.

According to another advantageous mode of carrying out said method, said microorganisms are further subjected to an additional marking with at least one substance selected from the group consisting of other markers of viability positive, vital dyes, fluorescent antibody and rendered rendered fluorescent nucleic probes.

According to the latter advantageous mode of carrying out said method, the antibodies are selected from polyclonal and / or monoclonal, mono or polyspecific, anti-yeast and / or anti-bacterial and / or anti-mold and / or anti parasites and polyclonal and / or monoclonal mono or polygénériques anti-yeast and / or anti-bacterial and / or anti-mold and / or anti-parasites.

According to the invention, the antibodies used are made fluorescent by an appropriate fluorochrome.

The viability markers have the function of selectively marking live microorganisms.

Antibodies have the role of specifically labeling a species or type of microorganism, that he is alive or dead.

The determination method according to the invention may advantageously be implemented in an installation comprising a cytometer measuring cell stream comprising a measuring chamber through which flows the product sample or fluid for analysis and a device recording of fluorescence signals emitted by said micro-organisms, the measuring cell being provided with a flushing device of the measuring chamber, which comes into action under the effect of sample withdrawal from the sample holder or under the effect of an appropriate external electronic control.

Besides the above arrangements, the invention also comprises other provisions which will emerge from the following description which refers to examples of implementation of the process according to the invention, with reference to Figures 1-12 are diagrams.

It should be understood, however, that the examples, drawings and the corresponding descriptive parts are given solely by way of illustration of the object of the invention, which they do not in any way constitute a limitation.

<b>example 1</b> : Search contaminant yeast yogurt.

It is possible, according to / the specie (s) of yeast to seek, choose the viability marker and / or the most suitable anti-yeast antibody to said yeast.

Yogurt is solubilized by means of suitable reagents, such as carbonate buffer pH 9.0 EDTA. The product is centrifuged for 5 minutes at 1500 g. The pellet was resuspended in sodium chloride at 9 g / l. Fluorescein diacetate, optionally in conjunction with suitable anti-yeast fluorescent antibody are added. Incubation for 5 to 10 minutes at 37 ° C allows chemical reactions to occur; the suspension is subjected to ultrasound for 10 seconds. The resulting suspension is then passed directly into the particle counter for continuous flow analysis, in a volume ranging from 0.2 to 2 ml. It is possible to count or count significantly to the nearest unit, fluorescent rendered microorganisms (Figures 1, 2 and 3).

Figures 1, 2 and 3 show population histograms of yeast grown in yoghurt.

in abscissa there is the fluorescence channel representing the intensity of light and the ordinate the number of cells per channel.

In the case of Figure 1, these yeasts <i>Saccharomyces</i> ; in the case of Figure 2, these yeasts<i>Rhodotorula</i> and in the case of Figure 3, it is <i>Debaryomyces hansenii</i>.

<b>example 2</b> : Yeast contaminant Search fruit pulp yogurt:    In the case where the sample shall be set up fruit pulp yoghurts, enzymes are added to promote the solubilization of the sample.

The sample is treated as in Example 1 except that prior to addition of the dye and / or antibody, pectinase is added to promote solubilization; the enzymatic reaction is carried out by incubating at 37 ° C for 5 minutes and the yoghurt sample in the presence of the enzyme.

Figures 4 (yogurt) and 5 (fruit yoghurt) show comparative results between the method of the invention and the conventional method of culture petri dishes.

These figures comprise abscissae the results obtained by the process according to the invention (log (counts / ml)) and the ordinate the results obtained by the conventional method (log (yeasts / ml)). a very good correlation between the two methods.

<b>example 3</b> : Research bacteria in beer or culture media used for fermentation of recombinant organisms (production of drugs).

Typically, 50 ml of cell suspension is centrifuged. The pellet is resuspended as described in Example 1. The remainder of the process is similar, viability markers or antibodies are however adapted to the specific problem. However, the viability marker having a fluorescence in the green, the antibodies are labeled with a fluorochrome emitting in the red, phycoerythrin for example; it is then possible, in this case, performing a counting microorganisms doubly labeled, allowing to identify the microorganism and simultaneously determine its viability and vitality.

6 shows a histogram population of bacteria of the genus <i>Lactobacillus</i> detected in a sample of naturally contaminated beer. In abscissa there is the fluorescence channel, and the ordinate the number of particles per channel. The most fluorescent particles are those with a very active metabolism.

<b>example 4</b> : Research bacteria and / or yeasts in prepared products (eg tomato paste, mayonnaise, bearnaise sauce, etc ...)    After aqueous extraction of these products, the resulting suspension was coarsely filtered through a filter of 15 .mu.m, for example. The filtrate is centrifuged as set forth in Example 1 above. The rest of the process is similar, however with the possibility to use a mixture of two viability markers having fluorescence in the green and two monoclonal antibodies having fluorescence in the red at different wavelengths, for example. The unit will include a measurement of 3 parameters, to differentiate and count and count accurately the meadows unit simultaneously live yeast of a given species, the dead yeast species, live bacteria of one species, dead bacteria of the same species, total live yeast of other species and the living species other bacteria.

7 shows a histogram of a population of <i>Candida parapsilosis</i> contaminant béarnaise sauce. The x-axis is the fluorescence channel, and the ordinate the number of microorganisms per channel.

<b>example 5</b> : Total bacteria Research in raw milk.

Raw milk is processed to destroy the casein micelles and is then centrifuged; the pellet was resuspended in a buffer as described in Example 1, by further adding a few drops of a suitable detergent such as Tween 20. The following process is similar. However, milk somatic cell can be colored. This is followed by a measurement and a simultaneous counting of the fluorescence and particle size by impedance, which allows to differentiate bacteria (less than 1 mm in size) of the somatic cells (about 10 microns or more in size).

<b>example 6</b> : Research of pathogenic bacteria in human body fluids such as cerebrospinal fluid or urine.

Early detection of meningococcal meningitis can be performed directly from the cerebrospinal fluid by centrifugation, and staining of the pellet by specific monoclonal antibodies commercially available. The culture of specific microorganism is no longer necessary, the sensitivity of the apparatus is sufficient. The positive viability marker is used simultaneously to ensure the metabolic state of the cells. Similarly, bacteria (bacteremia) or parasites can be searched for in the urine, for example.

8 illustrates the detection and enumeration of enterococci in a urine sample.

<b>example 7</b> Fast Enumeration of yeast in brewing:    The yeast suspension constituting the brew is diluted in NaCl 9 g / l to obtain a cell density of between 5000 and 50 000 / ml. A solution of adequate viability marker is added to a concentration of 1%. Incubation for 5 min at 45 ° C colored yeasts, which can then count or count accurately to the nearest unit, using the installation according to the invention, in less than a minute.

<b>example 8</b> Fast Enumeration of yeasts in the leaven:    The procedure is as in Example 7.

<b>example 9</b> : Mold spore germination test and quick count spores germinated:    Useful molds are generally supplied lyophilized with cheese industries. Germination and the time for obtaining thereof can be determined by placing them in a favorable germination buffer swelling of their wall, in particular phosphate buffered saline and added a energy substance such as glucose, sucrose, fructose etc ... or possibly food environment.

After 4 to 6 hours of incubation, the swelling of the wall of the spores makes them permeable viability markers, used in the same manner as in Example 7.

The analysis of the sample in the apparatus according to the invention allows, after appropriate dilution, within about a minute, to determine the percentage of germinated spores and their total number.

<b>example 10</b> : Monitoring of culturing a yeast population, and fermentation that results.

A yeast population is inoculated into a wort at a concentration of 10⁶ cells / ml. A viability marker solution, specifically labeling the yeast is added to a concentration of 1%. Incubation for 5 min at 45 ° C colored yeasts which can then count, using an appropriate flow cytometer. In this case, one can determine the one hand, the number of living cells decreases, and secondly the average metabolic activity of the population increases (mean fluorescence intensity of histograms) of overall activity population increases (successful fermentation) even if the number of cells does not increase (figure given by the conventional methods) (Figure 9).

Figure 9 shows the evolution of the viability and vitality of yeast population (<i>Sacchoromyces cerevisiae</i>) In a wort to 10 ° -12 ° C over time.

9a is a curve of the change in cell number over time. U n sampling and counting are performed at time 0 (Figure 9b), after 20 h (Figure 9c), after 60 h (Figure 9d) and after 160 hours (Figure 9e). It is seen that the number of cells decreases over time, while the histograms are shifted to the right, indicating an increase in the vitality of the cells present.

<b>example 11</b> : Monitoring the effect of heat treatment on a population growing yeast in a wort.

A yeast population is subjected to a heat shock at 60 ° C for 5 minutes (Figure 10). FIG 10 includes, in abscissa different strains of<i>Saccharomyces cerevisiae</i> (A, B, C, D) I and II representing two different worts, and the ordinate, the cell concentration / ml.

Populations are analyzed before and after treatment method according to the invention: Before treatment: column 1; after treatment: Column 2 (response in 15 minutes); Method Methylene Blue: before treatment: column 3; after treatment: Column 4 (response in about 30 minutes) and method of cultivation on Petri dishes: Column 5 (answer within 3 days). It is noted that the correspondence process of the invention methylene blue is satisfactory before heat treatment but not after this treatment, the reference used is the culture method boxes. The result is obtained in a significantly reduced time.

<b>example 12</b> : Yeast and bacteria in the orange juice searches.

Specific viability markers respectively bacteria and yeast are added simultaneously to the pellet to 10 ml of pulpy orange juice.

After a 10 minute incubation at 40 ° C, the sample is passed through the cytometer according to the invention. Figure 11 shows the result of an analysis in which the peak of the left represents the population of bacteria and the right peak of the yeast population. The device determines the total numbers of cells in each peak (Figure 11).

<b>example 13</b> : Searching for bacterial contamination in a cosmetic product.

The treatment is carried out and a similar staining as described previously. Figure 12 shows the result obtained with a contaminated product, so that when the product is not contaminated, it gets no fluorescence signal, so a negative result.