AU7775498A

AU7775498A - Method for couting viable cells

Info

Publication number: AU7775498A
Application number: AU77754/98A
Authority: AU
Inventors: Jean-Louis Drocourt; Pascaline Levesque
Original assignee: Chemunex SA
Current assignee: Chemunex SA
Priority date: 1997-06-04
Filing date: 1998-05-20
Publication date: 1998-12-21
Also published as: CA2289566A1; FR2764305A1; JP2002505578A; WO1998055861A1; FR2764305B1; EP0986752A1

Description

The invention relates to a process for detecting and/or counting viable cells/ in particular micro organisms, comprising a step of labelling with a viabi lity label and a step of counter-labelling with a 5 blocking agent, on labelling and blocking compositions as well as to a kit for implementing the process. The invention also relates to a composition containing one or more blocking agents capable of specifically labelling dead cells or particles, with 10 the exception of live cells whether they are in cellular or sporulating form, which allows differential counting of the live cells from the other particles including dead cells. The detection and/or counting of viable cells 15 in a sample is of interest both for ensuring the quality of inocula for the production of fermentation products such as beer, wine, dairy products, etc. and for controlling the sterility of food products or healthcare products or pharmaceutical products before 20 use or marketing. The term viable cells is understood to refer to any biological material in unicellular form or, more generally, any material containing genetic information which is self-reproducibl or reproducible in a 25 biological system. This includes all eukaryotic or prokaryotic cells, including sporulating forms; by way of example, mention will be made of bacteria, fungi and yeasts. Various methods are available to determine 30 cell viability, including the traditional method of -2 counting the increasing population in time on a Petri dish. The counting of microorganisms by this technique is often slow to carry out, the result is observed only after 2 days to 7 days, even 14 days, of incubation, 5 this delay being incompatible with the marketing of perishable foodstuffs, and it is often burdened with considerable errors if the number of inoculated dishes is not sufficient for statistical counting. Lastly, these techniques do not work for detecting and counting 10 live m icroorganisms which have a limited or zero capacity to grow on a Petri dish or in culture. Direct counting techniques have been developed to overcome the long response delay of traditional methods. This includes, in particular, the 15 DEFT technique (direct epifluorescence technique), counting using fluorescent labels and in particular acridine orange; the use of this technique, and its limitations, are described in a recent review published by Kenner and Pratt (Microbiological Review (1994) 8 20 603-6150. However, these techniques have the drawback of giving rise to a large number of non-specific adsorptions which lead to an overestimation of the count of the increasing population in time by counting false-positives in particular when this counting is 25 automated using systems such as flow cytometers One variant of this approach consists in using labels that are capable of giving a fluorescent signal after transformation by a cellular enzyme. The emission of the fluorescent signal is the consequence 30 of the existence of enzymatic activity, and it reveals the presence of a viable cell. This technique has the advantages of minimizing the number of false-positives, Of being able to differentiate between viable cells and dead cells by the enzymatic conversion of the 35 precursor, and of promoting the intracellular accumulation of labels by means of maintaining the membrane integrity. Nevertheless, this intracellular enzymatic conversion is associated with a nonspecific hydrolysis due to the labelling buffer, as with any -3 ester in aqueous medium. Although minimal, this hydrolysis may nevertheless give rise to a small amount of non-specific labelling, which may become critical, in the use of these labels for a test of sterility. 5 - Methods for labelling live strains of bacteria with fluorogenic esters, such as fluorescein diacetate (FDA) or carboxyfluorescein diacetate (CFDA), have been described, in particular by J.-P. Diaper et al. in Journal of Applied Bacteriology - (1994) - 77: 10 221-228 and by J. Porter et al. in Journal of Applied Bacteriology, (1995) - 79: 399-408. The term fluorogenic ester is understood to refer to any non-fluorescent molecule which, after the action of an enzyme, gives rise to a molecule which is 15 fluorescent when excitated by light. In general, fluorescent labelling, whether obtained directly or by activation by a cell enzyme, has the advantage of the speed with which results are obtained, which is incomparably better than in the case 20 of cell culturing, but has the drawback of being less reliable in quantitative terms on account of the limit signal. This limitation may give rise to the existence of false-positives or false-negatives. The reason for this is that, in this type of technique, there are 25 several causes of inaccurate estimation of the number of live cells: a) when the microorganism exhibits a sporulent form, it is difficult, if not impossible, for any compound, and in particular fluorescent labels, to 30 enter the spore and this leads to the number of live microorganisms being underestimated. b) spurious labelling of particles in suspension or dead cells may result in the counting of false-positives, especially in the case of fluorescent 35 labelling obtained directly. c) after transformation of the fluorogenic precursor by a characteristic enzyme of the viable cell, an efflux may take place by an active mechanism, this efflux obviously decreasing the intracellular -4 labelling and thus posing a problem of sensitivity of the method. The present invention is directed towards overcoming all the drawbacks of direct labelling by 5 fluorescein esters which, it should be recalled, are essentially of three types: - the active efflux of the ester hydrolysis products, - the generation of false-positives by the labelling of the particles in suspension or of dead cells, leading 10 to an overestimation, - the impossibility of detecting bacterial, yeast or fungal spores, leading to an underestimation of viable cells. To this end, the present invention relates to 15 a process for detecting and counting viable cells in a total flora by detecting and selectively measuring the number of live cells, of particles and of dead cells in a sample. More particularly, the present invention 20 relates to a process for detecting and/or counting viable cells in a sample and characterized by a succession of steps comprising at least: a) placing the specimen in contact with a composition containing agents for counterstaining or blocking 25 viability labels, b) placing the specimen in contact with one or more viability labels in a viability buffer, c) detecting and counting by recognition of the viability label the viable cells in the total flora. 30 In the process of the invention, step a) is prior to or simultaneous with step b). The process of the invention advantageously comprises an additional step which is the placing in contact of cells with a swelling medium, this step 35 being prior to or simultaneous with step b) . If it is simultaneous, the swelling medium and the viability label buffer may only consist of one and the same medium.

-5 The aim of this step is to allow the incorporation and conversion of fluorogenic labels into viable cells existing in the form of spores. The swelling medium will be a culture medium which is 5 capable of germinating the spores, and consequently containing germination inducers such as amino acids, peptides, sugars, etc. For example, mention may be made of SCT (soybean casein tryptone) which will more particularly induce the germination of bacterial 10 spores, or malt extract which will be more particularly suitable for inducing the germination of mould spores. When it is desired to measure an overall viable flora, an advantageous swelling medium thus contains a mixture of SCT and malt extract. 15 In the process of the invention, the specimen studied may originally be in liquid form, which is capable of being filtered, or may result from the placing in suspension of particles present on any solid or in any gas; the cells may be detected and counted, 20 either directly in a liquid sample or after filtering the liquid sample. The filter will obviously be chosen such that the pore size is small enough to retain all of the viable cells which it is desired to detect and/or 25 measure. Steps a) and b) are then carried out on the filter and step c) is applied by the use of a scanning cytometer of ChemScanO type so as to obtain, on the one hand, the detection and counting of the specific 30 fluorescence of the viable cells and, on the other hand, where appropriate, detection of the fluorescence of the inert particles and of the dead cells, on the basis of their fluorescence emission wavelength. The advantage of the embodiment of the 35 invention in which the liquid sample is prefiltered is to significantly increase the concentration of the components which it is desired to detect. In addition, the various steps are easier to carry out when the cells are deposited on a solid support.

-6 In the process according to the invention, the filtration step may be followed by a washing step. After filtration of the sample, the vacuum is broken and the composition of blocking agents as 5 described below is passed over the filter bearing the sample which is liable to contain cells, or added directly to the sample in the case of analysis in liquid medium. . The term blocking agent or counter-staining 10 agent is understood to refer to any molecule or mixture of molecules which is capable of interacting with the binding sites of the viability label- or labels on inert particles or dead cells present in the sample to be analysed and preventing non-specific binding of the 15 viability label or labels on the said particles or dead cells. A blocking agent or counter-staining agent may also consist of any molecule or mixture of molecules which is capable of interacting specifically 20 with inert particles or dead cells and having the characteristic of neutralizing any spurious fluorescence emission by the viability label or its derivatives, either by - absorption of the spurious light, by energy transfer, 25 - competition at the site of non-specific binding, - or both phenomena simultaneously or by combination of blocking agents. Advantageously, the blocking agents will be chosen from the range of fluorescent labels having 30 similar structural characteristics but which do not emit, or emit at wavelengths different from those of viability labels, and advantageously from among labels having a fluorescence-absorption wavelength equal to the emission wavelength of the viability labels used. 35 The viability labels used in step b) are advantageously fluorescent labels, such as fluorescein esters, carboxyfluorescein, BCEF, 5,6 carboxyfluorescein diacetate (CFDA) or fluorescein diacetate, or a mixture thereof. These viability labels -7 each have a target-specificity and require conditions of suitable labelling buffers. By way of example, CFDA is more particularly suitable for labelling bacteria, whereas FDA has a better capacity to accumulate in 5 fungi or yeasts under neutral pH conditions. A mixture of these two fluorescent esters will thus make it possible to detect and count a broad spectrum of microorganisms which are simultaneously present in a biological sample. 10 In general, the viability labels are chosen from the group formed by xanthenes, acridines, fluorones and aminoazides. It should be noted here that the process of the invention may also be used when fluorescein 15 isothiocyanate (FITC) is used as fluorescent label in combination with a specific ligand (mono- or polyclonal antibody and/or nucleic acid probe) both for the identification of microorganisms and animal cells. In this case, non-specific adsorption of the fluorescence 20 emitted is often observed. The use of a blocking agent composition prior to or at the same time as the labelling with the ligand may make it possible to prevent this artefact. The invention also relates to a blocking 25 agent composition which can prevent the binding of first fluorescent compounds to inert particles or non living cells, characterized in that it comprises: a) one or more second fluorescent compounds having a fluorescence-absorption wavelength which is 30 substantially equal to the fluorescence-emission wavelength of the first viability labels; fluorones such as erythrosin B, ethyleosin, methyleosin, eosin B or Y, phloxin B or a mixture of these are more particularly chosen; 35 b) one or more compounds liable to compete with the product resulting from hydrolysis of the label, which is the cause of the spurious labelling; C) a mixture of a) and b).

-8 The present invention also relates to a labelling composition consisting of one or more mixtures of viability labels in a buffer of high ionic strength. 5 The invention also relates to the use of the process for the detection and/or counting of the increasing population in time of viable cells in a sample, including sporulating forms, with the exclusion of dead cells; similarly, the invention makes it 10 possible to count the latter specifically. Lastly, the invention relates to a kit which allows viable cells in a total flora- to be detected and counted, this kit.comprising at least: - an agent for blocking spurious labelling by viability 15 labels or a viability label composition as described above; - a composition containing one or more viability labels as described above; - a viability buffer; 20 - where appropriate, a swelling medium; - an experimental procedure for the dilutions, prepared at the time of use, of the various compositions and buffers. Detailed description of the invention: 25 In the process of the invention, the blocking agents make it possible to differentiate, on the one hand, the live cells, and, on the other hand, the particles and the dead cells by differential labelling. When the labelling agents are fluorescein derivatives 30 and have an emission wavelength of 515 nm, the blocking agents will preferably be halo derivatives of. the xanthene family having an absorption wavelength in the same range, or derivatives thereof. In this case, the blocking agent is 35 preferably chosen from the group formed by fluorones which are substituted with at least one halogen atom, such as chlorine, bromine, fluorine and/or iodine; among the substituted fluorones, mention may be made, for example, of eosin B, eosin Y, phloxin B, erythrosin -9 B, ethyleosin or a mixture of two or more of the latter. The blocking agent is, in a particularly preferred manner, chosen from the group formed by fluorones which are substituted with at least 3 halogen 5 atoms, and preferably with 4 halogen atoms and in particular with 4 iodine atoms or 4 bromine atoms, such as, in particular, eosin Y, phloxin B, erythrosin, ethyleosin or a mixture of two or more of the latter. Good results have been obtained when the blocking agent 10 is chosen from the group formed by erythrosin B, ethyleosin or a mixture thereof. The similarity between. the labels is important insofar .as most of the stains tested prove to be inefficient in the desired research; in particular, 15 the blue stains, such as Evan's blue, trypan blue or methylene blue, are inefficient. Table 1 below summarizes the main stains which have been tested with their characteristics and their effects: - 10 Table 1: STAINS ACTION STAINS ACTION Blue stains: No action Congo red No action .Evans blue (0.005%) .Trypan blue .Methylene blue Rothenium red Little or no action Alizarin reds No action (0.005%) Nile red No effect Eosin B (0.005%) Little action (8 pg/mI) Fast red Not usable Phenosafranin Little action (30 .g/ml) (0.002%) Neutral red Active Eosin Y (0.01 %) Active (0.003%) Erythrosin B Active phloxin B Active (0.004%) (0.005%) Ethyleosin Active (0.002%) The active blocking agents may be used alone or 5 in combination. The advantage of using a combination is to increase the spectrum of action of these blocking agents. By way of example, ethyleosin, phloxin B and eosin Y which are bromo derivatives of fluorescein, 10 will preferably label killed microorganisms whereas erythrosin, which is an iodo derivative of fluorescein, will preferably label the inert particles in the medium to be analysed. A mixture of ethyleosin and erythrosin will therefore be of particularly good performance as 15 regards labelling the set of particles or cells which are not viable microorganisms in any sample. The invention also relates to a blocking agent composition which comprises fluorones chosen more - 11 particularly from the group formed by erythrosin B, ethyleosin, methyleosin, eosin B and Y, phloxin B or a mixture of these in concentration ratios by weight ranging from 10/1 to 1/10. The choice of fluorones and 5 of their relative concentrations will depend on the type of product which needs to be analysed, as well as its medium. For example, for the microbiological analysis of water, the erythrosin and ethyleosin are used in.ratios of between 5/1 and 1/5 and preferably, 10 for example, of 2/1, equivalent to respective final concentrations of 0.004 and 0.002. The solution comprises the- constituents at a concentration which is between 2 and 15 times that of the product of hydrolysis of the viability label 15 responsible for the non-specific fluorescence signals, given that the hydrolysis products represent between 1 and 10% of the weight of the viability label according to the type of sample analysed. The process of the invention comprises the use 20 of a labelling composition of universal type which is capable of labelling any type of cell present in the sample studied. The cells may be a prokaryote, a monocellular eukaryote, an animal cell in a biologically active form or in a sporulating form. When 25 the fluorescent signal is desired, a labelling composition will be one which the experimenter will dilute, at the' time of use, in the labelling buffer of high ionic strength. The labelling composition will preferably consist of a mixture of 30 5( 6 )carboxyfluorescein diacetate (CFDA) at a concentration of between 5 and 10 mg per ml, and of fluorescein diacetate (FDA) at a concentration of between 0.5 and 5 mg per ml in pure acetone (QS). The weight ratios of CFDA and of FDA will preferably be 35 between 5/1 and 15/1 and, under optimum conditions, 9/1, i.e., for 1 ml, 9 mg/ml (19.5 mM) of CFDA and 1 mg/ml (2.4 mM) of FDA in pure acetone. At the time of labelling of the possible desired microorganisms in the sample, the labelling - 12 composition as described above is diluted one hundredfold in a labelling buffer whose ionic strength is greater than 0.5 and comprising, in an aqueous solution: 5 - a nonionic detergent, - a chelating agent, - a buffer, and - a salt. :A preferred composition is presented in Table 2 10 below: Table 2: Concentration Preferred - limits concentration Tween 20 (polyoxyethylene sorbitan monolaurate) 0.005% - 0.1% 0.03% 3SS trisodium ethylene diaminetetraacetate 1 mM - 10 mM 5 mM Sodium acetate trihydrate 10 mM 50 mM Sodium chloride 0.5 mM - 1.5 M 1.13 M Ultrapure water qsp The sodium acetate buffer may indifferently be replaced by other buffers of phosphate, HEPES, citrate, 15 etc. buffer type. Similarly, the sodium chloride may be replaced by potassium chloride (KCl), (NH 4

)

2

SO

4 , etc. The composition consisting of a label, or of a mixture of labels, as described above and diluted in the labelling buffer also forms part of the invention. 20 The particularly noteworthy efficacy of the buffer in question is surprising on account of its very high ionic strength. The reason for this is that isotonicity was generally the approach adopted to con serve the integrity of the viable cell. Tests 25 performed by flow cytometry on bacteria in the stationary phase are presented in Figure 1.

- 13 Figure 1 represents the effect of the ionic strength on the number of cells detected. The x-axis indicates the fluorescence intensity and the y-axis indicates the number of cells detected. The left-hand 5 column represents the results with Bacillus subtilis and the right-hand column with Serratia marcescens. They show that an increase in the ionic strength is accompanied by an increase in the fluorescence intensity, resulting from an increased accumulation of 10 the label in the live cells. This experiment clearly indicates that an ionic strength of greater than 0.8 M is recommended for precise counting. In the process of the invention, a step of 15 swelling of the spores which may be present in the medium to be analysed is carried out by addition of a medium of SCT type, a malt extract or advantageously a mixture of the two. The sample, treated beforehand with the 20 blocking agent as described above, is placed in contact with the swelling medium, either by dilution or resuspension of the sample to be analysed in the swelling medium in the case of counting in liquid medium, or by transferring the membrane on which the 25 sample to be analysed has been filtered onto an absorbent support saturated with the swelling medium. The sample is then incubated for 40 min to 3 hours at 300 or 37 0 C depending on the desired application; a short 40 min incubation at 37 0 C will allow the detection 30 of the bacterial spores, whereas a longer incubation (3 hours at 30 0 C) will also allow the detection of mould spores. However, the temperature may be decreased and the duration of incubation prolonged in order to obtain 35 the same result. For the detection of the microorganisms, the samples are placed in the presence of the labelling solution as described above, in a similar manner to - 14 that of the swelling step, and are incubated for 30 minutes at 30*C plus or minus 3*C. In the process of the invention, the blocking agent or agents has or have the particular feature of 5 being in a concentration ratio with the viability label or labels of from 5/1 to 15/1, which is the reverse of the usual ratios used between stains and counter stains, which are of the order of 1/10.. An optimum ratio for fluorescein derivatives will be about 10/1. 10 Treatment of the sample with the blocking agent will always take place prior to or, at the very latest, simultaneously with the treatment with the composition containing the viability labels. In a variant of the invention, the hydrophilic 15 support may be dispensed with, either in the swelling step or in the labelling step, by depositing the swelling buffer or the labelling buffer directly onto the filter bearing the filtered microorganisms. Lastly, the process of the invention comprises 20 a step of analysis by any means which is suitable for measuring the signal emitted by the viability label. If, however, the analysis is not performed immediately after the incubation with the labelling composition, the samples must be placed at 8 ± 4 0 C and sheltered from 25 the light, without, however, exceeding a duration of 30 minutes. The detection and/or counting process according to the invention may be carried out in particular in the machine described in patent applications 30 EP 0,333,561 and WO 89/08714 and sold under the brand names ChemScan@ and ChemFlow@. The invention also relates to the implementation and use of the kit of the process of the invention in any application in which the presence of 35 live cells is investigated. The process and the kit according to the inven tion may be used in particular for detecting and/or counting possible microorganisms in healthcare, food or pharmaceutical products.

- 15 The process and the kit of the invention may also be used for controlling an industrial process such as sterilization, whether in agrifood, pharmaceutical or nutraceutical applications, and either in a step of 5 a manufacturing process or on the finished product. Similarly, the process and the kit may be used to control the bacterial charge before and after a sterilizing filtration (bioburden). Lastly, the process and the kit of the 10 invention may be used to reveal viable microorganisms that are not detectable by standard methods. This may involve, for example: - controlling the efficacy of an antibiotic, particularly for those which interfere with the cell 15 division of microorganisms and thus are not detectable with standard methods of Petri dish type; - searching for contaminations liable to produce nocosomial infections in a hospital environment; - establishing a possible relationship between a test 20 of pyrogenicity in the course of manufacture, for example for medicinal products, and the presence of microorganisms in the sample; - detecting lymphocytes in urines. A person skilled in the art will be able, as is 25 needed, to carry out the process for the desired use. For the first time, an invention is at hand which provides' a means of labelling, on one and the same sample, and discriminating viable cells from particles of substantially identical size to the viable 30 cells, which may be dead cells or particles in suspension, this being achieved with a reliability which is imparted by: - the elimination of false-negatives by the presence of a swelling medium and the labelling of spores, 35 - the elimination of false-positives by virtue of the blocking agent composition, - increasing the sensitivity by means of the use of a labelling buffer of high ionic strength more especially for microorganisms.

- 16 Besides the developments and advantages detailed above, the invention also contains other advantages and characteristics which emerge from the examples which follow, these being given by way of 5 illustration without, however, limiting the scope of the invention. Example 1: Implementation of the process in the absence of any filtration of samples: In a first stage, implementation of the process 10 consists in carrying out a pretreatment with a blocking agent prior to the treatment with the viability labels. The aim of this experiment is to show the efficacy of this treatment .in eliminating the false-positives resulting from a non-specific interaction between the 15 viability labels and the filtration membrane. We used a counter-staining composition containing erythrosin and ethyleosin in a weight ratio of 2, referred to as CSE (Counter-Stain E), having the following composition: 0.004% erythrosin B diluted in 20 Hepes and 0.002% ethyleosin also diluted in Hepes. Table 3 below compares the ChemScan-measured fluorescence results obtained with or without prior treatment with CSE: - 17 Table 3: Blocking agent Number of F + % of the membranes membranes giving 0 Without 36 membranes .9 membranes give 0 25% .14 membranes give I .3 membranes give 1 .2 membranes give 3 .2 membranes have an F+ >3 With CSE 42 membranes .34 membranes give 0 81 % (Erythrosin + .1 membrane gives I ethyleosin) - .2 membranes give 2 .1 membrane has an F+>3 The procedure used is as follows: a) preparation of the filtration supports: 5 The filtration supports are rinsed successively with three times 1 ml of 70% (v/v) ethanol and then with 3 times 1 ml of filtered water (filtered through a 0.22 pm filter). The filtration support used is a polyester membrane which is then placed on a wet 10 filtration support. b) Filtration of the CSE counter-stain: The CSE is then filtered through the polyester membrane positioned on the wet filtration support, in an amount of 1 ml of CSE. 15 c) Labelling with the viability labels: The viability label comprises 9 mg per ml (19.5 mM) of carboxyfluorescein diacetate (CFDA) and 1 mg/ml (2.4 mM) of fluorescein diacetate (FDA) in pure anhydrous acetone. The viability solution was prepared 20 and diluted at the time of use and then one-hundredfold in a buffer whose composition is as follows: - 0.03% (v/v) of Tween 20 (polyoxyethylene sorbitan monolaurate), - 50 mM sodium phosphate trihydrate, pH7 25 - 1.13 mM sodium chloride in ultrapure water. c.1) - 18 - draw up 20 ml of swelling medium into a sterile syringe, - prefilter the 20 ml of swelling medium in a sterile flask, 5 - keep this solution at room temperature. c.2) Place an absorbent support (PAD) in cellulose or in polypropylene in a Petri dish: - soak the absorbent cellulose support (cellulose PAD) with 650 pl of swelling medium, or soak the 10 polypropylene PAD with 300 pl of swelling medium, as described above, - transfer the polyester membrane onto the PAD, - place the closed and labelled dishes in the oven, sheltered from light 15 - incubate at 40mm (± 3 0 C) . c.3) During this incubation period, prepare the labelling solution. Example for 20 analyses: - add 200 pl of viability substrate to a sterile flask 20 containing 20 ml of labelling buffer which has been prefiltered through a 0.22 pm filter, taking care not to touch the wall of the flask during the addition, - homogenize, - wrap the flask in aluminium foil. Store the solution 25 thus prepared at 8*C ± 4 0 C (4 hours maximum). c.4) Place a new PAD (cellulose or polypropylene) in each Petri dish - soak the cellulose PAD with 650 pl of labelling solu tion, or the polypropylene PAD with 300 pl of a 30 labelling solution - transfer the polyester membrane of the PAD saturated with swelling medium to the PAD saturated with labelling solution, - incubate for 30 min (± 5 min) at 30 0 C (± 3*C), shel 35 tered from the light. c.5) Analyse each sample individually. If the analysis is not immediately after the incubation, place the samples (membranes on PAD) at 8*C ± 4 0 C, sheltered from the light (do not exceed 30 min).

- 19 The results obtained are given in Table II above. The 36 membranes not treated with the blocking agent did not undergo step b) above. 5 The results indicate that simply by treating with the blocking agent allows the number of membranes bearing false-positives to go from 75% to 19%. Example 2: Efficacy of the process on buffered peptone water: 10 Peptone water is a water used to make bacterial dilutions in order precisely to carry out counting, as is described in particular in patent -WO 89/08714. However, certain types of peptone water pose the problem of giving many false-positives when the 15 sample is treated with fluorescein esters, which probably results from an interaction between the fluorescein resulting from the hydrolysis of the label and which would attach to particle elements residing in the peptone water. The same procedure as in Example 1 20 was used, except that a step of filtration of 50 ml of sterile peptone water was introduced between step a) of preparation of the filter and step b) of filtration of the blocking agent. The results obtained are given in Tables 4.1, 4.2 and 4.3 below: - 20 Table 4.1: Counter-stains Primary count F+ Control 6835/7804/10677 8/4/11 Neutral red 699/1371/894 1/4/3 Control 2706/3142/2848 6/7/6 Erythrosin B 87/71/107 0/0/1 Control 7445/7588/5986 8/9/15 Nile red 178/678/343 10/4/7 Table 4.2: Counter-stains Primary count F+ Control 2674/2968/2997 69/95/111 Congo red 1425/1361/3387 72/64/71 Alizarin red S 1762/2030 56/82 Phenosafranine 214/252/273 18/25/37 5 - 21 Table 4.3: Counter-stains Primary count F+ Control 2554/2492/2299 59/57/56 Eosin B 1229/1471/1169 31/49/41 Eosin Y 278/451/3166 11/21/21 Phloxin B 122/110/129 6/4/16 Ethyleosin 82/66/97 5/2/3 Six filtrations were carried out in the absence of step b) and with step b). The blocking agent and the 5 viability label used are the same as those in Example 1. The sign F+ indicates the number of fluorescent particles detected by ChemScan@. The column indicating the "primary count" is in fact the fluorescence count before the step of discrimination carried out by 10 ChemScan@, which, it should be reminded, makes it possible to eliminate all the events not associated with a particle having the shape and size programmed into the machine. The interpretation of these experiments makes 15 it clear that the use of the blocking agent allows the elimination of about 90% of all of the fluorescence events counted in the primary count - and which are therefore artefacts probably due to the particles present on the membrane - and the vast majority of the 20 true fluorescence events obtained after discrimination by ChemScan@ and which are therefore false-positives (the figures are too low for the percentage decrease to be reasonably measured). It is observed in. particular that two membranes out of the six treated with the 25 blocking agent give no positive fluorescence with the viability label after pretreatment with CSE. Example 3: Process performed on a sterilized solution of antibiotics: The experiment is strictly identical to that of 30 Example 2, except that 10 ml of a solution of sterile - 22 antibiotic were filtered through the polyester membrane between steps a) and b) in Example 1. The results are given in Table 5 below: Table 5: Sterile product Sterile Ab solution Primary count F+ Without CSE 2 2206-2426 322-293 With CSE 2 79-152 5-12 Phloxin B 290 16 16-42 0.005% 566 44 Eosin B 1442 104 100 - 370 0.005% 2001 374 5 Here also, in the absence of counter-staining, ChemScan@ respectively detected 322 and 293 particles exhibiting fluorescence after discrimination, whereas on treatment with CSE, this figure comes down to 5 and 10 12 fluorescent particles respectively, i.e. a 98.5% decrease in the first case and a 96% decrease in the second case, as regards the number of false-positives. In conclusion, it is seen clearly that the process of the invention leads to a drastic reduction 15 in the false-positives obtained by direct use of viability labels on a sample which is liable to contain microorganisms, this decrease ranging from 80 to 99%. Moreover, this technology has the advantage of giving no false-negatives, in other words the blocking 20 agent does not decrease the number of viable micro organisms counted in a sample. The counter-stain and more particularly CSE moreover has the advantage of being able to be used in a kit since it has autoclaving and storage properties 25 which are compatible with commercial distribution. Not least among the advantages of the composition containing the blocking agent or agents is its broad spectrum of use since the mixture proposed allows both dead cells and non-organic particles to be 30 labelled. it goes without saying that depending on the - 23 sample which it is desired to test, one or other of these blocking agents will preferably be used such that it remains within the ratios desired with the viability labels used in the rest of the process. 5 If need be and depending on the sample which it is desired to analyse, a person skilled in the art will know how to select both its blocking agent composition and its viability label composition, and the weight ratio between the two. 10 Lastly, the final advantage of the process and of the compositions of the invention is that, in a single series of manipulations, they make it possible to count the live cells exclusively, including the sporulent forms, on the one hand, and the inert forms, 15 on the other. The process of the invention is of general application to other labelling agent/blocking agent couples. A person skilled in the art will still be able to determine the blocking agent having one or other of 20 the characteristics mentioned at the start of the text which are complementary to the characteristics of the viability label.

Claims

1. Process for detecting and counting viable cells in a sample, comprising a step of labelling with one or more viability labels, characterized in that it 5 comprises the following steps: a) placing cells in contact with a composition containing agents for blocking viability labels, b) placing cells in contact with one or more viability labels in a viability buffer, 10 c) detecting and counting the viable cells in the total flora, wherein step a) is prior to or simultaneous with step b).

2. A process according to Claim 1, wherein the 15 sample is prefiltered.

3. Process according to Claim 1 wherein step c) is performed by a flow cytometer or scanning cytometer machine.

4. Process according to Claim 1 wherein the 20 viability buffer has an ionic strength of between 0.5 M and 3 M.

5. Process according to one of Claims 1 to 4, in which the cells are also placed in contact with a swelling medium before or simultaneously with step b). 25

6. Process according to one of the preceding claims, characterized in that the blocking agents are chosen from the group formed by halogenated xanthenes or derivatives thereof, and in particular fluorones substituted with at least one halogen atom. 30

7. Process according to Claim 6, characterized in that the blocking agent is chosen from the group formed by erythrosin B, ethyleosin, Eosin B, Eosin Y or Phloxin B, or a mixture thereof.

8. Process according to Claim 7, characterized in 35 that the blocking agent is a mixture of erythrosin B and ethyleosin in proportions ranging from 10/1 to 1/10.

9. Process according to any one of the preceding claims, characterized in that the viability labels are - 25 chosen from the group formed by xanthenes, acridines, fluorones as well as from the group of aminoazides.

10. Process according to Claim 9, characterized in that the viability label is chosen from the group 5 formed by FDA, 6-CFDA, 5-CFDA, fluorescein dilaurate, 5- or 6-CFDA ester, N-hydroxysuccinimide or a mixture thereof.

11. Process according to Claim 9 or 10, characterized in that the viability label is a mixture 10 of FDA and 5-CFDA in proportions of between 5/1 and 15/1.

12. Blocking agent composition which is capable of preventing the binding of first fluorescent compounds to inert particles or non-living cells, characterized 15 in that it comprises one or more second fluorescent compounds having a fluorescence adsorption wavelength which is substantially equal to the fluorescence emission wavelength of the first viability labels.

13. Composition according to Claim 12, 20 characterized in that the second fluorescent agent is chosen from the group formed by erythrosin B, ethyleosin or a mixture thereof.

14. Composition according to Claim 13, characterized in that the blocking agent is CSE 25 consisting of erythrosin B and ethyleosin in respective final concentrations of 0.004% and 0.002%.

15. Method'for the detection of viable cells in a sample, with the exclusion of particles and dead cells, comprising the steps of contacting the sample with a 30 composition according to Claim 12, then contacting the sample with a viability label and detecting and counting the viable cells in said sample.

16. The method of Claim 15, wherein the cells are viable microorganisms comprising, where appropriate, 35 sporulent forms.

17. Kit for the detection and/or counting of the viable cells in a total flora and comprising at least: - one or more agents for blocking spurious labelling by viability labels; - 26 - one or more viability labels; - a viability buffer; - where appropriate, a swelling and/or germination medium. 5

18. Kit according to Claim 17, wherein the blocking agent or agents capable of preventing the binding of first fluorescent compounds to inert particles or non living cells, is characterized in that it comprises one or more second fluorescent compounds having a 10 fluorescence adsorption wavelength which is substantially equal to the fluorescence emission wavelength of the first viability labels.

19. Kit according Claim 17, wherein the viability label is chosen from the group formed of FDA, 6-CFDA, 15 5-CFDA, fluorescein dilaurate, 5- or 6-CFDA ester, N hydroxysuccinimide or a mixture thereof.

20. Kit according to Claim 17, wherein the swelling medium is a mixture of SCB and malt extract.