Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination

Definitions

• the invention relates to a method for detecting and / or counting viable cells, in particular microorganisms, comprising a step of labeling with a viability marker and a step of counter-labeling with a blocking agent, on the labeling compositions and blocking as well as on a kit for implementing the method.
• the invention also relates to a composition containing one or more blocking agents capable of specifically labeling dead cells or cells, excluding living cells, whether in cellular or sporulating form, allowing differential counting of living cells. other particles including dead cells.
• the detection and / or the counting of viable cells in a sample is of interest both to ensure the quality of the inocula for the production of products resulting from fermentation such as beer, wine, dairy products, etc. to check the sterility of food or health or pharmaceutical products before use or marketing.
• viable cells any biological material in single-cell form, or more generally any material containing genetic information which is self-reproducing or reproducible in a biological system.
• Direct counting techniques have been developed to overcome the long response time of traditional methods. These are in particular the DEFT technique (direct epifluorescence technic), counting by means of fluorescent markers and in particular of acridine orange; the use of this technique and its limitations are described in a recent review published by KENNER and PRATT (Microbiological Review (1994) 58 603-615). However, these techniques have the drawback of generating numerous non-specific adsorptions which lead to an overestimation of the count by counting false positives; especially when this counting is automated using systems such as flow cytometers.
• DEFT direct epifluorescence technic
• a variant of this approach consists in using markers capable of giving a fluorescent signal after transformation by a cellular enzyme.
• the emission of the fluorescent signal is the consequence of the existence of enzymatic activity, and reveals the presence of a viable cell.
• This technique has the advantages of minimizing the number of false positives, of being able to differentiate viable cells from dead cells by the enzymatic conversion of the precursor, and of promoting the intracellular accumulation of markers by maintaining membrane integrity.
• this intracellular enzymatic conversion is associated with non-specific hydrolysis due to the labeling buffer, like any ester in an aqueous medium. This hydrolysis, although minimal, can however, generate some non-specific labeling, which can become critical, in the use of these markers for a sterility test.
• 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: 221-228 and by J. Porter et al. in Journal of Applied Bacteriology, (1995) - 79: 399-408.
• fluorogenic ester any non-fluorescent molecule which, after the action of an enzyme, gives rise to a fluorescent molecule after excitation by light.
• fluorescent markings obtained directly or by activation with a cellular enzyme have the advantage of the speed of obtaining results which is incommensurate with cell culture, but have the disadvantage of being less reliable on the quantitative plan due to the existence of false positives or false negatives.
• the present invention relates to a method for detecting and counting viable cells in a total flora by detection and selective measurement of the number of living cells, particles and dead cells in a sample.
• the present invention relates to a method for detecting and / or counting viable cells in a sample and characterized by a succession of steps comprising at least: a) bringing the cells into contact with a composition containing agents for blocking viability markers, b) bringing the cells into contact with one or more viability markers in a viability buffer, c) detecting and counting viable cells in the total flora.
• step a) is prior to or simultaneous with step b).
• the method of the invention advantageously comprises a complementary step which is bringing the cells into contact with a swelling medium, which step is prior to or simultaneous with step b). If it is simultaneous, the swelling medium and the viability buffer can constitute only one and the same medium.
• the purpose of this step is to allow incorporation and conversion of fluorogenic markers into viable cells existing under form of spores.
• the swelling medium will be a culture medium capable of causing spores to germinate, and consequently containing germination inducers, such as amino acids, peptides, sugars, etc.
• germination inducers such as amino acids, peptides, sugars, etc.
• TCS tripton casein soy
• malt extract which will be more particularly suitable for inducing the germination of mold spores.
• An advantageous swelling medium when it is desired to measure an overall viable flora therefore contains a mixture of TCS and malt extract.
• the sample studied can be originally in liquid form, capable of being filtered, or result from the suspension of particles present on a solid or in any gas; cells can be detected and counted, either directly in a liquid sample, or after filtration of the liquid sample.
• the filter will obviously be chosen in such a way that the pore size is small enough to retain all of the viable cells that it is desired to detect and / or 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 the CHEMSCAN® type so as to obtain, on the one hand, detection and counting specific fluorescence of viable cells and, on the other hand, if necessary, the detection of the fluorescence of inert particles and dead cells, based on their wavelength of fluorescence emission.
• the advantage in the embodiment of the invention in which the liquid sample is previously filtered is to significantly increase the concentration of the elements which it is desired to detect.
• the implementation of the various steps is easier when the cells are deposited on a solid support.
• the filtration step can be followed by a washing step.
• the vacuum is broken and the composition of blocking agents as described below is passed over the filter carrying the sample which may contain cells or added directly to the sample in the case of 'analysis in liquid medium.
• blocking agent blocking agent or counter-dye
• a blocking agent, blocking agent or counter-dye can also consist of any molecule or mixture of molecules capable of interacting specifically with inert particles or dead cells having the characteristic of neutralizing a possible emission of parasitic fluorescence by the viability marker. or its derivatives, either by:
• the blocking agents will be chosen from the range of fluorescent markers, having similar structural characteristics, but not emitting or emitting at wavelengths, different from those of viability markers, and advantageously among the markers having a fluorescence absorption wavelength substantially equal to the emission wavelength of the viability markers used.
• the viability markers used in step b) are advantageously fluorescent markers, such as esters of fluorescein, carboxyfluorescein, BCEF, 5-6-carboxyfluorescein diacetate (CFDA) or fluorescein diacetate or a mixture of those -this. These viability markers each have a specificity of targets, and require suitable labeling buffer conditions.
• the CFDA is more particularly suitable for labeling bacteria while the FDA has a better capacity for accumulation in fungi or yeasts under neutral pH conditions.
• a mixture of these two fluorescent esters will therefore make it possible to detect and count a wide spectrum of microorganisms present simultaneously in a biological sample.
• the viability markers are chosen from the group formed by xanthenes, acridines, fluorones and aminoazides.
• the method of the invention can also be used when fluorescein isothiocyanate (FITC) is used as a fluorescent marker in association with a specific iigand (mono or polyclonal antibody and / or nucleic probe) that it s is the identification of microorganisms or animal cells. In this case, non-specific adsorption of the fluorescence emitted is often observed.
• FITC fluorescein isothiocyanate
• iigand mono or polyclonal antibody and / or nucleic probe
• the use of a composition of blocking agents prior or simultaneous to labeling with Iigand can make it possible to avoid this artefact.
• the invention also relates to a composition of blocking agents capable of preventing the attachment 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 length wavelength of fluorescence absorption substantially equal to the length emission wave of the fluorescence of the first viability markers; are more particularly chosen from fluorones such as Perythrosine B, ethyl eosin, methyl eosin, Eosin B, Y, Phloxin B or a mixture of these; b) one or more compounds capable of entering into competition with the product resulting from the hydrolysis of the marker, cause of the parasitic marking; c) a mixture of a) and b).
• fluorones such as Perythrosine B, ethyl eosin, methyl eosin, Eosin B, Y, Phloxin B or a mixture of these
• the present invention also relates to a labeling composition consisting of one or more mixtures of viability markers in a high ionic strength buffer.
• the invention also relates to the use of the method for detecting and / or counting viable cells in a sample including sporulating forms, excluding dead cells; it also makes it possible to specifically count these.
• the invention relates to a kit or kit allowing the detection and the counting of viable cells in a total flora, which kit comprising at least:
• the blocking agents make it possible to differentiate on the one hand the living cells and, on the other hand, the particles and dead cells by differential labeling.
• the labeling agents are derivatives of fluorescein and having an emission wavelength of 515 nm
• the blocking agents will preferably be halogen derivatives of the xanthene family having an absorption wavelength in the same window or their derivatives.
• the blocking agent is preferably chosen from the group formed by fluorones substituted with at least one halogen atom, such as chlorine, bromine, fluorine and / or iodine; among the substituted fluorones, there may be mentioned, for example, eosin B, eosin Y, phloxin B, erythrosin B, ethyl eosin or a mixture of two or more of these.
• halogen atom such as chlorine, bromine, fluorine and / or iodine
• the blocking agent is preferably chosen from the group formed by fluorones substituted at least by 3 halogen atoms, and preferably by 4 halogen atoms, and in particular by 4 iodine atoms or 4 bromine atoms, such as, in particular, eosin Y, phloxin B, erythrosin, ethylosin or a mixture of two or more of these. Good results have been obtained when the blocking agent is chosen from the group formed by erythrosine B, ethyl eosin or a mixture of these.
• the similarity of the markers is important since most of the dyes tested prove to be ineffective in the desired application, in particular the blue dyes are ineffective such as evans blue, trypan blue or methylene blue.
• Table 1 summarizes the main dyes that have been tested with their characteristics and effects:
• Active blocking agents can be used alone or in combination.
• the advantage of using a combination is to increase the spectrum of action of these blocking agents.
• ethyl eosin, Phloxin B and Eosin Y which are brominated derivatives of fluorescein will preferentially mark the killed microorganisms while erythrosine, an iodine derivative of fluorescein, will preferably mark inert particles in the medium to be analyzed.
• a mixture of ethyl-eosin and erythrosine will therefore be particularly effective in marking all of the particles or cells which are not viable microorganisms in any sample.
• the invention also relates to a composition of blocking agents comprising fluorones more particularly chosen from the group formed by erythrosine B, ethyl eosin, methyl eosin, Eosin B, Y, Phloxin B or a mixture thereof in concentration ratios by weight ranging from 10/1 to 1 / 10.
• fluorones and their relative concentrations will depend on the type of product to be analyzed, as well as its environment. For example, for the microbiological analysis of water, erythrosine and ethyl-eosin are used in ratios of between 5/1 and 1/5, and preferably, for example 2/1, that is respective final concentrations of 0.004% 0 and 0.002% o.
• the solution comprises the constituents at a concentration which is between 2 and 15 times that of the hydrolysis product of the viability marker responsible for the non-specific fluorescence signals, knowing that the hydrolysis products represent between 1 and 10% by weight of the marker viability according to the type of sample analyzed.
• the method of the invention comprises the use of a universal type labeling composition capable of labeling any type of cell present in the sample studied.
• the cells can be a prokaryote, a monocellular eukaryote, an animal cell, in a biologically active form or in a sporulating form.
• a labeling composition will be, when the fluorescent signal is sought, a composition of markers which the experimenter will dilute extemporaneously in the high ionic strength labeling buffer.
• the marker composition will preferably consist of a mixture of 5 (6) carboxyfluorescein diacetate (CFDA) at a concentration between 5 and 10 mg per ml, and fluorescein diacetate (FDA) at a concentration between 0.5 and 5 mg per ml in pure acetone (qs).
• the weight ratios of CFDA and FDA will be between 5/1 and 15/1 and optimally 9/1, ie for 1 ml, 9 mg / ml (19.5 mM) of CFDA and 1 mg / ml (2.4 mM) of FDA in pure acetone.
• the marker composition as described above is diluted to a hundredth in a marking buffer whose ionic strength is greater than 0.5 and comprising in an aqueous solution.
• the sodium acetate buffer can be replaced by other buffers of the phosphate, HEPES, citrate, etc. type.
• sodium chloride can be replaced by potassium chloride (KCL), NH4 ( 2) S04 etc ...
• composition consisting of a marker, or of a mixture of markers as described above and diluted in the marking buffer, also forms part of the invention.
• the particularly high efficiency of the buffer in question is surprising because of its very high ionic strength. Indeed, the isotonicity was generally the solution chosen to maintain the integrity of the viable cell. Tests performed in flow cytometry on bacteria in stationary phase are presented in Figure 1.
• Figure 1 shows the effect of ionic strength on the number of cells detected.
• the abscissa indicates the intensity of fluorescence, and the ordinate the number of cells detected.
• the left column represents the results with Bacillus subtilis and the right column with Serratia marcescens. They show that an increase in ionic strength is accompanied by an increase in the intensity of fluorescence, resulting from a better accumulation of the marker in living cells.
• a step of swelling the spores present, where appropriate in the medium to be analyzed is carried out by the addition of a TCS type medium, a malt extract or advantageously a mixture of of them.
• the sample treated beforehand with the blocking agent as described above is brought into contact with the swelling medium either by dilution or resuspension of the sample to be analyzed in the swelling medium in the case of enumeration in the middle liquid, either by transfer of the membrane on which the sample to be analyzed has been filtered, on an absorbent support saturated with the swelling medium.
• the sample is then incubated from 40 mm to 3 hours at 30 ° or 37 ° C depending on the desired application; a short 40 mm incubation at 37 ° C will allow the detection of bacteria spores, while a longer incubation (3 hours at 30 ° C) will also allow the detection of mold spores.
• the temperature can be lowered and the incubation time extended to obtain the same result.
• the samples are placed in the presence of the labeling solution as described above, in a similar manner to the swelling step and incubated for 30 minutes at 30 ° C to plus or minus 3 ° C.
• the blocking agent (s) has the particularity of being in a concentration ratio with the viability marker (s) from 5 to 15 to 1, which is the reverse of the usual ratios used between dyes and counter-dyes which are of the order of 1/10.
• An optimal ratio for fluorescein derivatives will be about 10 to 1.
• the treatment of the sample with the blocking agent will always be prior, or at the limit simultaneous with the treatment with the composition containing the viability markers.
• hydrophilic support either in the swelling step, or in the marking step by direct deposition of the swelling medium or of the marking buffer on or under the carrier filter. filtered microorganisms.
• the method of the invention finally comprises a step of analysis by any suitable means for measuring the signal emitted by the viability marker. If, however, the analysis is not carried out immediately after the incubation with the labeling composition, the samples must be placed at 8+ 4 ° C. protected from light, but without however exceeding a period of 30 minutes.
• the detection and / or counting method according to the invention can in particular be implemented in the apparatus described in patent applications EP 0 333 561 and WO 89/08714 and sold under the brands CHEMSCAN® and CHEMFLOW®.
• the invention also relates to the implementation and the use of the kit of the process of the invention in all the applications where the presence of living cells is sought.
• the method and the kit according to the invention can be used in particular for detecting and / or counting possible microorganisms in hygiene, food or pharmaceutical products.
• the method and the kit of the invention can also be used to control an industrial process such as sterilization, whether in food, pharmaceutical or nutraceutical applications, either in a stage of a manufacturing process or on the product. finished.
• the method and the kit can be used to control the bacterial load before and after a sterilizing filtration (bioburden).
• the method and the kit of the invention can finally be used for the detection of viable microorganisms not detectable by conventional methods. It can be for example:
• Example 1 Implementation of the process in the absence of any filtration of samples:
• the implementation of the method consists in carrying out a pretreatment with a blocking agent prior to treatment with the viability markers.
• the aim of this experiment is to show the effectiveness of this treatment in eliminating false positives resulting from a non-specific interaction between the viability markers and the filtration membrane.
• CSE Counter Staining E
• Table 3 compares the fluorescence results measured at CHEMSCAN and obtained without or with prior treatment by the CSE: / 55861
• the filtration media are rinsed successively with three times 1 ml of 70% ethanol (v / v) and then with 3 times 1 ml of filtered water (through 1 0.22 ⁇ m filter).
• the filtration medium used is a poyester membrane, which is then placed on a wet filtration medium.
• the viability marker 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 and diluted extemporaneously then to the hundredth in a buffer whose composition is as follows:
• Example for 20 analyzes: - In a sterile bottle containing 20 ml of prefiltered marking buffer over 0.22 ⁇ m, add 200 ⁇ l of viability substrate, taking care not to touch the wall of the bottle,
• Example 2 Efficiency of the Process on Peptone and Buffered Water: Peptone water is water used to make dilutions of bacteria in order to precisely count them as described in particular in patent WO 8908714.
• step b) Six filtrations were carried out in the absence of step b) and with step b). the blocking agent and the viability marker used are the same as those of Example 1.
• the sign F + indicates the number of fluorescent particles detected by CHEMSCAN®.
• the column which indicates "primary count” is in fact the fluorescence count before the step of discrimination performed by CHEMSCAN® which eliminates, we recall, all events not related to a particle of shape and size taken into account in the program of the device.
• Example 3 Process carried out on a sterilized antibiotic solution:
• Example 5 The experiment is strictly identical to that of Example 2 with the exception that 10 ml of a sterile antibiotic solution were filtered through the polyester membrane between steps a) and b) in Example 1. The results are presented in Table 5 below: Table 5:
• CHEMSCAN® detected respectively 322 and 293 particles exhibiting fluorescence after discrimination whereas, by treatment with CSE, this figure is reduced to 5 and 12 fluorescent particles respectively. 98.5% decrease in the first case and 96% in the second case in the number of false positives.
• the method of the invention leads to a drastic reduction of false positives obtained by direct use of viability markers on a sample likely to contain microorganisms; this decrease going from 80 to 99%.
• this technology has the advantage of not presenting any false negative, in other words the blocking agent does not decrease the number of viable cells enumerated in a sample.
• the counter-dye and more particularly the CSE also has the advantage of being able to be used in a kit since it has autoclaving and preservation properties compatible with commercial distribution.
• One advantage, and not the least of the composition containing the blocking agent (s), is its wide spectrum of use since the proposed mixture makes it possible to mark both dead cells and non-organic particles. It goes without saying that, depending on the sample that it is desired to test, one or other of these blocking agents will preferably be used in such a way that it remains in the desired relationships with the viability markers used in the continuation of the process. A person skilled in the art will know, as necessary and according to the sample he wishes to analyze, to choose both its composition of blocking agents and that of viability markers and the weight ratio between the two.
• the last 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 exclusively the living cells including the sporulating forms, on the one hand, and the inert forms, of somewhere else.
• the method of the invention is of general application to other labeling agent / blocking agent pairs. The skilled person will still determine the blocking agent having one or other of the additional features cited in text early characteristics of the viability marker.

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• 1997
  • 1997-06-04 FR FR9706890A patent/FR2764305B1/fr not_active Expired - Fee Related
• 1998
  • 1998-05-20 CA CA002289566A patent/CA2289566A1/fr not_active Abandoned
  • 1998-05-20 AU AU77754/98A patent/AU7775498A/en not_active Abandoned
  • 1998-05-20 WO PCT/FR1998/001019 patent/WO1998055861A1/fr not_active Application Discontinuation
  • 1998-05-20 JP JP50170099A patent/JP2002505578A/ja active Pending
  • 1998-05-20 EP EP98925763A patent/EP0986752A1/fr not_active Withdrawn

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