WO1995000660A1

WO1995000660A1 - Method for assessing microorganism viability

Info

Publication number: WO1995000660A1
Application number: PCT/FR1994/000770
Authority: WO
Inventors: Pieter Breeuwer; Jean-Louis Drocourt
Original assignee: Chemunex
Priority date: 1993-06-28
Filing date: 1994-06-27
Publication date: 1995-01-05
Also published as: FR2707665A1

Abstract

A method for assessing microorganism viability, including: (a) loading the microorganisms with a fluorescent substance, and (b) measuring the brightness of the intracellular fluorescence by flow cytometry. The method is characterised in that it includes the steps of: (i) incubating said microorganisms for 5-20 minutes with the marking substance in a concentration of 1-300 νM and in the presence of a marking buffer at a pH of 4-8, to give microorganisms loaded with a fluorescent substance (loading step (a)); (ii) removing all of the marking substance not incorporated into the microorganisms; (iii) adding a source of energy prior to step (b); and (iv) using flow cytometry to measure the time required for half of the intracellular fluorescence of the microorganisms to disappear, within a predetermined brightness range, at a pH of 4-8 (outflow buffer) and at a temperature of 10-40 °C, preferably 20-40 °C.

Description

METHOD FOR EVALUATING THE VITALITY OF MICROORGANISMS

The present invention relates to a method for measuring and evaluating the vitality of living microorganisms (bacteria, yeasts, recombinant microorganisms) and to its applications, in particular in assessing the quality of the inoculum ( evaluation of beer fermentation, lactic ferments) and monitoring of fermentation. In general, to assess the biological state (living vs dead) of microorganisms, there are many methods that assess cell viability (ability of microorganisms to reproduce, appreciation of membrane integrity) or cell vitality (for example , acidifying power).

Among these methods for assessing the viability or the vitality of microorganisms (TREVORS JT et al, Biotechnology Letters, 1983, 5, No. 2, _131-134..); MJ CHILVER et al., Amer. Soc. Bre ing Chemists, 1978, 26., 13-18; JC SLAUGHTER et al., Enzyme Microb. Technol. , 1992, 14 _. , 64-67; BV KARA et al., J. Inst. Bre., 1988, 94., 153-159; HM SHAPIRO, 1988, Practical Flow Cytometry, Allan R. LISS, Inc. New-York; Patent Application EP 333,560), we can cite in particular: A. Methods of assessing viability:

- the method of counting on nutrient medium, which defines viability as the capacity of living microorganisms to form visible colonies (TREVORS et al.; SHAPIRO et al., cited documents); this method, although reliable, has the disadvantage of requiring a long response time (24 to 72 hours), which is not suitable for the abovementioned applications

- The methylene blue exclusion test in which viability is defined as the ability of microorganisms not to stain with methylene blue; that is, only the dead cells are stained blue (TREVORS et al., cited document); however during this test has the drawback of overestimating viability;

- intracellular glycogen and trehalose evaluation; this test is based on the hypothesis that the intracellular concentration of glycogen (or treha¬ lose) is correlated with the viability of microorganisms (SLAUGHTER et al., document cited); however, it should be noted that there is no single correlation between the glycogen or intracellular trehalose content and the viability and that this test is therefore difficult to interpret; the fluorescein ester test (FDA in particular), based on the use of non-fluorescent precursors (fluorochromes such as fluorescein esters), which when they enter mammalian cells or microorganisms, are hydrolysed by non-specific esterases and produce a fluorescent product which remains mainly or totally trapped inside said cells or said microorganisms (B. ROTMAN et al., Proc. Natl. Acad. Sci. USA, 1966, J55., 134-141; SHAPIRO et al., Cited document), when the latter present an intact membrane (capacity to accumulate the fluorescent product).

Many Authors have applied this latter test to determine the viability of microorganisms, yeasts or other eukaryotic cells (lymphocytes) and bacteria in different environments (water, soil, etc.) (J. BRUNING et al., J. Immunol. Meth., 1980, 23, 33-44; MJ CHILVER, document cited; TH CHRZANO SKI et al., Microb. Ecol., 1984, 10., 179-185; JP TIAPER et al., Appl. Microbiol. Biotechnol 1992, 28, _268-272;.. BE SOTERSTRÔM Biol Biochem 1977 9_, 59-63;.. TALBOT T. et al, J Immunol Meth 1987, 99, _137-140.... ). The fluorescence intensity of the individual cells can, for example, be measured by cytometry flow (HM SHAPIRO, document cited) or by microscopy (TREVORS et al., document cited).

However, as SHAPIRO clearly demonstrated, the criterion of membrane integrity for assessing viability has limits; in particular, the distinction between marked cells (which are considered to be viable) and cells which are not marked (considered to be non-viable) is not absolute. Indeed, cells that have lost the ability to reproduce (i.e. cells considered to be non-viable), can nevertheless be stained if they have an intact membrane.

B. Method for evaluating cell vitality: - the acidification power test: this test is based on the assumption that viable yeasts are capable of acidifying the medium while dead yeasts are not (KARA et al., Cited document); this method has the disadvantage of being a global measure, which does not allow to assess the cellular state of the population studied.

All the methods for determining the viability or the vitality of the microorganisms of the prior art therefore have the major drawback of not being reliable or of having too long a response time (method of counting on nutrient medium ) and therefore do not, for the most part, effectively distinguish viable cells from non-viable cells (false positives, false negatives, difficulty in interpretation, long response time, etc.).

The Applicant has consequently to the aim of providing a method for assessing in more s ^* ure viability live microorganisms, which does not have the disadvantages of proce- dice the prior art (aspen positive, false negative, difficult interpretation, long response time, etc.) and which allows to follow in particular the evolution of a fermentation process, in a reliable manner.

The subject of the present invention is a method for evaluating and measuring the viability of microorganisms, comprising:

(a) loading the microorganisms with a fluorescent product, by incubating said microorganisms at a temperature between 20 ° C. and 50 ° C. with a marker (fluorochrome), and (b) measuring the intensity of intracellular fluorescence by flow cytometry, which method is characterized in that it comprises:

(i) the incubation of said microorganisms for 5 to 20 minutes with said marker at a concentration of between 1 μM and 300 μM, in the presence of a labeling pad at a pH of between 4 and 8, for obtaining microorganisms loaded with fluorescent product (loading step (a)),

(ii) elimination of the marker not incorporated in the microorganisms,

(iii) adding an energy source, prior to step (b) and

(iv) measuring the time necessary for the disappearance of half of the intracellular fluorescence of microorganisms, by flow cytometry, in a window of predetermined intensity, at a pH between 4 and 8 (efflux buffer) and at a temperature between 10 ° C and 40 ° C, preferably between 20 ° C and 40 ° C.

Unexpectedly, the time necessary for the disappearance of half of the intra¬ cellular fluorescence of microorganisms, in the presence of an energy source, is a function of the vitality of the microorganisms, defined as the capacity of the microorganisms to metabolize quickly after transfer from a nutrient-poor environment to a nutrient-rich environment. According to an advantageous embodiment of said process, the marker is chosen from FDA, 6-CFDA, 5-CFDA, 5-maleimide FDA, fluorescein dilaurate, fluorescein dibutyrate, 5 ( 6) 2 ', 7' dichloro-CFDA, 5 (6) sulfo-FDA, 5 (6) 2 ', 7' dichloro-FDA, 5,6-CFDA ester N-hydroxy succinimide, dipropionate of fluorescein, di-beta-D-galactoside fluorescein, 3-O-methylfluorescein phosphate, pentaacetoxy ester of 2 ', 7' -bis (carboxyethyl) -5 (6) - carboxyfluorescein (BCECF / AM), 2 ', 7' pentaacetoxy ester -bis (carboxyethyl) -5 (6) -fluorescein (BCECF / AM), azidofluorescein diacetate, cororomethylfluorescein diacetate, eosin diacetate, carboxyeosine diacetate , 4-methylumbelliferyl acetate, 4-methylumbelliferyl beta-D-galactoside, 4-methyl¬ umbelliferyl alpha-D-mannopyranoside, 4-methylhumbumbelliferyl nonanoate, 4-methylumbelli¬ feryl phosphate, 1 'a lanine-7-amino-4-methylcoumarine, glycine-7-amino-4-methylcoumarine, proline-7-amino-4-methylcoumarine, valine-7-amino-4-methylcoumarine, glycyl-L-proline- 7-amino-4-methylcoumarin, 1,4-diacetoxy-2,3-dicyanobenzene (ABD), hydroethidine, resorufin acetate.

Such a marker (fluorochrome) is, as specified above, a non-fluorescent or weakly fluorescent substance, which penetrates into the microorganism and is hydrolyzed therein, so as to provide a fluorescent product which is trapped inside said microorganism .

Preferably, said marker is carboxyfluorescein diacetate (cFDA).

During the loading step (a), the fluorescent product remains correctly trapped in the microorganisms stored at 0 °, but from 10 °, an efflux of marked marked product is observed, in the presence of said energy source, if microorganisms are vital. According to another advantageous embodiment of said process, said energy source is a sugar.

According to a preferred arrangement of this embodiment, said sugar is chosen from glucose, fructose, mannose and lactose.

According to a preferred form of this arrangement, said sugar is 10 M glucose.

As an indication, mention may be made, as labeling buffer and / or efflux buffer, of an acetate-NaCl buffer at pH 7, a PBS buffer at pH 7, a citrate-phosphate buffer at pH 4 or 7.3 or a buffer phosphate at pH 7.

The efflux buffer can either be of identical composition to the marking buffer (same pH), or of different composition (different pH).

The elimination of the non-incorporated marker is preferably carried out by rapid washing of the labeled microorganisms.

As a variant, the process in accordance with the invention comprises, after the removal of the non-incorporated marker (step (ii)),

(iii) the implementation of step (b) on two sets of samples of microorganisms:

- assembly A, in which the intensity of intracellular fluorescence of the microorganisms is measured at regular time intervals for 5 to 60 min, in an efflux buffer at a pH between 4 and 8 and at a temperature between 10 ° C and 40 ° C, preferably between 20 "C and 40 ° C; - assembly B, in which the intensity of intracellular fluorescence of the microorganisms is measured at regular time intervals for 5 to 60 min, in an efflux buffer at a pH between 4 and 8, to which an energy source has been added, and at a temperature between 10 ° C and 40 ° C, preferably between 20 'C and 40 "C; and (iv) evaluation of the vitality of the microorganisms as a function of the ratio of the intensities of intracellular fluorescence: set B / set A.

Measuring the fluorescence intensity at regular time intervals makes it possible to assess the output flux (efflux) of the fluorescent product; the higher the speed of efflux, the more metabolically active the microorganisms.

The differential assessment of the efflux of fluorescent product between the microorganisms of set B and the microorganisms of set A makes it possible to assess the vitality of living microorganisms reliably.

Indeed, the efflux buffer, associated with an energy source added to the microorganisms (set B), significantly stimulates the efflux of fluorescent product (at pH 7.3 or at pH 4, for example and at a tempe¬ rature between 20 ° C and 40 ° C), only if said microorganisms have a certain vitality. In general, the results obtained by flow cytometry are in the form of histograms.

The loss of fluorescence (efflux from 10 or 20 "C) is characterized by a shift down the fluorescence channels, in the histograms.

The addition of sugar (set B) causes displacement (loss of greater fluorescence of the microorganisms). It is this displacement towards the low fluorescence channels, increased by the addition of sugar, which makes it possible to appreciate, as specified above, the vitality of the population of microorganisms.

The process according to the invention has the advantage of assessing the viability of microorganisms reliably, by bringing in other parameters than membrane integrity. The present invention also relates to a kit or kit for implementing the method according to the invention, characterized in that it comprises: at least one marker for viable microorganisms (fluorescein esters),

- a marking buffer at a pH between

4 and 8,

- an efflux buffer at a pH between 4 and 8,

- a sugar.

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.

It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1 Measurement of the vitality of yeasts. A) Lavaσe of yeasts:

2 ml of a yeast suspension (approximately 5.10 'cells / ml) are centrifuged for 90 seconds at 13,490 g; the supernatant is removed by aspiration, then the pellet is resuspended in 2 ml of labeling buffer comprising, for example, Mcllvaine buffer at pH 4 (the Mcllvaine buffer is composed of 30.7 ml of citric acid (100 itiM ) and 19.3 ml of dihydrogen hydrogen phosphate dihydrate (200 mM)); the suspension is again centrifuged for 90 seconds at 13,490 g, then the supernatant from this second centrifugation is removed by suction; a second washing of the yeasts is carried out by resuspending the pellet in 2 ml of labeling buffer as defined above; the yeast sample thus washed is placed in ice until marking.

The washed yeast suspension should not contain less than 2.10 ⁶ cells / ml. When the initial cell concentration is high (> 2.10 ⁶ cells / ml), the washing step can be replaced by a dilution step.

B) Marσuaσe:

2 ml of washed yeast suspension are taken and shaken vigorously, then 30 μl of cFDA is added, so as to obtain a final concentration of 43 μM.

The suspension is then incubated for 15 min at 40 ° C. and placed in ice, to avoid any efflux. It should be noted that the fluorescence intensity of the yeasts after loading with cF depends on the concentration of marker fluorochrome.

Consequently, to obtain a good analysis, the concentration of marker must be adjusted so that the average fluorescence intensity of the yeast population is preferably to the right of channel 100 on the histogram.

C) Lavaσe after marσuaσe:

2 ml of suspension of labeled yeasts are centrifuged for 90 seconds at 13,490 g, then the supernatant is removed by suction; the pellet is resuspended in 2 ml of efflux buffer, maintained at 0 ° C. and constituted by an Mcllvaine buffer at pH 7.3; centrifugation is again carried out for 90 seconds at 13,490 g, the supernatant is removed by suction and the pellet obtained is resuspended in 2 ml of efflux buffer at 0 ° C. as defined above.

The sample is then placed in ice until the actual efflux measurement is made. D) Measuring the efflux:

* Differential method: a) Take a 1 ml aliquot from the suspension obtained in C) and dilute it in 30 ml of efflux buffer as defined above, shake it and analyze it directly using a flow cytometer (at room temperature). b) Take another 1 ml aliquot from the suspension obtained in C) and dilute it, either in 30 ml of efflux buffer as defined above (set A), or in 30 ml of buffer d efflux to which 200 μl of glucose were added (set B), so as to obtain a final glucose concentration of 10 mM; the suspension is stirred and placed in a water bath at a temperature between 10 and 40 ° C, preferably between 20 ° C and 40 ° C.

Aliquots of each of these suspensions (3 ml) are taken, at regular time intervals, which are analyzed directly by flow cytometer. Ideally, the yeast population which will be analyzed by flow cytometry should contain approximately 5.10 ^ to 10 ^ cells / ml (equivalent to approximately 1.10 ⁴ to 2.10 ⁴ cells on the histogram).

The volume of the sample analyzed and / or the dilution in the efflux buffer will be adapted according to the initial concentration of cells. The efflux temperature can vary between 0 ° (no efflux) and 40 'C

(significant efflux).

* Glucose only method: Under the conditions of b) above, at 40 ° C., the histogram of a suspension of yeasts put into culture overnight will "disappear" after approximately 25 min (when glucose is added.) At 30 ° C, the histogram of a viable yeast suspension will disappear after approximately 50 min (when glucose is added). * Results: a) Measurement of efflux:

Cells of S. cerevisiae were loaded, as specified above, at pH 4 and the retention of cF esl studied under different conditions (sets A and B).

As shown in FIG. 1 (which includes the time in minutes on the abscissa, and the ordinate, the percentage of trapped carboxyfluorescein), the cF is trapped in cells stored at 0 ° C., but at 30 °, an efflux rapid cF occurs, dependent on pH.

The addition of glucose to cells at pH 7.3 results in a significant stimulation of the efflux of cF, which suggests that the cF is released in an energy-dependent manner. Fructose and mannose stimulate the flow of cF in the same way as glucose, while galactose and glycerol have no effect.

Figure 1 shows the measurement of the efflux at pH 7.3, at 0 ° C (M), 30 ° C (Δ) and 30 "C in the presence of 10 mM glucose (A). The efflux at pH 4 was measured at 30 ° C (O) and at 30 ° C in the presence of 10 mM glucose (•).

For this experiment, cells of S. cerevisiae were loaded into cF by incubation at 40 "C with 0.22 mM cFDA; the cells were washed and resuspended in Mcllaine pH 4 buffer, as specified above. .

The stimulation of the transport of cF at pH 7, 3, after addition of glucose (FIG. 1), suggests that the transport is effectively active. The efflux of cF can be determined as shown above by flow cytometry; the histogram will undergo a shift down the fluorescence channels on the X axis as a result of the loss of intracellular fluorescence and this phenomenon is correlated with the capacity of the cells to create or maintain an active pro¬ tonic force. (set B). The intensity of intracellular fluorescence in cF of the microorganisms of set B at 15 min is lower than that of set A by a factor of between 2 and 4, the ratio of fluorescence intensity set B / fluorescence intensity set A being less than 1.

In the glucose-only method, the time necessary for the disappearance of half of the intracellular fluorescence is measured in a predetermined window (see FIG. 9). b) Demonstration of an active transport system:

- efflux of cF against a concentration gradient: To determine if the efflux of cF is linked to metabolic energy and not to a passive flux, the efflux is studied in the presence of a concentration of extracellular cF large (1 mM). Then, the efflux is calculated, after determining the concentration of intracellular cF.

The initial intracellular cF concentration is approximately 0.5 mM. The efflux, in the absence and in the presence of 1 mM extracellular cF is comparable, in the presence of an energy source; the efflux therefore takes place against the concentration gradient.

These results effectively suggest that the efflux of cF in S. cerevisiae is carried out using an active transport system.

- Inhibition of the efflux of cF:. the effect of different compounds on the efflux of cF has been studied in order to support the hypothesis of the active transport system of cF.

(i) effect of DNP and benzoic acid: FIG. 2 illustrates the effect of DNP (1 mM) and benzoic acid (10 mM) on the efflux of cF. To carry out this test, i, the cells are preincubated for 20 min at 40 ° C. with an Mcllvaine buffer pH 4, in the absence (•) or in the presence of 2.4 DNP 1 mM (A) or benzoic acid 10 mM (M). After washing, the cells are loaded into cF by incubation at 40 ° C with 43 μM cFDA in Mcllvaine buffer pH 4, washed and resuspended in Mcllvaine buffer pH 7.3. The efflux test is carried out at 30 ° C. This figure 2 shows that DNP is more effective in inhibiting the efflux of cF than benzoic acid.

These results confirm the presence of an energy-dependent efflux. (ii) effect of TPP ⁺ (tetraphenylphosphonium ions): FIG. 3 shows that TPP ⁺ , at a concentration of 10 M, completely inhibits the efflux of cF. The addition of TPP ⁺ does not cause a significant decrease in the intracellular ATP concentration. This suggests that the efflux of cF is related to the proton pump.

More precisely, FIG. 3 which shows, on the abscissa, the time in minutes and, on the ordinate, the percentage of trapped carboxyfluorescein, illustrates the effect of TPP ⁺ on the efflux of cF (A) and on the intracellular concentration of ATP (B) in energized S. cerevisiae cells, as follows: the cells are loaded into cF by incubation at 40 ° C with 43 μM cFDA in Mcllvaine buffer pH 4, washed and put back in addition - board in Mcllvaine buffer pH 7.3. The test is carried out at 30 ° C, the efflux is measured in the absence (•) or in the presence of TPP ⁺ 10 mM (A).

(iii) effect of ATP-ase inhibitors: Such compounds are in particular vanadate, DES and DCCD (N, N '-dicyclohexylcarbodiimide); the effect of these compounds on the proton pump in cells Energized whole yeast cells are illustrated in Figure 4.

The production of H ⁺ ions is measured continuously, with a pH electrode (determination directly from the slope (in nmol H ⁺ / min / mg of protein). After the addition of glucose (10 mM) (energized yeasts), the initial release of H ⁺ is increased to approximately 540 nmol H ⁺ / min / mg of proteins. Among the inhibitors tested, DES (150 μM) is the most effective: the production of H ⁺ in the presence of glucose (10 mM) is 122 nmol H ⁺ / min / mg of proteins. Incubation with DCCD (200 μM) decreases the production of H ⁺ approximately by 50% at around 330 nmol H ⁺ / min / mg of proteins, while vanadate, which is a phosphate analog, shows only a weak effect (436 nmol / min / mg of proteins).

A longer pre-incubation of the cells (45 min with DCCD or vanadate) does not change the effect obtained. Figure 4, which includes, on the abscissa, the time in minutes, and on the ordinate, the percentage of trapped cF, illustrates the effect on the efflux of cF (A) and on the intracellular concentration of ATP (B) of different compounds on cells of S. cerevisiae energized by different compounds.

As in the previous tests, the cells are loaded into cF by incubation at 40 "C with 43 μM cFDA in Mcllvaine buffer pH 4, washed and resuspended in Mcllvaine buffer pH 7.3. The test is carried out at 30 ° C. The curve (O) corresponds to the addition of no product, the curve (Δ) corresponds to the addition of DES (150 μM) or the curve (•) corresponds to the addition of DCCD (100 μM).

This figure shows that the inhibition of the proton pump is correlated with the inhibition of the ef¬ flux of cF. In energized whole cells, DES significantly inhibits the efflux of cF, • while DCCD shows only a weak effect.

The intracellular ATP concentration is not influenced by either DES or DCCD. The vandat (1 mM) shows no effect. c) Study of the kinetics of the efflux of cF: The cells are loaded with different concentrations of cF and the initial efflux speed is determined (FIG. 5). FIG. 5 comprises on the abscissa, the intracellular concen¬ tration in cF (mM) and on the ordinate, the efflux speed of cF (μmol / min), after loading of S. cerevisiae cells with cF, by incubation at 40 ° C, for variable times, with cFDA (0.22 mM) in Mcllvaine buffer pH 4, washing and resuspension in Mcllvaine buffer pH 7.3, as specified above; from the slope, in the Arrhenius block (Figure 6), an activation energy of 50 kJ / mol is calculated, which confirms the involvement of a transport system; apparently the transport system (cF output flow) obeys Michaelis-Menten kinetics.

The results are analyzed in the form of an Eadie-Hofstee plot and the K _m of the 0.25 mM cF transport is calculated. The results indicate that the cF is released using a membrane transport system, from loaded S. cerevisiae cells as described above (incubation at 40 ° C. with different concentrations of cFDA in Mcllaine pH 4 buffer. ), washing and resuspension in Mcllvaine buffer pH 7.3). The efflux is measured at 30 ° in the presence of glucose (10 mM).

The efflux, measured at different temperatures (Figure 7) shows that this efflux is dependent on the temperature. E) Analysis of the data obtained by flow cytometry:

The flow cytometer used is a Chemflow® flow cytometer. The fluidic system comprises a closed laminar flow with a maximum flow rate of 0.4 ml / min. For the enumeration by flow cytometry, a sample of 200 μl is analyzed. Under normal conditions, the maximum concentration of fluorescent particles which can be measured by this flow cytometer is limited to approximately 1.10-Vml.

The instrument includes a light source constituted by an arc of mercury HPO 100. The excitation wavelength is selected by the use of 2 band filters and is between 450 and 490 nm. The emission wavelength is 515 nm and is selected using a dichroic filter of 500 nm (eliminating signals less than 500 nm) and an interference filter (FI 515) at 515 nm. The data are presented in the form of a histogram in which the Y axis corresponds to the number of fluorescent particles and the X axis is divided into channels (0 to 255) corresponding to the fluorescence intensity of the particles. . Logarithmic amplification (logl mode) of the incoming signal is applied to directly measure a larger number of signals on a histogram; the data can be, if necessary, recalculated on a linear scale. Figure 8 illustrates the determination

(histogram) of the efflux of cF. The average intracellular cF concentration of a culture of S. cerevisiae loaded with cFDA is about 0.1 to 0.4 mM, which is approximately equivalent to 1.10 'fluorescent molecules / cell. The minimum intra-cellular fluorescein concentration that could be measured (channel 1 in log 1 mode of _. flow cytometer) is approximately between 0.05 and 0.1 mM.

FIG. 8 illustrates a shift to the left of the histograms due to the loss of fluorescence at 40 ° of a culture of yeasts loaded with cF. The addition of glucose leads to a significant additional shift to the left, of the fluorescence intensity of the cells.

To show that this additional displacement is indeed a parameter of the analysis of the vitality of the yeasts, comparative tests are carried out with yeasts S. cerevisiae stressed by a short incubation at high temperature (60 ° C., 1.5 min), DNP (1 mM) or benzoic acid (10 mM). The histograms obtained show that neither the initial number of fluorescent particles, nor the intensity of initial fluorescence of the cells charged with cF, are significantly reduced.

However, the decrease in CFUs after treatment at the temperature of the suspension of S. cerevisiae is greater than 4 log units and the shift to the left of the histogram is significantly reduced.

In this case, the addition of glucose has practically no effect; this therefore suggests that cells are not vital.

In contrast, washed cells preincubated with benzoic acid show no decrease in CFUs, but the reduction in the displacement of the histogram is significantly significant compared to the control.

However, the posterior addition of glucose induces a shift to the left (decrease in the intensity of intracellular fluorescence); this indicates that the cells are stressed, but still have some vitality. In the case of DNP, the cells are severely stressed, insofar as there is a decrease in the concentration of intracellular ATP and a decrease in CFUs, but the slight displacement of the histogram towards the left after addition of glucose indicates that at least a significant part of the population has lost much of its vitality.

In the histograms of FIG. 8, the cells were loaded by 15 minutes of incubation with 43 μM cFDA at 40 "C in Mcllvaine buffer pH 4, washed and resuspended in Mcllvaine buffer at pH 7, 3.

The loss of fluorescence at 40 ° C after 0, 20, 40 and 60 minutes is measured with a flow cytometer for non-energized cells (histograms A to D) and for cells energized with glucose (10 mM) (histograms E to F).

The cells were pretreated as follows: no treatment (histograms A and E), preincubation with DNP (1 mM) (histograms B and F), heat treatment at 60 ° C for 90 seconds (histograms C and G) and preincubation with benzoic acid (10 mM) (histograms D and H).

The viability-vitality relationship of the cells is illustrated in FIG. 9 which shows that there is an excellent correlation between the viable cells (method of counting on nutritive medium: counting after 5 days of incubation, on Sabouraud agar medium) and the fraction of yeast cells that have lost most of their cF after 30 min at 40 ° C, so that they can no longer be detected by the flow cytometer.

The results obtained in this FIG. 9 were carried out by loading the cells with cF by incubation with 43 μM cFDA in Mcllvaine buffer pH 4, washed and resuspended in buffer

Mcllvaine pH 7.3. The efflux test is carried out at 40 ° C. The fluorescent cells are counted at time 0 and after 30 min.

EXAMPLE 2 Measurement of the vitality of bacteria. A) Lavaσe of bacteria:

Lactococcus lactis bacteria cultivated overnight in an Ml7 medium are washed and resuspended in a 50 mM phosphate buffer, pH 7. B) Marσuaσe:

2 ml of suspension of washed bacteria are withdrawn and stirred vigorously, then cFDA is added, so as to obtain a final concentration of 43 μM. The suspension is then incubated for 10 min at

40 ° C and placed in ice (0 ° C), to avoid any efflux.

C) Lavaσe after marσuaσe:

2 ml of suspension of labeled bacteria are centrifuged for 4 to 5 min at 13,490 g, then the supernatant is removed by suction; the pellet is returned to sus¬ pension in 2 ml of efflux buffer, maintained at 0 ° C, and consisting of 50 mM phosphate buffer pH 7; optionally, again centrifugation is carried out for 4 to 5 min at 13,490 g, the supernatant is removed by suction and the pellet obtained is resuspended in 2 ml of efflux buffer at 0 "C. .

D) Measuring the efflux:

The efflux is measured, under the conditions of Example 1, in the presence of 10 mM glucose or 10 mM lactose, the efflux buffer being, as above, a 50 mM phosphate buffer pH 7.

The results are illustrated in FIG. 10 (abscissa: time in min, ordinate: percentage of cFDA retained). As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which can come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims

CLAIMS 1) Method for evaluating and measuring the viability of microorganisms, comprising:

(a) loading the microorganisms with fluorescent product, by incubation of said microorganisms at a temperature between 20 ° C. and 50 ° C. with a marker (fluorochrone), and

(b) measuring the intensity of intracellular fluorescence by flow cytometry, which method is characterized in that it comprises:

(ii) elimination of the marker not incorporated in the microorganisms,

(iii) adding an energy source, prior to step (b) and

(iv) measuring the time necessary for the disappearance of half of the intracellular fluorescence of the microorganisms, by flow cytometry, in a window of predetermined intensity, at a pH between 4 and 8 (buffer of efflux) and at a temperature between 10 ° C and 40 ° C, preferably between 20 ° C and 40 ° C.

2) Method for evaluating and measuring the viability of microorganisms, comprising:

(b) measuring the intensity of intracellular fluorescence by flow cytometry, which method is characterized in that it comprises: (i) the incubation of said microorganisms for 5 to 20 minutes with said marker at a concentration of between 1 μM and 300 μM, in the presence of a labeling pad at a pH of between 4 and 8, for obtaining microorganisms loaded with fluorescent products,

(ii) elimination of the marker not incorporated in the microorganisms,

(iii) the implementation of step (b) on two sets of samples of microorganisms:

- assembly A, in which the intensity of intracellular fluorescence of the microorganisms is measured at regular time intervals for 5 to 60 min, in an efflux buffer at a pH between 4 and 8 and at a temperature between 10 ° C and 40 ° C, preferably between 20 ° C and 40 ° C;

- assembly B, in which the intensity of intracellular fluorescence of the microorganisms is measured at regular time intervals for 5 to 60 min, in an efflux buffer at a pH between 4 and 8, to which an energy source has added, and at a temperature between 10 ° C and 40 ° C, preferably between 20 "C and 40 ° C; and

(iv) evaluation of the vitality of the microorganisms as a function of the ratio of the intensities of intracellular fluorescence: set B / set A.

3 °) Process according to claim 1 or claim 2, characterized in that the marker is chosen from fluorescein esters: FDA, 6- CFDA, 5-CFDA, 5-maleimide FDA, fluorescein dilaurate , fluorescein dibutyrate, 5 (6) 2 ', 7' dichloro-CFDA, 5 (6) sulfo-FDA, 5 (6) 2 ', 7' dichloro-FDA, ester of 5.6 -CFDA N-hydroxy succinimide, fluorescein dipropionate, di-beta-D-galactoside fluorescein, 3-O-methylfluorescein phosphate, pentaacetoxy ester of 2 ', 7' -bis (carboxyethyl) -5 (6) - carboxyfluorescein (BCECF / AM), pentaacetoxy ester of 2 ', 7' -bis (carboxyethyl) -5 (6) -fluorescein (BCECF / AM), azidofluorescein diacetate, chloromethylfluorescein diacetate, diacetate d eosin, carboxyeosin diacetate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl beta-D-galactoside, 4-methyl¬ umbelliferyl alpha-D-mannopyranoside, 4- methyllumbelliferyl nonanoate, 4- phosphate methylumbelli¬ feryl, alanine-7-amino-4-methylcoumarine, glycine-7-amino-4-methylcoumarine, proline-7-amino-4-methylcoumarine, valine-7-amino-4-methylcoumarine, glycyl-L-proline-7-amino-4-methylcoumarin, 1,4-diacetoxy-2, 3-dicyanobenzene (ABD), hydroethidine, resorufin acetate. 4 °) Method according to any one of reven¬ dications 1 to 3, characterized in that said marker is carboxyfluorescein diacetate.

5) Method according to any one of claims 1 to 4, characterized in that the energy source is a sugar.

6 ^* ) Process according to claim 5, caracté¬ ized in that the sugar is chosen from glucose, fructose, mannose and lactose.

7 °) Process according to claim 6, characterized in that the sugar is 10 mM glucose.

8 °) Kit or kit for implementing the method according to any one of Claims 1 to 7, characterized in that it comprises: at least one marker for viable microorganisms,

- a marking buffer at a pH between 4 and 8,

- an efflux buffer at a pH between 4 and 8, and - a sugar. 9 °) Application of the process according to any one of claims 1 to 7, to the evaluation of a fermentation.

10 °) Application of the process according to any one of claims 1 to 7, followed by a fermentation.