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Viability staining and flow cytometric detection of Listeria monocytogenes
Journal of Microbiological Methods 28 (1997) 35–43
Journal of Microbiological Methods
Viability staining and flow cytometric detection of Listeria monocytogenes Charlotte Nexmann Jacobsen*, Julie Rasmussen, Mogens Jakobsen Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 26 June 1996; revised 25 October 1996; accepted 28 October 1996
Abstract Viability staining and flow cytometric detection of viable Listeria monocytogenes, using five different fluorescent dyes, were investigated. The dyes employed were (i) 5(6)-carboxyfluorescein diacetate (CFDA), (ii) Chemchrome B, (iii) 29,79-bis(2-carboxylethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), (iv) rhodamine 123 (rh 123). The use of the LIVE / DEAD Baclight TM viability kit (BacLight) was also studied. The highest fluorescence intensity of L. monocytogenes, showing high reproducibility as well as good correlation between flow cytometric counts and colony forming units (CFU), was obtained using CFDA and Chemchrome B. BacLight showed variable results depending on the growth phase of L. monocytogenes. Unacceptable low fluorescence intensities were obtained under all conditions for BCECF-AM and rhodamine 123. For the most suitable staining reagents, CFDA and Chemchrome B, the fluorescence intensity increased during the exponential growth phase. The detection limit observed with these reagents in enrichment broths, was in the order of 10 4 cells / ml, and no reduction of fluorescence intensity was observed after addition of Listeria selective agents such as nalidixic acid, acriflavine and phosphomycin. Similar results were obtained for five different strains of L. monocytogenes. The results demonstrate the potential of combining viability staining and flow cytometry as a rapid technique for the detection and enumeration of viable L. monocytogenes in selective enrichment media. 1997 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Viability staining; Fluorescence intensity; Flow cytometry; Rapid method
1. Introduction Listeria monocytogenes is a major threat to public health worldwide. Traditional methods for the detection of L. monocytogenes are still time consuming and the development of more rapid methods is important in controlling the microorganism. Flow cytometry offers the possibility of rapidly counting *Corresponding [email protected]
author.
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and analysing particular characteristics of single cells in a suspension and a number of microbiological applications have been described (see [1] for a review). These include the counting of viable cells and the determination of viability based upon intracellular enzymatic activity [2,3]. For counting viable cells, compounds such as fluorogenic esters have the advantage, that few enzymes are needed to generate a large amount of fluorescence [4]. This makes the technique very sensitive for the counting of small bacterial cells such as those of L. monocyto-
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-7012( 96 )00960-8
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C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
genes compared to other staining methods like the fluorescent antibody technique [5]. Fluorescein diacetate (FDA) and derivatives of this compound have been reported for flow cytometric detection and enumeration of a wide range of bacteria [2,6–8]. This group of dyes also includes carboxyfluorescein diacetate (CFDA), Chemchrome B (Chemumex SA) and 29,79-bis(2-carboxyethyl)5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). In comparison with FDA and CFDA, BCECF-AM contains additional alkyl groups, thereby raising the pKa to 6.98, facilitating uptake and attainment of high intracellular concentrations of fluorescein [9]. Membrane potential sensitive dyes such as the lipophilic cationic dye rhodamine 123 (rh 123), have also been used for counting viable cells by flow cytometry [10]. For Micrococcus luteus, it has been reported [11], that the accumulation of rh 123 is dependent on the viability of the cells. Recently a new system for assessing bacterial cell viability using nucleic acid stains, LIVE / DEAD BacLight viability kit (BacLight), has become commercially available. This system contains a mixture of nucleic acid stains, that rapidly stain live bacteria with intact plasma membranes green fluorescent, to distinguish them from dead bacteria, which are stained red fluorescent. Initial trials carried out in our laboratory (non-published results) indicated that BacLight might be useful as a rapid staining reagent for flow cytometric analysis of L. monocytogenes. To our knowledge none of the above staining reagents have been reported for viability staining of L. monocytogenes. The aim of this study was to develop a sensitive flow cytometric method for rapid detection and enumeration of low levels of L. monocytogenes in selective enrichment media, using one or more of the reagents described.
2. Materials and methods
2.1. Cultures Listeria monocytogenes strains 11 116, 11 139 and 11 140 (serogroup 1), and strains 11 578 and 11 587 (serogroup 4), supplied by the Department of Veterinary Microbiology at the Royal Veterinary and Agricultural University, Copenhagen, Denmark,
were used. The cultures were maintained at 2408C in 15% glycerol and incubated in Brain Heart Infusion Broth (BHI), (Difco), at 378C for 20–24 h before use. Dilutions of cultures were made in sterile peptone water (PW) containing 8.5 g / l NaCl and 0.5 g / l Bactopeptone (Difco), pH 7.2.
2.2. Media and incubations Listeria monocytogenes was grown in various media being Brain Heart Infusion broth (BHI, Difco 0037-17-8), BHI enriched with 2.5% v / v egg yolk and 0.5 g / l magnesium sulphate (BHIEM), BHIEM with 0.1 g / l nalidixic acid (Sigma), 0.01 g / l acriflavine (Chroma-Gesellschaft, Schmid GmbH, ¨ Kongen, Germany) and 0.2 g / l phosphomycin (Sigma), BHIEM with 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.2 g / l phosphomycin and BHIEM added 0.1 g / l nalidixic acid and 0.02 g / l acriflavine. All cultures were incubated for 24 h at 378C, with the selective compounds being added after 6 h of incubation. In comparison to BHIEM with selective additions, University of Vermont (UVM) I and II [12] was used as a two step enrichment, comprising of 24 h incubation in UVM I, followed by transfer of 0.1 ml to 10 ml of UVM II, which was then incubated for another 24 h at 378C.
2.3. Staining procedures A modified version of the method for staining bacteria with FDA described by Fry [13], was used for staining with 5(6)-carboxyfluorescein diacetate (CFDA), (Sigma) and 29,79-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF-AM), (Sigma). After initial optimization with regard to dye concentrations, different incubation times, numbers of washings after staining and the influence of lowering the pH in the buffer either before or after the incubation, loading the cells with CFDA was carried out as follows. Cultures of Listeria monocytogenes were diluted as required in PW, centrifuged at 85003g for 10 min, resuspended in 0.05 M filtered (0.2 m m Minisart, Sartorius, Germany) Tris-HCl buffer, pH 7.4 and sonicated at 20 kHz for 20 s (Vibra Cell 72 434, Sonics and Materials Inc., Danbury, CT, USA) before staining. The sonication was carried out to obtain single cells and shown not to affect cell viability. The sonicated
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
cell suspension was then added 10 mM CFDA in acetone 10 m l / ml, followed by incubation at 378C for 30 min. After incubation, the sample was centrifuged as above and resuspended in Tris-HCl buffer, pH 7.4. The stained sample was kept on ice in the dark for no longer than 45 min, until the flow cytometric analysis was performed. For BCECF-AM, optimization of staining and pretreatment was performed as described above for CFDA, and 10 m l of 1 mM BCECF-AM in dimethylsulphoxide (DMSO) was added per ml of sonicated cell suspension. After 30 min of incubation at 378C, the pH of the cell suspension was adjusted to 4.0 with 1 N HCl (10 m l per ml of cell suspension), followed by mixing and washing twice in buffer before flow cytometry. Staining with Chemchrome B (Chemunex S.A., Maisons-Alfort, France), was performed according to the protocol for CFDA except that the incubation time at 378C was reduced to 10 min. Staining of L. monocytogenes with rhodamine 123 (rh 123), (Sigma), was performed by the method of Matsuyama [14], optimized with respect to concentration of dye, incubation time and numbers of washings after staining as follows. The cultures were diluted in PW and centrifuged at 85003g for 10 min, and the pellet resuspended in Tris-HCl buffer, pH 7.4, containing 0.013 mM rh 123, before sonication and incubation for 30 min at 378C. After incubation, the sample was centrifuged as above and resuspended in Tris-HCl buffer before flow cytometric analysis. Following the manufacturers’ instructions, the LIVE / DEAD BacLight TM Bacteria Viability Kit reagent mix a and b (Molecular Probes Europe BV, Leiden, Netherlands) and DMSO were mixed in a ratio of 1:1:2. This mixture was then added to cultures of L. monocytogenes, pretreated as described for CFDA, at a rate of 4 m l per ml of cell suspension, followed by mixing and incubation for 10 min at 378C before flow cytometric analysis.
2.4. Flow cytometry and determination of fluorescence intensity ( FI)
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nm. The flow rate corresponded to 200–500 cells / s in a sample volume of 200 m l. Coumarin-6 labelled fluorescent polystyrene latex beads with a diameter of 2 m m (Standard C, Chemunex S.A.), were used for calibration of the flow cytometer. The fluorescence intensity (FI) was defined as the ratio between the peak fluorescence intensity of the bacterial population and the peak fluorescence intensity of the latex beads (see Fig. 1).
2.5. Determination of colony forming units Colony forming units (CFU) of L. monocytogenes were determined by spreading 100 m l of appropriate dilutions in duplicate on Blood Agar (BA) (Difco, Tryptose Blood Agar Base, with 5% defibrinated calf blood). Plates were incubated for 24 h at 378C.
2.6. Fluorescence intensity during growth of L. monocytogenes The relationship between growth estimated by optical density measurements, and FI, was established in BHI at 378C. Optical Density (O.D.) was measured with a spectrophotometer (UV-1201, Shimadzu, Duisburg, Germany) at 600 nm. Tests were performed in duplicate.
2.7. Minimal cell concentration detectable by flow cytometry The lowest concentration of L. monocytogenes detectable by flow cytometry was estimated for each of the five staining reagents. For L. monocytogenes, grown in BHI at 378C for 24 h, 10-fold dilutions in PW were made in the range of 10 2 –10 7 cells / ml, with actual cell concentrations being determined as CFU. The flow cytometric detection limit was defined as the lowest concentration of Listeria monocytogenes in the analysed volume (200 m l), giving a histogram with a distinct peak. The examinations were carried out on two separate occasions.
2.8. Reproducibility of FI determinations Measurements were performed using a Partec PAS IIIi flow cytometer (Chemunex S.A.), equipped with an argon laser. Fluorescence was detected with an excitation wavelength of 488 nm and emission at 520
Ten separately grown cultures of L. monocytogenes (11 139) in the stationary phase, were stained with the five dyes in duplicate and analysed by flow
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Fig. 1. Fluorescence intensity profiles for Listeria monocytogenes (11 139), stained with the five staining reagents: carboxyfluorescein diacetate (CFDA), Chemchrome B, 29,79-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), rhodamine 123 and LIVE / DEAD BacLight viability kit (BacLight). The histograms show the culture stained immediately after inoculation, at the beginning (6 h) as well as at the end of the exponential growth phase (9 h). The x-axis shows the fluorescence intensity divided into 512 channels, and the y-axis the counts for each channel. The fluorescence intensity is defined as the ratio between the peak fluorescence intensity of the bacterial population and the peak fluorescence intensity of the latex beads, as indicated by channel numbers as well as vertical lines. The fluorescence intensity of CFDA stained bacteria at 0 h, could not be determined (ND).
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
cytometry. The average FI and standard deviation was calculated for each of the staining reagents.
3. Results For the five staining reagents examined, Fig. 1 shows the flow cytometric histograms for L. monocytogenes (11 139) obtained at the start of incubation (0 h), after 6 and 9 h of incubation at 378C in BHI. Optical density measurements showed that 6 and 9 h of incubation corresponds to the early and late exponential growth phase respectively (results not shown). With respect to CFDA it can be seen from Fig. 1 that the bacteria were stained weakly at 0 h, making it impossible to determine any FI value. With time, the FI value increased, reaching its maximum value in the early exponential growth, followed by a slight decrease. Similar results were obtained for Chemchrome B. Using BCECF-AM and rh 123, L. monocytogenes stained very poorly at all stages during growth in BHI (Fig. 1). The bacteria were stained well with BacLight at 0 h, but opposite CFDA and Chemchrome B, the highest FI value was observed during the lag phase, and followed by a pronounced decline of FI during the exponential growth with the lowest value observed in the late exponential growth phase (Fig. 1). Thereafter the FI increased again reaching the initial high level after 24 h (results not shown). The differences between the various staining reagents were confirmed for four other strains of L. monocytogenes as seen from Table 1, showing the FI of cells stained in the exponential growth phase. It can be seen that CFDA and Chemchrome B stained the five strains of Listeria monocytogenes uniformly
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with higher FI values than the other staining reagents and a low standard deviation between the stained strains. For exponential growth phase cells of L. monocytogenes, linear relationships were demonstrated between flow cytometric counts and CFU within the range of 10 4 –10 8 cells / ml, for CFDA, Chemchrome B and BacLight, with correlation coefficients being 0.94, 0.93 and 0.96 respectively (see Fig. 2). In all cases, the flow cytometric determination resulted in slightly lower cell numbers than determined by CFU. The flow cytometric detection limit for L. monocytogenes counted in 200 m l was found to be approximately 10 4 cells / ml sample for CFDA, Chemchrome B and BacLight stained cells. BCECFAM showed a higher detection limit corresponding to almost 10 6 / ml, and even 10 6 / ml of L. monocytogenes were not detectable when the culture was stained with rh 123 (results not shown). Table 2 shows the effect of selective agents on the FI value for L. monocytogenes (11 139), incubated in duplicate for 24 h in BHI, BHIEM and BHIEM added the selective agents, nalidixic acid, acriflavine and phosphomycin in different concentrations and in the selective media UVM I and II incubated for 48 h in total. The fluorescence intensity of L. monocytogenes stained with CFDA after incubation in various selective media, seemed to be slightly reduced in BHIEM with 0.1 g / l nalidixic acid and 0.02 g / l acriflavine as well as in UVM I 1 II. The FI of Chemchrome B stained cells were unchanged or even increased following enrichment in the presence of selective agents. Staining with BacLight showed a significant decrease in FI for cells of L. monocytogenes exposed to selective
Table 1 Fluorescence intensity of five strains (10 determinations) of L. monocytogenes stained in the early exponential growth phase (6–8 h) CFDA
Chemchrome B
BCECF-AM
Rhodamine 123
BacLight
mean6S.D. 0.8760.07
mean6S.D. 0.8760.08
mean6S.D. 0.2360.20
mean6S.D. 0.1560.18
mean6S.D. 0.3460.20
Results are the average of five strains, each stained at two separate experiments. S.D., Standard deviation. CFDA: 5(6)-carboxyfluorescein diacetate. BCECF-AM: 29,79-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. BacLight: LIVE / DEAD BacLight viability kit.
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with more unspecific signals and with less bacteria being detected (results not shown). To estimate the reproducibility of FI determinations, 10 repeated inoculations of L. monocytogenes (11 139) were stained after 24 h of incubation in BHI. A high reproducibility of FI was observed for CFDA, Chemchrome B and BacLight, with standard deviations of 0.01 (CFDA), 0.04 (Chemchrome B) and 0.03 (BacLight) respectively, indicating highly reproducible stainings. BCECF-AM showed the highest variation with a standard deviation of 0.13. Rh 123 was not investigated because of its very poor staining of L. monocytogenes.
4. Discussion
Fig. 2. Demonstration of linear relationships between flow cytometric counts and colony forming units of Listeria monocytogenes stained with a: CFDA, b: Chemchrome B and c: BacLight.
agents with the exception of the low level of acriflavine. The FI after staining with BCECF-AM and rh 123 remained low in all cases. The stability of the cells of L. monocytogenes obtained after staining with CFDA, Chemchrome B and BacLight was determined. It was found that storage up to 4 h on ice did not affect the FI of the stained cells and even after 24 h on ice the FI remained high, but the histograms were less distinct
For the three fluorogenic esters investigated, CFDA, Chemchrome B and BCECF-AM, different staining profiles of L. monocytogenes were observed. The highest FI was obtained for CFDA and Chemchrome B during exponential growth. The FI remained high for these substrates, when L. monocytogenes was grown in media containing Listeria selective agents and the detection limit was found to be approximately 10 4 cells / ml, with high correlation between CFU and the flow cytometric enumeration. The results presented indicate the potential use of CFDA and Chemchrome B for detection and enumeration of L. monocytogenes. The slight underestimation of the flow cytometric enumeration compared to CFU is likely to be explained by concurrent passage of two or more cells at the point of detection in the flow cytometer or by the fact that not all viable bacteria were stained. The low FI observed for the fluorogenic esters upon transfer of a 24 h old culture of L. monocytogenes into 378C fresh BHI (Fig. 1) was not seen when diluting directly into PW or buffer. A similar behaviour has been reported for FDA stained cells of Bacillus cereus [15]. The phenomenon was not observed for the other staining reagents investigated and it may be explained by increased active efflux of fluorescein from the cell [16] or by increased spontaneous formation of fluorescein in the fresh rich medium [15]. For the detection of L. monocytogenes in enrichment broths after a certain period of incubation this phenomenon
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
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Table 2 Fluorescence intensity of L. monocyogenes following incubation in non-selective and selective media followed by staining with five different staining reagents (individual values for two separate inoculations) Media
CFDA
Chemchrome B
BCECF-AM
Rhodamine 123
Baclight
BHI
0.76 0.81 0.81 0.84 0.89 0.89 0.89 0.89 0.60 0.71 0.57 0.60
0.74 0.77 0.75 0.72 0.90 0.92 1.00 1.00 0.82 0.82 0.80 0.83
0.07 0.10 0.11 0.11 0.10 0.13 0.16 0.16 0.07 0.07 0.16 0.19
0.04 0.04 0.05 0.05 0.05 0.07 0.07 0.07 0.09 0.10 0.05 0.05
0.62 0.62 0.61 0.68 0.55 0.68 0.08 0.10 0.21 0.24 0.13 0.11
BHIEM BHIEM1 1 sel.A BHIEM1 2 sel.A BHIEM1 3 sel.A 4
UVM I&II
For abbreviations of staining reagents, see Table 1. 1 sel. A: 0.1 g / l nalidixic acid, 0.01 g / l acriflavine, 0.2 g / l phosphomycin. 2 sel. B: 0.1 g / l nalidixic acid, 0.02 g / l acriflavine, 0.2 g / l phosphomycin. 3 sel. C: 0.1 g / l nalidixic acid, 0.02 g / l acriflavine. 4 UVM I&II [12].
is not considered important. The increase in FI observed for CFDA and Chemchrome B during the exponential growth phase is likely to be explained by increasing intracellular esterase activity as reported for other Gram-positive bacteria such as Brochothrix thermosphacta and certain lactobacilli [17], leading to higher intracellular formation of fluorescein. In comparison with CFDA and Chemchrome B, BCECF-AM behaved very differently. It stained L. monocytogenes poorly with low FI values giving a detection limit of 10 6 cells, indicating that only a minor fraction of the cells were stained. The reason is likely to be difficulties in loading the cells with BCECF-AM with subsequent low intracellular trapping of the fluorescent compound BCECF in agreement with earlier studies [18]. These workers succeeded in increasing the intracellular concentration of BCECF in Lactococcus lactis exposing the cells to a brief acid shock. In our hands, lowering of the pH, in the presence of the staining reagent, did not significantly increase the FI of BCECF-AM stained L. monocytogenes cells (unpublished results). In studies comprising various Gram-positive and Gramnegative bacteria, staining with CFDA, BCECF-AM and Chemchrome B was compared [7] finding that BCECF-AM and, in particular, Chemchrome B
stained a wider range of bacteria and with higher fluorescence intensities than CFDA. These findings do not agree with the results obtained for L. monocytogenes in the present study, and it appears that uptake and hydrolysis of fluorogenic esters as well as active release of the fluorochrome, vary among bacterial genera or even species in a way making BCECF-AM unsuitable for staining of L. monocytogenes. Several investigations have reported successful viability stainings of bacteria with the membrane potential sensitive dye rh 123 [2,6,11,19]. Diaper and Edwards [2] found that, compared to FDA, rh 123 was the most effective staining reagent for several bacterial species investigated, although of limited application since it failed to stain Bacillus subtilis and Aeromonas salmonicida, as for Pseudomonas fluorescens only 10% of the cells were detectable by flow cytometry. Differences in the ability of rh 123 to stain bacteria has been reported elsewhere [14], and in particular differences is observed for Klebsiella pneumoniae stained with rh 123 and Chemchrome B [7]. The authors suggested that the differences could be explained by different permeabilities for the two staining reagents. None of these studies included L. monocytogenes which, according to the present
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study, belongs to the bacteria poorly stainable with rh 123. The new and relatively unexplored viability kit, BacLight, gave fast and reproducible stainings with high FI for L. monocytogenes in lag phase and stationary growth, with high sensitivity and good correlation between CFU and flow cytometric determination. However FI was strongly reduced during the exponential growth phase. Such variations in the BacLight staining of L. monocytogenes, according to the physiological condition of the cells, may also explain the pronounced negative effects of the selective agents nalidixic acid, acriflavine and phosphomycin, when staining with BacLight. These findings make BacLight unsuitable for detection and enumeration of L. monocytogenes in selective enrichment media. The detection limit for L. monocytogenes in selective enrichment broths was in the order of 10 4 cells / ml, when stained with CFDA and Chemchrome B, and this compares favourably with other immediate detection techniques such as ELISA [20] and PCR [21]. With the intention to improve the specificity of the method, the flow cytometric method could be combined with other methods like immunomagnetic separation, using small paramagnetic particles (0.05 m m) [22]. This is now the subject of further investigation. For future use of the technique for determination of L. monocytogenes in foods, an initial enrichment step seems necessary, to restore stressed cells and increase cell numbers. Further work is still needed in this field. Cost of reagent for the analyses is marginal. Further the capacity of the flow cytometer is very high i.e. in the order of 20 samples per h, following 1 h of preparation. The method does not require particular skills. In conclusion, CFDA and Chemchrome B were found to be suitable for rapid, almost real time counting of pure cultures of L. monocytogenes by flow cytometry, after 6–24 h incubation in selective enrichment media.
References [1] Ueckert, J., Breeuwer, P., Abee, T., Stephens, P., Nebe von Caron, G. and ter Steeg, P.F. (1995) Flow cytometry applications in physiological study and detection of food borne microorganisms. Int. J. Food Microbiol. 28, 317–326.
[2] Diaper, J.P., Tither, K. and Edwards, C. (1992) Rapid assessment of bacterial viability by flow cytometry. Appl. Microbiol. and Biotechnol. 38, 268–272. [3] Riis, S.B., Pedersen, H.M., Sørensen, N.K. and Jakobsen, M. (1995) Flow cytometry and acidification power test as rapid techniques for determination of the activity of starter cultures of Lactobacillus delbrueckii ssp. bulgaricus. Food Microbiol. 12, 245–250. [4] Shapiro, H.M. (1990) Flow cytometry in laboratory microbiology: new directions. ASM News 56, 584–588. [5] Donnelly, C.W. and Baigent, G.J. (1986) Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52, 689–695. [6] Diaper, J.P. and Edwards, C. (1994a) Flow cytometric detection of viable bacteria in compost. FEMS Microbiol. Ecol. 14, 213–220. [7] Diaper, J.P. and Edwards, C. (1994c) The use of fluorogenic esters to detect viable bacteria by flow cytometry. J. Appl. Bact. 77, 221–228. [8] Jepras, R.I., Carter, J., Pearson, S.C., Paul, F.E. and Wilkinson, M.J. (1995) Development of a robust flow cytometric assay for determining numbers of viable bacteria. Appl. Env. Microbiol. 61, 2696–2701. [9] Tsien, R.Y. (1989) Fluorescent indicators of ion concentrations. Methods Cell Biol. 30, 129–156. [10] Diaper, J.P. and Edwards, C. (1994b) Survival of Staphylococcus aureus in lakewater monitored by flow cytometry. Microbiology 140, 35–42. [11] Kaprelyants, A.S. and Kell, D.B. (1992) Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. J. Appl. Bacteriol. 72, 410–422. [12] McClain, D., and Lee, W.H. (1988) Development of USDAFSIS Method for isolation of Listeria monocytogenes from raw meat and poultry. J. Asso. Off. Anal. Chem. 71, 660– 664. [13] Fry, J.C. (1990) Direct methods and biomass estimation. Methods Microbiol. 22, 41–85. [14] Matsuyama, T. (1984) Staining living bacteria with rhodamine 123. FEMS Microbiol. Lett. 21, 153–157. [15] Brunius, G. (1980) Technical aspects of the use of 39,69diacetylfluorescein for vital fluorescent staining of bacteria. Curr. Microbiol. 4, 321–323. [16] Breuwer, P., Drocourt, J., Rombouts, F.M. and Abee, T. (1994) Energy-dependent carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assesment of cell viability by flow cytometry. Appl. Environ. Microbiol. 60, 1467–1472. [17] Papon, M. and Talon, R. (1988) Factors affecting growth and lipase production by meat lactobacilli strains and Brochothrix thermosphacta. J. Appl. Bacteriol. 64, 107–115. [18] Molenaar, D, Abee, T and Konnings, W.N. (1991) Continous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator. Biochim. Biophy. 1115, 75–83. [19] Porter, J., Pickup, R. and Edwards, C. (1995) Membrane hyperpolarisation by valinomycin and its limitations for bacterial viability assessment using rhodamine 123 and flow cytometry. FEMS Microbiol. Lett. 132, 259–262.
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43 [20] Nørrung, B., Sølve, M., Ovesen, M. and Skovgaard, N. (1990) Evaluation of an ELISA test for detection of Listeria spp. J. Food Protect. 54, 752–755. [21] Makino, S., Okada, Y. and Maruyama, T. (1995) A new method for direct detection of Listeria monocytogenes from foods by PCR. Appl. Environ. Microbiol. 61, 3745–3747.
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[22] Clarke, C., Titley, J., Davies, S. and O’Hare, M.J. (1994) An immunomagnetic separation method using superparamagnetic (MACS) beads for large scale purification of human mammary luminal and myoepithelial cells. Epith. Cell. Biol. 3, 38–46.
Journal of Microbiological Methods
Viability staining and flow cytometric detection of Listeria monocytogenes Charlotte Nexmann Jacobsen*, Julie Rasmussen, Mogens Jakobsen Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 26 June 1996; revised 25 October 1996; accepted 28 October 1996
Abstract Viability staining and flow cytometric detection of viable Listeria monocytogenes, using five different fluorescent dyes, were investigated. The dyes employed were (i) 5(6)-carboxyfluorescein diacetate (CFDA), (ii) Chemchrome B, (iii) 29,79-bis(2-carboxylethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), (iv) rhodamine 123 (rh 123). The use of the LIVE / DEAD Baclight TM viability kit (BacLight) was also studied. The highest fluorescence intensity of L. monocytogenes, showing high reproducibility as well as good correlation between flow cytometric counts and colony forming units (CFU), was obtained using CFDA and Chemchrome B. BacLight showed variable results depending on the growth phase of L. monocytogenes. Unacceptable low fluorescence intensities were obtained under all conditions for BCECF-AM and rhodamine 123. For the most suitable staining reagents, CFDA and Chemchrome B, the fluorescence intensity increased during the exponential growth phase. The detection limit observed with these reagents in enrichment broths, was in the order of 10 4 cells / ml, and no reduction of fluorescence intensity was observed after addition of Listeria selective agents such as nalidixic acid, acriflavine and phosphomycin. Similar results were obtained for five different strains of L. monocytogenes. The results demonstrate the potential of combining viability staining and flow cytometry as a rapid technique for the detection and enumeration of viable L. monocytogenes in selective enrichment media. 1997 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Viability staining; Fluorescence intensity; Flow cytometry; Rapid method
1. Introduction Listeria monocytogenes is a major threat to public health worldwide. Traditional methods for the detection of L. monocytogenes are still time consuming and the development of more rapid methods is important in controlling the microorganism. Flow cytometry offers the possibility of rapidly counting *Corresponding [email protected]
author.
Fax:
145
35
283214;
e-mail:
and analysing particular characteristics of single cells in a suspension and a number of microbiological applications have been described (see [1] for a review). These include the counting of viable cells and the determination of viability based upon intracellular enzymatic activity [2,3]. For counting viable cells, compounds such as fluorogenic esters have the advantage, that few enzymes are needed to generate a large amount of fluorescence [4]. This makes the technique very sensitive for the counting of small bacterial cells such as those of L. monocyto-
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-7012( 96 )00960-8
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C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
genes compared to other staining methods like the fluorescent antibody technique [5]. Fluorescein diacetate (FDA) and derivatives of this compound have been reported for flow cytometric detection and enumeration of a wide range of bacteria [2,6–8]. This group of dyes also includes carboxyfluorescein diacetate (CFDA), Chemchrome B (Chemumex SA) and 29,79-bis(2-carboxyethyl)5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). In comparison with FDA and CFDA, BCECF-AM contains additional alkyl groups, thereby raising the pKa to 6.98, facilitating uptake and attainment of high intracellular concentrations of fluorescein [9]. Membrane potential sensitive dyes such as the lipophilic cationic dye rhodamine 123 (rh 123), have also been used for counting viable cells by flow cytometry [10]. For Micrococcus luteus, it has been reported [11], that the accumulation of rh 123 is dependent on the viability of the cells. Recently a new system for assessing bacterial cell viability using nucleic acid stains, LIVE / DEAD BacLight viability kit (BacLight), has become commercially available. This system contains a mixture of nucleic acid stains, that rapidly stain live bacteria with intact plasma membranes green fluorescent, to distinguish them from dead bacteria, which are stained red fluorescent. Initial trials carried out in our laboratory (non-published results) indicated that BacLight might be useful as a rapid staining reagent for flow cytometric analysis of L. monocytogenes. To our knowledge none of the above staining reagents have been reported for viability staining of L. monocytogenes. The aim of this study was to develop a sensitive flow cytometric method for rapid detection and enumeration of low levels of L. monocytogenes in selective enrichment media, using one or more of the reagents described.
2. Materials and methods
2.1. Cultures Listeria monocytogenes strains 11 116, 11 139 and 11 140 (serogroup 1), and strains 11 578 and 11 587 (serogroup 4), supplied by the Department of Veterinary Microbiology at the Royal Veterinary and Agricultural University, Copenhagen, Denmark,
were used. The cultures were maintained at 2408C in 15% glycerol and incubated in Brain Heart Infusion Broth (BHI), (Difco), at 378C for 20–24 h before use. Dilutions of cultures were made in sterile peptone water (PW) containing 8.5 g / l NaCl and 0.5 g / l Bactopeptone (Difco), pH 7.2.
2.2. Media and incubations Listeria monocytogenes was grown in various media being Brain Heart Infusion broth (BHI, Difco 0037-17-8), BHI enriched with 2.5% v / v egg yolk and 0.5 g / l magnesium sulphate (BHIEM), BHIEM with 0.1 g / l nalidixic acid (Sigma), 0.01 g / l acriflavine (Chroma-Gesellschaft, Schmid GmbH, ¨ Kongen, Germany) and 0.2 g / l phosphomycin (Sigma), BHIEM with 0.1 g / l nalidixic acid, 0.02 g / l acriflavine and 0.2 g / l phosphomycin and BHIEM added 0.1 g / l nalidixic acid and 0.02 g / l acriflavine. All cultures were incubated for 24 h at 378C, with the selective compounds being added after 6 h of incubation. In comparison to BHIEM with selective additions, University of Vermont (UVM) I and II [12] was used as a two step enrichment, comprising of 24 h incubation in UVM I, followed by transfer of 0.1 ml to 10 ml of UVM II, which was then incubated for another 24 h at 378C.
2.3. Staining procedures A modified version of the method for staining bacteria with FDA described by Fry [13], was used for staining with 5(6)-carboxyfluorescein diacetate (CFDA), (Sigma) and 29,79-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF-AM), (Sigma). After initial optimization with regard to dye concentrations, different incubation times, numbers of washings after staining and the influence of lowering the pH in the buffer either before or after the incubation, loading the cells with CFDA was carried out as follows. Cultures of Listeria monocytogenes were diluted as required in PW, centrifuged at 85003g for 10 min, resuspended in 0.05 M filtered (0.2 m m Minisart, Sartorius, Germany) Tris-HCl buffer, pH 7.4 and sonicated at 20 kHz for 20 s (Vibra Cell 72 434, Sonics and Materials Inc., Danbury, CT, USA) before staining. The sonication was carried out to obtain single cells and shown not to affect cell viability. The sonicated
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
cell suspension was then added 10 mM CFDA in acetone 10 m l / ml, followed by incubation at 378C for 30 min. After incubation, the sample was centrifuged as above and resuspended in Tris-HCl buffer, pH 7.4. The stained sample was kept on ice in the dark for no longer than 45 min, until the flow cytometric analysis was performed. For BCECF-AM, optimization of staining and pretreatment was performed as described above for CFDA, and 10 m l of 1 mM BCECF-AM in dimethylsulphoxide (DMSO) was added per ml of sonicated cell suspension. After 30 min of incubation at 378C, the pH of the cell suspension was adjusted to 4.0 with 1 N HCl (10 m l per ml of cell suspension), followed by mixing and washing twice in buffer before flow cytometry. Staining with Chemchrome B (Chemunex S.A., Maisons-Alfort, France), was performed according to the protocol for CFDA except that the incubation time at 378C was reduced to 10 min. Staining of L. monocytogenes with rhodamine 123 (rh 123), (Sigma), was performed by the method of Matsuyama [14], optimized with respect to concentration of dye, incubation time and numbers of washings after staining as follows. The cultures were diluted in PW and centrifuged at 85003g for 10 min, and the pellet resuspended in Tris-HCl buffer, pH 7.4, containing 0.013 mM rh 123, before sonication and incubation for 30 min at 378C. After incubation, the sample was centrifuged as above and resuspended in Tris-HCl buffer before flow cytometric analysis. Following the manufacturers’ instructions, the LIVE / DEAD BacLight TM Bacteria Viability Kit reagent mix a and b (Molecular Probes Europe BV, Leiden, Netherlands) and DMSO were mixed in a ratio of 1:1:2. This mixture was then added to cultures of L. monocytogenes, pretreated as described for CFDA, at a rate of 4 m l per ml of cell suspension, followed by mixing and incubation for 10 min at 378C before flow cytometric analysis.
2.4. Flow cytometry and determination of fluorescence intensity ( FI)
37
nm. The flow rate corresponded to 200–500 cells / s in a sample volume of 200 m l. Coumarin-6 labelled fluorescent polystyrene latex beads with a diameter of 2 m m (Standard C, Chemunex S.A.), were used for calibration of the flow cytometer. The fluorescence intensity (FI) was defined as the ratio between the peak fluorescence intensity of the bacterial population and the peak fluorescence intensity of the latex beads (see Fig. 1).
2.5. Determination of colony forming units Colony forming units (CFU) of L. monocytogenes were determined by spreading 100 m l of appropriate dilutions in duplicate on Blood Agar (BA) (Difco, Tryptose Blood Agar Base, with 5% defibrinated calf blood). Plates were incubated for 24 h at 378C.
2.6. Fluorescence intensity during growth of L. monocytogenes The relationship between growth estimated by optical density measurements, and FI, was established in BHI at 378C. Optical Density (O.D.) was measured with a spectrophotometer (UV-1201, Shimadzu, Duisburg, Germany) at 600 nm. Tests were performed in duplicate.
2.7. Minimal cell concentration detectable by flow cytometry The lowest concentration of L. monocytogenes detectable by flow cytometry was estimated for each of the five staining reagents. For L. monocytogenes, grown in BHI at 378C for 24 h, 10-fold dilutions in PW were made in the range of 10 2 –10 7 cells / ml, with actual cell concentrations being determined as CFU. The flow cytometric detection limit was defined as the lowest concentration of Listeria monocytogenes in the analysed volume (200 m l), giving a histogram with a distinct peak. The examinations were carried out on two separate occasions.
2.8. Reproducibility of FI determinations Measurements were performed using a Partec PAS IIIi flow cytometer (Chemunex S.A.), equipped with an argon laser. Fluorescence was detected with an excitation wavelength of 488 nm and emission at 520
Ten separately grown cultures of L. monocytogenes (11 139) in the stationary phase, were stained with the five dyes in duplicate and analysed by flow
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Fig. 1. Fluorescence intensity profiles for Listeria monocytogenes (11 139), stained with the five staining reagents: carboxyfluorescein diacetate (CFDA), Chemchrome B, 29,79-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), rhodamine 123 and LIVE / DEAD BacLight viability kit (BacLight). The histograms show the culture stained immediately after inoculation, at the beginning (6 h) as well as at the end of the exponential growth phase (9 h). The x-axis shows the fluorescence intensity divided into 512 channels, and the y-axis the counts for each channel. The fluorescence intensity is defined as the ratio between the peak fluorescence intensity of the bacterial population and the peak fluorescence intensity of the latex beads, as indicated by channel numbers as well as vertical lines. The fluorescence intensity of CFDA stained bacteria at 0 h, could not be determined (ND).
C. Nexmann Jacobsen et al. / Journal of Microbiological Methods 28 (1997) 35 – 43
cytometry. The average FI and standard deviation was calculated for each of the staining reagents.
3. Results For the five staining reagents examined, Fig. 1 shows the flow cytometric histograms for L. monocytogenes (11 139) obtained at the start of incubation (0 h), after 6 and 9 h of incubation at 378C in BHI. Optical density measurements showed that 6 and 9 h of incubation corresponds to the early and late exponential growth phase respectively (results not shown). With respect to CFDA it can be seen from Fig. 1 that the bacteria were stained weakly at 0 h, making it impossible to determine any FI value. With time, the FI value increased, reaching its maximum value in the early exponential growth, followed by a slight decrease. Similar results were obtained for Chemchrome B. Using BCECF-AM and rh 123, L. monocytogenes stained very poorly at all stages during growth in BHI (Fig. 1). The bacteria were stained well with BacLight at 0 h, but opposite CFDA and Chemchrome B, the highest FI value was observed during the lag phase, and followed by a pronounced decline of FI during the exponential growth with the lowest value observed in the late exponential growth phase (Fig. 1). Thereafter the FI increased again reaching the initial high level after 24 h (results not shown). The differences between the various staining reagents were confirmed for four other strains of L. monocytogenes as seen from Table 1, showing the FI of cells stained in the exponential growth phase. It can be seen that CFDA and Chemchrome B stained the five strains of Listeria monocytogenes uniformly
39
with higher FI values than the other staining reagents and a low standard deviation between the stained strains. For exponential growth phase cells of L. monocytogenes, linear relationships were demonstrated between flow cytometric counts and CFU within the range of 10 4 –10 8 cells / ml, for CFDA, Chemchrome B and BacLight, with correlation coefficients being 0.94, 0.93 and 0.96 respectively (see Fig. 2). In all cases, the flow cytometric determination resulted in slightly lower cell numbers than determined by CFU. The flow cytometric detection limit for L. monocytogenes counted in 200 m l was found to be approximately 10 4 cells / ml sample for CFDA, Chemchrome B and BacLight stained cells. BCECFAM showed a higher detection limit corresponding to almost 10 6 / ml, and even 10 6 / ml of L. monocytogenes were not detectable when the culture was stained with rh 123 (results not shown). Table 2 shows the effect of selective agents on the FI value for L. monocytogenes (11 139), incubated in duplicate for 24 h in BHI, BHIEM and BHIEM added the selective agents, nalidixic acid, acriflavine and phosphomycin in different concentrations and in the selective media UVM I and II incubated for 48 h in total. The fluorescence intensity of L. monocytogenes stained with CFDA after incubation in various selective media, seemed to be slightly reduced in BHIEM with 0.1 g / l nalidixic acid and 0.02 g / l acriflavine as well as in UVM I 1 II. The FI of Chemchrome B stained cells were unchanged or even increased following enrichment in the presence of selective agents. Staining with BacLight showed a significant decrease in FI for cells of L. monocytogenes exposed to selective
Table 1 Fluorescence intensity of five strains (10 determinations) of L. monocytogenes stained in the early exponential growth phase (6–8 h) CFDA
Chemchrome B
BCECF-AM
Rhodamine 123
BacLight
mean6S.D. 0.8760.07
mean6S.D. 0.8760.08
mean6S.D. 0.2360.20
mean6S.D. 0.1560.18
mean6S.D. 0.3460.20
Results are the average of five strains, each stained at two separate experiments. S.D., Standard deviation. CFDA: 5(6)-carboxyfluorescein diacetate. BCECF-AM: 29,79-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. BacLight: LIVE / DEAD BacLight viability kit.
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with more unspecific signals and with less bacteria being detected (results not shown). To estimate the reproducibility of FI determinations, 10 repeated inoculations of L. monocytogenes (11 139) were stained after 24 h of incubation in BHI. A high reproducibility of FI was observed for CFDA, Chemchrome B and BacLight, with standard deviations of 0.01 (CFDA), 0.04 (Chemchrome B) and 0.03 (BacLight) respectively, indicating highly reproducible stainings. BCECF-AM showed the highest variation with a standard deviation of 0.13. Rh 123 was not investigated because of its very poor staining of L. monocytogenes.
4. Discussion
Fig. 2. Demonstration of linear relationships between flow cytometric counts and colony forming units of Listeria monocytogenes stained with a: CFDA, b: Chemchrome B and c: BacLight.
agents with the exception of the low level of acriflavine. The FI after staining with BCECF-AM and rh 123 remained low in all cases. The stability of the cells of L. monocytogenes obtained after staining with CFDA, Chemchrome B and BacLight was determined. It was found that storage up to 4 h on ice did not affect the FI of the stained cells and even after 24 h on ice the FI remained high, but the histograms were less distinct
For the three fluorogenic esters investigated, CFDA, Chemchrome B and BCECF-AM, different staining profiles of L. monocytogenes were observed. The highest FI was obtained for CFDA and Chemchrome B during exponential growth. The FI remained high for these substrates, when L. monocytogenes was grown in media containing Listeria selective agents and the detection limit was found to be approximately 10 4 cells / ml, with high correlation between CFU and the flow cytometric enumeration. The results presented indicate the potential use of CFDA and Chemchrome B for detection and enumeration of L. monocytogenes. The slight underestimation of the flow cytometric enumeration compared to CFU is likely to be explained by concurrent passage of two or more cells at the point of detection in the flow cytometer or by the fact that not all viable bacteria were stained. The low FI observed for the fluorogenic esters upon transfer of a 24 h old culture of L. monocytogenes into 378C fresh BHI (Fig. 1) was not seen when diluting directly into PW or buffer. A similar behaviour has been reported for FDA stained cells of Bacillus cereus [15]. The phenomenon was not observed for the other staining reagents investigated and it may be explained by increased active efflux of fluorescein from the cell [16] or by increased spontaneous formation of fluorescein in the fresh rich medium [15]. For the detection of L. monocytogenes in enrichment broths after a certain period of incubation this phenomenon
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Table 2 Fluorescence intensity of L. monocyogenes following incubation in non-selective and selective media followed by staining with five different staining reagents (individual values for two separate inoculations) Media
CFDA
Chemchrome B
BCECF-AM
Rhodamine 123
Baclight
BHI
0.76 0.81 0.81 0.84 0.89 0.89 0.89 0.89 0.60 0.71 0.57 0.60
0.74 0.77 0.75 0.72 0.90 0.92 1.00 1.00 0.82 0.82 0.80 0.83
0.07 0.10 0.11 0.11 0.10 0.13 0.16 0.16 0.07 0.07 0.16 0.19
0.04 0.04 0.05 0.05 0.05 0.07 0.07 0.07 0.09 0.10 0.05 0.05
0.62 0.62 0.61 0.68 0.55 0.68 0.08 0.10 0.21 0.24 0.13 0.11
BHIEM BHIEM1 1 sel.A BHIEM1 2 sel.A BHIEM1 3 sel.A 4
UVM I&II
For abbreviations of staining reagents, see Table 1. 1 sel. A: 0.1 g / l nalidixic acid, 0.01 g / l acriflavine, 0.2 g / l phosphomycin. 2 sel. B: 0.1 g / l nalidixic acid, 0.02 g / l acriflavine, 0.2 g / l phosphomycin. 3 sel. C: 0.1 g / l nalidixic acid, 0.02 g / l acriflavine. 4 UVM I&II [12].
is not considered important. The increase in FI observed for CFDA and Chemchrome B during the exponential growth phase is likely to be explained by increasing intracellular esterase activity as reported for other Gram-positive bacteria such as Brochothrix thermosphacta and certain lactobacilli [17], leading to higher intracellular formation of fluorescein. In comparison with CFDA and Chemchrome B, BCECF-AM behaved very differently. It stained L. monocytogenes poorly with low FI values giving a detection limit of 10 6 cells, indicating that only a minor fraction of the cells were stained. The reason is likely to be difficulties in loading the cells with BCECF-AM with subsequent low intracellular trapping of the fluorescent compound BCECF in agreement with earlier studies [18]. These workers succeeded in increasing the intracellular concentration of BCECF in Lactococcus lactis exposing the cells to a brief acid shock. In our hands, lowering of the pH, in the presence of the staining reagent, did not significantly increase the FI of BCECF-AM stained L. monocytogenes cells (unpublished results). In studies comprising various Gram-positive and Gramnegative bacteria, staining with CFDA, BCECF-AM and Chemchrome B was compared [7] finding that BCECF-AM and, in particular, Chemchrome B
stained a wider range of bacteria and with higher fluorescence intensities than CFDA. These findings do not agree with the results obtained for L. monocytogenes in the present study, and it appears that uptake and hydrolysis of fluorogenic esters as well as active release of the fluorochrome, vary among bacterial genera or even species in a way making BCECF-AM unsuitable for staining of L. monocytogenes. Several investigations have reported successful viability stainings of bacteria with the membrane potential sensitive dye rh 123 [2,6,11,19]. Diaper and Edwards [2] found that, compared to FDA, rh 123 was the most effective staining reagent for several bacterial species investigated, although of limited application since it failed to stain Bacillus subtilis and Aeromonas salmonicida, as for Pseudomonas fluorescens only 10% of the cells were detectable by flow cytometry. Differences in the ability of rh 123 to stain bacteria has been reported elsewhere [14], and in particular differences is observed for Klebsiella pneumoniae stained with rh 123 and Chemchrome B [7]. The authors suggested that the differences could be explained by different permeabilities for the two staining reagents. None of these studies included L. monocytogenes which, according to the present
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study, belongs to the bacteria poorly stainable with rh 123. The new and relatively unexplored viability kit, BacLight, gave fast and reproducible stainings with high FI for L. monocytogenes in lag phase and stationary growth, with high sensitivity and good correlation between CFU and flow cytometric determination. However FI was strongly reduced during the exponential growth phase. Such variations in the BacLight staining of L. monocytogenes, according to the physiological condition of the cells, may also explain the pronounced negative effects of the selective agents nalidixic acid, acriflavine and phosphomycin, when staining with BacLight. These findings make BacLight unsuitable for detection and enumeration of L. monocytogenes in selective enrichment media. The detection limit for L. monocytogenes in selective enrichment broths was in the order of 10 4 cells / ml, when stained with CFDA and Chemchrome B, and this compares favourably with other immediate detection techniques such as ELISA [20] and PCR [21]. With the intention to improve the specificity of the method, the flow cytometric method could be combined with other methods like immunomagnetic separation, using small paramagnetic particles (0.05 m m) [22]. This is now the subject of further investigation. For future use of the technique for determination of L. monocytogenes in foods, an initial enrichment step seems necessary, to restore stressed cells and increase cell numbers. Further work is still needed in this field. Cost of reagent for the analyses is marginal. Further the capacity of the flow cytometer is very high i.e. in the order of 20 samples per h, following 1 h of preparation. The method does not require particular skills. In conclusion, CFDA and Chemchrome B were found to be suitable for rapid, almost real time counting of pure cultures of L. monocytogenes by flow cytometry, after 6–24 h incubation in selective enrichment media.
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