Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Rapid quantification of rice root-associated bacteria by flow cytometry G. Valdameri, T.B. Kokot, F. de O. Pedrosa and E.M. de Souza Department of Biochemistry and Molecular Biology, Federal University of Paran a, Curitiba, Paran a, Brazil
Significance and Impact of the Study: Determination of precise number of root-associated bacteria is critical for plant–bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.
Keywords A. brasilense, H. rubrisubalbicans, rice roots, epiphytic bacteria, plate count method and flow cytometry. Correspondence Glaucio Valdameri and Emanuel Maltempi de Souza, Department of Biochemistry and Molecular Biology, Federal University of Paran a, Cx.P. 19046 Centro Politecnico, Curitiba 81531-980, Parana, Brazil. E-mails: [email protected]; [email protected]. 2014/1692: received 15 August 2014, revised 21 October 2014 and accepted 24 October 2014
Abstract To understand the mechanism of plant–bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 109 CFU g 1). However, flow cytometry gave a number of viable cells of riceassociated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method.
doi:10.1111/lam.12351
Introduction Flow cytometry is a fast method for analysing individual cells that also allows the measurement of multiple parameters using the fluorescence emission produced by labelled cells (Fulwyler 1980). It is traditionally used to characterize the cellular components of blood or other tissue samples (Drouet and Lees 1993), although it has also been used for microbial analysis of milk (Gunasekera et al. 2000), detection of bacteria or yeasts in body fluids (Delanghe et al. 2000), identification of microorganisms (Davey et al. 1999), detection of Salmonellainfected cells (Th€ one et al. 2007), determination of the viability of encapsulated bacteria (Martin-Dejardin et al. 2013) and enumeration of probiotic strains (Davis 2014). Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Plant growth-promoting rhizobacteria (PGPR) are found in association with agro-economically important plants, and play a key role in promoting plant growth (James 2000). The colonization of host plants involves two types of interactions: (i) epiphytic, when the bacteria colonize the surface of plants, and (ii) endophytic, when internal tissues of the plant are colonized (Monteiro et al. 2012). For plant–bacteria interaction studies, it is critical to follow the time course variation of root-associated bacteria. Despite the established utility of manual epiphytic cell counting using agar plates, there are two major drawbacks associated with this technique (Wilson and Lindow 1992): (i) the long time required, and (ii) only the number of culturable cells is determined. Here, we present a rapid and reliable method, based on flow cytometry, for precise, 1
G. Valdameri et al.
Enumeration of epiphytic cells by flow cytometry
real-time counting of epiphytic bacteria colonizing host plants.
1010
Results and discussion 109
To investigate the feasibility of using flow cytometry to count the number of bacteria colonizing plant roots, two diazotrophic species were used, Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2. For both strains, dilution plate counts, performed using cultures with OD600nm values of 1, gave counts of around 6 9 108 CFU ml 1 (Fig. 1). Flow cytometry of the same cultures gave counts of 5 9 108 cells ml 1 for H. rubrisubalbicans and 4 9 108 cells ml 1 for A. brasilense. The counts obtained by the plate count method and flow cytometry were not significantly different. These data show that both methods can be used to enumerate viable cells of H. rubrisubalbicans and A. brasilense growing in liquid culture media (Fig. 1). Identification of epiphytic cells The next step was to identify the epiphytic populations by flow cytometry. For this purpose, we used Oryza sativa ssp. japonica cv. Nipponbare. At 7 and at 20 days after inoculation with either H. rubrisubalbicans or A. brasilense, the radicular system was removed and vortexed in buffer PBS and analysed by flow cytometry. The population of cells in suspension after vortexing was denominated as epiphytic. Figure 2a,b show clear separation of bacterial population from debris, with H. rubrisubalbicans cells being gated in R2 (Fig. 2b). This result shows that it is possible to identify epiphytic cells recovered from rice rhizosphere. Plate count method vs flow cytometry The number of epiphytic cells of H. rubrisubalbicans and A. brasilense on rice roots was obtained using the standard plating method and compared with the flow cytometry method. At both 7 and 20 days after inoculation with H. rubrisubalbicans, both methods gave counts of c. 109 UFC g 1 of moist root, with a slight increase of the cell number 20 days after inoculation (Fig. 2c). In the case of A. brasilense at 7 days, flow cytometry gave a count that was over 10-fold (P = 00008, unpaired t-test) greater than the count obtained with the dilution plate technique. At 20 days, the count given by flow cytometry was sevenfold (P = 00009) greater (Fig. 2c). Overall, the results show that flow cytometry can be used to quantify epiphytic bacteria on rice roots. In addi2
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Figure 1 Populations of Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2 grown in liquid media. Bacteria were grown at 30°C in a rotary shaker until an OD600 of 10. Cell numbers were determined as colony-forming units (CFU) by the plate count method or through flow cytometry using the BD Cell Viability Kit (with BD liquid counting beads). Key: (white bars) H. rubrisubalbicans M1Sm300; (black bars) A. brasilense FP2. Data are the mean SD of at least three independent experiments performed in duplicate.
tion to being more reliable by reducing human errors, a major advantage of using flow cytometry is that precise cell numbers are achieved rapidly and with minimum sample processing. It takes less than 5 minutes to count a sample by flow cytometry, while plate count method requires at least 24 hours, depending on the growth rate of the bacteria involved. The fact that the two methods gave different results for enumeration of A. brasilense on plant roots, whereas they did not give different results for H. rubrisubalbicans on plant roots, neither for enumeration of either strain from liquid suspension, can be explained by the principles governing the methods. The plate count method only detects culturable cells. In contrast, the flow cytometry method is able to detect and differentiate dead cells, viable but nonculturable and culturable cells. Differentiation into a viable but nonculturable state is a strategy adopted by several bacteria in response to adverse environmental conditions (Ramamurthy et al. 2014). This phenomenon has been reported for Escherichia coli (Xu et al. 1982), Campylobacter jejuni (Rollins and Colwell 1986), Vibrio cholera (Colwell et al. 1985) and Pseudomonas fluorescens (Normander et al. 1999). A rapid decline in culturable P. fluorescens population has been observed in the soil Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Figure 2 Number of epiphytic cells on rice roots colonized by Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2. Panels A and B represent the results obtained by adding 50 ll of BD liquidcounting beads, 5 ll of thiazole orange (TO) and 5 ll of propidium iodide (PI) to the suspension of bacteria detached from rice roots by vortexing. (a) Control condition using noninoculated plants. The graph shows the debris and particles of PBS in the region outside of gate R2. Counting beads are in gate R1. (b) Epiphytic cells of H. rubrisubalbicans M1Sm300 recovered from rice roots 20 days after inoculation. Counting beads are in gate R1 and H. rubrisubalbicans cells are in gate R2. (c) Number of epiphytic cells in rice roots 7 and 20 days after inoculation with H. rubrisubalbicans M1Sm300 (white bars) or A. brasilense FP2 (black bars) using the plate technique (CFU g 1 of fresh weight) and flow cytometry (cells g 1 of fresh weight). Data are the mean SD of at least two independent experiments performed in duplicate.
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environment, and the microcolony epifluorescence technique was proposed for determination of this bacterial in a viable but nonculturable state in soil samples (Binnerup et al. 1993). Studies of a gfp-tagged P. fluorescens in the rhizosphere of barley revealed that immediately after inoculation, around 75% of extracted cells were found in a viable but nonculturable state, in contrast to the 25% observed prior inoculation (Normander et al. 1999). Troxler et al. (1997) suggested that inoculated P. fluorescens persists as mixed populations of cells in different physiological states, in which nonculturable cells were predominant. Considerable attention has been given to resuscitation of bacteria from the nonculturable state, which is possible under appropriate conditions by a mechanism involving the resuscitation-promoting factor (Rpf) (Ramamurthy et al. 2014). We suggest that A. brasilense can be found in a nonculturable state, but further studies are needed to Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Flow cytometry
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unravel the mechanism of such transition. Whether the loss of culturability resulted from the depletion of nutrients in the habitats colonized by A. brasilense or a distinct stage of the interaction with plant, this finding is particularly relevant for monitoring this bacterium in plant colonization assays, where precise population measurement is needed to assess the success of the interaction. The use of only the plate count method may lead to lower numbers than the total of viable cells or apparent disappearance of A. brasilense. We have shown that flow cytometry can be used to enumerate the number of viable epiphytic bacteria thriving on plant roots. Epiphytic cells are regularly exposed to environmental stress factors and the enumeration of viable, but nonculturable is crucial. Moreover, in conjunction with labelling methods, flow cytometry is a rapid and high-throughput method for identifying and quantifying epiphytic populations without sacrificing precision 3
G. Valdameri et al.
Enumeration of epiphytic cells by flow cytometry
and complementing the information obtained by standard methods. Materials and methods Cultivation of bacteria Herbaspirillum rubrisubalbicans strain M1Sm300, a spontaneous streptomycin-resistant mutant of the wild-type strain M1, was cultivated in NFbHPN-malate medium (Klassen et al. 1997). Azospirillum brasilense (FP2) was cultivated in NFbHPN-lactate medium (Pedrosa and Yates 1984). The growth medium was supplemented with the antibiotic streptomycin (300 lg ml 1) for H. rubrisubalbicans M1Sm300, and streptomycin (80 lg ml 1) and nalidixic acid (10 lg ml 1) for A. brasilense (FP2). Cultivation of rice For gnotobiotic cultures of rice, seeds from Oryza sativa ssp. japonica cv. Nipponbare were dehulled and surface sterilized with 70% ethanol for 5 min, followed by 30 min incubation in 6% (v/v) sodium hypochlorite containing 01% (v/v) Triton X-100. The seeds were rinsed 20 times in sterile water and then incubated overnight in 0025% (v/v) fungicide (Vitavaxâ) solution at 30°C. Surface-sterilized seeds were germinated in 1% (m/v) sterile agar-water in the dark for 3 days at 30°C. Two axenic seedlings were transferred aseptically to a sterile glass tube containing propylene beads (55 g) and 25 mL of carbonand nitrogen-free Hoagland’s nutrient solution (Hoagland and Arnon 1950), with the following composition: 1 mmol l 1 KH2PO4, 1 mmol l 1 K2HPO4, 2 mmol l 1 MgSO4.7H2O, 2 mmol l 1 CaCl2.2H2O, 1 ml l 1 micronutrient solution (H3BO3 286 g l 1, MnCl2.4H2O ZnSO4.7H2O 022 g l 1, CuSO4.5H2O 181 g l 1, 1 008 g l , Na2MoO4.2H2O 002 g l 1) and 1 ml l 1 FeEDTA solution (Na2H2EDTA.2H2O 134 g l 1 and FeCl3.6H2O 6 g l 1), pH 65–70. For inoculation with H. rubrisubalbicans or A. brasilense, 72 h after germination, axenic seedlings were incubated for 30 min in the bacterial suspension (OD600nm = 1). Plants were cultivated at 26°C under 14 h light/10 h darkness for 7 or 20 days. Sample preparation and bacterial counting Method 1: plate count method. To enumerate bacterial cells in liquid cultures, serial dilutions were plated onto solid medium (NFbHP-malate or lactate medium containing 20 mmol l 1 NH4Cl) and incubated at 30°C for two days. The bacterial populations were expressed as CFU ml 1. For epiphytic bacteria, the rice plants were 4
carefully removed from the glass tubes; the roots of inoculated plants were separated, weighed and then agitated in a vortex for 1 min in 1 ml of sterile PBS (phosphate buffer saline) containing 1 mmol l 1 EDTA and 001% Tween 20. The suspension was serially diluted and counted as before. Bacterial populations were expressed as CFU g 1 of fresh roots. As negative controls, roots of noninoculated seedlings were submitted to the same process. Three independent experiments were performed in duplicate. Method 2: flow cytometry. Cultures were diluted 100-fold in buffer PBS containing 1 mmol l 1 EDTA and 001% Tween 20 and analysed using a FACS Calibur equipped with 488-nm laser excitation and BD CellQuestTM software. For determination of epiphytic cells, roots of inoculated plants were processed exactly as for plate counting, and the number of cells was determined by flow cytometry. Again, roots from the noninoculated seedlings were used as negative control. The number of cells was determined using the BDTM Cell viability kit with liquid counting beads according to the manufacturer’s instructions. The cells and beads populations were gated using FL1 and FL2 plot. Three independent experiments were performed in duplicate. Acknowledgements We thank Roseli Prado, Valter Baura, Alexsandro Albani and Marilza Doroti Lamour for technical assistance and David Mitchell for checking the English expression. We also thank the National Institute of Science and Technology of Nitrogen Fixation/CNPq/MCT and Fundacß~ao Araucaria for financial support. G.V. thanks PNPD/CAPES for postdoctoral scholarship. Conflict of Interest No conflict of interest declared. References Binnerup, S.J., Jensen, D.F., Thordal-Christensen, H. and Sørensen, J. (1993) Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique. FEMS Microbiol Ecol 12, 97–105. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nat Biotechnol 3, 817–820. Davey, H.M., Jones, A., Shaw, A.D. and Kell, D.B. (1999) Variable selection and multivariate methods for the
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identification of microorganisms by flow cytometry. Cytometry 35, 162–168. Davis, C. (2014) Enumeration of probiotic strains: review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods 103, 9–17. Delanghe, J.R., Kouri, T.T., Huber, A.R., Hannemann-Pohl, K., Guder, W.G., Lun, A., Sinha, P., Stamminger, G. et al. (2000) The role of automated urine particle flow cytometry in clinical practice. Clin Chim Acta 301, 1–18. Drouet, M. and Lees, O. (1993) Clinical-applications of flowcytometry in hematology and immunology. Biol Cell 78, 73–78. Fulwyler, M.J. (1980) Flow-cytometry and cell sorting. Blood Cells 6, 173–184. Gunasekera, T.S., Attfield, P.V. and Veal, D.A. (2000) A flow cytometry method for rapid detection and enumeration of total bacteria in milk. Appl Environ Microbiol 66, 1228– 1232. Hoagland, D.R. and Arnon, D.I. (1950) The water culture method for growing plants without soil. In California Agriculture Experimental Station Circular, California. Berkeley, CA: College of Agriculture, University of California, Circular 347. James, E.K. (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crop Res 65, 197–209. Klassen, G., Pedrosa, F.O., Souza, E.M., Funayama, S. and Rigo, L.U. (1997) Effect of nitrogen compounds on nitrogenase activity in Herbaspirillum seropedicae SMR1. Can J Microbiol 43, 887–891. Martin-Dejardin, F., Ebel, B., Lemetais, G., Nguyen, T.M.H., Gervais, P., Cachon, R. and Chambin, O. (2013) A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: interest of flow cytometry. Eur J Pharm Sci 49, 166–174.
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Enumeration of epiphytic cells by flow cytometry
Monteiro, R.A., Balsanelli, E., Wassem, R., Marin, A.M., Brusamarello-Santos, L.C.C., Schmidt, M.A., Tadra-Sfeir, M.Z., Pankievicz, V.C.S. et al. (2012) Herbaspirillum-plant interactions: microscopical, histological and molecular aspects. Plant Soil 356, 175–196. Normander, B., Hendriksen, .B. and Nybroe, O. (1999) Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere. Appl Environ Microbiol 65, 4646–4651. Pedrosa, F.O. and Yates, M.G. (1984) Regulation of nitrogenfixation (nif) genes of Azospirillum brasilense by nifA and ntr (gln) type gene-products. FEMS Microbiol Lett 23, 95– 101. Ramamurthy, T., Ghosh, A., Pazhani, G.P. and Shinoda, S. (2014) Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Front Public Health 31, 1–8. Rollins, D.M. and Colwell, R.R. (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol 52, 531–538. Th€ one, F., Schwanh€ausser, B., Becker, D., Ballmaier, M. and Bumann, D. (2007) FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen. J Microbiol Methods 71, 220–224. Troxler, J., Zala, M., Moenne-Loccoz, Y., Keel, C. and Defago, G. (1997) Predominance of nonculturable cells of the biocontrol strain Pseudomonas fluorescens CHA0 in the surface horizon of large outdoor lysimeters. Appl Environ Microbiol 63, 3776–3782. Wilson, M. and Lindow, S.E. (1992) Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae. Appl Environ Microbiol 58, 3908–3913. Xu, H.S., Roberts, N., Singleton, F.L., Attwell, R.W., Grimes, D.J. and Colwell, R.R. (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine-environment. Microb Ecol 8, 313–323.
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ORIGINAL ARTICLE
Rapid quantification of rice root-associated bacteria by flow cytometry G. Valdameri, T.B. Kokot, F. de O. Pedrosa and E.M. de Souza Department of Biochemistry and Molecular Biology, Federal University of Paran a, Curitiba, Paran a, Brazil
Significance and Impact of the Study: Determination of precise number of root-associated bacteria is critical for plant–bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.
Keywords A. brasilense, H. rubrisubalbicans, rice roots, epiphytic bacteria, plate count method and flow cytometry. Correspondence Glaucio Valdameri and Emanuel Maltempi de Souza, Department of Biochemistry and Molecular Biology, Federal University of Paran a, Cx.P. 19046 Centro Politecnico, Curitiba 81531-980, Parana, Brazil. E-mails: [email protected]; [email protected]. 2014/1692: received 15 August 2014, revised 21 October 2014 and accepted 24 October 2014
Abstract To understand the mechanism of plant–bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 109 CFU g 1). However, flow cytometry gave a number of viable cells of riceassociated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method.
doi:10.1111/lam.12351
Introduction Flow cytometry is a fast method for analysing individual cells that also allows the measurement of multiple parameters using the fluorescence emission produced by labelled cells (Fulwyler 1980). It is traditionally used to characterize the cellular components of blood or other tissue samples (Drouet and Lees 1993), although it has also been used for microbial analysis of milk (Gunasekera et al. 2000), detection of bacteria or yeasts in body fluids (Delanghe et al. 2000), identification of microorganisms (Davey et al. 1999), detection of Salmonellainfected cells (Th€ one et al. 2007), determination of the viability of encapsulated bacteria (Martin-Dejardin et al. 2013) and enumeration of probiotic strains (Davis 2014). Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Plant growth-promoting rhizobacteria (PGPR) are found in association with agro-economically important plants, and play a key role in promoting plant growth (James 2000). The colonization of host plants involves two types of interactions: (i) epiphytic, when the bacteria colonize the surface of plants, and (ii) endophytic, when internal tissues of the plant are colonized (Monteiro et al. 2012). For plant–bacteria interaction studies, it is critical to follow the time course variation of root-associated bacteria. Despite the established utility of manual epiphytic cell counting using agar plates, there are two major drawbacks associated with this technique (Wilson and Lindow 1992): (i) the long time required, and (ii) only the number of culturable cells is determined. Here, we present a rapid and reliable method, based on flow cytometry, for precise, 1
G. Valdameri et al.
Enumeration of epiphytic cells by flow cytometry
real-time counting of epiphytic bacteria colonizing host plants.
1010
Results and discussion 109
To investigate the feasibility of using flow cytometry to count the number of bacteria colonizing plant roots, two diazotrophic species were used, Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2. For both strains, dilution plate counts, performed using cultures with OD600nm values of 1, gave counts of around 6 9 108 CFU ml 1 (Fig. 1). Flow cytometry of the same cultures gave counts of 5 9 108 cells ml 1 for H. rubrisubalbicans and 4 9 108 cells ml 1 for A. brasilense. The counts obtained by the plate count method and flow cytometry were not significantly different. These data show that both methods can be used to enumerate viable cells of H. rubrisubalbicans and A. brasilense growing in liquid culture media (Fig. 1). Identification of epiphytic cells The next step was to identify the epiphytic populations by flow cytometry. For this purpose, we used Oryza sativa ssp. japonica cv. Nipponbare. At 7 and at 20 days after inoculation with either H. rubrisubalbicans or A. brasilense, the radicular system was removed and vortexed in buffer PBS and analysed by flow cytometry. The population of cells in suspension after vortexing was denominated as epiphytic. Figure 2a,b show clear separation of bacterial population from debris, with H. rubrisubalbicans cells being gated in R2 (Fig. 2b). This result shows that it is possible to identify epiphytic cells recovered from rice rhizosphere. Plate count method vs flow cytometry The number of epiphytic cells of H. rubrisubalbicans and A. brasilense on rice roots was obtained using the standard plating method and compared with the flow cytometry method. At both 7 and 20 days after inoculation with H. rubrisubalbicans, both methods gave counts of c. 109 UFC g 1 of moist root, with a slight increase of the cell number 20 days after inoculation (Fig. 2c). In the case of A. brasilense at 7 days, flow cytometry gave a count that was over 10-fold (P = 00008, unpaired t-test) greater than the count obtained with the dilution plate technique. At 20 days, the count given by flow cytometry was sevenfold (P = 00009) greater (Fig. 2c). Overall, the results show that flow cytometry can be used to quantify epiphytic bacteria on rice roots. In addi2
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Figure 1 Populations of Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2 grown in liquid media. Bacteria were grown at 30°C in a rotary shaker until an OD600 of 10. Cell numbers were determined as colony-forming units (CFU) by the plate count method or through flow cytometry using the BD Cell Viability Kit (with BD liquid counting beads). Key: (white bars) H. rubrisubalbicans M1Sm300; (black bars) A. brasilense FP2. Data are the mean SD of at least three independent experiments performed in duplicate.
tion to being more reliable by reducing human errors, a major advantage of using flow cytometry is that precise cell numbers are achieved rapidly and with minimum sample processing. It takes less than 5 minutes to count a sample by flow cytometry, while plate count method requires at least 24 hours, depending on the growth rate of the bacteria involved. The fact that the two methods gave different results for enumeration of A. brasilense on plant roots, whereas they did not give different results for H. rubrisubalbicans on plant roots, neither for enumeration of either strain from liquid suspension, can be explained by the principles governing the methods. The plate count method only detects culturable cells. In contrast, the flow cytometry method is able to detect and differentiate dead cells, viable but nonculturable and culturable cells. Differentiation into a viable but nonculturable state is a strategy adopted by several bacteria in response to adverse environmental conditions (Ramamurthy et al. 2014). This phenomenon has been reported for Escherichia coli (Xu et al. 1982), Campylobacter jejuni (Rollins and Colwell 1986), Vibrio cholera (Colwell et al. 1985) and Pseudomonas fluorescens (Normander et al. 1999). A rapid decline in culturable P. fluorescens population has been observed in the soil Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Figure 2 Number of epiphytic cells on rice roots colonized by Herbaspirillum rubrisubalbicans M1Sm300 and Azospirillum brasilense FP2. Panels A and B represent the results obtained by adding 50 ll of BD liquidcounting beads, 5 ll of thiazole orange (TO) and 5 ll of propidium iodide (PI) to the suspension of bacteria detached from rice roots by vortexing. (a) Control condition using noninoculated plants. The graph shows the debris and particles of PBS in the region outside of gate R2. Counting beads are in gate R1. (b) Epiphytic cells of H. rubrisubalbicans M1Sm300 recovered from rice roots 20 days after inoculation. Counting beads are in gate R1 and H. rubrisubalbicans cells are in gate R2. (c) Number of epiphytic cells in rice roots 7 and 20 days after inoculation with H. rubrisubalbicans M1Sm300 (white bars) or A. brasilense FP2 (black bars) using the plate technique (CFU g 1 of fresh weight) and flow cytometry (cells g 1 of fresh weight). Data are the mean SD of at least two independent experiments performed in duplicate.
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environment, and the microcolony epifluorescence technique was proposed for determination of this bacterial in a viable but nonculturable state in soil samples (Binnerup et al. 1993). Studies of a gfp-tagged P. fluorescens in the rhizosphere of barley revealed that immediately after inoculation, around 75% of extracted cells were found in a viable but nonculturable state, in contrast to the 25% observed prior inoculation (Normander et al. 1999). Troxler et al. (1997) suggested that inoculated P. fluorescens persists as mixed populations of cells in different physiological states, in which nonculturable cells were predominant. Considerable attention has been given to resuscitation of bacteria from the nonculturable state, which is possible under appropriate conditions by a mechanism involving the resuscitation-promoting factor (Rpf) (Ramamurthy et al. 2014). We suggest that A. brasilense can be found in a nonculturable state, but further studies are needed to Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Flow cytometry
Plate 7 days
Plate
Flow cytometry 20 days
unravel the mechanism of such transition. Whether the loss of culturability resulted from the depletion of nutrients in the habitats colonized by A. brasilense or a distinct stage of the interaction with plant, this finding is particularly relevant for monitoring this bacterium in plant colonization assays, where precise population measurement is needed to assess the success of the interaction. The use of only the plate count method may lead to lower numbers than the total of viable cells or apparent disappearance of A. brasilense. We have shown that flow cytometry can be used to enumerate the number of viable epiphytic bacteria thriving on plant roots. Epiphytic cells are regularly exposed to environmental stress factors and the enumeration of viable, but nonculturable is crucial. Moreover, in conjunction with labelling methods, flow cytometry is a rapid and high-throughput method for identifying and quantifying epiphytic populations without sacrificing precision 3
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Enumeration of epiphytic cells by flow cytometry
and complementing the information obtained by standard methods. Materials and methods Cultivation of bacteria Herbaspirillum rubrisubalbicans strain M1Sm300, a spontaneous streptomycin-resistant mutant of the wild-type strain M1, was cultivated in NFbHPN-malate medium (Klassen et al. 1997). Azospirillum brasilense (FP2) was cultivated in NFbHPN-lactate medium (Pedrosa and Yates 1984). The growth medium was supplemented with the antibiotic streptomycin (300 lg ml 1) for H. rubrisubalbicans M1Sm300, and streptomycin (80 lg ml 1) and nalidixic acid (10 lg ml 1) for A. brasilense (FP2). Cultivation of rice For gnotobiotic cultures of rice, seeds from Oryza sativa ssp. japonica cv. Nipponbare were dehulled and surface sterilized with 70% ethanol for 5 min, followed by 30 min incubation in 6% (v/v) sodium hypochlorite containing 01% (v/v) Triton X-100. The seeds were rinsed 20 times in sterile water and then incubated overnight in 0025% (v/v) fungicide (Vitavaxâ) solution at 30°C. Surface-sterilized seeds were germinated in 1% (m/v) sterile agar-water in the dark for 3 days at 30°C. Two axenic seedlings were transferred aseptically to a sterile glass tube containing propylene beads (55 g) and 25 mL of carbonand nitrogen-free Hoagland’s nutrient solution (Hoagland and Arnon 1950), with the following composition: 1 mmol l 1 KH2PO4, 1 mmol l 1 K2HPO4, 2 mmol l 1 MgSO4.7H2O, 2 mmol l 1 CaCl2.2H2O, 1 ml l 1 micronutrient solution (H3BO3 286 g l 1, MnCl2.4H2O ZnSO4.7H2O 022 g l 1, CuSO4.5H2O 181 g l 1, 1 008 g l , Na2MoO4.2H2O 002 g l 1) and 1 ml l 1 FeEDTA solution (Na2H2EDTA.2H2O 134 g l 1 and FeCl3.6H2O 6 g l 1), pH 65–70. For inoculation with H. rubrisubalbicans or A. brasilense, 72 h after germination, axenic seedlings were incubated for 30 min in the bacterial suspension (OD600nm = 1). Plants were cultivated at 26°C under 14 h light/10 h darkness for 7 or 20 days. Sample preparation and bacterial counting Method 1: plate count method. To enumerate bacterial cells in liquid cultures, serial dilutions were plated onto solid medium (NFbHP-malate or lactate medium containing 20 mmol l 1 NH4Cl) and incubated at 30°C for two days. The bacterial populations were expressed as CFU ml 1. For epiphytic bacteria, the rice plants were 4
carefully removed from the glass tubes; the roots of inoculated plants were separated, weighed and then agitated in a vortex for 1 min in 1 ml of sterile PBS (phosphate buffer saline) containing 1 mmol l 1 EDTA and 001% Tween 20. The suspension was serially diluted and counted as before. Bacterial populations were expressed as CFU g 1 of fresh roots. As negative controls, roots of noninoculated seedlings were submitted to the same process. Three independent experiments were performed in duplicate. Method 2: flow cytometry. Cultures were diluted 100-fold in buffer PBS containing 1 mmol l 1 EDTA and 001% Tween 20 and analysed using a FACS Calibur equipped with 488-nm laser excitation and BD CellQuestTM software. For determination of epiphytic cells, roots of inoculated plants were processed exactly as for plate counting, and the number of cells was determined by flow cytometry. Again, roots from the noninoculated seedlings were used as negative control. The number of cells was determined using the BDTM Cell viability kit with liquid counting beads according to the manufacturer’s instructions. The cells and beads populations were gated using FL1 and FL2 plot. Three independent experiments were performed in duplicate. Acknowledgements We thank Roseli Prado, Valter Baura, Alexsandro Albani and Marilza Doroti Lamour for technical assistance and David Mitchell for checking the English expression. We also thank the National Institute of Science and Technology of Nitrogen Fixation/CNPq/MCT and Fundacß~ao Araucaria for financial support. G.V. thanks PNPD/CAPES for postdoctoral scholarship. Conflict of Interest No conflict of interest declared. References Binnerup, S.J., Jensen, D.F., Thordal-Christensen, H. and Sørensen, J. (1993) Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique. FEMS Microbiol Ecol 12, 97–105. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nat Biotechnol 3, 817–820. Davey, H.M., Jones, A., Shaw, A.D. and Kell, D.B. (1999) Variable selection and multivariate methods for the
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identification of microorganisms by flow cytometry. Cytometry 35, 162–168. Davis, C. (2014) Enumeration of probiotic strains: review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods 103, 9–17. Delanghe, J.R., Kouri, T.T., Huber, A.R., Hannemann-Pohl, K., Guder, W.G., Lun, A., Sinha, P., Stamminger, G. et al. (2000) The role of automated urine particle flow cytometry in clinical practice. Clin Chim Acta 301, 1–18. Drouet, M. and Lees, O. (1993) Clinical-applications of flowcytometry in hematology and immunology. Biol Cell 78, 73–78. Fulwyler, M.J. (1980) Flow-cytometry and cell sorting. Blood Cells 6, 173–184. Gunasekera, T.S., Attfield, P.V. and Veal, D.A. (2000) A flow cytometry method for rapid detection and enumeration of total bacteria in milk. Appl Environ Microbiol 66, 1228– 1232. Hoagland, D.R. and Arnon, D.I. (1950) The water culture method for growing plants without soil. In California Agriculture Experimental Station Circular, California. Berkeley, CA: College of Agriculture, University of California, Circular 347. James, E.K. (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crop Res 65, 197–209. Klassen, G., Pedrosa, F.O., Souza, E.M., Funayama, S. and Rigo, L.U. (1997) Effect of nitrogen compounds on nitrogenase activity in Herbaspirillum seropedicae SMR1. Can J Microbiol 43, 887–891. Martin-Dejardin, F., Ebel, B., Lemetais, G., Nguyen, T.M.H., Gervais, P., Cachon, R. and Chambin, O. (2013) A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: interest of flow cytometry. Eur J Pharm Sci 49, 166–174.
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Enumeration of epiphytic cells by flow cytometry
Monteiro, R.A., Balsanelli, E., Wassem, R., Marin, A.M., Brusamarello-Santos, L.C.C., Schmidt, M.A., Tadra-Sfeir, M.Z., Pankievicz, V.C.S. et al. (2012) Herbaspirillum-plant interactions: microscopical, histological and molecular aspects. Plant Soil 356, 175–196. Normander, B., Hendriksen, .B. and Nybroe, O. (1999) Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere. Appl Environ Microbiol 65, 4646–4651. Pedrosa, F.O. and Yates, M.G. (1984) Regulation of nitrogenfixation (nif) genes of Azospirillum brasilense by nifA and ntr (gln) type gene-products. FEMS Microbiol Lett 23, 95– 101. Ramamurthy, T., Ghosh, A., Pazhani, G.P. and Shinoda, S. (2014) Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Front Public Health 31, 1–8. Rollins, D.M. and Colwell, R.R. (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol 52, 531–538. Th€ one, F., Schwanh€ausser, B., Becker, D., Ballmaier, M. and Bumann, D. (2007) FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen. J Microbiol Methods 71, 220–224. Troxler, J., Zala, M., Moenne-Loccoz, Y., Keel, C. and Defago, G. (1997) Predominance of nonculturable cells of the biocontrol strain Pseudomonas fluorescens CHA0 in the surface horizon of large outdoor lysimeters. Appl Environ Microbiol 63, 3776–3782. Wilson, M. and Lindow, S.E. (1992) Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae. Appl Environ Microbiol 58, 3908–3913. Xu, H.S., Roberts, N., Singleton, F.L., Attwell, R.W., Grimes, D.J. and Colwell, R.R. (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine-environment. Microb Ecol 8, 313–323.
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