Acta Biotechnol. 13 (1993) 3, 289-297 Akademie Verlag

Short Communication

Staining Procedures for Flow Cytometric Monitoring of Bacterial Populations

MOLL=, S., Loscm, A., BLEY, T

WIP Research Group “Biosignale” Permoserstr. 15 043 18 Leipzig, Germany

Summary

Dual or multiple parameter flow cytometric analysis is developing into a powerful method for characterizing microbial populations. The distinguishing of the populations only by assignment of size/shape measurements by scattered ligth renders as not satisfactory. To differentiate between the cells, the employment of a specific fluorescence marker is absolutely necessary. Methods are presented for the flow cytometrk determination of DNA and the polymer poly-8-hydroxybutyrate (PHB) content in three different bacterial strains. The measurement of the 38-hydroxysterol content enables the differentation between yeast and bacterial organisms in mixed microbial populations. Monitoring the ratio of live to dead bacterial cells in soil or water samples, e.g. in pure culture systems, is shown.

Introduction

Physical and morphological features of microbial cells can be detected by measuring their light scatter behaviour. On the other hand, detailed cytochemical techniques are found to be necessary for reproducible results in staining intracellular compartments of the cells [3]. The aim of our work was to develop methods for flow cytometric and fluorescence microscopic cellular characterization. The supposition for such examinations is a high instrumental sensitivity because of the small particle size (0.8 - 5 p), the similar surface structure and the low content of intracellular constituents of the cells. We have bypassed this problem by using a self-made flow cytometer with a variable optical system and software developed in our laboratory.

Materials and Methods

Flow Cytometry and Microscopy

The flow cytometer is constructed with a stream-in-air flow chamber and an argon ion laser as the light source [6]. Here we used the following measuring parameters: forward light scatter (3-9.6”) and fluorescence excitation at 488 nm. Optimal alignment was based on the optimized signal from 3.15 pm diameter fluorescent beads (Polysciences Inc. ref. 73957). Samples were viewed and photo-

290 Acta Biotechnologica 13 (1993) 3

graphed using bright-field, phase difference and fluorescence microscopy (OLYMPUS research microscope BHS). The mithramycin/ethidium bromide stained cells were excited using ultraviolet light from a 100 Watt mercury arc lamp. Fluorescence was viewed through a 590 nm bamer filter and the selective excitation filter BP545. The fluorescence from FITC-nystatin, Nile Red, fluoresceine diacetate (FDA) and erythrosine B stained cells was photographed through a 515 nm bamer filter and an excitation filter BJP495. Photomicrographs were done using Kodak daylight film (ASA 200)- Some photomicrographs were taken as double exposures of fluorescence and phase difference images of the same microscopic field. The samples were fixed on microscope slides brushed with freshly prepared 2 ml2% Bacto-Agar (DIFCO-LABORATORIES, Michigan, U.S.A.). Such slides were observed to reduce underground fluorescence and the movement of the cells.

Preparation and Staining of Cells

Cells of Pseudomonas$uorescens were grown in a pancreatic peptone medium, substituted with 0.3% yeast extract and 0.5% sodium chloride in a batch culture at 30 "C. Yeast cells of Saccharomyces cerevisiae S6 were cultivated as described earlier [8]. Cells of Methylobacter rhodesianum were kindly provided by Dr. J. U. ACKERMANN, WIP Research Group "Mikrobielle Leistungen", Leipzig. The Methylocystis GB 25 cells were obtained from the laboratories of Dr. D. WENDLANDT, Environmental Research Centre, Leipzig. For cytome- try, cells were collected and rinsed with phosphate-buffered saline (PBS), pH 7.2. Aliquots of cells suspended in PBS were preserved with 10% sodium azide in 0.9% sodium chloride for at least one hour or, in the case of Pseudomonasfluorescens, frozen at 20 degrees below zero. Subsequently, the cells were measured by the coulter counter technique to get a concentration of 3 x lo8 cells/ml followed by the realization of the staining prescriptions. For bacterial DNA staining, a method of SEEN et al. and SKARSTADT et al. [12, 131 was strictly modified. Cells were resuspended for 10 minutes in 1 ml denaturation buffer (0.5 M NaOH, 1 M NaCI, pH 13.5) at room temperature, followed by the treatment of 1 ml neutralization buffer (1 M TRIS-HCI, 1 M NaCl, pH 7.5, 10 minutes, at room temperature). The cells were washed once again in PBS, resuspended in the staining solution ( I 0 pg MJ, 2 pg EB/ml PBS pH 7.2 + 25 mM MgCI,) and left overnight. Staining of the 3j3-hydroxysterols was done as described by MULLER et al. [8]. Before cytometric measurements were made, samples had to be separated from the staining solution for avoiding quenching effects of the solvents and put into PBS to be measured immediately because of the changing binding equilibrium. Staining of the neutral lipids was modified according to a method of LINZ [5] and used to measure the bacterial PHB content. In this case the behaviour of the selected microbial strain had to be considered as well in finding the optimal staining conditions. Depending on the bacterial strain used, 20- 100 p1 of Nile Red solution (1 mg/ml acetone) was added to 3 x lo8 cells/ml PBS. To differentiate between live and dead cells, a method of JONES et al. [4] for the determination of the cell viability in a tissue culture and cytotoxicity studies was chosen and modified for staining bacterial cells. 25 p1 of the stock solution (70 mg FDA/100 ml acetone) was taken into 1 ml probe (3 x lo8 cells/mI a.d.) and left at room temperature for 30 minutes. The non-fluorescent fluorescein diacetate was absorbed by each cell. The active non-specific esterases of live cells catalyzed the hydrolysis of the diacetate to the fluorescent fluorescein. We observed a quickly release of the latter if the cells were rinsed with PBS. Therefore, we measured and photographed treated cells within the staining solution. The disadvantage of this technique is explicable by the uncertain ratio of the dead to the live cells. Cellular esterases may be active if proliferation and growth stagnate. But other methods, e.g. the staining with 5-cyano-2,3-ditolyl tetrazolium chloride [l I], are above all difficult to employ in detecting environmental probes. This dye is incorporated during cell growth in artificial

M ~ L L E R , S., Ldscm, A. et al., Flow Cytometric Monitoring of Bacterial Populations 29 1

systems. The use of acridine orange for determining the ratio of dead to live bacterial cells could not be recommended because the staining seems to be a function of many methodical factors and the state of the sample [l, 71. Here the measurement of dead bacterial or yeast cells is done by staining with the dye erythrosin B (2,4,5,7-tetraiodofluorescein-sodium, JENAPHARM, Apolda, Germany; 3 x lo8 cells/ml were treated with 50 J.II of erythrosin B stock solution (4 mg/ml PBS) for 10 minutes, washed with PBS and detected).

Results

In Fig. 1 several microphotographs of yeast and bacterial cells stained with these methods are presented. Differentiation of mixed bacterial and yeast cell populations was done by flow cytometric measurement of the 3fbhydroxysterol content (Fig. 2). These 3P-hydroxy- sterols are important membrane components of yeast cells necessary for proliferation and survival features [9]. Bacterial cells do not possess that substance and could not be stained with the nystatin - FITC conjugate. They are found to have membrane sterol surrogates with the same function as the 3fl-hydroxysterols in yeast cells [2], but this component cannot form complexes with the above described macrolide. For that reason differentiation between bacterial and yeast cells is in fact possible. In the first scatter plot (A) of Fig. 2 the pure yeast culture, stained with nystatin - FITC, is represented. That culture, combined with cells of Methylobacter rhodesianum, appears in the case of the scatter plot (B) in the same manner, but in the forward scatter histogram two populations are cleary distinguished. As shown in comparison with the second scatter plot, the first population (the Methylobacter rhodesianum strain) is not fluorescent after being stained with the macrolide complex. In Fig. 3, DNA staining of the strain Pseudomonas fruorescens with the dye MI/EB is presented. Differentiation between the cells of the logarithmic phase (A) or the resting state (B) is difficult. Two subpopulations were found by measuring logarithmically grown cells (A). In the case of cells in the resting state (B), it was often observed, (e.g. also Methylobacter

Fig. 1 A. Staining of DNA with a combination of MI/EB (Methylocystis GB 25)

Fig. 1 B. Staining of 3j3-hydroxysterols with FITC marked nystatin (Succhuromyces cerevisiue) (see page 4)

Fig. I C. Phase difference photomicrograph of Methylocystis GB 25 (see page 4)

Fig. 1 D. Double exposure of number C. The live bacteria appear with green fluorescence

Fig. 1 E. Phase difference combined with fluorescence microscopy Yeast cells were stained with erythrosin B. Dead cells appear with green fluorescence, live cells are stainless (Succhuromyces cerevisiue) (see page 5).

Fig. 1 F. Phase difference photomicrograph of Methylobucter rhodesiunum without PHB

Fig. 1 G. Fluorescence photomicrograph of the same culture (F) subsequent to staining with Nile Red (see page 6)

Fig. 1 H. Phase difference photomicrograph of Methylobucter rhodesiunum Enrichment of PHB is completed (see page 6).

Fig. 1 I. Fluorescence photomicrograph of the same culture (H) subsequent to staining with Nile Red (see page 6)

(see Page 4)

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292 Acta Biotechnologica 13 (1993) 3

MULLFX, S., Loscm, A. et al., Flow Cytometnc Monitoring of Bacterial Populations 293

294 Acta Biotechnologica 13 (1993) 3

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Fig. 4. Dual parameter (Nile Red fluo- rescence/light scatter) histrogram of Me- fhyfobacter rhodesianum cells harvested at the beginning of the exponential growth (A), during the exponential growth (B), after limitation of nitrogen and phosphorus (C) PHB equivalents are indicated on the fluorescence axis.

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rhodesiunum), that they possess the higher content of DNA [ 101. Their classification is possible by measurements of their behaviour in the forward scatter. The increase in the intracellular produced PHB of Methyfobucter rhodesiunum is measured by fluorescence marking with the dye Nile Red (Fig. 4). Samples were harvested at different times. In the fust scatter plot, cells are demonstrated without any PHB (A). The visible cellular fluorescence was caused by intracellular dissolved neutral lipids and membrane phospholipids as described in the case of yeast cells by LINZ [q. Ten hours later, at the

MOLLEX, S., L~SCHE, A. et al., Flow Cytometric Monitoring of Bacterial Populations 297

beginning of nitrogen and phosphorus limitation, particular granules arose in almost each cell (B). The cells became greater as measured by the forward light scattering. At the end of limitation, the fluorescence was detected for a distinct amount of PHB, in accordance with the number of intracellular granules and their fluorescence (C).

Conclusion

This preliminary study shows that specific qualified staining procedures make flow cytometry a powerful to for investigating bacterial populations, as well. It must be taken into consideration that this method, on the level of a particle size of 1-2 pm, is only expressive if the staining method regards the actual physiological properties of the cells investigated. A close collaboration between a biochemist elaborating on the preparation method and a measuring technician working with the cytometer is the key in successfully characterizing bacterial mixed populations.

Acknowledgements

The authors would like to thank Prof. W. BABEL and Prof. SCHEPER for stimulating discussions and Mrs. I. KONARSKI for her skilful, technical assistance throughout this work. They also gratefully acknowledge the support of the DFG.

Received 5 February 1993

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