Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli in seawater by flow cytometry

JOURNAL OF ELSEVIER

Journal of Microbiological Methods 22 (1995) 165-176

Use of two oxonols and a fluorescent tetrazolium dye to monitor starvation of Escherichia coli in seawater by flow cytometry R. Lbpez-Amorbs* Microbiology

Group,

, D.J.

Mason, D. Lloyd

School of Pure and Applied Biology, University of Wales College of Cardiff, P.O. Box 915, Cardiff, Wales CFl 3TL, UK

Received 2 September

1994; accepted 10 October 1994

Abstract

Flow cytometry was used to study starvation of Escherichiu coli in artificial seawater using two membrane potential sensitive dyes and redox dye. The oxonols, DiBAC4(3) and oxonol VI, enabled assessment of energization states. Parallel use of these two dyes suggests that oxonol VI is the more useful for detection of membrane depolarization. The NADPH reducibility of 5-cyano-2,3-ditolyl tetrazolium chloride can be used to estimate

loss of membrane integrity during survival in seawater. Keywords:

E.

coli;

Seawater;

Membrane

potential

dye;

Redox

dye;

Flow

cytometry;

Starvation.

1. Introduction It is well known that in natural microbial environments only a very small fraction of the microbes present can be enumerated using agar plate techniques. The existence of ‘viable but not culturable states’ has aiso been confirmed [1,2] using several methods; these topics assume special importance when considering nutrient starvation of bacteria. Flow cytometry provides a promising tool for the investigation and analysis of those complicated phenomena implicated in the starvation process [3,4]. During starvation, cells eventually become non-viable; typically they also show decreased * Corresponding author. Present address: Departament de Microbiologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain. Tel: 343-402.14.89; Fax number: 343-411.05.92. 0167-7012/5’51$09.50 @ 1995 Elsevier Science B.V. All rights reserved 016’7-7012(94)00073-5

SSDI

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light scatter [5], but decreased size and refractability do not allow a clear discrimination between viable and non-viable cells. An electrochemical potential gradient, typically from 10 to 100 mV (with the cytosol electrically negative), is maintained across the cytoplasmic membrane of eukaryotic cells [6]. In bacteria the maintenance of this potential is dependent on energy metabolism, and is reported to decrease within seconds following the removal of energy sources. The use of membrane potential fluorescent probes (cationic or anionic) can provide information about the polarization state of cells and this gives some indication of the viability state of the organism [7]. Rhodamine 123 has been the most widely used probe to monitor bacterial viability by how cytometry [8]. Slow growing chemostat cultures of Micrococcus Zuteus [9], and Staphylococcus aureus grown in laboratoria media [lO,ll] or starved in lakewater [12] have been monitored by using this dye. In the present study, two other membrane potential sensitive dyes previously used in the study of antifungal agents [13] and eukaryotic cells [14] have been applied to studies of bacterial starvation. These anionic lipophilic oxonols have increased binding affinities for depolarized membranes. The extent of membrane energization is not an absolute indication of respiratory actvity in mitochondrial and bacterial systems. At this point, redox dyes are useful [15]. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been employed for microscopic visualization of actively respiring bacteria in natural aquatic environments [16], and drinking water [17]. Flow cytometric aplication of this dye has been reported to be useful to complement information obtained by the use of rhodamine 123 [18,19]. In this paper we report the usefulness of the oxonols and CTC for assessment the starvation-survival of E. coli in seawater. Recently the use of an oxonol (DiBAC4(3)) m c 1inical microbiology has been described [20].

2. Material and methods 2.1. Starvation experiments E. coli 536, an isolate from a human urinary tract infection was used in this study [19]; 1 litre Erlenmeyer flasks containing 250 ml of artificial sea water (ADSA-Micro, Barcelona, Spain) sterilized by autoclaving and incubated in the dark at 20°C with shaking (120 rpm), were used for the starvation experiments. Organisms from an overnight culture (incubated in 10 ml of Luria-Bertani Broth at 30”(Z), centrifuged (10,000 rpm, 10 min, 20°C) and washed twice in sterile artifical sea water, were used to inoculate falsks to a final concentration of about 106-7 cells/ml. The artificial seawater medium contained (per litre distilled water): NaCl, 24.53 g; Na,SO,, 4.11 g; MgCl, 11.2 g; CaCl,, 1.16 g; SrCl,, 0.7 g; KHCO,, 0.1 g; KBr, 029 g; H,BO,, 0.0029 g; and NaF, 0.003 g. Standard plate count estimations used TSA (Tryptone Soya Agar) plates. Culture plates were incubated at 30°C for 48 h before counting; 4-5 plates were inoculated from the appropriate 1 in 10 dilutions to give the most significant estimates.

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2.2. Bacterial staining Staining with DiBAC,(3)(bis-(1,3_dibutylbarbituric Molecular Probes, Inc.)

acid)trimethine

oxonol),

Samples (1 ml) were withdrawn from cultures and cells permeabilized EGTA (Sigma Chemical Company, Poole, UK) at 1 mM final concentration min. The dye was added to a final concentration of 1 PM and samples maintained at room temperature for 2 min before processing in the cytometer. Where indicated, the uncoupler CCCP (Sigma Chemical Company, Poole, was added at 10 PM final concentration. Staining with Oxonol VI (bis-(3-propyl-5oxoisoxazol-4-yllpentamethine Molecular Probes, Inc.)

with for 2 were flow UK)

oxonol),

Samples (1 ml) of organisms were permeabilized as above, the final concentration of the dye was 1 PM for optimal discrimination between alive and dead cells. Samples were processed after 2 min incubation at room temperature. Staining with CTC

1 mM concentration was used. No measurable differences were observed after staining with 0.5, 1, 2. 4 or 10 mM final concentration for assessment of survival. Samples were previously treated with NADPH (1 mM, final concentration) for 1 h at room temperature, staining time was also 1 h. 2.3. Flow cytometric analysis

Measurements were performed using a Skatron Argus 100 instrument, (Skatron, Ltd, Newmarket, Suffolk, United kingdom). The instrument, mercury lamp based, contains low angle and large angle light scattering detectors plus a variety of fluorescent detectors that can be selected with appropriate optical filters. Deionize’d water was used as sheath fluid after passing through a 0.2 pm pore size filter. Sheath pressure was mantained at 1 kPa, and the flow rate was 5 pl/min. When staining with DiBAC4, a filter block with the following characteristics was selected: excitation at 470 to 560 nm, band stop at 510 nm and emission at 520-560 nm. When Oxonol VI and CTC were used a filter block with 530 to 550 nm excitation, 560 nm band stop and emission at > 580 nm was used. Forward angle light scattering was selected as the second parameter.

3. Resultri 3.1. Use Iof DiBA C4 in early stages of starvation The membrane potential sensitive dye DiBAC4 (Molecular Probes, Inc) was used to follow the effect of starvation on membrane potential of E. coli cultures

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previously grown in nutrient medium (Luria Bertani) and suspended in artificial seawater. Formaldehyde (2% v/v final concentration) was used to kill the cells used as controls. Dead cells stained with the dye showed high uptake of the dye by comparison with the live cells from an overnight culture. The relatively higher binding afinity of the dye for depolarized membranes produces a high percentage of fluorescent cells. Stationary (12 h) or exponential (3-4 h) phase cultures of E. coli 536 simultaneously stained with the dye resulted in organisms with hardly any fluorescence (Fig. lA), organisms from stationary phase cultures gave somewhat broader distributions of fluorescence intensity compared with those from exponential cultures (data not shown). Fig. 1 also shows the behavior of stained organisms from an exponential culture after washing and resuspension in seawater medium. It is clear that there is a progessive increase in the number of fluorescent cells (Fig. 1B) with time. Organisms from stationary phase cultures similary starved in sea-water did not show such rapidly progressive depolarization as measured by amount of dye uptake. Cultures starved in the presence of uncoupler CCCP showed a much more rapid depolarization compared with that of untreated starved cultures (Fig. 1C). A decline in viable counts accompanied the increased number of organisms

Fig. 1. Dual parameter histograms of forward angle light scatter (FALS) fluorescence of organisms from an exponential culture resuspended in seawater and with the addition of the uncoupler CCCP at 48 h (C).

versus DiBAC4(3) (A), after 48 h (B),

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Fig. 1. Continued.

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Table 1 Viable counts (cfuiml) during starvation of E. coli 356 in seawater with and without addition of the uncoupler CCCP

No

CCCP CCCP

Time zero

48 h

5 x loh 5 x lo6

1.2 x lo6 5 x 10’

in the depolarized state as measured by dye uptake (Table 1). A more rapid decline in viable counts occured in the presence of CCCP. Table 2 compares the percentage of fluorescent cells above channel 50 during early stages of starvation of exponential and stationary E. coli cultures. A higher depolarization rate was observed in the exponential culture after this had been resuspended in nonnutrient medium. 3.2. Coupled use of DiBAC4 in starvation experiments

and oxonol VI to follow membrane

depolarization

The aim of these experiments was to compare the relative sensitivity of two membrane potential sensitive dyes to detect loss of membrane potential during prolonged starvation in artificial seawater. Starvation samples were obtained from stationary phase cultures washed and resuspended in seawater medium to an initial population of 5 x lo6 cfu/ml. Samples were periodically taken from flasks maintained up to 22 days at 20°C and stained with either dye. Prolonged starvation in seawater resulted in a progressive increase on the number of fluorescent cells. Both dyes showed higher binding affinities to cell membranes as the time of starvation increased. Oxonol VI (Fig. 2) was found to be more sensitive than DiBAC4(3) (Fig. 3) for detection of subpopulations of organisms heterogeneous with regard to their membrane energization states. Thus the production of fluorescent cells is more clearly distinguishable with Oxonol VI rather than with DiBAC4(3). 3.3. Use of a redox dye with starvation samples

The redox dye CTC was normally used in combination with NADPH as a measure of membrane integrity. Formaldehyde killed cells from growing cultures Table 2 Percentage (%) of DiBAC4(3) fluorescent cells above channel 50 during early stages of starvation of exponential or stationary cultures released in seawater

Tu 5h 24h 5 davs

ExDonential

Stationarv

11.25 13.14 18 28.6

8.9 5.67 4.4 9.2

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2 hl

8 c-4

Fluorescence Fig. 2. Single parameter histograms showing Oxonol line) and starved for 20 days in seawater (thin line).

VI fluorescence

is

8

of unstarved

organisms

(thick

2 CJ

:

Fluorescence Fig. 3. Single parameter histogram showing DiBAC4(3) line) and starved for 20 days in seawater (thin line).

fluorescence

of unstarved

organisms

(thick

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Fig. 4. Dual parameter histograms of forward angle light scatter versus CTC fluorescence of live (A) and formaldehide treated organisms (B) in the presence of NADPH.

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or starvation experiments, showed a fast reduction of the redox dye in the presence of NADPH, (Fig. 4). No endogenous reduction to the formazan was found in any of the sample of organisms. Only when NADPH was added was reduction of the dye observed. The proportion of fluorescent cells detected after NADPI-I addition depended on the age of the starved culture used (Fig. 5).

4. Discussion Studies of cell viability by flow cytometry have often employed rhodamine 123 as a membrane potential sensitive indicator. This has been particulary successful when studying viability of M. futeus in conditions were the development of ‘dormant’ stages is likely to become a transient phase between life and death [8]. However, this dye is not useful for many other bacterial species [lo] and does not specifically indicate membrane potential. By using anionic lipophilic dyes such as those of the oxonol group, another approach to the assessment of bacterial viability is possible. In the present work, the dyes DiBAC4(3) and Oxonol VI have been found to be useful for the detection of membrane depolarization during starvation survival processes. The production of a subpopulation of stained cells during survival experiments in seawater has been shown with either of the oxonol dyes; this correlates with a decrease in viabilty determined by traditional plate counts in nutrient medium. As shown in Table 2, a different behavior has been observed when starving cells from exponential or stationary cultures. The transition from a log growth to starvation implies important biochemical and genetic changes as a consequence of the population adaptation to the new situation. However, cells that are in the stationary phase have already undergone such adaptations [21-231. This fact would explain the lower depolarization rate (according to percentage of fluorescent cells stained with DiBAC4(3)) observed during early stages of starvation compared with that of exponential cultures. Oxonol VI is somewhat preferable to DiBAC4(3) as its longer wavelength of emission. is further removed from autofluorescence emission. Depolarization of organisms by the uncoupler CCCP has also been demonstrated. The presence of this compound also leads to loss of normal transition to the depolarized state upon starvation in seawater. Membrane energization can be maintained in stressed bacteria even when respiratory activity is decreased to very low levels. Endogenous reduction of CTC to the fluorescent formazan has not been detected in our experiments during survival in seawater. Aged individuals of the copiotrophic bacterium M. luteus stored up to 20 days in spent growth medium are also unable to endogenously reduce this dye [15], an only after resucitation by addition of nutrient increasingly levels of CTC reduction can be observed. The ability to oxidize exogenous NADPH is correlated with loss of cell viability. Results obtained in this work confirm that long starvation periods in seawater result in a higher permeability of organisms to externally added NADPH that acts as an electron donor for reduction of CTC.

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Fig. 5. Dual parameter histograms of forward angle light scatter versus CTC fluorescence after addition of NADPH during a starvation experiment at 0 (A), 7 (B), and 20 days (C) in seawater.

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Fig. 5. Continued.

Forward angle light scatter (FALS) changes during starvation were hardly detectable. Although this parameter is associated with cell size in eukaryotes, recent reports provide evidence for only poor correlation between bacterial size and FAILS [5,12,24]. Consequently, information on the cell size variations during starvation cannot be directly or easily obtained.

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[61

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