Determining the physiological status of individual bacterial cells

10 Determining the Physiological Status of Individual Bacterial Cells H H Howard-Jones, HE Frischer and PGVerity Skidaway Institute of Oceanography, I 0 Ocean Science Circle, Savannah, Georgia 3141 I, USA eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeoeeeeeoeoeeeoeoe

CONTENTS Preface General introduction Vital stain and probe (VSP) method Nucleoid staining technique Microautoradiography and probes Live/dead BacLight TM BacteriaIViability Kit Direct viable counting: Kogure method Conclusions

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PREFACE O

During the past several decades the field of marine microbiology has blossomed, arguably due to the development and application of new methodological approaches. During this period our awareness of the importance, diversity, and complexity of microbial communities in marine systems has dramatically increased. This awareness has prompted the recognition that methods capable of assessing the identity and activity of individual cells are needed to augment those available to measure bulk community properties. Thus, an active research area has been the development of new methods to measure the physiological status and activity of individual cells in planktonic environments. Already application of these methods has led to potentially paradigm shifting observations regarding the composition and activity of marine microbial assemblages. For example, it is now reasonably well accepted that a significant fraction of planktonic bacteria at any given instance in space and time are either dead or relatively inactive. In this chapter we describe and provide detailed laboratory protocols for five techniques that can be used to estimate the physiological status of individual cells in marine and estuarine water samples. M E T H O D S IN MICROBIOLOGY, V O L U M E 30 ISBN 0 12-521530 4

C o p y r i g h t © 2001 A c a d e m i c Press Ltd All rights of reproduction m a n y form reserved

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eeeeee, GENERAL INTRODUCTION During the past quarter century, the appreciation of bacteria as important biological components in marine ecosystems has undergone a dramatic revolution. This epiphany has largely been stimulated by two methodological advancements; direct microscopic enumeration techniques to estimate bacterial abundance (Hobbie et al., 1977; for a recent review see Kepner and Pratt, 1994) and radiotracer methods for estimating the activity of bacterial communities (Brock and Brock, 1966; Carman, 1990). The application of these approaches to marine sciences has led to a paradigm shift regarding the importance of microbial communities in marine systems. For example, prior to the development of these techniques bacteria were thought to be relatively unimportant in marine systems. We now recognize that a majority of living biomass in the sea consists of bacteria and, on average, half of all photosynthetically fixed carbon is processed through heterotrophic bacteria and the microbial loop (Whitman et al., 1998; Ducklow, 1999). However, as the awareness of the importance, diversity, and complexity of microbial communities has developed, it is now recognized that an understanding of the ecology of microbial communities in nature requires information about the identity and activity of individual cells (Sherr et al., 2000). Such information would allow us to address questions such as: 'Which cells are active, why, when, and what are the controls?' Traditional counting and activity measurements cannot provide answers to these and similar questions; therefore, a considerable amount of recent research effort has focused on the development of new techniques that permit estimation of the activity of individual bacteria cells in marine samples. We predict that, just as direct enumeration and activity measurements transformed the field of marine microbiology in the last several decades, the capability of measuring the activity of individual bacteria cells will likewise propel the field to new levels of understanding. Thus, the object of this chapter is to describe several methodologies that are currently under development a n d / o r in use to investigate the physiological activity of individual bacteria cells in marine samples. Several cellular criteria have been proposed and targeted by specialized stains and probes as indicators of cellular activity. These vital stains and probes take advantage of a number of different cellular properties that can be associated with cellular physiology. Examples of criteria that have been used to assess the metabolic status of individual cells include membrane integrity (L6pez-Amor6s et al., 1995; Mason et al., 1995; Haugland, 1996; L6pez-Amor6s et al., 1997), cellular reducing potential (Zimmermann et al., 1978; Rodriguez et al., 1992; Epstein and Rossel, 1995), specific enzyme activity (Lundgren, 1981; Tsuji et al., 1995), and ribosomal RNA (rRNA) cell content (Schaechter et al., 1958; Bremer and Dennis, 1987; DeLong et al., 1989; Lee et al., 1993; Williams et al., 1998). Although several techniques are available and have been used to describe the activity of individual cells in a variety of environments, currently there is no clear consensus among the scientific community regarding the absolute validity of these methods, or even which methods 176

are the most reliable (but see Karner and Fuhrman, 1997). Therefore, at this time, regardless of the method used, it must be recognized that the physiological state of a cell is operationally defined and inferred from the general properties (specificity, sensitivity, etc.) of a particular method, and that the results produced from any single method may provide only relative rather than absolute information. Despite the recognized limitation in the methods available for determining individual cell metabolic status, a number of studies have examined the activity of cells in a variety of marine environments. A general review of this literature suggests that regardless of the method employed, it is clear that a significant fraction of cells visualized by direct epifluorescence microscopy methods are probably dead or relatively inactive (del Giorgio and Scarborough, 1995; Zweifel and Hagstrom, 1995; Heissenberger et al., 1996; Choi et al., 1999; Sherr et al., 1999; Sherr et al., 2000). However, the relative proportion of these groups of cells remains a subject of active debate. Estimates of the proportion of dead and low-activity cells range from 0.1 to 99% depending on which method was used and what environments were examined (Lovejoy et al., 1996; Karner and Fuhrman, 1997; Sherr et al., 2000). These results confirm the hypothesis that techniques that measure bulk community properties underestimate the complexity of microbial community structure and function, and that methods to assess the physiological status of individual cells in situ are required. The purpose of this chapter is to provide a summary and detailed protocols for several methods that show promise for measuring the activity of individual cells in marine samples. We have not discussed the use of CTC within this chapter since this method is described in an earlier chapter of this book (Sherr et al., Chapter 8, this volume). It is our hope that as these methods are improved, calibrated, and commonly used, a more consistent and coherent understanding of the activities of individual bacterial cells in situ will be elucidated.

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V I T A L S T A I N A N D PROBE (VSP) M E T H O D

Introduction The Vital Stain and Probe (VSP) method is an epifluorescent direct microscopy counting technique that combines the general DNA stain DAPI, the vital stain propidium iodide (PI), and 16S rRNA targeted oligonucleotide probes to identify bacterial cells in situ (Williams et al., 1998). This technique simultaneously assesses two independent criteria of cells that can be related to cell physiological status: (a) membrane integrity and (b) cellular ribosomal content. Membrane integrity is assessed using the vital stain PI. PI is a nucleic acid stain that accumulates in cells that have leaky or compromised membranes (Jernaes and Steen, 1994; Lopez-Amoros et al., 1995, 1997; Williams et al., 1998). Cells that stain positively with P1 are interpreted as being dead, although recently divided cells may also be stained by PI (Howard-Jones et al., unpublished). The activity status of microbial cells is determined by hybridiza177

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tion with 16S rRNA oligonucleotide-targeted probes (Lane et al., 1985; Braun-Howland et al., 1992; Spring et al., 1993; Amann et al., 1995). Cells that contain sufficient numbers of ribosomes to be detected by probe hybridization can be visualized with epifluorescent microscopy. Those that appear positively hybridized with oligonucleotide probes are considered to be active since activity and growth rate can be qualitatively related to ribosome content (Giovannoni et al., 1988; DeLong, 1989; Lee et al., 1993, 1994). The combination of DAPI, PI, and probes reveals the physiological status of individual cells. Thus, it is hypothesized that the VSP method can distinguish between four categories of cells: live and active, dead, dead but recently active, and cells that are living but relatively inactive (Table 10.1). Since two independent cellular criteria are simultaneously evaluated by the VSP method, this technique is considered fairly robust in comparison to methods that rely on single cellular criteria. Similar characterization of cells has been defined by Gasol et al. (1999) as follows: (1) large, rapidly growing cells, or cells defined as live and active by the VSP method, (2) dead cells with no potential for growth correspond to those characterized as dead by the VSP; and (3) cells that are growing at a very low rate, or cells that are inactive possibly due to inappropriate growth conditions defined as two separate groups by Gasol et al. (1999) correspond to cells that are live but express low activity as defined by VSP technology. Gasol et al. (1999) also defines a group of cells as cell fragments or ghost cells. This group of cells would be characterized by two different categories of the VSP: (a) cell fragments, if they contain DNA would be classified as dead, while (b) ghost ceils would be categorized as living but low activity cells.

Table I 0. I Proposed definitions of cell status as determined by the uptake or exclusion of propidium iodide (PI) and hybridization with 16S rRNA targeted oligonucleotide probes Cell status

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Live, active Dead Dead, recently active Live, low-no activity

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The VSP method uses 16S rRNA targeted oligonucleotide probes which are comprised of synthesized DNA oligomers, a fluorochrome, and a linker molecule. Oligonucleotides are custom-synthesized with a 5' aminoqink modifier and conjugated with a fluorochrome in the laboratory. Alternatively, fluor conjugated oligonucleotides can be synthesized directly. Oligonucleotide probes used in our laboratory are synthesized by Integrated DNA Technologies (Coralville, IA) and were conjugated with fluorescein isothiocyanate (FITC). However, a variety of other fluorochromes are available, including FluorX, the cyanine dyes (Cy3, Cy5), 178

tetramethvl-rhodamine isothiocyanate (TRITC) etc. (Braun-Howland et a/., 1992; Hicks et al., 1992; DeLong, 1993; Amann et al., 1995; Glockner et al., 1996). A wide range of phylogenetically-specific 16S rRNA targeted oligonucleotide probe sequences have been described in the literature that are suitable for use with the VSP method. Therefore, the VSP technique has the potential to measure the activity and viability of individual cells within the bacterial population, the activity of individual subgroups, or the activity of the entire community, depending on the specificity of the probes utili×ed.

Principle DAPI is a general fluorescent stain that complexes to A-T rich sequences in the major groove of dsDNA in bacterial cells. Under UV (Exciter filter BP 330-385, dichroic mirror DM 400, and barrier filter BA 420) excitation, DAPI stains the DNA of bacteria cells white-blue (emission wavelength for white blue is @ or above 390 nm) and differentially stains particulate matter (detritus) yellow (emission wavelength for yellow is below 390 nm). The vital stain propidium iodide (PI) binds to nucleic acids in cells that have lost membrane integrity (Jernaes and Steen, 1994; LopezAmoros cta/., 1995, 1997; Comas and Vives-Rego, 1997; Williams et aI., 1998). PI accumulates in cells that have compromised membranes and stains the cell a red color when visualized with Wide Green (WG~,) Excitation filter 510-550, dichroic mirror DM570, and barrier filter BA 590) excitation. Cells that are visualized in the Pl field are considered dead. Propidium iodide passively diffuses into cells (influx), a process driven by the dye concentration across tire cell wall. However, in cells with intact membranes this process is limited and possibly counter-balanced by an efflux system [driven by the electrochemical potential] in cells that are considered live by definition of an undamaged membrane (Jernaes and Steen, 1994). Active cells do not stain with PI (Lopez-Arnoros et al., 1995). The ribosomal RNA content of the cell is evaluated by employing 16S oligonucleotide targeted probes. Cells that contain sufficient RNA to be considered metabolically active are visualized green with Narrow Blue (NBX) Excitation filter BP 470-490, dichroic mirror DM 500, and barrier filter BA 515) excitation. The following method yields quantitative information regarding the viability and physiological status of individual bacterial cells iH sitzl.

Equipment and reagents Vacuum manifold, 25 m m vacuum filter holders. Epifluorescence microscope equipped w i t h a UV, Wide Green (WG), and Narrow Blue (NB) filter sets. • Vacuum pump (source of vacuum). • I n c u b a c o r able co maintain 37°C temperature. • •

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Coplin slide staining jars.

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1.5 and 4 ml Eppendorfcubes.

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Humidified chamber (covered tupperware dish with paper towels lining the bottom; soaked with distilled water). 0.2 ~tm black polycarbonate filter (Micron Separation, Inc.). 25 mm GF/F (Whatman). 99% glycerin. 100% analysis grade methanol. 10 mM MgSO4 (pH 6.5); this solution can be pre-made and stored at room temperature; prior to use, 10 mM MgSO4 is filtered through a 0.2 lum Acrodisc filter. Ix SET (I 50 mM NaCI, 20 mM Tris-HCI, I mM EDTA, pH = 8.0); this solution is pre-made and stored in an incubator at 37°C; Ix SET is filtered through a 0.2 lure Acrodisc filter. Ethanol: Formaldehyde (90:10) (100% EtOH, 37% formalin). Hybridization solution (contains the following: 0.2% w/v bovine serum albumin (BSA), 0.01% w/v polyadenylic acid, Ix SET, I1% dextran sulfate); hybridization solution can be pre-made and stored in individual I ml aliquots in the freezer at -20°C. DAPI (stock solution: 50 lug ml ');the stock solution is made by dissolving 5 mg of DAPI in 100 ml of distilled water; the solution is filtered through a 0.2 lum Acrodisc filter and stored in the dark at 4°C. Propidium iodide, PI (stock solution: 20 lug ml ');the stock solution is made by dissolving I mg of PI in 50 ml of distilled water; the solution is filtered through a 0.2 lum Acrodisc filter and stored in the dark at 4°C. Fluorochrome labeled oligonucleotide probes; this method employs three distinctly different oligonucleotides targeted to universally conserved regions of the bacterial ribosome. Only three oligonucleotides are used for this method, however, there are numerous sequences available for synthesizing 16S rRNA probes (Lane et al., 1985; Giovannoni et al., 1988; Lee et al., 1993; Amann et al., 1995; Frischer et al., 1996). Each of these is conjugated to the fluorochrome FITC (fluorescein isothiocyanate): (a) Primer A (5'-gwattaccgcggckgctg; Lane et al., 1985) 5 1 9 - 5 3 6 (b) Primer C (5'-acgggcggtgtgtrc; Lane et al., 1985) 1 3 9 2 - 1 4 0 6 (c) EUB 342 (5'-ctgctgcsycccgtag;Vescio and Nierzwicki-Bauer 1995) 3 4 2 - 3 5 8 The numbers in italics correspond to the nucleotide positions relative to the E. coli 16S rRNA gene.Ambiguous bases are w = a or u; r = a or g; s = c or g; k = g or u; y = c or u.The three oligonucleotide probes are combined (three probes per 1.5 ml Eppendorf tube; 340 ng each), dried in a vacuum dessicator and stored in the freezer at -20°C. Glass microscope slides. Glass coverslips. Microscope grade non-fluorescence immersion oil.

Protocol 1. Water samples (15ml) are collected in 20ml scintillation vials containing 5 ml of sterile glycerol (99%) for a final concentration of 25%; vortex well; allow the sample to equilibrate at room temperature for approximately 10 min before analysis or storage at -20°C. Water samples in 25% glycerol can be stored for 30-60 days at -20°C. 180

2. If necessary (culture work), cells are diluted appropriately in 10 mM MgSO~ (pH 6.5). 3. For sufficient statistical treatment, triplicate samples are prepared and enumerated. 4. Cells are fixed with analysis grade methanol (final concentration: 2~:). Methanol fixation stabilizes cell membranes so that cells remain intact throughout the protocol treatments. 5. Incubate cells for 30 min at room temperature, preferentially in the dark. 6. Stain cells simultaneously with 60 btlml' of sample DAPI (final concentration: 3 btg ml ~) and 10 btl ml ~PI (final concentration: 0.2 btg ml '). Stain samples in the dark for 30 min at room temperature. 7. Permeabilize the cells with ethanol: formaldehyde (90:10) (100 sample original volume). Oligonucleotide probes are 'large' molecules that need to enter the cell in order to hybridize with the ribosomal subunits. Permeabilization is required because it serves to 'poke holes' into the membrane, allowing the probe molecules to enter into the cell more efficiently and easily. 8. Incubate in the dark for 30 rain at room temperature. 9. Filter the samples onto a 0.2 btm black polycarbonate filter underlaid with a 25ram G F / F filter. Filter the samples under low vacuum (<15 m m Hg). The underlying G F / F filter helps to produce an even distribution of cells on the polycarbonate filter. 10. Wash the filters three times with 1 ml of 10mM MgSO~ [pH 6.5] to remove any non-specifically bound stains (under low vacuum < 15 m m Hg). 11. Place the filter onto a labeled glass microscope slide, cell side face up. Attach the polycarbonate filter to the glass slide with small drops of fingernail polish at the edges of the filter. 12. Place the slides into a humidified chamber and overlay the filters with 500 btl of the hybridization/probe solution. The hybridization/probe solution is a combination of the hybridization solution and the three oligonucleotide probes. When it is time to hybridize, one aliquot of hybridization solution is added to one probe (consists of three oligonucleotide, 340ng each) and vortexed well. For each 1 ml mixture, two slides can be overlaid with hybridization/probe solution. 13. Place chamber into an incubator equilibrated to the appropriate hybridization temperature (for the probe set described here, 37°C) and incubate tile slides overnight. A m i n i m u m of 8 h is required for hybridization. 14. Next day: remove the slides from the chamber and wash them for 30 min in three changes of lx SET (in a Coplin jar at the hybridization temperature). 15. Lay slides out to air dry (5 rain) at room temperature. 16. Place a small drop of mounting solution (50:50 lx SET, glycerol) on the filter and cover with a glass coverslip. 17. Enumerate cells with an epifluorescent microscope equipped with the following filter sets: 181

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(a) DAPI: Wide UV (WU). Olympus U-M536 contains exciter filter BP 330-385, dichroic mirror DM 400, and barrier filter BA 420. (b) PI: Wide Green (WG). Olympus U-M526 contains exciter filter BP 510-550, dichroic mirror DM 570, and barrier filter BA 590. (c) Probe (FITC or FluorX): Narrow Blue (NB). Olympus U-M536 contains exciter filter BP 470-490, dichroic mirror DM 500, and barrier filter BA 515. Enumeration is facilitated by using an imaging system equipped with a 100x Plan-apochromat oil objective (NA 1.3-1.4), a color integrating analog or digital CCD, and commercial (e.g. Image Pro Plus) or custom (Sieracki et al., 1989, Shopov et al., 2000) imaging software. Systematic evaluation of staining protocols during the development of the VSP method demonstrated that removing the P! stain (washing step) from the sample incubation, prior to permeabilizing the membranes for probe hybridization, did not influence the fraction of cells appearing dead or dead but recently active (Howard-Jones et al., unpublished). Therefore the protocol does not require removal of PI by washing. Because the fluoresence emission spectra of PI overlaps that of chlorophyll a, autofluorescent picoplankton may be identified as dead cells. However, in our experience, bacterial cell abundance is usually one to two orders of magnitude greater than the abundance of autofluorescent picoplankton cells in marine water samples. Therefore, confusion of PIstained bacteria and autofluorescent picoplankton is rarely a problem. Nevertheless environmental samples that have fewer bacterial cells, requiring larger volumes of water to be filtered for sufficient numbers of cells, might pose a problem. It is recommended that the number of autofluorescent picoplankton be determined in each sample prior to VSP analysis. An example of VSP staining of bacterioplankton from surface waters from the Barents Sea is shown in Plate 2. The micrographs represent cells that are stained with DAPI (left), propidium iodide (right), and universally-targeted 16S rRNA oligonucleotide probes conjugated to FITC (centre). Cells that stain with both DAPI and P! are considered dead while cells that appear in both the DAPI and probe fields are considered active. Cells that appear in both the probe and PI fields are cells that are categorized as dead but recently active, or cells that are undergoing division or recently divided. Cells that fall into the category of dead but recently active are those that have in all probability been active within a few days of sampling. These cells contain a sufficient amount of RNA to be detected with probes, however, their cell membranes have been significantly compromised. Cells that are undergoing division or have recently divided can stain positively for both PI and probe. Based on ongoing laboratory and field studies, recently divided cells persist only for minutes, while recently active cells are present for hours to days (Hong et al., 1998; Howard-Jones et al., unpublished). Advantages Method measures two independent criteria simultaneously. Individual cells are scored as active, inactive, dead or live.

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Accurately and reliably distinguishes between living and dead cells in manipulated and natural systems. • Samples can be collected and stored for up to 3 months. • Can be used to assess the activity of the total bacterioplankton community or specific fractions of the community depending on the probes used.

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Disadvantages Under some circumstances propidium iodide can yield variable staining of cells.Therefore, the scoring of Pl staining may require a subjective decision. Only cells that appear brightly stained with PI are considered dead. Use of a computer-aided image analysis system can minimize this difficulty. • Except in a few special cases, ribosome-targeted probes rarely provide functional information about the individual cells. • The relationship between ribosomal content and activity varies among cell types. •

.e~l, e e e N U C L E O I D S T A I N I N G T E C H N I Q U E Introduction In a healthy bacterial cell DNA is organized in a condensed structure called a nucleoid. The nucleoid can be observed with epifluorescence microscopy w h e n it is stained with a D N A fluorochrome such as DAPI. However, because DAPI is a general stain that can bind non-specifically to bacterial cells and debris1 counts using this technique are thought to overestimate the n u m b e r of cells that actually contain DNA (Zweifel and Hagstrom, 1995; Choiet al., 1996; Vosian and van Noort, 1998). The goal of enumerating nucleoid-containing cells (NUCC) is to count only the cells that contain visible DNA and exclude bacterium-like particles or dead cells ('ghosts') that might be counted after staining with standard DAPI m e t h o d s (Karner and Fuhrman, 1997). Cells that contain nucleoids are considered active, although NUCC bacterium counts probably represent m a x i m u m estimates of the n u m b e r of active bacteria since the presence of a nucleoid does not ensure that a bacterium is growing or even capable of growth (Zweifel and Hagstrom, 1995). For example, cells that are dormant, starved, or infected by viruses m a y contain nucleoids although they are likely to be relatively metabolically inactive. Conversely, cells that are inactive but alive m a y be identified as dead cells (Proctor and Fuhrman, 1990; Zweifel and Hagstrom, 1995; C h o i e t al., 1996, 1999). For example, Choi st ~71. (1996) found that starving, non-nucleoid cells maintained in the laboratory regained yellow-fluorescing nucleoids w h e n supplied with a nutrient source, while with natural samples, approximately 20~/~ of the non-nucleoid cells developed visible nucleoids following the addition of nutrients. These results suggest that non-nucleoid cells m a y still be viable but contain D N A in a less compact region of the cell or of insufficient concentration to cause DAPI-stained DNA to precipitate (Choiet al. 1996).

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The technique described here to visualize nucleoid containing cells was developed by Choi et al. (1999) and represents a modification of the method first described by Zweifel and Hagstrom (1995).

Principle DAP1 is a fluorochrome that binds to the A-T rich sequences in dsDNA. However, standard methods of using DAPI to enumerate bacteria are challenged by the demonstration that it binds non-specifically, particularly at salinities greater than 12 ppt. DAPI binding most likely occurs on reactive bacterial surfaces, rather than to the DNA within the cell membrane (Zweifel and Hagstrom, 1995). Staining bacterial cells in fresh water promotes effective binding of DAP! to DNA (Choi et al., 1996). To remove non-specifically bound DAPI, DAPI-stained cells are washed with a solution of 2-propanol. This washing step removes DAPI from a large but variable number of cells (Zweifel and Hagstrom, 1995; Choi et al., 1996; Vosjan and van Noort, 1998). Fixation prior to de-staining the cells is crucial because it provides physical rigidity to the bacterial cell membranes and allows fluorochromes to penetrate the membranes more readily. Following this protocol, nucleoid-containing cells are visualized as bright yellow spots within dimly blue-fluorescing cells (Choi et al., 1996). The following method yields estimates of viability and metabolic activity based on the presence of a condensed region of DNA known as the nucleoid.

Equipment and reagents • Vacuum manifold, 25 mm vacuum filter holders. • Epifluorescent microscope equipped with UV capability. • Temperature-controlled water bath. • 25 mm, 0.2 ~m black polycarbonate filters (Micron Separations, Inc.). • GF/C filters 25 mm (Whatman). • 0.2 lum pore-size filtered Millipore Milli-Q water. • Fresh sterile-filtered 37% borate-buffered formalin. • DAPI (stock solution: 0.1 mg ml-').The stock solution is made by dissolving 5 mg of DAPI in 50 ml of distilled water, filtered through a 0.2 pm filter, and stored in the clark at 4°C. • Pro-analysis grade 2-propanol warmed to 60-65°C. • Glass microscope slides. • Glass coverslips. • Microscope grade immersion oil.

Protocol 1. Collect water samples in 1 1bottles. 2. Separate sample into two aliquots: one that will be stained immediately to determine total cells and one that will be treated as stated 184

below. For the sample that is to be stained immediately, the cells are fixed with a 5% final concentration of 37% formalin, stained with 10 gg ml ~final concentration of DAPI for 10 min and filtered onto a 25 mm, 0.2 g m black polycarbonate filter. The filters are then m o u n t e d on slides u n d e r oil and coverslips, and e n u m e r a t e d by standard UV epifluorescence microscopy. 3. To visualize nucleoids, fix samples in glass containers with 10% final concentration of 37% formalin. Vortex the samples thoroughly and allow them to equilibrate at room temperature for 10 rain. The cells are fixed with formalin to avoid swelling of the cells and to avoid significant cell loss in the staining process. 4. Set u p the filter towers with 0.2 gm black polycarbonate filters underlain with G F / C filters. The G F / C filters help to produce an even distribution of cells on the polycarbonate filter. 5. Diluting the sample: (a) Low salinity samples (< 12 ppt). Pipette sample (1-2 ml of natural water) on to the filter tower. A d d 5 ml of 0.2-gm-filtered MilliQ water. (b) High salinity samples (> 12 ppt). Dilute the sample with 0,2 ~m Milli-Q water by pipetting 5 ml of 0.2-Hm-filtered MilliQ water to the filter tower, followed by the sample (1-2 ml of natural water). Another 5 ml (depending on the bacterial abundance) of filtersterilized water to the tower can be used as an additional rinse. The addition of distilled water is required for (i) lowering the salinity to less than 12 ppt and (ii) removing remaining a l d e h y d e fixative. 6. Filter the samples until dry at 10 m m Hg. 7. Add DAPI (final concentration: 10 gg ml ~ sample) to the filter tower and incubate for 1 0 m i n in the dark (i.e. cover the filter tower completely with a l u m i n u m foil). 8. Filter d o w n the samples to dryness at 10 m m Hg. 9. Apply three, 1 ml rinses of w a r m (60-65°C) 2-propanol to the filter towers. It is important to rinse gently to avoid dislodging cells from the filter. The propanol wash removes the non-specifically bound DAPI stain from the cells resulting in only nucleoids being stained with DAPI. 10. Once the filter is dry, remove and place it on a paper towel to allow excess propanol to evaporate. 11. Place the filter onto a microscope slide. Immersion oil is placed both u n d e r and on top of the filter before it is covered with a glass coverslip. If necessary, samples can be stored at -20°C for u p to several weeks. 12. Cells are e n u m e r a t e d with an epifluorescent microscope equipped with a UV filter set (e.g. Zeiss 4877-02, excitation filter 365 n m / b a r r i e r filter 420 nm). An example of nucleoid visualization of marine bacterioplankton is s h o w n in Figure 10.1.

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Figure 10.1. Nucleoid staining technique. DAPI-stained marine bacteria; total numbers compared with NUCC bacteria as viewed by epifluorescence microscopy. (A) Conventional DAPI stained sample (site NB1). (B) Bright NUCC bacteria in the same sample used in panel A, after de-staining (overexposure creates images that are larger than the original cell). (C) Nucleoids in a marine isolate (ZS2) visualized by superimposing a binary image of bright nucleoids onto the original negative to compensate for the color information that was lost in transforming to gray scale, Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts). Reprinted with permission from: Zweifel and Hagstrom (1995), Applied Euviroumeut Microbiolo~(ll.

Advantages • •

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Technique is comparatively simple and rapid. Theoretically provides information on total bacteria and active bacteria, regardless of cell type, although this assumption has not been rigorously tested. Data is more relevant to community dynamics than just total DAPI-stained cell abundance.

Disadvantages t

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Unable to discriminate living from low activity cells. Cells that are living but inactive (e.g. starved cells) can be misidentified as either living/active or dead cells. Cells that do not contain visible nucleoids are difficult to discern. Separate samples are required for determining total cell counts. Method relies on a single criterion.

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MICROAUTORADIOGRAPHYAND

PROBES

Introduction Microautoradiography is a technique that detects the cellular localization of a radiolabeled substance ill situ (Brock and Brock, 1966; Hoppe, 1976; Meyer-Reil, 1978). Specifically, microautoradiography can be used in radiotracer studies to determine the proportion of aquatic microorganisms that are metabolizing a given radiolabeled substrate (Carman, 1990). The labeled substrate should be a building-block molecule used bv the cell only to make the molecules of interest. For example, radiolabeled thvmidine is used as a measure of DNA synthesis, uridine for RNA synthesis, and amino acids for protein synthesis (Wolfe, 1983). Although it has been recognized that these assumptions are idealized and that the radiolabeled compounds can enter into numerous other biosynthetic pathways, for the present discussion, it is assumed that the majority of these molecules go directly into cell synthesis. Microautoradiography relies on the ernission of beta particles ejected during radioactive decay. Beta particles can expose crystals (silver grains) in a photographic emulsion that corresponds spatially to ceils that have incorporated the radiolabeled compound. After incubation and development, the cells can be examined microscopically to distinguish metabolically active from inactive cells (Brock and Madigan, 1988). The method described here combines microautoradiography with 16S ribosomal RNA-targeted oligonucleotide probes which allows simultaneous in situ identification and determination of substrate uptake patterns of individual cells (Lee eta/., 1999; Ouverney and Fuhrman, 1999; Cottrell and Kirchman, in press). Cells are labeled with a general stain (DAPI), fluorescently labeled oligonucleotide probes, and tritiated carbon sources. This technique incorporates two independent measurements: (a) cellular ribosomal content/identity and (b) substrate uptake. The combination of microautoradiography and phylogenetic-specific probes provides a means to identify cells that are actively incorporating a specific substrate. This approach promises to become an extremely valuable tool for determining the functional role of diverse microbial communities in nature.

Principle DAPI is a general DNA stain that binds to the A-T rich sequences in dsDNA and cells that are stained with DAP1 can be visualized by epifluorescence microscopy. DAPI staining provides a means to enumerate the total bacterial community regardless of identitv or physiological status. 16S rRNA oligonucleotide probes bind to ribosomal RNA in phylogenetically specific groups of cells that can be considered to be metabolically active (see VSP section of this chapter). Microautoradiography is used to identify cells that are actively incorporating a specific substrate. The method described here is based primarily on the STARFISH protocol developed by Ouvernev and Fuhrman (1999), although similar methods

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have been developed by other groups that incorporate several modifications and or improvements to this method (Lee et al., 1999; Cottrell and Kirchman, 2000). For example, in the MICRO-FISH method described by Cottrell and Kirchman (2000) a special slide having a viewing hole is not required. As described in their manuscript, Ouverney and Fuhrman (1999) utilized the STARFISH technique to examine the utilization of glucose and free amino acids by eubacteria c~-proteobacteria, and the Cytophaga-Flavobacterium sub-groups of the eubacteria. Cottrell and Kirchman (2000) utilized the MICRO-FISH method to examine the utilization of low and high molecular weight dissolved organic matter (DOM) by eubacteria, the Alpha-, Beta-, and Gamma-subdivisions of the proteobacteria, the Cytophaga-Flavobacter cluster of the CFB division, and the high G+C subdivision of the Gram positive phylum.

Equipment and reagents

• • • • • • • • • • • • • • • • • •

• • •

Refrigerator (+4°C). Freezer (-20°C). Temperature controlled water bath. Epifluorescence/transmission light microscope equipped with UV and Wide Green (WG) filter sets. Rotary shaker. Vacuum manifold, 25 mm vacuum filter holders. Vacuum source. Scintillation counter. Dark room. ITT NightVision scope. 15 W safelight. 250 ml acid washed dark bottles. Sterile conical tubes (minimum 50 ml volume). Micropipettes. Forceps. 25 mm 0.2 ~m Nuclepore polycarbonate filters. 25 mm 0.8 lum typeAA Millipore filters. Heavy Teflon glass slides with ten 7 mm diameter wells. Lightproof box wrapped in aluminum foil. Tritiated glucose, specific activity 50 Ci mmol ', final activity 10 pCi (Dupont, N ET100). Tritiated amino acid mixture, specific activity 50 Ci mmoF', final activity 10 IJCi (Amersham, St Louis, MO, TRK440). DAPI (stock concentration: 0.1 ~g ~1 '). The stock solution is made by dissolving 5 mg of DAPI in 50 ml of distilled water. The stock solution can be stored in the dark at 4°C. 0.2x SET: (I × SET is 150 mM NaCI, 20 mM Tris-HCI pH 7.8, and I mP1 EDTA) I × PBS (phosphate-buffered saline: 8 g NaCI, 0.2_g KCI, 1.44 g Na2HPO 4,0.24 g KH2PO4, pH = 7.4). Oligonucleotide probes labeled with the fluorochromeTRITC (5 ng ml ~each of); probes can be labeled with a variety of other available fluorochromes such as Cy3:

188

(a) Negative control [5'-cctagtgacgccgtcgac] (minimum of three mismatches with all rRNA sequences in the Ribosome Database Project) (b) Universal [5'-gwattaccgcggckgctg] 519-536 (c) Bacteria [5'-accgcttgtgcgggccc] 342-359 (d) (z-proteobacteria [5'-cgttcgytctgagccag] 19-35 (e) Cytophaga-Flavobacterium group [5'-tggtccgtrtctcagtac] 319-336 The numbers in italics correspond to the nucleotide positions relative to the E. coli 16S rRNA gene. Hybridization buffer: 5x SET,0.2% bovine serum albumin (BSA), 10% dextran sulfate, 0.01% polyadenylic acid, and 0. 1% sodium dodecyl sulfate. Deionized water. Mounting solution (50:50 (v/v) glycerol: I Ox SET). Ecoscint A cocktail (National Diagnostic, Atlanta, GA). Photographic emulsion (type NTB2). Gelatin solution (gelatin solution: 0.02% final concentration gelatin, 0.02% final concentration CrKSO, at a 50:50 (v/v) ratio). Kodak film developer and fixer (Dektol and fixer: Kodak# 146-4114).

Protocol 1. Water samples are collected in 250 ml acid-washed dark bottles. 2. Divide the water sample into four 40 ml equal subsamples in sterile conical tubes. 3. Kill two subsamples by adding 10% formalin and incubate for 1 h at ambient seawater temperature on a rotary shaker. 4. Add tritiated glucose (Dupont) to two replicate samples (final concentration: 10 nM; specific activity: 50 Ci mmole ~;final activity: 10 btCi). 5. Supplement all four of the samples with a tritiated amino acid mixture (final concentration: 5riM; specific activity: 50Cimmole'; final activity: 10 btCi) (Amersham). Incubate at ambient seawater temperature. 6. Sample from all of the live and killed samples over time by withdrawing 2 ml aliquots. 7. Filter the aliquot onto a 25 ram, 0.2 btm Nuclepore polycarbonate filter and rinse the filter four times with 2 ml lx PBS. 8. Measure the radioactivity using a scintillation counter and Ecoscint A cocktail (National Diagnostic, Atlanta, GA). Determine the cell concentration by DAPl staining. When samples reach saturation levels of radioactivity, terminate the uptake of nutrients by adding 10% formalin to live incubations. 10. In a darkroom, melt photographic emulsion (type NTB2) for 1 h at 43°C in a water bath. 11 • Mix the emulsion with gelatin solution at 43°C (gelatin solution: 0.02% final concentration gelatin, 0.02% final concentration CrKSO4 at a 50:50 (v/v) ratio). The emulsion alone has been found to peel off the glass slide during emulsion development o r in situ hybridization. This can be avoided by mixing the emulsion and gelatin solutions together• .

189

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12. Place an 1TT Night Vision scope and a 15 W safelight approximately 2 m away from all procedures performed in the darkroom. 13. Using the night vision scope to aid seeing in the darkroom, coat the wells on the glass slides containing ten 7-ram-diameter wells by dispensing 20 ~1 of the emulsion:gelatin solution into each well and immediately withdrawing as much solution as possible with a micropipette. Coating the wells separately is necessary to keep the Teflon areas around the wells hydrophobic. Coating the wells individually also prevents the hybridization buffer in one well from merging with buffer from adjacent wells. 14. Allow the slides to dry for 30 rain in total darkness. It is recommended to coat only three slides at one time to minimize background exposure of the emulsion in wells. 15. Set up the filtration tower with 25 ram, 0.2 ~tm Nuclepore filters placed over 0.8 ~tm type AA Millipore filters. 16. Outside the darkroom, under room light, add 10 ml aliquots of the formalin-fixed sample to clean sterile filtration towers. 17. Filter the volume down to approximately 2 ml. 18. Stain the cells with 100 ~1 of DAPI for 10 min in the dark (stock concentration: 0.1 ~tg~ll"). Staining in the dark can be accomplished by covering the filter tower with aluminum foil. 19. Rinse the filters four times with 2 ml lx PBS. Rinsing removes unincorporated radioactive glucose and amino acids. 20. With clean forceps, place the filters, cells side up, onto a drop (10 ~tl) of lx PBS in a Petri dish. 21. Cut the filters into eight equal pieces with a clean and sharp razor blade; carry them to the dark room where they will be transferred to Teflon slides that have been pre-coated. 22. Immediately transfer filters onto coated Teflon slides in the dark room by peeling each of the eight sections of the Nuclepore filter off the Millipore filter and placing it upside down (cells facing down) onto a single well. Only three filters are prepared at any one time to prevent the filters from drying out before being transferred to slides. 23. Allow the slides to dry for 30 min in complete darkness. 24. Place slides in a lightproof box wrapped in aluminum foil and placed in a cardboard box at 4°C for 3 days (for emulsion exposure). 25. Develop the emulsion using Kodak specifications: 2 rain in Dektol developer; 10 s stop in deionized water, 5 min in fixer. 26. Wash the slides with deionized water for 2 rain. 27. Peel off the Nuclepore filters and allow the slides to dry. 28. Hybridize the filters with oligonucleotide probes: (a) Hybridization buffer: 5× SET (lx SET is 150 mM NaC1, 20 mM TrisHC1 pH 7.8, and 1 mM EDTA), 0.2% bovine serum albumin (BSA), 10% dextran sulfate, 0.01% polyadenylic acid, and 0.1% sodium dodecyl sulfate. (b) Probe concentration is 5 ng ml 29. Incubate the slides at 43°C for 3 to 16 h. 30. Rinse the slides with distilled water at 43°C and immerse them three times in 0.2× SET at 43°C for 10 min each time. 190

31. Allow the slides to air dry. 32. Mount the slides with glycerol: 10x SET (50:50 (v/v)) and store at -20°C for at least 1 h. 33. Detect probe hybridization with fluorescence and uptake of radiolabeled substrates by transmitted light microscopy. (a) Total DAPI counts with UV excitation (fluorescence microscope equipped with a UV filter (Olympus type U-MWU)). (b) Probe fluorescence counts with green excitation (fluorescence microscope equipped with a chroma type TRITC U-M41002 filter). (c) Microautoradiography counts represented by small dark silver grains (transmitted light microscopy). (d) Counts for cells labeled with fluorescent probes and simultaneously labeled with microautoradiography. An example of microautoradiographs generated using the MICRO-FISH method (Cottrell and Kirchman, 2000) is shown in Plate 3. The MICROFISH method identifies bacterial cells that incorporate tritiated compounds by the presence of silver grains adjacent to the blue DAPIstained cells (Panel A). The phylogenetic classification of bacterial cells is determined by hybridization with 16S rRNA oligonucelotide probes conjugated to Cy 3 (Panel B).

Advantages Simultaneously determines the phylogenetic identity and specific metabolic activity of individual cells. Results from the triple labeling STARFISH protocol are consistent with standard fluorescent in situ hybridization (FISH) protocols. The scope of the technique application is broad with respect to the identity of target microorganisms and metabolic activity of interest.

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•

Presence of silver grains in the photographic emulsion that do not correspond to a cell in either the DAPI or probe fields may result in ambiguous analysis. • Slow uptake and incorporation of labeled substrates may result in cells appearing active in the probe field (based on rRNA content) and not active in the autoradiographic emulsion. • Procedure is tedious and labor intensive.

4,41,4,~,4,~ L I V E / D E A D B A C L I G H T VIABILITY KIT

TM

BACTERIAL

Introduction Microbial viability can be assessed by monitoring biological factors that are altered during loss of viability including variations in membrane permeability (Jepras et al., 1995). A bacterium is assumed to be viable and 191

potentially active if the membrane is not damaged and assumed to be dead if the membrane is compromised (Decamp and Rajendran, 1998a,b). Membrane integrity analysis is based on the capacity of the cells to exclude compounds, such as fluorescent intercalating dyes, which when used at low concentrations do not normally cross intact membranes (Jepras et al., 1995). A membrane-impermeant stain that can passively diffuse through a cell wall can act as an indicator of loss in membrane integrity and thus as an indicator of cell viability. The LIVE/DEAD BacLight'" bacterial viability kit (Molecular Probes, Oregon) provides a two-color fluorescent assay of bacterial viability. The kit utilizes mixtures of two nucleic acid stains: SYTO 9 and Propidium Iodide (PI). These stains differ in their spectral characteristics and in their ability to penetrate healthy bacterial cells. SYTO 9 is a green fluorescent stain that generally stains all bacteria in a population regardless of their viability (Molecular Probes, Product Information). Propidium iodide is a membrane-impermeant stain that penetrates only bacteria with damaged membranes (Jernaes and Steen, 1994; Lopez-Amoros et al., 1995; Comas and Vives-Rego, 1997; Williams et al., 1998). When combined, the two stains rapidly distinguish between live bacteria with intact membranes from dead bacteria with compromised membranes. Although not initially used with marine samples for technical reasons, recently the BacLight" protocol has been modified and applied with apparent success to marine samples (Decamp and Rajendran, 1998a,b; Gasol et al., 1999). In addition, because of its simplicity and potential applicability with flow cytometry and epifluoresence microscopy (Molecular Probes, Product Information), the LIVE/DEAD BacLighV" kit remains an attractive methodology for exploring the physiological status of marine bacteria.

Principle SYTO 9 is a nucleic acid stain that penetrates the cell wall of intact live cells. The dye can stain both DNA and RNA and is permeable to virtually all cell membranes. SYTO 9 accumulates inside all cells and can be visualized with Narrow Blue (NB) excitation (Molecular Probes, Product Information). Propidium iodide is a fluorescent nucleic acid dye, which stains by intercalating into nucleic acid molecules (Jepras et al., 1995; Lopez-Amoros et al., 1995, 1997). PI binds to both DNA and RNA and is non-specific with respect to base sequence. PI accumulates inside cells that have compromised or leaky membranes and stains the nucleus light to dark red in color when visualized with Wide Green (WG) excitation (Williams et al., 1998). Accumulation of PI inside the cell causes a reduction in the SYTO 9 stain fluorescence when both dyes are present. Propidium iodide competes for nucleic acid binding sites with SYTO 9 and displaces the already bound dye (Virta et al., 1998). With an appropriate mixture of SYTO 9 and PI stains, bacteria with intact membranes stain green, whereas bacteria with damaged membranes stain fluorescent red (DeCamp and Rajendran, 1998a,b). These two stains when used in combination can be visualized simultaneously or separately.

192

Equipment and reagents • Vacuum manifold, 25 mm vacuum filter holders. • Epifluorescence microscope equipped with a Narrow Blue (NB) and aWide Green (WG) filter set. • Vacuum source. • 1.5 ml Eppendorf tubes. • 0.2 Mm black polycarbonate filters (Micron Separations, Inc). • Distilled water. • Formalin. • LIVE/DEAD BacLight ~' Kit: (a) ComponentA: 300 MI solution (in DMSO) of 3.34 mM SYTO 9 dye. (b) Component B: 300 MI solution (in DMSO) of 20 mM Propidium Iodide. (c) Component C: BacLight mounting oil; I 0 ml, for bacteria immobilized on membranes. Refractive index at 25°C is 1.517+0.0003. NOT IMMERSION OIL. The manufacturer recommends the following: • These components should be stored frozen a t - 2 0 ° C and protected from light. • The reagents should be warmed to room temperature and centrifuged briefly each time they are removed from the freezer. • Vials should always remain tightly sealed. Under these conditions the components are stable for at least one year. • Glass microscope slides. • Glass 18 mm square coverslips.

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Protocol 1. Water samples collected in the field can be fixed and stored in 4% final concentration of buffered formalin and stored at 4°C in the dark until analyzed. 2. Bacterial suspensions: remove traces of growth media before staining bacteria with kit reagents. Nucleic acids and other media components can bind the dyes in unpredictable ways. A single wash step is usually sufficient to remove significant traces of interfering media components. Phosphate buffers are not recommended because they appear to decrease staining efficiency (Haugland, 1996). 3. Combine equal volumes of Component A and Component B in a microfuge tube and mix thoroughly. 4. Add 3 ~tl of the dye mixture for each 1 ml of the bacterial suspension or aquatic sample. When used at the recommended dilutions, the reagent mixture will contribute 0.3% DMSO to the staining solution. Higher DM50 concentrations may adversely affect staining. 5. Mix thoroughly and incubate at room temperature in the dark for 15 rain. 6. Filter the suspension through a 0.2~tm black polycarbonate filter placed onto a manifold set-up. Wash with 2ml of distilled water, mount onto a glass microscope slide with the mounting oil supplied with the BacLight kit, OR

193

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Trap 5 gl of the stained bacterial suspension between a slide and an 18 m m square coverslip. . E n u m e r a t e stained cells with an epifluorescence microscope e q u i p p e d with a N a r r o w Blue (NB) filter set to visualize SYTO-9-stained cells and a Wide Green (WG) filter set to visualize PI-stained cells. Molecular probes r e c o m m e n d s the use of O m e g a Optical filter sets (for SYTO 9 - - O-5715 or O-5716; for PI - - 0-5723, 0-5724, 0-5733) which are available through their catalog.

Advantages • All cells are visualized, not restricted to specific groups of bacteria. • SYTO 9 dye coupled with PI allows for cells to be visualized as dead or live simultaneously. • Both SYTO 9 and PI express fluorescence enhancement upon binding nucleic acids making the differentiation of live and dead cells uncomplicated. • The BacLight T~'kit can be coupled with flow cytometry. • Rapid and simple protocol.

Disadvantages • Technique provides information about potential viability only, not activity of cells. • BacLight Tr' is unable to indicate dead cells that have already lost their cytoplasmic content (Lawrence et al., 1997). • Relies on a single cellular criterion. • Staining by PI and SYTO 9 is highly dependent on salt concentration. Haugland (1996) reported that BacLight '~ may not be suitable for marine samples although it has been used successfully in some marine environments (Decamp and Rajendran, 1998a, b; Gasol et al., 1999).

eeeeee

DIRECT VIABLE COUNTING METHOD

[DVC]: K O G U R E

Introduction A cell that is actively g r o w i n g and dividing can be considered viable and active. Thus, the ability of a cell to g r o w and divide can be used as a p r i m a r y characteristic of living, active cells (Joux and LeBaron, 1997). During the late 1970s, Kogure et al. (1979) d e v e l o p e d the Direct Viable Counting (DVC) m e t h o d to identify and e n u m e r a t e cells that were actively growing. This m e t h o d utilizes the antibiotic nalidixic acid to interfere with normal cell division. In the presence of this antibiotic, sensitive cells g r o w but do not divide and can therefore be easily identified microscopically as elongated or enlarged cells.

194

One of the limitations in applying the original Kogure DVC method (1979) to complex communities is the presence of bacteria in the environment that are resistant to nalidixic acid and are therefore able to grow and divide normally in the presence of this antibiotic. This limitation has led to modifications of the initial procedure (Kogure et al., 1984, 1987; Tabor and Neihof, 1984; Coallier et al., 1994). Recent improvements have utilized multiple antibiotic cocktails that act similarly to nalidixic acid (Kogure ct tTl., 1984; Servis et al., 1993; Joux and LeBaron, 1997). In general DVC results are well correlated with other measures of cell activity, i.e. cell reducing potential (Maki and Remsen, 1981; Tabor and Neihof, 1984).

Principle Nalidixic acid (nal) is an antibiotic that affects the cell by inhibiting the bacterial enzymes DNA gyrase and topoisomerase II by binding to the DNA gyrase complex (Prescott et a[., 1997). DNA gyrase inhibition disrupts DNA synthesis and replication due to improper coiling of the DNA molecule. While DNA synthesis is disrupted by nal, synthesis of RNA, protein, and cellular components continue (Maki and Remsen, 1981). Active cells that are unable to divide will continue to grow and become elongated. The visible enlargement of the cell is the basis for recognizing active bacteria. Other antibiotics that have been used with this method include piromidic acid, pipemidic acid, ciproflaxin, and cephalexin (Joux and LeBaron, 1997). The first three antibiotics listed inhibit DNA gyrase as well, however, cephalexin inhibits the transpeptidase and transglycosylase reactions, which serve to cross-link the cell wall in bacteria. Cephalexin blocks the formation of the septum during cell division and in st) doing, produces elongated cells. These antibiotics working together have been found to inhibit 96(7~: of all isolates tested (Joux and LeBaron, 1997).

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Equipment and reagents

•

•

Epifluorescent microscope equipped with a UV filter set. Vacuum manifold, 2_5 mm vacuum filter holders. Vacuum source. 20°C incubator. Sterile yeast extract solution (final concentration: 50 mg ml '). 37% pro-grade formalin. Antibiotics: see Table 10.2_. DAPI (stock concentration: 2_00 ~tg ml '). Sigma Chemical (St Louis, MO). DAPI stock solutions are dissolved in distilled water and can be stored in the refrigerator at 2 ~ ° C in the dark until needed. Acridine Orange (final concentration: 0.01%). Sigma (St Louis, M O ) . A O stock solutions are dissolved in distilled water and can be scored in the refrigerator at 2-4°C in the dark until needed. 0.5 M NaOH.

195

Table 10.2 List of the antibiotics used for inhibiting cell division with the direct viable counting (DVC) technique.These antibiotic solutions are filter sterilized through 0.2 ~tm membrane filters and can be pre-made and stored in the dark at 4°C Antibiotic

Stock conc.

Final conc.

Solvent

Nalidixic acid

500 big ml '

20 ~g ml ~

Pipemidic acid

500 ~lg ml '

10 btg ml '

Piromidic acid

500 btg ml '

10 btg mt '

Cephalexin

500 btg ml '

10 btg ml '

Ciproflaxin

500 ~lg ml ~

0.5 btg ml '

Sterile water Sterile water Sterile water Sterile water Sterile water

• • • • • • • •

Distributor

MilliQ MilliQ MilliQ MilliQ MilliQ

Sigma St Louis, MO Sigma St Louis, MO Sigma St Louis, MO Sigma St Louis, MO Bayer Pittsburgh, PA

0.2 IJm filtered distilled water. 25 mm, 0.2/Jm Nuclepore black polycarbonate filters. Screw cap glass bottles (amber or clear). 15 ml glass tubes (test tubes) and caps. Glass microscope slides. Glass microscope cover slips. Microscope grade immersion oil. Non-fluorescing immersion oil.

Protocol

1. Samples are collected and placed into screw cap amber or clear glass bottles. 2. Fill sterile 15 ml glass tubes with 9 ml of sample (duplicate samples are recommended). 3. For each sample, a control (1-2 ml d e p e n d i n g on the bacterial biomass of the sample) is fixed in f o r m a l d e h y d e (2% final concentration with 37% formalin) and stained as described below in step 8. 4. Aseptically enrich each of the samples with 1 ml of yeast extract (final concentration: 50 mg 1 ~) 5. Add the antibiotic cocktail; see Table 10.2. 6. Incubate the samples at 20 ° C for 6 to 8 h. 7. Fix the samples with 0.2~7, f o r m a l d e h y d e (final concentration). Fixing the samples terminates synthesis of cellular materials and growth. 8. Stain cells with Acridine Orange (AO). Stain cells with a 0.01% final concentration of AO. Mix 1 ml of the water sample with 1 ml of dilution water and 0.2 ml of 0.1% AO (stock solution). Vortex and incubate for 2 minutes, OR 4'6-diamidino-2-phenylindole (DAPI). Stain cells with 2.5 ~g of DAPI per 1 ml of sample. Vortex and incubate the sample in the dark for 30 rain. 196

9. Place a 25 mm, 0.2 pm Nuclepore black polycarbonate filter onto a tower set-up. Pipette solution of cells and stain onto a filter tower and wash the glass bottle with 2 ml of filtered water (0.2 ~tm pore-size filter). Pipette this volume onto the filter tower and filter until dry with gentle vacuum. 10. Remove the filter from the column and place it onto a glass slide, cell side up. 11. Place a drop of non-fluorescing immersion oil on top of the filter and cover with a coverslip. 12. Enumerate the total cells (AO or DAPI) and active cells (elongated) using an epifluorescent microscope equipped with a blue excitation filter and UV filter set, respectively. Joux and LeBaron (1997) recomm e n d the use of a Zeiss UV filter set (4877-02). Excitation 365 nm, barrier 420 nm. An example of bacteria cells before and after incubation with a combination of antibiotics (nal, pipemidic acid, piromidic acid, cephalexin, and ciproflaxin) that inhibit cell division is presented in Figure 10.2.

Advantages The assessment of viability is based on indicators of DNA, RNA, and protein synthesis and not on the concentration of those molecules within the cell. Thus, cells must be actively growing and dividing in order to be detected. The DVC method is theoretically not restricted to particular cell types. Protocol is easy to perform, and has a short incubation time. i

Disadvantages •

• •

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Technique necessitates the in vitro growth of cells and therefore is subject to cultivation bias. The extent of this bias is unknown and likely to be variable between samples. The addition of a growth substrate (e.g. yeast extract) may introduce artefacts associated with cultivation. The natural occurrence of antibiotic resistant cells in natural samples may bias results.The extent of this bias is unknown and likely varies among samples.

CONCLUSIONS In this chapter we have described five methods available to assess the physiological status of individual cells in marine water samples. A sixth method 'CTC' is described elsewhere in this volume (Sherr et al., Chapter 8). The general advantages and disadvantages of each of these methods are summarized in Table 10.3. In general, we have considered and compared methods based on their simplicity, reliability, specificity, versatility, and the degree that they have been used in published reports. 197

°4 ca.

198

Table 10.3 Summary of technical aspects of the five methods employed for determining the physiological status of bacteria, described in detail in the text

VSP

Microautoradiography Nudeoid and probes

Kogure

Live/Dead

Simplicity

Intermediate 2 hours

Difficult ~ 2 days

Simple < 1 hour

Simple ~ 10 hours

Simple < 1 hour

Versatility, reliability

Good Two independent criteria to assess the physiological status

Good Two independent criteria to assess the physiological status

Fair One criteria

Fair One criteria

Fair One criteria

Cell specificity

Total community or specific groups of bacteria

Total community or specific groups of bacteria

Total community

Total Total community community

Key references

° This chapter * Williams et al., 1998

• Ouverney and Fuhrman, 1999 • Cottrell and Kirchman, 1999

• Choietal., * J o u x a n d *Gasolet 1996 LeBaron, al., 1999 • Zweifel 1997 ° DeCamp and ° Kogure and Hagstrom, et al., 1987 Rajendran, 1995 1998 a,b

The d e v e l o p m e n t of n e w m e t h o d o l o g i e s in the field of microbial ecology has b e c o m e a science unto itself (Paul, 1993). A s p e c t r u m of alternative methodological a p p r o a c h e s is n o w either u n d e r d e v e l o p m e n t or available for m e a s u r i n g single cell attributes of aquatic bacterioplankton. These techniques and protocols target specific cellular criteria that can be associated with the physiological status of an individual bacterial cell. As these m e t h o d s are d e v e l o p e d , used, and c o m p a r e d in various natural systems, it is clear that different m e t h o d s can yield different estimates of the p r o p o r t i o n of bacterial cells that are alive, viable and metabolically active (Choi et al., 1999). The active fraction of bacterial p o p u l a t i o n s that h a v e been reported in the literature to date range from nearly 0% to nearly 100% d e p e n d i n g on the e n v i r o n m e n t and the technique used to estimate this parameter. H o w e v e r , e m e r g i n g from these disparate data sets is the general conclusion that, in m o s t marine e n v i r o n m e n t s and at any given point in time, a significant fraction of the bacteria e n u m e r a t e d b y direct microscopic counting m e t h o d s are either dead or inactive. To complicate these conclusions there is a relative sparseness of studies that directly c o m p a r e m e t h o d s in a systematic fashion. Karner and F u h r m a n (1997) conducted a rare e x a m p l e of such a study. These investigators c o m p a r e d

F i g u r e 10.2. Fluorescence micrographs of DAPI-stained bacteria from a marine sample before incubation (A), after 6 h incubation with nalidixic acid (20 gg ml ~) (B), and after 18 h incubation with the antibiotic cocktail (C). Substrate responses are identified by arrows. Bar, 10 btm.

199

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results obtained from a variety of m a r i n e w a t e r s a m p l e s using the CTC, nucleoid staining, microautoradiography, 16S r R N A oligonucleotide probes, and DAPI staining methods. Although the results obtained from these c o m p a r i s o n s varied, based on their s t u d y Karner and F u h r m a n reached the conclusion that m i c r o a u t o r a d i o g r a p h y and ribosomal RNA targeted probes were m o s t likely the m o s t reliable indicators of physiological activity since these two m e t h o d s were self-consistent. Extending this logic, it seems reasonable that the m o s t useful methodological a p p r o a c h for estimating the physiological status of an individual cell will combine c o m p o n e n t s that s i m u l t a n e o u s l y target multiple cellular targets that can act as indicators of physiological status. Of the m e t h o d s described in this chapter, several allow the simultaneous evaluation of multiple criteria of physiological status (Williams et al., 1998; Lee et al., 1999; O u v e r n e y and F u h r m a n , 1999; Cottrell and Kirchman, 2000). Therefore, these m e t h o d s are considered to be the m o s t reliable m e t h o d s available today. However, all of the m e t h o d s available h a v e limitations and therefore results m u s t be interpreted cautiously and with respect to the specific cellular criteria examined. Future m e t h o d d e v e l o p m e n t should concern itself with m e t h o d s that are straightforward, simple to p e r f o r m and reproduce, and h a v e universal application to a variety of cell types. The need for these d e v e l o p m e n t s is undeniable.

References Amann, R. I., Ludwig, W. and Schleifer, K. H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143-169. Braun-Howland, E. B., Danielsen, S. A. and Nierzwicki-Bauer, S. A. (1992). Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes. Bioteclmiques 13, 928-934. Bremer, H. and Dennis, P. P. (1987). Modulation of chemical composition and other parameters of the cell by growth rate. In: Escherichia coli and Sahnonella typhinturium: Celhdar and Molecular Biology (E C. Weidhardt, Ed.), pp. 1527-1542, American Society for Microbiology, Washington, DC. Brock, T. D. and Brock, M. L. (1966). Autoradiography as a tool in microbial ecology. Natmv 209, 734-736, Brock, T. D. and Madigan M. T. (1988). Biology q(Microotxanisms, 5th edn. Prentice Hall, Englewood Cliffs, NJ. Carman, K. R. (1990). Radioactive labeling of a natural assemblage of marine sedimentary bacteria and microalgae for trophic studies: an autoradiographic study. Microbial Ecol. 19, 279-290. Choi, J. W., Sherr, E. B. and Sherr, B. E (1996). Relation between presence/absence of a visible nucleoid and metabolic activity in bacterioplankton cells. Limnol. Ocemrogr. 41, 1161-1168. Choi, J. W., Sherr, B. E and Sherr, E. B. (1999). Dead or Alive? A large fraction of ETS-inactive bacterial cells, as assessed by reduction of CTC, can become ETSactive with incubation and substrate addition. Aquatic Microbial Ecol. 18, 105-115. Coallier, J., Prevots, M. and Rompre, A. (1994). The optimization and application of two direct viable count methods for bacteria in distributed drinking water. Can. J. Microbiol. 40, 830-836.

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ACKNOWLEDGMENTS This work was supported by grants from the National Science foundation to M.E.E and P.G.V. OCE-9617884 and OCE-9906734, and a National Science Foundation CIRE Activity award OCE-9872694 also to P.G.V. and M.E.E The Department of Energy also provided support for this work DEFG02-88ER62531. We wish to acknowledge the important contribution of Ms. Samanthia Williams for the initial development of the VSP method and Y. Hong, J. Danforth, A. Shopov, and V. Ballard for technical assistance. We especially thank L. Cowden for critically critiquing the manuscript and A. Boyette for preparing the figures.

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