Using Flow Cytometry for Counting Natural Planktonic Bacteria and Understanding the Structure of Planktonic Bacterial Communities

Contributors: Josep M. Gasol and Paul A. del Giorgio
E-mail: pepgasol@icm.csic.es
Affiliation: Departament de Biologia Marina i Oceanografia, Institut de Ciencies del mar, CSIC
URL: http://www.icm.csic.es/bio/index_bio.html

This is an excerpt of a paper that will appear in June 2000. References can be found in the document entitled "How to count picoalgae and bacteria with the FACScalibur flow cytometer"

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Counting Bacterioplankton, in practice

In this section we will explore practical aspects of the flow cytometric enumeration of bacteria, focusing on three key steps of the protocol: Cell fixation, cell staining and data processing and interpretation. In table 1 we have summarized the different protocols currently used by researchers, to highlight the diversity of approaches that have been taken. .

Fixation of samples is needed whenever the samples cannot be processed fresh immediately after sampling. But fixation may in addition be required to permeabilize cells and thus facilitate the penetration of certain stains into the cell (Bullock, 1984). The ideal fixation protocol should be fast, should effectively preserve nucleic acids, and protect autofluorescence without altering the size and the light scatter properties of the cells. Fixatives currently used include ethanol (70%), formaldehyde, diluted as formalin or methanol-free as paraformaldehyde (PFA), glutaraldehyde (Glut) and even TCA (Rice et al., 1997) and cold shock followed by metabolic inhibition (to block stain efflux pumps, Wallberg et al., 1998). Paraformaldehyde (the solid form of formaldehyde, as opposite to the commonly used hydrolysed form, which is 40% formaldehyde and has methanol) quickly penetrates the cells and is assumed to be the most effective fixative of nucleic acids and proteins. Glutaraldehyde penetrates slowly and may not permeate all gram negative bacteria (Bullock, 1984). However, 1% Glut was found to protect microbes from cell lysis and loss of autofluorescence upon rapid freezing in liquid nitrogen and long-term cryogenic storage (Vaulot et al., 1989). 1% PFA has been seen to offer similar protection (Monger and Landry, 1993) and fluorescence protection was even better when the samples were frozen after fixation (Hall, 1991; Zubkov et al., 1999). Campbell et al. (1994), however, did not find any differences between fixation with PFA and with Glut. Glutaraldehyde, unless of very good quality, may produce an autofluorescence signal in FC that can be very annoying (Booth, 1987). And formalin is known to negatively affect cell fluorescence (Crissman et al., 1978; Lebaron et al., 1998; Troussellier et al., 1999).

Some degree of post-fixation cell disappearance and cell alteration has been reported with most common fixatives. Marie et al. (1993) reported that 0.5% PFA produced a 9% loss of Prochlorophyte cells, and Troussellier et al (1995) reported a similar value. del Giorgio et al. (1996) found that fixation with formalin and glutaraldehyde decreased the forward scatter and green fluorescence and increased side scatter of fixed cells relative to live cells. They assigned those changes to post-fixation cell shrinkage that seemed to be particularly important in the case of formalin.

We tested some of these fixatives in two marine samples, with or without, freezing in liquid nitrogen. The protocol labeled PFA+G consists in PFA 1% + 0.05% Glut (Marie et al., 1996). The two samples, one from an oligotrophic site and the other from eutrophic waters, responded differently to some of the treatments. There was some degree of cell loss even in the fixed and frozen samples, but loss was greater for formalin and Glut treatments. Side scatter increased in all treatments, especially if no freezing was involved. A 10% decrease in green fluorescence occurred with the PFA+G fixation while a stronger reduction in fluorescence was produced by formalin and freezing. With the present data, and given that PFA is the fixative of choice for fluorescent in situ hybridization (Wallner et al., 1993) and that the PFA+G protocol has been seen to reduce the variability in DNA analyses of the microbes (Jacquet et al., 1998b), we tend to recommend that protocol for cell fixation of prokaryotes.

 

Staining. The length of incubation of cells with fluorochromes to attain optimal staining prior to FC analysis varies with each type of compound. The recommended incubation time for DAPI and Hoescht 33342 is at least 1 h (Robertson & Button, 1989) or more (Campbell et al., 1994; and Monfort & Baleux, 1994, stained for 2 h), although one of the advantages of Hoescht over Dapi was its lower staining time. The newer blue stains require much lower times, usually less than 15 min (del Giorgio et al., 1996; Marie et al., 1996; Veldhuis et al., 1997). The behavior of some of these stains is quite interesting. For example, Li et al. (1995) used ToPro-1 to stain and count all bacteria, but because this fluorochrome is marketed as cell-impermeant by the manufacturers (Haugland, 1999), these authors used fixed and permeabilized cells which quickly took up the stain. But on fresh samples, ToPro-1 stains only a fraction of the cells in the first minute, and this number slowly increases until most of the population has been stained within the next 15-20 min. The mean fluorescence per cell is very high for the cells that have been stained in the first minutes and decreases exponentially afterwards (del Giorgio et al., in press). The interpretation is that only cells with damaged membranes allow the stain to enter the cell and bind to the nucleic acids, while later all cells have their outer membranes stained with less fluorescence.

Staining is sometimes done with the addition of buffers (acting also as cell permeants), such as Triton X-100 (Button & Robertson, 1993; Li et al., 1995), TE buffer (Marie et al., 1996), EDTA or EGTA (Kaprelyants and Kell, 1992; López-Amorós et al., 1995b). The reason would be that some of the dyes are very sensitive to ionic strength (Marie et al., 1997; Veldhuis et al., 1997). Some authors, however, have found these treatments unnecessary and even detrimental because Triton X-100 generates background fluorescence (Monger & Landry, 1993) and reduces cell autofluorescence (Marie et al., 1996). Interestingly, Marie et al. (1999) report the need for Triton pretreatment to stain live samples with Syto 13, but not to stain fixed samples, while Comas & Vives-Rego (1997) found no need for pretreatment to stain bacteria with Syto 13. Finally, Lebaron et al. (1998) report increased cell-specific fluorescence of the Syto stains when incubated in the presence of 30 mM potassium citrate. Given that many authors have successfully stained and counted bacteria using Syto 13 without any pretreatment of the kind discussed here, it is up to each researcher to decided whether he/she has to use it or not. Other fluorochromes, such as To-Pro 1, that are inherently cell-impermeant, will require some kind of permeabilization pretreatment.

Some authors have suggested that samples should undergo RNAse treatment before the addition of the nucleic acid stains, to eliminate the confounding effect of RNA-induced fluorescence. This is mandatory if one is interested in the cell cycle of the prokaryotes and wants to infer growth rates from those data (e.g. Vaulot et al., 1995), but it is not necessary for regular enumeration of chemotrophic bacteria. Since these organisms do not seem to divide at once, cell cycle analysis for growth rate determination seems not to be possible (Jacquet et al., 1998b). Furthermore, staining with Syto 13, TOTO-1, TOPRO-1 and YOYO-1 of planktonic bacteria seem to be dependent only on the amount of DNA, with little RNA interference (Li et al., 1995; Guindulain et al., 1997). This is possibly not due to the binding affinities of the stains to DNA and RNA, but probably to the low amounts of RNA in planktonic bacteria or to the physical unavailability of rRNA to the dyes.

 

Bacterial discrimination. Stained bacteria are detected and discriminated from other non-bacterial particles with a combination of light scatter, green and orange or red fluorescence. In addition, the combination of these parameters allows better resolution of the different subpopulations within the mixed bacterial assemblage. It also lets easily identify particles that can interfere with the counts. The instrument threshold defines the minimum scatter or fluorescence intensity needed to trigger an event that will be processed by the system software. The threshold allows the reduction of both electronic noise as well as unwanted, non-target particles, and it is usually set on the same primary parameter used to discriminate bacterial cells (i.e. green, or blue, or red, depending on the stain used or the cell autofluorescence). But there will inevitable be some "noise" particles that have a fluorescent level above that threshold. In our experience, relatively large particles with weak autofluorescence can be discriminated well in the Side scatter — Green fluorescence plot. Particles with low fluorescence and low side scatter have a greater potential to interfere with the actual determination of the bacterial density, but these can easily be taken apart in the Red vs. Green fluorescence plot where they appear in a diagonal line with relatively more red fluorescence than that of bacteria (as long as no electronic compensation has been applied).

 

Counting. A few cytometers, such as the Coulter XL and the Ortho Cytoron Absolute, are equipped with devices that exactly control and record the volume of sample that circulates in front of the laser. But most cytometers have no way of exactly controlling the flux of sample, and therefore, the number of particles detected in a cytometric analysis cannot be directly related to a given sample volume to obtain an estimate of particle density. There are at least three ways of obtaining absolute counts in that case: i) a known amount of reference beads can be added (Cantineaux et al., 1993), ii) the flow can be calibrated each day of work, or iii) the samples can be weighted before and after the run. The last alternative is very time consuming, and in addition, it may be less accurate, because there may be some backflow of sheath fluid into the tube that confounds the actual sample volume processed. Alternative ii) (daily calibration) gives good results but requires an extremely stable instrument. Calibration of the flow can be done easily by weighting a tube containing water, processing various volumes through the cytometer, estimating the time needed for each volume to go through, and then weighing the tube again. Many researchers, however, use alternative i) (reference beads), because it is accurate, fast and in addition to allowing absolute counts, it also provides an internal standard that can be used to assess instrument performance and to standardize scatter and fluorescence measurements for quantitative applications. However, the beads have to be counted each day of work, sometimes get contaminated with bacteria, and have to be sonicated to avoid aggregation. In our laboratory, the first two of the methods cited above (reference beads and flow calibration) offer highly similar estimates of bacterial abundance. The bead stock is dispensed to each sample to a final bead density that is about 1-10% of the expected density of target cells. For a regular bacterioplankton sample with an abundance of 106 cells ml-1, a final bead density in the sample of 1 to 5x105 beads ml-1 is appropriate. An accurate measurement of the reference bead density in the stock solution is of key importance and must be done on a routine basis. Larger beads (>2.0 痠) can be counted in a Coulter particle analyzer, but this method is less effective for the smaller beads which are generally used for bacterial work. Alternatively, bead density can be determined using regular epifluorescence microscopy, but this is time consuming and not particularly accurate. A more effective approach is to use a primary reference bead solution where the bead density is precisely known, and to compare this to the working bead solution using the flow cytometer. Primary reference bead solutions are commercially available (i.e.TrueCount, Becton Dickinson).

There are other issue to consider when counting cells, in addition to estimating the volume of sample processed. Bacteria are found in plankton in concentrations varying from 105 up to 107 cells ml-1. A reasonable sample rate of 10 痞 min-1 (see Table 1), translates into a rate of cell passage through the laser of hundreds to thousands of cells. We often add beads to the sample, and there are other particles which are not bacteria ("noise") in the same sample, all of which contribute to the events detected by the instrument. The light scattered and emitted by each particle must be collected and converted to an electrical current which must then be digitized by the electronic system, and there are limits to how many of these events a system can effectively handle. In addition, when too many particles go through the cytometer, there is a greater probability that two particles will pass together and be considered by the electronic system as a single larger particle. This phenomenon is called coincidence, and tends to become significant at concentration levels above 2.5 106 cells ml-1 (Marie et al., 1996) which translate to count rates of 1000 - 1400 events s-1 (del Giorgio et al., 1996; Marie et al., 1999; Cristina et al., submitted). Samples with higher concentrations have to be diluted either in filtered water, in buffer (Marie et al., 1999) or in dH2O if the samples are fixed (authors’ obs.). We have been succesful at enumerating bacteria from solar salt ponds where they are at concentrations above 107 ml-1 in salinities around 250‰ (J.M.Gasol and C.Pedrós-Alió, unpublished). Dilution of the PFA-fixed sample in dH2O served here two purposes: to reduce coincidence and simultaneously to reduce salinity so that salt did not interfere with the nucleic acid stain. Some researchers, however, are routinely counting at rates at or above 2000 s-1 (e.g. Porter et al., 1993). One way of empirically determining the level of coincidence for a given instrument is by means of a bead solution serially diluted to mimic varying particle concentrations. By increasing the bead concentration and, thus, the rate of particle passage, the amount of doublets (two particles seen as one) increases exponentially. By relating then the observed particle concentration to the expected concentration, we were able to find out the limits of a FACScalibur and of a Coulter XL, which were very similar and broke out at particle passage rates of around 2000 s-1, equivalent to total particle concentrations of several million particles per ml. Even though this procedure can be used to find out the limits of any machine, it will always be safe to keep the rates of particle passage below the 1000 s-1.

Table 1
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Description: Some technical details offered in most of the papers to date where natural planktonic bacteria have been enumerated by FC.

Reference

Instrument

Laser

(power)

Flow rate

痞 min-1

Sheath

fluid

Fixative

Stain

Pretreatment

Robertson & Button 1989

Ortho Cytofluorograph IIS

UV 5W

5

dH2O

Ethanol 75%

DAPI (2.5 痢 ml-1)

-

Button & Robertson 1989

Ortho Cytofluorograph IIS

UV 5W

5

dH2O

Ethanol 75%

DAPI (2.5 痢 ml-1)

-

Monfort & Baleux 1992

ACR-1400-SP Bruker*

UV 100W

-

-

Formalin 3.7%

DAPI (2.5 痢 ml-1)

-

Button & Robertson 1993

Ortho Cytofluorograph IIS

UV 5W

5

dH2O

Formalin 0.5%

DAPI (0.5 痢 ml-1)

Triton X-100 (0.1%)

Troussellier et al. 1993

ACR-1400-SP Bruker*

UV

-

-

PFA 0.5-4%

DAPI (-)

-

Monger & Landry 1993

Coulter EPICS 753

UV 200 mW

25 — 40

-

PFA 1%

Hoechst 33342 (0.5 痢 ml-1)

-

Heldal et al. 1994

Argus 100-4

UV

20

-

Glut 2%

DAPI (20 痢 ml-1)

-

Campbell et al. 1994

Coulter EPICS 753

UV 200 mW

-

-

PFA 0.2 %

Hoechst 33342 (0.5 痢 ml-1)

-

Troussellier et al. 1995

ACR 1400-SP Bruker*

UV

5

-

several

DAPI (2.5 痢 ml-1)

-

Li et al. 1995

FACSort

Blue

12

-

Glut 1%, PFA 1%

TOTO (0.3-0.5 然)

TO-PRO (3-5 然)

Triton X-100 (0.1%)

del Giorgio et al. 1996

FACScan

Blue 15 mW

12

Hematall

Glut 1%, FA 3%

Syto13 (2.5 然)

-

Binder et al. 1996

Coulter EPICS 753

UV 200 mW

-

-

Glut 0.12 %

Hoechst 33342 (0.5 痢 ml-1)

-

Landry et al. 1996

Coulter EPICS 753

UV 200 mW

-

-

PFA 0.9%

Hoechst 33342 (0.8 痢 ml-1)

-

Button et al. 1996

Ortho Cytofluorograph IIS

UV 5W

5

dH2O

Formalin 0.5%

DAPI (0.5 痢 ml-1)

Triton X-100 (0.1%)

Jellett et al. 1996

FACSort

Blue 15 mW

12

Filt Seawater

PFA 1%

TO-PRO-1 (3 然)

Triton X-100 (0.1%)

Buck et al. 1996

Coulter EPICS 753

UV 200 mW

-

-

PFA 0.2 %

Hoechst 33342 (0.5 痢 ml-1)

-

Pile et al. 1996

Coulter EPICS 753

UV 225 mW

-

-

-

Hoestch 33342 (0.5 痢 ml-1)

-

Campbell et al. 1997

Coulter EPICS 753

UV 200 mW

-

-

PFA 0.2 %

Hoechst 33342 (1 痢 ml-1)

-

del Giorgio et al. 1997b

FACScan

Blue 15 mW

12

Hematall

-

Syto13 (2.5 然)

-

Marie et al. 1997

FACSort

Blue 15 mW

-

-

Glut 0.1%

SybrGreen I (10-4)

RNAse, 30 mM potassium citrate

 

Coulter EPICS 541

UV 500 mW

-

-

Glut 0.1%

Hoestch 33342 (0.45 痢 ml-1)

 

Wallner et al. 1997

FACStar Plus

UV 200 mW

-

-

PFA 3%

DAPI (1 然)

-

Guindulain et al. 1997

Coulter EPICS Elite

Blue 15mW

-

-

PFA 2%

Syto13 (2.5 然)

Triton X-100 (0.1%)

Pile 1997

Coulter EPICS 753

UV 225 mW

-

-

-

Hoestch 33342 (0.5 痢 ml-1)

-

Zubkov et al. 1998

FACSort

Blue 15mW

45

-

Glut 0.2%

TOTO (0.4 然)

Triton X-100 (0.1%)

Peters et al. 1998

FACSCalibur

Blue 15mW

19

dH2O

PFA 1% + Glut 0.05%

Syto13 (2.5-5 然)

-

Campbell et al. 1998

Coulter EPICS 753

UV 200 mW

-

-

PFA 0.2 %

Hoechst 33342 (1 痢 ml-1)

-

López-Amorós et al. 1998

Coulter XL

Blue 15mW

-

-

PFA 0.2%

Syto13 (0.125 然)

Triton X-100 (0.1%)

Jacquet et al. 1998b

FACSort

Blue 15mW

-

Filt Seawater

PFA 1% + Glut 0.05%

SybrGreen I (10-4)

30 mM potassium citrate

Lebaron et al. 1998

FACSCalibur

Blue 15mW

-

-

Formalin 2%

(several)

sodium or potassium citrate

Ribes et al. 1998a, 1998b

Coulter EPICS 753

UV 225 mW

-

-

-

Hoestch 33342 (0.5 痢 ml-1)

-

Marie et al. 1999

FACSort

Blue 15mW

50

-

PFA + G / Glut 0.1-0.5%

SybrGreen I (10-4)

(several)

Gasol & Morán 1999

FACSCalibur

Blue 15mW

19

dH2O

PFA 1% + Glut 0.05%

Syto13 (2.5-5 然)

-

Gasol et al. 1999

FACSCalibur

Blue 15mW

19

dH2O

PFA 1% + Glut 0.05%

Syto13 (2.5-5 然)

-

Sherr et al. 1999

FACScan

Blue 15mW

12

Hematall

-

Syto13 (2.5 然)

-

Sieracki et al. 1999

FACScan

Blue 15mW

20 - 50

-

PFA 1%

PicoGreen (10-2)

-

Vives-Rego et al. 1999

Coulter XL

Blue 15mW

-

-

none

Syto 13 (2.5 然)

-

Mostajir et al. 1999

ACR 1400-Sp Bruker*

UV 100W

-

-

Formalin 3.7%

DAPI (2.5 痢 ml-1)

-

Chatila et al. 1999

ACR 1400-Sp Bruker*

UV 100W

-

-

Formalin 3.7%

DAPI (2.5 痢 ml-1)

-

Karl et al. 1999

Coulter EPICS 753

UV 200 mW

-

-

PFA 1 %

Hoechst 33342 (1 痢 ml-1)

-

Gin et al. 1999

Coulter EPICS 753

UV 200 mW

-

-

Glut 0.1 %

Hoechst 33342 (0.5 痢 ml-1)

-

Troussellier et al. 1999

FACSCalibur

Blue 15mW

-

-

-

Syto 13 (5 痢 ml-1)

-

Servais et al. 1999

FACSCalibur

Blue 15mW

-

-

-

Syto 13 (5 痢 ml-1)

-

Lebaron et al. 1999

FACSCalibur

Blue 15mW

-

-

-

Syto 13 (5 痢 ml-1)

-

* Arc-Lamp machines

The Argus-100 (Skatron), the BioRad Bryte and the Bruker ACR 1400-Sp are essentially the same machines.

The FACSort evolved to the FACSCalibur, while the FACScan evolved to the FACSVantage





Josep M. Gasol and Paul A. del Giorgio. 2000. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Scientia Marina, 64 (2). Esp. Issue "Achievements and Prospects in flow cytometry of aquatic microbes", M. Reckermann & F. Colijn (eds.)

1 Departament de Biologia Marina i Oceanografia, Institut de Ciencies del Mar, CSIC. Passeig Joan de Borbo s/n. E08039 Barcelona, Catalunya. Spain. pepgasol@icm.csic.es 2 Horn Point Laboratory. University of Maryland Center for Environmental Science PO. Box 775 Cambridge, MD 21613 USA