FEMS Microbiology Reviews 24 (2000) 429^448

www.fems-microbiology.org

Current and future applications of £ow cytometry in aquatic

microbiology
a;
J. Vives-Rego *, P. Lebaron b , G. Nebe-von Caron c

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a
Departament de Microbiologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain
b
Observatoire Ocëanologique, Universitë Pierre et Marie Curie, UMR 7621 CNRS, Institut National des Sciences de l'Univers, P.O. Box 44,
66651 Banyuls-sur-Mer Cedex, France
c
Unilever Research, Colworth Laboratory, Sharnbrook, Bedfordshire MK44 1LQ, UK

Received 5 February 2000 ; accepted 7 April 2000

Abstract

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to
determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the
heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study
the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers,
which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical,
biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is
now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of
heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a
new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow
cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters.
The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes
be difficult to accomplish. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.

Keywords : Flow cytometry; Aquatic microbiology ; Sorting; Phytoplankton ; Bacterioplankton; Protozoon; Virus

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
1.1. Start-up di¤culties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
1.2. The unique capacities of £ow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
2. Enumeration of microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
2.1. Fixation and permeabilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
2.2. Unicellular phototrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
2.3. Bacterioplankton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
2.4. Protozoa and grazing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
2.5. Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
3. Cell size measurements and scatters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
4. Determination of cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
4.1. Membrane integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
4.2. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
4.3. Direct viable count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
4.4. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

* Corresponding author. Fax: +34 (93) 411-0592; E-mail: jvives@porthos.bio.ub.es

0168-6445 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 0 0 ) 0 0 0 3 3 - 4

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430 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448

5. Metabolic activity measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

5.1. Membrane potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
5.2. Esterase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
5.3. Dehydrogenase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
6. Di¡erentiation based on probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
6.1. Di¡erentiation by `non-speci¢c' probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
6.2. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
6.3. Nucleic acid probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
6.4. Multiparametric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
7. Assessment of bacterial starvation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
8. Biodegradation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
9. Solid phase cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

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10. Cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
10.1. Common applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
10.2. Cell sorting and molecular microbial ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
11. Controls and validations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
12. Summing up the limitations of £ow cytometry for potential users . . . . . . . . . . . . . . . . . . . 443
13. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

1. Introduction richment for the detection of rare events in aquatic sam-

Rapid methods to enumerate and size cells are an evi-
dent need in aquatic and environmental microbiology.
Flow cytometry has become a valuable tool in aquatic
and environmental microbiology that combines direct
and rapid assays to determine numbers, cell size distribu-
tion and additional biochemical and physiological charac-
teristics of individual cells, revealing the heterogeneity
present in a population or community [1^3]. However,
counts and cell size measurements can also be achieved
by image cytometers [4^7]. Image cytometry in the context
of confocal microscopy is ideal to obtain spatial informa-
tion and it is a de¢nitive method to study bio¢lms. The
main advantages of £ow cytometry are the high sample
throughput and automation, the analysis of thousands of
cells per second and the possibility of cell sorting.
In £ow cytometry, individual cells con¢ned within a
rapidly £owing jet of water pass a measuring window, in
which several parameters can be simultaneously measured
for several thousand cells per second with high precision.
Although it is di¤cult to detect bacterial cells at concen-
trations lower than 100^1000 cells ml31 of aquatic sample,
this limit is mainly dependent on the presence or absence
of electronic and £uorescence background, on the hetero-
geneity of the sample, on the £uorescence distribution and
on the £uorescence signal-to-noise ratio. Flow cytometric
analyses are commonly performed at a £ow rate of 10^100
Wl min31 and detection of up to 10 000 events s31 , when
the microbial concentration in the processed sample is
su¤ciently high. Even at a high £ow rate of 100 Wl
min31 , the detection of 100 targeted cells present in a
sample at a concentration of 10 cells ml31 requires 100
min. Thus, although this technique is geared for numerical
resolution, it still requires physical or biological pre-en-
ples.
Although phytoplankton and bacterioplankton have
been extensively studied, protozoa and viruses are still to
be properly detected and studied by £ow cytometry. The
study of viruses in aquatic samples is di¤cult due to their
small size, low nucleic acid content and their unique struc-
ture and biology. We wish to emphasise that £ow cyto-
metry o¡ers unique capabilities that are summarised in
Section 1.2 and discussed throughout the text. Flow cyto-
metry and its applications in environmental microbiology
have been the subject of several previous publications [1^
3,8,9], and a recent review on routine £ow cytometric es-
timation of abundance, biomass and activity in natural
planktonic systems has been published [10]. We focus
our review on today's aquatic applications, paying special
attention to current techniques and their limitations.
1.1. Start-up di¤culties
Flow cytometric measurements of microorganisms are
hindered by their small size and the reduced contents of
structural molecules, specially DNA, RNA and proteins.
The DNA content of Escherichia coli is about 4^5U106
bp, and is 1000 times smaller than a human cell. The size
di¡erences become more important when we consider that
bacteria in aquatic ecosystems exhibit a much smaller size
and lower content of speci¢c macromolecules than labo-
ratory cultures. These limitations are especially critical
when working on viruses. Interactions of £uorescent
dyes with viruses and the interactivity of the external viral
layers (peptide capsomers or lipoprotein layers) with £uo-
rochromes remain unknown to date.
A second obstacle in microbial £ow cytometry is that
the used £uorescent dyes are relatively well understood

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 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
when applied to mammalian cells, but their interaction
with algae, bacteria or viruses is poorly known. This be-
comes more complicated when considering the increase in
commercially available £uorochromes which need to be
checked for their potential applications [11]. Prokaryotes
do not share the permeability or the uptake kinetics of
mammalian cell envelopes. Usually, bacteria are less per-
meable to £uorochromes than mammalian cells and this is
even more pronounced since many bacteria have very e¤-
cient e¥ux pumps. In such cases, the dye may be pumped
out of the cell very rapidly [12^16], hampering interaction
with the target molecule. These extrusion pumps interfere
not only with £uorescent staining methods, but also with
radioactive probes [17^19]. The extrusion of rhodamine
123 by Listeria for example is not only dependent on en-
ergising but also on the bu¡er system chosen for the stain-
ing [20,21]. Although dye extrusion is a major source of
staining interference, it also provides valuable physiologi-
cal information for the detection of pump activities which,
in the case of the ethidium bromide proton antiport sys-
tem, can be used to detect a proton gradient [22,23]. Dye
extrusion speeds depend on several parameters such as dye
in£ux, pump activity, pump a¤nity to the £uorochrome
and availability of the £uorochrome close to the mem-
brane.
Another concern is associated with the microbial mor-
phology, which is usually far from the ideal homogeneous
spherical shape for cytometry. Non-spherical cells, rod-
shaped bacteria, elongated or ¢lamentous microorganisms
and in general non-axial symmetry may cause orientation
artefacts where the £uorescence and light scatter signals
depend on the orientation of the cell relative to the direc-
tion of the excitation light. However, all these di¤culties
associated with the nature of aquatic microorganisms also
represent an excellent opportunity to gain further insight
into the still poorly understood aquatic microbial ecosys-
tems.
Finally, auto£uorescence and abiotic particles may be of
special interest to environmental cytometrists. Typical
problems encountered when applying £ow cytometry to
marine or continental waters may be high levels of auto-
£uorescence and non-speci¢c dye binding. Auto£uores-
cence results from phototrophic pigments and some organ-
ic compounds but it also depends on certain £uorochrome
physicochemical properties or lipophilic capacity. Flow cy-
tometry is of limited application in aquatic systems with
high particulate and solid phase content. The more par-
ticles and aggregates the sample contains, the greater the
di¤culty of £ow cytometric analysis. Abiotic particles
such as clay, inorganic and organic complexes and detritus
of various origins represent an additional obstacle since
they may also interact with £uorochromes which are sup-
posedly speci¢c for biological macromolecules. In such
cases, the chemical speci¢city of the £uorochrome is coun-
teracted or blocked by the physical a¤nity (absorption,
clustering, etc.) between the £uorochrome and the abiotic
FEMSRE 685 29-8-00
431
particles. All these potential di¤culties indicate the need to
use calibrations, controls and validations which must be
speci¢cally chosen for each cytometric application.
1.2. The unique capacities of £ow cytometry
Despite the limitations of £ow cytometry when applied
to aquatic microbiology (see Section 1.1), it is evident that
£ow cytometry exhibits three unique technical properties
of high potential to study the microbiology of aquatic
systems : (i) tremendous velocity to obtain and process
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data; (ii) the sorting capacity of some cytometers that
allows the transfer of speci¢c populations or even single
cells to a determined location, thus allowing further phys-
ical, chemical, biological or molecular analysis; (iii) high-
speed multiparametric data acquisition and multivariate
data analysis at high speed.
As analysed and discussed by Davey and Kell [8], it is
clear that both natural populations and laboratory axenic
cultures are heterogeneous, in both cases the origin of
their heterogeneity is genetic, phenotypic and environmen-
tal [8,24]. In aquatic microbiology, overcoming the prob-
lem of population heterogeneity is crucial. Today the only
available methodology to assess such heterogeneity is by
analysing individual cells by £ow cytometry and cell sort-
ing.
2. Enumeration of microbes
The quanti¢cation of both prokaryotic and eukaryotic
cells is essential to characterise natural ecosystems and the
enumeration of microorganisms has undoubtedly been the
most frequent and successful application of £ow cytometry
in the study of aquatic microbial systems. Flow cytometry
can be used to analyse mixed populations of bacteria, to
distinguish between classes of picophytoplankton in terms
of size, physiological status and apparent DNA content
and to detect £agellates [10,25,26], ciliates, yeasts and vi-
ruses. Although small pigmented £agellates (2^5 Wm) may
be detected more or less easily through their speci¢c pig-
ment signals (Section 2.2), they are generally included to-
gether with the phototrophic picoeukaryotes. Methods for
detection and quanti¢cation of viruses and heterotrophic
nano£agellates are still under development. Direct quanti-
¢cation of microbe concentration in a sample is possible in
instruments which determine the processed volume. If the
volume of a processed sample cannot be measured, the
common indirect method is the addition of a known con-
centration of £uorescent polystyrene beads. The number
of beads enumerated in the sample is proportional to the
processed sample volume thereby allowing the calculation
of bacterial concentration. Alternatively, the volume ana-
lysed can be determined from the time analysis at a given
£ow rate or, although this technique is less accurate, by
weighing the sample before and after analysis. Absolute
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432 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
counts may also be directly obtained by separate sample
runs using a cytometer-coupled Cytek module (Cytek De-
velopment, CA, USA) [24,27^29]. Cytek's £ow module for
absolute counts is a device that measures particle concen-
tration and £ow rate by injecting a ¢xed volume of the
sample in the £ow cytometer. Although the unit is very
precise, this equipment has received little attention in en-
vironmental studies.
2.1. Fixation and permeabilisation
A previous and basic ¢rst step in most £ow cytometric
studies and in all £ow cytometric enumeration is the ques-
tion of whether or not to ¢x and how. Fixation can pro-
vide both advantages and disadvantages. The main advan-
tages are: (i) ¢xed samples may be stored before staining
for months or even longer; (ii) usually ¢xation also per-
meabilises the cell, improving the entrance of the £uoro-
chrome or making the staining quicker. The drawbacks of
¢xation are: (i) viable, membrane potential- and enzy-
matic-based stains cannot be applied in ¢xed cells; (ii)
sorting aiming to verify any physiological function such
as culturability is not possible; (iii) scatter properties usu-
ally decrease; (iv) £uorescence after association with the
structural molecules (nucleic acids for instance) is lower
than in non-¢xed cells; (v) conformational changes in cel-
lular macromolecules may lead to di¤culties if subsequent
work is needed. Three classical ¢xatives at 2% ¢nal con-
centration are mostly used in microbial £ow cytometry : (i)
glutaraldehyde; (ii) formaldehyde, derived from paraform-
aldehyde by heating, to be used fresh ; and (iii) formalin,
which is 35% formaldehyde+10% methanol for stabilisa-
tion to avoid acid formation. Paraformaldehyde does not
¢x as it is already polymerised and it requires heat to be
dissolved into formalin. Fixation with glutaraldehyde fre-
quently leads to intense auto£uorescence which limits its
use with £uorescent probes. Auto£uorescence is minimised
by ¢xation with formaldehyde but £uorescence assessment
before and after ¢xation is specially needed when using
£uorescent probes. Cold ethanol at 70% may be needed
for special purposes such as labelling the protein content
with FITC, but this ¢xation is usually problematic in ma-
rine environments because it may produce precipitates. In
marine studies the most commonly used ¢xative is para-
formaldehyde. A comparison of the ¢xation and storage
techniques suggests that for marine bacteria and phyto-
plankton, the ¢xatives of choice are formaldehyde or para-
formaldehyde [30]. Fixed cells should be stored frozen,
preferentially in liquid nitrogen, when analysis cannot be
performed in a short time after sampling. For a more
complete discussion on ¢xatives we suggest the article by
Gasol and del Giorgio [10]. In addition to ¢xation, many
£uorochromes (which usually are membrane-impermeable,
especially in Gram-negatives) require permeation to opti-
mise dye penetration. In addition to the permeation e¡ect
obtained by some ¢xatives, two classical permeates may be
FEMSRE 685 29-8-00
useful : EDTA or EGTA at 1 mM and Triton X-100 at
0.1%. However, further development of more appropriate
¢xation and permeation methods to optimise cell labelling
for £ow cytometry is still required, especially when using
nucleic acid probes and quantifying the chemical compo-
sition of the cell.
2.2. Unicellular phototrophs
Flow cytometry was initially used for quantifying auto-
trophic picoplankton on the basis of their distinctive pig-
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ment £uorescence, orange for chlorophyll and red for the
cyanobacterium-speci¢c phycoerythrin [31^33], the speci¢c
detection of autotrophic microorganisms from abiotic par-
ticles being solved by their pigment signature. Algal pop-
ulations and cyanobacteria are routinely determined in
marine samples in this way and picoplanktonic prokary-
otes such as Prochlorococcus [32] and eukaryotes such as
Ostreococcus [34] have also been identi¢ed by their £ow
cytometric signature. Flow cytometric apparent quantita-
tive detection of cellular DNA in marine phytoplankton
by Pico Green and SYTOX Green is a way to approach
taxonomic and ecological implications in seawater [35].
2.3. Bacterioplankton
Non-pigmented prokaryotes are di¡erentiated from
abiotic particles by staining with speci¢c nucleic acid £uo-
rochromes. A frequent approach is to label with a DNA-
speci¢c £uorochrome (Hoechst 33342) at 0.5 Wg ml31 .
Hoechst staining has been shown to be more useful than
DAPI because the response provides a higher signal-to-
noise ratio and lower coe¤cient of variation according
to the protocol of Monger and Landry [36]. Comparisons
between several instruments have given similar results [37],
but the combination of UV excitation and DNA-speci¢c
£uorochromes remains problematic primarily due to: UV
source problems especially with mercury lamps, low quan-
tum e¤ciency and background responses especially when
phototrophic pigments are present.
The more recently developed £uorochromes, which can
be excited with the 488 nm line of an air-cooled argon ion
laser and result in a much higher quantum yield, show less
interference with phototrophic pigments and have im-
proved the analysis. Examples of these £uorochromes
are: SYTO 13 [38^40]; YOYO-1, YO-PRO-1 and Pico
Green [38,41]; TOTO and TO-PRO [42]; and the SYTO
and SYBR series [43]. Recent intercalibrations between
epi£uorescence and £ow cytometric counts have been pub-
lished, showing good correlations in coastal waters [27,44].
However, these dyes are not as DNA-speci¢c as Hoechst
33342 and DAPI, and may also label RNA (for a more
detailed discussion, see [10,38]). Consequently, the use of
these nucleic acid dyes may require further validation to
verify the circumstances in which the resulting £uorescence
may be associated with cellular DNA or RNA, for in-
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 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
stance by pretreatment with RNase and DNase digestion
[38,42]. However, such validations highlight drawbacks
such as high background noise, scatter changes, cell deg-
radation, formation of vesicles from cell membranes and
molecular aggregates [38]. One of the main obstacles when
analysing DNase- or RNase-treated bacteria by £ow cyto-
metry is the lack of positive, negative and intermediate
controls. In other words, it is not possible to assess the
amount of DNA or RNA degraded inside the cells by the
enzyme treatment. Of special interest is a recent analysis
on the high and low apparent DNA content in bacterio-
plankton in relation to its dead or alive status [44]. The
e¤ciency of ¢lters with which studies of bacterial activity
and size fractionation are performed may be di¡erent,
depending on the material the ¢lters are made of (poly-
carbonate, cellulose, aluminium oxide or glass ¢bre) and
must be assessed by £ow cytometry [25]. In recent years
speci¢c DNA and RNA double- or single-stranded £uo-
rochromes have been commercially available [11], but their
usefulness has not been assessed to date.
2.4. Protozoa and grazing
Flow cytometry in the enumeration of non-pigmented
protozoa is much more limited. Nano£agellates and cili-
ates usually occur at densities ranging from 102 to 103
ml31 and from 1 to 10 ml31 respectively in many aquatic
habitats. As pointed out in Section 1, low frequency events
require some form of pre-enrichment or previous concen-
tration. Speci¢c detection and quanti¢cation of non-pig-
mented protozoan populations present the following limi-
tations: insu¤ciency of the scatter value to adequately
di¡erentiate protozoa, low absolute densities and low pro-
portion related to the bacterial or algal populations.
Although many heterotrophic nano£agellates are around
2^10 Wm and ciliates range between 10 and 40 Wm, their
quanti¢cation simply by scatter is hindered by their usu-
ally low densities and the presence of large, elongated or
aggregated bacteria. However, their detection and quanti-
¢cation has been achieved by in situ hybridisation with
speci¢c £uorescent oligonucleotide probes [45,46]. Grazing
rates and the clearance e¡ects of protozoa on predated
bacteria or algae can also be assessed using several ap-
proaches: (i) prey accumulation inside the predator in
the case of phototrophs; (ii) prey increments in samples
previously passed through ¢lters of 0.8^2 Wm for chemo-
trophic prokaryotes ; (iii) reduction of the targets (grazed
bacteria) corrected with the bacterial production; (iv) re-
duction of the targets in ¢xed samples with the £uores-
cence-labelled bacteria ; and (v) prey elimination to follow
the consumption of picoplankton by ciliates and/or £ag-
ellates [47].
2.5. Viruses
Viruses are abundant, active components of aquatic
FEMSRE 685 29-8-00
433
ecosystems, but many di¤culties are encountered in their
enumeration, infectivity determination and classi¢cation.
Although several £ow cytometric techniques to count vi-
ruses have been published, they have not been adapted
for aquatic samples. The following £uorochromes and
techniques have been used to detect viruses in aquatic
systems : SYBR Green I in epi£uorescence microscopy
[48], YO-PRO in epi£uorescence microscopy [49] and ¢-
nally a £ow cytometric assessment using SYBR Green I
has been published [50]. Although this last application
requires some further development in instrument sensitiv-
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ity and labelling protocols to improve the discrimination
of viruses, this ¢rst attempt to enumerate viruses in natu-
ral waters by £ow cytometry is promising.
3. Cell size measurements and scatters
Flow cytometric cell sizing is mostly estimated from the
intensity of forward light scatter, which is used in prefer-
ence over the 90³ scatter because of its high signal inten-
sity but also because of its insensitivity to subcellular
structure. However, only in un¢xed cells is it possible to
obtain reliable values of scatter to be converted to size
values, since ¢xation usually reduces the scatter, refractive
index and also cell size [2]. Forward scatter is assumed to
be proportional to bacterial size [26,51^55]. However,
many authors have reported that a correlation between
narrow-angle light scatter and cell size is not observed
[56^58]. Recently the Rayleigh^Gans theory has been ap-
plied to predict biomass in dry weight terms from forward
scatter values [51,59]. Light scattering is a complex func-
tion of the cell size and also depends on the shape, struc-
ture, presence of inclusion bodies, chemical composition,
refractive index, angle of detection and on several instru-
ments (even belonging to the same model). All these fac-
tors may give signi¢cantly di¡erent scatter values from the
same sample, simply because of small changes in geometry
of the detection. We conclude that a linear or universal
correlation between bacterial size and scatter does not ex-
ist, but mathematical adjustments may be practical in
some speci¢c cases as for instance the linear correlation
between standardised scatter values and mean cell length
[26] or second-order relationships between scatter and bac-
terial diameter [60].
4. Determination of cell viability
A basic question that microbiologists have been asking
(and still ask) since the beginning of microbiology is
whether microorganisms are alive or dead. Surprisingly,
this question is not always easily answered and during
the last two decades a permanent controversy on this topic
has been going on [61^64]. This review does not intend to
further this debate. However, bacterial viable plate counts
Cyaan Magenta Geel Zwart
434 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
in aquatic systems are between 0.1 and 10% of the total
microscopic counts and new techniques are currently re-
quired to determine the viability and activity of such bac-
terioplankton populations. Cytometry and £uorescent
probes allow the di¡erentiation of stages far beyond the
classical microbiological de¢nition of cell growth. Most of
the existing techniques to assess viability or activity by
£ow cytometry have two limitations: lack of universality
and atypical responses when applied to environmental
water samples. This review describes some of the available
techniques that may provide information on the physio-
logical state of bacterioplankton and microbial cultures.
These techniques may be applied to microbial cultures
but are of limited use in natural aquatic samples and fur-
ther improvement is needed.
4.1. Membrane integrity
Membrane integrity demonstrates the protection of cell
constituents and its potential to generate gradients that
endow ability to live, but does not guarantee cell replica-
tion. It is also a way to discriminate bacteria from other
organic matter and debris. Cells with a damaged mem-
brane cannot sustain any electrochemical gradient and
are normally classi¢ed as dead cells. As their structures
are freely exposed to the environment they eventually de-
compose and consequently viability measurements should
be aimed at di¡erentiation of bacteria from other organic
matter and demonstrate a biological function. The high
ranking of membrane integrity measurements in the as-
sessment of physiological state is based on three argu-
ments: (i) cells that have lost membrane integrity cannot
maintain any of the electrochemical gradients necessary to
remain functional. Unless temporary permeability is delib-
erately caused, for example by electro- or chemoporation
for inserting genetic elements, such cells can be classi¢ed
as dead, since their cellular structures are exposed to the
environment, and will eventually decompose ; (ii) the pres-
ence of a membrane with selective permeability indicates
that the cytoplasm is still a separate entity from the envi-
ronment and therefore has the potential to give rise to
metabolism or proliferation. This can be critical in risk
assessment studies and also comes closer to a more appro-
priate viability de¢nition for viruses that do not show
direct proliferation or metabolism ; (iii) membrane integ-
rity measurement does not require any cell activity, thus it
is suitable for starved, dormant or injured cells that may
require reactivation or `resuscitation' to recover more
complex functions.
The presence of DNA in all bacteria makes this an ideal
staining target. If used alone it can be confusing as signals
can also be generated from other biological debris and
dead cells. The combination of DNA staining with the
test for a selectively permeable membrane surrounding
and protecting the DNA is a more robust approach. The
exclusion properties of membranes to the distinct stains
FEMSRE 685 29-8-00
can be tested by azide inhibition of extrusion pump cells
and by counterstaining with complementary stains as well
as cell sorting techniques [20,65]. Propidium iodide (PI)
may be chosen as the dye exclusion marker [66^68] and
can be combined with ethidium bromide as a supravital
DNA stain but the spectral di¡erentiation of the two
stains requires careful gating and controls. The more re-
cently introduced dyes from Molecular Probes [10] do not
allow a third colour application but give much brighter
signals. Other possible combinations are DAPI or Hoechst
33342 with PI or TO-PRO 3 with PI, but additional light
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sources are required [69,70].
4.2. Biosynthesis
Biosynthesis is the most stringent measure towards com-
plete cell functionality as it requires the highest complexity
in cellular metabolism. It has also been the ¢rst parameter
to which £ow cytometry was applied on microorganisms
in the form of nucleic acid synthesis and content [71,72]. It
has been used for monitoring toxicity and antibiotic sus-
ceptibility [73,74], and is also used for process control and
monitoring in microbial biotechnology [75,76]. It has been
established in environmental microbiology under the con-
cept of direct viable count discussed in Section 4.3. In
aquatic microbiology this approach may be especially in-
dicated during bloom periods, but is of limited use during
the starvation phases.
4.3. Direct viable count
The most stringent measurement for biosynthesis is the
growth of bacteria in volume. The cell enlargement assay
is commonly called the direct viable count (DVC) assay
and was initially developed by Kogure et al. in 1979 [77].
Antibiotics inhibiting cell division such as quinolones and
nutrients are added to the sample, which is then incubated
until growth can be detected. The procedure leads to the
development of elongated cells, which result from the
growth of metabolically active cells that are unable to
divide in the presence of quinolones. It is often considered
that enlarged cells have retained the potential to generate
energy and coordinate macromolecular synthesis. This as-
say su¡ers from some limitations such as antibiotic resis-
tance, the selectivity of the added nutrients and the possi-
ble induction of the SOS response by quinolones [78]. The
presence of some antibiotic-resistant species may result in
the growth of some cells whereas the growth and thus the
elongation of very sensitive cells may be inhibited. This
assay has been improved for applications in natural waters
in combination with £ow cytometry analysis [76] by using
a mixture of antibiotics, obtaining a high proportion of
elongated cells (up to 40%) while culturable counts were
below 3%.
The DVC assay generally requires preliminary optimi-
sation of the range of incubation times and antibiotic con-
Cyaan Magenta Geel Zwart
 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
centrations in environmental studies. The DVC assay has
not been used widely in the natural environment, this may
be because elongated cells have a growing potential but
not real activity within the natural communities at the
time of sampling. This assay is more generally applied in
laboratory studies working with a given species for which
antibiotic sensitivity can be easily optimised. The DVC
method can be advantageously combined with cell sorting
to provide further information such as identi¢cation by
molecular techniques of the cells with the ability to elon-
gate.
4.4. Reproduction
Reproductive growth as the most stringent proof of vi-
ability requires metabolic activity and in turn membrane
integrity. In many cases this function cannot be detected
due to irreversible cell damage or fastidious growth con-
ditions. This type of assay can be used to demonstrate
antibiotic susceptibility within 2 h from sampling [79]. An-
other way to detect replication is by cell tracking, where
covalently labelled cells halve their £uorescence upon cell
division [80]. Such labelled cells can also be introduced
into complex environments to follow their progress. It is
also possible to compare competitive growth in cultures of
two species with distinct scatter pro¢les [81]. The same
approach has been taken to study cell^cell or host^patho-
gen interactions [82]. However, there is evidence that some
labels can a¡ect the growth properties of cells.
5. Metabolic activity measurements
Detection of metabolic activity is less stringent than
culturability but suggests the absence of cell death. Whilst
it does not warrant reproductive growth, this function
may be su¤cient to detect bacterial e¡ects such as biode-
gradation, organic matter transformation or accumulation
of toxins. In cases of injury, dormancy or extreme starva-
tion, metabolic functions might be below the detection
limit. Metabolic activity does not warrant reproductive
growth if evaluated by energy-independent methods such
as esterase or dehydrogenase activity, but it is the next
best criterion, especially if performed by energy-dependent
methods, such as membrane potential.
5.1. Membrane potential
This is an obvious candidate for energy-dependent mea-
surements, and is widely used but sometimes abused
[66,67,83^87]. The principles of this measurement are
best described in the book by Howard Shapiro [2], also
holding a patent on its application to the measurement of
bacteria [88]. Commonly used probes for estimating mem-
brane potential are charged lipophilic dyes, such as carbo-
cyanines, rhodamine 123 or oxonols. These dyes, which
FEMSRE 685 29-8-00
435
can readily cross the membrane and therefore follow the
membrane potential according to the Nernst equation, are
called distributional probes. However, care must be taken
as the £uorescence intensity depends not only on mem-
brane potential, but also on the number of lipophilic bind-
ing sites within the cell, the volume of dye concentration
and the number of binding sites in solution. Other com-
plications arise from active dye extrusion and the need to
make the outer cell membrane permeable [89]. Recently, a
ratiometric method for membrane potential measurements
was described that overcomes some of these limitations
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[90] ; although this method can be applied well to Gram-
positive bacteria, at its present stage, it is of limited use for
Gram-negatives. However, the lack of appropriate con-
trols and validations for mixed populations may explain
why no method has been successfully applied to natural
communities and at best, they were applied to study spe-
ci¢c species in laboratory microcosms.
5.2. Esterase activity
While the synthesis of enzymes requires energy, en-
zyme^substrate reactions usually do not. Esterase activity
is probably the most common form of metabolic activity
measurement used [23,28,91^95]. It can be classi¢ed as
energy-independent as the enzyme will remain functional
in cells as long as it is retained by the intact membrane
and protected from the environment. Our own unpub-
lished studies have shown that H2 O2 -killed cells maintain
esterase activity for over a week (Nebe-von Caron, person-
al communication). Colin Fricker's team at Thames Water
in the UK has shown the same for Q-radiated cells, where
esterase activity could be detected for over 2 weeks thus
indicating its long persistence (Nebe-von Caron, personal
communication). Esterase activity measurements are based
on non-£uorescent compounds that become £uorescent
when cleaved by the enzyme inside the cell. In addition,
the molecules increase in charge and polarity, causing dye
retention, which indicates membrane integrity. A number
of esterase substrates and their applicability to several or-
ganisms have been compared [96]. Apart from di¤culties
with dye loading, staining problems arise from dye extru-
sion [18,19,97] as mentioned above or from the pH depen-
dence of £uorescent signals. The use of dichlorocarboxy-
£uorescein diacetate [98] or calcein-acetomethyl ester [28]
may overcome this drawback. Since esterase activity
probes do not easily enter into Gram-negatives, or they
are just removed actively, this application in water samples
is uncertain unless adequate permeation methods are de-
veloped.
Dye extrusion can be overcome either by using dyes that
cross-link inside the cell [79] or by using appropriate met-
abolic inhibitors. Thus, staining procedures such as the
Chemchrome kit by Chemunex (Maisons-Alford, France)
can achieve a good staining for a large spectrum of patho-
gens [99]. This protocol has been used in combination with
Cyaan Magenta Geel Zwart
436 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
the Chem£ow or in the ChemScan system in pharmaceut-
ical industries for real-time quanti¢cation of active cells in
processed water [100].
5.3. Dehydrogenase activity
Tetrazolium salt reduction has been used as an indicator
of respiratory activity in non-culturable cells. These re-
agents capture electrons from oxidative metabolism and
produce a localised formazan deposit which can be de-
tected by light microscopy. The £uorogenic tetrazolium
salts such as 5-cyano-2,3-ditolyl tetrazolium chloride
(CTC) have been increasingly used during recent years
as a measure of cell-speci¢c metabolic activity in bacteria
with and without £ow cytometry [68,101^104] since it acts
as an electron acceptor in the electron transport system of
bacterial cells. As such, it is taken to be a direct measure
of abundance of those cells that are actively engaged in
respiration [104]. The CTC assay remains controversial
since the interpretation of results is often di¤cult. For
instance, the number of respiring bacteria is often lower
than viable counts determined by the DVC method [105]
or by cultural methods [106]. Therefore, there is clear evi-
dence that the CTC assay cannot be considered a viability
assay. Furthermore, respiring cell counts are also often
lower than those obtained by other methods used for ac-
tivity assessment such as microautoradiography or the de-
tection of esterase activity and membrane potential
[104,107]. Discrepancies are due to the fact that each assay
addresses di¡erent cellular functions and has speci¢c lim-
itations. However, the CTC assay is increasingly used in
microbial ecology because active cell counts seem to vary
in an ecologically interpretable manner [108^111]. The re-
cent application of £ow cytometry for the detection of
CTC+ cells has resulted in a signi¢cant improvement of
the method [111] since £ow cytometry is less sensitive to
the fading of CTC+ cells which represents the main limi-
tation for microscopic observation and counting. Thus,
respiring cell counts should be used to enumerate the
most active cells within a natural community but further
studies are needed to better understand what the results
mean. For this reason, CTC counts should be di¡erenti-
ated from active cell counts determined by other methods.
We suggest the use of `actively growing cells' or `active
and productive cells' when counts are used to determine
speci¢c growth rates from production measurements. In
all cases, comparison of results is very di¤cult and a clear
description of the staining procedure and instrumentation
used for the detection of CTC+ cells is important with
regard to the diversity of factors in£uencing the results.
6. Di¡erentiation based on probes
Aquatic autochthonous or allochthonous microbial spe-
cies may be present at low concentrations and thus detec-
FEMSRE 685 29-8-00
tion methods in water require high quantitative sensitivity.
The methods must have a very low detection limit and
should be applicable to large volumes of water samples,
as speci¢ed in water quality guidelines or standards. In
addition to high sensitivity, high speci¢city for the enu-
meration of speci¢c microorganisms ranges is also re-
quired. Such detection methods should also be rapid, sim-
ple to perform and cost-e¡ective. This section proposes
several £uorescence strategies depending on the £uoro-
chromes and sensitivity required for analysis, and de-
scribes their limitations. Immunological and molecular
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methods show the extraordinary advantages of detecting
and identifying bacteria without a culture step. Although
these methods still require further development, their ap-
plications in aquatic microbiology are promising.
6.1. Di¡erentiation by `non-speci¢c' probes
One of the basic classical di¡erentiation criteria is Gram
staining. This can be achieved by using stains impermeable
to the outer membrane in the presence and absence of
EDTA or through labelling with £uorescent lectins
[112,113]. A £uorescent Gram stain for un¢xed organisms
describes the use of hexidium iodide (HI) and SYTO 9
[113] that is also available as a mixture from Molecular
Probes. Fig. 1 shows the application of this kit and also
illustrates the phenomena of energy transfer between dyes
that are mentioned in Section 6.4. Although this Gram
stain assessment is correctly applied to axenic cultures
and even to Gram-variable bacteria, its potential useful-
ness in aquatic microbiology remains to be assessed. For
many species, this technique works well when applied to
axenic cultures, however, the method does not work with
some Vibrio and Pseudomonas species, since these Gram-
negative bacteria are characterised as Gram-positive bac-
teria when this £uorescent probe-based method is applied
(Lebaron, personal communication).
A number of attempts have been made to di¡erentiate
bacteria based on relatively non-speci¢c measurements
such as nucleic acid and light scatter. In fact, one of the
early publications demonstrated separation of three spe-
cies based on base pair composition [64]. Improved £ow
cytometric detection and di¡erentiation of microbial par-
ticles can be achieved by using additional parameters such
as protein and other cellular molecules, multiple stains and
neural networks [114]. However, in the light of the com-
plexity and heterogeneity of natural samples and the ten-
dency of some organisms to aggregate under stress or
become ¢lamentous at low temperatures, the application
of these methods is limited. The use of speci¢c markers
such as antibodies, rRNA or genetic probes is more ap-
propriate in this context.
6.2. Antibodies
Immuno£uorescence allows the visualisation of bacteria
Cyaan Magenta Geel Zwart
 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
Fig. 1. Energy transfer between SYTO 9 and hexidium iodide (HI) as
obtained using the BacLight Gram Stain kit (Molecular Probes).
Although Gram-negatives should not take up HI, Vibrio metschnikovii
living cells are stained with both dyes combined or not. The green £uo-
rescence from SYTO 9-stained bacteria is recorded in the FL1 channel
and the orange £uorescence from HI-stained cells is recorded in the
FL2 channel. A gate with reference beads of 1 Wm is included. Note
that the double stain application results in a decrease in green £uores-
cence and in the increase in orange £uorescence as a consequence of the
energy transfer between dyes. Analyses were performed with a FacsCali-
bur £ow cytometer (Benton Dickinson, San Jose, CA, USA) equipped
with an air-cooled argon laser (488 nm, 155 mW). Green and red emis-
sions were measured at 530 and 586 nm, respectively.
at the single-cell level under non-destructive conditions
and independently of the cell growth rate. It can be ap-
plied to environmental samples [115] but requires prior
isolation of the target organism to generate antibodies.
Antibody molecules can be made £uorescent by covalently
attaching them to £uorescent organic compounds such as
£uorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), Texas Red, phycobiliproteins,
the recently developed cyanine dyes such as the indocar-
bocyanine dyes (CY3, CY5 and CY7), and more recently
the ALEXA dyes. FITC is the most frequently used dye,
because it can be excited at the 488 nm line of an argon
laser, is easily conjugated to antibodies, lectins and other
compounds, has a relatively high £uorescence, is water-
soluble, and has a long record of successful use.
Staining methods can be divided into direct staining
(direct labelling of the bacteria with speci¢c antibodies)
and indirect staining consisting of non-£uorescent mono-
clonal or polyclonal primary antibodies followed by £uo-
rescent polyclonal secondary antibodies directed against
the non-£uorescent antibody. One of the advantages of
indirect staining is that it is not necessary to make a £uo-
rescent antibody for each antigen of interest. When neces-
sary, reporter molecules can also be attached to primary
or secondary antibody molecules to amplify the £uores-
cence signal [116]. Direct staining with FITC-labelled anti-
bodies has been reported to have limitations [117] but
FEMSRE 685 29-8-00 Cyaan Magenta Geel Zwart
437
reagents and instruments have undergone substantial im-
provements. Magnetic separation technology can also be
used to make the assays faster and more speci¢c, and
overcome the presence of non-cellular particles [118^
120]. Apart from the lack of existing antibodies, their lim-
ited application to natural aquatic environments is also
due to the rapid development of molecular probes based
on complementary DNA sequences targeted against
rRNA and their increased use in the detection and classi-
¢cation of bacteria in the environment.
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6.3. Nucleic acid probes
Fluorescent in situ hybridisation (FISH) is a recognised
tool for phylogenetic identi¢cation of bacteria at the indi-
vidual cell level. This method is based on the hybridisation
of ¢xed cells with oligonucleotide probes, generally tar-
geted to rRNA. The rRNA is the major cellular RNA
component (more than 80%). It is unclear whether the
correlation between growth rates and rRNA content de-
termined for cell suspensions also holds for natural com-
munities and there is evidence that the classic correlation
does not apply to carbon-starved cells [121]. Ribosomes
are degraded during starvation [121^124], and it has been
shown that the RNA content of Salmonella typhimurium
cells starved in arti¢cial seawater decreases sharply during
the ¢rst days of starvation concomitantly with the de-
crease of culturability and then stabilises until the disrup-
tion of membrane integrity [61]. Similarly, Fukui et al.
[125] have shown that within a starved population of De-
sulfobacter, some cells are able to maintain intracellular
rRNA and they suggested that a fraction of rRNA is
more resistant to starvation. Thus, although their detec-
tion may be limited by the sensitivity of our methods and
instruments, starved cells are able to maintain enough ri-
bosomes and may remain detectable at any time of their
life cycle. However, the RNA content of individual cells
detected by £uorescent probes cannot be considered a re-
liable indicator of cellular viability. Several dyes have been
proposed for RNA analysis but most lack speci¢city and a
DNase treatment is generally required before analysis.
Fluorescent rRNA probes for FISH labelling are com-
monly used in microcosms and in situ studies with or
without signal ampli¢cation techniques [116].
Whole-cell hybridisation of bacterial cells with £uores-
cently monolabelled rRNA-targeted oligonucleotide
probes has widespread applications in several areas of mi-
crobiology [116]. The standard probes are rRNA-targeted
oligonucleotides with a single £uorescent dye molecule
attached to the 5P end via a linker molecule. The most
frequently used dyes are similar to those reported for im-
muno£uorescence techniques. However, £uorescently
monolabelled probes cannot be used for the detection of
cells containing a small number of ribosomes such as those
encountered in most aquatic systems [123,126]. Although
several signal ampli¢cation methods have been developed
438 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
based on indirect assays, their application to natural bac-
teria remains limited [123,126]. For the probe to enter a
bacterium, paraformaldehyde-¢xed cells are permeabilised
with a cell wall-degrading enzyme such as lysozyme. This
is essential when large probes labelled with high-molecu-
lar-mass enzymes such as horseradish peroxidase are used.
Most of the procedures based on reporter molecules work
well on gelatin-coated slides after a permeabilisation step
but cannot be applied after concentration of cells on a
¢lter or in suspension because the signal is signi¢cantly
reduced or lost, being then not applicable to £ow cytome-
try. Moreover, permeation procedures are often species-
dependent and cannot be applied to complex communities.
The new generation of epi£uorescence microscopes
equipped with a £uorite lens combined with carbocyanine
dyes such as CY5 and an infrared CCD (charge-coupled
device) camera is very promising and has increased the
£uorescence detection limits (Lebaron, unpublished re-
sults). When nucleic acid probes are applied in clinical
isolates or axenic cultures the validation of the probing
is inherent in the use of single, culturable and well-known
bacteria. However, when nucleic acid probes are applied in
aquatic samples, which contain heterogeneous popula-
tions, unknown and non-culturable species and abiotic
particles, the validation of the probing is a basic need
that is usually not performed.
6.4. Multiparametric analysis
A usual goal in £ow cytometry is to select two or more
probes with contrasting wavelengths for the measurement
of multiple parameters. Contrasting wavelengths means a
combination of excitation and emission wavelengths which
allows discrimination of each probe in the presence of the
others, thus allowing the simultaneous use of four anti-
bodies [127] or the combination of immuno£uorescence
with rRNA probes [128]. Physiological or taxonomic
probes are often used in double staining procedures and
include a nucleic acid dye for the quanti¢cation of total
bacteria. With antibody £uorescence for example, it is es-
sential to ensure that only antibody £uorescent events as-
sociated with nucleic acid are analysed [79,129,130].
The combination of both physiological and taxonomic
information in a single assay is probably one of the main
goals in the development of rapid methods. Fluorescent
antibody staining has also been successfully combined
with the DVC method (see Section 4.3) for the detection
of Vibrio cholerae [131,132], Pseudomonas £uorescens [133]
and Salmonella [134,135]. FISH techniques have also been
combined with the DVC procedure and the intracellular
RNA content was used as a criterion for viability [136].
Similarly, Kalmbach and co-workers [137] have combined
FISH techniques with in situ reduction of CTC to analyse
the development of bacteria during the formation of
drinking water bio¢lms. Enzyme activity has also been
combined with antibody £uorescence in image [138] and
FEMSRE 685 29-8-00
£ow cytometric studies [139] and also with membrane in-
tegrity [58]. Considering the limitations of metabolic activ-
ity measurements and the crucial importance of membrane
integrity, a combination of total cell staining with mem-
brane integrity and immuno£uorescence provides a basic
combination for a rapid detection method. The whole pro-
cess can be realistically accomplished in less than 45 min
including sample preparation (sonication) and two anti-
body labelling steps. Fig. 2 gives an example of such a
multiparametric analysis and also shows how cell sorting
can be used to verify staining speci¢city or to collect cells
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for further molecular analysis.
The selection of £uorophores in multiple-staining pro-
cedures may take into account molecular interactions
which lead to quenching. Quenching is a reduction in £uo-
rescence intensity which can be due to displacement by
other dye molecules, i.e. modi¢cations in the excited states
of the £uorophores resulting in energy dissipation by non-
radiative transitions. These physico-chemical changes may
be caused by the interaction between £uorophores. Such
an interaction can also result in energy transfer between
£uorophores. This has been exploited for many years by
using mythramicin with ethidium bromide [74,140] and
also by combining DAPI or Hoechst with PI [70]. If per-
meable bacterial cells are labelled with SYTO 9 and HI, a
decrease in the green £uorescence emission of SYTO 9 and
in increase of the orange £uorescence of HI is observed
compared to staining with HI or SYTO 9 only (see Fig. 1).
7. Assessment of bacterial starvation
Allochthonous bacteria and most enteric and pathogen-
ic microorganisms are released directly or through waste
waters into rivers and coastal areas. In the aquatic ecosys-
tem, their survival is a¡ected by complex environmental
stresses and killing agents, which trigger cellular responses
that are still poorly understood. The survival of bacteria in
aquatic ecosystems has been the subject of many studies
[57,58,61,141] and £ow cytometry has also provided new
insights, especially into the evaluation of population het-
erogeneity, DNA changes and membrane potential varia-
tions. The multicolour staining used to detect transitional
energetic states in cultured organisms [23] may not be ap-
plicable to aquatic samples, but shows similar sub-group-
ings prior of cell integrity as postulated by Joux et al.
[142,143]. The actual challenge in microbial ecology is to
in situ determine the heterogeneity of physiological states
within speci¢c populations. This is due to (i) the quanti-
tative limit of £ow cytometry and epi£uorescence micros-
copy for the detection of rare events and (ii) the di¤culty
of combining taxonomic and physiological probes in the
same assay. For instance, FISH techniques require the
permeability of cell membranes and therefore, physiolog-
ical probes cannot be applied. In contrast, speci¢c anti-
bodies can be combined with physiological probes but
Cyaan Magenta Geel Zwart
 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448 439

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Fig. 2. Antibody-based analysis of dental plaque and its veri¢cation by cell sorting. Dental plaque was collected in Dulbecco's bu¡ered saline containing
0.05% Tween 20 and 0.1% sodium azide, spiked with reference beads, sonicated and labelled with antibody against Streptococcus sanguis 4715 followed
by rabbit-anti mouse FITC F(ab)2. Propidium iodide (PI) and ethidium bromide (EB) were added to the sample, EB in order to stain all DNA-contain-
ing events orange and red, PI to quench the orange EB £uorescence in the cells that have lost membrane integrity. Cells were brie£y sonicated after
staining to minimise the risk of aggregation. Bacteria were sorted based on membrane integrity and the absence or presence of antibody label. The sort
pattern re£ects two rows of three and two cells per location with single cells on the 30 locations on the right side of the agar plate. Selected colonies
were identi¢ed by API based on morphology and haemolytic capacity. Of the antibody-positive cells, 99.4% were correctly identi¢ed and 3.6% appeared
false-negative due to the sonication treatment required to disaggregate the antibody-labelled cells.

their speci¢city is sometimes low and limited to those spe-
cies for which antigens have been characterised. Another
approach is the use of green £uorescent protein to tag
bacteria and to monitor their gene expression under envi-
ronmental conditions [144^146].
8. Biodegradation studies
Flow cytometry represents a novel approach to study
biodegradation processes in natural waters, since repro-
duction, changes in microbial size and metabolic activity,

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440 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
together with the monitoring of the microbial consortia
associated with the degradation of a particular compound
may be carried out simultaneously. A novel application of
the tetrazolium salt CTC coupled with the nucleic acid
stain SYTO 13 in the determination of total counts and
number of respiring bacterioplankton in natural seawater
has been reported [29]. During linear alkylbenzene sulfo-
nate (the most widely used surfactant in the world) bio-
degradation in seawater microcosms, two distinct pools of
cells could be di¡erentiated by double SYTO 13^CTC
stain : one was inactive (with no measurable electron trans-
port activity) while the other comprised metabolically ac-
tive cells. This double-stain procedure allowed the deter-
mination of correlation between the total number of cells
and the proportion of these, which were metabolically
active and the correlation between nucleic acid staining
and CTC reduction. Monitoring the formation and evolu-
tion of bacterial consortia during degradation of surfac-
tants in coastal water has also been lately approached by
£ow cytometry [101].
9. Solid phase cytometry
Solid phase cytometry and laser scanning cytometry are
terminologies which overlap and can be confused, as it is
di¤cult to fully clarify the conceptual di¡erences and their
applications, especially in the context of solid state tech-
nology. These terminologies describe a technology where-
by cells are analysed or counted while being ¢xed on a
solid support such as a glass slide or a ¢lter membrane.
Instead of moving the cells through a stationary laser
beam for excitation as in £ow cytometry, the laser beam
is moved over the immobilised cells on their support. Solid
phase cytometry and laser scanning cytometry should not
be confused with image cytometry systems, which may
also work on a solid support, or with solid state technol-
ogy. For a recent review of the potential applications of
solid or scanning cytometry see Darynkiewicz et al. [147];
however, few applications have been developed in the
aquatic ¢eld at present.
Two commercial instruments are based on this concept.
The ¢rst is a microscope-based system generally called a
laser scanning cytometer, developed by Kamentsky and
Kamentsky [148] and commercially known as LSC, Com-
pucyte, Cambridge, MA, USA. A specimen placed on a
computer-controlled stage of a microscope is scanned by a
laser beam of a resonant galvanometer with a 2.5, 5 or 10
Wm spot size, depending on the magni¢cation used. For-
ward angle scatter and multiple wavelength £uorescence
collected at a 180³ angle are measured, and cells are de-
tected using thresholds on each of the measurements. Data
are displayed in similar fashion to £ow cytometric 2D dot
plots and include, among other values, peak and integral
from each cell, the time of measurement and the exact x/y
location on the slide. The events inside a region of interest
FEMSRE 685 29-8-00
can therefore be directly located to obtain high-resolution
images for further image analysis (virtual sorting) as the
system is also equipped with a camera system and stand-
ard epi£uorescence. This research instrument is normally
equipped with dual laser excitation and four photomulti-
plier detectors and a solid state detector. The instrument
has been used for bacterial detection and growth studies as
well as ribosomal probe work (Nebe-von Caron, personal
communication) but was originally designed for the path-
ology^clinical market and optimised for use with mamma-
lian cells. Using the instrument in high-resolution mode
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increases the scanning time per area and therefore reduces
sample throughput.
The second instrument is commercialised as ChemScan
by Chemunex France. This system was developed primar-
ily for rapid detection in industrial and environmental
microbiology for ¢lterable products, and is therefore opti-
mised for microbiological applications with standardised
protocols. Particles are concentrated by membrane ¢ltra-
tion and £uorescently labelled by a proprietary method
before being transferred to the analyser which then laser
scans the surface of the membrane and detects £uorescent
signals via photomultipliers. Using innovative digital sig-
nal processing, the system is able to di¡erentiate between
labelled microorganisms and auto£uorescent particles
[149]. This scanning procedure takes only a few minutes
and ensures a fully overlapping scan of the entire surface
of the ¢lter membrane. Thus a single cell present on the
membrane can be detected by the multiple detection chan-
nels and the position of each detected event is displayed.
This procedure solves quanti¢cation of the cells in case of
rare events if samples can be concentrated by ¢ltration.
The ChemScan sample holder with the membrane in place
can be transferred to a motorised stage driven by the sys-
tem and mounted on an epi£uorescence microscope, which
also allows an immediate visual control and con¢rmation
of the results. The ChemScan system has been successfully
used for the detection of viable microorganisms in potable
and process waters [145,149,150], and for speci¢c detection
of Cryptosporidium and Giardia cysts in river and drinking
waters [151].
10. Cell sorting
In recent years, the number of applications for separat-
ing and analysing bacterial types has increased enormously
as a consequence of the improvement in available sorting
technology. Because this is a relatively new area of £ow
cytometry applications, the limits do not lie in what has
been accomplished or not, but rather in what has yet to be
tried or explored with the existing instrumentation. The
basic sorting principles are based on (in order of sorting
speed) either the destruction of unwanted cells in a £ow
path (zapper), physical deposition of a cell contained in a
droplet (droplet sorter) or the mechanical change of a
Cyaan Magenta Geel Zwart
 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448 441

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Fig. 3. In this ¢gure we illustrate how to assess the percentage of viable bacteria that are present in a prede¢ned cytometric population. Plate counts
were obtained from natural (A) or incubated (B) samples or from sorted bacteria of a prede¢ned cytometric gates. A is a freshly collected coastal water
sample and B is the same sample previously incubated on a shaker (100 rpm) with of 5 mg l31 of tryptic soy broth at 20³C during 4 days. R1, R2, R3,
R4 and R5 are the de¢ned gates used in sorting operations. The percentages of viable or culturable bacteria (2) were calculated according the total
counts (1) obtained by £ow cytometry and plate counts. The sorted population from a de¢ned gate was dropped on agar marine plates on 100 Wl of
NaCl 0.9% (freshly deposited on the plate surface just before the sort) and incubated for 7 days at 20³C. SSC = 90³ side scatter; FALS = 1.5^19³ for-
ward light scatter (3). Cells were sorted with a Coulter Epics ELITE (Coulter Corp., Miami, FL, USA) £ow cytometer, using a de£ected droplet elec-
trostatic system. Calibrator beads of 1 Wm are detected in the circular gate and illustrate that after the incubation of the sample, a part of the popula-
tion increased in size. Also the percentage of viable bacteria in the ¢ve de¢ned gates is higher in the incubated sample and its gates.

£owpath of cells in a liquid stream (switch sorter). Classi-
cal image cytometry as well as solid phase cytometry allow
cell discrimination based on cell location. The latter can be
of particular importance when examining cells needing
complex environments for growth. Droplet sorting is the
commonest form of cell separation. It involves the use of a
£ow cell vibrating at high frequencies (usually 30^50 kHz)
for the generation of uniform droplets from the liquid
stream containing the cells. Sorting can be used either to
deposit single cells (cloning mode) as seen in Fig. 2, or to
collect high numbers of cells with similar properties (en-
richment mode). Flow cytometric sorting application for
microbial aquatic samples has an additional drawback
since natural water contains a high heterogeneity of biotic
and non-biotic particles most of which are unknown.
10.1. Common applications
Fluorescent activated cell sorting has been used for
some time in the ¢eld of microbiology for: (i) determina-
tion of culturability; (ii) isolation of cellular clones or
prede¢ned subpopulations; (iii) molecular and immuno-
logical detection ; and (iv) selection of genetically altered
or mutated bacteria. Fig. 2 gives an example of cell sorting
from a bio¢lm (dental plaque) to assess reproductive
growth and isolated strains. Fig. 3 shows an example of
cell sorting to assess culturability of light scatter based
clustering from marine water samples. Probably the ear-
liest applications in microbiology were the puri¢cation of
spores by £uorescent activated cell sorting [152]. Another
use is the selection of mutants [153^160], which is still
routinely used in the pharmaceutical industry today for
strain improvement (Nebe-von Caron, personal communi-
cation). Cell sorting based on the expression of £uores-
cently labelled lectin or antibody is the next commonest
use [161^164]; Cryptosporidium detection [165] is probably
the most established application. The use of cell sorting to
evaluate culturability [23,65] has led to an improved
understanding and interpretation of physiological states
and staining methods. Sorting can also be used to study
correlations between bacterial size and activity [166].
10.2. Cell sorting and molecular microbial ecology
Molecular biological techniques are now routinely used
in microbial ecology. The nucleic acid techniques o¡er
approaches to questions that were previously di¤cult or
impossible to answer in microbial ecology. Information on
the identity of species can be obtained from natural pop-
ulations that cannot be identi¢ed or cultured (for an over-
view see [167]). However, most of these techniques are
destructive and cannot be applied at the cellular level.
Hybridisation with rRNA-targeted oligonucleotide probes
can be used to identify individual bacterial cells in natural
ecosystems [116,128,168]. However, as most aquatic envi-
ronments are oligotrophic systems, the detection sensitiv-
ity of £uorescent oligonucleotides is often limited by low
ribosome content or restricted cell accessibility.
Although molecular techniques provide information on
the taxonomic diversity of natural communities, a straight-

FEMSRE 685 29-8-00 Cyaan Magenta Geel Zwart

442 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
forward method that reliably indicates what species are
viable or active at the time of sampling would be of great
help and most useful in aquatic microbial ecology. At
present, both physiological and taxonomic information
at the cellular level can be obtained by combining cell
sorting and molecular techniques. Porter and collabora-
tors [152] for example sorted antibody-stained cells, and
Wallner et al. [128] sorted cells stained with rRNA oligo-
nucleotide probes for such purposes.
Cell sorting also allows the isolation of cells depending
on their physiological state as determined by physiological
measurements. The principal assays that can be applied to
natural communities as positive correlates of cell function
are the DVC and CTC assays, respectively. Active cells, as
judged by these methods, can be sorted and subsequently
identi¢ed by using molecular biological techniques such as
denaturing gradient gel electrophoresis (DGGE) (P. Leb-
aron, personal communication). Although this approach
cannot be used routinely to analyse the diversity of species
present in the fraction of viable or active cells, it has been
very useful to investigate the heterogeneity of the physio-
logical state of given species in natural environments. It is
also of great interest to examine the presence of active but
non-culturable cells of pathogenic species. Since cell sort-
ing is done in open compartments and subjected to micro-
bial contamination from the environment, a routine ster-
ility control is needed. Three methods may be used to
assess the reproductive growth of sorted cells and the ster-
ility of the sorting process. The ¢rst consists of directly
sorting cells for reproductive testing or sterile beads for
sterility assessment in microtitre wells and then transfer-
ring and streaking onto agar plates. The second is to sort
cells or beads on agar plates and streaking before incuba-
tion. The third system consists of sorting 42 single cells in
a 6U7 matrix directly onto agar TSA plates.
11. Controls and validations
Standardisation, calibration, control and validation pro-
vide consistency and reliability in £ow cytometric analysis.
These four actions are especially needed in aquatic and
environmental studies since aquatic ecosystems are com-
plex, heterogeneous, very variable and poorly known. An
excellent review has been published on the basic de¢ni-
tions, terminology, materials and proceedings for stand-
ardisation, calibration and control [169]. Instruments are
unable to control reagents, data analysis and samples,
which have to be properly prepared, maintained and
used. Urgent attention is currently required to obtain ad-
equate cell biology validations. A few examples of these
validations are the following: (i) in order to estimate cell
size by scatter, cells of known di¡erent sizes are needed;
(ii) to approach genome size or DNA or RNA content, the
classical calibration plots with pure DNA or RNA are
insu¤cient, because the topology and accessibility of the
FEMSRE 685 29-8-00
molecules may only be approached by using several types
of bacteria with known content of DNA or RNA. The
same should be valid for the quanti¢cation of protein or
lipid content ; (iii) ¢nally, when assessing general metabolic
or speci¢c enzyme activities, cells with well-known activity
need to be used as ¢nal validation.
In the case of sorting applications the needed controls
and validation are more numerous and complex since the
following information is needed for conclusive analysis:
(i) sorting e¤ciency for calibrators and also cells; (ii) con-
trols for biological contamination; (iii) controls for molec-
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ular contamination. The last two steps are crucial for sort-
ing applications in reproductive and molecular assays. A
basic requirement for sorting is to establish and verify the
sterility and alignment of the cytometer. Technical solu-
tions for these two aspects have been proposed [170].
However, if the sorting process is long enough, sterility
and alignment/recovery need to be checked at intervals
and at the end of the sorting process. The initial require-
ments for sorting microorganisms are: (i) the preparation
of the cell sorter to eliminate and control microbial con-
tamination introduced into the system by the sheath £uid
and the environment and (ii) to reduce the noise from
small particles in the £uid or even in the sample.
The most convenient positive recovery or alignment
control is to sort single cells from an early stationary cul-
ture directly onto agar plates using a cloning system. Re-
covery should be greater than 98%, and ideally 100%.
Pipetting from solutions of sorted cells tends to lead to
complications due to the high possibility of losing cells
that stick to surfaces, especially with low cell numbers.
For an instant check of the alignment it is advisable to
sort £uorescent beads directly onto glass slides (about 20
per location) as it is too late to identify a misalignment by
lack of plate recovery the following day. Sorting sterility
can be tested by sorting heat-killed bacteria or sterile
beads in a similar fashion. Stringency of the test can be
improved by carrying out the most probable number
counts on sorting populations, e.g. sorting plates with
1000, 100, 10 and 1 cell per location either on Petri dishes
or in 96-well plates with recovery medium. The length of
the sort run is also critical as it increases the likelihood of
contamination. Contamination from the sheath liquid can
be easily avoided by the incorporation of a disposable in-
line sheath ¢lter cartridge close to the £ow cell. Other
contamination sources may be stains, which can be ster-
ile-¢ltered by centrifugation through inert ¢lter mem-
branes (for example Whatman Anapore ¢lter tubes).
When sorting for molecular analysis the requirements for
all liquids used become even more stringent to avoid con-
taminating nucleotides. Therefore, the entire liquid han-
dling system requires thorough cleaning procedures.
The initial requirements for sorting microorganisms are:
(i) the preparation of the cell sorter to eliminate and con-
trol microbial contamination introduced into the system
by the sheath £uid and the environment ; (ii) to reduce the
Cyaan Magenta Geel Zwart
 J. Vives-Rego et al. / FEMS Microbiology Reviews 24 (2000) 429^448
noise from small particles in the £uid or even in the sam-
ple. Recovery should be estimated using immunocheck
calibration beads (Coulter Corporation, Florida). Re-
peated sorts of 20 £uorescent beads were done on a slide
and checked with an epi£uorescence microscope. The
mean recovery obtained with beads was 98.46%
(S.D. = 3.16) after 54 calibration operations done before
and after the sorting sessions (Nebe-von Caron, personal
communication). A similar procedure was done sorting
cultured bacteria on a slide, and microscopically con-
¢rmed.
12. Summing up the limitations of £ow cytometry for
potential users
Cost is the ¢rst limitation when scientists in aquatic
microbiology consider the incorporation of £ow cytometry
in the group or institution. However, a relative decrease or
non-increased prices have already been seen, furthermore
cytometers tailored to speci¢c operations are now avail-
able. For sophisticated instruments such as cell sorters,
skilled operators are usually required to obtain optimal
performance and also to avoid failures and misuse of
equipment. Sophisticated equipment also needs su¤cient
room, appropriate air quality, a clean atmosphere and
adequate refrigeration systems if high-power lasers are
used. Once the equipment and trained technicians are
available, the next obstacles are associated with the choice
of the most suitable £uorochromes among the hundreds
existing and especially how to pinpoint those £uoro-
chromes that could be of innovative use in the study. To
obtain the best possible controls and validations is the
¢nal critical step.
13. Perspectives
The most attractive perspective in aquatic and environ-
mental microbiology lies in the combination of £ow cyto-
metry with molecular techniques. Flow identi¢cation and
sorting using FISH techniques o¡er great potential. Sepa-
ration of populations and subsequent use for PCR ampli-
¢cation, image cytometry, biological activity, electron mi-
croscopy with X-ray analysis or Raman spectroscopy,
culturability and identi¢cation are options of great interest
today. Neural networks can be trained to recognise sub-
populations of microorganisms from previous data to ex-
amine and sort aquatic microbes [171]. Spectral ¢nger-
printing can be used to determine the spectral properties
of particles at hundreds of wavelengths simultaneously
[172] and lectins can help to improve discrimination of
cells from abiotic particles [173]. The exposure of front-
line soldiers or civilian populations to biological warfare
agents needs extremely rapid detection. Flow cytometry is
now a well-established method in the armed forces (Nebe-
FEMSRE 685 29-8-00
443
von Caron, personal communication) since it can di¡er-
entiate between biological and non-biological particles and
more importantly determine whether or not the detected
microorganism is potentially virulent. This type of detec-
tion may be superior to molecular techniques that are at
present unable to determine the functional state of bacte-
ria. However, £ow cytometry has recently been used to
size DNA fragments in bacterial ¢ngerprinting [174].
The development of modern environmental and micro-
bial ecology requires the incorporation of the most ad-
vanced and sophisticated methods from biochemistry, ge-
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netics, molecular and microbial biology. However, the
knowledge obtained using these methods can only be con-
sistent if rigorously applied, this therefore implies perma-
nent calibrations and validations. The environmental mi-
crobiology of the future need high-quality methods to be
available in order to encourage people to perform experi-
ments that may allow us a better comprehension of Na-
ture. It is the duty of £ow cytometrists to endeavour it.
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