FEMS Microbiology Ecology 53 (1988) 315-324
Published by Elsevier
315
FEC 00179
An investigation of staining methods to determine total cell
numbers and the number of respiring micro-organisms in samples
obtained from the field and the laboratory
Richard P.J. Swannell
a and Francis
A. Williamson
b
’ Depariment of Agrinrltural and Environmental Science, The Uniuersiiy, Newcastle-upon-Tyne,
and b Shell Research Limited
Sittingbourne
Research Centre, Sittingbowne,
U.K.
Received 26 January 1988
Revision received 5 April 1988
Accepted 5 April 1988
Key words: Microbial viability; Ethidium bromide; INT; Fluorescence
1. SUMMARY
Dyes were evaluated in combination with 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyl
tetrazolium chloride (INT) to enable total cell numbers
and the numbers of respiring cells to be determined on the same preparation. Malachite green
and 4’,6-diamidino-2-phenylindole
(DAPI) were
unsuitable counter-stains. Cells which contained
INT formazan crystals could be stained with
ethidium bromide or auramine. At high concentrations of INT formazan, auramine fluorescence was
reduced, although this effect was partially rectified
by prior fixation with glutaraldehyde. Staining
with ethidium bromide produced a strong fluorescence in cells containing crystals of INT for-
Abbreviations:
INT, 2-(4Godophenyl)-3-(4_nitrophenyl)-5phenyl tetrazolium chloride; FDA, fluorescein diacetate; DAPI,
4’,6-diamidino-2-phenylindole;
PTFE,
polytetrafluoroethylene; PBS, phosphate-buffered saline.
Correspondence to (present address): R.P.J. Swannell, Department of Biology, University of Essex, Wivenhoe Park, Colchester CO4 3SQ Essex, U.K.
0168~6496/88/$03.50
microscopy
mazan. This observation was developed into a
procedure which allowed total cells to be determined and provided a useful estimate of the
number of respiring cells in samples obtained
from the laboratory and the field.
2. INTRODUCTION
To determine the effect of physical or chemical
perturbations on microbial communities it is important to know both the changes in total cell
numbers and the numbers of viable cells. Many
intact cells seem to be metabolically inactive, particularly in environmental samples [1,2]. Several
methods for determining the viable/non-viable
ratio have been described in the literature [3,4].
A useful method for estimating viability is to
use a cytochemical procedure which leads to the
accumulation of the product of a metabolic reaction. Two substrates have been widely used, fluorescein diacetate (FDA) and 2-(4-iodophenyl)-3(4-nitrophenyl)-5-phenyl
tetrazolium
chloride
(INT). FDA is uncharged and non-fluorescent
and is passively transported into cells. Endoge-
0 1988 Federation of European Microbiological Societies
316
nous enzymes then hydrolyse the FDA to fluorescein which is charged and fluorescent. In viable
cells, the membrane is impermeable to the charged
molecules which therefore accumulate intracellularly and can be detected by fluorescence.
Organisms with leaky membranes do not retain
the fluorescein.
This method is very reproducible for plant cells
[5,6]. However, results with micro-organisms appear rather variable. Successful use of FDA for
viability determination
has been reported for
mycobacteria [7] and ‘soil bacteria’ [8] but in
general FDA has been found unsuitable for
bacterial viability determination ([g-11], and Williamson, unpublished observations).
An alternative measure of viability is detection
of respiration by assays of dehydrogenase activity
of the electron transport chain. The formazan
produced by the reduction of INT is insoluble in
water. It is therefore accumulated in actively respiring cells, and can be detected by its red colour
under bright field [12].
A combination of INT and acridine orange was
successfully used to determine the respiring/nonrespiring ratio in environmental samples [l]. The
advantage of this staining procedure is that it is a
relatively easy and reproducible method for determining viability compared with other more
time-consuming methods, e.g. most probable number techniques, and the uptake of 14C- and 3Hlabelled organic solutes [2,13]. However, acridine
orange fluorescence can be difficult to distinguish
from auto-fluorescence of non-living particles [14].
The aim of this work was to find an alternative
staining procedure to determine total cell numbers
which could be used in conjunction with INT.
Several of the published staining methods using
ethidium bromide [15], auramine [16], 4’,6-diamidino-2-phenylindole
(DAPI)
[14],
and
malachite green [17] were evaluated.
3. METHODS
3.1. Source of micro-organisms
Samples of micro-organisms were obtained from
stream, salt marsh and pond sediment, freshwater,
sea water, and pure cultures of Pseudomonas
aeruginosa strain 10662 NCTC and Pseudomonas
mendocina strain 11687 NCIB. The sediment and
freshwater samples were initially suspended in
sterile double-distilled water at a concentration of
10% (w/v) and mixed for 1 min using a vortex
mixer. The salt marsh sediment and sea water
were serially diluted in filter-sterilised water (0.2pm cellulose nitrate filter, Sartorius) obtained from
the same sampling point. Field samples taken for
viability determinations were transported to the
laboratory at 4 o C. Triplicate samples were taken
from the top 10 mm of the sediment cores and
from the sea water. The sea water was mixed
thoroughly before the subsamples were taken. Each
subsample was incubated for 12 h with INT in a
cooled orbital shaker (150 rpm) at 16 + lo C. This
12 h period was longer than that used routinely
(see section 3.2) but it was found to give optimal
results for these particular samples which were
removed in late December 1987. The length of
incubation has to be a compromise between being
long enough for organisms with a low metabolic
rate to deposit enough INT to be visible, and
short enough to prevent significant growth within
the incubated sample. There has been no evidence
that INT can be utilized as a growth substrate.
The incubation began within 3 h of the removal of
the material from the field.
3.2. Preparation of cells stained with INT
Samples of micro-organisms
were incubated
with filter-sterilised INT (Sigma) at a final concentration of 0.04% (w/v) for 30 min at room
temperature in sterile universal bottles (modified
from [27]). Preparations were either allowed to dry
on microscope slides, or filtered under vacuum
through a 0.2 pm PTFE filter (25 mm diameter,
Fluoropore FG, Millipore). A 10 ~1 volume was
used for air-dried preparations and 4 ml were used
for filtration. This was carried out with sterile
pipettes. Each filter was washed with 1.5 ml
acetone (GPR Grade, BDH) prior to use, to wet
the hydrophobic pores. The acetone was removed
under vacuum. It was thought that any remaining
acetone residues were washed away during sample
filtration, reducing any toxic effects of the solvent
on the microbes. Both the prepared PTFE filters
mounted on microscope slides and the air-dried
317
samples were covered with 10 ~1 phosphate buffer
(pH 9.2) and a cover-slip sealed on with a mixture
of lanolin, Vaseline and paraffin wax (1: 1 : 1).
This seal prevented the INT formazan from being
dissolved by immersion oil used during microscopic examination [18]. Slides were viewed under
bright field using oil immersion on a Leitz Ortholux II microscope.
3.3. Preparation of cells counter-stained with DAPI
A mixed culture of micro-organisms obtained
from sediment was used to evaluate DAPI (Sigma)
as a potential counter-stain for INT. Samples of
micro-organisms were incubated in triplicate with
INT as related previously (section 3.2). The preparations (10 ~1) were fixed either by air drying,
by gentle warming on a hot plate [19] or by
formaldehyde treatment (4% (v/v) for 30 min)
followed by air drying.
In each case the dry samples were stained with
DAPI [14]. The preparations were examined using
bright field and epifluorescence (excitation filter:
365 nm; barrier filter: 397 nm) on a Zeiss Axiomat
microscope with Neofluar objectives. This procedure was repeated five times to confirm the observations with DAPI concentrations of 0.01 pg.
ml-’ and 0.1 pg. ml-‘.
3.4. Preparation
of cells counter-stained
with
malachite green
A mixed culture of micro-organisms obtained
from sediment was used to evaluate malachite
green (Sigma) as a potential counter-stain for INT.
Samples of micro-organisms were incubated in
triplicate with INT as related previously (section
3.2). Samples counter-stained with malachite green
were prepared [17]. The microscope slides were
examined under bright field using a Leitz Ortholux II microscope. The staining was repeated five
times to confirm the observations.
3.5. Preparations
of cells. counter-stained
with
auramine
Micro-organisms from a variety of sources were
used to evaluate auramine (Sigma) as a potential
counter-stain for INT. Samples were incubated in
triplicate with INT as described above (section
3.2). A cell suspension (10 ~1) from each source
was then combined with 10 ~1 of auramine (0.1
rngein-’
in double-distilled water) on a glass
slide and allowed to dry. The preparations were
mounted in distilled water and viewed under bright
field and epifluorescence on a Leitz Ortholux II
microscope and Ploemopak epifluorescence
illuminator, fitted with an 12 filter block (excitation
filter: BP 450-490 nm; suppression filter: LP 515
nm; beam splitting mirror: RKP 510 nm).
Auramine was then used to counter-stain various samples on PTFE filters. The micro-organisms
were treated with INT as before. All of the groups
of organisms mentioned earlier were used in triplicate as samples. Volumes (4 ml) were filtered
under vacuum through 0.2 pm PTFE filters as
above and washed with 1 ml sterile distilled water.
Auramine (0.5 ml, 0.1 mg - ml-‘)
was added and
left for 12 min. The filter was rinsed with sterile
phosphate-buffered
saline (PBS; Dulbecco “A”
Oxoid (BR14a); NaCl 8.0 g - l-‘, KC1 0.2 g. l-‘,
Na,HPO,
1.15 g. l-l, KH,PO,
0.2 g. l-‘, pH
7.3) to remove background stain and mounted on
a glass slide with 20 ~1 of double-distilled water
and a cover-slip sealed on with a mixture of
lanolin, Vaseline and paraffin wax (1: 1: 1). The
preparations were examined using epifluorescence
and bright field optics. This procedure was repeated using cells stained with INT and fixed in
glutaraldehyde (2.5% (v/v) final concentration,
pH 7.2) for 10 min prior to filtration. Photographs
of preparations were taken using a Zeiss Axiomat
microscope.
3.6. Preparation
of cells counter-stained
with
ethidium bromide
A wide range of micro-organisms were also
used to evaluate ethidium bromide as a potential
counter-stain for INT. Cells were prepared on
glass slides and on PTFE filters in a identical
manner to that used for auramine except that all
the preparations were mounted in filter-sterilised
phosphate buffer (pH 9.2) and not in distilled
water. The preparations were examined microscopically under bright field and epifluorescence
using a Leitz Ortholux
II microscope
and
Ploemopak epifluorescence illuminator using filter
block N2 (excitation filter: BP 530-560 nm; suppression filter: LP 580 nm; beam splitting mirror:
318
RKP 580 nm). Stock concentrations of ethidium
bromide (0.1 mg - ml-’ in double-distilled water)
were stored at 4O C for 3 weeks without obvious
loss of efficacy [15].
3.7. Counting procedure
Cells were enumerated using the method described by Roser et al. [20,21]. This ‘plotless’
technique is based on measuring the distance of
cells from a fixed point in a field of view. Each
field was divided into four quadrants and the
distance from the third nearest cell to the centre of
the slide was recorded. Ten fields of view were
counted on every slide resulting in forty measurements per sample. From these measurements a
best estimate of the cell densities can be ,obtained
from the equations described by Roser et al.
[20,21]. Using these mathematical relations D.B.
Nedwell and A.C. Upton have written a computer
program to rapidly convert the measurements into
cell densities. This program was subsequently
modified for environmental samples by R.P.J.
Swannell. All cell densities were calculated using
such a program run on a BBC microcomputer.
The best estimates were determined for three samples (see section 3.1) and the mean and standard
deviations were calculated from these results.
4. RESULTS
4.1. Membrane filters
In preliminary experiments, filters made from a
variety
of materials
were evaluated:
polycarbonate, cellulose and PTFE filters (obtained
from both Nucleopore and Millipore). All of these
except the PTFE (fluoropore FG) adsorbed the
dyes to such a high degree that it was difficult to
distinguish the stained cells. Washing the stained
preparations with a saline solution (PBS; Oxoid)
greatly reduced the adsorption of dyes to fluoropore membranes, and was therefore used routinely.
The only disadvantages of these membranes are
the need for initial treatment with acetone or
methanol to wet the hydrophobic pores and the
rather uneven surface caused by the network of
the backing necessary to support these fragile
membranes. This made photography difficult and
necessitated frequent re-focusing whilst counting.
4.2. Samples stained with INT
In all samples tested, it was found that many
micro-organisms contained red intracellular INT
formazan crystals. Apparently, both eukaryotes
and prokaryotes were stained. Eukaryotes tended
to contain several discrete foci of INT whereas
prokaryotes tended to contain single granules (Fig.
la, b). Samples taken from an axenic P. aeruginosa strain 10662 NCTC culture in log phase
were more intensely stained than those obtained
from sea water.
4.3. Samples counter-stained with DAPI
Samples counter-stained with DAPI produced
little contrast with each method used. A concentration of 0.01 pg. ml-’ DAPI gave marginally
better blue fluorescence than 0.1 pg * ml-‘. Cells
containing large INT formazan crystals seemed to
further decrease the DAPI fluorescence.
4.4. Samples counter-stained with malachite green
Malachite green was found to stain microorganisms well, but intracellular INT formazan
granules were difficult to distinguish against the
green background. It was particularly difficult to
distinguish the red INT formazan crystals in small
cells, e.g. small prokaryotes, whereas in larger
cells, e.g. fungi and actinomycetes the red crystals
were seen much more clearly.
4.5. Samples counter-stained with auramine
Organisms stained with auramine on glass slides
were found to yield a clear yellow fluorescence.
Under bright field the red INT formazan crystals
could be seen clearly. However, the intensity of
the yellow fluorescence was dependent on the
deposition of INT formazan, i.e. as the deposition
of the formazan crystals increased the intensity of
the auramine fluorescence decreased. Auramine
fluorescence faded with time and yellow fluorescence of non-living matter was noted.
On PTFE filters the auramine fluorescence
could clearly be seen (Fig. 2a) and the cells enumerated. However, the intensity of the fluo-
Fig. 1. Samples treated with INT on WFE filters. Bright field transmitted light illumination. (Field width, 93 pm). (a) Sediment
sample. Red INT formazan is deposited in discrete granules in eukaryotic cells. (b) P. aemginosa 10662 NCTC. Most cells are
completely filed with INT formazan.
rescence was sharply reduced when a large amount
of INT formazan was deposited intracellularly
(Fig. 2b), although this was partially reversed by
prior treatment with glutaraldehyde. Again, the
auramine fluorescence of non-living material was
noted. Crystals of INT formazan could be seen
clearly in auramine-stained cells (Fig. 2b).
4.6. Samples counter-stained with ethidium bromide
Organisms stained with ethidium bromide on
glass slides were found to yield an intense red
fluorescence. Again, under bright field the red
INT formazan crystals could be clearly seen. Preparations stained with ethidium bromide alone
were found to show no red colour under bright
field. The intensity of ethidium bromide fluorescence was not apparently effected by the presence of INT formazan granules.
The fluorescence of ethidium bromide could be
seen clearly on PTFE filters (Fig. 3a). Counting
the micro-organisms
in a field of view was
straightforward. On PTFE filters the intensity of
320
Fig. 2. Sediment samples stained with auramine on PTFE
Auramine-stained only. (b) INT-treated and auramine-stained.
ethidium bromide fluorescence was apparently unaffected by the presence of intracellular INT formazan crystals (Fig. 3b) but a little obviously
non-living material could be seen. The presence of
glutaraldehyde in the ethidium bromide preparations increased the background fluorescence, making the cells less distinguishable.
Samples which had a high metabolic activity,
e.g. log phase P. aeruginosu, were enumerated
simply, as the number of viable cells could be
determined by counting cells containing the INT
filters. Epifluorescence
illumination. (Field width, 93 pm).
(a)
formazan crystals under bright field. The total
number of cells in the aqueous sample could then
be determined in the same field by changing to
epifluorescence optics and counting the number of
cells stained with ethidium bromide. In samples
which had low metabolic activity, e.g. samples
from sea water and freshwater, there were fewer
actively respiring cells as a proportion of the total
counted using ethidium bromide. In this case two
separate preparations of differing dilutions may
have to be made, one for analysis under bright
321
Fig. 3. P. mendocina strain 11687 NCIB stained with ethidium bromide on F’TFE filters. Epifluorescence illumination. (Field width,
104 pm). (a) Ethidium bromide-stained only. (b) INT-treated and ethidium bromide-stained.
field and one for observation under epifluorescence. Samples obtained from the serial dilution
of sediment did contain small pieces of detritus
which did not stain with ethidium bromide but
were difficult to distinguish from cells containing
crystals of INT formazan. Less detritus particles
were seen in preparations incubated with INT and
then treated with Triton X-100 (0.1% (v/v)) to
encourage micro-organisms to fall off the sedi-
ment particles [20]. The samples were then serially
diluted and prepared for observation, as described
previously. The standard method decided upon as
a result of these studies is shown in Table 1.
This method was used to determine the ratio of
respiring to non-respiring micro-organisms in a
saltmarsh sediment and in the overlying water.
The samples were taken from Colne Point, Essex,
U.K. The results obtained from these samples are
322
5. DISCUSSION
Table 1
Procedure for the determination of total and respiting cell
numbers
_
1. Incubate the sample with INT at a final concentration of
0.04% w/v for 30 min.
2. Dilute a portion of the sample so that on filtration 3-4 ml
of the resulting suspension leaves no more than a monolayer
on the filter in order that the slide may be easily counted.
3. Soak a PTFE filter (0.2 pm Millipore Fluoropore FG, 25
mm diameter) with 1.5 ml acetone (GPR grade, BDH). Excess
acetone should be removed by vacuum leaving the filter slightly
damp.
4. Pour 3-4 ml of the diluted sample over the filter and apply
a vacuum until the liquid is just removed.
5. Add 0.5 ml of ethidium bromide (0.1 mg.ml-‘)
and leave
on the filter for 12 min. Remove the remaining dye by vacuum
filtration (caution! ethidium bromide is a possible mutagen).
6. Wash the filter with l-2 ml of sterile phosphate-buffered
saline (Difco) and place the damp filter on a glass microscope
slide. Add 1 drop of sterile phosphate buffer (pH 9.2), and seal
with hot wax mixture.
7. View the preparation under bright field to note those cells
containing INT formaaan deposits and under epifluorescence
to count the total number of cells (Leitz filter block N2).
given in Table 2 and are expressed as the number
of cells in 1 ml of sample. The dry weight of the
sediment sample was 0.16 g - ml-’ wet sediment.
Table 2
The respiring/non-respiring
from a saltmarsh pan
ratio of micro-organisms obtained
Three separate dilution series were prepared for each sample
and each was counted in the same manner.
Sample
Mean number of cells
(ml-’ of sample + S.D.)
Number of
respiring cells
Total number
Percentage
respiring
cells ( W)
CdlS
Water
8.13 x 10’
+2.12x106
1.65 x 10’
+4.71x105
49.5
f 1.5
Sediment
8.27 x 10”
+1.41x10”
3.42 x 10”
f 1.42 x 10”
24.2
*1.0
The INT formazan was deposited intracellularly and could be satisfactorily viewed under the
microscope using bright-field optics. As eukaryotes
apparently deposited a number of discrete crystals
in each cell, it was important to scrutinise any cell
containing INT formazan carefully to prevent the
same cell being counted more than once. This is
not so important when dealing with samples containing prokaryotes only.
Malachite green was found to be unsuitable as
a counter-stain for small prokaryotes containing
INT formazan. This method has been successfully
used with filamentous bacteria [17], but the intracellular INT formazan crystals were difficult to
distinguish
from the malachite
green-stained
membrane of smaller micro-organisms.
DAPI
staining was weak, which may have been due to
the barrier filter (397 nm) used on the Zeiss
Axiomat microscope. A barrier filter ‘at or > 390
nm’ was recommended [14]. The weak DAPI fluorescence, however, appeared to be inhibited by the
presence of INT formazan crystals. Similarly, the
fluorescence of auramine was inhibited by the
presence of INT formazan, although glutaraldehyde improved penetration of the probe. Even
with the glutaraldehyde the low intensity of the
emitted light coupled with the rapid loss of the
fluorescence with time, for a given field of vision,
made accurate counting of the cells difficult.
Ethidium bromide fluoresced in cells even when
INT formazan granules were present. The stain
has high affinity for DNA but it does lightly stain
some non-living matter [15]. The intensity and the
persistence of the red fluorescence enabled cells to
be satisfactorily enumerated, and made it difficult
to confuse ethidium bromide fluorescence with the
autofluorescence of non-living matter.
The incubation conditions used in any experimental work, whether for laboratory or field samples, have a profound effect on the deposition of
INT formazan. A higher intracellular deposit of
INT formazan has been noted with lo-fold increases in INT concentration, and with changes in
the diluent, the time of incubation, the addition of
auxiliary carbon sources and the temperature
[12,18]. Thus, if this method is to be used routinely
323
preliminary work must be undertaken to optimize
the conditions of incubation to suit the type of
sample. Some cells, owing to their poor metabolic
state, may not reduce sufficient INT whilst in the
incubation mixture to be visible under bright field,
yet may still be viable under different conditions
[LIgl.
The ability of micro-organisms to convert INT
to INT formazan appears to be very widespread
[1,12,22-241 but not universal [25]. Furthermore,
owing to the effects of the incubation and the low
metabolic state achieved by some live microorganisms, INT may not provide an absolute in
situ enumeration of respiring cells. This is not to
say that the use of INT is invalid; it is merely
noting that the technique has some recognised
limitations. However, for measuring metabolic activity there does not appear to be a more suitable
alternative currently available. Ethidium bromide
provides a good estimate of the total number of
cells in a sample owing to its high affinity for
DNA [15]. When the number of viable microorganisms is a small proportion of the total population, two slides of differing dilution may have to
be prepared, one for examination of organisms
stained with INT formazan and one for examination of organisms stained with ethidium bromide.
The processing of soil and sediment samples
presented some difficulties, particularly of achieving realistic incubation conditions [4], removal of
micro-organisms
from particles, distinguishing
INT formazan-stained cells from inanimate debris, and uniform distribution of the substrate
(INT). In this work sediment was mixed thoroughly in a solution of INT in double-distilled
water or sterile sea water, to try to dislodge cells
from particles. This encouraged substrate distribution and minimized the impact of disruption on
resident microbes. In the resulting preparations it
was sometimes difficult to distinguish INT formazan-stained cells from non-living material. In
such circumstances, the same field of view was
scrutinised under epifluorescence to note whether
the matter was stained with ethidium bromide.
Samples prepared using Triton X-100 produced
slides apparently less contaminated
with inanimate debris but it was not clear what effects
the treatment may have on cells containing INT
formazan crystals. The use of ultrasonication to
remove attached bacteria has been suggested [26],
but again the effects on INT formazan-stained
cells are difficult to assess. The advantage of this
rapid technique is the simple staining procedure
compared with other more time-consuming protocols (e.g. autoradiography [4]).
For work in the laboratory which aims to look
at the effects of stress, e.g. addition of toxicant,
changes in temperature and substrate levels on the
viability of micro-organisms in pure and mixed
culture, this combination of INT and ethidium
bromide appears suitable. Similarly, this procedure could be employed, in conjunction with other
activity measurements [4], when studying the ecological effects of stress on samples obtained from
the field.
ACKNOWLEDGEMENTS
We are grateful to Colin Nicholson for assistance with photomicrography. We would like to
thank the Science and Engineering
Research
Council for the award of a CASE studentship to
R.P.J.S.
and Shell Research Limited for their
financial backing and permission to publish this
work.
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