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Applied and Environmental Microbiology, October 1998, p. 3900-3909, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Determination of the Biomasses of Small Bacteria at Low
Concentrations in a Mixture of Species with Forward Light Scatter
Measurements by Flow Cytometry
B. R.
Robertson,1,*
D. K.
Button,1,2 and
A. L.
Koch3
Institute of Marine
Science1 and
Department of Chemistry and
Biochemistry,2 University of Alaska
Fairbanks, Fairbanks, Alaska 99775, and
Biology Department,
Indiana University, Bloomington, Indiana 474053
Received 27 February 1998/Accepted 23 July 1998
 |
ABSTRACT |
The forward light scatter intensity of bacteria analyzed by flow
cytometry varied with their dry mass, in accordance with theory. A
standard curve was formulated with Rayleigh-Gans theory to accommodate
cell shape and alignment. It was calibrated with an extinction-culture
isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter
intensity due to formaldehyde accumulation in preserved cells was
included in the standard curve. When differences in the refractive
indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell
mass obtained by flow cytometry for various bacterial species and cell
mass computed from Coulter Counter volume and buoyant density. This
agreement validated the standard curve and supported the assumption
that cells were aligned in the flow stream. Several subpopulations were
resolved in a mixture of three species analyzed according to forward
light scatter and DNA-bound DAPI (4',6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 µg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus
containing cells with a single chromosome. Calculations from
measurements of dry mass, Coulter Counter volume, and buoyant density
revealed that the dry weight of the isolate was 14 to 18% of its wet
weight, compared to 30% for Escherichia coli. The method
is suitable for cells with 0.005 to about 1.2 pg of dry weight at
concentrations of as low as 103 cells/ml and offers a
unique capability for determining biomass distributions in mixed
bacterial populations.
| |
INTRODUCTION |
Measurements of bacterial biomass
are essential for determining growth rates and cell yields and provide
a base for formulations that relate rates of growth and nutrient
accumulation to substrate concentration (12, 13). Accurate
values are difficult to establish for dilute or mixed populations or
for very small bacteria. Among the available methods (61),
optical density measurement is commonly used and can be related to dry
weight with light scatter theory (35). However, the method
is insufficiently sensitive for analyzing very dilute concentrations of
cells and cannot differentiate subpopulations or discriminate organisms
from debris. Concentrations of some bacteria can be determined from
plate counts or most-probable-number determinations (31),
but many bacteria resist growth in laboratory cultures (64).
The biomasses of such populations are often determined from cell number
and size by microscopy (55, 56, 71), but the optical sizing
of small cells appears to lack precision (45) and the dry
matter content used is often inappropriately assumed to be similar to
that measured for Escherichia coli (48). Coulter Counter impedance (40, 42) provides a fairly accurate
estimate of cell volume, but the method lacks sensitivity for small
bacteria, does not account for changes in the density of the cell
material (28), and is subject to interference from debris.
Flow cytometry can determine biomass from the intensity of light
scattered by single cells (52). Advantages include
simultaneous multiparameter analysis, adequate sensitivity for
organisms <0.1 µm3 in size at <1,000 cells/ml,
favorable statistics due to analysis rates approaching 104
particles/min (14), resolution of individual species in a
mixture (67), and ability to determine organism
concentrations (14). Bacterium-sized particles scatter light
mainly in the forward direction (35), within the detection
range of most flow cytometers. Small size and an index of refraction
only 3 to 6% higher than that of the surrounding medium minimize phase
change and allow the use of Rayleigh-Gans theory to relate bacterial
biomass to light scatter intensity in flow cytometric analyses
(37).
The size of bacterial cells has been computed from flow cytometry data
by Mie theory (8), with the results being reported in terms
of diameters of polystyrene spheres (1). Davey et al.
(20) related forward light scatter intensity from
polystyrene spheres to particle size and calibrated the resulting
polynomial with microscopic measurements of bacteria. They attributed
an underestimate of cell volume by this method to a difference in the
ways in which cells and polystyrene standards scatter light. Other
attempts to relate forward light scatter to cell size have depended on
measurements of cell volume by electrical impedance (3, 50).
However, calibration of the light scatter intensity from the very small
bacteria resolvable by flow cytometry with the signal from larger
organisms measurable with a Coulter Counter required a long
extrapolation, and variation in dry weight content was not addressed.
Light scatter was reported to reflect the protein content (9,
62) of E. coli because of a constant
protein-to-dry weight ratio, but the range of values examined was too
small to establish the shape of a standard curve. Most flow cytometry
data for cell size are reported in arbitrary units, such as channel number (2, 6, 22), which is nonlinear with cell volume or
mass.
An earlier investigation (37) applied Rayleigh-Gans theory
to flow cytometry by integrating the predicted intensity of light scattered at a specified angular range from the path of the laser beam
by cells of a specified size, axial ratio, and orientation in the
flow stream and illuminated at a particular frequency. Here, we
explore limitations of the theory and compare predictions with data
from standard spheres. A theoretical curve is produced and
calibrated with measurements of dry weight obtained for a small marine
isolate. Dry mass values of various bacterial species are
interpreted in terms of their relative refractive index
m and are verified by independent measurements of buoyant
density and Coulter Counter volume. Forward light scatter and DNA-bound DAPI (4',6-diamidino-2-phenylindole) fluorescence intensity are used to
obtain the biomass of each of several subpopulations resolved from a
mixture of species.
| |
MATERIALS AND METHODS |
Cultures.
An extinction-culture isolate (16) of
the marine organism Cycloclasticus oligotrophus
(68) (formerly oligobacterium RB1 [16]) was
grown in synthetic seawater medium (54). Growth rate and
cell size range were controlled by acetate concentrations ranging from
12 to 200 mg/liter. Enrichment culture isolates of Marinobacter (formerly Pseudomonas
[49]) sp. strain T2 and RB95-4 were grown in synthetic
seawater medium with amino acids and glucose, respectively, as the sole
carbon sources. E. coli DH1 (ATCC 33849) and
Pseudomonas diminuta (ATCC 19146) were grown in M9 medium (23) supplied with glucose.
Standards.
Fluorescent polystyrene microspheres
(Fluoresbrite Microparticles; Polysciences, Inc.), 0.3 to 2.02 µm in
diameter, with a refractive index of 1.6 and with a density of 1.05 g/cm3 (47), were used. A mixture of 0.90- and
0.60-µm spheres provided internal standards (14). The
concentration of the larger spheres was adjusted at 105
cells/ml with a Coulter Counter, and the ratio of their frequency to
that of bacteria was used to compute the populations of bacteria. Forward light scatter and blue fluorescence intensity of the smaller spheres were used to normalize intensities among samples.
Sample preparation for analysis by flow cytometry.
Samples
were preserved with filtered formaldehyde (0.2-µm-pore size; as
formalin; 0.5% [wt/vol]), stored at 5°C in the dark for at least
16 h unless otherwise noted, vortexed, diluted to about
106 cells/ml, treated with Triton X-100 (0.1%), stained
with DAPI (0.5 µg/ml) at 10°C in the dark for 1 h, amended
with the standard microspheres, and kept at 10°C during analysis
(14).
Flow cytometry.
Instrumentation consisted of an Ortho
Cytofluorograf IIS equipped with a flat-sided quartz flow cell, a 5-W
argon laser tuned to UV emission (351.1 and 363.8 nm) at 100 mW of
power, and a focusing lens to reduce the beam width to 44 µm for
better resolution of small bacteria (50). Light scattered by
bacteria in the forward direction past a vertical 1.5-mm beam blocker
bar was reflected by a 424-nm long-pass dichroic filter through a 310- to 370-nm band-pass filter and focused onto the plane of a shielded
fiber-optic cable leading to the photomultiplier detector. Orthogonal
light scatter, used to evaluate the frequency of internal
0.9-µm-diameter standard spheres for population counts
(14), was isolated with similar optical filters. Blue
fluorescence from DNA-bound DAPI was collected orthogonally through the
424-nm dichroic filter and a 450- to 490-nm band-pass filter.
Logarithmic amplifiers with a dynamic range of 3.5 decades were
calibrated (53) to establish the range of linearity between signal input and numeric response (14). Data acquisition,
analysis, and storage were done with a PC-based Cicero system and
Cyclops software (Cytomation, Inc.). Acquisition was triggered by
DAPI-DNA fluorescence to eliminate the forward light scatter signals
from nonfluorescent debris.
Conversion of 256 channels resolved by the logarithmic amplifiers to
10
3.5 linear channels was accommodated by the software, and
mean values
(linear) for the forward light scatter and DNA-bound DAPI
fluorescence
intensity of each population were recorded. The ratio of
the mean
forward light scatter or fluorescence intensity of a
population
to that of the 0.6-µm internal standard spheres was used
to normalize
among samples for calculations of cell mass and DNA
content.
Light scatter theory.
Expected intensities of light
scattered by bacteria of a given mass, shape, and orientation were
obtained from computer programs (37) based on Rayleigh-Gans
theory. To verify the formulations, calculations for spherical
particles were compared with those based on the more complete but
complex Mie theory (34) as programmed by Tsay and Stephens
(63), since the Mie theory is not easily applied to
nonspherical particles. Input for all programs included an excitation
wavelength of 360 nm and a forward light scatter collection angle of
0.5 to 20°. Error from signals excluded by the beam blocker bar was
assumed to be minimal (37). Mie calculations were performed
for m values of 1.04 and 1.19 to compare the curves obtained
for bacteria with those for polystyrene standards.
Standard curve for dry mass.
A curve relating dry weight to
forward light scatter intensity was calculated for particles modeled as
ellipsoids of revolution with an axial ratio of three and aligned
perpendicular to the laser beam (37). Curves were also
computed for particles with different axial ratios and random or
perpendicular alignment for comparison to validate the use of the input
parameters. The curves were fitted with the Marquardt-Levenberg
algorithm and a volume weighting factor of
1/V2.3 (where V is volume)
(SigmaPlot; Jandel Scientific) to give the dry weight of a particle as
a function of its forward light scatter intensity.
The proportionality constant required to normalize forward light
scatter intensities obtained from the flow cytometer with
values
calculated from theory (
37) was computed by determining
the
dry weight and the forward light scatter intensity of
C. oligotrophus grown on
14C-acetate as the sole carbon
source and by determining the ratio
of carbon to dry weight.
1,2-
14C-acetate (58.2 mCi/mmol; DuPont NEN) was diluted
with unlabeled
anhydrous sodium acetate (analytical reagent grade;
Mallinckrodt)
to a specific activity of 2.19 × 10
6
dpm/mg of sodium acetate or 6.83 × 10
6 dpm/mg of
carbon. Populations were grown to attain at least a
14-fold increase in
biomass so that, assuming an endogenous metabolism
rate of 0.007 h
1, more than 98% of the cell carbon would be
labeled. Dry weights
were determined at a range of acetate
concentrations and computed
from the radioactivity (~10
4
to 10
6 dpm) collected on a polycarbonate membrane filter
(0.2-µm-pore
size; Nuclepore Corp.) and counted with a scintillation
spectrometer,
the specific activity of the substrate, and the cell
population
determined by flow cytometry. To determine the ratio of cell
carbon
to dry weight, a culture was grown on acetate and pelleted by
centrifugation. The 27-mg pellet was washed with 8 ml of saline
to
reduce the carryover of medium solutes to 28 µg (<0.8 µg of
acetate), transferred into tared tin boats, dried along with cell-free
controls overnight at 100°C in a vacuum oven, weighed, and analyzed
for carbon content with a CHN600 analyzer (Leco Corp.) calibrated
with
a coal standard.
DNA content.
DNA was determined from DAPI-DNA fluorescence
intensity and standardized with the signal from E. coli
with a known genome size of 4.7 Mbp (38, 51), or 5.17 fg,
and a GC content of 50 mol% (39). To account for the
effects of medium salinity on DAPI fluorescence intensity
(70), which amounted to a 10 to 30% reduction with
increasing salt concentration, depending on the species measured
(unpublished data), preserved E. coli was stained and
analyzed in both M9 and seawater media. Linear regression analysis of
modal values of DAPI-DNA fluorescence intensity of E. coli subpopulations containing cells with integral numbers of
chromosome copies gave the intensity associated with a single chromosome copy relative to the intensity of the internal-standard 0.6-µm fluorescent microspheres. Cellular DNA content for the other
strains or subpopulations within a culture was obtained from mean
fluorescence intensity with a correction for the AT bias of DAPI
(70) based on their G+C contents: 52.7 mol% for Marinobacter sp. strain T2 (25) and 41.6 mol%
for C. oligotrophus (68).
Cell size.
The volumes of the organisms were measured with a
Coulter Counter (model ZBI with a Channelyzer; Coulter
Electronics, Inc.) with 16- and 30-µm apertures. Instrument
calibration was done with 1.942-µm-mean-diameter spheres
(Coulter lot no. 6179; National Institute of Standards and
Technology traceable standard) and corroborated by data from a Coulter
Multisizer II. Volumes were corrected for cell shape according to
theory (27, 32). Cell dimensions were determined from
electron micrographs. For scanning electron microscopy, cells were
harvested by centrifugation, washed with basal medium, concentrated on
polycarbonate filters, dehydrated in an ethanol series, and
critical point dried. For transmission electron microscopy, harvested
cells were applied to a Formvar grid stabilized with carbon on 100-Mbar
copper (1 bar is 105 pascals) (Ted Pella, Inc.), stained
with 2% uranyl acetate, rinsed, and air dried.
Buoyant density.
Cell densities were determined by
equilibrium centrifugation in Percoll gradients made iso-osmotic with
culture medium by the addition of sodium chloride. Mixtures of Percoll
(Sigma Chemical Co.) and culture fluid at 70:30 for M9 medium and 40:60
for synthetic seawater medium were centrifuged at 30,000 × g in a fixed-angle rotor for 1 to 2 h at 4°C. Values
were determined from the positions of cells and density marker beads
(Sigma). The densities of the marker beads were corrected for medium
salinity based on their position in a gradient of Percoll-synthetic
seawater solution and the assumption that the density of the green
beads of 1.099 g/cm3 remained constant (46). The
refractive index of the medium at the location of the beads was
determined from fluid sampled above and below the band by measurement
with a refractometer (Abbe model 3L; Bausch & Lomb, Inc.). The
densities of the remaining standard markers were computed from assumed
linearity between the refractive index and the medium density
(46) at the location of the green beads and the refractive
index and measured density (by hydrometer) of Percoll-free medium.
Refractive index.
Organisms were suspended at approximately
107 to 108 cells/ml in basal medium
containing 0 to 30% bovine serum albumin (66) and with
adjustments of the sodium chloride concentration to maintain osmolarity. The refractive index of the organisms was taken as the
refractive index of the medium adjusted with bovine serum albumin to
give a minimal absorbance (UV-1201; Shimadzu) of the bacteria at 700 nm. The refractive index of cells as measured by a refractometer was
normalized to that of basal medium to obtain m.
Calculations based on cell composition.
For comparison with
dry mass determined from forward light scatter intensity, dry mass was
computed from the product of cell volume, buoyant density, and the
ratio of dry weight to wet weight (Xdry/Xwet). Buoyant
density (
cell) is given as the density of dry matter
(dry),
Xdry/Xwet, and the
density of water (water) (4):
|
(1)
|
The value of
dry was computed from the
composition of the organisms and the density of each component taken
from the literature.
A value of 1.35 g/cm
3 was obtained for
E. coli from the composition of cells growing
at a rate
of 0.011 h
1 on glucose in M9 medium (
17):
protein at 54% of total dry weight,
with a density of 1.3 g/cm
3 (
44); nucleic acid at 18.3%, with 1.7 g/cm
3 (
60); lipid at 9.1%, with 0.9 g/cm
3 (
60); and the remaining constituents at
13%, with 1.4 g/cm
3. A
dry value of 1.39 g/cm
3 was used for
C. oligotrophus, with protein
at 44% of dry weight,
nucleic acid at 36%, lipid at 14%, and others
at 6%. Organisms
of a smaller size and a higher surface-to-volume
ratio, compared
to
E. coli, were expected to have
a higher proportion of nucleic
acids and lipids in their dry matter.
The refractive index of the organisms relative to their medium
(
m) was computed as follows (
35):
|
(2)
|
In this equation, 0.18 is the specific refractive index
increment factor (
66), based on the weighted average of
values
for protein (0.186), nucleic acid (0.16), and other dry-matter
constituents (0.178).
For comparison with Coulter Counter volume measurements, cell volume
(
V) was computed as follows:
|
(3)
|
| |
RESULTS |
Theoretical predictions.
Mie theory specifies an increase in
forward light scatter intensity with particle size and refractive
index. The intensity of light scattered from small particles is a
linear function of refractive index when expressed as
(m 
1)2 (35) (Fig.
1A). This relationship is particularly
useful for anticipating the effects of changes in m on
forward light scatter intensity (see below). Curvature results at large
particle sizes because of a greater proportion of light being scattered
at angles broader than 20°. For very small particles and those such
as bacteria for which m is 1.04 (35), forward
light scatter intensity can be predicted by the Rayleigh-Gans
approximation, as shown by the similarity between the results of Mie
and Rayleigh-Gans calculations for spheres in Fig. 1B. Due to the low
refractive index of bacteria, signal from cocci as large as 1.5 µm3 would be expected to lie in the region where the two
curves appear identical, justifying the use of the simpler
Rayleigh-Gans theory in calculations for cells of this size.
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FIG. 1.
Dependence of forward light scatter intensity on
particle characteristics. (A) Intensities calculated from Mie theory
for three sizes of spheres as a function of m. Ordinate
scales increase with particle size. (B) Intensities for latex
microspheres of various sizes as predicted by Mie theory when
m is 1.04 ( ··)
or 1.19 (······)
and by Rayleigh-Gans (37) theory (). Data are measured
intensities for microspheres with volumes and indicated standard
deviations (error bars) computed according to diameters from electron
microscopy reported by the supplier ( ) and volumes determined with a
Coulter Counter ( ). The inset extends the particle size range. (C)
Effect of axial ratio on forward light scatter intensity over a range
of cell volumes as determined by Rayleigh-Gans theory.
In/I1 compares the intensity of a particle with
an axial ratio of n to that of a sphere of the same
volume.
|
|
Figure
1B (inset) shows the expected difference between curves
calculated for bacteria (
m, 1.04) and polystyrene particles
(
m, 1.19) larger than 0.3 µm
3 (0.83-µm
diameter). Data for standard polystyrene spheres were
difficult to
interpret due to differences between the volumes
computed from
measurements by electron microscopy as specified
by the supplier and
those measured here with a Coulter Counter.
However, they were
consistent with Mie predictions at volumes
greater than 1 µm
3.
The forward light scatter intensity of spheres of a specified size
analyzed over a wide range of photomultiplier gain settings
varied less
than 9% (
n = 18) when normalized to that of an
internal
standard (data not shown). This fact allowed extension of the
3.5-decade dynamic range of the amplifier to include the full
range of
signals encountered from various particles.
Forward light scatter intensity increased with axial ratio (Fig.
1C).
Since nonspherical particles tend toward alignment in
the flow stream
because of hydrodynamic forces (
33), greater
forward light
scatter for the same volume was expected due to
decreased interference
and smaller phase shifts for light passing
through smaller-diameter
particles (
37). The effect increased
with size, but
calculations showed that for particles of 0.05
µm
3, there
was only a 13% increase in forward light scatter intensity
with
elongation from an axial ratio of one to three, whereas for
cells of 2 µm
3, there was a 79% increase.
Effects of preservatives.
Formaldehyde treatment of the cells
during sample preparation resulted in additional light scatter, as
expected from reports of increased refractive index with preservation
of Streptococcus faecalis (19) and E. coli (66). The increase in intensity for C. oligotrophus was 68%, which corresponded to a 35% increase in
dry weight (Table 1). The effect was the
same for cells containing a single chromosome (see below), so clumping
was not a contributing factor. Other data (not shown) indicated that
overnight fixation at 5°C was sufficient to stabilize light scatter
properties for about 2 months. Effects on E. coli were
less pronounced, amounting to a 9 to 15% increase in apparent biomass
(Table 1).
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TABLE 1.
Effect of formaldehyde on forward light scatter intensity
and dry mass determined from flow cytometry and on Coulter
Counter volume
|
|
Buoyant density measurements showed an apparent 50% increase in dry
material for
C. oligotrophus following preservation
(Table
2) but only a 10% increase for
E. coli, in agreement with the
light scatter data.
Similar increases were computed for
m. Effects
on Coulter
Counter volumes were negligible over the 49 days observed.
This finding
indicates that preservation by formaldehyde can affect
the cell
mass and light scatter properties of organisms in a manner
that
is dependent on the species observed but that determination
of the
apparent sizes of cells from electrical impedance is less
sensitive to
the addition. The differences in the apparent addition
of mass to the
two organisms by formaldehyde may be related to
differences in media
and sample dilution. Binding is pH sensitive,
maximal at pH 7.5 to 8.0, and reversible (
29). The slightly
lower pH of M9 medium (pH
7.5 versus 8) and the higher dilutions
used for high-biomass cultures
before staining to obtain appropriate
populations for flow cytometry
and for equilibration with DAPI
may favor greater formaldehyde loss
from
E. coli.
Preservation with 0.5% glutaraldehyde for 2 h gave a sixfold
increase in the apparent biomass of
C. oligotrophus
(data not
shown). Half of this increase can be accounted for by the
larger
molecular mass of the preservative. Differences in response to
the fixation process among species may also affect observations,
since
formaldehyde penetrates rapidly as methylene glycol but
reacts slowly
(
24), while the opposite has been reported for
glutaraldehyde (
29). Valkenburg and Woldringh
(
66) showed
that glutaraldehyde treatment caused an increase
in the buoyant
density of
E. coli from
1.093 to 1.138, which amounted to a 50%
increase in apparent cell
mass, according to equations 1 and 2
and as shown in Fig.
1A.
Standard curves for dry mass.
Equation 4 (37) was
used to express the relationship between the cell mass and
forward light scatter intensity of bacteria as calculated from
Rayleigh-Gans theory:
|
(4)
|
K is the proportionality constant converting instrument
intensity (
I) to intensity expected from theory
(
KI), and the constants
a,
b,
c, and
d are 1.62 × 10
4,
0.0144, 0.480, and 0.274, respectively, for ellipsoidal cells
with an
axial ratio of three and oriented in the direction of
flow. An axial
ratio of three was used for the calculation because
it agreed with the
dimensions of
E. coli,
Marinobacter sp.
strain
T2, and
C. oligotrophus measured by electron
microscopy (axial
ratio, 2.7 ± 0.3;
n = 7 samples; 168 cells). Equation
4 fits the
computed values to within 5%
for cells with up to 40 pg of dry
mass (Fig.
2, upper inset; data shown to 15 pg/cell). The divergence
of the theoretical curves for cells aligned in
the flow stream
and cells with a random orientation showed the
anticipated increase
(
37) in light scatter intensity with
orientation. Experimental
values were 83% ± 12% (
n = 22) predicted values for aligned cells,
whereas values for bacteria
with a large mass, presumed to be
more strongly influenced by
hydrodynamic forces, were only about
50% those expected for randomly
oriented cells. The better fit
of experimental data to the curve for
oriented cells is consistent
with the expectation (
33) of
alignment in the flow stream. The
effects of orientation increase with
cell mass, as shown by the
difference between the shapes of the two
curves.
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FIG. 2.
Standard curves for dry mass. Curves were computed for
cells with an axial ratio of three and with linear () and random
(·····)
orientations in the flow stream (37) and were calibrated
with the dry mass measurement for C. oligotrophus
( ). Data are from light scatter intensities of C. oligotrophus ( and ), Marinobacter sp. strain T2
( and  ), and E. coli ( and  ), with dry
weights computed from Coulter Counter volumes corrected for cell
density on the basis of equations 1 and 3. Closed symbols are for data
in Table 4; open symbols are for additional data. The upper inset shows
data generated from Rayleigh-Gans theory ( ) and fitted curves and
extends computations to larger particle sizes. The lower inset expands
data from small organisms.
|
|
Dry weights determined for cultures of
C. oligotrophus
grown on
14C-acetate were used to obtain
K for
calibrating the curve (Table
3). CHN
analysis determined cell carbon content to be 47% ± 1%
(
n = 3) dry weight, similar to values reported for
other bacterial
strains (
11). Agreement was within 6%
between populations measured
by flow cytometry (
14) and with
the Coulter Counter for various
cultures of
E. coli,
Marinobacter sp. strain T2, and
C. oligotrophus (
r2 = 0.995;
n = 15; data not shown), so that mean dry weight could
be obtained on a per-cell basis. The carbon content calculated
from
14C-acetate incorporated by
C. oligotrophus
was 10.3 ± 1.8 fg/cell
(
n = 4) over a range of
populations, for a dry weight of 22 ±
4 fg/cell. Experimental
data were fit to the theoretical curve
at a
K value of
1.61 × 10
5 (Fig.
2).
Since the standard curve was based on
C. oligotrophus
in seawater medium, for bacteria analyzed in freshwater medium,
K was
multiplied by 0.80 to correct for the difference
between the refractive
index of M9 medium (1.3331) and that of seawater
medium (1.3380).
For
E. coli, for which
m is
1.037 in M9 medium (see below) (Table
4),
the expected
m in seawater would be 1.037 × 1.3331/1.3380,
or 1.033. Based on the linearity between forward light
scatter
intensity and (
m 1)
2 from Fig.
1A,
E. coli would produce
(0.033)
2/(0.037)
2, or 0.80 as much forward
light scatter intensity if it were in
seawater. To account for the
smaller effect of formaldehyde on
the light scatter intensity of
E. coli,
K was further multiplied
by 1.46 to
give a
KM9 of 1.88 × 10
5 for
organisms grown in M9 medium.
Dry mass measurements.
Samples of bacteria for dry mass
analysis represented a variety of species and culture conditions at the
time of preservation to generate a range of mean cell sizes for method
evaluation. Values for the dry masses of C. oligotrophus, Marinobacter sp. strain T2, and
E. coli measured by flow cytometry were generally within 15% those computed from Coulter Counter volume and buoyant density and from
Xdry/Xwet (equations 1 and 3). Agreement supported the use of an axial ratio of three in the
calculations, which set the shape of the standard curve
(37). Buoyant densities measured here were within the range
reported for numerous bacterial strains, including E. coli (5, 28).
The mean cell dry mass for additional cultures was computed from
Coulter volumes and buoyant densities given in Table
4 (1.07
for
C. oligotrophus) and plotted against forward light
scatter
intensity determined from flow cytometry (Fig.
2). For all of
the samples, linear regression analysis between the computed values
and
cell dry mass determined from forward light scatter intensity
gave an
r2 of 0.979, with cell dry mass
determined by flow cytometry being
20% larger. Error is expected due
to changes in cell density with
growth conditions and to Coulter
Counter volume measurements of
C. oligotrophus near the
limits of the instrumentation. In addition,
the assumption of cell
alignment in the aperture of a Coulter
Counter in which a sample is not
hydrodynamically focused adds
to the uncertainty of Coulter Counter
volume measurements that
are corrected for the axial ratio of these
rod-shaped bacteria
(
27,
32). Measurements from electron
micrographs of the organisms
gave axial ratios of about three, subject
to error from shrinkage
during sample preparation (
20). The
error expected in mass measured
by flow cytometry due to variation in
axial ratios of two to four
was less than 10% (Fig.
1C).
Variation in refractive index.
Buoyant density measurements
demonstrated large differences in
Xdry/Xwet and, therefore,
m for the organisms (equations 1 and 2). The results in Fig.
3A show the separation between the marine
isolates and E. coli. This distribution was in an 0.85 osM gradient to provide a direct comparison between the
organisms. However, data were consistent with band locations of
single species in separate iso-osmotic gradients (not shown) that gave
Xdry/Xwet values of 14 to
18% for C. oligotrophus and 29% for E. coli. The diluteness of C. oligotrophus was
corroborated by optical density (OD) measurements which gave
Xdry/Xwet values of 19 to
21% from the proportionality between Xdry and
OD0.75 (36), the biovolume (Coulter Counter
volume × population) of the two cultures, and Xdry/Xwet values of 27 to
30% reported for E. coli (11, 72). Also,
the values of m determined for C. oligotrophus from buoyant density measurements (Table 4) agreed
with the measurement of 1.025 in Fig. 3B and were significantly lower
than the m value of 1.037 determined here and reported
elsewhere (66) for E. coli.
View larger version (17K):
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|
FIG. 3.
Physical properties of test organisms. (A) Buoyant
densities of C. oligotrophus, Marinobacter
sp. strain T2 (M. T2), and E. coli from
their positions (arrows) in a 70:30 gradient of Percoll-seawater
medium. Data are for standard density marker beads. (B) m of
C. oligotrophus determined by the minimal-absorbance
method (solid line). The broken line is the least squares fit to the
data for an optical density of >0.06 and yields an m of
1.025. (C) Cell volume computed from dry mass by flow cytometry,
buoyant density, and
Xdry/Xwet (equation 3)
with respect to Coulter Counter volume measured for the three species.
The inset shows cell volumes of C. oligotrophus
determined by flow cytometry compared with those determined by
transmission ( ) and scanning () electron microscopy (EM).
|
|
Cell volume.
Measurements of dry mass and buoyant density gave
cell volumes (equation 3) which agreed with measurements made by
Coulter impedance, as shown in Fig. 3C. These values were 1.14 times Coulter Counter volumes (r2 = 0.936; n = 28). Measurements made by electron
microscopy gave volumes only half those obtained from forward light
scatter intensity measurements (Fig. 3C, inset), consistent with
significant shrinkage due to dehydration during sample
preparation.
Biomass in a mixture of species.
Nine subpopulations were
resolved within three major clusters when preserved samples of
E. coli, Marinobacter sp. strain T2, and
C. oligotrophus were combined and analyzed by flow
cytometry (Fig. 4A). Species were
identified by comparison with data from analyses of pure cultures.
Concentrations in the subpopulations were as low as 104
cells/ml, with a dry weight of 1 ng/ml, compared to a total of 106 cells/ml and 0.3 µg of dry mass/ml for the mixture
(Table 5). Coefficients of variation (CV)
for forward light scatter intensity were 40 to 55% for the species
clusters and 27 to 36% for the subpopulations. Since CV for forward
light scatter intensity were only 5% for the internal standard
spheres and less than 2% for the spheres used for instrument
alignment, distributions for cell mass (Fig. 4B to D) were thought to
reflect real variation in the light scatter properties of the cells
rather than analytical error. While the concentrations of cells in the
mixture were about the same for E. coli and
C. oligotrophus, the total biomass of the E. coli culture was 10-fold higher (Table 5), reflecting the higher
dry weight of that organism.
View larger version (28K):
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[in a new window]
|
FIG. 4.
Cell mass and DNA content of subpopulations in a mixture
of three species. (A) Bivariate histogram of forward light scatter
versus DAPI-DNA fluorescence intensity for the mixture along with the
0.6- and 0.9-µm-diameter microspheres. Whole numbers give the number
of chromosome copies contained by cells in various subpopulations. Dim
indicates cells of E. coli showing very low
fluorescence, and C indicates cells of C. oligotrophus
undergoing chromosome replication. M.,
Marinobacter. (B, C, and D) Distributions of dry mass and
DNA content for E. coli, Marinobacter sp.
strain T2, and C. oligotrophus, respectively.
|
|
The mean cell volume of
Marinobacter sp. strain T2
calculated from dry mass (Table
5) and from
Xdry/
Xwet and buoyant
density
(Table
4) was 0.172/(0.192 × 1.075), or 0.83 µm
3; the Coulter Counter volume determined 11 years
earlier, when
the culture was preserved, was 0.73 µm
3.
However, according to equation 1, an increase in buoyant density
to
1.17 g/cm
3, consistent with the observed 56% decrease in
Coulter Counter
volume over the duration, gives a volume of 0.77 µm
3, within 5% of the initial value. Contrary to reports
that apparent
populations of DAPI-stained cells decrease
exponentially with
preservation time (
65),
populations determined with the Coulter
Counter remained unchanged at
2.3 × 10
9 cells/ml. The data suggest that during
extended refrigerated
storage of preserved cells, the changes in
properties affecting
electrical impedance are most likely related to
cell shrinkage
rather than significant changes in dry matter
composition and
dry.
Over 90% of the cells of each species were within a threefold range in
biomass (Fig.
4B to D); the remainder appeared as large
particles and
extended the range to over 10-fold. Clumps of cells,
which have been
observed in some substrate-depleted cultures of
C. oligotrophus and other bacteria (
21,
26), would cause
an
underestimate of total biomass. However, an examination of data
from
off-scale signals (data not shown) indicated their insignificance
here.
Most subpopulations had narrow distributions of DNA content. CV of only
6 to 9% for DAPI-DNA fluorescence intensity facilitated
separation of
the nine groups resolved. The predominance of cells
containing two
chromosomes and the very low incidence of cells
with three or four
chromosomes in the
E. coli culture suggest
a doubling
time of 40 to 60 min based on cell cycle analysis (
18,
59).
However, the absence of cells in the C phase of the cell
cycle, in
which chromosome replication occurs, is consistent with
chromosome
runout with inhibition of protein synthesis (
58).
About 15%
of
C. oligotrophus cells were in the C phase and 70%
were about equally apportioned between the B and D phases,
respectively,
in which chromosome replication has not been initiated
and in
which replication has been completed but cell division has not
occurred. These data are consistent with cell cycle analysis of
the
organism at its maximal doubling time of 4 h on acetate.
About 10% of the population of
E. coli was comprised
of "dim" cells with DAPI-DNA fluorescence intensity only one-third
that
observed for cells containing a single chromosome. However, their
light scatter properties were the same as those of the main population
(Table
4). Dim cells are commonly observed in samples from natural
aquatic systems (
15) and may represent organisms undergoing
nucleic acid degradation due to stress (
69). The low
fluorescence
intensity and the lack of structure in the DNA profile of
Marinobacter sp. strain T2 (Fig.
4C) were very likely a
result of nucleic acid
degradation during the extended duration of
sample storage, since
DNA profiles of the organisms observed within 6 months of preservation
were similar to those seen here for
C. oligotrophus (unpublished
data).
Estimated buoyant densities of other bacteria analyzed by flow
cytometry.
When the buoyant density of an organism was not
measured, it was calculated from the dry mass determined by flow
cytometry and Coulter Counter volume (equations 1 and 3) and taken to
be the value which gave the smallest error in computed mass and volume. For P. diminuta and marine isolate RB95-4, values of 1.05 and 1.055 g/cm3, respectively, gave that agreement for each
parameter to within 5% and appeared reasonable, since they were not
very different from the buoyant density determined for C. oligotrophus.
| |
DISCUSSION |
Our data demonstrate that the biomass of small bacteria at low
concentrations in a mixture can be determined from measurements of
forward light scatter intensity by flow cytometry. Constraints imposed
by Rayleigh-Gans theory on the theoretical curve (37) give
an upper limit for the method of 1.2 pg (dry weight) per cell (about 6 µm3 in size) for bacteria with an axial ratio of three,
assuming that m is 1.03 and that elongated cells are aligned
in the flow stream, as the data suggest. The lower limit is set by
instrument capability. For the Cytofluorograf IIS, it is about 0.005 pg
(0.025 µm3) per cell at the highest gain setting that
shows no evidence of photomultiplier saturation. Although the dynamic
range of the amplifiers gives only about 2 decades of cell mass (Fig.
4B, C, and D), the data show that the full theoretical range can be
accommodated with an error of only 5% when internal standards for
signal normalization are used along with gain changes. Concordance
between population counts determined by flow cytometry and those
determined with a Coulter Counter allowed total biomass for each
subpopulation to be computed on the basis of population, cell mass, and
sample volume. Since 100 cells in a 0.1-ml sample can be resolved
(14), biomass as low as 0.5 pg can be measured by this
method.
Agreement between dry mass measurements obtained from forward
light scatter intensity and values computed from Coulter Counter volume and buoyant density (cell) measurements (Fig. 2)
depended on corrections of m for medium composition and
species-specific effects of formaldehyde absorption and on estimates of
the density of dry matter. The value of dry, 1.39 g/cm3, for C. oligotrophus reflects the
greater contribution of high-density nucleic acids to the dry matter of
small cells and is within the range reported by others for cultures of
marine bacteria (57). The influence of growth conditions on
the composition of dry matter (17) and dry
was likely to have contributed to the differences between dry mass
determined from flow cytometry and values computed from Coulter Counter
volume and buoyant density. Buoyant density can vary with growth
conditions (72) and is affected by cellular inclusions
(4), capsules and gas vacuoles (28), and medium osmolarity (5) but not by growth rate, at least for
E. coli in continuous cultures (41).
Although cells were harvested at different stages of the growth
cycle, intraspecies variations in density were expected to be modest in
this study, since there was constancy in medium preparation and culture
incubation conditions. Changes in the specific refraction increment
factor based on variations in the compositions of bacteria were less
than 1% and so should not have significantly influenced
dry. Error due to variations in axial ratio from 2:1 to
6:1 among the rod-shaped bacteria was less than 10%, based on
Rayleigh-Gans calculations (37).
A dry weight content of 15 to 20% determined for C. oligotrophus contrasts with values of 50 to 60% reported for
marine bacteria (10, 43, 57). However, due to small
cell size, cell shrinkage during preparation for microscopy, the
presence of debris in natural aquatic samples, and the large error seen
in measurements of standard microspheres observed here, there is
general agreement that reliable data for the cell volumes needed for
such assessments are not easily obtained (10, 30, 45).
Agreement between the dry weight content determined here for
E. coli and values in the literature and the
differences between the buoyant densities observed here for
E. coli and C. oligotrophus support the
dilute nature of the marine isolate.
Greater precision in dry mass measurements from forward light scatter
can be obtained by developing standard curves for specific bacterial
strains. In this study, we used C. oligotrophus for standardization. Since there was little variation in the axial ratios of the organisms, the shape of their standard curves
remained the same. K for E. coli was
adjusted to accommodate differences in the absorption of formaldehyde,
dry, and the growth medium. Reduced precision is to be
expected in an analysis of undescribed organisms. Based on differences
in cell shape, an overestimate of 30% for dry mass could occur if the
standard curve used here were applied to spherical cells with an
equivalent mean cell volume. For cocci larger than 1.5 µm3, it would be useful to compute a new curve with an
axial ratio of one. The small size of bacteria in aquatic systems
minimizes error due to differences in cell shape (Fig. 1C).
If buoyant density is known and constant, biomass in terms of wet
weight can be obtained from dry mass by flow cytometry and Xdry/Xwet or, as
previously suggested (37), a standard curve calibrated
according to Coulter Counter volume can give organism size for
computing wet weight from cell volume and density.
The large difference between m values for bacteria and the
latex microspheres often used as size standards as well as
internal standards for comparison among samples is noteworthy. Due to
much greater forward light scatter intensity from microspheres (Fig. 1A) and inconstancy in the dependence of light scatter intensity on the
size of large particles with a high refractive index (Fig. 1B, inset),
the use of latex particles as size standards for biological cells may
be problematic, as suggested by others (7, 20).
The method presented here is an improvement over other flow cytometric
methods for determining bacterial biomass (3, 20, 50)
because it is more general, due to its foundation in light scatter theory, it accounts for differences in cell shape and relative refractive index (37), and it extends analysis to
very small bacteria and to mixed populations. It is particularly
valuable when biomass is low and cell numbers are limited, as in
experiments for quantifying viability (16) and evaluating
the nutritional requirements of new species isolated by extinction
culture techniques (54). In combination with DNA analyses,
biomass measurements for resolved subpopulations offer improved
evaluation of bacterial growth according to cell cycle theory (18,
59). This ability to quantify light scatter intensity in terms of
biomass enables inquiry toward an improved understanding of microbial
processes with unprecedented detail.
| |
ACKNOWLEDGMENTS |
We thank Knut Stamnes and Yong-Xiang Hu of the Geophysical
Institute, University of Alaska Fairbanks, for helpful discussions and
the Mie calculations; Pham X. Quang of the Mathematics Department, University of Alaska Fairbanks, for help with formulating the standard
curve; and Michael Dowling of the Mineral Industry Research Laboratory
for CHN analyses.
This work was supported by grants from the Biological
Oceanography and Metabolic Biochemistry sections of the National
Science Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Marine Science, University of Alaska Fairbanks, Fairbanks, AK
99775-1080. Phone: (907) 474-7709. Fax: (907) 474-7204. E-mail:
brrob{at}ims.uaf.edu.
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Applied and Environmental Microbiology, October 1998, p. 3900-3909, Vol. 64, No. 10
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
