Northeast Fisheries Science Center Reference Document 03-11
Accuracy
enhancement of microscope enumeration of picoplankter Aureococcus
anophagefferens
by John B. Mahoney1, Dorothy
Jeffress2, John Bredemeyer3,
and Kari Wendling1
1National Marine Fisheries Serv., James J. Howard Lab.,
74 Magruder Rd.., Highlands, NJ 07732
2National Marine Fisheries
Serv., Milford Lab., 212 Rogers Ave., Milford CT 06460
3Bureau
of Marine Resources, Suffolk County Dept. of Health Serv., Evans K. Griffing
County Ctr.,
Riverhead NY 11901
Print
publication date August 2003;
web version posted August 29, 2003
Citation: Mahoney, J.B.; Jeffress, D.; Bredemeyer, J.; Wendling, K. 2003. Accuracy enhancement of microscope
enumeration of picoplankter Aureococcus anophagefferens. Northeast Fish. Sci. Cent. Ref. Doc. 03-11;
18 p.
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ABSTRACT
Two main types of enumeration error were experienced in the use of
an immunofluorescence protocol for identification of the picoplankter Aureococcus
anophagefferens from western New York Bight coastal waters. Microscopist
discrimination was affected when cells were too numerous to count reliably
or too lightly stained to be identified. This was remedied by sample
dilution, and increase of antisera and stain concentration and incubation
time. The second type, extensive clustering of A. anophagefferens cells
in monospecific clumps, and as embeds in floc matrices, was much more
problematic. To remedy this, cell disaggregation was sought
through mechanical and chemical treatments. A single treatment effective
for all samples was not found but various treatment combinations greatly
improved enumeration. Varied efficacy of treatment regimes among samples
suggests that cell binding strength was variable.
KEY WORDS: Aureococcus anophagefferens enumeration, harmful algal
blooms, brown tide, New York Bight.
INTRODUCTION
Northeast United States "brown tide" species, Aureococcus anophagefferens, cannot
be distinguished reliably from similar 2-3 µm diameter picoplankters
with phase contrast or epifluorescence microscopy (Sieburth et al.,
1988). For more than a decade an immunofluorescence protocol (Anderson
et al.,1989) has been the main means to identify and enumerate the
species. The method is, or has been, relied on in approximately a half
dozen laboratories (Kulis, personal communication). The NMFS James
J. Howard Marine Sciences Laboratory used the method in 1997-2001 to
determine distribution of A. anophagefferens in the western
New York Bight and track bloom development in the New Jersey Barnegat
Bay-Little Egg Harbor estuarine system. During a 1999 bloom, and subsequently,
serious error in enumeration of many samples was experienced, primarily
due to tenacious cell clumping. A. anophagefferens embedding
in floc matrices of amorphous unidentified material, plankton spp.,
and detrital particles, contributed to clustering error. Enumeration
accuracy also was affected when A. anophagefferens concentration
was high, and when cell staining was insufficient due to high cell
number and abundance of particulates. These enumeration problems were
not present or were not recognized in samples from the Barnegat Bay-Little
Egg Harbor system during a 1997 bloom, or in 1998, a non-bloom year
when A. anophagefferens maximal abundance was ~104 cells
ml-1. This paper outlines a series of sample treatments
and immunofluorescence protocol changes to overcome or minimize these
error sources.
BASIC METHODS
The immunofluorescence protocol for A. anophagefferens enumeration
as developed by Anderson et al.(1989) is: (1) A. anophagefferens in
water samples is preserved with 0.6-1.0% glutaraldehyde; samples are
stored at 4°C until processed; (2) When processed, a small aliquot
(e.g., 200 µl) is incubated with 1.0 ml of 3% normal goat serum
for 40 min. in a 12 x 75 mm test tube; (3) The aliquot is then rinsed
with10 ml of phosphate buffered saline (PBS) onto a 0.2 µm pore
25-mm diameter black polycarbonate membrane filter, backed by a 25-mm
glass fiber filter, in a 25-mm micro-filtration funnel; the filter
is rinsed three times with 10 ml of PBS; (4) One ml of A. anophagefferens antiserum
(1:3200 dilution) is applied; after incubation for 40 min., the filter
is PBS-rinsed as before; (5) A 1:800 dilution of FITC conjugated goat
anti-rabbit antiserum is applied, incubated for 20 min., and the filter
PBS-rinsed as before; (6) The filter is gently dried, placed on a slide,
and covered with a drop of 9:1 glycerine/PBS and a cover slip; (7)
Slides are examined at 400X using an epifluorescence microscope with
a FITC filter set; A. anophagefferens is identified by fluorescent
labeling around the cell perimeter, resembling a green ring or halo.
Using a cross pattern over the membrane filter, 50 fields are enumerated.
Processing of a 200-µl sample aliquot provides an estimated detection
limit of 100 cells ml-1. Subsequently, Anderson et al. (1993)
recommended 1.0-µm pore polycarbonate membrane filters, and application
of glycerine/PBS to the cover slip, rather than the filter.
Protocol modifications made by J. Bredemeyer (unpublished), N. Y.
Suffolk County Department of Health Services (SCDHS), and adopted
by the James J. Howard Marine Sciences Howard Laboratory (HL), include
doubling the salinity of PBS, from 8.7 to 17.4 PSU, to make it more
isotonic with the samples; change of the polycarbonate membrane filter
pore size to 0.8 µm; incubation with goat serum on the membrane
filter rather than in a test tube; increase of incubation time of
goat serum, primary anti-serum and secondary anti-serum to 45, 45,
and 30
min., respectively; decrease of PBS rinsing following secondary anti-serum
incubation to one rinse; and change of glycerine/PBS ratio to 5:1.
Eventual increases in anti-sera concentration and incubation time
at HL are discussed below. HL personnel were trained in the immunofluorescence
procedure by SCDHS personnel. SCDHS personnel had been trained by
the
Anderson Laboratory, Woods Hole Oceanographic Institution (WHOI).
At HL, slides routinely were prepared on one day, refrigerator-stored
in covered trays, and examined the next day using a Zeiss Axiovert
microscope. Prepared slides could be refrigerator-stored for at least
several days with no apparent reduction of cell numbers or fluorescent
stain brightness. Enumeration of A. anophagefferens in each
sample was done at least twice, by two microscopists when possible,
or by the same microscopist at different times. Initially, 60 fields
per count were enumerated; later this was extended to 100 fields per
count. If counts were within 20% of each other, they were assumed to
be representative and were averaged. If initial counts were not in
such agreement, counting was repeated. A cross pattern (as on a clock
face:12 to 6, 9 to 3, etc.) was used in counting; in a traverse, the
stage was advanced field-to-field in a random manner. Distributing
enumeration effort uniformly helps to minimize the effect of non-random
cell distribution on the membrane, especially concentration at the
periphery (Scientific Committee on Oceanic Research, UNESCO, 1974).
During slide examinations, besides cell enumeration, cell staining
level and overall cells/background contrast was noted, as well as the
presence of A. anophagefferens cells in clumps or detrital aggregates. "Clump" refers
to aggregation of A. anophagefferens cells into a monospecific
cell mass. "Embed" refers to aggregation of cells (usually not contiguous)
in a matrix of unidentified apparently organic material, other phytoplankton
spp., and various particulates. Clumps were identified by their fluorescence
and form (they resemble a cluster of grapes). Numbers of embeds and
trapped cell numbers; numbers of clumps and approximate observable
clump dimensions, including diameter or length and width, were noted.
Cells in small clumps, <10 cells, were counted; counting
of cells in larger clumps is considered unreliable. Focusing at different
planes was done for each field; this was especially necessary when
counting cells in small clumps.
Cell disaggregation tests were done at HL, initially in consultation
with SCDHS. Field samples rather than standardized material were used
for the tests. In consequence, test sample character varied. Some of
the test samples, collected by SCDHS from various sites in the Long
Island Peconic Bay system, were previously counted by SCDHS microscopist
J. Bredemeyer. These data provided inter-lab count reference. Some
multiple replicate SCDHS Long Island samples were pooled for treatment
tests. HL samples collected during a 1999 Barnegat Bay-Little Egg Harbor
brown tide also were used in disaggregation tests. SCDHS samples are
identified by permanent station number and collection date, HL samples
by a dedicated number.
RESULTS AND DISCUSSION
Causes of Enumeration
Error; Some Remedies
High A. anophagefferens concentration in some New Jersey samples
increased count difficulty and, depending on the microscopist, to a
greater or less degree contributed to count inaccuracy. Preparations
considered "too numerous to count" (TNTC) were encountered for Barnegat
Bay-Little Egg Harbor bloom samples in mid-May 1999, and subsequently.
This fairly routine problem was addressed in reprocess by reducing
the amount of sample filtered and/or diluting the sample. Similarly,
when Long Island A. anophagefferens samples are enumerated by
SCDHS, reprocess is done when counted cells exceed 600. Insufficient
staining also was encountered in New Jersey sample preparations, associated
with high numbers of A. anophagefferens and/or abundance of
particulates. The latter apparently sequestered stain. This reduced
ability to discriminate A. anophagefferens cells. In addition
to sample dilution and/or reducing the volume of sample filtered, insufficient
staining was remedied by doubling the concentrations of the primary
and secondary anti-sera (advised by D. Kulis, WHOI). Another measure,
advised by K. Milligan who participated in testing of the immunofluorescence
protocol while at the Marine Sciences Research Center, SUNY, Stony
Brook, NY, increase of primary and secondary anti-sera incubation an
extra 15 min., to 60 min. and 45 min., respectively, also was adopted.
These measures greatly improved counting of some samples. For example,
counts (cells ml -1) in two samples rose from 86,020 and
500,711, to 376,541 and 1,079,024, respectively, increases of 337%
and 115%, respectively. SCDHS did not incorporate these changes because
the staining problem was deemed less serious in Long Island samples.
Erroneously low A. anophagefferens enumeration due to cell
aggregation was far more problematic than cell number TNTC or insufficient
staining. Cell clustering can greatly compound non-random dispersion
of cells on the filter membrane, and is considered a major cause of
microscope enumeration error (Scientific Committee on Oceanic Research,
UNESCO,1974). Referring to enumeration error caused simply by the presence
of diatoms in chains, Holmes and Widrig (1956) reported the difficulty
of obtaining accurate estimates of abundance when cells were so clustered.
In our study, extensive clumping, with pronounced non-random distribution
of A. anophagefferens cells was first noted in a May 1999 Little
Egg Harbor, NJ, sample; this sample had been stored five days prior
to processing. Subsequently, clumps of A. anophagefferens cells
(sometimes of 75-100 cells), and cells embedded in matrices of varied
composition, were frequently observed in samples from the Barnegat
Bay-Little Egg Harbor system, especially during blooms.
The occurrence or degree of A. anophagefferens aggregation
in nature is undetermined. Cell aggregation was not assessed in unpreserved/unprocessed
samples because of unreliability of distinguishing A. anophagefferens from
other picoplankters using light microscopy. Suggesting that aggregation
of live cells in a natural population can occur, at HL clumping in
older cultures of an axenic A. anophagefferens strain (Center
for Culture of Marine Plankton 1984) is common. Rigorous mixing, e.g.,
30 sec. or more of vortexing, is required to disperse cell floc in
the latter but even then smaller aggregates (1 mm diameter) often remain.
This clumping was not seen in bacterized strains (CCMP 1784 and 1794
- 1784 is the parent culture of 1984); presumably associated bacterial
culture contaminants metabolized the binding material. One of the authors
(JB) observed that clumping was more likely in samples from certain
Long Island locales than others, raising the question of whether it
may be linked to cell metabolism and/or physiological status. Clumping
appeared more prevalent in New Jersey samples with higher A. anophagefferens concentrations
than in samples with lower concentrations (it expectedly would be more
noticeable with higher cell concentration). Clumps of preserved A.
anophagefferens cells were found in some sample preparations soon
after collection, e.g., within a day. However, cell clumping can initiate
or advance during storage of preserved samples in which cells apparently
were in, or close to, single cell suspension a short time earlier.
Cell count reductions of 20-30% were found for certain samples reprocessed
after an additional week of storage. This suggested cell loss, but
when reprocessed with cell disaggregation treatment counts close to
the original ones were obtained.
Counts routinely are done of only a relatively small number of microscope
fields, i.e., 25-50 by SCDHS, 120-200 by HL, out of approximately 4,000
fields for SCDHS and 6000 fields for HL (field size in the respective
laboratories is determined by the Whipple count disc being used). Clumping/embedding
lowers the likelihood of a small part of the total filter membrane
being representative. It might be thought that accuracy could be increased
by greatly increasing the number of fields counted, thereby increasing
potential for encountering cell clumps. Fournier (1978) believed the
only certain method is to count all the cells on the membrane. These
measures would not be practical or sufficient with A. anophagefferens due
to the large numbers of cells frequently encountered and the need,
at least during a bloom, to process many samples. Even great expansion
of membrane area counted likely would not be a sufficient solution
when cell aggregation is pronounced. Microscope counting of cells embedded
in floc matrices is difficult but doable. Estimation of cell numbers
in larger clumps (>10 cells) is highly error prone, however. Although
cells in surface planes of A. anophagefferens clumps are countable,
discrimination of cells at all planes would be problematic at best.
Another complication is varied clump shape. Clumps that may have formed
in nature from cells aggregating in the water column likely would be
globular. Cells aggregating during storage in the collection vessel
likely formed layered clumps. We suspect we have seen both types but
were unable to view clumps adequately; they could not be rotated to
view all their dimensions.
Counting of cells in a single plane in larger clumps obviously would
consider an inadequate fraction of aggregated cells. Illustrating how
this could affect enumeration, approximate length and width of four A.
anophagefferens clumps (variously having slightly bulbous sides
or incomplete corners) were measured with an ocular micrometer. Cells
in a single surface plane in the four clumps were counted, providing
a total of 388. If these clumps are assumed roughly cylindrical, with
depth approximating diameter (assuming cylindrical shape is conservative
because a cuboidal shape would contain more cells), the estimated cell
total would be 3019. The two estimates vary by a factor of 7.78.
According to Lund et al. (1958), the accuracy of results needed for
estimating algal populations is not normally very large; algal population
estimates with an accuracy of +50% may suffice for investigations
concerned with generations, i.e., change in abundance of 100%. Greater
accuracy than this is needed for a species that causes highly detrimental
effects. Bricelj et al. (2001) reported that A. anophagefferens concentrations
as low as 3.5 x 104 cells ml-1 significantly
reduce northern quahog, Mercenaria mercenaria, feeding, and
clearly A. anophagefferens enumeration can be subject to serious
error when its cells are aggregated. Single cell suspension is essential
for accurate counting of microorganisms (Nebe-von-Caron et al., 2000).
The material causing tenacious cell binding of A. anophagefferens or
its embedding in floc matrices has not been identified. Hypotheses
explaining mucilage formation in the sea assume accumulation of colloidal
and gel-like polysaccharide from phytoplankton exudates (Alldredge
and Crocker, 1995, in Najdek et al., 2002). Larger aggregates (defined
as clouds 0.5-5 m) may be formed directly by the coagulation of gels
entrapping plankton cells and other particles (Degobbis et al., 1993,
in Najdek et al., 2002). A. anophagefferens has an exocellular
layer of organic material, probably mucopolysaccharide, which is sometimes
copious (Sieburth et al.,1988). If this excreted material has adhesive
properties it could be the binding material in clump formation, and
it possibly is the material in which cells were seen to "embed" in
field samples. Middlebrook and Bowman (1964) remarked that purification
of cultures of algae having mucopolysaccharide capsules is particularly
difficult because bacteria stick tenaciously to them. At HL, cultures
of an axenic strain of A. anophagefferens (CCMP 1984) regularly
form an extensive concentration of cells in a mucus-like material at
culture tube bottoms. Evidenced by resistence to dispersion of cells
by vigorous vortex mixing, this material has binding strength. Whether
degree of binding might be based on amount or quality of the binding
material, and whether cell binding strengthens or weakens over time,
likewise is unknown. Therefore, disaggregation of A. anophagefferens was
approached empirically, using a variety of treatment options employed
in various similar microbiology/cytology applications.
Cell Disaggregation Protocol
Development
To achieve disaggregation of A. anophagefferens we assumed
high integrity of the original cell complement despite prolonged storage.
Glutaraldeyhde is an excellent fixative for phytoplankton in general,
and is known to maintain cells for long periods. Anderson et al.(1993)
found that counts of A. anophagefferens in glutaraldehyde-preserved
field samples were constant over an extended period of time (six months).
Cell numbers of glutaraldehyde-preserved phytoplankton, assessed by
epifluorescence microscopy, were unchanged after a year in storage
(Booth et al., 1993).
Sample mixing was examined first. Routine A. anophagefferens immunofluorescence
protocol mixing procedure, i.e., inverting the sample vials 50 times,
was insufficient when cells were aggregated. Anderson et al. (1993)
reported no difference in counts whether samples were shaken vigorously
or mixed gently; apparently A. anophagefferens cells in the
samples they compared were not aggregated. Vortex mixing (suggested
by J. Bredemeyer) was tested as a replacement. Cause for caution about
vigorous mixing was that A. anophagefferens lacks a cell wall
(Sieburth et al., 1988); and Anderson et al. (1989) reported fragility
of A. anophagefferens cells unless fixed for several weeks.
Kulis (personal communication) expressed concern that vortexing might
disrupt cells. At HL vortexing was optimized with the particular mixer
in use, a Vortex-Genie with variable power setting, and an on-off switch
activated by pressure on the mixing head. Mixing was done by repeatedly
pulsing the mixer long enough to get a vortex, and presumably considerable
shear force. Madrigal et al. (1993), in addition to other treatments,
recommended shear force through agitation to separate cells for flow
cytometric analysis. Long-term glutaraldehyde-preserved A. anophagefferens cells
proved relatively robust, and a 100 pulse vortexing (this took about
60 sec.) eventually was adopted as standard. Longer-term vortexing
(as a sole treatment) was not additionally beneficial. Pulsed vortexing
is assumed to be more effective for cell disaggregation than continuous
vortexing but relative efficacy was not determined. Different mixer
power settings were tried and a moderate setting ("3.5") was adopted
- approximately one third power. Higher power than this ("4") resulted
in cell loss in two test samples. Eventually, multiple vortexing applications
as described were used during sample cell disaggregation treatments.
Although an improvement over sample vial inversion (data not shown)
vortex mixing, as is shown in Table 1, did
not adequately remedy cell aggregation.
Detergent in combination with other treatments commonly is used to
lyse cells, e.g., for harvesting of subcellular constituents (Buetow,
1973). However, McDaniel et al. (1962) used detergent (ARKO Hospital
and Laboratory Detergent) to separate bacteria from mucoprotein-capsulated
blue-green algae to which they were strongly attached, and (Kutkuhn
(1958) had general success using detergent (unspecified) to remedy
phytoplankton cell aggregation due to centrifuge compaction. R. Guillard
(personal communication) recommended Aquet detergent (Bel-Art Products)
for cell disaggregation. Results with Aquet, tested at 0.5, 1.0, 1.5
and 2.0 % with 3-10 min. exposure, varied with concentration and sample
(data not shown). For some samples enumeration was improved; at minimum,
clump size was reduced. In two of four samples 1.0% had no more effect
on cell separation and cell count than 0.5%, but in another the higher
concentration was additionally beneficial and there was very dramatic
benefit in the remaining sample. For the latter sample, when treated
with 0.5% Aquet, large clumps and embeds and uneven staining persisted,
whereas 1.0% Aquet provided better cell dispersion, greatly reduced
cell aggregation, and improved staining. Aquet at 2.0% lowered count
levels with some samples so 1.0 %, the concentration recommended by
Guillard as a starting level, was adopted. Combination of Aquet and
vortexing was more beneficial than Aquet singly. Long-term Aquet treatment
(17, 26, 41 and 47 hr, respectively) showed some additional cell disaggregation
but at the cost of reduced cell staining. Limited trials were made
of some other detergents: FL-70 (Fisher SF 105-1) reduced clumps and
improved enumeration, but apparently was disruptive to cells and filter
membranes. Micro-90 (International Products Corp.) reduced cell aggregation
in one test, but caused clogging of filter membranes in two other tests.
BRIJ 35 (Sigma P 1254) was less effective than Aquet.
Because combined vortexing and Aquet was only partially effective,
a protocol developed by Velji and Albright (1986) to free bacteria
associated with particulates, was adapted for A. anophagefferens.
Their procedure includes fixation with an aldehyde, treatment with
a sequestering/deflocculating agent (tetra sodium pyrophosphate), and
ultrasound. In their study, this treatment combination provided more
random dispersion of bacteria on the filter membrane, and increase
in cell number enumerated. The pyrophosphate concentration they recommended
for sea water samples, 0.001M, lowered A. anophagefferens cell
enumeration by approximately half, so 0.0001M was adopted. Pyrophosphate
was effective at the lower level, and a large margin of safety was
considered necessary. The 0.001M pyrophosphate treatment time of 15
min.Velji and Albright (1986) recommended eventually was at least doubled
for our treatment with 0.0001M. This treatment minimum of 30 min. was
extended incrementally, but usually not past 60 min., for samples in
a batch because 15 were processed sequentially. Sonication was approached
carefully to avoid cell disruption. Velji and Albright (1986) used
their BioSonik II sonicator, with a 4-mm probe, at a power level of
100 W for 30 sec.. At HL, sonication was optimized for A. anophagefferens with
a Misonix Model XL2015 sonicator having a 12.7-mm probe. Sonication
of 10 ml samples was done in polystyrene Coulter Counter cups with
the probe immersed to about half sample depth. To minimize heat build-up
in the sample, sonication was pulsed for 0.05 second per second. Power
setting was tested incrementally, and power setting "4" was adopted,
although the cells generally could tolerate the next level of sonication.
Sample-to-sample difference in what sonication power level cells could
tolerate was apparent. Various exposure times were tested, with 70
sec. (this includes non-pulse time) adopted finally. Results of longer
sonication varied, from a net loss to a gain of cells available for
enumeration. Presumably, if most cells initially were in single suspension
these might be especially vulnerable to disruption by prolonged sonication,
whereas many cells in clumps and embeds could be partially shielded
temporarily, and then disaggregated. Nebe-von-Caron et al. (2000) described
optimal recovery of aggregated bacteria cells with a particular regime
of sonication amplitude and time, and cell destruction when exposure
time was five times the optimal. A. anophagefferens sonication
term maximum tolerance is far less; as little as 15% increase of exposure
term beyond apparent optimal could result in destruction of cells.
Table 1 shows results of
tests of vortex mixing, pyrophosphate treatment and sonication on a
group of 10 Long Island samples. In these tests vortexing was done
for 30 sec.; pyrophosphate treatment for 15 min.; and sonication for
30 sec.. The data illustrate that vortex mixing as sole treatment was
inadequate. Pyrophosphate treatment coupled with vortexing was sufficient
to recover (within 20%), or exceed, the SCDHS counts in tests 5, 7,
8, 9. In tests 1, 2, 3, 6, 10, this treatment was inadequate. The latter
group had the higher cell levels, which suggests a link between cell
concentration and treatment adequacy. Sonication combined with vortexing
and pyrophosphate was effective (within 22%) in restoring, or exceeding,
the SCDHS counts in all tests. For most samples SCDHS/HL count differences
were less than 50%, but for two samples (tests 9,10) increases were
slightly over 100%. Because the various treatments in these tests did
not incorporate the more effective final treatment times (mentioned
above) HL count accuracy might be questioned. However, cell aggregation
likely was not serious in these samples; it was seen pre-treatment
in only two samples of the group. Furthermore, if A. anophagefferens cell
excretion is stress-associated, as has been shown for other phytoplankton
(Myklestad, 1995), it should be noted that the test samples were collected
in December when A. anophagefferens would be advantaged due
to its ability to grow at low temperature (5.0oC) (Cosper
et al., 1989), absence of heat stress, a presumably ample nutrient
supply, and reduced competition from other phytoplankton including
picoplankon that typically are summer dominants. Cell levels in most
of these samples were only 3.0 x 10 5 cells ml-1 or
lower, whereas levels encountered during a spring/early summer bloom
often were 106 cells ml-1 or greater. In the
Barnegat Bay-Little Egg Harbor system, cell aggregates were common
in bloom samples but not in winter samples. Besides showing utility
of certain treatments, the varied results in these tests were an early
indication that cell aggregation alleviation could differ sample to
sample. That is, a treatment regime that apparently could disaggregate
cells, or at least reduce aggregation below our level of detection,
in one sample could be far less effective with another sample. This
suggests cell binding strength difference. The Long Island test samples
were collected at different times from sites with different water qualities,
e.g., salinity, nutrient level. It is likely that the cells in these
samples at the time of collection had varied health and metabolic state,
and possibly varied type and amount of cellular excretion.
SCDHS processed the Long Island samples used for the tests discussed
above (Table 1) 3-7 weeks after collection
in December 1999. HL reprocessed these samples 12-19 weeks after collection.
In the time between the two processings cells had so aggregated that
mechanical and chemical treatments were required to disperse them.
How effective were the treatments? A long standing enumeration guideline
is that 100 cells are counted to give a 95% confidence interval of
the estimate within +20% of the mean value, and 400 cells for +10%
of the mean (Lund et al., 1958); this is still commonly accepted (Throndsen,
1995). Of the HL cell counts only the test 4 count was under 400 cells,
all others were 500 to over a thousand. Assuming a 10% confidence interval,
considering just HL counts, the maximum percent count difference in
tests 1-4 is 14; for tests 6-8 the maximum percent count difference
is 30; and percent differences for tests 9 and 10 are 52 and 55, respectively.
SCDHS cells counted ranged approximately 200 to 400, with the majority
in the 300 to 400 range. With confidence interval for half of the SCDHS
counts at, and the other half greater than, +10% of the mean,
differences between HL and SCDHS cell levels would be additionally
lessened. The general agreement between the SCDHS and HL post-sample
treatment counts suggests that the mechanical and chemical treatments
applied were highly effective, and that cell aggregation was not a
serious problem when SCDHS processed these winter samples. A. anophagefferens cell
aggregation in Long Island samples during other seasons remains to
be assessed.
The modified Velji and Albright (1986) protocol
could be very effective in restoration of single cell suspension, but
refractory cell aggregates in some post-treatment sample preparations
indicated need for additional treatment. Aquet detergent previously
had proved to have benefit so was tested (1.0%; 15 min. exposure) on
aliquots of the same sample, in combination with vortexing for ~30
sec., pyrophosphate for ~15 min., and sonication times that varied
from 30 to120 sec.(Table 2). Aquet addition
resulted in considerable increases in enumeration levels in six of
seven tests. The seventh test showed the least increase in cell level
with Aquet treatment. Presumably the 90 sec. sonication term used in
this test alone was sufficient to disaggregate cells. This was noticeable
but less so with test six (80 sec. sonication). Sonication for 120
sec. was the only term over 90 sec. tested and resulted in pronounced
cell destruction (not shown). A 90 second sonication term was beneficial
for this sample but cell loss was experienced with previous test samples
when sonication term was over 70 sec. It is considered preferable to
retain an apparently safe sonication term, and employ Aquet as an additional
disaggregation treatment. It should not be concluded from these results
that the four-treatment combination necessarily will provide dramatic
enumeration change; rather simply that it is effective. A disadvantage
of the four-treatment combination is that it results in clearing of
the black polycarbonate filters. To minimize this, filters were flushed
with 5.0 ml of PBS immediately after sample aliquot placement on the
filter. The clearing reduced contrast but did not prevent accurate
identification and enumeration. In all but occasional preparations
cells showed adequate to bright staining. Necessity to verify some
counts by reprocessing treated samples revealed generally detrimental
effect of prolonged chemical treatment. Lowered cell enumeration was
obtained for four of five samples reprocessed six days after treatment.
It probably is best to assume that treated samples are no longer useable
more than one day after initial process. If aliquots of a treated sample
are diluted (we used 3:7/2:8 sample/diluent, routinely), or reprocessed < a
day after treatment, cell disruption likely will be avoided.
Reprocessing of HL samples progressed from those collected during
non-bloom, to bloom initiation, and finally bloom, conditions. Cell
aggregation in the sample collection ranged from none or minimal to
very cell-aggregated. Cell disaggregation protocol development reflects
this, with treatment options added, and exposure times lengthened,
as increasingly more problematic samples were encountered. Combined
vortexing, Aquet, pyrophosphate, and sonication treatment sufficed
until bloom samples were processed. Refractory aggregates in these
necessitated more treatment. Enzyme preparations including hyaluronidase
(Sigma H 2126) (recommended by S. Merlin, Becton Dickinson Biosciences,
personal communication) and Accumax, a commercial multi-enzyme product
for dissociating cell clumps (Innovative Cell Technologies, Inc.) were
evaluated. Hyaluronidase was tested at 1, 2, 3, and 4 x 10-4 %.
The lowest concentration was approximately the concentration Merlin
(personal communication) employed to suppress clumping of human and
rat intestinal epithelial cells during flow cytometry analysis. These
cells, as reported for A. anophagefferens (Sieburth et al.,
1988), secreted large amounts of mucopolysaccharide. The concentration
Merlin employed was used as a starting and lowest level for A. anophagefferens disaggregation
because separation of preserved cells in formed clumps likely would
be more demanding than clumping suppression (for extrapolation an A.
anophagefferens level of 106 cells ml-1 was
assumed). Initial tests showed hyaluronidase at 10-4 % to
be very beneficial, raising enumerated cell levels substantially (22-69%)
in five of seven samples. Results of tests with higher concentrations
were varied, with 2 or 3 x 10-4 % usually more effective
than the lowest concentration, and the highest concentration not additionally
beneficial. Concentrations of 2 and 3 x 10-4 % were incorporated
in the final treatment regime. In combination with the above treatments,
Tween 80 (Sigma P 4780), a surfactant commonly used as a dispersing
agent, was tested at 0.5, 1.0, 1.5 and 2.0 %; 1.0 % was most effective
most often and this level was adopted. The higher concentrations were
beneficial and/or non-disruptive with some samples but apparently disrupted
cells in others. Interestingly, sonication, vortexing, Tween 80 and
detergent, sodium dodecyl sulfate, are all employed in an immunofluorescence
protocol for detection of Cryptosporidium and Giardia cysts
in surface fresh water (LeChevallier et al., 1991). Another enzyme
preparation, Accumax, was tested for A. anophagefferens disaggregation
at 0.5, 1.0, 2.0, 3.0 and 4.0 %. Either 0.5 or 1.0% was beneficial;
concentrations > 2.0% resulted in lowered cell counts. As
with the other options, it could not replace combined treatments. Compared
with combined treatments, 1.0% Accumax treatment of 15 test samples
with just vortex mixing provided cell counts lower for 9, equivalent
for 3, and higher for 3 (not shown). In routine sample processing it
was employed at this level with one of the treatment combinations.
When beneficial in sample processing, Accumax could raise enumeration
level considerably, e.g., for several samples increases of 23-45%.
Although beyond the scope of this study, enzyme treatment of clumped A.
anophagefferens deserves additional exploration. The mechanical
and chemical treatment options adopted are considered to be at or
close to their safe limits for A. anophagefferens but may
not be sufficiently effective. Increased cell disaggregation with
modest increase of pyrophosphate concentration is a possibility,
if serious contrast reduction can be avoided. Also, a detergent more
effective as a disaggregant might be found. However, testing for
more effective enzyme treatment could be the most profitable approach.
Considerable breakdown of macromolecules in natural waters is through
the action of extracellular enzymes, principally those of bacteria
(Price and Morel, 1990, in Leppard, 1995).
Cell Disaggregation Protocol
(1) 10-ml samples in 15-ml screw-cap polypropylene centrifuge tubes
first are vortexed (100 pulses, or about 60 sec). (2) Desired dilutions,
usually sample:diluent 2:8 or 3:7, are made; dilution depends on the
general anticipated cell level, e.g., stage of bloom development, and
the number of required treatment combinations. Diluent is bay water
filtered with a 0.2-µm pore membrane, and having glutaraldehyde
addition at the usual level for sample preservation. (3) All subsamples,
usually three, are treated with 0.0001M pyrophosphate, 1.0 % Aquet,
and 1.0 % Tween 80; the latter requires vortex mixing to dissolve.
(4) One subsample additionally is treated with 2 x 10 -4 %
hyaluronidase; the third subsample additionally is treated with hyaluronidase
at 3 x 10 -4 % and 1.0 % Accumax. (5) Subsamples are revortexed,
and incubated for 30 min. or until process of a series is complete,
usually not exceeding 60 min. (6) Each subsample is again vortexed
immediately before being decanted into a polystyrene 20 ml Coulter
Counter sample cup, sonicated (at HL, a Misonix Model XL2015 / 5 %
pulse / power setting "4" / 70 sec.), and returned to the sample tube.
(7) The aliquot to be filtered is immediately dispensed on the filter,
and rinsed with 5.0 ml of PBS. It is desirable to have two people working
together at this stage. (8) The Anderson et al. (1989, 1993) protocol,
is then followed as outlined previously with the changes already noted.
Assessment of Cell Disaggregation
Protocol
Use of a heterogenous collection of field samples for disaggregation
tests rather than standardized material complicated protocol development.
However, use of diverse test samples revealed that varied combinations
of treatments were best instead of a single protocol. The treatment
regime that resulted in the highest cell number is assumed most effective
for the particular sample. The highest cell number obtained in the
treatment series is assumed to be the most accurate. Enumeration obtained
when cell aggregates were not detected in a post-treatment preparation
is assumed to approximate best the actual level. Cell aggregates were
not detected in most of the post-treatment preparations. Especially
because microscope scan for cell aggregates is a qualitative check,
not a quantitative measure, absolute accuracy is not claimed. Cell
enumeration with the Anderson et al. (1989, 1993) protocol routinely
is done with counts of 50 fields, and this is generally accepted as
sufficient. However, scan of even 200 fields for cell aggregates may
not be sufficient to determine presence or absence of such. Nevertheless,
there must be some practical limit to microscope observation when many
samples must be processed. Finding a small clump or two of cells in
200 fields of several thousand possible fields suggests that more exist
in the preparation, but the associated count error is undetermined.
Likewise, certainty about absence of cell aggregates in the preparation
when none were visually detected was not attained. In some instances,
judging from post-treatment enumeration level increase, aggregates
apparently were present although visually undetected. Low abundances
of cell aggregates (e.g., one per 100 fields) were seen in some samples
even after the most rigorous treatment. The treatment regime that was
necessary to disaggregate cells in some samples, without discernible
cell disruption, apparently could cause cell disruption in some other
samples. The latter suggests an accurate count may not be unattainable
for a minority of samples, i.e., ones that may have an aggregation
of relatively fragile cells.
For some samples, post-treatment counts were relatively
unchanged from the original; cell clustering likely was never serious
in these. For many samples, especially bloom samples, disaggregation
treatments resulted in considerable increase in cell counts. Table
3 shows enumeration changes for representative samples from the
Barnegat Bay-Little Egg Harbor system, following application of the A.
anophagefferens disaggregation protocol. In bloom initiation samples
cell numbers were generally low, and effects of treatments were relatively
unimportant. Cell aggregation was not detected visually in these. Severe
cell aggregation was encountered first in some bloom development samples,
and continued through bloom duration. Generally, sample treatment to
restore single cell suspension was effective, presumably to a high
degree. For most of the Table 3 examples, treatment resulted in at
least 100% higher cell level, and for the sample showing greatest change
(660) the increase was over 35 times. Unlike the relatively slight
enumeration change (50% or less) with treatment of those Long Island
samples that initially had cell abundance estimates ~ < 200
x 103 cells ml-1 (Table
1), some Barnegat Bay-Little Egg Harbor samples with initial counts
at this cell level showed post-treatment dramatic count increases (e.g. Table
3, 645, 660, 683). Illustrating varied degree of cell binding,
resulting benefit of sample reprocess soon after initial process, with
dilution and additional mixing but no other treatment, for sample 606
was none, slight for sample 660, intermediate for sample 677, and almost
as much benefit as full cell disaggregation treatment for sample 643.
SUMMARY AND CONCLUSIONS
The focus of this study was to achieve increased accuracy of microscope
enumeration of A. anophagefferens populations in the western
New York Bight. Especially during post-initiation bloom development
in this area cell aggregation had rendered enumeration of many water
samples highly inaccurate.
Whether or not A. anophagefferens aggregates in nature in
this area has not been determined, but a certainty is that it can to
a high degree in glutaraldehyde-preserved water samples within a day
of collection. The question of importance of this factor in other areas
has not been resolved. Long Island winter samples reprocessed in this
study had considerable cell aggregation after prolonged storage (12-19
weeks), but apparently not when initially processed 3-7 weeks after
collection. Possible cell aggregation in Long Island samples in other
seasons, e.g., the Spring growth season, was not assessed.
To correct enumeration of HL samples, restoration of single cell
suspension was sought. A single totally effective cell disaggregant
was not found, instead varied combinations of mechanical and chemical
treatments provided best results sample-to-sample. The reported A.
anophagefferens disaggregation protocol is a compromise between
effectiveness and need to not disrupt cells, given apparent wide sample
variability in terms of cell treatment tolerance and type of cell aggregation.
Preserved A. anophagefferens cells can withstand a variety of
disaggregation treatments if they are properly adjusted.
Treatment combinations can largely or completely restore single cell
suspension, although refractory cell aggregates remained in a minority
of samples even after the most stringent regime. Presumably, cell binding
was strongest in these.
The sample reprocessing and present treatment protocol satisfy the
goals of this study, but the treatment regime might be further refined/simplified.
For example, could vortexing be eliminated when sonication is employed?
Could a more suitable surfactant be found to replace the two used in
this study? Enzyme treatment appears deserving of further exploration.
RECOMMENDATIONS
If a microscope scan of A. anophagefferens preparations reveals
cell aggregation, reprocess with disaggregation measures should be
considered. Because the protocol we report was developed for a particular
sample set and may not be universally appropriate, treatment should
be appropriate for the samples being processed. Part or all of our
protocol could be employed, depending on such factors as cell concentration,
cell binding strength, and likely sample time in storage.
Multiple treatments to disaggregate cells greatly extends the time
and effort required to process a given number of samples, so this could
be limited to when most necessary. Accurate population enumeration
may not be necessary in routine monitoring, e.g., to simply determine
presence or absence of the species in high abundance, whereas it is
required when assessing bloom development, and levels for detrimental
effects on the biota. Non-bloom samples, i.e., when cell numbers are < 200
x103 cells ml-1, may not require treatment beyond
vortexing.
Treating a percentage of non-bloom samples should be considered as
a check on count accuracy. Careful general microscope exam of slide
preparations can suggest when additional sample treatment is required,
although this should not be considered an absolute gauge.
Extra caution is recommended regarding a possible secondary pulse
in the fall, an overwintering population, and when a bloom may be initiating.
Collection of multiple samples is recommended at least during bloom
development and duration.
ACKNOWLEDGMENTS
The authors thank R. Guillard, D. Kulis, S. Merlin, and K. Milligan
for helpful technical advice. We thank A. Calabrese, G. Wikfors, R.
Guillard, and T. Smayda for editorial comment. We thank D. Kulis, Woods
Hole Oceanographic Institution, for providing the primary anti-serum
and C. Kevin Becker, Innovative Cell Technologies, Inc., for providing
the Accumax enzyme preparation.
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