Parasitol Res (2002) 88: 326±331
DOI 10.1007/s004360100504
O R I GI N A L P A P E R
Satomi Kato á Dwight D. Bowman
Using ¯ow cytometry to determine the viability
of Cryptosporidium parvum oocysts extracted
from spiked environmental samples in chambers
Received: 19 June 2001 / Accepted: 1 August 2001 / Published online: 15 December 2001
Ó Springer-Verlag 2001
Abstract Cryptosporidium parvum oocyst viability was
determined by a dye permeability assay using a ¯ow
cytometric method. Oocysts were inoculated into small
chambers with soil and biosolids. Oocysts extracted
from soil and biosolids were then stained with propidi-
um iodide (PI) and labeled with a ¯uorescein isothio-
cyanate (FITC)-conjugated monoclonal antibody. The
oocyst population in each sample was determined using
forward and side scatter plots, then further analyzed
with ¯uorescence. A red and green ¯uorescence detector
using gates established single populations of unstained,
PI-stained, or FITC-labeled oocysts. No statistical dif-
ference was observed between viability of oocysts ex-
tracted from soil and biosolids as determined by either
¯ow cytometry or microscopy. The location of excysted
oocysts was changed in forward and side scatter plots.
Results indicated that, although oocysts are not identi-
®ed if they excyst, the ¯ow cytometric method could be
used to determine oocyst viability from spiked envi-
ronmental samples.
Introduction
In vitro excystation methods and dye permeability assays
using microscopy have been used e€ectively to measure
the viability of Cryptosporidium parvum oocysts that have
been inactivated by formalin ®xation, increased temper-
atures, freezing, and ammonia treatment (Fayer and Leek
Presented at the American Association of Veterinary Parasitolo-
gists 45th Annual Meeting. Salt Lake City, Utah, July 2000
S. Kato (&) á D.D. Bowman
Department of Microbiology and Immunology,
College of Veterinary Medicine,
Cornell University, Ithaca, NY 14853, USA
E-mail: sk89@cornell.edu
Tel.: +1-607-2533394
Fax: +1-607-2533384
1984; Jenkins et al. 1997). Although these in vitro viability
assays have proven at the same time less than perfect for
treatments where oocysts are inactivated by processes
such as UV (Bukhari et al. 1999) or ozone (Bukhari et al.
2000) that disrupt the DNA of organisms without dam-
aging the oocyst wall, propidium iodide (PI) staining is a
good indicator of oocyst viability for measuring the
e€ects of environmental stresses. However, the dye
permeability assay using microscopic observation is un-
fortunately a time-consuming process. Flow cytometry
provides a means for faster counting of larger sample
volumes and has been used to detect oocysts of C. parvum
in environmental samples (Vesey et al. 1994), human stool
samples (Valdez et al. 1997), water supplies (Ho€man
et al. 1997), and with PI staining to detect dead oocysts in
environmental samples of water, sewage, and secondary
e‚uent (Medema et al. 1998). Using ¯ow cytometry, large
numbers of samples can be processed (Vesey et al. 1997),
which allows the rapid comparison of the e€ects of envi-
ronmental and soil factors on the viability of oocysts.
In this study, the viability of C. parvum oocysts ex-
tracted from soil and biosolids (processed municipal
wastewater sewage sludges) inoculated within chambers
(the sentinel chambers of Jenkins et al. 1999) was de-
termined using ¯ow cytometry. Comparisons were made
between the viability of oocysts determined by ¯ow cy-
tometry and by epi¯uorescence microscopy. The goal of
this work was to develop a means by which the viability
of oocysts recovered from sentinel chambers could be
rapidly monitored for the purpose of assessing the e€ects
of various substrates on the dye staining of oocysts in
chambers that have been placed within a speci®c matrix
or test vessel.
Materials and methods
Oocyst preparation
Cryptosporidium parvum oocysts were obtained from naturally in-
fected, 7- to 14-day-old calves in Tompkins County, New York. A
sucrose/Percoll(Pharmacia, Uppsala, Sweden) ¯otation method

was used to extract oocysts from calf feces (Jenkins et al. 1997).
After the extraction, oocysts were stored at 4°C in water with an-
tibiotics (100 U of penicillin G sodium ml±1, 100 lg of strepto-
mycin sulfate ml±1, and 0.25 lg of amphotericin B suspension ml±1)
and were used within 1 month of collection.
Oocyst treatment
In the examination of puri®ed oocysts for the purpose of determining
settings on the ¯ow cytometer, puri®ed oocysts were treated with
1%, 25%, and 90% ammonia (0.01 M, 0.29 M, and 1.1 M of am-
monia, measured by an Ammonia Electorode, Orion, Beverly,
Mass.) for 1 h at room temperature. The oocysts were then washed
4 times with 0.1 M PBS using centrifugation. This ammonia treat-
ment is known to inactivate oocysts (PI positive) (Jenkins et al. 1998).
Oocyst excystation
A modi®cation of a previously described excystation method was
used (Campbell et al. 1992; Jenkins et al. 1997). Initially, a 100 ll
aliquot of a suspension of oocysts in a 1.5 ml microfuge tube was
washed 3 times by centrifugation with 1 ml of Hanks' Balanced
Salt Solution (HBSS, Sigma, H8264, St. Louis, Mo.). Approxi-
mately 107 oocysts were pretreated in a microfuge tube with 1 ml of
0.01 N HCl in HBSS (pH 2.3) for 1 h at 37°C on a heating block.
After pretreatment, the oocysts were centrifuged at 13,000 g for
3 min. The supernatant was removed and discarded. Then 100 ll of
excystation ¯uid [0.022 M sodium bicarbonate (NaHCO3) and
0.0002 M sodium deoxycholate (C24H39O4Na) in HBSS, pH 9.0]
was added to the microfuge tubes, and the mixture was incubated
for 3 h at 37°C. After incubation, the oocysts were washed with
0.1 M PBS 3 times and stained with PI and ¯uorescein isothiocy-
anate (FITC).
Oocyst extraction from chambers
The oocysts were inoculated into soil using the sentinel chamber
method (Jenkins et al. 1999). Each chamber contained 1.75 g of
sandy loam soil (5% clay content) or 1 ml of primary biosolids (4%
solid concentration, obtained at Onondaga county wastewater
treatment plant, Syracuse, N.Y.), to which 107 oocysts suspended in
distilled water were inoculated. The inoculated oocysts were then
extracted using a sucrose/Percoll ¯otation method (Brush et al.
1998). In order to extract oocysts from the sentinel chambers, soil
was removed from the chambers and put into a 15 ml polypropyl-
ene centrifuge tube containing 7 ml of a 0.5% 7X detergent (Linbro
7X, Thomas Scienti®c, Swedesboro, N.J.) in phosphate bu€ered
saline (PBS). A small amount of Zirconia/silica beads (0.5 mm) was
added (BioSPec Products, Bartlesville, Okla.), and the soil, beads,
and detergent were mixed for 20 s using a vortex mixer. The 7 ml
mixture was then underlaid with about 7 ml of cold sugar solution
(speci®c gravity 1.18). After the samples were centrifuged at 1,800 g
for 20 min at 4°C, about 3 ml of the interface was gently removed
and transferred to a second tube. Distilled water, 12 ml, was added
to the sample; the diluted sample was mixed thoroughly and cen-
trifuged again at 1,800 g for 20 min at 4°C. The supernatant was
aspirated leaving approximately 1 ml in which the pellet was re-
suspended. Half of each of the six samples was analyzed via ¯ow
cytometry, while the viability of the oocysts in the other half of each
sample was determined using epi¯uorescence microscopy.
PI staining
A modi®ed dye permeability assay (Jenkins et al. 1997) was per-
formed immediately after the oocysts were extracted. A stock so-
lution of PI (Sigma), 1 mg PI ml±1 in 0.1 M phosphate-bu€ered
saline was added to aliquots of oocyst suspensions at the ratio of
1:10 (vol/vol), mixed gently, and the samples were incubated for 2 h
at 37°C in the dark.
327
Fluorescent staining of oocysts
The method was as described previously (Jenkins et al. 1997). After
the oocysts were stained with PI, they were labeled with monocl-
onal antibody speci®c for the oocyst wall of Cryptosporidium
followed by a reaction with a FITC-labeled secondary antibody
(Hydro¯uor, Strategic Diagnostics, Newark, Del.). A 1:20 volume
of the primary antibody was added, and samples were incubated for
2.5 h at 37°C in the dark. Oocysts were washed once using cen-
trifugation in 0.1 M PBS, and then a 1:50 dilution of the secondary
antibody was added. The samples were then incubated in the dark
at room temperature for 2.5 h, after which oocysts were washed
twice in PBS using centrifugation and resuspended in the original
volume with 0.3 M 1,4-diazabicyclo [2.2.2] octane (DABCO) in
0.1 M PBS (DABCO-PBS). The dyed and stained oocysts were
stored at 4°C in the dark until microscopic examination.
Flow cytometry
A ¯uorescent-activated cell sorter (FacsCalibur, Becton and
Dickinson,) set to count at least 10,000 oocysts was used. A green
¯uorescence detector, with a 530‹30 nanometer band pass ®lter,
and a red ¯uorescence detector, with a 650 nm long pass ®lter, were
used. Control samples were oocysts treated with 90% ammonia for
1 h at room temperature. Three controls, unstained oocysts, PI-
stained oocysts, and FITC-stained oocysts, were used for each run.
Readings from the control oocysts were used to divide the detected
oocysts into four categories: PI-negative, FITC-negative oocysts
(gate R2); PI-positive, FITC-negative oocysts (gate R3); PI-nega-
tive, FITC-positive oocysts (gate R5); and PI-positive, FITC-pos-
itive oocysts (non-gates R2, R3, and R5). Oocysts were ®rst
detected by forward scatter using the size of the particles and side
scatter examining the granularity of the particles. A gate was cre-
ated to select at least 10,000 control oocysts using the unstained
control oocyst population (Fig. 4a). This population was analyzed
with the green and red ¯uorescence detector. In the di€erent runs,
the voltage was adjusted to improve the visualization of separation
and gates were reset each time based on the three populations of
controls. The change in voltage gives an apparent linear shift along
the FSC-H axis; however, the use of the control oocysts guaranteed
that the correct oocyst populations were identi®ed. Using the gates
set for each run, the percentage of viable oocysts was determined as
follows: [(counts of PI-negative, FITC-positive oocysts, i.e.,
gate R5)/(counts of PI-negative, FITC-positive oocysts, i.e., gate
R5)+(counts of PI-positive, FITC-positive oocysts, i.e., non-gates
R2, R3, and R5)]´100%.
Microscopic observation
Samples were also examined by epi¯uorescence and di€erential
interference contrast microscopy (Eclipse, E600, Nikon, Japan).
Excitation bands of 330±380, 546/10, and 450±490 nm were used.
At least 100 oocysts in each sample were examined by the micro-
scopic method.
Statistical analysis
Di€erences in viabilities determined by the ¯ow cytometric and
microscopic methods were statistically compared by a paired t-test
using Minitab (Minitab 12, Minitab, State College, Pa.).
Results
Oocysts treated with ammonia
Ninety percent ammonia-treated oocysts appeared as a
distinct population in the forward and side scatter
328
plots (Fig. 1). In preparation for analysis with ¯uo-
rescence (Fig. 2a±c), gates were set using 90% ammo-
nia-treated oocysts that were either unstained (gate
R2), PI-stained (gate R3), or FITC-labeled (gate R5).
Non-viable oocysts that were PI-positive were found in
the area outside of gates R2, R3, and R5 (Fig. 3a, b;
dot plot shown only for oocysts treated with 90%
ammonia). Thus, the percentage of viable oocysts was
100 multiplied by the number of oocysts in gate R5
divided by the total number of oocysts in gate R5 and
in the area outside of the three gates. When oocysts
were treated with 1%, 25%, or 90% ammonia for 1 h,
78.4%, 31.0%, and 6.6%, respectively, of the oocysts
were still viable (Table 1). There was no statistical
di€erence between the percentages of viable oocysts
detected by either the ¯ow cytometric or the micro-
scopic observation of oocysts after ammonia treatment
(Table 1).
Excysted oocysts
After excystation, the location of the oocyst popula-
tion changed in the forward and side scatter plots
Fig. 1 Forward and side scatter plot of Cryptosporidium parvum
oocysts treated with 90% ammonia for 1 h. The X-axis (FSC-H) is
forward scatter, and the Y-axis (SSC-H) is side scatter. The gate
that is set is gate R1
(Fig. 4). Again, untreated oocysts formed a distinct
population that was described by gate R1 (Fig. 4a).
After excystation (93% of oocysts excysted as deter-
mined by microscopic observation), the treated oocysts
were found outside gate R1 (Fig. 4b). When oocysts
were excysted without acid pretreatment, only 28% of
the oocysts excysted as determined microscopically,
and a larger proportion of the oocysts was still found
in gate R1 during the ¯ow cytometric analysis
(Fig. 4c).
Oocysts extracted from soil
When oocysts were extracted from soil, the results were
similar to those for puri®ed oocysts in that they formed
a distinct population on the generated dot plots
(Fig. 5a). The analysis using ¯uorescence revealed that
81% of the extracted oocysts were viable (Fig. 5b), and
there was no statistical di€erence between the percent-
ages of viable oocysts detected by this method and by
routine microscopy where 83% of the oocysts were
found to be viable (Table 2).
Oocysts extracted from biosolids
The ¯ow cytometric analysis revealed that 26% of the
oocysts extracted from biosolids were viable, which did
not di€er statistically from the 29% found to be viable
using the routine microscopic method (Table 2).
Discussion
In this study, ¯ow cytometry was found to be a satis-
factory alternative method to microscopy for determin-
ing oocyst viability. Flow cytometry has the advantages
that samples were examined more rapidly and a larger
Fig. 2a±c The ¯ow cytometric analysis of C. parvum oocysts,
treated with 90% ammonia for 1 h. Setting the gates for ¯ow
cytometric analysis of ¯uorescent C. parvum oocysts. The X-axis
(FL1-H) is a green ¯uorescence detector, and the Y-axis (FL3-H) is
a red ¯uorescence detector. a The ¯uorescence analysis of unstained
oocysts, used to set gate R2. b The ¯uorescence analysis of
PI-stained oocysts, used to set gate R3. c The ¯uorescence analysis
of FITC-labeled oocysts, used to set gate R5
 329

Fig. 3a, b The ¯ow cytometric analysis of C. parvum oocysts,
treated with 90% ammonia for 1 h. a Forward and side scatter plot
of oocysts [the X-axis (FSC-H) represents forward scatter, and the
Y-axis (SSC-H) represents side scatter]. b The ¯ow cytometric
analysis of oocysts. The oocysts that are PI-positive and FITC-
positive (non-viable) appear in the area bounded by the 3 gates,
R2, R3, and R5. The X-axis is a green ¯uorescence detector, and
the Y-axis is a red ¯uorescence detector
number of oocysts were counted (10,000 oocysts are
typically counted by ¯ow cytometry and only 100±
200 oocysts are counted in microscopic analyses). Flow
cytometry, however, requires the presence of a large
number of oocysts in the original sample. The sentinel
chamber method (Jenkins et al. 1999) provides a method
to obtain concentrated oocysts for viability testing after
di€erent inactivation treatments. Thus, combining
the sentinel chamber method with the ¯ow cytometry
would be advantageous for viability determination
because of the rapidity of sample analysis. However, if
the process that inactivates the oocysts causes a per-
centage of the oocysts to open and lose their contents,
the FACS method would be inappropriate because the
empty oocysts would not appear in the initial gated
population.
Cross-talk between the green and red ¯uorescence
detection channels with the present choice of ¯uoro-
chromes and staining procedure precluded absolutely
separating the live and dead populations using simple
gates on red and green ¯uorescence. The green was
much brighter than the red, at even relatively low
FITC concentrations. When oocysts were very brightly
labeled with FITC, there was a tendency for them to
also be detected to some extent on the red channel even
though they were determined microscopically to be
viable. Alternative strategies that could be used to re-
duce the amount of cross-over in the brightly staining
oocysts would be to use di€erent ¯uorochromes for the
labeling of total and viable oocysts or to increase the
separation between oocyst types using either narrower
gates or a two-laser system with ¯uorochromes having
more widely spaced spectra. As currently performed,
however, giving careful attention to the setting of the
gates with control samples seems to produce very high
correlations between microscopic observations and
FACS counts.
This work has shown that a process that causes the
excystation of a large percentage of oocysts would not
be successfully monitored by the ¯ow cytometric method
described here. The location of the majority of excysted
oocysts was changed within the gates due to changes in
their forward and side scatter. Thus, if a disinfection
method inactivates oocysts by causing them to excyst,
methods other than the ¯ow cytometry should be used
for viability determination.
In conclusion, ¯ow cytometry using oocysts from
spiked chambers from environmental samples appears

Table 1 The comparison of

¯ow cytometric and micro- Treatment Method Number Percentage of
scopic methods for determining of samples viable oocysts
the viability of Cryptosporidium (mean‹standard error)
parvum oocysts treated with
1%, 25%, and 90% ammonia 1% ammonia Flow cytometry 5 78.40‹2.27
for 1 h. Statistical analysis was Microscopy 5 81.80‹1.46
performed using a paired t-test Di€erence 5 ±3.40‹2.71
t=±1.25, P=0.28
25% ammonia Flow cytometry 5 30.98‹5.11
Microscopy 5 30.14‹4.12
Di€erence 5 0.84‹5.42
t=0.15, P=0.89
90% ammonia Flow cytometry 5 6.599‹0.93
Microscopy 5 6.012‹0.83
Di€erence 5 0.587‹0.70
t=0.84, P=0.45
330

Fig. 4a±c The ¯ow cytometric analysis of C. parvum oocysts. The
X-axis (FSC-H) is a forward scatter, and the Y-axis (SSC-H) is a
side scatter. a Forward and side scatter plot of untreated oocysts,
setting the gate R1 around the distinct oocyst population. b
Forward and side scatter plot of excysted oocysts. 93% of the
oocysts were excysted according to microscopic observation, and
very few oocysts are found in gate R1. c Forward and side scatter
plot of oocysts excysted without an initial acid pretreatment. 28%
of the oocysts were excysted according to microscopic observation,
and only a small proportion of oocysts remain in gate R1 indicating
that a percentage of oocysts did not excyst without the acid
pretreatment
to be a promising means for examining a large number
of samples in a relatively short period of time. It is an
advantage that routinely 10,000 oocysts rather than
100 oocysts can be rapidly examined for viability in each
processed sample. Also, when large numbers of samples
are processed, ¯ow cytometric analysis would be faster
than the microscopic method for a viability examina-
tion.
Acknowledgements This material is based upon work supported by
the Cooperative State Research, Education, and Extension Service,
U.S. Department of Agriculture, under Agreement No. 98-35102-
6503. The authors thank Dr. Andrew Yen, Department of
Biomedical Sciences, Cornell University, for his valuable assistance
with the speci®es of ¯ow cytometric analysis. The authors also
thank E.A. Fogarty for technical assistance.
References
Brush CF, Walter MF, Anguish LJ, Ghiorse WC (1998) In¯uence
of pretreatment and experimental conditions on electrophoretic
mobility and hydrophobicity of Cryptosporidium parvum
oocysts. Appl Environ Microbiol 64:4439±4445
Bukhari Z, Bolton JR, Dussert B, Clancy JL (1999) Medium-
pressure UV for oocyst inactivation. J Am Water Works Assoc
91:86±94
Bukhari Z, Marshall MM, Korich DG, Fricker CR, Smith HV,
Rosen J, Clancy JL (2000) Comparison of Cryptosporidium
parvum viability and infectivity assays following ozone treat-
ment of oocysts. Appl Environ Microbiol 66:2972±2980

Fig. 5a, b The ¯ow cytometric

analysis of C. parvum oocysts,
extracted from soil in a cham-
ber. a Forward and side scatter
plot of oocysts showing the
distinct oocyst population
within the gate set using control
oocysts (treated with 90%
ammonia for 1 h). The X-axis
(FSC-H) is a forward scatter,
and the Y-axis (SSC-H) is a side
scatter. b The ¯ow cytometric
analysis of oocysts. The X-axis
is a green ¯uorescence detector,
and the Y-axis is a red ¯uores-
cence detector

Table 2 The comparison of

¯ow cytometric and micro- Matrix Method Number of Percentage of viable
scopic methods for determining samples oocysts Mean‹standard error
the viability of C. parvum
oocysts after extraction from Soil Flow cytometry 5 81.14‹2.05
soil and biosolids matrices. Microscopy 5 83.00‹0.97
Statistical analysis was per- Di€erence 5 ±1.86‹1.77
formed using a paired t-test t=±1.05, P=0.34
Biosolids Flow cytometry 5 26.01‹6.61
Microscopy 5 28.82‹3.06
Di€erence 5 ±2.80‹4.27
t=±0.66, P=0.55

Campbell AT, Robertson LJ, Smith HV (1992) Viability of Cryp-
tosporidium parvum oocysts: correlation of in vitro excystation
with inclusion or exclusion of ¯uorogenic vital dyes. Appl
Environ Microbiol 58:3488±3493
Fayer RR, Leek G (1984) The e€ects of reducing conditions, me-
dium, pH, temperature, and time on in vitro excystation of
Cryptosporidium. J Protozool 31:567±569
Ho€man RM, Standridge JH, Prieve AF, Cucunato JC, Bernhardt
M (1997) Using ¯ow cytometry to detect protozoa. J Am Water
Works Assoc 89:104±111
Jenkins MB, Anguish LJ, Bowman DD, Walker MJ, Ghiorse WC
(1997) Assessment of a dye permeability assay for determina-
tion of inactivation rates of Cryptosporidium parvum oocysts.
Appl Environ Microbiol 63:3844±3850
Jenkins MB, Bowman DD, Ghiorse WC (1998) Inactivation of
Cryptosporidium parvum oocysts by ammonia. Appl Environ
Microbiol 64:784±788
331
Jenkins MB, Walker MJ, Bowman DD, Anthony LC, Ghiorse WC
(1999) Use of a sentinel system for ®eld measurements of
Cryptosporidium parvum oocyst inactivation in soil and animal
waste. Appl Environ Microbiol 65:1998±2005
Medema GJ, Schets FM, Ketelaars H, Boschman G (1998) Im-
proved detection and vital staining of Cryptosporidium and
Giardia with ¯ow cytometry. Water Sci Technol 38:61±65
Valdez LM, Dang H, Okhuysen PC, Chappell CL (1997) Flow
cytometry detection of Cryptosporidium oocyst in human stool
samples. J Clin Microbiol 35:2013±2017
Vesey G, Hutton P, Champion A, Ashbolt N, Williams KL,
Warton A, Veal D (1994) Application of ¯ow cytometric
methods for the routine detection of Cryptosporidium and
Giardia in water. Cytometry 16:1±6
Vesey G, Griths KR, Gauci MR, Deere D, Williams KL, Veal
DA (1997) Simple and rapid measurement of Cryptosporidium
excystation using ¯ow cytometry. Int J Parasitol 27:1353±1359