Isolation, Identification of the Water-borne Protozoan Parasites
349
2 Methodology
2.1 C. parvum Oocysts and G. iamblia Cysts Used in Concentration
Methods
Cryptosporidium parvum oocysts from two sources were evaluated. GCHI isolate
was kindly donated by Prof. S. Tzipori, Division of Infectious Diseases, School of
Veterinary Medicine, Tufts University, MA, U.S.A. A second isolate was
purchased from Moredun Research Institute, Edinburgh, U.K. Giardia lamblia
cysts were isolated from faecal material of infected patients (kindly supplied by
the parasitology Laboratory, Hillel Jaffe Hospital, Hadera, Israel) concentrated
and purified by sucrose gradient. Both oocysts and cysts were stored in 2.5%
potassium di-chromate solution at 4°C. Some aliquots were fixed in 10% buffered
formalin.
2.2 Monoclonal Antibodies Used for Fluorescent Staining of Cysts
and Oocysts
Fluorescent monoclonal antibodies (MAB) AquaGlo G/C (Waterborne Inc., New
Orleans, LA, U.S.A.) against both Giardia and Cryptosporidium were used
according to the manufacturer's instructions for simultaneous detection in water
samples, or Crypt-A-Glo (Waterborne Inc.) against Cryptosporidium only.
2.3 Concentration Methods for Cysts and Oocysts in Small and
Large Volumes of Surface Waters
Concentration of parasites from 10 to 800 L of surface water was performed by
two concentration methods: (1) large volume filtration (polypropylene wound 10"
long cartridge filter, 1 /lm porosity rating, Filterite, MEMTEC America Corp.,
U.S.A.) (Rose et al. 1991; APHA 1995) and (2) inorganic flocculation as already
described (Vesey et al. 1993). Briefly, the inorganic flocculation concentration
method was performed as follows: 100 ml CaCl2 (1 M) and 100 ml NaHC03
(lM) were added to 10 L of sample (raw sewage, effluent, vegetable elution
solution) in plastic containers, and mixed well. The pH was adjusted to 10 by
addition of approximately 100 ml NaOH (l N). The solution was left for 4 hours
to allow formed flocs to settle on the bottom of the plastic container. The
supernatant was aspirated and discarded while the flocs were dissolved by
addition of 100 ml 10% sulphamic acid (w/v) and centrifuged at 1800 g for 12
minutes. The pellet was transferred to 50 ml centrifuge tubes and centrifuged at
4000 g for 15 min. The supernatant was decanted and the final pellet was reacted
with FITC-labeled specific MAB and observed under a fluorescent microscope.
349
2 Methodology
2.1 C. parvum Oocysts and G. iamblia Cysts Used in Concentration
Methods
Cryptosporidium parvum oocysts from two sources were evaluated. GCHI isolate
was kindly donated by Prof. S. Tzipori, Division of Infectious Diseases, School of
Veterinary Medicine, Tufts University, MA, U.S.A. A second isolate was
purchased from Moredun Research Institute, Edinburgh, U.K. Giardia lamblia
cysts were isolated from faecal material of infected patients (kindly supplied by
the parasitology Laboratory, Hillel Jaffe Hospital, Hadera, Israel) concentrated
and purified by sucrose gradient. Both oocysts and cysts were stored in 2.5%
potassium di-chromate solution at 4°C. Some aliquots were fixed in 10% buffered
formalin.
2.2 Monoclonal Antibodies Used for Fluorescent Staining of Cysts
and Oocysts
Fluorescent monoclonal antibodies (MAB) AquaGlo G/C (Waterborne Inc., New
Orleans, LA, U.S.A.) against both Giardia and Cryptosporidium were used
according to the manufacturer's instructions for simultaneous detection in water
samples, or Crypt-A-Glo (Waterborne Inc.) against Cryptosporidium only.
2.3 Concentration Methods for Cysts and Oocysts in Small and
Large Volumes of Surface Waters
Concentration of parasites from 10 to 800 L of surface water was performed by
two concentration methods: (1) large volume filtration (polypropylene wound 10"
long cartridge filter, 1 /lm porosity rating, Filterite, MEMTEC America Corp.,
U.S.A.) (Rose et al. 1991; APHA 1995) and (2) inorganic flocculation as already
described (Vesey et al. 1993). Briefly, the inorganic flocculation concentration
method was performed as follows: 100 ml CaCl2 (1 M) and 100 ml NaHC03
(lM) were added to 10 L of sample (raw sewage, effluent, vegetable elution
solution) in plastic containers, and mixed well. The pH was adjusted to 10 by
addition of approximately 100 ml NaOH (l N). The solution was left for 4 hours
to allow formed flocs to settle on the bottom of the plastic container. The
supernatant was aspirated and discarded while the flocs were dissolved by
addition of 100 ml 10% sulphamic acid (w/v) and centrifuged at 1800 g for 12
minutes. The pellet was transferred to 50 ml centrifuge tubes and centrifuged at
4000 g for 15 min. The supernatant was decanted and the final pellet was reacted
with FITC-labeled specific MAB and observed under a fluorescent microscope.