350
R. Armon et al.
Filtration was used for concentration of G. lamblia and C. parvum from large
volumes of water (200 to 800 L) and inorganic flocculation for C. parvum
concentration from 10 L volume.
2.4 Microscopical Identification and Viability Testing of Cysts and
Oocysts
Ten-microliter aliquots of cyst/oocyst suspension were stained with monoclonal
antibodies (Waterborne Inc., USA) and placed on a 12 microwell slide for
microscopic examination. Using a Zeiss epifluorescence microscope, the oocysts
were identified and enumerated by blue filter block (excitation 490 nm, emission
510 nm). When necessary, viability was assessed by inclusion or exclusion of
fluorogenic vital dyes (DAPI and PI); UV filter block for DAPI (excitation 335
nm, emission 450 nm) and green filter block for PI (excitation 488 nm, emission
610 nm) (Campbell et al. 1992). A minimum of 100 cysts/oocysts was counted in
each well. All experiments were performed in duplicates.
2.5 Concentration Method of Oocysts and Cysts from Soil Samples
A low-cost method for recovery of Cryptosporidium oocysts from soil samples
irrigated with treated effluents was developed (Zilberman and Armon 2001).
Briefly, the method is a modification of an earlier procedure previously used for
extraction C. perfringens spores from bottom sediment samples (Emerson and
Cabelli 1982) that comprise phase separation of PEG (60%) and sucrose (25%).
The overall recovery of this method ranged from 60 to 70%.
2.6 Elution and Concentration Method of Oocysts and Cysts from
Various Vegetables Irrigated with Effluents
Postharvest vegetables were brought to the laboratory in plastic bags kept in
coolers at 4°C. Half a kilogram of each vegetable type was introduced in 1.8 L of
elution solution (sterile distilled water containing 0.1% Tween 80, v/v) in large
plastic vessels and shaken for 20 minutes at room temperature. The elution
solution was further subjected to inorganic flocculation concentration method as
already described (Vesey et al. 1993; Shepherd and Wyn-Jones 1996). The final
precipitate was layered upon a sucrose gradient (Armon et al. 1995) for further
cleaning of the final concentrate, and finally stained with MAB for microscopic
examination as described above. The recovery of this method ranged from 40 to
50%.
R. Armon et al.
Filtration was used for concentration of G. lamblia and C. parvum from large
volumes of water (200 to 800 L) and inorganic flocculation for C. parvum
concentration from 10 L volume.
2.4 Microscopical Identification and Viability Testing of Cysts and
Oocysts
Ten-microliter aliquots of cyst/oocyst suspension were stained with monoclonal
antibodies (Waterborne Inc., USA) and placed on a 12 microwell slide for
microscopic examination. Using a Zeiss epifluorescence microscope, the oocysts
were identified and enumerated by blue filter block (excitation 490 nm, emission
510 nm). When necessary, viability was assessed by inclusion or exclusion of
fluorogenic vital dyes (DAPI and PI); UV filter block for DAPI (excitation 335
nm, emission 450 nm) and green filter block for PI (excitation 488 nm, emission
610 nm) (Campbell et al. 1992). A minimum of 100 cysts/oocysts was counted in
each well. All experiments were performed in duplicates.
2.5 Concentration Method of Oocysts and Cysts from Soil Samples
A low-cost method for recovery of Cryptosporidium oocysts from soil samples
irrigated with treated effluents was developed (Zilberman and Armon 2001).
Briefly, the method is a modification of an earlier procedure previously used for
extraction C. perfringens spores from bottom sediment samples (Emerson and
Cabelli 1982) that comprise phase separation of PEG (60%) and sucrose (25%).
The overall recovery of this method ranged from 60 to 70%.
2.6 Elution and Concentration Method of Oocysts and Cysts from
Various Vegetables Irrigated with Effluents
Postharvest vegetables were brought to the laboratory in plastic bags kept in
coolers at 4°C. Half a kilogram of each vegetable type was introduced in 1.8 L of
elution solution (sterile distilled water containing 0.1% Tween 80, v/v) in large
plastic vessels and shaken for 20 minutes at room temperature. The elution
solution was further subjected to inorganic flocculation concentration method as
already described (Vesey et al. 1993; Shepherd and Wyn-Jones 1996). The final
precipitate was layered upon a sucrose gradient (Armon et al. 1995) for further
cleaning of the final concentrate, and finally stained with MAB for microscopic
examination as described above. The recovery of this method ranged from 40 to
50%.