in vitro culture and isoenzyme analysis of giardia lamblia

40C for further processing on subsequent days to replicate experiments. At the
early stages of this work, cyst numbers that were used ranged between 100 and
10 000 cysts/ml, depending on the number of cysts obtained after purification.
Later, cyst concentrations were adjusted to have at least 100 000 cysts/m I in
0,1 ml of distilled water and stored at 4°C in microfuge tubes (Nash, personal
communication). Where low numbers were obtained, (as determined in 2.2.4) the
gradient purification method was repeated and a larger volume of stool was used
for replicate purifications and the replicate cyst concentrates were pooled to obtain
larger numbers of cysts.
2.2.5 Determination of Cyst Viability
To ascertain whether cysts would be capable of excysting, their viability was
assessed by the eosin exclusion method (Bingham et aI, 1979). Viable cysts
inhibit dye penetration while non-viable ones absorb the dye and appear pink
internally.
Equal volumes of the washed cyst suspension and 0,0025% aqueous eosin
solution were mixed in a 5ml-polycarbonate tube. Ten microlitres of the
suspension was transferred to a haemocytometer with a coverslip in position and
the preparation incubated in a moist chamber at 22°C for 15 min. Cysts were then
examined under a light microscope at 400x magnification for dye uptake and
enumerated. Percentage viability was determined by enumerating unstained and
stained cysts in the five large squares of the counting chamber and expressing
these as percentage viability as follows:
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