in vitro culture and isoenzyme analysis of giardia lamblia

emulsified in the stain and a 22 x 40mm coverslip applied. The entire area was
systematically examined on the microscope at 100x magnification for the
presence of Giardia cysts. A higher magnification (400x) was also employed to
confirm suspicious objects identified using the 100x objective.
2.2.3 Purification of Cysts
It is essential to separate the cysts from the faecal matter as soon as possible to
minimise overwhelming fungal and bacterial multiplication, and to prevent
undesirable drying effects. If and when separation could not be undertaken
immediately, stools were diluted 1:3 in O,2M Phosphate Buffered Saline (PBS),. pH
7,0 and stored at 4°C until purification. Three methods were assessed for
optimum recovery of cysts from faecal material as outlined below:
2.2.3.1 The discontinuous gradient method (AI-Tukhi et al., 1991)
Approximately O,5g of stool samples (in duplicate) were emulsified in, and
vigorously shaken with, 10ml of distilled water and filtered through 2 layers of wet
gauze. The filtrate was layered onto a density gradient made with 5ml each of
O,4M and O,85M sucrose solutions and centrifuged at 400xg for 10min at 4°C in a
50ml polypropylene centrifuge tube. The material at the water/sucrose interface
was aspirated, resuspended in cold distilled water and washed by centrifuging
twice at 400xg for 5min. Lipid debris was removed by resuspending the cyst pellet
in a mixture of 5ml cold distilled water and 7ml ethyl ether, vortexing, and
centrifuging at 400xg for 10min at 4°C. The resultant cyst pellet was collected,
washed and resuspended in O,5ml of normal saline. Cysts were enumerated as
described in section 2.2.4.
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