in vitro culture and isoenzyme analysis of giardia lamblia

3.2.3 The modified Acid Pepsin method of Bingham and Meyer (1979)
One percent Pepsin (Sigma, St. Louis, USA.) was made in 0, 15M NaCI solution
and the pH adjusted to 2,0 with 10 M HCI (Polychem, Durban, SA). A purified cyst
suspension (as described in Chapter 2) containing at least 1x 10 5 cysts/m I in 0,1ml
normal saline was incubated with 0,9ml of 1 % Acid Pepsin at 37°C for 30 minutes
in a microfuge tube. The tube contents were centrifuged at 200xg for 2min at
22°C. The supernatant was discarded and pelleted cysts aseptically added to
Kimax tubes containing TYI-S-33 medium with antibiotics. The culture tubes were
incubated at a 5° angle at 35,5°C for up to 7 days. All experiments were
performed in duplicate. One hour after incubation, the tubes were examined with
an inverted microscope for the presence of excysted trophozoites. Thereafter, one
tube was retrieved from the preparation, chilled for 20 minutes and centrifuged at
400xg at 4°C for 5mins. A 1 O~I aliquot of the pelleted trophozoites (resuspended
in 0,1 ml of TYI-S-33) was fixed in 0,09ml of 1 % formalin and the number of
excysted cysts was determined using a haemocytometer under a light microscope.
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