in vitro culture and isoenzyme analysis of giardia lamblia

3.2 MATERIALS AND METHODS
Three excystment techniques were assessed as detailed below:
3.2.1 The Acid Induction method (AI Tukhi et al., 1991).
Cysts purified as described in Chapter 2 (2.2.3.3) were aliquoted in duplicate
(where sufficient numbers were available, triplicate aliquots were made). A 0,1 ml
aliquot of the purified cyst suspension, containing between 1 x1 0 2 and 18,3 x 10 4
cysts/m I was suspended in 0,9 ml of 1 M Hydrochloric acid (Hel) (Polychem,
Durban, SA.) at pH 2 in a microfuge tube. The tubes were incubated upright at 37°
C for one hour and the mixture then centrifuged at 600xg for 10 minutes at 22°C.
Supernatant acid was gently decanted and the preparation was washed free of
acid by resuspending in an alkaline TYI-S-33 medium (Appendix 7) and
centrifuging as described above. The pellet was aseptically harves
ted and inoculated into an 8ml Kimax (Kimble Glass Inc., USA) glass tube
containing 7ml of pre-warmed, modified TYI-S-33 medium with bile and antibiotics.
The tubes were incubated at 35,5°C at a 5-degree angle (to allow emerging
trophozoites, if any, to adhere to glass) for at least 7 days to allow complete
excystation. At daily intervals, the tubes were examined on an inverted
microscope, and an aliquot was aseptically transferred to a slide and examined for
the presence of excysted trophozoites.
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