in vitro culture and isoenzyme analysis of giardia lamblia

3.2.2 The Modified Acid Induction Method of Rice and Schaefer (1981)
(Hamilton and Jackson 1990).
Washed cyst pellets as obtained in Chapter 2 (section 2.2.3.3) containing at least
10 5 cysts were transferred to a 5ml polypropylene tube. The tubes were filled with
freshly prepared pre-warmed acid induction medium (Appendix 6a) and tightly
capped to keep the contents anaerobic. The mixture was incubated at 37°C for
30min and thereafter centrifuged at 400xg for 1 min at 22°C. The acidic
supernatant was gently aspirated and discarded. The pellet was resuspended in
5ml excystment medium (HSP-1, Appendix 6b) and centrifuged at 400xg for 1 min
at 22°C and the supernatant medium was discarded. The resultant sediment was
incubated at 37°C in 3ml of prewarmed excystment medium into which 1 ml of
0,3M Glutathione (SDH, Poole, England) was added. A liquid paraffin overlay (0,5
ml) was added. After one hour, the tubes were gently centrifuged at 200xg for
2min at 22°C and the pellet transferred to Bml Kimax glass tubes (in duplicate)
containing 7ml TVI-S-33 medium supplemented with serum, bile and antibiotics
(Appendix 7). The tubes were then incubated at 35,5°C at a five-degree angle for
at least 7 days. Following examination for excysted trophozoites by inverted
microscopy; fresh medium was added to the tubes at 4Bh intervals. If excysted
trophozoites were detected, one of the duplicate tubes was retrieved, chilled on
ice for 20min and centrifuged at 400xg at 4°C for 5mins. The deposit was
resuspended in 0,1 ml of sterile TVI medium, diluted 1 in 10 in 1 % formalin (to fix
motile trophozoites) and transferred to a haemocytometer. Intact cysts and
excysted trophozoites were enumerated and the percentage excystation was
determined.
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