in vitro culture and isoenzyme analysis of giardia lamblia

APPENDIX 6
Preparation Of Excystation Media (Hamilton & Jackson, 1990)
Appendix 6a
Induction Medium
Twenty-five ml of Hanks salt solution was made up to 100 ml with distilled water,
autoclaved for 10 min at 103 kPa, cooled to 37°C and 0.40g L-cysteine
hydrochloride (Polychem, Durban, South Africa) and 0.42 g sodium bicarbonate
(Associated Chemicals Enterprise cc, Gauteng, South Africa) added. The pH was
adjusted to 2,0 with 10M HCI (Polychem, Durban, and SA).
Appendix 6b
Excystment medium
Forty millilitres of Hanks solution was made up to 100 ml with distilled water. One
gram of Trypticase (Merck, Darmstadt, Germany) and 0,5g glucose (Polychem,
Durban, SA) were added. The solution was autoclaved at 103 kPa for 10 min.
Following cooling to 37°C, the following were added: 0,4 g L-cysteine
hydrochloride (Polychem, Durban, SA.); 0,25 g Trypsin (Merck, Darmstadt,
Germany) and 15 ml decomplemented horse serum *(Highveld Biological, JHB,
SA.) The pH was adjusted to 7,5 with 1M NaOH (Polychem. Durban, South
Africa) and the medium distributed in 3ml aliquots in glass tubes, overlaid with
0.5mlliquid paraffin and incubated at 37°C. Excess medium was used for
washing cysts following the induction step.
~
*Later, fetal calf serum (Sigma, St Louis, USA) was used in place 0/ the Highveld brand
0/ horse serum.
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