in vitro culture and isoenzyme analysis of giardia lamblia

7.2 MATERIALS & METHODS
7.2.1 Preparation of Iysates
Seventy two-hour confluent cultures were ice-chilled for 20 minutes and
centrifuged at 400xg for 10 min at 4°C. Supernatant medium was gently
discarded and the pelletted cells were transferred to Eppendorf microtubes using
a sterile Pasteur pipette. The cells were treated with equal volumes of 1 M each of:
ethylene diamine tetra acetic acid di-sodium salt, (Polychem, Durban, SA); 6-
caprionic acid, (BDH, Poole, England); Dithiothreitol (Sigma, St. Louis, USA) to
stabilise the target enzymes and inhibit proteolytic activity.
The mixture was centrifuged in a microfuge (Beckman Instruments) at 15000xg for
2.5 min and frozen at -20°C for 24hours. The samples were subsequently thawed
at 22°C and re-centrifuged as before. Using a sterile Pasteur pipette, supernatant
was collected and formed into beads by adding it dropwise into liquid nitrogen in a
plastic beaker. The settled beads of lysate were collected and placed in Nunc
cryogenic vials and stored in liquid nitrogen until electrophoresis was undertaken.
7.2.2 Electrophoresis
7.2.2.1 Enzyme systems
The enzyme systems that were used are based on methods modified from Harris
and Hopkinson (1976). Seven different enzymes were employed to determine the
banding patterns of the local isolates. Their buffer systems are described in
Appendix 8 and the preparation of buffers and quantities of substrates, co-factors
and enzymes added are outlined in Appendices 9(b) & 10 respectively. Several
preliminary runs were performed to determine optimum electrophoresis conditions
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