in vitro culture and isoenzyme analysis of giardia lamblia

idge buffers for each enzyme (Appendix 7) were introduced (in 75ml volumes)
into each chamber of the electrophoresis apparatus (LKB, Biochrom, Model
2103). Inoculated plates were placed in their respective tanks and saturated wicks
adjoining the anodal and cathodal compartments were laid onto each end of the
inoculated plate. A cooling platform was incorporated into the system to maintain
the temperature at 11°C during electrophoresis. The surface of the cooling plate
was insulated with plastic sheeting and inoculated gels were electrophoresed for 2
hours in the electrophoresis apparatus at 240V. Following electrophoresis the gels
were laid on a flat surface and a Perspex frame covering the whole migration zone
was placed around the edges of the gel to retain the staining solution. Agar (as
prepared in Appendix 8a) was melted by heating over a Bunsen burner and mixed
with a staining solution specific for each enzyme. The agar/stain mixture was
overlaid onto the gel and the plates were incubated at 37°C for 1 hour in the dark.
The formazan reaction was used to visualise the banding patterns of the enzymes.
CONTROLS:
1. Entamoeba histolytica zymodeme II (strain HM1) was included as a control in
every run. The H7 and WB reference strains were used as standard Giardia
controls.
2. Uninoculated TYI-S-33 medium was included as a control in every
electrophoresis run.
3. Lysates were prepared from different subcultures of the WB reference strain to
assess the stability of isoenzymes during serial subculture of trophozoites.
Lysates made from serial subcultures of the local isolates were also inoculated
within runs.
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