in vitro culture and isoenzyme analysis of giardia lamblia

6.2 MATERIALS AND METHODS
6.2.1 Cooling of Cultured Trophozoites
Two techniques of cooling were assessed for their efficacy in achieving long term
preservation of trophozoites. Subsequent success in retrieval of viable cultures
was also evaluated. In both, the same cryopreservant was used. A 9% (v/v)
solution of Dimethyl sulphoxide (DMSO) (Saarchem, Durban, and SA.) was made
in sterile TYI-S-33.
6.2.1.1 Preparation of samples
Tubes of cultures at early stationary phase were chilled on ice for 20 minutes to
detach the trophozoites from the glass. After mixing by gentle inversion they were
centrifuged at 400xg for 5 minutes at 4°C and supernatant medium was
discarded. Pelletted trophozoites were resuspended in 1 ml of fresh TYI-S-33
medium. The organisms in a 1 ml aliquot were enumerated in a haemocytometer.
One ml of suspensions containing 1 x1 0 6 trophozoites fml were then transferred
with a sterile Pasteur pipette to a Nunc cryotube (Nalge Nunc International, USA).
Immediately before cooling, 1 ml of 18% DMSO was gradually introduced (by
dropwise addition) to the cryogenic vial to give a final concentration of 9% DMSO.
6.2.2 Cooling Techniques
6.2.2.1 Electronically controlled cryofreezing apparatus (modification of
Phillips et al., 1984 method)
Vials containing cryopreservant and cells were promptly (within 5 minutes)
transferred to a Liquid Nitrogen, programmable controlled freezer, Planer model
Xb634 (Planer Products, Sanbury-On-Thames, England). The cooling rate was
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