in vitro culture and isoenzyme analysis of giardia lamblia

adjusted to 5°C per minute from ambient temperature to OOC; 1°C per minute from
o to -25°C and then 5°C per minute from -25 to -135°C. At completion of the
cooling cycle, the cryotubes were transferred to a liquid nitrogen (LN) storage
Dewar flask (Taylor-Wharton, British Oxygen, UK) until required for retrieval.
6.2.2.2 Mechanically insulated freezing container
The procedure was carried out according to the manufacturer's instructions as
follows. Isopropanol (250ml) (Merck, Darmstadt, Germany) was added into the
Nalgene Cryo 1°C Freezing Polycarbonate Container (Nalge Nunc International,
U.S.A, Cat.No.51 00-0001). An insulating foam insert was immersed into the
alcohol. Samples prepared as described in section 6. 2.1.1 were placed into the
vial holder, the unit was promptly placed into a -70°C mechanical freezer and left
undisturbed for at least 4 hours. This system allows a gradual rate of cooling at
1°C/min from room temperature to -70°C. The frozen vials were transferred for
long-term storage into a liquid nitrogen Dewar flask until retrieval was necessary.
6.2.3 Retrieval Of Frozen Cultures.
Cryopreserved cultures were retrieved from liquid nitrogen and thawed quickly at
37°C for 2 minutes without agitating the tubes. Using a sterile Pasteur pipette, the
contents were gently mixed and transferred to a sterile 8ml glass Kimax tube
containing 7ml of TYI-S- 33 medium. The cultures were examined on an inverted
microscope and the percentage of motile trophozoites (indicating viability) was
128