in vitro culture and isoenzyme analysis of giardia lamblia

4.2.3 In vivo Excystation
This method was based on that of Dr TE Nash & JT Conrad of the N.I.H
(Bethesda, Maryland) (personal communication) and modified for local
experimentation.
Suspensions containing 1000 cysts/m I in 0,1 ml aliquots of distilled water were
injected into each of the stomachs of 1-2 day old suckling mice in a litter (a litter
consisted of at least 4 up to 9 pups). A total of 53 litters were inoculated. Depending
on the availability of neonatal mice and the number of cysts in a sample, the
inoculations were replicated either within the same strain of mice or between the two
strains. Seven to ten days after inoculation, the infected mice were sacrificed by
cervical dislocation. The proximal 8-cm of small intestine was removed and
macerated with scissors in 1 ml of cold TYI-S-33 medium in a Petri dish. The mixture
was transferred to a sterile 9' 0 x 10mm Kimax glass tube filled to 80% capacity with
the TYI-S-33 culture medium containing antibiotics and its cap was immediately
screwed on. Inoculated tubes were chilled for 30 minutes to detach trophozoites from
the macerated intestines and then incubated at 35,5° C for 1 hour at a five-degree
angle to allow the trophozoites to adhere to glass. Medium containing intestine
fragments was filtered through a double layer of gauze to eliminate the toxic
intestines. The filtrate, which contained the unattached trophozoites, was transferred
to a sterile 8ml Kimax tube and the latter filled to 80% capacity with fresh TYI-S-33
medium with antibiotics. All the tubes were incubated at 35,5°C for 1 hour and
supernatant medium gently decanted and replaced with an equal volume of fresh
TYI-S-33 with antibiotics. All tubes were then incubated for 1 hour and the medium
was changed one more time. The tubes were examined on an inverted microscope
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