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A simplified method for in vitro excystation of Giardia muris.

Excystation of Giardia lamblia was first induced experimentally by Hegner (1927, American Journal of Hygiene 7: 433-447) who injected isolated cysts into the stomach of a laboratory rat and harvested excysted organisms from the small intestine. Bingham et al. (1979, Experimental Parasitology 47: 284-291) induced excystation of G. lamblia in vitro by first exposing cysts to an acidic aqueous solution (pH 2.0), followed by a wash in distilled water and incubation in a culture medium. Schaefer et al. (1984, Transactions of the Royal Society of Tropical Medicine and Hygiene 78: 795-800) used this method to induce excystation of Giardia muris but were unable to achieve rates higher than 5%. An alternate procedure was proposed which used an induction medium of Hanks' balanced salt solution (HBSS) with reducing agents, sodium bicarbonate and an oxidation-reduction potential (Eh) of + 120 mV, and an excystation medium of Tyrode's solution containing crude trypsin. High rates (> 90%) of excystation were reported within the first 30 min of incubation. Although this method yields consistently high excystation rates, the Tyrode's-trypsin medium is difficult and tedious to prepare, because the trypsin does not completely dissolve and must be removed by centrifugation at 21,000 g for 10 min followed by filtration through a 0.45 um membrane (Schaefer et al., 1984, loc. cit.). The role of the trypsin in that medium is not understood. Purified trypsin, when substituted for the crude trypsin in the medium, does not yield high excystation rates (Meyer and Schaefer, 1984. In Giardia and giardiasis, S. L. Erlandsen and E. A. Meyer (eds.). Plenum, New York, pp. 131-144). Heat inactivation of the trypsin does not prevent excystation but decreased excystation rates to 78% (Schaefer et al., 1984, loc. cit.). A simplified method for excystation of G. muris that produces high rates of excystation and uses easily prepared media has been developed in this laboratory. This method is a slight modification of the technique of Bingham et al. (1979, loc. cit.). Feces were collected from G. muris infected CF1 mice (Roberts-Thompson et al., 1976, Gastroenterology 71: 57-61), and a suspension was prepared by mixing the feces with distilled water, using a laboratory stirrer and stir bar. It was then filtered through 1 layer of gauze and the cysts isolated on a sucrose gradient as described by Bingham et al. (1979, loc. cit.). The isolated cysts were then incubated at 37 C for 30 min in HBSS that had been adjusted to pH 2.0 with 2 N HC1 immediately prior to use. The HBSS was prepared daily from a 10 x stock solution. Sodium bicarbonate was added to bring the pH to 7.2. A portion of this was then prepared for the acid induction step by the addition of 2 N HC1 to adjust it to pH 2. The Eh of this solution was + 600 mV as measured with the aid of a platinum electrode. The cysts were then concentrated by centrifugation and washed briefly in HBSS (pH 7.2). The cysts were again concentrated by centrifugation and then placed in 2-3 ml of TYI medium (pH 7.2-7.4) supplemented with serum and bile described by Keister (1983, Transactions of the Royal Society of Tropical Medicine and Hygiene 77: 487-488). The cysts were incubated in uncapped conical centrifuge tubes at 37 C. Excystation began within the first 5 min of incubation. Rates at 15 and 30 min were determined by the method of Bingham et al. (1979, loc. cit.) and were 96.2% (n = 23, SD = 1.7) and 97.3% (n = 17, SD = 2.1), respectively. Excystation proceeded in a similar fashion to that reported by Meyer and Schaefer (1984, loc. cit.). Mature trophozoites were observed after 30 min. This method differs from the method of Bingham et al. (1979, loc. cit.) by the use of acidic HBSS and physiologic HBSS in the induction and washing steps, and TYI medium instead of HSP medium. No trypsin or reducing agents were added. Excystation of G. muris may be easily produced in vitro with this simplified medium. The mechanism of excystation is not completely understood. Schaefer et al. (1984, loc. cit.) reported optimal excystation with the aforementioned induction solution of pH 2.0 and Eh of + 120 mV followed by a wash and incubation in a Tyrode solution containing trypsin. The results