Excystation of the metacercarial cysts of Echinostoma caproni in a frozen and thawed trypsin–bile salts–cysteine medium

Saxton et al. (2008) recently described the use of a trypsin– bile salts–cysteine (TBC) medium to excyst the metacercarial cysts of Echinostoma caproni and Echinostoma trivolvis. The medium was prepared as described by Irwin et al. (1984) to excyst Himasthla leptosoma, except that bile salts were substituted for sodium taurocholate. The medium was used fresh by combining two solutions, A and B, just before use. Solution A consisted of trypsin and bile salts, whereas solution B consisted of the reductant L-cysteine. Details of the formulation and use of this medium were described by Saxton et al. (2008). A frozen and thawed TBC medium has not been used previously for work on E. caproni, although brief mention of this medium for the excystation of E. trivolvis was given by Saxton et al. (2008). The life cycle of this echinostome is maintained in at least six laboratories (see Toledo and Fried 2005), and encysted metacercariae of this echinostome are usually available for teaching and research purposes. One of us (BF) supplies colleagues with encysted metacercariae of this echinostome, and other workers mentioned in Toledo and Fried (2005) also provide this echinostome to colleagues. Some workers request BF to supply a medium for excystation of cysts of E. caproni. In the past, a frozen trypsin and bile (TB) medium of Fried and Roth (1974) was sent to investigators on dry ice to be used for the excystation of E. caproni. As explained by Saxton et al. (2008), the inability to get certain batches of the original trypsin stocks made it difficult to prepare and supply an effective TB medium. The newer TBC medium described in Saxton et al. (2008) has now replaced the old TB medium. Because of the advantages of making up large amounts of medium, e.g. 100 ml, and freezing it for long times, e.g., up to 2 years (see Fried 1994), we recently initiated work to describe the efficacy of a frozen and thawed TBC medium. The purpose of this report is to provide the results of our studies on excysting E. caproni metacercarial cysts in a frozen and thawed TBC medium. Cysts of E. caproni were obtained from the kidney/ pericardium of experimentally infected Biomphalaria glabrata snails and used within 1 to 14 days after removal from the snails (Fried and Huffman 1996). Cysts were placed, 25 to 30, in a 3.5-cm diameter Petri dish containing 3 ml of the frozen and thawed medium. The TBC medium was prepared as described in Saxton et al. (2008), and then 5 ml of solution A and 5 ml of solution B were frozen individually at either −10°C or −60°C for 24 h, 1 week, or 4 weeks before defrosting the medium and then combining solutions A and B. Defrosting of the medium was done under tap water at 22–24°C. Just before use, the two solutions were combined, and since no particulate matter was seen in the thawed medium, filtration was not necessary. The Petri dish containing the cysts was placed in an incubator at 41±0.5°C for up to 60 min. In preliminary trials, observations made at 60 min revealed a very high (approaching 100%) percentage of excystation. In subsequent trials, to determine the rate of excystation in this medium, observations were made on 25 to 30 cysts per datum point at 15, 20, 25, and 30 min. As seen in Fig. 1, excystation approached 100% within 30 min, and the rate of excystation was greatest between 15 and 20 min. Parasitol Res (2008) 102:809–810 DOI 10.1007/s00436-007-0850-y