1. A technique is described for the preparation of axenic cultures of protoscolices of Echinococcus granulosus.2. Protoscolices removed aseptically from hydatid cysts are treated with 0·02% crystalline pepsin at pH 2·0 for 10 min. at 38°C. in order to remove (a) daughter cyst germinal membranes, and (b) dead or degenerating protoscolices.3. Protoscolices may be maintained for more than 100 days in a variety of media. These include media containing various proportions of some of the following: chick embryo extract, beef embryo extract, hydatid fluid, bovine amniotic fluid, Parker 199, bovine serum.4. 10–20 ml. of culture media was used at 38°C., either in roller tubes or shaken in a water-bath. Media were renewed every 48 hr.5. Protoscolices developed into cysts by two routes, either (a) by becoming vesicular, gradually growing in size, and finally becoming enclosed in a laminated envelope, or (b) by forming a ‘posterior bladder’ which became relatively enormous and ultimately absorbed the scolex region, the whole forming a cyst within an envelope as in (a).6. In the majority of media, after about 31–48 days' culture, a thin envelope was secreted which developed later into a thick laminated membrane.7. Cysts with laminated membranes ultimately underwent fatty degeneration and became cytologically abnormal.8. In the most successful experiments, cells or clusters of cells formed within the cysts and these may have represented the anlagen of the protoscolices.9. In no case was segmentation, or any other sign of strobilar development, obtained.10. These results show that the laminated membrane in E. granulosus is initially of parasite origin and not of host origin as believed by some workers to be the case.11. Since there is evidence from the literature that E. multilocularis does not form such an envelope in vitro, a clear developmental difference appears to exist between the two species.12. It is suggested that this difference offers an explanation for the formation of unilocular or multilocular cysts. Since E. granulosus secretes a laminated envelope, its development is limited to a single or unilocular type of cyst. In the case of E. multilocularis no such limitation occurs, so that the invasive multilocular type of cyst could more easily develop.
The Daughter Cyst
