Modifications in Media and Surface Sterilization Methods for In Vitro Cultivation of Hymenolepis diminuta

Abstract

The efficacy of defibrinated horse and sheep bloods and sheep serum was compared to that of defibrinated rabbit blood for the in vitro cultivation of Hymenolepis diminuta (Cestoda: Cyclophyllidea). Weight gains and reproductive success of worms cultured with sheep blood in the medium were not significantly different from those of worms cultured in the presence of rabbit blood. Horse blood was less effective, and sheep serum was poorest in this regard. Growth of penicillinand streptomycin-resistant bacteria in the cultures was controlled with tetracycline, and the presence of the antibiotic did not affect the development of H. diminuta at the concentrations tested. However, tetracycline-resistant bacteria were encountered, and the feasibility of complete sterilization of the surface of the worms with chemical agents was investigated. A brief rinse in 0.0625% or 0.00625% Zephiran chloride? (benzalkonium chloride) or 0.1% Phisohex? (hexachlorophene) before placing the worms in cultivation was effective and produced no detectable effect on subsequent development in vitro. Schiller (1965) described a method for in vitro cultivation of Hymenolepis diminuta in a biphasic medium. The phases consisted of a blood-agar base to which an equal volume of Hanks' balanced salt solution was added 24 hr before worm inoculation. Certain modifications were described by Roberts and Mong (1969), and although the method remains less than ideal, it is simple enough to be of considerable use for investigating some aspects of cestode development (Roberts and Mong, 1969, 1973; Bolla and Roberts, 1971). Among the disadvantages of the technique are (1) the inclusion of 30% defibrinated rabbit blood in the agar base, and (2) the time-consuming and costly requirement for daily transfer of the worms. The first of the foregoing is a disadvantage not only because the blood is an undefined component in the medium, but also because rabbit blood is expensive and often not available conveniently close to the user. If the worms are cultivated from the cysticercoid stage (Schiller, 1965), more transfers, medium, etc., are required than if the worms are allowed to begin growth in vivo. In the belief that some other blood product may be less expensive and more widely available commercially than rabbit blood, certain alternatives have been tested. Also, as indicated below, the young tapeworms often carry bacterial conReceived for publication 12 January 1973. * This investigation was supported by grant AI06153 from the NIH, U. S. Public Health Service. tami ants from the rat gut that are difficult to control in vitro. Thus, additional means for control of bacterial contamination have been