The effects of different atmospheres and various supplements on the in vitro survival and growth of Ascaris suum larvae.

The in vitro cultivation of Ascaris larvae was carried out by supplementing either Eagle's medium or medium 199 with 20% calf or bovine serum. Growth was observed only when an initial concentration of 20,000 or more larvae/10 ml of medium were cultured at 37.5 C under partially anaerobic conditions. By this procedure a small percentage of the larvae reached sizes which made their isolation feasible. Continued development of the isolated larvae occurred only after the normal atmosphere was replaced by one of a nitrogen-5% CO2 mixture. Larvae were maintained for as long as 110 days and reached sizes up to 2,300 by 83 ,. In a previous paper (Cleeland and Laurence, 1962) a simple method for hatching large numbers of viable Ascaris larvae was described and the cultivation of these larvae in vitro in medium 199 (Morgan et al., 1950) supplemented with 20 to 30% bovine serum was reported. Survival for periods of 60 days at 37 C and 100 days at room temperature was observed along with growth of the larvae (up to 600 by 30 4) at the higher temperature when cultivation was carried out under partially anaerobic conditions. It has subsequently been found possible to increase both the time of survival and growth of the larvae. The present paper reports the results of these studies along with the effect of different atmospheric environments on their development and the initial attempts to define their nutritional requirements. MATERIALS AND METHODS Collection and preparation of infective eggs The collection and preparation of infective eggs were carried out as previously described (Cleeland and Laurence, 1962). However, most of the eggs used in the present study were obtained from the posterior two-thirds of the uteri of female worms, and a majority of these eggs never developed to the vermiform embryonic state. Consequently, to obtain enough larvae for study and eliminate the large amount of contaminating egg material, the following procedure was adopted. Hatching and preparation of the larval inoculum The egg suspensions containing approximately 10 to 20% infective eggs were washed several times with 0.01 M phosphate buffered saline, then resuspended in 20 ml of medium 199 to a concentration of 10,000 to 20,000 eggs/ml and transferred to a 125-ml Erlenmeyer flask. Hatching was carried out as reported earlier (Cleeland and Laurence, 1962) with a magnetic stirrer for a period of 24 hr at 22 to 25 C. Over 90% of the infective and a majority of the noninfective eggs were ruptured by this treatment, and most of the free larvae appeared to be undamaged and viable. The contents of two or three flasks were combined, centrifuged at 200 g for 10 min, and the supernatant fluid removed. The sediment was resuspended in 10 ml of fresh medium 199 and transferred to a 15-ml centrifuge tube. After centrifugation at 200 g for 10 min, the supernate was decanted leaving a sediment consisting of two distinct layers. The careful addition of 1 ml of 199 followed by gentle agitation of the tube caused most of the fluffy white upper layer to resuspend in the medium leaving the light brown lower layer intact. The resuspended material was decanted and the process repeated until only the brown layer remained. This layer, consisting mainly of live hatched larvae free of much of the eg material and all of the shell debris, was resuspended in 199 at room temperature until used (never more than 4 to 6 hr after hatching). Examination of the material in the white fluffy layer revealed that it consisted of damaged larvae, eggshells, egg debris, and from 20 to 40% of the viable larvae, depending on how carefully separation was carried out.