Acridine orange was used to stain the gramnegative anaerobic bacilli (Bacteroides), the round boides and the amebae in the SF, MSF, CLG, and CLVG media. Evidence was found which indicates the round bodies are actually protoplasts.
The protoplasts, when first formed, exhibited a red fluorescence on staining with acridine orange. As they enlarged, this material was localized as beads around the periphery. After 24 to 48 hours, all red fluorescence disappeared. When the protoplasts were ingested by trophozoites of Entamoeba histolytica, the red fluorescence disappeared more rapidly. It is suggested that this red fluorescence is indicative of the presence of RNA.
The use of lysozyme to speed up production of protoplasts resulted in more rapid propagation of the amebae. This was interpreted as evidence of the importance of srotoplasts in the propagation of E. histolytica in SF type media. However, when culture S5, which does not support propagation of E. histolytica in the SF medium, was induced to produce protoplasts by using lysozyme, it still did not support propagation of the amebae.
As a follow-up on the observations of the disappearance of the red fluorescence from the protoplasts, commercial RNA was added to E. histolytica cultures in a modified CLG medium. This resulted in a definite enhancement of propagation.
Present address: The Lutheran General Hospital, 1775 Dempster Street, Park Ridge, Illinois.