Purification of the endospores and sporangia of Rhinosporidium seeberi on Percoll columns

Human rhinosporidial tissue was used as the source of the various developme ntal stages of Rhinosporidium seeberi - endospores with electron dense bodi es, juvenile, and immature sporangia. After homogenisation in phosphate buf fered saline (PBS) and removal of tissue fragments by centrifugation, the r hinosporidial bodies were isolated on centrifuged Percoll columns with grad ients of densities or on triple-layered columns of varying density. The sep arated bands, after repeated washing in PBS gave bodies free from human tis sue as shown on Leishman and PAS staining and indirect immunofluorescence w ith rabbit and human patients' anti-rhinosporidial sera. Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining. Percoll columns were s hown to be capable of isolating these stages of R. seeberi, free from human tissue and contaminating bacteria.