USE OF THE PCR AND FLUORESCENT-PROBES TO RECOVER SSU RIBOSOMAL-RNA GENE-SEQUENCES FROM SINGLE CELLS OF THE CILIATE PROTOZOAN SPATHIDIUM

A two-stage heminested PCR approach was developed to amplify small sub unit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucaryotes. The method was evaluated using the cili ate protozoon Spathidium when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments con tained the same sequence in a region of SSU rDNA which is normally hig hly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidium. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background autofluorescence. The Spathidium sequence is closely related to that from the haptorid Homa lozoon vermiculare.