New Assay Procedure for Separation of Mycoplasmas from Virus Pools and Tissue Culture Systems

Abstract

Presence of mycoplasma organisms in tissue culture systems and virus pools was detected by titration of the contaminated material on agarose-suspended BHK21 / 13S cells. The use of this method permitted isolation of mycoplasmas which could not be detected by standard assay methods. Mycoplasma colonies at concentrations ranging from 104 to 106 colony-forming units/ml in agarose-BHK21/13S media could be distinguished from virus plaques, and the two populations of inicroorganisms could be easily disassociated either by electron microscopy or by biological methods. All isolated mycoplasmas were identified in growth inhibition tests as belonging to the GDL group. The growth inhibition test on agarose-BHK21/13S cell suspension plates could also be applied directly to those strains which could not be isolated by standard assay procedures.

Rapid Detection and Isolation of Mycoplasmas From Cell Cultures

“…Hopps et al (1973) identified Mycoplasma hijorhinis in cell cultures by immunofluorescence, though they failed to culture it on agar medium. Similar results were reported by Zgorniak-Nowosielska et al (1967), Schneider et al (1974b) and House and Waddell (1967). Thus, an obvious need for an improved medium for the isolation of mycoplasmas exists.…”

Reproducible Plaquing System for Rabies, Lymphocytic Choriomeningitis, and Other Ribonucleic Acid Viruses in BHK-21 / 13S Agarose Suspensions

“…In addition, an accompanying paper (10) describes the use of this method for the growth of mycoplasma contaminants of virus pools which formed easily visible colonies. This fact enabled us to derive pure rabies and LCM clones from two virus stocks which had become contaminated with mycoplasma (10).…”

Description of a Viral Agent Found in Blood Obtained from Patients with Infectious Hepatitis

“…Incorporation of kanamycin, streptomycin, or tylosin into maintenance media did not prevent plaque formation. The plaques formed by agent Y lacked the morphological characteristics recently described for PPLO (7,11), and known cultures of PPLO did not plaque in the systems described above. Adsorption of antisera against agent Y with concentrated preparations of PPLO isolated from contaminated tissue culture did not inhibit the formation of characteristic precipitin patterns in immunodiffusion tests.…”