A Method for Eradication of Mycoplasma Infections in Cell Cultures

Abstract

Investigators using cell cultures have become increasingly aware of the problems introduced by parasites such as Mycoplasma.

In the past few years many have attempted to eliminate Mycoplasma in tissue cultures, using different methods and with varying degrees of success. Heat treatment (Hayflick (7)), use of specific antiserum (Pollock and Kenny(2)) or antibiotic treatment (Hearn, Officer, Eisner, and Brown(3); Pollock, Kenny and Syverton(4); Koehler(5)) have all been suggested. In our hands these methods have not been consistent, in that they may have been successful only in particular cell lines and with particular strains of Mycoplasma contaminants, or on account of more sensitive methods (Hayflick(l)) used in our laboratory to isolate aerophilic and micro-aerophilic Mycoplasma (Table I). As the morphology of isolates suggested that a variety of Mycoplasma was infecting the screened cell lines, the preparation of polyvalent anti-sera was not considered practical, and it was decided instead to explore fully the usefulness of antibiotic treatment. Simple antibiotic treatment, even at near cytotoxic doses and for periods of up to 5 months, proved only capable of depressing the severity of infection, approximately from 100 or more to 10 or less colonies per million cells, without effecting sterilization (Table I). One obvious difficulty was the fact that Mycoplasma infection can be and usually is an intracellular infection (Edwards and Fogh (6)) : Hayflick and Stinebring(7); Carski and Shepard(8)) . In consequence, antibiotics or specific antisera may have eliminated the Mycoplasma present in culture fluids without affecting those nested inside cells. A possible approach was to increase the permeability of the cells and, of the several possible methods, a simple hypotonic treatment was selected.

Materials and Methods. Antibiotic-Hypotonic treatment. Previous studies (Blyth(9); Hearn, Officer, Elsner, and Brown(3); Fogh and Hacker(l0); Pollock, Kenny and Syverton(4); Koehler (5)) indicate that Mycoplasma are highly sensitive to aureomycin