Contamination Of Cell Cultures Research Articles

Detection and treatment of contaminating mycoplasmas in cell culture.

SummaryAspects of the problem of mycoplasma contamination of cell cultures have been examined. Particular attention has been paid to the effective eradication of mycoplasma contaminants. The effects of tylosin, a new antibiotic, are examined in detail.The work also describes attempts to eradicate a particularly resistant strain of M. orale type I, which has been isolated from contaminated cell lines in this laboratory.

Control of Pleuropneumonia-like Organisms in Cell Culture

Mammalian cell culture systems were maintained free of mycoplasmas by using a 3-day agar plate test as a weekly routine to monitor the conditions of the cells. If contaminated cell cultures were found, they were discarded and replaced from a pleuropneumonia-like organism (PPLO)-free cell bank. PPLO-free lines were established by treatment with various antibiotics. The KB cell line was freed of mycoplasmas by treatment for 1 week with a mixture of chlortetracycline, kanamycin, and chloramphenicol. L-929 cells were cleared of contamination with either spectinomycin or tylosin, and a synovial cell line was cleared with lincomycin or tylosin. Each cell line, after eradication of the contaminant, was stored in liquid nitrogen. A number of agents were tested to determine minimal inhibitory concentration against three known and three unidentified mycoplasmas. Chlortetracycline and tetracycline were found to be highly active against all strains, whereas tylosin, spectinomycin, and lincomycin, though less active, were equally useful because of their low toxicity against cells. Kanamycin was highly active against three strains, but inactive at high levels against the KB cell contaminants. A disc plate test was used to check isolated cell contaminants for sensitivity to various agents.

Characteristics of Avian Mycoplasma (PPLO) in Tissue Cultures of Human and Avian Cells

Mycoplasma organisms (PPLO) are ubiquitous; therefore, it is no surprise that they have been a real problem as contaminants in cell cultures. A report of Robinson et al. (6) in 1956 alerted researchers to the problem of PPLO contamination in tissue cell cultures. Hayflick et al. (4) reported that a human synovial fluid cell line (McCoy) was particularly susceptible to human strains of PPLO. No gross effects on the cells were reported, but intracytoplasmic inclusions considered to be PPLO were demonstrated. The widely used HeLa cell line has frequently been found to be contaminated. Collier (2) checked eight sub-lines and found only one that was not contaminated. Serologic tests showed all contaminants to be similar to PPLO isolated from human genital infections. Morphologic changes in tissue cells were observed by Collier, but he pointed out that such changes were not observed in other laboratories using the same contaminated sub-lines. Rothblat et al. (7) emphasized that cell cultures contaminated with PPLO may show no turbidity and no outstanding cytopathogenic effects; thus, their presence may be detected only by exhaustive culturing. Those authors demonstrated that cell cultures, in the presence of antibiotics, may be negative when cultured, but again become positive after passage in the absence of antibiotics. Rothblat et al. (8) found that 22 of 37 cell lines tested were contaminated with PPLO. None of the cell lines had

A Rapid Chemical Method for Detecting PPLO Contamination of Tissue Cell Cultures

A rapid, simple and routine chemical method for detection of PPLO contamination in cell cultures is reported. The method is based on the presence of arginine deiminase activity in all tissue culture PPLO strains, but absence from all mammalian tissue and PPLO-free cell cultures. In a total of 73 cell cultures examined, the detection of PPLO determined by presence of arginine deiminase activity agreed entirely with the most sensitive cultural procedure which requires 14 days.

Mammalian cell cultures contaminated with PPLO III. Elimination of PPLO with specific antiserum.

SummaryA procedure for control of PPLO contamination in mammalian cell cultures has been presented as an alternative to the employment of antibiotics. Six cell lines have been freed of PPLO by comparatively short-term exposure to antiserum specific for the contaminant in question. For destruction of 2 of the mycoplasma species a heat-labile serum component was required; the remaining 4 lines were successfully treated with inactivated serum. Because control of these contaminants is of utmost importance in the use of cell cultures as experimental systems application of this technic may be advantageous when antibiotic resistance is encountered.

Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures.

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs. Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84:130-136. 1962-A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics.

The vacuolating virus, S.V. 40.

SummaryThe newly discovered vacuolating virus, S.V.40, appears to be a common and essentially ubiquitous contaminant of rhesus monkey kidney cell cultures and a likely common contaminant of cynomolgus kidney cultures in which it develops to high titer without evident cytopathic change. The virus grows readily in grivet, vervet and patas kidney and in rhesus testicle cultures causing a unique cytopathology characterized by ballooning and intense vacuolation. Some biological and biophysical properties of the virus are described. Principal interest in and importance of the vacuolating virus arise from its demonstrated presence in all 3 types of Sabin's live poliovirus vaccines and in numerous respiratory virus seed lines employed in human volunteer inoculation experiments. Demonstration of the virus has raised the question of safety following administration to human subjects, especially infants, and of possible lack of validity of conclusions reached from experiments to measure neurovirulence or “genetic” ma...

Detection, elimination, and prevention of contamination of cell cultures with pleuropneumonialike organisms.

Detection, elimination, and prevention of contamination of cell cultures with pleuropneumonialike organisms.

Detection and possible source of contaminating pleuropneumonialike organisms (PPLO) in cultures of tissue cells.

Summary1) A total of 37 tissue cell lines growing in the presence of horse, calf, human, rabbit or dog serum have been tested for contamination by PPLO. Of these, 22 or 59% contained PPLO. Human, monkey, and mouse cell lines were contaminated. All first and second passages of cell lines were negative. PPLO were isolated from tissue cells growing in human, horse or calf serum. Nineteen positive cell lines were, at some time, grown in presence of human serum. Three positive lines growing in horse serum had never been in contact with human serum. 2) All 25 samples of sera, including pooled lots of human, calf, and horse sera, were negative for PPLO. 3) Possible sources of PPLO contamination of tissue cell cultures are discussed.

Contamination of human cell cultures by pleuropneumonialike organisms.

Contamination of human cell cultures by pleuropneumonialike organisms.