MOLECULAR CHARACTERIZATION OF MYCOPLASMA CONTAMINATING TISSUE CULTURES

Dina Y.h Elshafey ,Abo-Shama U.h ,Saad A.a

doi:10.21608/avmj.2015.170246

Abstract

One of the main problems in cell culture unit is mycoplasma contamination. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production, cell therapy and its importance for diagnostic routine work in selected labs, so more concerns about mycoplasma contamination will arise. In our study, a total of 50 cell cultures from Animal Health Research Institute, Dokki, Giza., VACSERA and Veterinary Serum & Vaccine institute Abbasia were monitored for mycoplasma using culture and PCR methodology. The contamination was detected in the cell culture collected from all laboratories. Mycoplasmas were detected by culture in 29/50 (58%) of the cell culture samples which subsequently identified with PCR using Mycoplasma group specific primer detecting all species of mycoplasma which gave bands at 280 bp in all positive culture samples, the most frequent species was M. arginini (30%), followed by M. orale (28%). MDBK were positive only for M. arginini which gave band at 326 bp, while VERO and BHK cells were infected only with M. orale which gave bands at 87bp. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture unit along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture.

Highlights

Mycoplasma is the smallest free-living organisms that, unlike other bacteria, lack a cell wall
Mycoplasma contamination of cell cultures is a serious problem across the world because of the infection of cell cultures with mycoplasma can have different cytogenetic effects (Rottem and Barile, 1993)
The cell culture were examined for any changes or effects before uses, due to mycoplasma contamination many cytogenetic effects were detected, such as decrement of viability, detachment of adherent cells from the cell culture vessel surface (Photos 1&2), inhibition of proliferation, cell growth interference, morphological changes (Photos 3&4)

Summary

Introduction

Mycoplasma is the smallest free-living organisms that, unlike other bacteria, lack a cell wall. The outer layer is instead, a three layered membrane containing sterols. Diameters of these organisms may range from 0.2-0.3 μm and, due to their plasticity, are able to pass through the pores of a 0.2 micron filter with applied pressure. Solid and liquid media that will support the growth of small numbers of tested organisms such as typical contaminating organisms Acholeplasma laidlawii, Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and M. synoviae should be used. The nutritive properties of the solid medium should be such that no fewer than 100 CFU should occur with each tested organism when approximately 100–200 CFU are inoculated per plate. An appropriate colour change should occur in the liquid media when approximately 20–40 CFU of each tested organism are inoculated. The ability of the culture media to support growth in the presence of product should be validated for each product to be tested, and for each new batch or lot of culture media. (OIE Terrestrial Manual, 2012)

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Conclusion