Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations.
Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.
Kumar
Ashok
A
Cell Biology Laboratory, Institute of Pathology (ICMR), P.O. Box 4909, Safdarjung Hospital Campus, New Delhi 110029, India.
Yerneni
Lakshmana K
LK
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D001330:Electronic Data Processing / Q000295:instrumentation / Q000379:methods*; D004866:Equipment Contamination / Q000706:statistics & numerical data; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D008253:Macaca mulatta; D051379:Mice; D009174:Mycoplasma / Q000166:cytology* / Q000302:isolation & purification; D012984:Software; D014709:Vero Cells