The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures.

SummaryMycoplasma hyopneumoniae , the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparinbinding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages.

without detectable blast infiltration. At diagnosis of MDS, interface cytogenetic and RT-PCR analyses, respectively, showed trisomy 8 and absence of AF9-MLL rearrangement in Introduction the bone marrow (BM). On readmission with florid leukemia, his peripheral white blood cell count was 13 200/mm 3 , with A subtle, reciprocal translocation exchanging the terminal 56% leukemic blasts. His hemoglobin concentration was short and long arm segments of chromosomes 9 and 11, 15.0 g/dl, and his platelet count was 22 000/mm 3 . The bone respectively t(9;11)(p21-22;q23), is associated with acute marrow aspiration showed 92% leukemic blasts, and the mormyeloid leukemia (AML)-M5, 1 particularly the M5a subtype. 2 phological diagnosis was made as AML-M5a. Cytogenetic Ascertainment may be difficult in suboptimal preparations analysis was interpreted as: 47, XY, +8, t(9;11)(p22;q23). and, despite being regarded as the 'standard' cytogenetic Immunophenotyping analysis of fresh leukemia blasts change in acute monoblastic leukemia, its overall incidence revealed no significant expression of CD antigens associated and pattern of associations remain uncertain. 2 A recent study with the myelo-monocytic lineage. CD34 was 30% positive comparing AML-M1 and -M5 patients, analyzed simuland non-lineage-associated HLA-DR was found to be positive taneously by reverse transcriptase-polymerase chain reaction at 70%; whereas those indicative of lymphoid lineage includ-(RT-PCR), Southern blotting and fluorescence in situ hybridizing CD3, CD4, CD8, CD10, CD19 were absent or weakly ation (FISH) with an MLL-specific yeast artificial chromosome expressed. Myeloperoxidase activity was weakly detected on (YAC) probe, suggests the overall incidence of MLL rearrangethe fresh leukemic blasts. Despite receiving chemotherapy, he ment in AML-M5 may be as high as 60%. 3 It was apparent in succumbed to rapidly progressive leukemia on 15 August that study that approximately half the cases with MLL 1995. rearrangement went undetected by cytogenetic methods, including a cryptic t(6;11)(q27;q23) resulting from a cytogenetically invisible insertion juxtaposing AF6 and MLL.Materials and methods A case of AML-M5a with de novo MLL-AF9 fusion evolving from MDS with trisomy 8 present at all phases has recently Cell culture been described. 4 We describe a pair of cell lines, MOLM-13 During relapse, after chemotherapy, a heparinized peripheral blood specimen, obtained with informed consent, was proCorrespondence: Y Matsuo,

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.

The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures.