Prevention and control of mycoplasma contamination in cell. The main source of mycoplasma infection, in the majority of cases, is cross contamination by a previously mycoplasma infected cell culture which is used in the same laboratory. It has been shown that invasion of hela cells by m. However, a multitude of effects were described for infected cell cultures and a variety shall be mentioned in the following. Very effective at removing mycoplasma from infected cell cultures at a low concentration 0. Here, we examined the effectiveness of the new antimycoplasma compound.
We regularly stain cells on coverslips with dapi to test for mycoplasma. These organisms are resistant to most antibiotics commonly employed in cell cultures. Mycoplasma contamination in cultured versus noncultured cells. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients. Assessing the prevalence of mycoplasma contamination in. Elimination of mycoplasma contamination of virus stocks. Note the impressive penetration of the mycoplasma cells into the eukaryotic cell surface a and the. Nov 21, 2007 ive been culturing hela cells for a while. This organism can modify many aspects of cell physiology, rendering experiments that are conducted.
The cells grew fine then over two days the culture was covered with tiny. The cancer cells, now called hela cells, grew rapidly in cell culture and. Treatment of mycoplasma contamination in cell cultures with. Mycoplasma contamination of murine embryonic stem mes. Mycoplasma contamination is difficult to detect and therefore its presence can go undiscovered for months. Mycoplasma contamination of cell cultures ncbi nih. It requires a full understanding of the nature and causes of the problems followed by the development. Chronic infections usually cause decreased cell proliferation, while acute infections result in total destruction of the cell culture. Hence, we posited that noncultured samples in our study would be underrepresented for mycoplasma contamination and provide a baseline for the expected number of mycoplasma mapped reads in a. Contamination of cell cultures and sera used for animal virus propagation with mycoplasmas represents a serious problem, especially in virology. In fixed cells, the mycofluor reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear. Mycoplasma contamination of cultured cells poses a serious. Prevention and detection of mycoplasma contamination in.
Contamination of both unrelated cell lines and of bacteria pose the gravest threat to cell cultures. The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Detection of mycoplasma contamination in cell cultures sissada tannukit supreya wanichpakorn abstract mycoplasma contamination is one of the most common contaminants that can cause. All mollicute contaminants originated from a small number of primary sources. Treatment of mycoplasma contamination in cell cultures. Mar 10, 2010 despite the passing of nearly 50 years since the problem first surfaced of hela cell contamination of tissue cultures and despite the explosive advances in molecular biology, cell culture contamination remains an important issue for the scientific community. Mycoplasma species are often found in research laboratories as contaminants in cell culture. Part of the cellular senescence and somatic cell genetics book series csscg. Despite the passing of nearly 50 years since the problem first surfaced of hela cell contamination of tissue cultures and despite the explosive advances in molecular biology, cell culture contamination remains an important issue for the scientific community. The cells grew fine then over two days the culture was covered with tiny objects just floating around. Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation.
Henrietta lacks, hela cells, and cell culture contamination. Hence, mycoplasma contamination in cell culture is a longstanding and persistent problem. Many mycoplasmas exhibit the typical polymorphism of mycoplasmas, with the most common filamentous, flask shapes or ovoid structures figure 1, ref. Guidelines for the use of cell lines in biomedical research nature. Eradication of mycoplasma contaminations from cell cultures. It presents a major challenge for research, as well as in the manufacturing of bioproducts.
Mycoplasma is a genus of bacteria that lack a cell wall around their cell membranes. We grow humanmouse cell lines in culture hela, hek293, mefs in the absence of antibiotics. Mycoplasma contamination of cell cultures researchgate. Mycoplasma can skew your data before you even know its there, so it is smart to test cultures routinely. A high percentage of cell lines are chronically infected with various mycoplasma species. The main source of contamination is, in many cases, infection by previouslycontaminated cell.
Photomicrographs of mycoplasma free cultured cells panel a and cells infected with mycoplasma panels b and c. Murine embryonic stem cells mescs inoculated at passage p with the mycoplasma species m. Clean your cultures with these mycoplasmaelimination tools. There are six species that account for 95% of all detected mycoplasma contaminations in continuous cell line cultures. Mycoplasma cells are physically small less than 1 m, so are difficult to detect with a conventional microscope. Cell culture is also fraught with problems that confound reliable data, such as mycoplasma contamination. The serum provides mycoplasma with the cholesterol and longchain fatty acids required for growth. Mycoplasma have high nutritional demands and can grow on pplo agar with beef heart infusion and 1020% horse serum. Detection of mycoplasma contamination in cell cultures and. Experimental cell research 57 1969 374384 mycoplasma contamination of cell cultures interference with plabelling pattern of rna g.
Mycoplasma removal agent is a derivative of the quinolone family of antibiotics, developed to combat the widespread problems caused by contamination of cell cultures with mycoplasma through inhibiting mycoplasma dna gyrase. Understanding and managing cell culture contamination. In our attempt to find an antimycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities. Features 0 20 40 60 80 100 % 84 5 11 77 14 9 64 22 bmcycline 95 ciprofloxacin 114 mra 157 cured resistant culture death 49 contents figure 1 outcome of treatment of mycoplasmapositive cell cultures with either bmcyclin, ciprofloxacin, or mra. Frozen and cultured hel cells had only one type of mycoplasma m. According to the reagent manufacturers instructions, cells. Elimination of mycoplasma contamination in cell cultures. The bacterium plagued early cultures of hela cells, a widely used human cell line established in the 1950s. Mycoplasma contamination thermo fisher scientific kr. Test the cultures for mycoplasma contamination see unit 28. Here we provide a concise overview of the current knowledge on. Hela cells used in their lab robinson and wichelhausen 1956.
Mycoplasma contamination of cell lines is a major problem in cell culture technology. How can i avoid mycoplasma contamination and other serious. Mycoplasma contamination is predominantly found in cultured samples e. Photomicrographs of mycoplasmafree cultured cells panel a and cells infected with mycoplasma panels b and c. How to remove mycoplasma contamination in cell lines. Contamination of tissue cultures by mycoplasmas intechopen. This unit presents protocols involving either the polymerase chain reaction pcr or fluorescent in situ hybridization fish to provide independent, fast, and sensitive techniques to monitor mycoplasma contamination in laboratory cultures. Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Mycoplasma contamination thermo fisher scientific us. Tsanev1 biochemical research laboratory, bulgarian academy of sciences, 2cell culture laboratory, department of virology, research institute of epidemiology and microbiology.
In 1956, researchers from johns hopkins reported mycoplasma con tamination of hela cells. Sep 26, 2008 murine embryonic stem cells mescs inoculated at passage p with the mycoplasma species m. Materials and methods for mycoplasma detection and elimination the maintenance of contaminationfree cell cultures is fundamental to cellbased research. This organism can modify many aspects of cell physiology, rendering experiments that are. There are no secret tricks to avoiding mycoplasma contamination or other serious cell culture problems. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis. Host cell responses to persistent mycoplasmasdifferent stages in infection of hela cells with.
The lab i am working in has encountered a major issue. Even if the mycoplasma has not altered your cells, the treatments to remove it may have. Antimycoplasma activity of daptomycin and its use for. Elimination of mycoplasma contamination from infected. About frontiers institutional membership books news frontiers. Apr 22, 2010 mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. The presence of mycoplasma nucleases may account for some of the chromosome damage observed in mycoplasma infected cells and has been suggested as a possible cause for the failure of reverse transcriptase assays in contaminated cells. Nonetheless, contamination from exogenous sources also occurs during cell propagation and continuous cell cultures are the most frequently contaminated. Mcgarrity 1976 clearly demonstrated the efficiency and rapidity by which mycoplasma infection could spread in a cell culture laboratory. The contamination of cell cultures by mycoplasmas remains one of the major problems encountered in biological. Learning materials in biosciences cell culture technology. Over the last week, it seems that my cells have become contaminated.
Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. In fixed cells infected with mycoplasma, the mycofluor reagent stains both the nuclei and the. Detection of mycoplasma contamination in cell cultures sissada tannukit supreya wanichpakorn abstract mycoplasma contamination is one of the most common contaminants that can cause serious problems in cell cultures. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. We can toss most of the affected cell lines because we can purchase them from atcc, but we have several stable cell lines that people are not willing to throw away. Mycoplasmas are notorious contaminants of cell culture and can have profound effects. Reichelova veterinary research institute, brno, czech republic abstract. Mycoplasma contamination of cell cultures has been known for decades 1 and disturbingly, has become widespread, threatening academic labs to biopharmaceutical production facilities 2. In fixed cells, the mycofluor reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that the culture is free from mycoplasma contamination panel a. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. We studied the effectivity of a commercial antibiotic kit mycokill ab for the elimination of mycoplasma. Assessing the prevalence of mycoplasma contamination in cell.
I tried washing the culture out but the contamination seems to keep growing. The incidence of single mycoplasma contamination is still high, being 15 to 35% worldwide with extreme incidences of 65 to 80%, whereas incidences of multiple mycoplasma. It requires a full understanding of the nature and causes of the problems followed by the development of a comprehensive program to actively manage your cell cultures, laboratory facility and personnel coecke et al. This unit presents protocols involving either the polymerase chain reaction pcr or fluorescent in situ hybridization. Among the most pervasive concerns is contamination with mycoplasma, a genus of bacteria whose members are typically submicron in size and lacking a cell wall, making them difficult or. Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. Are tiny dots in cell culture a mycoplasma contamination. Detection of mycoplasma contamination in cell cultures. Scanning electron micrograph of critical pointdried cell cultures infected with infected cell line grown on coverslips. Most mycoplasmas are typical extracellular microorganisms able to adhere to the surface of tissue culture cells. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. Has anyone had luck eliminating mycoplasma in cell lines like hela, u2os, and hek293 cells. Detection and antibiotic treatment of mycoplasma arginini. Detection of mycoplasma in cell cultures nature protocols.
Detection of mycoplasma contamination in cell cultures and bovine sera h. Jan, 2015 given the widespread yet unpredictable effects of mycoplasma on cells in culture, researchers have every reason to avoid contamination 1. Nonetheless, contamination from exogenous sources also occurs during cell propagation and. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsiainfected cells. Are there mycoplasma contaminations in cell cultures that. Mycoplasma contamination of your cell cultures can seriously impact the reliability, reproducibility, and consistency of your experimental results.
The results obtained from these cells might not be indicative of the cell line or the cells in general. Crosscontamination of mouse and human cells, xmrv infection and cell. Mycoplasma contamination of murine embryonic stem cells. Grow the cells without antibiotics for 2 weeks, then repeat mycoplasma test the gpcf accepts no liability for the success of this protocol. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a. Mycoplasma is the smallest selfreplicating prokaryote characterized by lack of cell wall. Materials and methods for mycoplasma detection and elimination the maintenance of contamination free cell cultures is fundamental to cellbased research. This characteristic makes them naturally resistant to antibiotics that target cell wall synthesis like the betalactam. Thus, mycoplasma has infected onto mainly noncancer serum with nutrition deficiency, but has not infected onto cancer cells, which seem rich in the nutritions. In fact, depending on the laboratory, anywhere from 10% to 85% of cell lines may be contaminated 3. Positive identification of mycoplasma typically requires cells. The original discovery of mycoplasma growth in cell cultures was in 1965.
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