Contamination of tissue cultures by mycoplasmas intechopen. We regularly stain cells on coverslips with dapi to test for mycoplasma. Nov 21, 2007 ive been culturing hela cells for a while. Elimination of mycoplasma contamination of virus stocks h. 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. Mycoplasma contamination is predominantly found in cultured samples e. All mollicute contaminants originated from a small number of primary sources. 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 contamination thermo fisher scientific us. The cultures were tested using the mycofluor mycoplasma detection kit, following the kit protocols. Photomicrographs of mycoplasmafree cultured cells panel a and cells infected with mycoplasma panels b and c. The main source of contamination is, in many cases, infection by previouslycontaminated cell.
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. Prevention and detection of mycoplasma contamination in. Murine embryonic stem cells mescs inoculated at passage p with the mycoplasma species m. Photomicrographs of mycoplasma free cultured cells panel a and cells infected with mycoplasma panels b and c. Mycoplasmas are notorious contaminants of cell culture and can have profound effects. Mycoplasma contamination in cultured versus noncultured cells. Assessing the prevalence of mycoplasma contamination in. The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. 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. 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. It requires a full understanding of the nature and causes of the problems followed by the development. Mycoplasma contamination of cell lines is a major problem in cell culture technology. The original discovery of mycoplasma growth in cell cultures was in 1965.
Detection of mycoplasma in cell cultures nature protocols. Mycoplasma is the smallest selfreplicating prokaryote characterized by lack of cell wall. Understanding and managing cell culture contamination. 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. 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. It has been shown that invasion of hela cells by m. Mycoplasma contamination is difficult to detect and therefore its presence can go undiscovered for months. Hence, mycoplasma contamination in cell culture is a longstanding and persistent problem. Scanning electron micrograph of critical pointdried cell cultures infected with infected cell line grown on coverslips. Detection of mycoplasma contamination in cell cultures and.
Nonetheless, contamination from exogenous sources also occurs during cell propagation and. The results obtained from these cells might not be indicative of the cell line or the cells in general. We grow humanmouse cell lines in culture hela, hek293, mefs in the absence of antibiotics. Mycoplasma is a genus of bacteria that lack a cell wall around their cell membranes. Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Detection of mycoplasma contamination in cell cultures sissada tannukit supreya wanichpakorn abstract mycoplasma contamination is one of the most common contaminants that can cause. Very effective at removing mycoplasma from infected cell cultures at a low concentration 0. 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. Learning materials in biosciences cell culture technology. Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. These organisms are resistant to most antibiotics commonly employed in cell cultures.
In fixed cells infected with mycoplasma, the mycofluor reagent stains both the nuclei and the. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a. How can i avoid mycoplasma contamination and other serious. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients. Host cell responses to persistent mycoplasmasdifferent stages in infection of hela cells with. Mycoplasma contamination of cultured cells poses a serious. Treatment of mycoplasma contamination in cell cultures. Assessing the prevalence of mycoplasma contamination in cell. The cells grew fine then over two days the culture was covered with tiny objects just floating around. Mycoplasma contamination thermo fisher scientific kr.
Spectral karyotypic analysis of mycoplasmainfected mescs showed a number of nonclonal chromosomal aberrations which increased with the. Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. 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. Mycoplasma contamination of murine embryonic stem mes. The bacterium plagued early cultures of hela cells, a widely used human cell line established in the 1950s. Many mycoplasmas exhibit the typical polymorphism of mycoplasmas, with the most common filamentous, flask shapes or ovoid structures figure 1, ref. A high percentage of cell lines are chronically infected with various mycoplasma species. Detection and antibiotic treatment of mycoplasma arginini. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Sep 26, 2008 murine embryonic stem cells mescs inoculated at passage p with the mycoplasma species m. 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.
Mycoplasma contamination of cell cultures researchgate. Detection of mycoplasma contamination in cell cultures and bovine sera h. The cancer cells, now called hela cells, grew rapidly in cell culture and. About frontiers institutional membership books news frontiers. Nonetheless, contamination from exogenous sources also occurs during cell propagation and continuous cell cultures are the most frequently contaminated. Clean your cultures with these mycoplasmaelimination tools. Mycoplasma cells are physically small less than 1 m, so are difficult to detect with a conventional microscope. Grow the cells without antibiotics for 2 weeks, then repeat mycoplasma test the gpcf accepts no liability for the success of this protocol. Thus, mycoplasma has infected onto mainly noncancer serum with nutrition deficiency, but has not infected onto cancer cells, which seem rich in the nutritions. Even if the mycoplasma has not altered your cells, the treatments to remove it may have. Jan, 2015 given the widespread yet unpredictable effects of mycoplasma on cells in culture, researchers have every reason to avoid contamination 1.
The following clues may help signal deterioration of a culture affected by mycoplasma contamination. 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. Mcgarrity 1976 clearly demonstrated the efficiency and rapidity by which mycoplasma infection could spread in a cell culture laboratory. However, a multitude of effects were described for infected cell cultures and a variety shall be mentioned in the following. Positive identification of mycoplasma typically requires cells. Hela cells used in their lab robinson and wichelhausen 1956.
Materials and methods for mycoplasma detection and elimination the maintenance of contamination free cell cultures is fundamental to cellbased research. Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted. It presents a major challenge for research, as well as in the manufacturing of bioproducts. Mycoplasma have high nutritional demands and can grow on pplo agar with beef heart infusion and 1020% horse serum. In our attempt to find an antimycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities. Frozen and cultured hel cells had only one type of mycoplasma m.
This characteristic makes them naturally resistant to antibiotics that target cell wall synthesis like the betalactam. This organism can modify many aspects of cell physiology, rendering experiments that are. Request pdf henrietta lacks, hela cells, and cell culture contamination henrietta. Henrietta lacks, hela cells, and cell culture contamination. Elimination of mycoplasma contamination in cell cultures. Materials and methods for mycoplasma detection and elimination the maintenance of contaminationfree cell cultures is fundamental to cellbased research. Eradication of mycoplasma contaminations from cell cultures. This unit presents protocols involving either the polymerase chain reaction pcr or fluorescent in situ hybridization. Experimental cell research 57 1969 374384 mycoplasma contamination of cell cultures interference with plabelling pattern of rna g. Here we provide a concise overview of the current knowledge on. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsiainfected cells. The contamination of cell cultures by mycoplasmas remains one of the major problems encountered in biological. According to the reagent manufacturers instructions, cells. Mycoplasma contamination of murine embryonic stem cells.
Detection of mycoplasma contamination in cell cultures. The lab i am working in has encountered a major issue. Mycoplasma contamination of your cell cultures can seriously impact the reliability, reproducibility, and consistency of your experimental results. 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. The serum provides mycoplasma with the cholesterol and longchain fatty acids required for growth. Treatment of mycoplasma contamination in cell cultures with. How to remove mycoplasma contamination in cell lines. I tried washing the culture out but the contamination seems to keep growing. Test the cultures for mycoplasma contamination see unit 28. Contamination of cell cultures and sera used for animal virus propagation with mycoplasmas represents a serious problem, especially in virology. Elimination of mycoplasma contamination from infected. Chronic infections usually cause decreased cell proliferation, while acute infections result in total destruction of the cell culture. Cell culture is also fraught with problems that confound reliable data, such as mycoplasma contamination.
Are tiny dots in cell culture a mycoplasma contamination. In 1956, researchers from johns hopkins reported mycoplasma con tamination of hela cells. 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. The cells grew fine then over two days the culture was covered with tiny. 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. Over the last week, it seems that my cells have become contaminated.
Reichelova veterinary research institute, brno, czech republic abstract. There are six species that account for 95% of all detected mycoplasma contaminations in continuous cell line cultures. Apr 22, 2010 mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. Mycoplasma contamination of cell cultures ncbi nih. Part of the cellular senescence and somatic cell genetics book series csscg. Antimycoplasma activity of daptomycin and its use for. Here, we examined the effectiveness of the new antimycoplasma compound. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis. Tsanev1 biochemical research laboratory, bulgarian academy of sciences, 2cell culture laboratory, department of virology, research institute of epidemiology and microbiology. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect.
In fact, depending on the laboratory, anywhere from 10% to 85% of cell lines may be contaminated 3. Mycoplasma can skew your data before you even know its there, so it is smart to test cultures routinely. 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. There are no secret tricks to avoiding mycoplasma contamination or other serious cell culture problems. Most mycoplasmas are typical extracellular microorganisms able to adhere to the surface of tissue culture cells. We studied the effectivity of a commercial antibiotic kit mycokill ab for the elimination of mycoplasma. Prevention and control of mycoplasma contamination in cell. Are there mycoplasma contaminations in cell cultures that.
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. 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. Note the impressive penetration of the mycoplasma cells into the eukaryotic cell surface a and the. Contamination of both unrelated cell lines and of bacteria pose the gravest threat to cell cultures. Mycoplasma species are often found in research laboratories as contaminants in cell culture. Has anyone had luck eliminating mycoplasma in cell lines like hela, u2os, and hek293 cells. Elimination of mycoplasma contamination of virus stocks. Guidelines for the use of cell lines in biomedical research nature.
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