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 cultures were tested using the mycofluor mycoplasma detection kit, following the kit protocols. A highly sensitive colorimetric microplatebased assay designed to detect the eight mycoplasma species known to cause 95% of eukaryotic cell culture contamination. Detection of mycoplasma contaminations 17 positive cultures should either be discarded and replaced by clean cultures or cured with specific antibiotics see chapter 3. Thus, mycoplasma infected cell cultures are themselves the single most important source for further spreading of the contamination table 1. Mycoplasma contamination of cell cultures ncbi nih.
A mycoplasma infected cell culture is a major source of mycoplasma contamination of other cell cultures in the lab. Apr 22, 2010 mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. Eradicating mycoplasma contamination 20 cell culture destroy contaminated cell cultures antibiotic therapy acquire fresh cells media destroy contaminated media use media guaranteed to be mycoplasmafree sterilize media via filtration or uv irradiation laboratory disinfect all laboratory surfaces and equipment biosafety. Data provided by the dsmz german collection of microorganisms and cell cultures also shown mycoplasma resistance within the range of 7 to 24% drexler and uphoff, 2002. Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. Mcgarrity department of microbiology institute for medical research camden, new jersey i. Google scholar drexler hg, gignac sm, hu zb, hopert a, fleckenstein e, voges m, uphoff cc. Vyzkumny ustav veterinarniho lekarstvi, brno czech republic. Mycoplasma contamination of cell cultures has been known for decades 1 and disturbingly, has become widespread, threatening academic labs to biopharmaceutical production facilities 2. The elimination of mycoplasma contaminations from cell cultures with antibiotics has been proven to be a practical alternative to discarding and re. Detection of mycoplasma in cell cultures global health laboratories. Drexler 1leibnizinstitute dsmzgerman collection of microorganisms and cell cultures, braunschweig, germany abstract mycoplasma contaminations have a. The pcr technology in the mycoplasma detection kit is fast results are typically obtained in less than 3 hours and easy to use. Detection of mycoplasma contamination in cell cultures.
Only definitely clean cultures should be used for research experiments and for the production of biologically active pharmaceuticals. Prevention and detection of mycoplasma contamination in. Pcr detection of multiple mycoplasma inf ecti on in cell cultures dilution was inoculated onto solid medium. How can i eliminate mycoplasma from infected cell cultures. Identity of 466 mycoplasma isolates from cell cultures species no. The quickest and most reliable way to eliminate mycoplasma is to autoclave the contaminated cultures and any bottles of media or sterile reagents that were used with them figure 8a. The microorganism was presumptively identified based. Prevention and detection of mycoplasma contamina tion in cell culture article pdf available in cell journal 4. A mycoplasmainfected cell culture is a major source of mycoplasma contamination of other cell cultures in the lab. Prevention and control of mycoplasma contamination in cell. Here, we examined the effectiveness of the new antimycoplasma compound.
A total of 104 cell cultures were tested by using microbiological culture, dna fluorochrome staining, dnarrna hybridization, and pcr techniques. Importance of the mycoplasma tests cell cultures offer ideal living c onditions to parasitic mycoplasma. 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. Several species are pathogenic in humans, including m. Mycoplasma detection protect your continuous cell lines. Although using good technique in the cellculture hood is perhaps the simplest preventive measure against mycoplasma, it is also the most difficult to implement. Contamination of both unrelated cell lines and of bacteria pose the gravest threat to cell cultures. Mycoplasma detection and prevention in cell cultures. Mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. For example, parameters inherent to slight differences in n2a cell lines across laboratories due e. Request pdf detection of mycoplasma in cell cultures mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures.
Detection of mycoplasma in cell cultures request pdf. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. A number of mycoplasma species commonly found in cell cultures hydrolyse arginine as an energy source. One of the main problems in cell culture is mycoplasma infection. Mycoplasma contamination of vaccines presents a potential health hazard. Drexler 1leibnizinstitute dsmzgerman collection of microorganisms and cell cultures, braunschweig, germany abstract mycoplasma contaminations have a multitude of effects on cultured cell lines that may. Mycoplasma removal agent is a broad spectrum antibiotic reagent containing a 4oxoquinoline3carboxylic acid derivative which is highly effective at killing and removing mycoplasma from cell cultures. Pdf prevention and detection of mycoplasma contamination in. Detection of mycoplasma contamination in cell cultures and. Immediately following mixing gluc with the media on these cells time 0h and 24 h later, 50. Jan, 2015 mixed observations are reported in the literature regarding the safety of antibiotics for cultured cells, though those may have pertained more to earlier generations of the drugs 2. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. Rapid detection methods using pcr and other techniques can achieve dramatic time savings and are ideal for screening cell culture in the laboratory and in industrial processes.
Cell cultures are used every day as a part of research methods. May 20, 2014 mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. Feb 26, 2019 mycoplasma contamination is a bacterial infection of cell cultures in laboratories. Prevention and detection of mycoplasma contamination in cell. 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. However, despite the presence of resistance, in subsequent studies at their institution evaluating the effectiveness of antibiotic regimens, they were able to cleanse 66 to. Pdf detection of mycoplasma contamination in cell cultures. To minimize these risks, routine testing of cell cultures and reagents is recommended.
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. In addition to contaminated incoming cultures, another main source of mycoplasma contamination is poor aseptic cellculture technique by lab members. Mycoplasma pneumoniae is one of the species of mycoplasmas which could contaminate cell cultures. This was the first report on the detection of mycoplasma in cell cultures 5. A simple and sensitive assay for mycoplasma detection in. How to keep your cultures mycoplasmafree biocompare. The original discovery of mycoplasma growth in cell cultures was in 1965. Detection of mycoplasmas in cell culture by fluorescence. In fact, depending on the laboratory, anywhere from 10% to 85% of cell lines may be contaminated 3. A high percentage of cell lines are chronically infected with various mycoplasma species. Pdf prevention and detection of mycoplasma contamination. Many cells support low levels of contamination that can only be detected. The mycodect mycoplasma detection kit is designed to detect mycoplasma infection in cell cultures in less than two hours. We recommend treating each affected cell line in parallel using the following three antibiotics.
These bacteria are difficult to detect and can easily spread from one culture to another once within a laboratory. Test cultures for mycoplasma it is not that difficult it is worth your peace of mind and reputation 4. Mycoplasma removal in cell cultures the following protocol is designed to remove mycoplasma from cell cultures. Detection of mycoplasma contamination in cell cultures by.
Treatment of mycoplasma contamination in a large panel of cell cultures. A myriad of detection methods exist for mycoplasmas in cell culture. Mycoplasma positive cell cultures are themselves the major source of infection 7. Afterwards, replace them with fresh culture stocks which have been certified as mycoplasmafree. Dec 22, 2011 other mycoplasma contaminated cell cultures. Materials and methods for mycoplasma detection and elimination the maintenance of contaminationfree cell cultures is fundamental to cell based research. Understanding and managing cell culture contamination.
A subculture of each culture was performed if the initial culture showed no mycoplasma. Mycoplasma contamination is a longstanding problem that can lead to erroneous data, misinterpreted results, and extensive costs. Treatment of mycoplasma contamination in cell cultures with. Detection of multiple mycoplasma infection in cell cultures. Materials and methods for mycoplasma detection and elimination the maintenance of contaminationfree cell cultures is fundamental to cellbased research. Mycoplasma is the smallest selfreplicating prokaryote characterized by lack of cell wall. Treatment of mycoplasma contamination in cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted. Pcr detection of multiple mycoplasma infection in cell cultures dilution was inoculated onto solid medium. To avoid mycoplasma contamination in cell cultures, it is recommended to test the new cell lines which are obtained from an outside source. Eradication of mycoplasma contaminations from cell cultures.
Mycoplasma contamination affects many cell functions including metabolism, morphology, protein synthesis and cell proliferation all of which lead to unreliable results as well as lost. Detection of mycoplasma contamination in cell cultures by a mycoplasma groupspecific pcr article pdf available in applied and environmental microbiology 601. In order to achieve reproducible results from cells. The suitability of a 16s rrnabased mycoplasma groupspecific pcr for the detection of mycoplasma contamina tion in cell cultures was investigated. These organisms are resistant to most antibiotics commonly employed in cell cultures. To avoid mycoplasma contamination in cell cultures, it is recommended to test the. Assessing mycoplasma contamination of cell cultures by. Mycoplasma plural mycoplasmas or mycoplasmata is a genus of bacteria that lack a cell wall around their cell membranes. The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the national cell bank of iran. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction.
In this presentation, we will discuss the most frequently used mycoplasma testing methods, and will expand on the products and services offered by atcc that support the early detection of mycoplasma as well as the development and validation of novel test methods. We suggest the same reason for both types of contaminations. Therefore, it is important to use efficient detection methods to in. The antibiotics see below should not be applied together as this will substantially harm your cells. Detection of mycoplasma in leukemialymphoma cell lines using polymerase chain reaction. Mycoplasma contamination thermo fisher scientific us.
Mycoplasma contaminations in the cell culture background. Elimination of mycoplasma contamination in cell cultures. However, all mycoplasma species contaminating cell cultures cannot be detected by a single test due to their limited sensitivity or specificity uphoff et al. Here we provide a concise overview of the current knowledge on. The sensitivity is up to 1020 copies of target dna, which translates to less than 10 mycoplasma species. Clean your cultures with these mycoplasmaelimination tools. The kit incorporates polymerase chain reaction pcr to amplify the conserved 16s ribosomal rna coding region within the mycoplasma genome, thereby providing an extensive, highly sensitive, and efficient detection method. Effects of mycoplasmal infection of cell culture methods to detect cell culture mycoplasmas a.
It can extensively affect cell physiology and metabolism. It is true, though, that cell health can sometimes suffer while youre trying to rid cultures of mycoplasma. Figure 8a autoclaving is the only guaranteed method for mycoplasma elimination. This characteristic makes them naturally resistant to antibiotics that target cell wall synthesis like the betalactam antibiotics. Photomicrographs of mycoplasmafree cultured cells panel a and cells infected with mycoplasma panels b and c. Mycoplasma removal agent is a derivative of the quinolone family of antibiotics, developed to combat the widespread problems caused by. Recently, several methods of detection of mycoplasma contaminations of cell cultures based on the polymerase chain reaction pcr technology have been developed. Survey of mycoplasma infections in cell cultures and a. It can detect mycoplasma from both cell lysates and cell culture media.
Mycoplasma contamination of cell lines is a major problem in cell culture technology. 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. Detection of mycoplasma in cell cultures nature protocols. Detection of mycoplasmal infection of cell cultures. Rely on a tested cell repository as the source of all of your cells atcc, dsmz, etc.
Page 2 of 4 tb100 description the southernbiotech mycoplasma detection kit is designed to specifically detect potential mycoplasma contamination in cell cultures. 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. Uses and standardization of vertebrate cell cultures in vitro monograph. Be careful of freecell lines they are often worth what you pay for them 3. Mycoplasma contamination can cause various effects on cultured cells such as change in growth rate and morphology, alteration of gene expression, and. The kit is also highly sensitive and can detect as little as 25 femtograms of mycoplasma dna in 100 l of test sample supernatant.
Conventional methods to detect mycoplasma contamination in cell cultures are technically demanding, labourintensive and take up to five weeks to produce a result. Consequently, mycoplasmas can be difficult to detect in routine cell lines culture work1. Eukaryotic and bacterial dna from cell culture supernatant is not amplified by the kit. Mcgarrity 1976 clearly demonstrated the efficiency and rapidity by which mycoplasma infection could spread in a cell. Mycoplasmapositive cell cultures are themselves the major source of infection 7. You rely on these cultures to be healthy, yet up to 35% may be contaminated due to mycoplasma. 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. Pdf isolation and detection of mycoplasma pneumoniae. Arginine depletion has also been linked with the chromosomal aberrations observed in contaminated cell cultures. Detection of multiple mycoplasma infection in cell. The best protection against these bacteria is to quickly identify contaminated cultures and reagents through routine testing.
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