Here are some things to do:. So you found mycoplasma contamination, what do you do next? The best way is to discard the infected cell culture flasks. In cases where the culture is irreplaceable or very expensive, there are some ways to rescue the cultures.
Elimination is a very time-consuming process and increases the risk of secondary contamination to other healthy cell lines. Mycoplasma Removal Agents MRA are derivatives of the quinolone family of antibiotics and are broad-spectrum antibiotic agents. These agents can be used to wash the cell cultures.
The treatment can take a few weeks to a couple of months depending on the severity of the infection. Plasmocin is a widely used drug that can clear most of the mycoplasmas present in the culture media.
Also, drugs like BM Cyclin, fluoroquinolone ciprofloxacin, ciprobay, zagam, baytril, tetracycline, etc. MRAs are non-toxic, effective in the removal of most of the mycoplasma contamination, easy to use, and effective for wide range of mycoplasma. These methods cannot guarantee complete removal of mycoplasma but, can get rid of most of mycoplasma contamination and are the last alternative.
There are some drawbacks while considering this treatment as the duration of treatment can affect the cell growth and viability. If mycoplasma has made permanent damage to the cell culture it is irreparable.
Some cells may die due to longer incubation periods and affect the total cell viability. To sum up, it is always important to prevent contamination rather than eradicating it later. Educate others when you can and help them understand the importance of preventing contamination. Be aware and cautious rather than learning it the hard way!
All cell culture contaminants can be problematic, but in comparison to other types of bacterial and fungal contaminants, mycoplasma contamination is usually more difficult to detect. This is because Mycoplasma are very small and replicate at a slower rate compared to many other bacterial species.
Discarding your contaminated cell cultures may seem like an obvious response to mycoplasma contamination. However, if you are using a culture that you can no longer obtain, or that is particularly valuable, it could be worth trying to eliminate the contamination. Keep in mind that treatment to get rid of Mycoplasma can take weeks, if not longer.
Elimination of mycoplasma contamination can be carried out by using specially formulated reagents, such as MycoZap TM Mycoplasma Elimination Reagent. This tool combines antibiotic and antimetabolic agents to eliminate detectable mycoplasma contamination in as little as four days.
It has been optimized to clear the contamination with minimal effects to your cell culture. More details on the elimination of Mycoplasma species can be found in the next section. There are various methods for eliminating mycoplasma contamination. These fall into one of four categories: chemotherapeutic, chemical, immunological, and physical.
Elimination techniques include antibiotic treatment, exposure to detergents, exposure to complement, cell cloning, heat treatment, and filtration 1. Unfortunately, many of these techniques have been shown to be unreliable, as they cannot kill all Mycoplasma species. They are also very time-consuming and are not very efficient at removing contamination.
Antibiotic treatment, for instance, has shown promising results in mycoplasma-contaminated leukemia-lymphoma cell lines. If antibiotics are unable to clear the mycoplasma contamination, bacteriostatic antimicrobial agents might help to inhibit the growth of the bacteria. Elimination of mycoplasma contamination can also be achieved by using specially formulated reagents, such as MycoZap TM Mycoplasma Elimination Reagent.
This reagent combines antibiotic and antimetabolic agents to eliminate detectable mycoplasma contamination in as little as four days. We have optimized it to clear mycoplasma contamination with minimal effects to your cell cultures. Leukemia — Mycoplasma contamination, transmission, and prevention require a three-pronged approach to ensure you are working as cleanly as possible 1 :.
It is essential that your facility offers suitable equipment, and that the laboratory is kept clean and tidy. This includes having a certified laminar-flow biological safety cabinet, performing regular inspections and cleaning of incubators, and ensuring the proper disposal of consumables and cell culture materials.
Use reliable mycoplasma detection methods and test cultures on a regular basis to help limit the impact that contaminants have on your facility, as well as on your work.
Keep incoming cultures in quarantine until proven to be mycoplasma free, and immediately discard contaminated cultures if possible. Purchase cultures from reputable suppliers. Although overuse of antibiotics is not recommended, for precious cell cultures the continuous use of antibiotics that are effective against Mycoplasma species, such as MycoZap TM Antibiotics, can be a way of preventing contamination.
Thorough washing and disinfecting of your hands is key to working as cleanly as possible. Wear personal protective equipment PPE , such as a clean laboratory coat and appropriate gloves. All but two series are associated with publications. These articles have been generally well cited with half of them receiving at least 51 citations since or later, suggesting their importance to their respective fields. Two of the series have over citations.
We looked up the publication information for the indicated series including the journal name, year of publication and the number of citations according to Google Scholar.
This study was inspired by a recent incident of mycoplasma contamination in our lab. We wondered how often this occurred in other labs. There have been several such surveys over the last three decades 5—7.
These studies used DNA fluorescent staining or polymerase chain reaction-based methods to detect mycoplasma contamination in collected samples. This study represents one of the most extensive surveys for mycoplasma contamination. Importantly, these entries were comprised of various sample types under different experimental conditions and originated from multiple institutions.
However, this is likely an underestimate. For one, in our analysis, we discarded mycoplasma-mapped reads that were not unique to mycoplasma. Some of these included reads that mapped to other closely related bacteria and hence could not be unambiguously associated with mycoplasma.
Secondly, Figure 1 suggests that a cutoff of 10 mycoplasma-mapped RPM was equally powerful to discriminate between cultured and non-cultured samples. Poly A -selection should eliminate most host and mycoplasma rRNA.
In those series with contamination, it is likely that polyA-selection was inefficient. In our hands it often takes multiple rounds of selection to remove a sufficient amount of rRNA. At such, many of the samples that appeared contamination-free might have simply masked contamination by thoroughly selecting for polyadenylated transcripts. Nonetheless, no matter the exact rate of contamination, our study suggests mycoplasma contamination remains a significant problem.
So what do we do with all these contaminated studies? Should we discard them altogether? Are any of their results valid? These are tough questions that will unfortunately require examination on a case-by-case basis. It should be noted though that mycoplasma contamination does not necessarily invalidate the findings of a study.
We can imagine scenarios were certain cellular responses are sufficiently robust even in the presence of such perturbations if we could call contamination such. However, we cannot selectively pick which studies we choose to believe.
At some point, all contaminated experiments need to be repeated. In the mean time, we suggest GEO and other sequence repositories develop a standard for flagging studies with evidence of mycoplasma contamination. We mapped sequence reads to four complete mycoplasma genomes: M. As such, A. These four species, along with M. In our study, we found A. This may be indicative of the reduced likelihood of contamination from bovine serum as commercial products are now routinely 0.
We identified 61 genes associated with mycoplasma contamination in a single cell RNA-seq dataset. Unfortunately, we could not identify series containing comparable contaminated and non-contaminated samples. Therefore, we measured the association between the number of mycoplasma-mapped reads per sample and host gene expression in a contaminated series.
Consequently, directionality of effect could not be determined. That is, differences in the extent of mycoplasma contamination may have driven host gene expression or differences in host gene expression especially as it relates to variability of single cells may have conferred resistance relatively speaking to contamination or impacted mycoplasma gene expression.
It is also possible that the associations seen here are not biological per se , but instead indicative of some technical artifacts associated with the presence of the AT-rich mycoplasma transcripts in the sample.
Nonetheless, this is equally problematic as it effects the interpretation of the results. Hence, further controlled studies are needed to fully understand the effect of contamination on the quality of RNA-Seq data and possibly how to account for this in data analysis.
Although beyond the immediate scope of this study, we could not help but wonder what interesting aspects of mammalian and mycoplasma biology could be gleamed from this vast, varied and complex nature of the samples comprising these studies. These studies consisted of cell lines of all types, with knockdowns, knockouts and overexpression of many different genes under complex drug treatments. How did the cellular pathways of interest change with the presence of mycoplasma?
Does this give us any insight into the biological system? Perhaps more importantly how did these conditions impact the ability of mycoplasma to contaminate cells? After all, mycoplasma is pathogenic in humans and such insight may be helpful in developing therapeutics for diseases such as atypical pneumonia.
We hope future studies can tackle some of these questions. Lastly, our study has broader implications for the analysis of high-throughput sequencing data. Too often in our analysis pipelines, we disregard unmapped reads as technical artifacts or attribute them to problematic or unsequenced regions of the reference genome.
While this may be true most of the time, this study suggests such reads warrant further investigation—particularly when they constitute a large number of the total reads. We focused on mycoplasma in this study as a proof of concept due to its known prevalence. However, the methods used here are easily applicable to other known and unknown contaminants of cell culture including other bacteria, yeast and viruses. Google Scholar.
Google Preview. Oxford University Press is a department of the University of Oxford. Mycoplasma mess: High-throughput detection sheds light Evidently, mycoplasma is a serious and potentially long-standing and widespread problem in both research and biopharmaceutical settings.
For biologics production, mycoplasma infection can inhibit cellular proliferation or induce death. It can also adversely influence the biological properties of cell cultures, which could impair their ability to express recombinant proteins, or impact their susceptibility to viral infections. From a safety perspective, mycoplasma may pose a health hazard in the biologics product. Even the final biologics product must be tested, according to regulatory requirements. This makes them hard to spot visually by microscopy among the host cells.
The problem with attempting to detect mycoplasma by culturing is that some species are not readily cultivable. Alternatively, mycoplasmas can be amplified in a specialized indicator cell line followed by nonspecific Hoechst DNA staining.
Although these methods detect live mycoplasma specifically, they require specialized staff and are time consuming due to the culturing step.
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