Mycoplasma Troubleshooting Tips & FAQ

Mycoplasma FAQs & Troubleshooting Tips

Our Mycoplasma FAQs & troubleshooting guide is designed to help improve and troubleshoot the common problems that laboratories encounter when performing cell culture steps and mycoplasma contamination occurs. Optimising cell culture techniques and removing common mistakes can dramatically decrease the likelihood of mycoplasma contamination. In this troubleshooting guide we have detailed the common areas where mycoplasma infection occurs and how it can be eliminated.

Mycoplasma Contamination Frequently Asked Questions

1. What is Mycoplasma?

Mycoplasma is a type of bacteria. The primary difference between mycoplasma and other bacteria is the lack of cell wall and their flexible membrane. The size of mycoplasma bacteria is typically 0.15-0.3µm. Mycoplasma escape through filtering systems in cell culture facilities occurs due to its small size. These organisms are resistant to most antibiotics commonly employed in cell cultures such as such as penicillin and streptomycin.

2. What are the most common Mycoplasma species?

A. laidlawii*
M. hominis*
M. arginini*
M. fermentans*
M. hyorhinis*
M. salivarium*
M. pirum*
M. orale*
A. granularum
M. alkalescens

A. pleciae
M. neophronis
M. timone
M. caviae
M. alvi
M. bovis
M. primatum
M. leopharyngis
M. maculosum

A. oculi
M. iners
M. gallinarum
M. sphenisci
M. bovigenitalium
M. auris
M. columbinum
M. lipophilum
M. falconis

* More than 95% mycoplasma contaminations in cell culture are caused by these 8 kinds of mycoplasma

3. Why is Mycoplasma testing required?

Mycoplasma testing is required to detect the presence of mycoplasma contamination in biopharmaceutical laboratories. Mycoplasma contamination is one of the main problems in cell culture. Mycoplasma are ubiquitous in the environment. In cell culture laboratories, the majority of mycoplasma infections in continuous cell lines are caused by as little as six contaminant species of bovine, swine, and human origin. These microbial agents easily propagate from an infected cell culture batch to another via aerosol transmission or inadequate aseptic techniques. If undetected, mycoplasma contamination can have significant effects on the quality and reliability of your cell culture preparations.

4. What are the sources of Mycoplasma contamination?

  • One of the main sources of mycoplasma contamination is infected cultures obtained from other research laboratories or commercial suppliers.
  • Cell culture media and animal products used in cell culture are a major source of mycoplasma contamination. Mycoplasmas can pass into the filter membranes used in sterilizing cell culture media, sera and other reagents since they are very small and pliable due to the absence of a cell wall.
  • The most common mycoplasma cell culture contaminates are human, bovine or swine in origin, with human being the largest.
  • Laboratory personnel can lead to mycoplasma contamination. Mycoplasma can spread with a single sneeze or even by talking.

5. What is the impact of Mycoplasma contamination on cell culture?

Description

1.

Increases competition for nutrients in cell culture – hinders cell growth and proliferation

2.

Exposes cells to unwanted metabolites

3.

Leads to mutations and chromosomal changes

4.

Alters the levels of protein, RNA, or DNA synthesis

5.

Alters gene expression, cell signaling and morphology

6.

Harms membranes and organelles

6. Why is routine Mycoplasma testing recommended?

Mycoplasmal infection can often dwell in cell cultures for a long period of time without any visible cell damage. Therefore, it is vital to routinely use effective detection methods to test for the presence of mycoplasma contamination. Mycoplasma contamination represents a serious issue for academic and biopharmaceutical laboratories due to their effects on cell properties and on safety of biological products.

Regular mycoplasma testing and control procedures help avoid the costly consequences of a cell culture contamination and ensure safe biopharmaceuticals and reliable scientific results.

7. Can mycoplasma contamination be visually detected?

Mycoplasmas cannot be detected by the naked eye, but our MycoGenie Rapid Mycoplasma Detection Kit, can detect the presence of mycoplasma in 1 hour from 1μl of cell culture supernatant by visual determination. This eliminates the requirement for PCR, qPCR, electrophoresis or ELISA.

8. How is mycoplasma contamination detected?

  • It is important to note that mycoplasma infection is usually undetectable by microscope. A number of techniques - direct and indirect tests - have been developed for detecting mycoplasma contamination in cell cultures.
  • Direct culture requires the use of one or more complex nutritionally enriched mycoplasmal media and controlled environmental conditions and can offer the highest sensitivity for vital mycoplasma. The method is slow and usually requires a month for completion.
  • There are various indirect tests of varying sensitivity and convenience, including DNA fluorescence, RNA hybridization, immunofluorescence, electron microscopy, ELISA, DNA probes, biochemical assay. Indirect tests offer two advantages over direct culture methods: first, they can detect so-called "non-cultivable" mycoplasmal strains that direct culture may miss; second, they are faster, usually taking only a few hours to complete. In the past, two independent methods were typically used to ensure that cultures were mycoplasma contamination free.
  • PCR technology has been adapted for mycoplasma detection and has proven to be a reliable tool for mycoplasma contamination control.
  • At Assay Genie we provide the MycoGenie Rapid Mycoplasma Detection Kit which enables the rapid and simple detection of mycoplasma contamination in cell culture. The results of using this kit are highly consistent with the most sensitive and accurate PCR kits for Mycoplasma detection.

9. What is the MycoGenie Rapid Mycoplasma Detection Kit?

The MycoGenie Rapid Mycoplasma Detection Kit enables the rapid and simple detection of mycoplasma contamination in cell culture. This novel system detects mycoplasma in 1 hour from 1µl of cell culture supernatant by visual determination eliminating the requirement for PCR, qPCR, electrophoresis or ELISA. Compared with PCR, the MycoGenie Rapid Mycoplasma Detection Kit is more resistant cell culture inhibitors thus avoiding false positive and false negative results. The results are highly consistent with the most sensitive and accurate PCR kits for Mycoplasma detection.

The MycoGenie Rapid Mycoplasma Detection Kit can detect up to 28 mycoplasma species, including 8 of the most common species associated with cell culture contamination. It is suitable for mycoplasma detection in a wide range of suspension and adherent cells including CHO, Vero, hybridoma, Sf9, HEK293 and many more. Furthermore, the kit is compatible with a comprehensive suite of cell culture media and serum.

The MycoGenie Rapid Mycoplasma Detection Kit is an excellent choice for routine mycoplasma detection in biopharma, vaccine/monoclonal antibody production, cell therapy/embryo laboratories and other scientific research laboratories.

10. What are the applications of the MycoGenie Rapid Mycoplasma Detection Kit?

MycoGenie Rapid Mycoplasma Detection Kit is suitable for many kinds of suspension and adherent cells with a wide range of media and serum compatibility. These include but are not limited to:

Application Example

Suspension cells

CHO, NS0, 293F, mouse hybridomas, Sf9, BHK21, etc.

Adherent cells

Vero, MDCK, SP2/0, 293T, HepG2. HeLa, A549, MB-MDA231, L929, MEF, etc.

Medium

CD FortiCHO, CDM4, Expi 293 Medium, CD Hybridoma, Grace, DMEM, 1640, F12, etc.

Serum

Fetal bovine/calf serum, horse serum, Gibco KSR serum replacement, etc.

11. What are the advantages of the MycoGenie Rapid Mycoplasma Detection Kit?

Advantage Information

Rapid detection time

Within 1 hour

Small sample required

Only 1μl is needed

Easy identification of contamination

Visual observation mycoplasma contamination

Simple kit components

PCR, qPCR, electrophoresis or ELISA is not required

High sensitivity & accuracy

Consistent with conventional PCR methods

More resistant to inhibitors in cell culture models
(compared with conventional PCR)

Avoids weak positive and false negative results

Can detect up to 28 mycoplamsa species

Including the 8 most common species associated with cell culture contamination

Mycoplasma detection in a wide range suspension & adherent cells

e.g. CHO, Vero,hybridoma, Sf9, HEK293 etc.

12. How can Mycoplasma contamination be prevented?

  1. Always wear personal protective equipment. This includes a lab coat, gloves (be sure to change them often), and a mask if working outside of the hood.
  1. Know the origin of your cells. Be sure to obtain cells from a reliable source. Mycoplasma survive in liquid nitrogen, so it is important to quarantine new cells and screen them for mycoplasma before introducing them into the lab.
  1. Good sterile techniques are necessary. Watch for drips from pipettes and clean any spills quickly. It is vital to change pipette tips and avoid talking around cells.
  1. It is important to keep the lab space clean. This includes regularly sterilizing work areas. Also it is important to service lab equipment on schedule to ensure all items are working properly.

13. How is Mycoplasma eliminated?

One option for mycoplasma elimination is treatment with antibiotics, which do not damage or alter cells. Antibiotics such as penicillin, which attacks bacterial cell walls, are ineffective in this instance, since mycoplasma lacks a true cell wall. Several antibiotics eliminate mycoplasma effectively, such as: Tylosin, Neomycin, Tetracycline and Gentamicin. However, the efficacy of these antibiotics is restricted to specific mycoplasma species and frequently only reduce the concentration of mycoplasmas rather than disinfect the cell culture. Hence, as soon as treatment is concluded, contamination will recur.

Our more effective option for Mycoplasma Elimination is the MycoGenie MycoPlasma Elimination Kit. This kit is a very robust method for removing Mycoplasma in cell culture, serum, and medium. Unlike conventional antibiotics, this kit kills mycoplasma by disrupting the membrane structure, and can remove antibiotic-resistant mycoplasmas, so as to achieve more effective elimination of mycoplasma rather than inhibiting proliferation. This kit is also effective on Gram negative and positive bacteria.

14. What is the MycoGenie Rapid Mycoplasma Elimination Kit?

The MycoGenie MycoPlasma Elimination Kit is designed to eliminate Mycoplasma from contaminated cell culture media and serum.

Unlike conventional antibiotics used to treat mycoplasma contamination, this kit removes mycoplasma by disrupting membrane structure allowing elimination of even antibiotic-resistant mycoplasma. This is a much more effective method of eliminating mycoplasma versus other common methods that simply inhibit proliferation. It is also very effective at removing Gram-negative and positive bacteria. This kit successfully eliminates intracellular and extracellular mycoplasma after a 3-7 day treatment protocol. Furthermore, it is non-toxic to cells and has been shown to kill most mycoplasma species. The MycoGenie MycoPlasma Elimination Kit has been validated with most commonly used cell lines including mouse and human embryonic stem cells, iPS cells, HEK293, Hela, HepG2, HCT116, COS-7, Vero, Huh-7, MDCK, PANC-1, SW620 and U2OS.

Mycoplasma Detection Kit Troubleshooting Tips

15. What is the sensitivity of the MycoGenie Rapid Mycoplasma Detection Kit?

The MycoGenie Rapid Mycoplasma Detection Kit is suitable for most cell culture experiments. It can accurately detect at least 500 cfu of mycoplasma from 1 μl of culture supernatant (5 × 105 cfu/ml). Typically, the mycoplasma content in culture supernatant is between 106-108cfu/ml. According to published literature, one single mycoplasma in cell culture can grow to 106 cfu/ml in 3-5 days. Therefore, it is highly recommended to detect after the third day after cell passage or after replacing medium.

16. What to do if the detection color changes immediately after supernatant is added or different colors (not blue or purple) appear during the reaction?

In rare cases, ingredients in the medium interfere with the color of the MycoGenie reagent. For example, Cell Boost 5 (Hyclone) makes the MycoGenie reagent appear pink. To avoid this:

Steps Description

1.

Collect a small amount of culture supernatant or cell suspension and centrifuge at 500 × g for 5
min. Collect the supernatant.

2.

Centrifuge again at high speed (> 12,000 × g) for 5 min to precipitate mycoplasma in the supernatant. Discard most of the supernatant and leave about 50μl in the tube. Add 950μl of sterile water and mix gently by pipetting.

3.

Repeat Step (2) for three times. Discard most of the supernatant and leave about 50μl in the tube.

4.

Take 1μl of supernatant for detection.

17. How to protect cells from mycoplasma contamination?

If mycoplasma contamination occurs, it is recommended to discard the cells to prevent other cells from contamination. If a mycoplasma positive result is detected, the same batch of cells should be discarded. Also, consider MycoGenie MycoPlasma Elimination Kit (Cat. No. MORV0195) as an excellent choice to remove mycoplasma from cell culture.

18. How to avoid false positives?

Generally, no false positives will occur during proper usage. The reaction tube should never be reopened, due to possible false positives resulting from aerosol contamination. Change tips between samples and always add the positive control last.

19. How to treat the cells with severe mycoplasma contamination?

For severely contaminated cultures, it is recommended to extend the MycoGenie Mycoplasma Elimination Kit treatment to 7 days or even longer.

20. How to minimize the toxicity of mycoplasma-removing reagents to my cells?

During validation, it has been shown that the MycoGenie MycoPlasma Elimination Kit is non-toxic to most cell types. However, it is recommended to increase the dilution factor from 1:1000 to 1:2000 or even higher if cell growth is reduced during treatment.

21. Is this MycoPlasma Elimination Kit product applicable to the primary cell cultures?

Yes. Compared with similar products from other resources, the MycoGenie MycoPlasma Elimination Kit demonstrates is higher efficiency in eliminating mycoplasma from MEF (mouse embryo fibroblast) and other primary cell cultures, which are at high risk of mycoplasma contamination.