Mycoplasma Testing - Detection & Elimination

What is Mycoplasma?

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

Assay Genie & Mycoplasma Testing

SKU Assay Genie Mycoplasma Kits Size

MORV0011

20/50 tests

MORV0012

100μL/500μL

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.

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 avoiding the costly consequences of a cell culture contamination and ensuring safe biopharmaceuticals and reliable scientific results.

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.

MycoGenie Rapid Mycoplasma Detection Kit Applications

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.

Why should Mycoplasma be eliminated?

Mycoplasma elimination from lab work areas, safety cabinets, incubators as a routine cleaning measure can save time, money, and the laboratory reputation. Also timely decontamination of infected cell culture materials can prevent further spreading of mycoplasmas within the cell culture laboratory and contamination of other cell lines.

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.

MycoGenie MycoPlasma Elimination Kit

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 & Elimination Troubleshooting

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 -108 cfu/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 media.

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 media 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.

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.

How to avoid false positives?

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

How many mycoplasma species can be detected by MycoGenie Rapid Mycoplasma Detection Kit?

There are 28 species of mycoplasma that can be detected accurately by MycoGenie Mycoplasma Detector:

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