Mycoplasma testing

Please Note: Testing of Mycoplasma by culture isolation has been suspended until further notice. Please be advised that once the service is available again, we will update this page as appropriate.

 

Mycoplasma are the smallest (0.2 - 2µm in diameter) and most unusual of the prokaryotes.  We can test your cell lines, cell culture media and reagents to determine if mycoplasma is present. The effects of mycoplasma contamination on cell lines can be wide ranging and are often underestimated and can include:

• alteration of growth rate

• induction of morphological changes

• chromosomal aberrations

• altered cell metabolism

• decreased viability upon resuscitation of frozen ampoule.

• induction of transformation, apoptosis, cytokines, signal transduction and oxidative radicals

• macrophage activation

• inhibition of antigen presentation

In addition, mycoplasma can also interfere with membrane receptor function and penetrate host cells.

All laboratories carrying out cell culture should test for mycoplasma contamination on a regular basis, and we recommend that a combination of methods is used to determine whether a sample is free of mycoplasma.  This is because the different methods have different levels of detection sensitivity and some strains of mycoplasma do not grow in vitro. These can be detected by DNA stain and PCR.

Three different mycoplasma detection methods are routinely used by our scientists:

 

1. qPCR

This assay consists of a closed tube qPCR detection format and uses TaqMan fluorogenic probes and primers derived from the 16SrRNA region; these were designed manually in-house and consist of three sense primers and four TaqMan fluorogenic probes targeted to anneal to specific regions common in many bacteria.

Specificity of the assay is conferred by the 53 anti-sense primers used, which overlap a broadly conserved region in the 16SrRNA gene of mycoplasma but differ significantly from other (non-mycoplasma) bacterial sequences to enable differentiation from phylogenetically related bacteria. The assay also incorporates an inhibition control that is detected in parallel to the mycoplasma target in a duplex, single-tube reaction.

 

2. Indirect Hoechst Stain

This test is used to detect mycoplasma by Hoechst stain using an indicator cell line (Vero ECACC 84113001) and meets the requirements of the FDA Points to Consider 1993.

The test sample is inoculated onto Vero cells which have been grown on coverslips in tissue culture dishes. Both negative and positive controls (Mycoplasma hyorhinis NCTC 10112 and Mycoplasma orale NCTC 10130) are also set up. All samples are then incubated for 3-5 days after which the samples are fixed to the coverslip and stained using the fluorescent dye (Hoechst 33258). The fixed samples are then examined under UV epi fluorescence at x100 magnification. Positive samples are identified by their characteristic particulate or filamentous pattern of fluorescence on the cell surface or if contamination is heavy in the surrounding area. Negative samples should demonstrate no evidence of fluorescing DNA outside of the cell membrane against the fluorescing Vero cell nuclei.

 

3. Culture Isolation

Please Note: Testing of Mycoplasma by Culture Isolation has been suspended until further notice. Please be advised that once the service is available again, we will update this page as appropriate. 08/09/2023

Often regarded as the gold standard method for mycoplasma detection, the culture isolation detection method uses a combination of selective growth media and incubation conditions to positively enrich for any mycoplasma present in a sample. Most mycoplasma contaminants can be detected by growth on standardised agar, with the exception of certain strains of M.hyorhinis.

Colonies of mycoplasma exhibit a distinctive "fried egg" morphology when viewed under a plate microscope. The assay requires a 28 day test interval before a definitive result can be obtained.

. This test meets the requirements of the FDA Points to Consider 1993.

 

How to submit samples for mycoplasma and sterility testing

Please note that prior approval of QC testing GMO2 samples is required. Please contact CultureCollections.Quality@ukhsa.gov.uk  for more information.

Any biological material can be mycoplasma and/or sterility tested if it is cell culture compatible. Please complete the Sample Submission form and the Biohazard Risk Assessment form (see below) and send both forms to us prior to arranging shipment of samples. Once the forms have been received, reviewed and accepted by ECACC, you will be issued with a reference number that will need to be quoted in all future correspondence (and on your samples). A Purchase Order number is also required before any sample can be processed.

If sending growing cultures:

• growing cultures should have been passaged twice in the absence of antibiotics. They should have been growing for three days since the last medium change. ECACC need to be notified of the time of the last medium change so that, if necessary, the minimum growth time in the absence of antibiotics can be completed at ECACC

• growing cultures should be sent in flasks topped up with medium. The flask cap should be of the non-vented variety and wrapped e.g. in parafilm, to prevent leakage. The flask should then be packed in a leak proof bag or primary container with adequate absorbent material to contain the contents of the flask if there is any breakage or leak (see below)
  
  

If sending frozen cultures:

• a handling fee will be charged to cover costs of establishing the cell lines in culture

• details of the cell line growth media and culture conditions must be supplied

Flasks and ampoules should be adequately packed and transported according to UN 602 regulations. Approved UN 602 packaging may be obtained from several suppliers, the details of which are available from any Royal Mail Sales Centre.

If you use a courier service to ship your samples, please contact the company involved for advice on their own approved packaging.

 

Parcels should be addressed to: QC Testing Department, Culture Collections, UK Health Security Agency, Porton Down, Salisbury, SP4 0JG, UK

Testing is initiated on Tuesdays and Fridays. Notification of dispatch of samples to ECACC is very much appreciated.

 

Price

 

Sample submission form and biohazard risk assessment form

Sample submission form

Biohazard risk assessment form - cell line

 

Sterility testing

ECACC offers a sterility testing service, .