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Mycoplasma Test FAQ

Frequently Asked Questions

  • How to Prepare Samples

  • Sample and Media Control Preparation and Submission:



    • Culture cells ­> 1 week in media FREE of antibiotics

    • Collect 0.5 ml media from a 100% confluent live cell culture

    • Transfer to a STERILE microfuge tube

    • Spin out cells (e.g., spin 200 x g for 5 min) but not the mycoplasma

    • Transfer 0.4 ml to new STERILE microfuge tube

    • Discard cell pellet

    Media Control (Negative Control)

    • When culturing cells to be tested, take some of the media used to grow the cells and incubate it in parallel

    • Collect 0.5 ml at the same time you collect the aliquot from the cells being tested

    • Transfer to a STERILE microfuge tube (one tube for each)

    Collecting samples:

    • The best option is to collect the samples the morning of the test

    • Samples can also be collected prior to the day of the test, but needed to be stored at - 80°C
      until tested

    • Drop samples off in the facility BY 10 AM Wednesday morning

    • NOTE: If an MSKCC holiday falls on Wednesday, contact thefacility as to when the test will be performed that week.

  • When and Where to Drop Off Samples

    • Before depositing samples:

      • Write the test code provided at the bottom of this confirmation on the top of your vials.

      MSKCC Requestors: Where and When To Drop Off Samples at MSKCC

      Fresh Samples:

      • When: Before the 10 AM deadline on Wednesday (usual testing day).

      • For weeks with mid-week holiday occurrences, please call/email the facility to find out about our alternate testing day.

    • Where:

      • Memorial Sloan Kettering Cancer Center's Zuckerman Research Center (ZRC)

        • 15th Floor, South Wing

        • Enter through Lab Door Z-15C1C/15 South.

        • Place samples in the facility refrigerator marked "Mycoplasma Testing - Fresh Samples" in the designated box.

  • Frozen Samples:

    • When:

      • Before the 10 AM deadline on Wednesday (usual testing day).

    • Where

      • Memorial Sloan Kettering Cancer Center's Zuckerman Research Center (ZRC)

        • 15th Floor, South Wing

        • Enter through Lab Door Z-15C1C/15 South.

        • At the end of the hall next to a lab door will be three freezers.

        • The third freezer will have a sign stating "Mycoplasma Testing - Frozen Samples Only."

        • Samples are to be placed on the second shelf from the top in the designated box.

        • The Rockefeller University Requestors: Where and When To Drop Off Samples at RU

          • Before coming to drop off samples at RU, please call 212-327-7030 (RU Site)or 646-888-2331 (MSK Site) to ensure someone is there to accept your samples.

          • Once confirmed, please come to drop off your samples at:

            • Bronk Laboratories, 4th Floor, Room 415.

        • Viewing Results

          1. Go to our website:

          2. On the left-hand side, click on Log-In > and choose your User Type (e.g. MSKCC users, RU Users, Other)

          3. Enter your email. Note:

            • MSKCC Users: without the domain (i.e. smithj)

            • RU & Other Users: enter your entire email address.

          4. Enter your password. Note:

            • MSKCC Users: this is your email password

            • RU & Other Users: this is the password you chose upon your first log-in.

          5. Click Submit

          6. Once logged in, click on the Mycoplasma Test link on the left-hand side menu

          7. Click on View Results

          8. Select the correct date of testing.

          9. You will see your results posted there.

    • Can you test a sample prepared from a culture grown in the presence of antibiotics?

    • Yes, but you need to know that you may want to question a negative result. Although most mycoplasma are resistant to penicillin and streptomycin, their growth may be retarded and thus not grow to a titer high enough to ensure detection.

      So if you have something that you really suspect is contaminated, test it knowing that if it comes up negative you should really re-test after growing it under the recommended conditions (see FAQ #3).

    • What sources of mycoplasmal contamination should you be concerned about?

    • The two most likely sources of mycoplasma contamination are:

      * Untested cell lines from your own frozen stocks or ones you acquired from other sources

      Please note:

      • ATCC does carry and distribute cell lines that are positive for mycoplasmal contamination. Check their web site to see if the culture you want to purchase harbors mycoplasma.

      • Unfortunately, many laboratories do not check their own cultures for mycoplasma and thus spread this menace unknowingly.

      • YOU, from poor sterile techniques.

      • What do you do if your cells are contaminated with mycoplasma?

      • FIRST - Do not get too stressed out !

        Mycoplasmal contamination can be eliminated with some time and effort.

        Here are a few questions to help you decide the best course of action:

        Q1. Do you have a cryopreserved stock of the cells that you know is free of mycoplasma?

        YES - do a little dance, toss what you have and start a new culture

        NO - curse out the person who handled the cells before you and go to question 2

        Q2. Can you easily re-acquire a stock that is mycoplasma free?

        YES - then buy it or get it from the group who sent it to you

        You want to do this whenever it is not too much of a hassle because the co-culturing of your cells and the mycoplasma causes your cells to permanently change much faster than they normally would have. Thus, why clean up something that may not be what you need in the end, when you can re-acquire it?

        NO - take a deep breathe and go to number 3

        Q3. Are the cells worth the effort needed to clean them ?

        NO - Chalk it up to experience and move on

        YES - choose one of the following treatments that are commercially available to eradicate mycoplasma from ...

        Established cell cultures:

        * MycoZap™ Mycoplasma Elimination Reagent .... Lonza (Cat# LT07-818)
        2 drug treatment - alternate between each drug 3x
        Days of treatment = 21

        * BM-cyclin ........ Roche Applied Science (Cat #10799050001)
        2 drug treatment - alternate between each drug 3x
        Days of treatment = 21

        * Plasmocin ....... Invivogen (Cat # ant-mpt)
        Days of treatment = 14

        * Ciprofloxacin ... Fisher Scientific (Cat # MT61277RF)
        Days of treatment = 12 - 20

        Primary cells,

        * Primocin ........ Invivogen (Cat# ant-mp1)
        Days of treatment = 7 - 14

        No matter which treatment is utilized, one needs to ...
        (1) Consider that these drugs can be toxic to the cells – thus intially titer the drug on your cell line and identify the highest concentration that is not deliterious to your cells - and continue treatment at that concentration -
        (2) The culture should be retested 1 week and then 1 month post treatment to CONFIRM that cells are "clean"

      • What are some good steps one can take to minimize an outbreak of mycoplasma in the lab?

      • Depending upon how much your science relies upon a good source of tissue culture cells, you could incorporate any combination of a these precautions:

        1. Monthly testing:

        2. For those labs where cell culture is at the center of their science and/or have many people employing it, you might want to test every month. To limit the cost, you could submit a pooled sample from each person. This can be done by having each person pool equal volumes of their various cultures into one flask, ensuring that there is fresh media for any potential mycoplasma to amplify. Grow the cells as usual and process the pooled sample just as you would a regular one.

        3. "Quarantine" untested cells:

        4. Whether you just thawed the cells out from your cryogenic system or got them in from someone else, if you do not know that they are free of mycoplasma, handle them as is if they are not. Specifically,

          test them as soon as possible

          have them be the last culture you process during the day and if space permits

          have a dedicated areas for suspicious cultures

        5. Review good sterile technique:

        6. Once a lab has mycoplasma contamination under control and are putting suspicious culture in quarantine, future outbreaks tend to occur within the same people's samples. Thus, it is always good to review good sterile technique and remind the experienced people that just because they did not have bacteria or yeast in their cultures does not mean that they have good technique even if they use antibiotics and /or antimyotics.

      • Is the test performed by the ABCF clinical grade?

      • No. All the reagents clearly state that they are for research purposes only

      • What is mycoplasma?

      • Mycoplasma , Ureaplasma, Acholeplasma, etc.

        • are the smallest known self-replicating life forms

        • are evolutionarily distinct from bacteria and viruses

        • lack a cell wall

        • are undetectable under a normal light microscope

        • are < 0.2 µm, but do tend to grow in colonies - thus they are still in your media even after you use the standard filter systems

        • can grow (1) inside or outside cells without killing them, (2) under standard tissue culture conditions, with or without cells as long as the proper nutrients are available (i.e. fatty acids, amino acids, nucleic acid precursors and cholesterol) and (3) in the presence of commonly used antibiotics

        • survive cryopreservation

      • Why should you care about mycoplasma, with respect to your experiments?

      • Very often, mycoplasma is the root of unrepeatable results whether you work in an in vitro or in vivo system.

        1. Tissue culture studies. A broad spectrum of cellular processes are altered by inadvertently co-culturing cell lines with mycoplasma. A few examples are:

          • cell growth - i.e. doubling time, viability, premature senescence

          • signaling pathways - i.e. growth stimulation/inhibition and differentiation

          • metabolic pathways

          • adherence

          • morphology

          • viral propagation

          • DNA transfectability

          Many of these changes may be permanent due to chromosomal aberrations that naturally accumulate as any cell line is passaged, but occurs at a much higher frequency when mycoplasma is sharing the flask.

        2. Animal studies. Mycoplasma can also skew your results if you introduce it into your animals, for example by injecting your mycoplasma positive cell line into the animal to form a tumor. Whether or not you are introducing a pathogenic strain, you are at least adding another variable to your system.