Frequently Asked Questions
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, pre-mature senescence
* signaling pathways - i.e. growth stimulation/inhibition and differentiation
* metabolic pathways
* 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.
- How does one identify mycoplasmal contamination in their tissue culture stocks?The ABCF offers a weekly mycoplasmal contamination screening service in tissue culture samples with a selective biochemical assay that detects the presence of conserved mycoplasmal enzymes (MycoAlertTM Plus, Lonza). This system is very sensitive, detecting < 50 cfu/ml and covers wide variety of species of Mycoplasma, Acholeplasma, Ureaplasma.
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)
- 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
- 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.
- Place an order online.
- Label the sterile microfuge lids with the codes on the order confirmation page and in the email confirmation.
- Freshly collected samples should be placed in the facility refrigerator and frozen samples are to be placed in the - 80C freezer. At MSKCC both are found in the linear equipment space outside ZRC 1553/1551. Please call 212-327-7030 before bringing samples to Bronk 415 to ensure someone is there to accept them.
- To submit the samples for mycoplasma testing before 10 AM the day of the test (usually Wednesday). For holiday scheduling changes, contact the facility at (212) 327-7030.
Note: “Off-Schedule” testing can usually be arranged. Inquiries should be sent to firstname.lastname@example.org. A set up charge will apply.
- 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
- 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
* 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:
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.
2. "Quarantine" untested cells:
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
3. Review good sterile technique:
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