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FAQ Cell Culture free of Mycoplasma

Background Information Mycoplasma free Cell Culture

 

Frequently Asked Questions

► What is the difference between the MycoSPY® and the MycoSPY® Master Mix kit?

Regarding the performance of the kits there is no difference. The MycoSPY® kit is a classical PCR kit with single components (Taq-Polymerase, PCR Buffer with dNTPs, Primer Mix), which have to be mixed before PCR. There is no loading buffer and tracking dye for gel electrophoresis included. MycoSPY® Master Mix kit is easier to handle and to ship. The lyophilisized Mastermix contains all neccessary components premixed for PCR and subsequent gel electrophoresis. After reconstitution with water (included in the kit) the working flow is very simple (see MycoSPY® Master Mix). The Mastermix Kit is more stable and can therefore be shipped at room temperature.

► Must one run a mycoplasma detection test after each standard application of MycoRAZOR® (3 passages)?

Yes. It is important to check that mycoplasmas have been completely eliminated by MycoRAZOR® (e.g. by using MycoSPY® or MycoSPY® Master Mix) to prevent the establishment of resistance. As resistance can be built up in the same way as in all use of antibiotics, complete elimination of mycoplasmas is vital.

► After one application cycle (3 passages) of MycoRAZOR® a mycoplasma test showed positive results. What action should I take?

First check whether two passages without the use of MycoRAZOR® were conducted between the last use of MycoRAZOR® and the current test. If this was not the case, dead mycoplasmas may have been detected, particularly by highly sensitive methods such as PCR. If the minimum time between the last application and the test was observed, use MycoRAZOR® in the next five passages of your cells, gradually increasing the dose of MycoRAZOR® up to a maximum dilution of 1/25. Please note that depending on the cell type, toxic effects may result from increasing the concentration of MycoRAZOR®. If you observe toxic effects (lower proliferation rate; changes in cell morphology) in your cells, apply the last-used dose in the remaining cycles. It is essential to test for results of each treatment by using a mycoplasma detection kit (e.g. MycoSPY® or MycoSPY® Master Mix)!

► After the successful use of MycoRAZOR® and testing free of mycoplasma, a culture has developed further mycoplasma contamination. How did this happen?

The animal products used in cell culture are primary sources of mycoplasma contamination. To avoid this risk, use only fetal bovine serum (FBS) and trypsin that are guaranteed mycoplasma free.
Mycoplasmas belong to the class of Mollicutes and thus lack cell walls, they are resistant to many antibiotics that attack cell wall synthesis. The user itself is thus an important source of contamination in routine use of this type of antibiotic (e.g. penicillin/streptomycin) for cell culture. In this case, non-sterile working conditions go unnoticed, as the addition of antibiotics prevents the growth of most bacteria – and thus macroscopic effects – while allowing mycoplasmas to multiply unhindered.
In addition, cross-contamination from another cell culture is possible. For this reason always test all cultured cells and replace any potentially contaminated cell culture material (medium, FBS, trypsin, buffer).

► Does MycoRAZOR® contain doxy- or tetracycline-like antibiotics?

No. MycoRAZOR® does not contain any doxy- or tetracycline-like antibiotics.

► Many commercial PCR-based mycoplasma test kits include a positive control and internal control. What is the difference?

Most kits for PCR-based detection of mycoplasmas contain a plasmid as positive control that encodes for the 16S rRNA of mycoplasmas. This ensures the functionality of the primers. This plasmid produces a DNA amplicon of equal or similar size to that expected if mycoplasma contamination were present. Therefore, the PCR with the positive control must be carried out in a separate tube. An internal control is usually included additionally, to make sure that the PCR is not inhibited by proteins and cell debris from the cell culture supernatant. The internal control produces a DNA amplicon of a different length from a mycoplasma-contaminated cell sample. Therefore the internal control can be carried out in each approach.

► Why is no positive control included in the MycoSPY® Kit or MycoSPY® Master Mix Kit?

The internal control included in the MycoSPY® Master Mix kit and the MycoSPY® kit is a plasmid, which encodes for a shortened form of the 16S rRNA of mycoplasmas. The internal control thus serves as a positive control and ensures for each PCR approach that no inhibitors are present from the cell culture supernatant. It thus excludes false-negative results.

► What is the sensitivity of the MycoSPY® or MycoSPY® Master Mix Kit?

Both the MycoSPY® Master Mix kit and the MycoSPY® kit detect at least 80 copies of a mycoplasma genome. The internal control reduces the sensitivity slightly. Since mycoplasmas reproduce relatively quickly after contamination of a cell culture, this sensitivity is easily sufficient.
We therefore recommend adding the internal control in every PCR to ensure the absence of any existing inhibitor (proteins, cell debris) originating from the cell culture supernatant.

► The cells to be examined with the MycoSPY® or MycoSPY® Master Mix Kit were cultivated with antibiotics. Would it be better to use antibiotic-free media when performing the assay

It is not necessary to avoid conventional antibiotics such as penicillin, streptomycin or gentamicin, as these do not inhibit the assay of MycoSPY® Master Mix Kit the or the MycoSPY® kit.

► Is it possible to store cell culture supernatants to be examined with the MycoSPY® or MycoSPY® Master Mix Kit?

Yes. We recommend storage at-20°C without additives (e.g. DMSO). After thawing, the cell culture supernatants should be prepared as described in the manual.

► What is the recommended waiting time before performing the assay with the MycoSPY® or MycoSPY® Master Mix using freshly thawed cells?

We recommend cultivating the cells for at least 48–72 hours or until at least 80% coverage of the growth area is achieved. For suspension cells, cell density should be approximately in the range of the cell number recommended for subculturing. Since the mycoplasmas are detected in the cell culture supernatant, the medium should not be changed in the interim.

► How often should I test for mycoplasmas?

We recommend performing the PCR for mycoplasmas (e.g. with MycoSPY® or MycoSPY® Master Mix) least every 1-2 months, particularly when cells are cultured in the presence of antibiotics (e.g. pen/strep). These antibiotics prevent the discovery of unsterile working techniques but allow mycoplasma contamination of cell cultures.