Protocol - Mycoplasma Removal Agent

Mycoplasma Removal Agent (MRA)

Procedures

  1. Add Mycoplasma Removal Agent to cell cultures contaminated by mycoplasma at the recommended concentration of 0.5 µg/ml and incubate for 1 week
  2. Mycoplasma Removal Agent concentration should be raised up to 1 µg/ml only if the recommended concentration is ineffective in removing the mycoplasma
  3. For media replacement or culture transfer (passage), use a medium containing Mycoplasma Removal Agent at this same concentration
  4. Transfer the cell cultures several times without Mycoplasma Removal Agent and confirm that regrowth of the contaminating mycoplasma has not occurred

Mycoplasma Removal Agent | Tips

  • If mycoplasma in serum or trypsin cannot be ruled out, Mycoplasma Removal Agent can be added to the media at a concentration of 0.5 µg/ml to prevent contamination of cell cultures exposed to these products
  • Mycoplasma Removal Agent has a long shelf life of 5 years from date of dispatch, is also stable at room temperature, although it should be protected from light to prevent decomposition
  • The cytotoxicity of Mycoplasma Removal Agent is low but, cell cultures which are highly contaminated with mycoplasma may exhibit altered cellular characteristics or sensitivity to cytotoxic antibiotic effects when treated with Mycoplasma Removal Agent, therefore in these cases it is recommended that expected cellular characteristics are checked following the treatment

Example data

The following tables show that the level of infection, cell type and mycoplasma strains may influence specific results. We encourage researchers to use this data as a helpful guide to indicate the effective Mycoplasma Removal Agent concentration needed with their specific cell line and mycoplasma strain.

Mycoplasma Removal Effect – Spontaneous Infection

Concentration of MRA (ug/ml) Days*
    0 7 14 21
Human-derived cell-A 0.39 + - - -
  0.2 + - - -
  0.1 + - + +
  0 + + + +
Human-derived cell-B 0.78 + - - -
  0.39 + - - -
  0.2 + - - -
  0.1 + + + +
  0 + + + +

(+) = Mycoplasma positive, (-) = Mycoplasma negative, (Days*) = Mycoplasma status at stage of culture (days).

Comparisons with other antibiotics on the market such as Tiamulin and Minocycline shows that Mycoplasma Removal Agent is comparable for its minimum inhibitory concentration (MIC), with MICs from as low as 0.1 µg/ml for all the tested bacteria (see Table 2). This shows that if you are looking to inhibit the growth of Mycobacteria within your cultures, you can reliably use Mycoplasma Removal Agent because it is just as effective as other antibiotics which target mycobacteria.

Where Mycoplasma Removal Agent out performs the other tested antibiotics is in its low Minimum Microbicidal Concentration (MMC) which shows consistently low concentrations required for microbicidal effect (see Table 2). This enables you to confidently kill and remove mycoplasma from cultures at low concentrations of antibiotic. This reduces the chance that the antibiotic will have any inhibitory effects upon other cells in your culture.

Efficacy Comparison of MRA and Pharmaceuticals on the Market

  MRA Tiamulin Minocycline
  MIC* MMC** MIC MMC MIC MMC
Mycoplasma orale CH-19299 0.05 0.1 0.0031 3.13 0.05 25.0
Mycoplasma arginini G-230

 

0.1 0.2 0.0063 12.5 0.2 (>)100
Mycoplasma hyorhinis BST-7 0.05 0.1 0.0031 0.39 0.0031 0.39
Acholeplasma laidlawii PG-8> 0.0125 0.025 0.05 (>)100 0.05 (>)100
MMC/MIC 2   128 - (>) 2048   512 - (>) 2048  

*MIC = Minimum inhibitory concentration (µg/ml), **MMC = Minimum microbicidal concentration (µg/ml)

References:
  • Nakai, N. et al. (2000). Detection and elimination of contaminating microorganisms in Transplantable Tumors and Cell Lines.
    Exp. Anim. 49: 309 – 313.
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