Mycoplasma Contamination

Mycoplasma contamination is difficult to detect by eye. Moreover, they can easily pass through standard filters. Visible indicators such as turbidity or pH changes are rare. Detection of Mycoplasma by analytical methods is therefore essential. The most sensitive method is therefore the direct culture method.

Mycoplasma are parasites that obtain important nutrients not only from the cell culture medium, but also from cells. The release of toxins damages the cultured cells.

Detectable effects are: 

• Reduction of host cell metabolic activity.
• Inhibition of cell proliferation due to nutrient deficiency 
• pH changes due to glucose degradation
• Inhibition of differentiation
• Alteration of membrane properties and protein expression profile
• Chromosome aberrations 
• Arginine depletion 
• Insensitivity to antibiotics, e.g., penicillin, streptomycin
• Impairment of the transfectability of cells 
• Modulation of cellular cytokine production

For more information, see also "Effects of a Mycoplasma contamination".

In addition to the problems already listed above regarding the metabolic activities of the cell, identification of Mycoplasma contamination is extremely difficult. Macroscopic detection using a microscope is not possible. Also, Mycoplasma kill their host cells very slowly, which affects the live cell count only at a later stage. Another problem is the decade-long presence of Mycoplasma in cell lines, which could falsify important research results.

An increasing number of established cell lines are showing Mycoplasma contamination. Currently, 15-35% of cultures in cell culture banks are found to be contaminated. An even higher number of unreported cases in selected populations is assumed. Less than 20% of laboratories regularly test for Mycoplasma.

So before using a cell culture from another lab, test it for purity. This provides assurance that the cell culture is sterile and free of contamination.

See also "Effects of a Mycoplasma contamination".

In general, a distinction is made between direct and indirect contamination.

Direct contamination usually occurs through the addition of contaminated media or animal products (e.g. FBS, growth factors, coating proteins). Indirect contamination is mostly caused by non-sterile working techniques or lack of quality control.

The use of antibiotics makes it difficult to detect bacterial contamination and Mycoplasma. Due to this, transfer/cross-contamination between cell lines is frequently encountered.

For further information, see also "What causes a Mycoplasma contamination".

As persistent and robust organisms, they can survive for several days on surfaces and sterile workbenches. Within a media droplet, their lifetime can even be up to 6 weeks at RT.

We recommend testing every 1-2 months.

Regular testing is essential, especially during the use of antibiotics. Standard antibiotics (e.g. penicillin) lead to the elimination of bacteria with cell walls, but are not effective against Mycoplasma. Thus, contamination resulting from non-sterile working techniques does not cause turbidity. It appears that the cell culture is sterile, but still has Mycoplasma.

In case of contamination, the infected cell culture should be discarded and a new stock thawed.

To date, there is no patent remedy for the removal of Mycoplasma; the choice of method depends on the particular cell line and, if present, antibiotic resistance.

Nevertheless, antibiotic treatment with macrolides, tetracyclines and quinolones has been proven to be suitable.

You can find everything you need to know here: "Mycoplasma Removal".

Yes, transmission to humans is possible.

Mycoplasma can survive both commensally and parasitically in humans. In the human body, they live on the surface of epithelial cells or in the gastrointestinal tract. Pathogenic species are mainly transmitted via droplets. By changing their surface proteins with high frequency, they are difficult for the immune system to detect. Among other things, mycoplasmas are associated with atypical pneumonia.

Optimally, the supernatant should be processed directly. If the samples are processed within one day, storage at 4 °C is required.

Alternatively, the samples can be stored at -80 °C for up to 6 months. It is important that the supernatant is frozen immediately after centrifugation. Thawing of the supernatant (without exposure to heat) for at least 15 minutes at room temperature is recommended prior to testing. 

Due to the lack of a cell wall, macroscopic detection is very difficult. Commercial kit systems or published standard methods are suitable for checking cell cultures.

The main methods used for the detection of Mycoplasma contamination are: 

1. Mycoplasma cultures:
   
   A special liquid medium is inoculated with your sample and later grown on Mycoplasma agar plates.
    
2. DAPI or Hoechst staining:
   
   DAPI (4',6-diamidino-2-phenylindole) intercalated with the DNA of the Mycoplasma leads to a blue staining which can be visualized by UV excitation (460 nm) using a fluorescence microscope. Negative cell cultures show only a blue staining of the cell nuclei. 
    
3. PCR:
   
   The polymerase chain reaction (PCR) leads to amplification of the bacterial DNA, i.e. an amplification of the Mycoplasma DNA. PCR allows routine detection at early stages and at low concentrations. 

The European Medicines Agency (EMA) offers guidelines for the detection of Mycoplasma contamination.

For more information, see also "How to detect Mycoplasma contamination?".

In most cases, cell-free culture supernatant from cultures incubated for 48-72 h is used for testing. It is therefore important that the medium is not changed or discarded during cultivation. The culture supernatant is then centrifuged so that no cell residues remain. Cell residues would lead to a falsification of the results.

DAPI staining

A simple way to test for the presence of Mycoplasma in your cell culture is to use DAPI staining. DAPI (4',6-diamidino-2-phenylindole), is a fluorescent dye that intercalates with AT-rich region of mycoplasma DNA. This results in a blue staining that can be visualized by UV excitation (460 nm) using a fluorescence microscope.

Sample preparation

Culture the indicator cell line in a 25 cm² cell culture flask (glass bottom dishes, confluency 60-80 %). Passage the cell line as usual, without using an antibiotic. Then add 1 ml of your sample to the cell culture and culture the cells for three days at 35-38 °C.

Protocol

Fix and permeabilize the cultured cells using a protocol suitable for your samples. The performance of DAPI staining differs depending on the protocol and experimental setup. A detailed execution from the EMA can be found here.

Example of execution

1. Remove the cell culture medium
2. Incubate the cells with 4 % PFA for 15 min at RT 
3. Wash the cells 2 times with 1x PBS 
4. Add the DAPI solution (1:5000 dilution)  
5. Incubate for 1 min in the absence of light
6. Wash the cells 2-3 times with 1x PBS
7. Resuspend the cells in 1x PBS 
8. Fluorescence microscopic evaluation at 400x magnification

Evaluation

Compare your microscopic image with your reference controls and check for extranuclear florescence. Mycoplasma form characteristic filamentous structures over the cytoplasm of the indicator cell. In addition, they can produce filaments in intracellular spaces. A sample is considered negative if no expressions characteristic of Mycoplasma are detected.

The test is invalid if: 

• No growth is seen on the positive control or
• Growth is seen on the negative control

PCR detection

This method is based on the specific amplification of a DNA segment of the Mycoplasma 16S rRNA gene. Total DNA is extracted from the obtained cell culture supernatant using designated DNA extraction and purification test kits or other methods. PCR allows routine detection at early stages and use of low concentrations. In addition, detection of live and dead mycoplasmas is performed. For further specification of the detected DNA, further restriction analysis or sequencing of the PCR product is required. All work is to be performed in an S2 laboratory and under safety cabinets.

Sample preparation

Culture the cell line to be analyzed in a 25 cm2 cell culture flask until confluent growth (60-80 %) has been achieved. Passage the cell line for further analysis (1/5 at 90 % confluence) and take at least 3 ml supernatant per cell culture in the meantime. If the analysis is not performed on the same day, it is best to store your samples at a minimum of 4 °C.

Protocol

From cell culture supernatants

1. Centrifuge 1 ml cell culture supernatant at 15,000 x g for 6 min.
2. Discard supernatant
3. Wash pellet 2x with 1 ml each of 1x PBS buffer
4. Centrifuge at 15,000 x g for 6 minutes 
5. Resuspend cell pellet in 200 µl PBS 
6. Inactivate the suspension for 15 minutes at 95 °C
7. Sample input for DNA extraction: total sample volume

Cell pellets from cell cultures

1. Discard supernatant
2. Wash 80 % confluent cells 2x with PBS
3. Dissolve adherent cells with trypsin for 5 minutes beforehand
4. Inactivate trypsin by adding 5 ml culture medium  
5. Centrifuge suspension mixture for 5 minutes at 200 x g 
6. Resuspend cell pellet in 1.8 ml PBS 
7. Centrifuge at 15,000 x g for 3 minutes 
8. Discard supernatant and resuspend cell pellet in 400 µl PBS 
9. Inactivate suspension for 15 minutes at 95 °C 
10. Sample input for DNA extraction: 200 µl

DNA extraction and PCR

Commercially available extraction test kits and analyzers are suitable for DNA extraction from cell cultures and for PCR analysis. For further procedure, please contact your respective manufacturer or send your samples to an analytical laboratory of your confidence.

MycoXpert is provided as a 50x concentrate.

Recommended working concentration: 1x.
Therefore, dilute the stock solution 1 : 50 (Dilution 1)
Example: Add 10 ml MycoXpert to 500 ml base medium.

If required, increase MycoXpert concentrations as follows in different subsequent steps:
Dilution 1 → 1 : 50
Dilution 2 → 1 : 40
Dilution 3 → 1 : 30
Dilution 4 → 1 : 25

For the different concentrations, please see "Preparing different dilutions of MycoXpert".

Use the following protocol, starting with Dilution 1 (1:50):

1. Remove culture medium from culture vessels, wash cells with PBS and detach cells using Trypsin or Accutase.
2. Count cells and plate in fresh medium (with MycoXpert).
3. Cultivate cells for 2 to 3 days in the usual way.
4. Repeat the passaging cycle at least twice.
5. Check for Mycoplasma contamination (Staining method, cultivation or ELISA).
6. If mycoplasma positive, check methods (positive and negative controls and evaluate).
7. If required, repeat treatment with increased MycoXpert concentrations (Dilution 2, Dilution 3 or Dilution 4).