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In summary?

1. Why is it important to keep your cells healthy?
2. How do I recognize a Mycoplasma bacteria contamination?
3. How does Mycoplasma affect the cells?
4. How can you prevent this?
   

What is bias?

Why is it important to keep your cells healthy?

Cells undergoing physical or genetic changes during population doubling can behave differently in comparable experiments. And there is the problem described in the reproducibility crisis blog—poor practice in cell biological research results in untrustworthy conclusions.

Whereas bacteria, yeast, or fungal contamination is visible in any cell culture, and thus most of the time treated on time, Mycoplasma is not.

Contaminations can lead to physical cell changes, resulting in differential behavior between experiments and unreliable data.

Just like you promise to back-up important files, you do the same for checking your cell culture for Mycoplasma. But with PCR, it takes hours to get the results. You promise the next time you will do it. What if you can have reliable Mycoplasma detection within half an hour? And eliminate or prevent it if necessary.

Recognizing it

How do I recognize a Mycoplasma bacteria contamination?

Mycoplasma is one of the simplest bacteria that can infect different body parts. This bacteria has no cell wall and is as tiny as 0.15-0.3μm, making it impossible to identify by microscopy. To make things worse for cells in culture, antibiotics that target cell walls, like penicillin and streptomycin, don't prevent the growth of Mycoplasma. Being smart and using a filter system is a lost case too. Most can escape through filtering systems of 0.22µm.

Mycoplasma is known for causing respiratory disorders. But researchers should not underestimate the effect on cell culture.

Causes

How does Mycoplasma affect the cells?

Cross-contamination by infected cell cultures, present in culture reagents, transmitted by laboratory personnel, or in original isolate tissue are the primary sources of contamination. Mycoplasma can quickly appear in your cell culture, slowly utilizing nutrients from the host's cells.

Mycoplasma shows no cell culture symptoms when grown in high concentrations. But it seriously impacts experimental results' reliability, reproducibility, and consistency,  representing a significant problem for basic research and the manufacturing of bioproducts.

A conservative estimate states that Mycoplasma contaminates 15–35% of all continuous cell cultures. 5% of early passage cell cultures and 1% of primary cells. Since contaminations are very difficult to detect or prevent, the presence of Mycoplasma can remain undiscovered for months.

Going unnoticed, Mycoplasma affects various cellular parameters, such as inhibition of cell growth and metabolism, induction of chromosomal aberrations, stimulation of changes in cell membrane antigenicity, and sometimes even cell death.

It is a pity when months of work go wasted when you could have detected the problem much sooner.

Our solutions

How can you prevent this?

The most common method is PCR, applying primer sets to detect Mycoplasma species. Various suppliers offer PCR-based detection kits with constraints to a limited number of targets. The hands-on time, PCR thermocycler run, and data analysis together will busy you with half the working day.

If you wish never to skip this quality check again, a more convenient and time-saving method could be your rescue. A simple and exceptionally sensitive 2-step luminescent assay will screen your cell culture for mycoplasma contamination within 20 minutes. This bioluminescence assay measures the activity of enzymes found in all common Mycoplasma and Acholeplasma contaminants (44 species).

This process is more sensitive and faster than PCR which uses primer sets that do not detect all types of mycoplasma and related organisms. The primers of the PCR-based method will detect living and dead Mycoplasma DNA, which could produce false. You can avoid false positives with the MycoAlert kits, which only detect live Mycoplasma, thus giving an accurate contamination assessment. In addition, this process takes at least four to five hours. Compared to other methods, the MycoAlert detection kit is very user-friendly and sensitive.

Want to know what to do when you can't afford any risk and need to prevent Mycoplasma? Continue reading here.

The principle of MycoAlert further explained:

1. Two enzymes found only in Mycoplasma and related organisms are detected. Then ATP is synthesized, resulting in the conversion of luciferase to oxyluciferin.
2. The whole process, handling, detection, and analysis, takes 30 minutes.
3. A luminometer will enable the detection of the light emitted by this process.
4. It detects over 44 mycoplasma species tested so far can be detected with this kit.
5. We recommend the use of the MycoAlert control set. These positive and negative controls will help assess the reliability of the measured values. And test the sensitivity of the illuminometer.
6. The signal of the positive control is powerful. It is best to keep the adjacent wells empty to prevent false positives.
7. It is also crucial to follow the duration as indicated in the manual for reading the samples because this is the peak of the chemiluminescence reaction. Before using the reagents, it is advisable to equilibrate them for 15 minutes at room temperature. Once thawed, use them immediately. Keep the reagents optimal by storing the kit at 4°C. Never freeze the reagents until reconstituted. Reconstituted reagents can be aliquoted and kept at -20°C or -80°C for six months.

This webinar explains how Mycoalert helps combat cell culture's worst enemy.