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Detection of Mycoplasma Contamination in Cell Culture

Overview

Post By : Bijoy Varma

Source: ABDOS Life Sciences

Date: 09 Nov,2021

Mycoplasma is responsible for up to 60% of the cell culture contamination (Uphoff,2002). Mycoplasmas are supposed to be the simplest self-reproducing prokaryotes devoid of a rigid cell wall. They fall in the family of mollicutes, and the genome is only 1/5^th that of E. coli. The cell varies from a spherical or pear shape (0.3 to 0.8 micrometers [0.0000117 to 0.0000312 inch]) to that of a slender branched filament (up to 150 micrometers [0.00585 inch]). Mycoplasma species are mostly facultative anaerobes, colonial microorganisms that are parasitic in nature.

Method	Principle	Required Material and Efforts
6-MPDR- Test	Biochemical Activity	Indicator Cell line, Time Consuming, Low Sensitivity


Luminiscence	Biochemical Activity	Luminometer, good sensitivity, quick protocol


Microbiological culture	Culture of living Mycoplasma	No special Instruments, Good sensitivity, Time consuming

By virtue of this, Mycoplasmas are resistant to antibiotics and antibacterial drugs like penicillin and streptomycin. Mycoplasma can pass through filtration methods because of its ability to change shape and the absence of a rigid cell wall.

Mycoplasma Contamination Source

orale 20-40% (human epithelium-by human contamination)
fermentens 10-20% (human origin)
hominis 10-20% (human origin)
hyorhinis 10-40% (Animal origin-through supplements)
laidlawii 5-20% (Animal origin-through supplements)
arginini 20-30% (Animal origin-through supplements)
Others up to 5% (Animal origin-through supplements)

ILL-Effects of Mycoplasma Contamination

Mycoplasma contamination can lead to inhibition of growth, inhibition of cell metabolism, cell death, morphology, disturbance in virus production, up to 30% of total protein or DNA, membrane integrity, chromosome abrreations etc.

Detection Methods

Even high titers of mycoplasma can not be seen with a light microscope and would not lead to turbidity of cell culture media.
Some of the common methods for detection of Mycoplasmas are as follows:
By biochemical activity of the living mycoplasma.

By Nuclear Stain:

Direct DNA detection of the living mycoplasma by nuclear stain like the Hoechst or DAPI. By this the extra-chromosomal DNA can be seen outside the cell.

PCR method:

Is the most sensitive and shortest tests, detect mycoplasma specific genes and are done elaborate manner. Only a few microliters of the cell culture supernatant is required and subject it to a PCR reaction and either run it on a gel or have it as a quantitative PCR

Real Time PCR:

This is a more quantitative approach, here beside the positive control, the different titers of mycoplasma can be quantified very well.

Mycoplasma Detection Interval:

Whenever there is a mycoplasma contamination, we have to test weekly until there is no contamination any more. (at least 2-4 times per month)
Keep new cell lines separate until it is tested.
Whenever there are problems of reproducibility of experiments or cellular morphology think about testing and be safe.
After this keep on testing every 3 months
Before transfer in to liquid N₂for cell banking by Cryopreservation, testing is recommended.

Conclusion:

Hence a lab personnel who handles the cell culture work must wear proper PPE and conduct experiments inside a validated LAF and make periodic checks to rule out Mycoplasma contamination.

If Mycoplasma Contamination is not controlled it can affect the cellular morphology and activities and can lead to loss of productive time and cost the scientist dearly. There is a need to ensure that heat labile components added to Cell Culture media are free from Mycoplasma.

Biofill bottle top filtration system of Abdos Life Science may be used to clarify, sterilize and also remove Mycoplasma from the heat labile components such as sera that are used in Cell culture media. The 0.1 micron membrane filters are capable retaining mycoplasma and hence are ideal for cell culture work.

Biofill Bottle Top filtration System –The New Generation Vacuum Filtration System is useful in Sensitive Lab applications such as stem cell, Mammalian cell culture, Drug discovery and Cosmetic product testing.