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Mycoplasma Contamination | Cell Culture

We include here for cell culture labs, information on mycoplasma contaminations and the latest mycoplasma forum discussions for all your contamination questions and discussions.

Mycoplasma Background

Mycoplasmas are a genus of bacteria that lack a cell wall. Due to a lack of a cell wall, they are resistant to many antibiotics including penicillin or other beta-lactam antibiotics that target cell wall synthesis.

Mycoplasma are either parasitic or saprophytic. Several Mycoplasma are disease-causing in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases. They may cause or contribute to some cancers.

Genus Mycoplasma

The genus Mycoplasma is one of several genera within the class Mollicutes. Mollicutes are bacteria which have small genomes, lack a cell wall and have a low GC-content (18-40 %).

There are over 100 recognized species of the genus Mycoplasma. Their genome size ranges from 0.58 1.38 megabase-pairs. Mollicutes are parasites or commensals of humans, animals (including insects), and plants; the genus Mycoplasma is by definition restricted to vertebrate hosts. Cholesterol is required for the growth of species of the genus Mycoplasma as well as certain other genera of mollicutes. Their optimum growth temperature is often the temperature of their host if warmbodied (e.g. 37 degrees Celsius in humans) or ambient temperature if the host is unable to regulate its own internal temperature. Analysis of 16S ribosomal RNA sequences as well as gene content strongly suggest that the mollicutes, including the mycoplasmas, are closely related to either the Lactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto).

Cell Lines and Mycoplasmas

In cell cultures, mycoplasmas may induce cellular changes, including chromosome aborations, changes in metabolism and cell growth. Severe mycoplasma infections may destroy a cell line or render expensive or precious cell lines useless.

Cell Culture Contamination with Mycoplasma

Mycoplasmas are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients.

Mycoplasma Detection Methods

Mycoplasma cells are usually smaller than 1 µm and therefore quite difficult to detect with conventional microscopy methods. Detection techniques include:

• PCR
• Real-time PCR
• Plating on sensitive Agar
• DNA stains including DAPI or or Hoescht Stain

Hoechst Stain Mycoplasma Contamination Detection Protocol

A Hoechst (Sigma, catalog #:H6024) staining method for Mycoplasma:

1. For Adherent cells, place a sterile cover glass in petri dish. Add 2-3 drops of cell suspension in the middle of coverglass (depending on the growth rate of your cells).
2. Then carefully add 2-3 ml of medium without disturbing the cells. Grow the cells until they are 50 -70% confluent.

Fixing Cells:

1. Prepare cell Fixative which is a mix fo 3:1 Methanol : glacial acetic acid (M:AA). Make sure you prepare this fresh before use.
2. Discard medium from growing cells. Add 2 ml of the Methanol : glacial acetic acid M:AA solution dropwise onto the side of the plate (not directly onto the cells). Swirl the plate and discard solution.
3. Add 2 ml of M:AA, swirl and leave on for 2-3 minutes at room temperature.
4. Discard M: AA solution and rinse with water
5. Leave to air dry (you can keep the plate until you are ready to stain) Hoecht stain (cat# 33258 from Sigma) stock concentration = 0.1 mg/ml in water You can filter sterilize at this stage using a 0.2uM filter Wrap container in aluminium foil Store at 4 oC Working solution Prepare fresh each time as necessary Make 1:1000 dilution (0.1 µg/ml) of stock in water

Staining Cells:

1. Add enough Hoechst stain to cover the entire cover slip (use 1-2ml for a small Petri dish).
2. Leave the stain on for 45 minutes - 1 hour (in the dark).
3. Rinse well with water.
4. Mount on a microscope slide (mountant mix: 22 mM Citric Acid; 55.6 mM disodium phosphate; 50% glycerol, pH mix to 5.5) place a drop or two on cover slip (cells up) and place cover slip (cells down) on slide.
5. Visualise under fluorescence microscope

Note: store Hoechst stain at 4oC at least, or aliquot into the freezer for long-term storage.