Mycoplasmas

Beware of Mycoplasmas: the most serious, widespread, and devastating culture contaminants.

Any handling of cell cultures poses the risk of contaminations with microorganisms. While bacterial and fungal infections of cultures are relatively easy to detect, to preventand to treat, contamination with mycoplasmas represents a much bigger problem interm of incidence, detectability, prevention, eradication and effects. 
More than 20 different mycoplasma species were isolated from cell cultures butthe most frequently isolated, representing 80 - 90% of isolates are M. arginini, M. fermentans, M. hyorhinis, M. orale and Acholeplasma laidlawii.

Why is the mycoplasma contamination rate so high?

  1. Mycoplasmas are the smallest self-replicating microorganism known (0.2 to 2μm indiameter). For this reason, mycoplasmas have the ability to to go through the filters used to sterilize cell culture media and sera, resulting in low levels of these organisms beingaccidentally introduced into cultures during routine feeding.
  2. Mycoplasmas lack a rigid cell wall then, commonly used antibiotics that act on this structure like penicillin and streptomycin will not be effective.
  3. Their small size and lack of a cell wall allow mycoplasmas to grow to very high densities in cell culture (107 to 109 colony forming units/ml are common) often without any visible signs of contamination - no turbidity, pH changes or even cytopathic effects. Microscopic observation of live cell cultures cannot detect their presence.

What are the consequences of mycoplasma contamination?

  1. Adverse effects on the cultures: alteration of growth characteristics, cell membrane composition, chromosomal structure etc...
  2. Inaccurate or erroneous experimental results
  3. Loss of valuable cell cultures
Therefore, regular testing for mycoplasmas is the only way to be sure of working withmycoplasma-free cell cultures leading to confidence in the quality and validity ofdata obtained.



Mycoplasma Stain Kit

Kit contents

  • Hoechst Stain 33258 - 1x10 ml
  • 10X HBSS without phenol red and sodium bicarbonate - 1x10 ml
  • Mounting Medium - 1x10 ml
  • Positive/Negative fixed control slides – 5 slides

Advantages

  • Reliable: Use of the Hoechst fluorescent stain method cited by the Tisseu Culture Association (TCA procedure no.75361).
  • Versatile: Detection of mycoplasma and bacteria, yeast, fungi.
  • Rapid: The test takes less than 2 hours.
  • Complete: Stain, diluent, mounting medium and controls are included in the kit.
The Mycoplasma Stain Kit uses a DNA fluorochrome staining method. A cell sheet between 50-80% confluent is fixedand stained with the Hoechst dye and examined under fluorescent microscopy.
The nature of the contaminants may be determined by its morphology, size and relationship to the cells. Mycoplasmawill appear as very small bright extranuclear dots or rods. Other microbial contaminants will be larger.

Results

Specimens should be observed by fluorescent microscopy (excitation wavelength : 360 nm and emissionwavelength : 490 – 500 nm) at 400-1000X with oil immersion. Mycoplasma can be easily identified bycomparing the results obtained with the positive and negative control slides.

Negative culture

A negative culture will show only nuclear staining. Occasionally micronuclei or other nuclear fragments from dead ordisrupted cells will appear as spherical bodies. They may be distinguished from mycoplasma by their large size andbrighter fluorescence.

Positive culture

A positive culture will show the cellular nuclei surrounded by small pinpoint dots of fluorescence. They will havea uniform size ranging from 0.1 – 0.3 μm in diameter and may be pleomorphic. The morphology may range fromspherical bodies to filamentous-like forms.
Cat. No.
DescriptionAmountBrand
093030000
Mycoplasma Stain Kit
1 kit
MP Biomedicals



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Brands: MP Biomedicals
Product groups: Assays