This kit is designed for in situ detection of mycoplasma and other prokaryotic organisms, such as bacteria, fungi and yeast in cell culture

• Uses Hoechst stain method
• Detects mycoplasma and bacteria, yeast, and fungi
• Takes less than 2 hours

It utilizes DNA fluorochrome staining technology featuring Hoechst stain 33258. This technology is both rapid and highly sensitive. Non-nuclear staining is readily apparent and contaminants will stand out boldly against a black background. The nature of the contaminant is then determined by morphology, size and relationship to the cultured cells. The test is rapid; the results are available in two hours. Mycoplasmas are small and simple parasitic prokaryotes that reside in the endosomes of mammalian cells and are widespread contaminants found in cell culture. They alone have been shown to alter the growth rate of cells in culture, induce chromosomal aberrations, influence amino acid and nucleic acid metabolism and cause membrane aberrations. Many methodologies exist which are used to isolate and identify mycoplasma contaminants. Among these are direct growth on agar, broth or semisolid media, enzymatic procedures, RNA labeling, autoradiography, and staining with DNA fluorochromes. All of the above tests with the exception of the DNA staining require time, expertise and a substantial amount of equipment and reagents.

Mycoplasma Hoechst Stain Kit is a DNA fluorochrome used to isolate and identify mycoplasma contaminants, it is the only known method which is sufficiently rapid and sensitive to allow frequent testing at each passage. Several DNA fluorochromes such as DAPI, quinacrine mustard and quinacrine dihydrochloride have been used in the same technique but none of the above fluorochromes perform as well as the Hoechst stain with respect to fluorescent effect, slow quenching and minimum background fluorescence. It selectively binds minor grooves of the DNA and hence allows for efficient staining of the nuclei. It is designed for in situ detection of mycoplasma and other prokaryotic organisms in cell cultures.

Components
1. Hoechst Stain (0.5 μg/mL) 10 mL
2. 10X HBSS without Phenol Red and Sodium Bicarbonate 10 mL
3. Mounting Medium 10 mL
4. Fixed Control Slide 5/pk

Dilute the 10X HBSS reagent 1/10 for use with this kit. The Hoechst Stain should give maximum fluorescent intensity when diluted 1:10 with Hanks Balanced Salt Solution, pH 7.0, achieving a final concentration at 0.05 ug/ml. A lesser or greater dilution may be necessary depending on the microscope being utilized. It is suggested that the investigators determine their own optimum working dilution by testing various dilutions with the known positive specimens included in the kit.

The Hoechst Stain should give maximum fluorescent intensity when diluted 1:10 with Hanks Balanced Salt Solution, pH 7.0, achieving a final concentration at 0.05 ug/mL.

Handle the stain carefully since it is a mutagen and does bind well to DNA. Keep all solutions as sterile as possible. If any turbidity or precipitation is noted in the stain or diluent, discard the solution and begin with fresh material.

This stain is highly specific for DNA. It is thought to occupy one of the grooves of the DNA double helix, binding by intercalation. The stain will cause cellular nuclei to fluoresce; however, the other major DNA cytoplasmic components - mitochondria - will not. All prokaryotic organisms (Mycoplasmas, bacteria, yeast, fungi) will give off fluorescence. The identification of the contaminant must be made by noting size, morphology and relationship to the cell.

Control Slides: Zone 1 - Vero cell line, a continuous monkey kidney cell line. Zone shows typical results for genetically stable adherent cells demonstrating uniform nuclear staining with little extranuclear staining. Zone 2 - Vero cell line co-cultivated with Mycoplasma hyorhinis represents the ideal picture of a cytoadsorbing pleomorphic mycoplasma contaminating a stable continuous cell line. Zone 3 - Mouse-mouse hybridoma, an example of a genetically unstable nonadherent cell line, demonstrates the pleomorphism of nuclear structure and extranuclear staining often observed with hybridomas, tumor-derived cell lines and/or cells growing under non-optimal conditions. Zone 4 - Mouse-mouse hybridoma co-cultivated with Acholeplasma laidlawii represents the more frequently encountered situation of having to discern normal nuclear and extranuclear staining from contamination with a pleomorphic, non-cytoadsorbing mycoplasmal species.

Materials required (not supplied):
1. Sterile distilled water for dilution of 10X HBSS.
2. Incubator (37 °C)
3. Refrigerator
4. Various glassware
5. Fluorescent microscope
6. Carnoy's fixative or 1 part glacial acetic acid to 3 parts absolute methanol.