Mycoplasma Stain Assay Kit

The mycoplasma stain detection kit is specifically designed for the in situ detection of mycoplasma or other prokaryotes in cultured cells. Its main purpose is to identify the presence of mycoplasma contamination in cultured cells, as this type of contamination is not visible under an optical microscope and requires a specific detection method. While bacterial contamination, yeast contamination, or mold contamination can be observed under an optical microscope, mycoplasma contamination cannot be detected through visual examination alone.
There are various methods available for detecting mycoplasma contamination, such as mycoplasma isolation and culture, mycoplasma-specific enzyme detection, RT-PCR detection, and DNA fluorescent staining detection. The mycoplasma stain detection kit utilizes Hoechst staining, which offers a quick, effective, and highly sensitive means of detecting mycoplasma contamination. In the event of mycoplasma contamination, it is recommended to replace the contaminated cells with non-contaminated ones for further culture.
The kit is suitable for testing samples in a six-well plate format, enabling the testing of at least 100 samples. It contains three components: Component A, which is the fixing solution (100mL); Component B, which is the Hoechst staining solution (10mL); and Component C, which is the anti-fluorescence quenching mounting solution (10mL).
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The mycoplasma stain detection kit is specifically designed to detect mycoplasma or other prokaryotes within cultured cells through in situ staining. Its primary purpose is to identify the presence of mycoplasma contamination, an issue that cannot be visually observed under an optical microscope. Unlike bacterial, yeast, or mold contamination, mycoplasma contamination necessitates a specialized detection method.
Multiple techniques are available for mycoplasma detection, including mycoplasma isolation and culture, mycoplasma-specific enzyme detection, RT-PCR detection, and DNA fluorescent staining. This particular kit employs Hoechst staining as an efficient, sensitive, and rapid means of detecting mycoplasma contamination. In the case of contamination, it is advisable to replace the contaminated cells with uncontaminated ones for further cultivation.
The kit is designed for use with six-well plate samples, allowing for the testing of no less than 100 specimens. It comprises three main components: Component A, the fixing solution (100mL); Component B, the Hoechst staining solution (10mL); and Component C, the anti-fluorescence quenching mounting solution (10mL).

Instructions for use:
1. Preparation of cell samples:
When culturing adherent cells, it is best to fill six-well plates or other multi-well plates or coverslips to only 50%-80% capacity. Over-culturing the cells can make it challenging to detect whether there is a mycoplasma contamination present. Therefore, it is essential to culture the cells appropriately to ensure accurate detection of any contamination.
To prepare suspended cells for further experiments, start by centrifuging them to pellet the cells. Then, take a small amount of the pellet to make cell smears and let them fully dry in the air. This will allow for a better visualization of the cells and facilitate any subsequent analyses that may be necessary. Make sure to handle the cells carefully during this process to avoid any damage or contamination, and always follow the appropriate safety precautions to ensure the integrity of the experiment. With proper preparation, suspended cells can yield valuable insights into various biological processes.
To ensure proper usage, it is recommended to dilute the Hoechst staining solution with PBS at a ratio of 1:10 (1 volume of Hoechst staining solution to 9 volumes of PBS). It is important to note that the diluted Hoechst staining solution should be utilized within 24 hours for optimal results.
To prepare your sample, you need to add enough fixative to cover it adequately. For one well of a six-well plate, you should add 1 mL of fixative. It's essential to ensure that the fixative is enough to cover the sample and not to wash the cells before you fix them. For adherent cells, it's necessary to remove the culture medium before fixation. Leave your samples to fix for 10-20 minutes, adding the correct amount of fixative, and you'll be ready to move on to your next step.
4. Remove the fixative and dry in the air.
To stain your samples with Hoechst, add an appropriate amount of 10-fold diluted Hoechst staining solution and allow it to stain at room temperature for 10-30 minutes. For a six-well plate, you should add 1 mL of staining solution to one well. Ensure that the staining solution fully covers the sample and protect it from light by using aluminum foil. It is important not to expose the sample to light during staining.
6. Remove the dyeing solution and dry in the air.
7. Add dropwise the anti-fluorescence quenching mounting solution provided in the kit, and observe the blue fluorescence under a fluorescent microscope after mounting. Fluorescence microscope observation requires 400x or 1000x magnification observation (oil lens is required). Cell samples without mycoplasma contamination or other prokaryotic contamination, only the blue fluorescence of the nucleus is observed, and the mitochondrial DNA will not be stained.

Cell samples contaminated with mycoplasma can be observed in particulate or filamentous blue fluorescence around the nucleus. A large amount of particulate or filamentous blue fluorescence can be observed in cells with severe mycoplasma contamination.

In the case of bacteria, yeast or mold contamination, although Hoechst staining will be positive, bacteria, yeast or mold contamination can be observed under an optical microscope, but mycoplasma cannot be observed, which can be preliminarily distinguished from mycoplasma or other microorganisms.

If further identification is necessary, further testing can be carried out by inoculating mycoplasma and RT-PCR.

1. Hoechst dyeing reagent is harmful to human body, please pay attention to proper protection.
2. The fixing solution contains glacial acetic acid and has pungent odor. It is appropriate to conduct fixing operation in the fume hood.
3. Fluorescent dyes all have the problem of quenching. It is suggested to complete the test on the same day as possible after dyeing.
4. It is better to use the culture medium without antibiotics for 2 to 3 generations before detection of mycoplasma, so it is easier to detect mycoplasma, because some antibiotics can inhibit the growth of mycoplasma.

Store at 2~8° C away from light for 12 months.

Mycoplasma Stain Assay Kit