Detecting Mycoplasma Cell Culture Contamination

[dnamolecule]

Hey guys how does one detect mycoplasma contamination in cell culture?
our cells are growing slower than usual and we want to check it out...
thanks in advance

[mickey07]

Hi
I routinely check my cells for mycoplasma contamination with Hoechst staining solution (Sigma, cat# H6024). It's a nuclear stain, so it's rather non-specific for mycoplasma (ie. regarding species). There are quite a few kits on the market, however, most of them are PCR-based. The Hoechst staining is relatively easy and convenient (no need for DNA/RNA extraction, RT, etc), but you need a flourescent microscope for visualisation.

[Narumi]

Does the Hoechst stain store well? I've read mostly to freeze it, but how long will it last? Is it ok for multiple freeze/thaws?

[mickey07]

Hi Narumi

I keep my Hoechst stain at 4oC (solution from Sigma). It should be fine for up to 6 months (or longer; I know someone who kept their home-made-up stain for more than a year ! at 4oC and it still worked fine). Freezing should be fine, but make single-use aliquots and wrap the tubes in foil. I think if exposure to light is limited, the stock solution should keep for quite a while. Hope this helps.
Ciao

[binxue]

what is the concentration of Hoechst 33258 you used? working solution and storing solution concentrations ? thanks in advance.

[proatpcr]

Hello guys,
found the protocol for in a 8-well chamber slide.

Hoechst 33258 direct DNA stain for Mycoplasma

This Hoechst 33258 direct DNA stain protocol is rapid (can perform in less than 30 minutes), however requires heavy contamination (10^6 mycoplasma/ml) to produce a clear positive result.

If the suspected cells are co-incubated for 2–4 days with an “indicator” cell line (such as 3T3) that is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased.

Cell cultures are stained with Hoechst 33258, a fluorescent stain, which binds specifically to DNA. Mycoplasma contain DNA and can be detected readily by their characteristic particulate or filamentous pattern of fluorescence in the cytoplasm or between cells. Mycoplasma-negative cells will show only brightly fluorescent cell nuclei. The Hoechst stain also detects bacterial and fungal contamination, although these are usually obvious from the visible turbidity of the culture medium of infected cells.

0.1 ml Hoechst 33258 stain in BSS-PR is added to each well, ensuring that the stain covers the complete surface of the well, and leave for 10 minutes at room temperature.

See here for more information:
4.5 Hoechst 33258 direct DNA stain for Mycoplasma

cheers

[mickey07]

Hi there
Here's the Hoechst stainingmethod I use:
1. MYCOPLASMA TESTING (adherent cells):

- Place sterile cover glass in petri
- Put 2-3 drops of cell suspension in the middle of coverglass (depends on growth rate of cells)
- Carefully add 2-3 ml of medium without disturbing the cells
- grow cells until 50 -70% confluent

Cell Fixative:
- 3:1 Methanol : glacial acetic acid (M:AA)
- Prepare fresh before use
- Discard medium
- Add 2 ml of the M:AA solution drop wise on the side of the plate (not directly onto the cells), swirl plate and discard
- Add 2 ml of M:AA, swirl and leave on for 2-3 minutes at RT
- Discard M: AA and rinse with water
- 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

For staining:
Add enough stain to cover entire cover slip (we use 1-2ml for a small Petri)
Leave on 45'' - 1' (in dark)
Rinse well with water
Mount on 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
Visualise under fluorescence microscope
Hope this helps.
Ciao

[Alyaa Mahmoud]

I read in your post that some of ur friends prepared home-made Hoechest stain ? Is it possible to reach the exact ingredients they used to prepare it by themselves ?
Thank you soo much

[mickey07]

Hi Alyaa

I use the Hoechst 33258 solution from Sigma, so I actually don't have the protocol they used to make up their Hoechst stain, but I think they probably just dissolved a powder. Unfortunately, I don't have any of those details.
Best regards

[Alyaa Mahmoud]

Hi Mickey

Thanks a lot for your reply
Have a good day : )
Alyaa