Fluorescence microscopy for mycoplasma detection

Summary

There are two different strategies for detecting mycoplasma: the first method, which will be described in the following scheme, utilizes the fluorescent dye Hoechst 33258, which stains DNA for specific substances around the nucleus (on the cell surface or in the cytoplasm) and can reveal mycoplasma contamination; the second method utilizes a DNA probe that recognizes mycoplasma-specific sequences of DNA in cellular extracts for mycoplasma contamination detection. The second method is to use DNA probes to recognize the DNA specific sequences of mycoplasma in cell extracts for the detection of mycoplasma contamination.

Operation method

Fluorescence microscopy for mycoplasma detection

Principle

There are two different strategies for detecting mycoplasma: the first method, which will be described in the following scheme, utilizes the fluorescent dye Hoechst 33258, which stains DNA for specific substances around the nucleus (on the cell surface or in the cytoplasm) and can reveal mycoplasma contamination; the second method utilizes a DNA probe that recognizes mycoplasma-specific sequences of DNA in cellular extracts for mycoplasma contamination detection. The second method is to use DNA probes to recognize Mycoplasma DNA-specific sequences in cell extracts for the detection of Mycoplasma contamination.

Move

1) Prepare the following liquids, then sterilize and refrigerate.

Phenol red free HBSS

NaCl 8 g
KCl 0.4 g
_CaCl2 0.14 g
_MgSO4-7H2O 0.1g
_MgCl2-6H2O 0.1g
Na 2 _{HPO4-2 H2O} 0 .06 g
KH 2 _PO4 0.06 g
Glucose 1.0 g
_NaHCO3 0.35 g
_dH2O 1000 ml

Hoechst 1000x storage solution

Citric acid/phosphate buffer (2X)

0.1 mol/L citric acid 22.2 ml
0.2mol/L Na 2 _HPO4 2 7.8 ml
Adjust pH to 5.5 with citric acid or Na 2 _HPO4.

2) Apply 1~2 drops of trypsin to digest the cells on a Labtek slide or coverslip in a Petri dish.

3) Add 1~2 ml of culture medium to cover the surface and let the cells grow for 2~3 days.

4) Prepare Camoy's fixative (should be prepared fresh before each use).

Camoy Fixative
3 parts methanol
1 glacial acetic acid

5) Pipette the culture medium without washing the monolayer and add Camoy's Fixative directly to the Labtek chamber or petri dish. Fill the dish with Camoy's fixative (approx. 5 ml) and leave the dish at room temperature for 2 minutes.

6) Remove liquid from dish and add fresh fixative to fill chamber or dish (~5 ml). Leave the dish at room temperature for 5 minutes.

7) Repeat step 6.

8) Remove the fixative, remove the plastic jacket from the Labtek chamber or remove the slide from the petri dish and allow the cells to air dry at room temperature for 30 minutes or longer.

9) Prepare Hoechst Working Solution with Hoechst 1000x Stock Solution (step 1).

Hoechst Working Solution

The working fluid should be prepared fresh each time.

Dilute 0.1 ml Hoechst stock solution (0.5ug/ml) with 10ml HBSS.

Dilute 0.5 ml Hoechst stock solution (0.05ug/ml) with 5 ml HESS.

or

Dilute 50ul of Hoechst 1000x stock solution with 50 ml of HBSS; add 40 ml per Coplin tank.

10) Stain slides for 10 minutes at room temperature in Hoechst working solution prepared in HBSS (final concentration 0.05ug/ml).

11) Rinse twice with _dH2O.

12) Mix equal volumes of glycerol, catalase/phosphate buffer (prepared in step 1) to prepare a closed culture solution to cover the cells.

13) Add 2 drops of Closure Solution between the slide and coverslip and gently blot out the excess liquid with a paper towel.

14) Close the slide and coverslip with rubber adhesive.

15) Observe the specimen with a fluorescence microscope. Apply excitation wavelength of 330/380nm and grating filter LP440nm.

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