Giemsa stain

Summary

Cultures were fixed with methanol and stained directly with undiluted Giemsa's solution, then the stain was diluted 1:10, the cultures were washed, and observed in a humid state.

Operation method

Scheme 16.2 Giemsa staining

Principle

Cultures were fixed with methanol and stained directly with undiluted Giemsa's solution, then the stain was diluted 1:10, the cultures were washed, and observed in a humid state.

Materials and Instruments

D-PBSA D-PBSA:Methanol (1:1)
Undiluted Giemsa stain Methanol Deionized water

Move

This procedure assumes that monolayer cells are used for staining, whereas fixed suspension cells can also be used, but starting at step 6.

1. Remove the culture medium.

2. Rinse monolayer cells with D-PBSA to remove rinse solution.

3. Add 5 ml/25 ^cm2 of D-PBSA/Methanol solution, let stand for 2 min, then remove D-PBSA/Methanol.

4. Add 5 ml of new methanol and leave for 10 min.

5. Discard the methanol solution, use anhydrous methanol, rinse the monolayer cells, and remove the methanol.

6. At this point, the flasks are dried for storage or can be stained directly. It is very important to stain directly after rinsing with fresh anhydrous methanol, even if the flasks are dry. If the methanol is poured out and the culture flask has been left to sit for some time, the residual methanol will absorb moisture, thus preventing the next step of staining, even if the dried monolayer cells can absorb moisture from the air.

7. Add pure Giemsa Staining Solution, 2 ml/ 25 ^cm2, making sure to maintain coverage of the entire layer of cells.

8. After 2 min, dilute the stain with 8 ml of water and shake gently for 2 min.

9. Displace the staining solution with water so that the scum that forms runs off and does not remain to cover the cells, and rinse the cells with tap water until the lamellar pink background (deposit) is removed, but do not bleed off the staining of the cells (usually 10-20 s).

10. pour out the water, rinse with deionized water, observe the cells under a microscope while the monolayer is still wet, dry and preserve the cells, and rewet them for further observation.

Caveat

Giemsa staining is a simple procedure that provides a contrasting, polychromatic stain, but precipitates out the dye, giving the cells a speckled appearance. Due to oxidation, a precipitate forms on the surface of the staining solution, and when water is added, a scum forms throughout the solution, especially on the surface of the slide; the staining solution is rinsed by lifting the level of the liquid upwards, instead of pouring out the liquid or removing the planted slides, and in the final step, in order to prevent contact between the cells and the scum, it is rinsed thoroughly in order to remove the scum that remains in the preparation.

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