G'day Katie --- Giemsa staining of cell nuclei, assuming it looks a nice purple colour, is due to a two dye-complex of azure B and eosin. This complex is unstable in methanol or ethanol, and azure B is soluble in these solvents ... so why not try extracting in one of those?

I don't know if you can solubilize GIEMSA. However, this can be achieved by using Crystal violet, you can then solubilize if with acetic acid and check it with a specific wavelength (which I don't remember but easy to find)

Giemsa is a polychromatic stain and not really suitable for quantitation. It also tends to precipitate during staining and that would also dissolve and add error. David is right; use Crystal Violet. Wikipedia gives you the absorbance maxima 590 nm in water and 420 nm in strong acid.

Dear Kate,

the two postings above (G'day, dear Richard!) point you to certainly good and feasible possibilites.

Nevertheless I have a problem of understanding your method since maybe I have missed or misunderstood an important detail of your study: your cell colonies are alive or have been "fixed" somehow prior to (or - at least by) Giemsa staining?

IF they have been fixed either by air drying, or some kind of alcohol (as this is done e.g. for staining chromosomal G-banding, then there perhaps are some possibilities.

I have found the following (text below are citations out of papers; if you will see the bibliographic sources, please let me know and I shall send them to your e-mail adress):

i) "Destaining of Giemsa (obviousely from paraffin sections?): xylene, xylene-100% EtOH; 2 min EtOH 100%, 2 min 95% EtOH, 30 “ 70% EtOH /1% HCl,

2 min 70 EtOH, air drying…..==> iterative Giemsa-Staining &destaining up to 6 x possible" (1973).

ii).".. Giemsa staining, the cells on cover slips are placed in buffered Giemsa (0.5 ml in 10 ml of phosphate buffer, pH 6.9) for 30 minutes then destained for a few seconds in acidified distilled water (a loopful of glacial acetic acid in 10 ml of water)".(1977)

iii) "Slide destained" [after Giemsa]...

only the Reference is given: “after: ROWLEY JD, Potter D and Mikuta J. Stain Technol. 46, p97f (1971)"

iv) "Preparations stained with Giemsa were destained in an alcohol series: five minutes each in absolute, 70%, and 30% ethanol. " (1984);

v) (2006)"…some of the Giemsa and May-Grünwald-Giemsa–stained slides were destained in methanol/glacial acetic acid (3:1) overnight (Ref.7)

Ref:[7]: Casartelli G, Monteghirfo S, De Ferrari M, et al. Staining of micronuclei in squamous epithelial cells of human oral mucosa. Anal Quant Cytol Histol 1997;19:475–81 "

vi) (2012)"...Eye ball pieces (Chicken & Japanese Quail embryos) fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence"

vii) ex wwweb:

"Metaphase slide preparation :

"Age the air-dried slide overnight in a drying oven at 55◦ to 60°C.

Destaining:

Rinse slide in autoclaved distilled water until the stain no longer discolors the water, using the same agitation technique as in step 2. CAUTION: Long exposure to water will result in destaining of the slide." -

and finally: (1976): (Differential Staining Giemsa in plants) "destaining in methanol". I should be glad to know about your consideration and progress with the destaining method...

I don't understand the relation between Giemsa staining and cell survival. But I would suggest an other staining. If your clonogenic assays are performed in 96 wels plates, stain your colonies by adding 100 µL of MTT in your wells (250 µg/mL ). The color develop in one hour and depends on the cell viability. You can then count the dark blue colonies and, when necessry, dissolve the formazan precipitates in 10% SDS or DMSO.

My apologies on the delayed response but I wanted to give you all an update. Yes, Wolfgang, the cell colonies were fixed using a PBS/Methanol gradient, so you know.

I had an old 6-well plate with Giemsa-stained colonies and used that to experiment on. I've found that using 0.5N HCl does re-dissolving the dye, giving a blue-light purple color. The color does fade over time (as well as the solution evaporated) so reading the absorbance values will have to be done immediately after. I plan testing the 0.5N HCl in the experiment I originally described. Hopefully I'll be able to get consistent and accurate results!

Thanks for updating us, appreciated. Richard.

I have exactly the same problem than you.

So, the 0.5N HCl incubation is effective ? Do you have any precipitate with this method ? Have you buffured the 0.5N HCl to a specific pH ?

What wavelenght do you read?