Salzburg, 14-08-06

in RE to: https://www.researchgate.net/post/What_is_the_protocol_for_Wright_staining?

What is the protocol for Wright staining ?

Can anybody help me to get the protocol for wright staining? I want to stain the different cell present in
sputum.
Dear Subas Bishwal,
having some troubles with automatic info-forwarding for new answers and replies via Researchgate for
the last days I found your answer just recently when joining RG for today.
In answer to your questions, - and not to use much space in the answering form - I add here a pdf
containing information you perhaps might have no access via Internet.
I found the following Sources for AMRESCO Wright stain (0687) only:
i) http://www.amresco-inc.com/WRIGHTS-STAIN-0687.cmsx
(no data for Molecular Weight: N/A; Molecular Formula: N/A ) but indicating <MeOH> was the diluent for Absorbance
measurement.
ii) http://www.thomassci.com/Chemicals/Stains-Dyes/_/Wright-Stain?q=Wright-Stain: no access since: <<This
product is a restricted product and can only be purchased by approved account holders. Click here to login to your account or
register for a new account.>>
iii) http://www.mandelscientific.com/products/microbiology/Dyes_Stains/Selection_Chart/index.htm:
<<Wrights Stain: Certifiable; A stain used for blood and bone marrow films. Useful for staining malarial parasites in blood film.
CI: not applicable, Use in: Histology>>

Since one can guess therefore that the substance you have is WRIGHTs pure dye powder (certifiable,
BUT NOT certified by the Biological Staining Commission,or is it?) you IMHO -can do/try only the
following:
If you read the TDS of Polysciences (as mentioned above, most of it copied and pasted in section
below), http://www.polysciences.com/SiteData/poly/Assets/DataSheets/350.pdf) youll find
a possible solution.
Usually, those dyes, including Giemsa, are used in concentrations between 0.1 to maximally 1.0% (w/v).

I personally would consider to mix up at least 3 solutions with varying dye concentration (just to be on
the safe side):
per 1000 ml end volume (or calculate dividing by 10 for e.g. each solution = 100ml end volume)
Wrights Stain:
1.0 g
3,0 g
6.0 g
Glycerin [NB: usually delivered as 85%solution) 10% (v/v)
100.0ml
100.0ml
100.0ml
Fill up with absolute Methanol to End volume of 1000 ml
(I am not sure about Wrights dye easy soluble in PBS! But for exerting the staining MeOH will be the right solvent)

From TDS POLYSCIENCES for WRIGHT STAIN 801

(cf: http://www.polysciences.com/sitedata/docs/801/2d1cfb49695ef129bbe941ed25feb2fc/801.pdf):

FIXATION
Streak thin smears across a sterile slide by means of a second slide or cover glass placed at an angle. Air-dry completely. Make
sure all slides are clean prior
to making the blood or bone marrow smear. If slides are not completely clean the stain will not absorb into the smear.

PROCEDURE
1. Place 1.0 ml of the Wright Stain Solution upon the smear approximately 1-3 minutes.
2. Add 2.0 ml of distilled water or Cat. #24989 - Wright Stain Phosphate Buffer pH 6.8 and let the slide stand twice as long as in
step 1.
3. Rinse off the slide with water or Cat. #24989 Wright Stain Phosphate Buffer pH 6.8 until a faint pinkish red appears on the
edges.
If a Giemsa appearance is desired, stain 10 minutes in one volume of Wright Stain Solution and 4 volumes of Wright Stain
Phosphate Buffer pH 6.8.
4. Dry thoroughly using bibulous paper to blot dry.
Allow the slide to dry at room temperature before examination to determine if adjustments are needed in the dilution or timing.

./.

STAIN RESULTS
Red blood cells red to pink
Neutrophils dark purple nuclei, pale pink cytoplasm, reddish-lilac small granules
Eosinophils blue nuclei, pale pink cytoplasm, red to orange-red large granules
Basophils purple to dark blue nucleus, dark purple, almost black large granules
Lymphocytes dark purple to deep bluish purple nuclei, sky blue cytoplasm
Platelets violet to purple granules
REFERENCES
Conn, J I , Biological Stains, 8th ed., Williams and Wilkins Co., Baltimore, 1969
Clark, G , (ed.) Staining Procedures, Williams and Wilkins Co.. Baltimore, 3rd Ed., c 1972, p. 122

OR, as From TDS POLYSCIENCES for WRIGHT STAIN 801

(cf: http://www.polysciences.com/sitedata/docs/801/2d1cfb49695ef129bbe941ed25feb2fc/801.pdf):

you modify staining solution to <WRIGHT-GIEMSA> stain solution; In both cases you could follow the
staining instructions there and as pasted here:
Reagents g / L
1.53g
2.50g
100,00 to 118 ml
personal NB (117,65 ml = definitively 10%w/v if considering Glycerol usually is delivered
as 85% solution).
Add absolute methanol up to 1000 ml end volume

Wrights Stain, certified

Giemsa Stain, certified
Glycerin(Glycerol) 10% (v/v)

from the TDS:

Safety Precautions:
Danger! Poison! May be fatal or cause blindness if swallowed.
Flammable. Keep away from heat, sparks and open flames. In case of fire, use water spray, C02, foam or dry
chemical.
Since you perhaps are not able to download the Polysciences TDS, I enclose the subsections by C&P here:
Storage Instructions:
Solution should be kept in the original bottle, tightly closed, to prevent contamination with water. It should be stored at
room temperature at 15-30C (59-86F). When stored in this manner, solution has good stability. To check usefulness
of solution, stain a known specimen in the usual manner and compare with usual results.
Specimen Preparations:
Blood films must be made properly to ensure correct morphology and staining. In particular, blood films must be
uniformly thin so as to obtain unvarying quality in staining from slide to slide.
Procedure:
a) Using any of the conventional techniques, smear a small drop of blood on a clean microscope slide. Allow to air dry.
b) Fix by immersing in methanol for at least 5 minutes.
c) Prepare pH 6.8 phosphate buffer solution consisting of potassium phosphate, monobasic (=KH2PO4*), 50.1%
(w/w) and sodium phosphate,dibasic (=Na2HPO4**), 49.9% (w/w).
[NB: this is obviously calculated for EV=100ml!)
[NB: According to LIAO et al, 1981: Phosphate buffer (0.008 M, pH 6.6 - 6.9) will be used as
washing buffer]
Weigh out accurately 0.501 grams of potassium phosphate, monobasic and 0.499 grams of sodium phosphate,
dibasic. Dissolve in 1000 ml of distilled water.
[NB: this now is obviously calculated for EV=1000ml!)
[personal NB: this in black within the rectangle - is the original text writing in the Polysciences TDS.
But at least one part of the calculation dissolving (0.501 g / 0.499 g) in 1000 ml of distilled water seems to be
wrong here!
[Disparity of molarity /concentrations of the two particulars of recipe]:
First you mix up 50,1% and 49,9% w/v) (=for <100 ml>, then the description says weigh out accurately 0.501 g
KH2PO4 and 0.499 g Na2HPO4 and fill up to 1000 ml of distilled water, which results in concentrations of 5.01%
and 4.99%, respectively . The notation does not make any sense for me without a recalculation, also since a
molarity of the buffer solution is not given)
*) KH2PO4(acidic component): 0.1M= 1,361%; **) Na2HPO4 (water-free, basic component): 0.1M= 1,420%
[NB: According to LIAO et al, 1981: Phosphate buffer (0.008 M, pH 6.6 - 6.9) will be used as washing buffer]. Also
be sure, when adding the second PO4 component = sodium phosphate, dibasic, that the first salt, potassium
phosphate, monobasic, has solved to 100%].

d) Mix equal parts of Wright-Giemsa Stain and buffer solution immediately before use.

e) Immerse blood films in Coplin jar or staining dish of solution prepared in Step d for 2 to 4 minutes, longer if
preferred.
f) Rinse in pH 6.8 buffer, three changes, 10 dips each.
g) Wipe back of slide. Blot dry or stand on end and allow to dry.
h) Examine smear by microscope, with or without coverslipping.
If using a coverslip, use the least amount of mounting medium and a No. 1 thickness coverglass if the blood is spread
on the slide. If the blood is spread on a coverglass, the thickness should be No. 1 1/2.
i) Discard the stain and the buffer rinses after each use.
Results:
Erythrocytes appear yellowish-red to tawny.
Polymorphonuclear neutrophils cytoplasm appears pale pink to tan; the granules, lilac; the nuclei, reddish-purple.
Eosinophils granules appear red-orange; the nuclei, reddish-purple.
Basiphils granules appear purple to dark bluish-purple; the nuclei, reddish-purple.
Lymphocytes cytoplasm appears sky blue; the nuclei, red dish-purple.
Platelets appear violet to purple.
Limitations:
The information obtained from the blood smear is dependent on the method of specimen collection, the preparation of
the film, the drying, the fixation and the final staining of the smear. To this end, slides should be free of all dirt and oily
film. Care should be taken in the staining and washing, as it can result in a precipitate, the wrong colors or excessive
coloring. This excessive staining can also be due to thick smears.
References:
1) Romanowsky, D.L., St. Petersburg Med. Wshr. 16, pp. 297-302, 307-315 (1891).
2) Lillie, R.O., Biological Stains, 8th Edition, Williams & Wilkins Co., Baltimore, MD, 1969, pp. 350-357.
3) Ibid., pp. 355-360.
=================End of pasting from TDS Polysciences =======================
Hope this elucidates your questions as well as the staining matter probably regards,
Wolfgang MUSS,PhD
SALZBURG-Austria