Sodium naphthyl phosphate 10 mg 0.2M Tris buffer (Stock solution A) pH 10 mL Diazonium salt (fast red TR) 10 mg The final pH of the incubating medium should be between 9.0 and 9.4. The sodium naphthyl phosphate is dissolved in the buffer, the diazonium salt is added and the
i. Preparation of May-Grünwald stain: 0.3 g of powdered
dye is weighed out and transferred to a conical flast of
200–250 mL capacity. A volume of 100 mL of methanol
is added and the mixture is warmed to 50°C. The flask
is then allowed to cool to room temperature and is
shaken several times during the day. After standing
for 24 hours, the solution is filtered. It is then ready
for use, no ripening being required.
ii. Preparation of solution of Giemsa powder is dissolved
in 54 mL of glycerol and after cooling, mixed with
84 mL of methanol GR and filtered.
Air-dried smears are fixed in a jar of methanol for
5 minutes. Fixed smears are stained as follows:
1. With May-Grünwald stain, freshly diluted with an
equal part of phosphate buffer for 5 minutes.
2. With Giemsa’s stain, diluted with 9 parts of phosphate
3. Washed with phosphate buffer (pH 6.8).
5. Mounted by a rectangular cover glass using DPX as
Papanicolaou’s Stain with EA-36
The hematoxylin is dissolved in absolute alcohol and
potassium alum in distilled water with the aid of heat. The
two solutions are mixed together. The mixture is boiled,
removed from the flame and mercuric oxide is added bit
by bit. The flask containing the solution is then immersed
into cold water bath. After cooling, it is filtered and stored
Orange G-6 solution is prepared as follows:
The stock solutions of light green SF yellowish (A), eosin
yellow (B), are prepared as follows:
FIGS. 26.3A TO D: Two-step technique. Line drawing summary. (A) Finger
position. (B) Collection of aspirate, (C) Particle concentration smear,
and (D) Preparation of a monolayer smear are illustrated for a righthanded operator
814 Concise Book of Medical Laboratory Technology: Methods and Interpretations a. Light green—(SF)
Light green SF yellowish 0.5 g
From the stock solution, the working solution of EA-36
Light green SF yellowish (A) 45 mL
All these solutions are kept in the refrigerator when not
Automatic stainer can process a large number of smears
Autostainer: Slides held in rack are automatically rotated
around baths containing stains and other reagents. Time
schedule for staining as mentioned previously in manual
process can be obtained by calibrating a timing dial.
The reagents are renewed weekly. The instrument has
the advantage that it can easily be adapted for staining
techniques. It uses laboratory prepared reagents and it
allows for complete adaptability in staining, times. After
staining, the slides are mounted with DPX.
The fixed slides are transferred directly from the fixative
5. Harris’s hematoxylin 1 minute
7. Hydrochloric acid (0.5%) 5 dips
9. Dilute solution of lithium carbonate 1 minute
10. Running tap water 1 minute
21. Absolute alcohol 4 minutes
Slides are then mounted with DPX.
Cytoplasm of superficial cell— pink
Cytoplasm of intermediate cell—bluish green
Papanicolaou’s staining schedule for automated stainer
(total staining time about 30 minutes)
Papanicolaou’s staining procedure for manual set-up
(total staining time under 7 minutes)
2. Harris’s Hematoxylin 60 dips 30
5. 0.1 % HCl in 70% Ethanol 5 dips Not Necessary
7. 1% NH4OH in 70% alcohol 30 dips 30
Combined Alcian Blue—PAS Technique
Acid mucin and neutral mucin are clearly separated by
this technique. It is also useful as a routine demonstration
technique for the presence of any mucin. The acid mucins
are first stained with alcian blue and are not available for
PAS reaction. Only the neutral mucin is stained by PAS
reaction which follows. In this way, a good color distinction
can be made between acid and neutral mucins.
1. Wash the fixed smears in distilled water.
2. Stain smear with alcian blue solutions for 5 minutes.
4. Treat with 1% periodic acid for 5 minutes.
6. Place in Schiff’s reagent for 10 minutes.
7. Wash in running water for 10 minutes.
8. Stain nuclei with hematoxylin.
10. Dehydrate, clear and mount.
Naphthol ASBI Phosphate Method for
Acid phosphatase is demonstrated by an Azodye coupling
technique, which depends upon the hydrolysis of a
substrate containing Alpha-naphthoxl phosphate. As
hydrolysis occurs, the liberated naphthol couples with
a diazotized amine and forms an insoluble colored
Burstone (1958), recommended naphthol ASBI phosphate
as substrate—the primary reaction product, produced by the
enzyme hydrolyzing this substrate is extremely insoluble.
d. Pararosanilin hydrochloride stock solution
Pararosanilin hydrochloride 1 g
Heat gently, cool to room temperature and filter
e. Distilled water preparation of incubating solution
Preparation of incubating solution
together and allowed to stand for 2 minutes before adding
The pH should be between 4.7 and 5.0 it is adjusted with
1. Incubate smears at 37°C for 60 minutes.
3. Counterstain in 2% methyl green (chloroform
Acid phosphatase activity : Red
Alkaline Phosphatase: Azo Dye Coupling Method
Using Alpha Naphthyl Phosphate
Preparation of Incubating Medium
Sodium naphthyl phosphate 10 mg
0.2M Tris buffer (Stock solution A) pH 10 mL
Diazonium salt (fast red TR) 10 mg
The final pH of the incubating medium should be
between 9.0 and 9.4. The sodium naphthyl phosphate is
dissolved in the buffer, the diazonium salt is added and the
solution well mixed. The solution is then filtered and used
a. After fixation, incubate the smears at room temperature
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