The document discusses staining techniques used in histology and cytology. It provides details on the preparation, components, and use of common stains including Hematoxylin, Giemsa stain, Papanicolaou stain, and Periodic acid–Schiff stain. The stains are used to differentially color structures like nuclei, cytoplasm, muscles, bones, parasites and glycogen under the microscope to enable examination of tissue samples and identification of cells and microorganisms.
4. Preparation
Dissolve 0.2 g of powdered Leishman’s dye in 100 ml of
acetone-free methyl alcohol in a conical flask.
Warm it to 50°C for half an hour with occasional shaking.
Cool it and filter it.
Giemsa stain – add 1:9 dilution (distilled water and
Giemsa)
Procedure for staining
Pour Leishman’s stain drop wise (counting the drops) on the
slide and wait for 2 minutes. This allows fixation of the PBF
in methyl alcohol.
Add double the quantity of buffered water(PH 6.8) drop wise
over the slide (i.e. double the number of drops).
Add Giemsa stain in 4 to 5 drops and mix.
Mix by rocking for 8 minutes.
Wash in water for 1 to 2 minutes.
Dry in air and examine under oil immersion lens of the
microscope.
6. The Hematoxylin and Eosin stain is the most
widely used histological stain because……
Its comparative simplicity
Ability to demonstrate clearly an enormous
number of different tissue structures.
The hematoxylin stains cell nuclei blue / black
Eosin stains cell cytoplasm and most connective
tissue fibers.
7. Principle
H and E are principle stain for demonstration of nucleus
and cytoplasm.
Alum acts as a mordant and the hematoxylin containing
alum stains the nucleus light blue which turns red in the
presence of acid.
The cell differentiation is achieved by treating the tissue
with acid solution. The counterstaining is performed
using eosin which imparts pink color to cytoplasm
9. Dissolve the hematoxylin in absolute alcohol
and ammonium alum
in hot water. Mix the two solutions and heat
to boiling. Remove from flame, and
add mercuric oxide and cool rapidly. Glacial
acetic acid if added gives brisk
nuclear staining, but life of the solution is
reduced. Hence if acetic acid is to be
added, it should be added in working
solution.
10. Ingredients
Hematoxylin 1.0gm
Distilled water 1000ml
Ammonium alum 50gm
Sodium iodate 0.2gm
Citric acid (reduces pH) 1.0gm
Chloral hydrate (preservative) 50gm
Method - Hematoxylin is dissolved in distilled water using gentle
heat. Then
alum is added and dissolved. Then sodium iodate, citric acid and
chloral hydrate
are added respectively.
11. Eosin is used as the counterstain that stains the
cytoplasm rose coloured. The
intensity of the eosin is individual choice. The most
widely used eosin is “eosin
Y”. The “Y” stands for yellowish. It is available in
either water soluble or alcohol
soluble form. Most laboratories use the water soluble
form of eosin Y in an
alcohol-water solution which is described here.
Eosin Y (water soluble) 1.0gm
Distilled water 80ml
95% alcohol 320ml
Glacial acetic acid 0.4ml
12. Dissolve eosin in water and then add this to
95% alcohol (one part eosin solution with 4
parts alcohol). To the final mixture add a few
drops of acetic acid (0.4ml). The acetic acid
increases the staining intensity of eosin.
When ready to use, the stain should be
cloudy; if clear, add a few drops of the acetic
acid. The solution should be standardized by
staining the control slides.
13. 1. Deparaffinize sections in xylene, 10-20
minutes. Filter Hematoxylin.
2. Rehydrate sections:
100% alcohol for 1-2 minutes
95% alcohol for 1-2 minutes
3. Rinse in tap water
4. Rinse in distilled water
5. Stain with Hematoxylin for 3-5 minutes
6. Wash in tap water
14. 7. Differentiate section with 1% HCl in 70%
alcohol 1-2 dips and check under
microscope. If necessary, return slides to HCl for
further differentiation.
8. Wash slides in running tape water for 15
minutes
9. Stain slides in Eosin for 1-4 minutes
10. Dehydration and Differentiation:
95% alcohol 5-6 dips
100% alcohol 5-6 dips
11. Clear slides in xylene 2 times
12. Mount slides with mounting media (Permount
or DPX
15. Removal of paraffin wax (Deparaffinization)
◦ Removed with xylene (impermeable to stains)
◦ 2-3min of xylene immersion sufficient for sections of
10 µ thickness
◦ First facilitated by warming the slides at 60 degrees
oven to melt the wax
Removal of xylene
◦ Xylene is not miscible with water or low grade
alcohols, hence dipped in two changes of absolute
alcohol
16. Hydration(High to low)
After removal from xylene sections are transferred to
absolute alcohol for 1-2min until it becomes opaque
Sections rinsed in second bath of alcohol, drained and taken
to water
Any pigments or deposits should be removed at this stage
17. Staining
◦ Slides immersed in hematoxylin (Mayer s,
Harris, Gills)
◦ If regressive stain is used longer time is used
to over stained the structures
Differentiation
◦ Sections are dipped in Acid alcohol, agitated
and washed in tap water
Observed under microscope
◦ If underdifferentiated- returned to acid
alcohol
◦ If overdifferentiaited – returned to
hematoxylin and differentiated again
18. Blueing
◦ Slides after draining off hematoxylin is transferred to
ammonia water for 2 min. Sections when removed from
hematoxylin or acid alcohol are pink in color.
◦ Washing turns them blue.
Counterstain(Eosin)
◦ Transfer the slides to 1% aqueous Eosin for 30 sec. Wash
in running water.
Dehydration( low to high)
Slides are taken through 3 stages of Alcohol.
19. 11. Clearing
◦ Sections transferred to xylene and left until clear.
◦ Tested for clarity by being held against a dark background.
12. Mounting
◦ Surplous xylene wiped off from slide surface.
◦ This step completed quickly to avoid section drying.
◦ Whole operation takes 5-10 seconds.
20. Results
Cell nuclei – Blue
Muscle fibers – Red
Collagen fibers – Pink
RBC – Bright red
21. Hematoxylin (Mayer's) is recommended for
Immunohistochemical and cytochemical
Staining (as Nuclear Counter Stain) (PAS
Staining Procedure). It may also be used for
routine Hematoxylin and Eosin Staining.
22.
23. Periodic Acid Schiff
Periodic acid + Glycogen
oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na
metabisulfite)
> Red deposit
24. Periodic acid specifically oxidizes 1–2 glycol
groups to produce stable dialdehydes.
These dialdehydes give a red reaction product
when exposed to Schiff's reagent (leucobasic
fuchsin).
In haemopoietic cells, the main source of
positive reactions is glycogen.
Reagents
1) Fixative . Methanol
2) Schiff’s reagents
3) Counter stain . Aqueous hematoxylin
25. Test for Schiff reagent: Pour 10 ml of 37%
formalin into a watch glass.
To this add a few drops of the Schiff reagent
to be tested. A good Schiff reagent will
rapidly turn a red-purple color.
A deteriorating schiff reagent will give a
delayed reaction and the color produced will
be a deep blue-purple
26. Periodic acid–Schiff stain.
A: Dysplastic micro megakaryocytes with diffuse cytoplasmic
staining and some coarse granules;
B: Dyerythropoiesis with diffuse staining in a tri nucleate
normoblast and coarse granular and diffuse staining in a pro
erythroblast.
C: Acute lymphoblastic leukemia with blasts showing block
positivity
The reaction product is red, with intensity ranging from pink to bright red .
27. Stains Include:-
Nuclear staining: Hematoxylin
Two cytoplasmic counter staining:
• Orange G (OG)-6, OG-5 and OG-8 is acidic
dye, stains keratin a bright, intense orange.
• Eosin Azure (EA) , EA-36,EA-50 Including
three stains-
-Eosin Y
-Light Green
-Bismarek Brown Y
28. Principle :
Papanicolaou staining is applied to vaginal
exudates for the detection of uterine or vaginal
cancer. The technique uses a high number of dyes
in its procedure.
• Hematoxylin: is the chosen nuclear staining,
basically allows to reveal the nuclei of the cells
present in the sample. Harris Hematoxylin is
usually used.
• Orange G: is a synthetic acid dye that reveals
basic compounds such as prequeratine (that stains
pink) or keratin (that stains bright orange).
29. • Yellowish Eosin: stains cytoplasm of
mature squamous cells, hair cells and
erythrocytes into pink-orange.
• Green SF Yellowish light: stains squamous
non-superficial cells (immature or partially
mature) into greenish-blue.
• Bismark Brown R: does not stain the
cellular cytoplasm but does mucin.
• Phosphotungstic acid: has a mordant
function, especially important for Green
Light SF
30. Procedure
1. Fix the sample with spray.
2. Submerge successively in alcohol 80%,
alcohol 70%, alcohol 50% and water, 1
minute in each liquid.
3. Stain with Hematoxylin Harris solution
for approximately 5 minutes.
4. Immerse in water 6 times for 1 second.
31. 5. Submerge in 0.5% Hydrochloric Acid, 8
times for 1 second.
6. Rinse with tap water for 5 minutes, and
pass the sample through successive grade
alcohols, 50%, 70%, 80% and 96% for 30
seconds in each of them.
7. Stain with Pap Smear or OG 6 for 1 to 1.5
minutes.
8. Wash the excess dye in two 96% Ethanol
baths by immersing the preparation 2 times
in each of 3 to 4 seconds.
32. 9. Stain with Pap Smear or EA 50 for 1.5 to 2 minutes.
10. Wash in 3 different containers of Ethanol 96% v / v
by immersing the preparation 2 times of 3 to 4 seconds
in each of them.
11. Wash in absolute ethanol for 30 seconds.
12. Immerse the preparation for 4 minutes in a 1: 1
bath of Xylene, mixture of isomers and absolute
ethanol.
13. Rinse with Xylene, mixture of isomers by immersing
the preparation for 3 minutes in a bath.
14. Mount with Mounting medium DPX
15. Observe under a microscope.
33. Choice of fixative and technique is
dependent on specimen types analyzed in the
laboratory. The stains offered are suitable for
both Gyn (EA-36, EA-50) and Non-Gyn (EA-
50, EA-65) specimens as determined by the
user.
35. PRINCIPLE
This method is a modified version of the original
Giemsa technique used for hematological smears
and gives good results for sections.
Giemsa is a Romanowsky stain which contains
azure B and eosin Y and is capable of making
subtle distinction in shades of staining.
The acidic groupings of the nucleic acids and
proteins of the cell nuclei determine their uptake of
the basic dye azure B and the presence of basic
groupings result in an affinity for acidic dyes and
their staining by eosin.
37. Hematoxylin (Harris) is used for general purpose
nuclear stain and for histological studies
Composition
Ingredients
Hematoxylin -5.000 gm
Ammonium or potassium alum - 100.000 gm
Mercuric oxide -2.500 gm
Alcohol 95% -50.000 ml
Distilled water -1000.000 ml
38. It is a multichromatic cytological stain
Composition
Ingredients
Orange G-6 Certified -0.3 gm
Phosphotungstic acid -0.015gm
Denatured alcohol -100.0ml
39. Papanicolau-EA-50 is use for routine diagnostic
cytology to aid in the identification and classification
of exfoliative cells.
Composition
Ingredients
Eosin Y Certified - 0.23gm
Bismark Brown Certified - 0.05gm
Fast green FCF Certified - 0.08gm
Phosphotungstic acid - 0.2gm
Denatured alcohol - 100.0ml
40. It can be used for histopathological diagnosis
of malaria and some other spirochete
protozoan, and blood parasites.
Results
Micro-organism, fungi - purplish-blue
parasites
Nuclei- blue to violet
Erythrocytes- salmon pink
cytoplasm - light blue
Collagen, muscle and bone - pale pink
