MODULE Cytology : Staining Methods

Histology and Cytology

25
Notes
CYTOLOGY : STAINING
METHODS

25.1 INTRODUCTION
Consistency and reliability are most important in cytological interpretation.
Cytologists rely heavily on the quality and appearance of the stain. The
Papanicolaou stain is recommended for the staining of alcohol fixed cytology
slides. Romanowsky stains may also be used for wet fixed slides, but are
primarily applied to air-dried smears.
Special stains are used as per requirements: Modified Ziehl Neelson (for acid
fast bacilli), Gram staining (Bacteria), Mucicarmine (mucins), PAS (for
glycogen, fungal wall, lipofuscin, etc), Oil red O (lipids), Perls Prussian blue
(iron), modified Fouchets test (bilirubin), etc. Recently, immunocytochemistry
is also being increasingly used in cytology specimens. These special stains and
immunocytochemistry will be discussed along with respective sections in
histopathology as the principles and methods remain the same.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the principle of cytology stains
z explain the methods of staining cytology specimens.

25.2 STAINING IN CYTOLOGY

The universal stain for cytological preparations is the Papanicolaou stain. Harris
hematoxylin is the optimum nuclear stain and the combination of OG6 and EA50
give the subtle range of green, blue and pink hues to the cell cytoplasm.

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Papanicolaou stain Histology and Cytology

Papanicolaou formula
1. Harris hematoxylin
Hematoxylin 5g
Ethanol 50ml
Potassium alum 100g Notes
Distilled water (50C) 1000ml
Mercuric oxide 2-5g
Glacial acetic acid 40ml
2. Orange G 6
Orange G (10% aqueous) 50ml
Alcohol 950ml
Phosphotungstic acid 0-15g
3. EA 50
0.04 M light green SF 10ml
0.3M eosin Y 20ml
Phosphotungstic acid 2g
Alcohol 750ml
Methanol 250ml
Glacial acetic acid 20ml
Filter all stains before use.
Original Papanicolaou staining method:
1. 96% ethyl alcohol 15 seconds
2. 70% ethyl alcohol 15 seconds
3. 50% ethyl alcohol 15 seconds
4. Distilled water 15 seconds
5. Harris hematoxylin 6 minutes
6. Distilled water 10 dips
7. Hydrochloric acid 0.5% solution, 1-2 quick dips
8. Distilled water 15 seconds
9. Few dips in 0.1% ammoniated water. The smear turns to blue.

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Histology and Cytology 10. 50% ethyl alcohol 15 seconds

11. 70% ethyl alcohol 15 seconds
12. 96% ethyl alcohol 15 seconds
13. OG-6 (orange) 2 minutes
14. 96% ethyl alcohol 10 dips
Notes 15. 96% ethyl alcohol 10 dips
16. EA 50 eosin yellowish 3 minutes
17. 96% ethyl alcohol (10 dips)
18. 100% ethyl alcohol (10 dips)
19. Xylene (10 dips)
20. Mount: in DPX using coverslip

Results:
The nuclei should appear blue/black
The cytoplasm (non-keratinising squamous cells) blue/green
Keratinising cells- pink/orange
Precautions:
1. Use stains only after filtering them
2. Change stains frequently
3. Check staining under microscope for good quality control

25.3 MAY-GRNWALD GIEMSA STAIN

This is one of the common Romanwsky stains used in cytology. It is useful for
studying cell morphology in air-dried smears. It is superior to Papanicolaou to
study the cytoplasm, granules, vacuoles, basement membrane material etc. For
nuclear staining Papanicolaou is superior.
Contents of the staining reagents:
May-Grnwald solution 0.2%
Methanol 99 %
May-Grnwalds eosin-methylene blue 0.2 %
Contains: Eosin G, Methylene blue

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Giemsa solution Histology and Cytology

Methanol 73 %
Glycerol 26 %
Giemsas Azur-Eosin-Methylene blue 0.6 %
Contains: Azur I, Eosin G, Methylene blue
Notes
Phosphate buffer
Potassium dihydrogen phosphate/ disodium hydrogen phosphate x 2H2O
67.0 mmol/l

Storage
Giemsa solution, May-Grnwald solution: protected from light at 2-25C.
Unopened reagents may be used until the expiry date on the label.
Phosphate buffer: at 2-8C. Unopened reagents may be used until the expiry date
on the label.

Preparation of working solutions

1. Buffered water: Dilute phosphate buffer with deionised or distilled water
1:20, e.g. 30 ml phosphate buffer + 570 ml deionised or distilled water.
2. Giemsa working solution : Mix 84 ml of Giemsa solution into 516 ml of
buffered water.
3. May-Grnwald working solution: Mix 360 ml of May-Grnwald solution
into 240 ml of buffered water.

Staining method
1. Fix the air-dried smear specimen in methanol for 10 -20 minutes
2. Stain with May-Grnwald working solution for 5 minutes
3. Stain with Giemsa working solution for 12 minutes
4. Wash with clean buffered water for 2, 5 and 2 minutes
5. Dry the slides in upright position at room temperature
6. Mount the slides with a coverslip using DPX
Any modifications to the staining procedure/working solutions may affect the
staining result, and are subject to precise method validation

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Histology and Cytology Sources of errors

Irregular distribution of the blood smear on a glass slide may result in an
erroneous cell counts. Alcohols used for wiping the skin may cause hemolysis
and artifacts. Do not let the specimens dry at any stage of the staining procedure.
Wash properly to avoid dye artifacts. Buffered water is strongly recommended
for washing. Staining result is dependent on pH. Alkaline pH increases blue and
acidic pH pink or reddish tinge in the stained specimen.
Notes

Ziehl-Neelsen stain
Reagents
(1) Carbol Soft Fuchsin
Basic Fuchsin 1 gm
Absolute alcohol 10 ml
Add the basic fuchsin to the alcohol in a 100 ml flask and mix, on a
magnetic stirrer for 30 minutes. Add 100ml of 5% aqueous phenol. Mix
well. Filter and store in a brown glass bottle.
(2) Acidified Methylene Blue
0.25% methylene blue in 1% acetic alcohol
(3) 0.5% Acid Alcohol
Distiller water 700 ml
Absolute alcohol 300 ml
Hydrochloric acid 5 ml
(6) 5% Sulphuric Acid
Distilled water 475 ml
Sulphuric acid 25 ml

Staining Method
Place fixed slides on the staining rack in serial order, smeared side up. Slides
should be separated by a 1 cm gap, and should never touch one another. Cover
slides individually with filtered Ziehls carbol fuchsin working solution. Heat
slides from underneath with the flame of a Bunsen burner, an alcohol lamp or
an alcohol soaked cotton swab until vapour starts to rise. Staining solution
should never be allowed to boil. Do not allow the stain to dry. Keep slides
covered with hot, steaming carbolfuchsin for 5 minutes by re-flaming as needed.
Rinse slides gently with water to remove excess carbolfuchsin. Drain off excess
rinsing water from slides. Sputum smears appear red in colour.

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Decolourising: Cover slides with 25% sulfuric acid or acid-alcohol solution and Histology and Cytology
allow to stand for 3 minutes, after which the red colour should have almost
completely disappeared. If needed, repeat sequence until the red colour
disappears, but do not overdecolourise. Gently wash away the sulfuric acid or
acid alcohol and the excess stain with water. Drain off excess rinsing water from
slides.
Counterstaining: Cover slides individually with 0.3% methylene blue Notes
counterstaining solution and allow to stand for 1 minute. Rinse slides individually
with water. Drain water off the slides, which are then allowed to air dry.
A properly stained smear should show a light blue colour due to methylene blue.
Results: Tubercle bacilli, hair shafts, Actinomyces, some fungal elements- red.
Background: pale blue.

INTEXT QUESTIONS 25.1

1. ................... stain is recommended for stains of alcohol fixed cytology
2. ................... stain is used for wet fixed slides
3. ................... stain is used for identification of Glycogen, Fungal Wall
4. ................... stain is used for identification of lipids
5. ................... test is used for identification of Bilirubin
6. The universal stain for cytological preparations is the ...................

WHAT HAVE YOU LEARNT

z Consistency and reliability are most important in cytological interpretation.
Cytologists rely heavily on the quality and appearance of the stain.
z Special stains such as Modified Ziehl Neelson for acid fast bacilli, Gram
staining for Bacteria, Mucicarmine for mucins, PAS for glycogen, fungal
wall, lipofuscin, Oil red O for lipids, Perls Prussian blue for iron, modified
Fouchets test for bilirubin
z The universal stain for cytological preparations is the Papanicolaou stain.
Harris hematoxylin is the optimum nuclear stain
z May-Grnwald Giemsa Stain is one of the common Romanwsky stains used
in cytology. It is useful for studying cell morphology in air-dried smears.

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Histology and Cytology z Irregular distribution of the blood smear on a glass slide may result in an
erroneous cell counts. Alcohols used for wiping the skin may cause
hemolysis and artifacts

TERMINAL QUESTIONS
Notes
1. What are the various stains used commonly in cytology.
2. Write the basic principle of Papanicolaou staining.
3. What cell components are better seen in MGG staining?
4. Name the sources of error in Papanicolaou & MGG staining.
5. Enumerate the substances that get stained red with Ziehl Neelsen staining.

ANSWERS TO INTEXT QUESTIONS

25.1
1. Papanicolaou stain
2. Romanowsky
3. PAS
4. Oil red O
5. Modified Fouchets
6. Papanicolaou stain

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