This document discusses various staining techniques used in cytology, including both routine and special stains. It provides details on the principles, procedures, and applications of stains such as May-Grunwald Giemsa, Diff-Quik, Papanicolaou, hematoxylin and eosin, periodic acid Schiff, mucicarmine, Alcian blue, Oil red O, Congo red, Feulgen, and Ziehl-Neelsen. The stains are used to demonstrate cellular and extracellular components, identify infectious organisms, and examine DNA content. Proper staining allows visualization of structures like glycogen, mucin, lipids, amyloid, and acid-fast bacteria under the microscope.

1. STAINS IN
CYTOLOGY
Presenter : Dr Suma H V
Moderator : Dr Siddegowda M S

2. Contents
 Introduction , principle, procedure and
application of
-Routine stains
-Special stains

3. Routine stains in cytology
 May Grunwald- Giemsa stain
 Diff-quik stain
 Papanicoloau stain
 Haematoxylin and Eosin stain

4. Special stains
 Periodic acid schiff stain
 Mucicarmine stain
 Alcian blue stain
 Oil red O stain
 Congo red stain
 Fuelgen stain
 Ziehl Neelsen stain

5. May-Grunwald Giemsa stain
 It is a mix of two neutral stains.
-May-Grunwald stain: composed of an
acidic stain (eosin) and a basic
stain(methylene blue).
-Giemsa stain : is composed of acidic stain
(eosin) and basic stain (azure of methylene)
Principle:

6. Procedure
 Stain the air dried smear with may-grunwald
solution for 5min
 Wash under running water for 1min
 Stain with giemsa solution for 15min
 Wash under running water for 1-2min

10.  Application :
To study cytoplasmic details in air dried
preparations

11. Diff-quik stain
 Quick and easy to perform
 Principle :
The azures are basic dyes that bind acid
nuclei and gives blue to purple color. The
acidic dye, eosin binds with alkaline cytoplasm
giving pink to red color.

12. Procedure
 Air-dry smears
 Fix in methanol for 30sec
 Stain with Diff-Quik solution II for 30sec
 Counterstain with Diff -Quick solution I for
30sec
 Rinse in tap water
 Rapidly dehydrate in absolute alcohol

15.  Application :
Cytoplasmic detail can be well noted.

16. Papanicolaou stain
 Multichromatic cytological staining technique.
 Developed by George Papanicolaou.
Principle:
 Hydration, dehydration and clearing

17. Procedure
 Smears fixed in 95% ethanol for 15min, rinse
 Harris hematoxylin for 1-3min, rinse
 95% ethanol 10 dips
 OG-6 stain for 1.5min
 95% ethanol 10 dips
 Eosin stain for 2.5min
 2 changes of 10dips of 95% ethanol
 100% ethanol for 1 min

19.  Application :
1. Good nuclear detail
2. Identification of keratin- normal squamous
cell V/S dyplastic/ carcinoma

22. Haematoxylin and eosin stain
Principle :
Hematoxylin stains the acidic part of the cell,
ie. Nucleus(nuclear stain).Eosin acts as a
acidic stain and binds with basic part of the
cell, ie. Cytoplasm of the cell

23. Procedure
 Fix the smears in methanol for 10min
 Wash with water
 Immerse in haematoxylin for 5min
 Wash in water
 Immerse the slide in eosin for 30-40sec
 Wash in water
 2 changes of alcohol

25. Special stains in cytology
1. To demonstrate cellular products
- Periodic Acid Schiff(PAS) stain
eg-Glycogen in mesothelioma
-Mucicarmine stains
eg- mucinous adenocarcinoma
-Oil red O stain
eg- lipid in renal cell carcinoma

26. 2. To demonstrate extracellular substance with
special importance.
Eg- Amyloid by Congo red stain
3. To demonstrate infective organisms.
Eg -Acid-fast bacteria by Ziehl Neelsen
stain.
4. To demonstarte DNA content and ploidy.
Eg- Feulgen stain

27. PERIODIC ACID SCHIFF’S
STAIN
 McManus, 1946
 Glycogen
 Glycoprotein, sialomucin and neutral mucin
 Deep magenta colour

28. Principle
 The hydroxyl group attached to carbon atom in
carbohydrate is oxidised to two free aldehyde
groups by periodic acid.
 Aldehyde group reacts with Schiff’s reagent
and produces deep magenta colour.

29. Procedure
 Alcohol fixed smears are treated with graded
lower concentration of alcohol and kept under
running water.
 Periodic acid (1%) for 10min-water wash
 Schiff’s reagent for 30min
 Wash under running tap water -5min
 Counterstain with hematoxylin
 Rinse in graded increasing concentration of
alcohol

33. Applications of PAS stain
 To demonstrate glycogen, mucin in the cells of
adenocarcinoma, fungal capsule,
mucopolysachharide substance, basement
membrane material.

34. MUCICARMINE STAIN
 To demonstrate acid mucins

35. PRINCIPLE
 The active dye molecule in stain is a chelate
complex of aluminium ions and carminic acid
which is positively charged. This positively
charged carmine reacts with the anionic
groups of acid mucins.

36. Procedure
 Hematoxylin for 5-10min
 Water wash
 Mucicarmine solution for 60min
 Water wash
 Metanil yellow solution for 30sec
 Rinse in water
 Rinse in graded increasing concentration of
alcohol

38.  Application:
To demonstrate -Acid mucin in the malignant
cells of adenocarcinoma.
-capsule of cryptococcus

39. Alcian blue
 Basic metachromatic stain
 Stains sulphated and carboxylated acid
mucopolysachharides

40.  Principle:
Alcian blue is a group of water-soluble
polyvalent basic dye. The blue colour is due to
presence of copper which is positively
charged. This binds with negatively charged
sulfated and carboxylated acid
mucopolysachharides and sialomucin.

41. Procedure
 Rehydrate of smear by graded decreasing
concentration of alcohol
 Rinse in water
 Keep the smear in alcian blue solution for
30min
 Rinse in water
 Counterstain with hematoxylin for 5min
 95% ethyl alcohol, absolute alcohol

43.  Application:
To stain acid mucin in adinocarcinoma cells .

44. Oil red O
 Stains lipid material
 Principle :
It is a liposoluble stain and an azo compound
use to visualize neutral triglycerides and lipids

45.  Procedure:
-Keep the slide in Oil red-O solution for 20-
30min
-Rinse in running water
-Counterstain with hematoxylin for 30sec
-Rinse in water
-Mount in glycerin

46.  Application:
To demonstrate lipid, lipoblasts.

49. Feulgen stain
 Specific for DNA and it demonstrates
deoxyribose

50.  Principle:
With the help of hydrochloric acid, the purine-
deoxyribose bond is broken exposing the
aldehyde group. This aldehyde group reacts
with Schiff’s reagent.

51. Procedure
 Rinse in water
 1M HCL(preheated at 600C) for 10min
 Keep in schiff’s reagent for 10min
 Immerse in 0.05M metabisulfite for 2min three
times
 Counterstain by 0.01% fast green
 Deydrate in absolute alcohol

52.  Application:
Helpful for DNA ploidy examination

55. Congo Red Stain
 To demonstrate amyloid

56.  Principle:
Congo red is an acidic diazo dye. The dye
molecules bind to amyloid by hydrogen bond
and have an oriented arrangement on the
amyloid fibrils. This binding of dye-amyloid
complex causes absorption of certain
wavelengths of light and produces greenish
birefringence under polarized light.

57. Procedure
 Slides are rinsed in water
 Hematoxylin stain for 5min
 Alkaline sodium chloride solution for 20min
 Alkaline congo red for 20min
 Rinse in alcohol
 Dried & mounted

58.  Application:
Amyloid in various tissue and organs in
amyloidosis and in tumors like medullary
carcinoma of thyroid

61. Ziehl-Neelsen staining
 Mycobacterial group of organisms are all
stained by Ziehl-Neelsen staining

62. Principle
 Mycobacterial cell wall contains mycolic acid.
Phenolic acid and high temperatures increase
the porosity of the membrane and helps in the
penetration of the dye. Mycolic acid resists the
removal of the stain by acid and alcohol.

63. Procedure
 Smears are air dried
 Hot carbol fuchsin for 6min
 2-3dips in acid alcohol for differentiation
 Counterstain with methylene blue for 1min
 Clean with water and dry

65. References
 Dey P. Diagnostic cytology. New Delhi:
Jaypee;2014.
 Bibbu M, Wilbus D C. Comprehensive
cytopathology. 3rd edition. Saunders. Elsevier.
 Orell S R, Sterrett G F. Fine Needle Aspiration
Cytology. 5th edition.India: Elsevier;2012.
 Kocjan G. Fine Needle Aspiration Cytology;
Diagnostic principles and dilemmas. Germany.
Springer; 2006.

66. THANK YOU