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Histopathology Staining and Cytopathology Notes

STAINING METHODS






Indirect method:
- Action of the dye is intensified by either a mordant or
accentuator
- Mordant: bridge or link between the tissue & dye
- Mordant + dye = lake
- Lake + tissue = tissue-mordant dye complex
- Example: potassium alum with hematoxylin in Erlich's
hematoxylin
- Accentuator: not essential to the chemical union of
the tissue and dye
-Does not participate in the staining reaction
- Speeds up reaction (increasing staining power)

3
...
Chloride) – used for
demonstration of Glycogen
Orcein – From Lichens,
Recommended for
dermatological studies
Saffron – From plant Crucos
sativus
...

Routine H and E Staining (Regressive type staining)
Results:
Nuclei: Blue to blue
black
Karyosome: Dark blue
Cytoplasm, proteins in
edema fluid: Pale pink

Direct Staining:
- Giving color to the sections by using aqueous or
alcoholic dye solutions
- Example: methylene blue, eosin, etc
Progressive staining:
- Follows a definite sequence
- Applied in a specific period (timed) or until the
desired intensity of coloring of different tissue
elements
- No decolorization/differentiation or washing

Natural Dyes
Hematoxylin - extracted from
heartwood of the Mexican
tree (Hematoxylin
Campechianum)

Synthetic Dyes
- A
...
A “Coal tar dyes”
- Derived from benzene
and collectively known as
aniline dye

1
...
Quinoid ring
2
...
Xanthene
4
...
) Iron Hematoxylin – used as
oxidizing agents and mordant
3
...
) Copper Hematoxylin –
Used for study of
Spermatogenesis
Cochineal Dyes – From female
Coccuc cacti
Carmine – Chromatin stain
Derivatives:
1
...
Hydroxide) –
demonstration of mucins and
C
...
Picrocarmine (Carmine +
Picric acid) – For
neuropathological studies

Auxochrome – Responsible
for the dyeing property of
the dye, imparts color
almost permanently
1
...
Anionic Auxochrome:
Hydroxyl and Carboxyl
Groups

Calcium and calcified
bone: Purplish blue
Muscle fibers: deep
pink

STAINS
Alum Hematoxylin
Ehrlich's solution
(ESI2)




Delafield's
hematoxylin




Natural ripening (2 months):
Chemical ripening with
sodium iodate
Regressive staining à for
acidic tissues
Natural ripening: 4- 6
months
Similar longevity with
Erhlich’s hematoxylin

Harris solution





mercuric chloride
For routine nuclear staining
Exfoliative cytology and
stain for sex chromosomes

Mayer's
hematoxylin





sodium iodine
Short staining time
Short storage

Cole's Hematoxylin




alcoholic iodine
Used after Celestine blue

Carazzi's
hematoxylin




potassium iodate
Short staining time
(progressive staining)
Does not stain cytoplasmic
organelles



Iron Hematoxylin
Weigert's Solution
(Weiferidic)




Ferric chloride
Used when acidic
solutions are to be
applied (nucleus resist
decolorization w/ acids)

Heidendain's Solution
(CHeFAS)



Ferric ammonium
sulfate
Regressive staining of
thin sections, also stains
cytoplasmic inclusions

OSMIC ACID STAIN
FOR FATS

NILE BLUE SULFATE
METHOD




Ferric ammonium
sulfate
Demonstrate myelin
Can be applied to
paraffin, frozen, or
nitrocellulose sections




Ferric chloride
Photomicrography



Loyez Hematoxylin
(Loyelin)

Verhoeff's solution
(VP)



Fixation: Fresh frozen or frozen
sections post-fixed in neutral
buffered formalin
Sections: 5 um on superfrost/ plus
slides
Results:
Fat – Brilliant red
Nuclei – Blue
Fixation: 10% formalin
Sections: Cryostat sections
Results:
Fat – Black
Nuclei – Yellow orange
Fixation: 10% formalin
Sections: Frozen sections
Results:
Neutral Fat – pinkish red
Cholesterol esters & fatty acids: Light
red
Cerebrosides – light blue
Fatty acids & soap – Deep blue to
violet

Staining for Lipids:
Sudanophilia – Property of tissue to be stained with fat or oil
soluble dyes
Groups:




Other stains:
Benzidine



Eosin




Basic acryl amine with very low water solubility
- Sudan black B (most sensitive)
- Sudan red VII B
B-napthols such as the original diazo dyes
- Sudan III
- Sudan IV

SUDAN BLACK

- Most sensitive
- Black color on intracellular lipids
and recommended for paraffin
sections
Fixation: Formaldehyde calcium with
post-chroming
Sections: Unfixed cryostat sections
or frozen sections post-fixed in
formol calcium
Results:
Lipids – Blue black
Nuclei – Red

SUDAN IV

Crystal Violet





Gentian Violet



Congo red



Acridine orange



- Recommended for staining
triglycerides
Fixation: 10% formalin
Sections: Frozen sections
Results:
Lipids – red (TAG)
Nuclei – Blue black

OIL RED O



- Used as a stain for neutral lipid
- Rapid and simple stain
- Useful in identifying emboli in lung
tissues or clot sections of peripheral
blood

Iodine





Malachite Green




Used for staining
hemoglobin
A red acid dye
Routinely used in
histopathology as a
counterstain after
hematoxylin and before
methylene blue
Three forms:
- Eosin Y (Yellowish) – Most
commonly used
- Eosin B (Bluish) – Deeper
red color
- Ethyl eosin S (Ethyl eosin)
DNA (Green fluorescence)
RNA (Red fluorescence)
For staining amyloid in
frozen sections and platelets
in blood
Formed by the mixture of
crystal violet + methyl violet
+ dextrin
Stains axis cylinders in
embryos
Used as a 4% aqueous
solution in Krajian’s method
of staining elastic tissues,
amyloid and myelin
The oldest stain
Staines amyloid, cellulose,
starch, carotenes, and
glycogen
Widely used for removal of
mercuric fixatives pigments
Contrast stain for staining
Ascaris eggs and
erythrocytes
Used also as bacterial spore
stain

Janus Green B



Night blue



Victoria blue



Lysochromes (Oil
soluble dyes)




Periodic Acid-Schiff





Used to demonstrate
mitochondria during
intravital staining
Used as substitute for carbol
fuschin in acid-fast staining
Used for demonstration of
neuroglia in frozen sections
...
George N
...


Fixatives:
 50% Alcohol: pleural and peritoneal fluids (can also be
replaced by saccomano preservative (50% alcohol +
carbowax)
 Equal parts of 95% ethyl alcohol & ether: BEST
FIXATIVE
 95% Ethyl Alcohol: ROUTINE PREP (urine, bronchial
brushing or gastric aspirates)
 Note: Spray fixatives  slide should be kept at a
distance of 1 foot from the spray

Gynecologic Specimens (PAP SMEARS)


Cytohormonal Maturation Index (CHMI)
 A quantitative evaluation done under vaginal
hormonal cytology
 General steps:
- Count 100 cevico-vaginal cells
- Classify as: Parabasal, Intermediate, Mature
superficial cells
- Report findings P/I/S
- Observe following:
> Shift to the left: Increased no
...

> Alveolar macrophages: Sputum(deep cough)
> Absence of alveolar macrophages: Saliva
- Bronchial washing/ Bronchoalveolar Lavage (BAL
> Performed in patients with AIDS to rule out
Pneumocystis carinii (P

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