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INDEX
S.n
o
1
2

Chapter
CYTOLOGY STAINING
Introduction
Clinical Significance

Page No.
3
4

Sampling Techniques in cytology1. Exfoliation of cells noabrasive

methods
2. Collections of cells removed by
brushing or similar abrasive
technique
3. Washing or lavage
4. Fine needle aspiration cytology
(FNAC)

4
5

Preparation of smear
Fixation

6
6

Types of stains
1. Paps stain
2. Romanowsky stain
3. Cresyl violet stain
4. Repid stain
5. Special stain
6. Hematoxylin & Eosin stain
7. Shorrs stain

7
8
9
10

Maintenance of the stains

Consolation
Reference
Annexure

7 to 15

16
17
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1. INTERODUCTION:- Cytology is a branch of diagnostic

medium it deals with study of free cells from shade from
body. Itself or aspirated by tube of needles, therefore
effusion secretion, aspirates & scraping all are use in
diagnostic cytology.
It can be referred to as cell biology or cytopathology
according to the specific use of cytology. Cytopathology is a
branch of pathology that steadies and diagnoses diseases on
the cellular level.

2. CLINICAL SIGNIFICANCE:- Cytopathology is usually

used to aid in the diagnosis of cancer. However it also
help in the diagnosis of certain infection discuses and
other inflammatory conditions. It is also commonly used to
investigate thyroid lesion, disease involving slerile body
cavities.
Which studies whole tissues a common application of
Cytopathology is the pap smear used as a screening tool, to
detect precancerous cervical lesion and prevent cervical
cancer.

3. SAMPLING TECHNIQUES IN CYTOLOGY: Exfoliation of cells nonabrasive Methods:- This is

spontaneous shedding of cells derived from the linling of
an organ into the body cavity. For example vaginal
smears, sputum, urine and other body fluids.
Collection of cells removed by brushing or similar
abrasive technique:- The method allows direct sampling
of the targets such. As surface of the uterine cervix or
bronchus. The sampling of organs of the respiratory &
gastrointestinal tract is possible
Washing or lavage:- Normal saline or similar solution are
installed in the target organ under visual control.
This solution are aspirated & collected in small
container
Fine needle aspiration cytology (FNAC):- Any palpable
or non palpable lesion in the body can be sample suing
either palpitation or imaging techniques . the technique
can be adopted as an outpatient procedure. FNAC is raip
& cast effective methodology.

4. PREPARATION OF SMEAR:- Smear are made

immediately form swab by:- Smear should by thin evenly spread without clump.
- Other smears are prepared by smearing or streaking.

5. Fixation: Smear should be immediately fixed when it is

still moist.
Label the no. on slide it will take about 1 hr of
fix.

6. TYPES OF STAINS:a. Paps stain (papinoculo stain)

b. Romano sky stain
c. Cresyl violet stain
d. Rapid stains
e. Hematoxylin & Eosin Stains
f. Shorrs staining
Paps stain (papinoculo stain):- The most commonly
used stain in the qynac cytology is the papinoclo stain the
important of these stain is suitable for cervical smear.
Method:- This staining Method can be perform either
manually or automatic.
Solution:a. Herrys Hematoxline.
b. Orange G-6
c. Eosin Anure-50
Procedure :a. Remove smear from fixative.
b. Rinse in descending grade of alcohol 80%, 70% & 50%
and water for 10sec. it.
c. Stain in Herrys Hematoxline for 2 minutes.
d. Rinse in water for 2 minutes.
e. Differentiate in 1% acid alcohol until only the nuclear retain
the stain.
f. Wash & Blue in tab water for 3-5 minutes.
g. Transfer to 70% alcohol for few seconds.
h. Transfer to 95% alcohol for a few seconds.
i. Stain in organ G-6 for 2minuts.
j. Rinse in 2 changes 95% alcohol.
k. Stain in eosin Azure 50 for 2-4 minutes.

l. Rinse in 2 changes 95% Alcohol.

m. Dehydrate in Absolute Alcohol clear in xylene and Mount
in natural synthetic modicum.

Result:

Nuclei
Acidophilic
Basophilic
Canada
Tricomonus Veginitis

Blue
Superficial cell
Blue
Red pale pink
Grayish Green

Romanowsky Stain :- Following Fixation in either alcohol

smears may be any of the Romanowsky stain.
The most popular Romanowsky stains are the Giemsa
and the Leishman stain.
Methylene Blue:- This signal stain rapid & simple
method is useful for the screening of fresh specimens
especially sputum for malignant cells.

Solution:Methylene
1 gm
Distilled water
100ml
PROCEDURE: Place a small amount of fresh purulent sputum or two
drops of centrifuged deposit of the body fluid on a
clean microscope slide.
Add one drop of the stain to the specimen on the
slide and mix the two together thoroughly.
Cover the mixture with a clean cover slip and spread
by gentle pressure.
Examine immediately
Result :-

Nuclei : Shades of blue

CRESYL VIOLETSIN: Importance:- Many laboratories use this method since it

is simple fast and gives clear morphologic cellular details.
Reagents: 0.1 gl (v/v) cresyl violet
1% acetic acid
Procedure: Prepare the smear and fix in ether alcohol fixative
Hydrate the smear in the following
o 70 % (v/v)
o 50 % (v/v) ethanol
o Distilled water
Dip in 1%(v/v) acetic acid for 30 sec.
Rinse in distilled water (2 changes)
Stain in freshly filered cresyl violet solution for 3 min.
Drain and dehydrates by rinsing in acetone (briefly
for 10 and 5 seconds respectively)
Clear in xylene for 5 minutes each
Mount in Canada balsam (DPX)

Additional information:- Other staring procedures

such as feulgen reaction Method, Silver Stein method
and periodic acid-schiff (PAS) method are also followed
in exfoliative cytological investigations.
Hematoxylin & Eosin Stain: Reagents:-

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Harris hematoxlin
1.0% (v/v) hydrochloric acid in 70% ethonal (acid
alcohol reagat)
Eosin (y) : 1% (w/v) aqueous.
Procedure: Treat with 95% alcohol for 2 min.
Wash in running tap water for 2-3 min.
Stain in Harris hematoxylin for 5-10 min.
Wash in running tap water for 5 min.
Differentiate in acid alcohol solution for 5-10 min.
Wash in running tap water for 5-10 min.
Treat with eosin solution using 2 dips
Blat and dehydrate in absolute alcohol
Clear in xylene and fix cover slip by using a drop of
DPX

Result :Nuclei

Blue

Cytoplasm

Pink

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Rapid Stains:- These stains are used mainly to check

adequacy of fine needle aspirates, Rapid stein can also be
used for immediate evolution to control cross contamination.
The dyes used in rapid steaming procedures are Methylen
blue. Thionin blue & Toluieline blue.
Touidine blue stain is prepared as follows:Toluidine blue

0.5 g

95% ethyl alcohol

20 ml

Distilled water

80 ml

Dissolve dye in alcohol & ass water filter & store in dark
bottle.
Toluidine blue staining procedure:- It is performed as
follows.
Fix the smear for 10 to 15 seconds in 95%
alcohols.
Treat with toluidine blue use 10 dips
Rinse in water to remove excess of stain.
Put a cover slip on wet smear & submit for
microscopic examination

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Special Stain:- These are widely use in histopathology lab can

be applied to cytological preparation.
In cytological examination which is use for the
investigation called HIV created inves & bronchial alvelar
lavage for pneumocytis is carinei.
This is used to quite easu to make a diagnosis of
MGG papinoculo stained smear.
Solution:1. Methylamine:- Silver nitrate (Stock solution and their
working solution)
- 5% of chromium trioxyle
- 1% of sodium bisuffite
- 0.2% of Gold chloride
- 12% of sodium thyosulfate
2. Procedure:a. Wash slide in D.W (10-20 dips)
b. Transfer to cooplin for with 5% chromium trioxide
place 1ur in a warn water 43-580 c 20 min
c. Rinse in tap water 10-20 dips
d. Transfer to sodium bisulphate 30sec
e. Running tap sodium bisulphate 30 sec
f. Running tap water 15-20 sec
g. Several rinse of D.W
h. Ethylamine silver main water
i. Rinse in D.W 3 times 10 to 15 days each
j. 0.2% Gold chloride 20-30 dip
k. Rinse in D.W 3 times 10-15 dip each
l. 2% Sodium thyorulfate for 1 min
m.Running tap water 15-20 sec
n. Fast grain 30-35 sec
o. Rinse in D.W lwice 15-20 dips
p. 95% ethyle alcohol twice 15-20 dip
q. Absolute alcohol twice 15-0-20 dips

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r. Xylene (Clearing)
s. Mount
Result:- Pnwmocytisis corinic & fungal element - Black
Musine & glycogen - pale gray
Back ground

- pale green

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Shorrs Staining:- This method uses a single differential

staining solution. It gives less cytoplasmic transparency and
poorer nuclear definition than the papanicolou.
It is not very useful in the diagnosis of malignant
cells.

Staining Solution:50 % ethyl alcohol

100 ml

Biebrich Scarlet (ClNo. 26905)

Water Soluble

0.5 g

Orange G(Cl No. 16230)

0.25g

Fastgreen FCF (Cl no. 42053)

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7. Maintainece of the stains:- Following precautions are

taken while using the steins:a. Stain used must be certified by Biological stein
commission.
b. Dye content on the certified label of dye must be
considered in stein preparation
c. Contamination control Gynec & Non-gynec slides are
stained separately
d. Fitter stains & reagents daily
e. Non-Gynec Specimens have high potential for cross
contamination during staining procedures.
f. Solutions and stains are changed from time to time
depending on the volume of work
g. Staining dishes should be kept covered
h. Alcohol used during rehydrating and dehydrating
process are changed on a rotation basis and if needed
also filtered prior to staining procedure
i. Daily microscopic check are documented for the quality
control of stains.

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8.

CONCLUSION:-

The focused ultrasound therapy is safe

and effective Method for treatment of chronic
cervicitis and is working to be popularized.

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9.
1.

REFRENCE:-

Text book of Medical laboratory Technology

3rd, Praful B.Godkar, Darshan P.Godkar,

2.

Bhalni publishing House, Mumbai, 2014

Medical Laboratory Science, J. Ochei,
A.Kolhatkar, Tata Mc Graw Education private
Limited New Delhi-2010