ROUTINE STAINING IN CYTOLOGY LABORATORY
PREPARED BY MR. ABHIJIT DAS
PAP STAINING
Pap Staining
(Papanicolaou Stain) is a multi-step staining technique used in cytology to highlight cellular details.
It uses a combination of dyes to differentiate cell components, aiding in the
detection of abnormalities like cancer cells.
Dyes Used
1.
Hematoxylin:
Stains the nuclei a blue-purple colour.
2.
Orange G:
Stains keratinized cells, used to identify mature squamous cells (orange).
3.
EA (Eosin Azure):
A mixture of Eosin Y and Light Green, stains cytoplasm in different shades
depending on the cell type (pink to green).
Principle
The Pap stain relies on differential staining, where different dyes selectively stain
cellular components. It allows clear visualization of cell nuclei,
cytoplasm, and other structures, helping to identify cellular abnormalities.
Steps
1.
Fixation:
Cells are fixed on a slide, often with 95% ethanol.
2.
Nuclear Staining:
Hematoxylin is
applied to stain the nuclei.
3.
Rinsing and Bluing:
Rinse and apply a bluing
solution to enhance the hematoxylin stain, making nuclei appear blue.
4.
Counterstaining:
Orange G and EA are
applied to stain cytoplasm
and other cell parts.
5.
Dehydration and Mounting:
Slide is dehydrated
through alcohols, cleared in xylene, and mounted with
a cover slip.
Bluing Solution
A bluing solution (mild alkaline solution, like
ammonium water) is used after hematoxylin to change the colour of the stain
from red-purple to blue,
enhancing nuclear detail.
Precautions
- Ensure
Adequate Fixation: Prevents cell shrinkage and
artifacts.
- Avoid
Overstaining: Excess stain can obscure cellular
details.
- Handle
Bluing Solution Carefully: Too much can reduce
contrast in nuclear staining.
- Proper
Dehydration: Incomplete drying can lead to
blurred or uneven staining.
Pap staining provides high contrast, making it easier
to detect abnormalities, essential for screening cytological specimens.
MAY GRUNWALD GIEMSA STAINING
May-Grunwald Giemsa (MGG) Stain
is a differential stain widely used in cytology and haematology for examining
blood smears, bone marrow samples, and cytological preparations. It highlights different cellular
components, making it easier to identify cell types and detect
abnormalities.
Principle
The MGG stain combines May-Grunwald and Giemsa
stains, both of which contain acidic and basic dyes. These dyes stain different cell
components based on their pH:
- Acidic
structures (like DNA in the nucleus) are
stained by basic dyes
(blue).
- Basic
structures (like certain cytoplasmic proteins)
are stained by acidic
dyes (pink to
red).
Procedure
1.
Fixation:
Slide is air-dried, and cells are fixed with methanol.
2.
May-Grünwald Staining:
o Apply
May-Grünwald stain.
o Leave
for a few minutes, then rinse gently with buffer.
3.
Giemsa Staining:
o Apply
Giemsa stain.
o Leave
for 10–15 minutes, then rinse with buffer.
4.
Drying and Mounting:
Allow the slide to air dry, then examine under the microscope.
Results
- Nuclei:
Blue to purple.
- Cytoplasm:
Pink, blue, or gray depending on the cell type.
Uses
MGG stain is effective for identifying blood cells,
bone marrow cells, and certain bacteria and parasites in clinical sample.
DIFF-QUICK STAINING
Diff-Quick is a rapid, two-step staining method commonly used in cytology
and histology. It is particularly useful for staining smears from fine needle
aspirations, bone marrow etc.
Principle:
Diff-Quick staining uses a combination of basic and acidic dyes to stain cellular
components:
- Basic
dyes (e.g., methylene blue) stain acidic structures
like the nucleus.
- Acidic
dyes (e.g., eosin) stain basic structures like the
cytoplasm.
Procedure:
1.
Prepare the Slide:
Collect the sample and air-dry the smear.
2.
Fixation:
Dip the slide in the fixative (usually methanol) for 10-15 seconds to preserve the cells.
3.
Staining:
o Eosin
(Acidic Dye): Dip in eosin for 30 seconds to stain the
cytoplasm pink.
o Methylene
Blue (Basic Dye): Dip in methylene blue for 30 seconds to
stain the nucleus
blue.
4.
Rinse and Dry:
Rinse with distilled water and allow the slide to air dry.
Results:
- Nucleus:
Blue to purple.
- Cytoplasm:
Pink to red.
Diff-Quick provides a quick and effective way to differentiate cellular components.