UNIVERSITI KUALA LUMPUR

INSTITUTE OF MEDICAL SCIENCE TECHNOLOGY

(MESTECH)

LAB REPORT: Exfoliative Cytology:

Buccal Smear Preparation, Staining &
Examination

COURSE CODE
HDB 30303

COURSE NAME
CLINICAL LABORATORY CYTOPATHOLOGY

SEMESTER
SEPTEMBER 2022

GROUP NUMBER
L01-B01

LECTURERS NAME
MADAM AZLINA MUHSIN

NAME & STUDENT ID

IZZAH HAZWANI BINTI MOKTAR (12213120097)
FARAH WAHIDA BINTI AZMAN (12213120084)
Introduction

Exfoliative cytology is the examination of specimens that contain cells exfoliated from body cavities and
surface such as the cervix, abdominal fluid, pleural fluid, oral mucosa, etc. It is useful for the detection
of precancerous or cancerous lesion in the body. For staining of cytological preparations, Papanicolaou
staining is used as a universal staining. It is a polychrome staining which enables to differentiate cells
according to their maturity and metabolic activity. When performed properly, the stained specimen
would display hues from the entire spectrum of red, orange, yellow, green, blue, and violet. The staining
results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.

Objectives

1. To prepare buccal smear

2. To perform Papanicolaou staining on prepared buccal smear

3. To examine the morphology of cells in the prepared smear

Methodology

Equipment and Materials

1. Wooden applicator

2. Glass slide

3. 95 % alcohol (fixative)

4. DPX (mounting medium) and applicator

5. Coverslip

6. Gloves

7. Staining rack

8. Pap stain:

a. 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol

b. Harris’ hematoxylin

c. 1% acid alcohol

d. Scotts tap water (bluing solution)

e. Tap water

f. Orange G-6

g. Eosin azure-50

h. Xylene
Procedures

(a) Buccal swab collection and smear preparation

1. A glass slide is labeled with the following: Name; Date of Collection; Type of Specimen. Using a
pencil.

2. Prior to sample collection, rinsed mouth with clean water.

3. At one side of the mouth, a wooden applicator is placed.

4. The applicator is pressed firmly and twirled against the inside of the inner cheek using an up and
down motion

5. The wooden applicator is removed from the mouth while avoiding saturate the wooden applicator
with excess saliva.

6. The collected sample is smeared onto the labeled glass slide by spreading over a large area,
preventing clumped cells.

7. Using 95% ethanol, the smear is immediately fix for 30 minutes.

(b) Papanicolaou (Pap) staining procedure

1. The prepared buccal smear is stained according to the following Pap staining protocol:
(c) Mounting and coverslipping

1. The DPX is dropped 1 or 2 drops onto the slide.

2. One side of the coverslip is placed against the slide making contact with outer edge of the mounting
medium at an angle approximately 45°.

3. The coverslip is lowered slowly, avoiding air bubbles over the slide.

4. Covering the whole area beneath the coverslip, allowed the mounting medium to spread.

5. The slide is air dried.

Safety and precaution

Avoid drying of smear before fixation as dryness leads to alterations in the cellular morphology
Results

Result Type of smear Description

Magnification: 10x
objective lens

Superficial cells:
Superficial Cells
• Have polygonal cell
shape (35 – 45 m
diameter)
• Small pyknotic
nucleus
• Low NCR
• Eosinophilic
cytoplasm

Intermediate cells:

• Have polygonal cell

shape (30 – 40 m
diameter)
• Nuclear size and
shape are round or
Buccal smear: oval
• Low NCR
• Superficial cells • Fine vesicular
appear in pink chromatin
colour •
Intermediate Cells The cytoplasm is
• Intermediate usually cyanophilic
cells appear in sometimes
blue-green eosinophilic
colour
Magnification: 40x
Superficial Cells objective lens

Superficial cells:

• Have polygonal cell

shape (35 – 45 m
diameter)
• Small pyknotic
nucleus
• Low NCR
• Eosinophilic
cytoplasm

Intermediate cells:

• Have polygonal cell

shape (30 – 40 m
diameter)
Intermediate Cells
• Nuclear size and
shape are round or
oval
• Low NCR
• Fine vesicular
chromatin
• The cytoplasm is
usually cyanophilic
sometimes
eosinophilic
Discussion

Based on the result, the buccal smear was examined under the microscope using a magnification of
10x and 40x objective lens. The type of cells that can be seen are superficial cells and intermediate
cells. Superficial cells appeared in pink colour while intermediate cells appeared in blue-green colour.
From the table above, superficial cells show polygonal cell shape in 35 – 45 m diameter. Besides that,
superficial cells also consist of a small pyknotic nucleus, low NCR, and eosinophilic cytoplasm.
Intermediate cells show polygonal cell shape in 30 – 40 m diameter. Moreover, nuclear size and shape
are round or oval, low NCR, fine vesicular chromatin, and the cytoplasm are usually cyanophilic
sometimes eosinophilic in intermediate cells.

The purpose of cytological fixatives is to maintain the cell's cytomorphologic characteristics and
diagnostically essential elements. The fixative especially recommended for cytological preparations is
ethanol. Fixation sharpens the characteristics of the nuclear chromatin pattern and coarsens the cell
structures. Wet fixatives and dry fixatives are two frequently used for cytological fixatives. The fixative
for routine cervicovaginal and fine-needle aspiration (FNA) cytology smears at the G.M.C., Nagpur,
cytology laboratory is 95% ethanol. The slides should be rapidly fixed in the fixative solution because
even a small amount of air drying might change the cytomorphology of the smear and affect diagnostic
accuracy. For proper fixing, the smear should be left on for at least 20 to 30 minutes. However, a
prolonged fixation that lasts for several days or even weeks will not change the characteristics of the
cells. In order to reduce evaporation, the smear and a fixative solution may be refrigerated together
(Sathawane et al., 2022).

The most commonly used nuclear stain is hematoxylin. The natural oxidation or ripening of
hematoxylin in an aqueous solution occurs gradually over time by keeping the stain bottle in the sunlight
area for at least a month. Hematoxylin stains the cell's DNA and RNA. A cytoplasmic counterstain is
orange G-6. It reduces precipitation as well as filtering and staining time. Eosin Azure-50 can also be
used as a cytoplasmic counterstain. Eosin is a pure acidic dye that primarily binds to protein and
enhanced green stain components. The most commonly used bluing agents are Scott's tap water
substitute (STWS) which dilutes an aqueous solution of ammonium hydroxide and lithium carbonate. If
the pH of the water is higher than 8, tap water can be used as a bluing agent. Moreover, xylene is used
as coatings and acts as filtration to remove contaminant cells (Sathawane et al., 2022).

Therefore, the advantages of this Papanicolaou stain preparation are simple, quick, and cost-
effective. It is common knowledge that most cytological samples may be obtained easily. Almost all
institutions and healthcare professionals are now aware of the technology and normal investigative and
diagnostic patient work now includes using various sampling procedures, which are becoming more
and more common. A description of the many types of samples will follow. The procedure takes only a
few minutes, and diagnostic results can be given right away if required, or within the next 24-48 hours.
The cost-effectiveness of cytological examination has been well demonstrated in the literature, which
is becoming increasingly important given the current high healthcare expenses. When compared to
surgical biopsies, the cost savings are significant (Al-Abbadi, 2011).
Conclusion

In a conclusion, this buccal smear preparation cytology is simple, quick, and cost-effective. The type of
cells that can be seen in this buccal smear cytology are superficial cells which appeared in pink colour
and intermediate cells which appeared in blue-green colour. Thus, this buccal smear slide was observed
using light microscope in magnification of 10x and 40x objective lens. Therefore, the objectives of the
experiment have been achieved.
References

Al-Abbadi, M. A. (2011). Basics of cytology. Avicenna Journal of Medicine, 1(1), 18.

https://doi.org/10.4103/2231-0770.83719

Sathawane, P., Kamal, M. M., Deotale, P. R., & Mankar, H. (2022). Nuances of the Papanicolaou stain.
CytoJournal, 19. https://doi.org/10.25259/CMAS_03_18_2021