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Parasitology Overview

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52 views3 pages

Parasitology Overview

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kariukifrancis.com
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FRANCIS KARIUKI

BSLT/2020/91071

PARASITOLOGY PRACTICALS REPORT

Practical 1: LEISHMAN'S STAINING

Introduction:

Leishman stain is a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted
with buffer or distilled water during staining procedure. Leishman stain, also known as Leishman's stain,
is used in microscopy for staining blood smears. It is generally used to differentiate between and identify
white blood cells, malaria parasites, and trypanosomas.Leishman stain is a differential stain that is used
to variably stain the various components of the cells and it can be used to study the adherence of
pathogenic bacteria to the human cells.It stains the different components of blood in a range of shades
between red and blue. It is based on a methanolic mixture of "polychromed" methylene blue (i.e.
demethylated into various azures) and eosin. The methanolic stock solution is stable and also serves the
purpose of directly fixing the smear eliminating a prefixing step. If a working solution is made by dilution
with an aqueous buffer, the resulting mixture is very unstable and cannot be used for long. Leishman
stain is named after its inventor, the Scottish pathologist William Boog Leishman. Leishman’s staining
method for thin and thick smears is a good alternative to Giemsa’s stain for identifying Plasmodium
parasites. The Leishman method is superior for visualization of red and white blood cell morphology.

Materials:

Leishman Stain (Stock Solution)

Microscopic Glass Slide

Phosphate buffer (pH 6.8)

Graduated pipettes

Measuring cylinder

Distilled Water

Pasteur pipette

Coplin Jar

Blood Specimen

Procedure:

Prepare a thin blood smear on a clean and dry microscopic glass slide and air dry it
Now, cover the well dried, thin blood smear with undiluted Leishman Stain solution by counting the
drops of Leishman stain.

⇒ Let it stand for 2 minutes, the methanol present in the stain fixes the smear onto the glass slide.

⇒ After 2 minutes, add twice the amount of distilled water or Phosphate buffer solution and mix the
content by swirling or by blowing gently. Incubate the slides for at least 10 min at 37 °C. This will stain
the blood cells.

⇒ Rinse the slides thoroughly with Phosphate buffer solution up to 2 minutes or until it acquires a
purple-pinkish tinge.

⇒ Air dry the slides in a tilted position so that the water easily remove out of the slides.

⇒ Now you can mount the smears with mounting media, e.g. Gurri’s neutral mounting media or any
other mounting medium which do not decolorizes the smear. Do not use Canada balsam as it may
decolorize the smear.

⇒ Let it dry in air for few hours and then observe the slides under oil immersion objective lens of the
microscope.

Results:

Erythrocytes stain yellowish red.

Lymphocytes nuclei stain deep purple, cytoplasm of lymphocytes stain light blue.

Eosinophil nuclei blue, red to orange granules, blue cytoplasm.

Basophil stain purple to dark blue nuclei, dark purple to black granules.
Polymorphs stain dark purple nuclei, reddish violet granules, pale pink cytoplasm.

Platelets stain violet granules.

Malarial parasites stain red, cytoplasm blue. Trypanosomes stain chromatin red.

Discussion

Leishman's stain is for the general differentiation of blood cells for malaria and trypanosomes in
prepared slides from clinical specimens. Leishman's stain was discovered in 1901 and is used for staining
blood smears. It is generally used to differentiate and identify leucocytes, malarial parasites and
trypanosomes.It is recommended for thin smears for identification of species.Leishman stain provides
clear visualization of the nuclear chromatin pattern of cells and is used for staining blood.In that case the
buffer solution is essential for preparation of diluted Leishman solutions and for rinsing stained samples
without causing destaining of stained cells. Buffer solutions are solutions of weak acids and theirs salts
or weak bases and their salts.

Conclusions:

Leishman's staining method for thin and thick smears is a good alternative to Giemsa's stain for
identifying Plasmodium parasites. The Leishman method is superior for visualization of red and white
blood cell morphology.

Practical 2

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