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This document provides instructions for preparing a blood smear slide and examining it under a microscope. It describes how to collect a blood sample, make a smear on a slide by spreading the blood in a thin layer, and stain it using Leishman's stain. A good blood smear is uniform in thickness and contains evenly distributed blood cells. The stained slide is then examined under the microscope using low and high powers to identify and characterize red blood cells and white blood cells. The staining quality and cell morphology are evaluated according to standard criteria.

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102 views

Files PDF

This document provides instructions for preparing a blood smear slide and examining it under a microscope. It describes how to collect a blood sample, make a smear on a slide by spreading the blood in a thin layer, and stain it using Leishman's stain. A good blood smear is uniform in thickness and contains evenly distributed blood cells. The stained slide is then examined under the microscope using low and high powers to identify and characterize red blood cells and white blood cells. The staining quality and cell morphology are evaluated according to standard criteria.

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Абу Джуд
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‫المرحـــلــــة ‪ :‬الثـــانـــيـــة‬ ‫الجـــامـــــعـــة االســـــالمــــيــــــــة‬

‫المادة‪ :‬الفسلجة البشرية العملي‬ ‫قسم تقنيات التحليالت المرضية‬


‫للعام الدراسي ‪2018 - 2017 :‬‬
‫التدريسي ‪:‬م‪.‬م نور هاني‬

‫‪Practical of physiology‬‬
‫‪2nd stage‬‬
Practical of physiology

Lab (5): Preparation Of Blood Smear

By: M Sc.Noor Al-Naji

Apparatus and Reagents


1. Compound microscope.
2. 2 Glass slides.
3. Sterile pricking needle (disposable).
4. Leishman’s stain.
5. Cotton and spirit.
6. Water.

Leishman’s Stain
It is Romanosky group of stain consisting of:

10% solution of methylene blue prepared in 0.5% Na2CO3.


Equa1 volume of 0.1% solution of eosin is added.
The solution is dried and powdered.
0.15 gm of the powder is dissolved in 100 m1 of acetone free methyl alcohol.
*Methylene blue: It is a basic dye, stains RNA of cytoplasm, DNA of nuclei and
granules of basophiles.

*Eosin: It is an acidic dye and stains granules of eosinophils and hemoglobin of RBC.

*Acetone free methyl alcohol: It is fixes the smear to the glass slide.

Slide Method of Spreading a Blood Smear


 2 clean glass slides, one to cover with blood sample & one used as spreader.
(Never put your fingers on the surface of the slide).
 Clean the finger with alcohol, allow it dry & then prick it with a disposable
lancet to obtain a drop of blood (peripheral or capillary blood collection).
 Place a small drop of blood near end of glass slide.
 Place the spreader at approximately 40-45o in front of the drop and move the
spreader back to the right until it just touches the drop. The drop immediately

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runs along the edge of the spreader. Then move the spreader slide to the left
direction.
 Thick, viscous, blood should be spread rapidly with the spreader at a lower
angle. Thin, anaemic, blood should be spread slowly with the spreader held at a
greater angle (up to 50-60ᵒ).
 Allow the smear to dry in air at room temperature; unheated table fans can be
used to hasten the process.
 Stain the smear with Leishman's stain.

Criteria of Good Blood Smear

 Smear should occupy 3-4 cm length of the slide/middle 2/3rd of slide.


 It should be tongue shaped with no tails at the end (It has three parts head, body
and tail).
 Thickness should be uniform, it should not be too thick or too thin, no
longitudinal or cross striations should be visible under microscope.
 There should be no interruptions in between.
 There should be no collection of blood at either ends.
 It should be one cell thick when visualized under microscope and cells should
be uniformly distributed.


Staining of blood Smear
 The slide is placed on stand/rod in horizontal position.
 Leishman's stain (8-12 drops) is poured on slide so that it just covers the smear
fully.
 It is kept for two minutes ,this is to fix the smear on the slide this is known as
(fixation time).

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 Waiting time of (5-10) minutes is given. During this time the smear is actually
stained This is known as (staining time).
 Proper staining procedure gives a green scum over the stain.
 The slide is washed with tap water (taking care that water stream should not
strike smear directly).
 The slide is kept vertically for drying.
 Properly stained slide should look bluish pink.

Criteria of Well-stained Smear

 A Well stained smear looks bluish pink on naked eye examination.


 Under low and high power objectives of the microscope, we can examine for
quality of the smear.
 RBCs: Look orange pink/buff color, one cell layer thick and uniformly
distributed.
 WBCs: At least one WBC per high power field.
 Overstaining: RBCS look blue.
WBCS look bluish black (dark).

 Understaining: RBCS are pale.


WBCS are colorless.

Examining blood smear


 The examining of a well-made, well stained smear is a very important part of
the full blood examination. Every blood smear should be examined first by low
power (X10) objective to find the body of the smear and to check the total white
cell count to see that there is nothing abnormal or unusual about the red cells,
observe abnormalities of red cells such as size, shape, Hb content & inclusions.
 After these observations, rack around to high power oil immersion to confirm
findings.

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