Staining of peripheral blood film
Prepared by
Hadeel Al Sadoun
Introduction
 Types of staining:
 lishman
 Wright
 May Granuled stain stain
Gimsa or Gimsa
derivatives
 Used for:
 Peripheral blood film
 Bone marrow
 Purpose:
 To examine the blood cell’s number and morphology
objectives
 To stain the blood film properly in order to examine it under
the microscope
 To see the proper color reflection form cells and cellular
component.
Figure 1: Gimsa stained blood smear
Main component of Gimsa stain
 Gimsa stains are considered polychrome stain (why?)
 Composed from : *eosin
*Azure B
 Staining reaction is acid base reaction where acidic
component in the cells react with basic component in the
satin and vice versa
 Eosin: is acidic component of the satin and satin basic and (acidic)
component of the cells like Hemoglobin
 Azure B Mythylene Blue: is basic component and stains the acidic
and (basic) component of the cell like DNA and RNA (nucleus of WBC)
Materials
 Slides holder or rack
 Lishmen stain reagent (the satin used on this practical
session)
 Methanol:
 Fix the cells on the slide
 Buffer:
Figure 2: material for blood staining method
 Actual staining of the slide dose not occur until buffer is added
 0.05M Na2Po4 (PH 6.4)
 Aged dis.H2O : is a dis.H2O that remained in a glass bottle for at least 24
hour. (PH 6.4-6.8)
 Accurate PH is critical to obtain good quality staining.
Methodology
 Put the smear into mehthanol jar and fix it for 1 minute.
 Remove excess mehtanol from the smear
 Insert the smear into with Wright’s stain jar and leave the stain
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for 2 minutes.
Insert smear into a buffer jar and allow to stand for 4-8 minutes
Rinse thoroughly with a steam of distilled water
Allow to air dry
Note: time varies from manufacturers, thus insure to follow the exact time
in the banflet of each procedure.
Factors influence smear staining method
 Blood smear may be under or over stained based on the following:
 Concentration of the stain used
 Low concentration: plae coloerd cells (under stainig)
 High concentration: dark stained smear (over stained)
 Time of exposer to the stain and the buffer
 Too long: overstainig
 Too short: understainig
 Washing step
 Over washed: pale staning
 Under washed  over exposure dark stained smear