Blood Film Technique

Making Blood film

Practical Parasitology

2nd StageLab 12: Blood Film

University of Sulaimani

School of ScienceDepartment of

Biology

University of Sulaimani

School of ScienceDepartment of

Biology

OBJECTIVES

• Students should be able to:– Make a good blood film– Stain blood film – Identify the diagnostic stages of a

blood-dwelling parasites

Blood Film• The most commonly used technique for blood

examination is blood film.• A very thin layer of blood spread over a

microscope slide• Allows the various types of blood cells to be

seen and identified. • Blood smear plays an important role in

diagnosing a wide range of illnesses.• Includes detection of blood-borne parasites, like

malaria.

Collection of Blood Smears

5.Touch the drop of blood to the slide from below.

4.Slide must always be grasped by its edges.

2.Puncture at the side of the ball of the finger.3.Gently squeeze toward the puncture site.

1.The second or third finger is usually selected and cleaned.

Preparing thick and thin films1. Touch one drop of blood to a clean slide.2. Spread the

first drop to make a 1 cm circle.3. Touch a fresh drop of blood to the edge of another slide.

6. Wait for both to dry before fixing and staining.

5.Pull the drop of blood across the first slide in one motion.

4.Touch the drop of blood by spreader slide at 45degree angle.

Blood Film Preparation

Thick Blood Film preparation

Thin Blood film preparation

Thick and Thin Films

• THICK FILM– lysed RBCs– larger volume– 0.25 μl

blood/100 fields– more difficult to

diagnose species– parasite density

• THIN FILM– Intact RBCs– smaller volume– 0.005 μl

blood/100 fields– good species

differentiation– low density

infections can be missed

Staining

• Staining is a biochemical technique of adding a specific dye to a substrate (DNA, proteins, lipids, carbohydrates) to qualify or quantify the presence of a specific compound.

• Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.

Staining• Romanowsky stains are based on a

combination of eosine and methylene blue

• Wright's stain, Leishman stain and Giemsa stain.

• All are used to examine blood or bone marrow samples.

• Nuclei stained dark blue/violet, erythrocytes pale pink & cytoplasm pale blue

• All are also suited to examination of blood to detect blood-borne parasites like malaria.

Staining Procedure • Thin smear are air dried.• Overflow the smear with Leishman stain

(1 ml) for 1-5 min. • Add a double amount of distilled water

and mix the stain by blowing the fluid.• Leave the mixture on the slide for 10-15

min. • Wash off by slow-running water to

remove the extra liquid stain.• Stand slide on end, and let dry in air.• Examination

Methylene blue• Basic dye• Blue-purple colour• Stains nuclei,

ribosomes & rough ER (DNA & RNA - acidic)

• Structures that stain with methylene blue are termed basophilic

Eosin• Acid dye• Pink-red colour• Stains most

cytoplasm proteins which are mostly basic

• Structures that stain with eosin are termed eosinophilic

Leishman stain

Examination

Microfilaria Microfilaria

References

• http://www.dpd.cdc.gov/dpdx/ • http://www.stanford.edu/group/parasites/Parasit

es2006/• http://www.bsieducation.org/Education/14-19/to

pic-areas/applied-science/standard-procedure/sp-0002-1.shtml

• http://www.ruf.rice.edu/~bioslabs/studies/studies.htm

• Clodfelter, R.L. (1986). The peripheral smear. Emerg. Med. Clin. North. Am. 4(1):59-74.

Next LabCiliates:

Balantidium coli