Unit 3: Blood film preparation and staining
Basic Haematology & Haematological Techniques-I notes · PTU syllabus (BMLS301-18)
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Unit summary
Stained blood films reveal cell morphology and parasites. This unit covers types and methods of blood film preparation — thin and thick smears — and the principle, composition, reagent preparation and procedure of Romanowsky stains: Giemsa, Leishman, Wright's, Field's and JSB stains.
After this unit you can
- Prepare thin and thick blood films
- Explain the principle of Romanowsky staining
- Prepare Leishman, Giemsa and Wright's stains
- Use Field's and JSB stains for malaria
PTU syllabus topics
- Types and methods of blood film preparation (thick and thin smear)
- Romanowsky staining principle
- composition
- reagent preparation and procedure — Giemsa
- Leishman
- Wright's
- Field's and JSB stains
- 1
Place a drop of blood near one end
- 2
Spreader at 30–45°
Push smoothly
- 3
Air dry
Feathered tail = good smear
- 4
Leishman stain
2 min: fixes the smear
- 5
Add buffered water
Dilute and stain about 8–10 min
- 6
Wash, dry and examine
Oil immersion
Topic 1
Blood film preparation
Preparation
Drop spread with a spreader at 30–45°
Small drop spread in a 1 cm circle
Fixation
Methanol fixed
Not fixed — red cells lysed by water in the stain
Use
Cell morphology, differential count, species of malaria parasite
Detecting parasites (more sensitive — 20–40 times)
Features of a good film
Tapering tail, smooth, two-thirds of the slide, uniform thickness
Even, newsprint just readable through it when wet
- Cover-glass method
- Two coverslips pulled apart
- Spinner method
- Automated monolayer films
- Buffy coat film
- Concentrates white cells and parasites
- Faults: too thick (large drop, steep angle), streaks (dirty slide or spreader), holes (grease), no tail (drop too large).
Topic 2
Romanowsky staining: principle
- Romanowsky stains combine eosin (acidic dye, orange-red) with methylene blue and its oxidation products (azures) (basic dyes, blue). Acidic components (nuclei, cytoplasmic RNA) take up basic dyes; basic components (haemoglobin, eosinophil granules) take up eosin — the Romanowsky effect gives purple chromatin.
- pH matters: buffer at pH 6.8 (7.2 for malaria parasites) to get correct colours — too acidic gives pink films; too alkaline gives blue films.
Topic 3
Leishman stain
- 1Reagent: 0.15 g Leishman powder in 100 mL acetone-free methanol; mature for a few days
- 2Cover the air-dried film with stain for 2 minutes (methanol fixes)
- 3Add double volume of buffered water (pH 6.8); mix by blowing; leave 5–10 minutes
- 4Wash with buffer; dry; examine under oil
- Results: red cells pink; nuclei purple; neutrophil granules lilac; eosinophil granules orange-red; basophil granules dark blue; platelets violet.
Topic 4
Giemsa stain
- Reagent: Giemsa powder dissolved in glycerol and methanol (stock); diluted 1 : 10 with buffer (pH 7.2) before use. Procedure: fix thin films with methanol; stain 20–30 minutes (thick films unfixed, 3% stain for 30–45 minutes). Best for malaria parasites, Leishmania, Borrelia, and bone marrow.
Topic 5
Wright's stain
- Eosin and polychromed methylene blue in methanol; used widely in the USA; procedure similar to Leishman (stain 1–3 minutes, then buffer). Also used in automated stainers.
Topic 6
Field's stain
- A rapid water-based stain for thick films: Field's A (methylene blue and azure) and Field's B (eosin). Dip in A for 3–5 seconds, rinse, B for 3 seconds, rinse — results within 1 minute. Used for quick malaria diagnosis.
Topic 7
JSB stain
- Jaswant Singh–Bhattacharji (JSB) stain, developed in India for malaria under the national programme: JSB I (methylene blue with potassium dichromate, acidified) and JSB II (eosin). Thick film dipped in JSB I, washed, then JSB II — parasites show red chromatin and blue cytoplasm.
Leishman
Methanol
Routine thin films, differential count
Giemsa
Methanol and glycerol; diluted before use
Malaria and other parasites, marrow
Wright's
Methanol
Routine films
Field's
Aqueous; rapid
Thick films for malaria
JSB
Aqueous
Malaria in national programmes
Key terms
- Thin film
- Monolayer smear for morphology
- Thick film
- Concentrated smear for detecting parasites
- Romanowsky effect
- Purple staining of chromatin by eosin and azure dyes
- Buffer water
- Water at a fixed pH for staining
- Fixation
- Preserving cells before staining, usually with methanol
Quick revision
- Thin vs thick films; features of a good film; faults.
- Romanowsky principle; role of pH.
- Leishman preparation and procedure; colours of cells.
- Giemsa for parasites; Wright's; Field's and JSB rapid stains for malaria.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Distinguish thin and thick blood films.
- Q2.What is the Romanowsky effect?
- Q3.Why is buffered water used in staining?
- Q4.Why is a thick film not fixed?
- Q5.Which stain is best for malaria parasites?
- Q6.What is JSB stain?
Long-answer questions
- Q1.Describe the preparation of thin and thick blood films.
- Q2.Explain the principle of Romanowsky staining.
- Q3.Describe the preparation and procedure of Leishman and Giemsa stains.
- Q4.Describe Field's and JSB stains for malaria diagnosis.
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