A Step-by-step Guide to Field’s Stain Procedure

Introduction

Principle

Storage and stability of Field’s stain

• All reagents are stable at room temperature until the expiry date stated on each label.
• Do not spray fixative onto open fire.
• Avoid inhaling the spray.

Safety precautions while handling Field’s stain

• Field’s stain are for In Vitro Diagnostic Use only.
• Field’s stains are  professional use only.
• Directions should be read and followed carefully.
• Do not use beyond the stated expiration dates.
• Microbial contamination may decrease the accuracy of the staining.
• Safety precautions should be taken in handling, processing and discarding all
  clinical specimens.
• Samples should be processed in the correct containment level conditions.

Type of Specimen:

Clinical specimen:

• Blood samples.

Materials required

• Clean grease-free glass slide,
• Staining rack,
• Blotting paper,
• Immersion oil
• Microscope.

Procedure

Staining procedure for thick film Field’s stain

• Prepare a thick blood film.
• Fix blood film with Heme-Fixative for 1 minute and air dry.
• Wash off fixative with tap water and air dry.
• Cover the blood smear with Field B stain and keep for 1 minute.
• Gently wash the slide with tap water.
• Immediately add an equal volume of Field A stain and stain for 1 minute.
• Rinse the slide with tap water and place upright to drain and dry.
• Observe under microscope, 40X and 100X under oil immersion lens.

Staining procedure for thin film using Field’s stain

Prepare a thin blood film on a microscope slide.

• Air dry the film
• Fix in methanol for one minute.
• Cover the slide in with 1 mL Field Stain B (1 in 4 in buffered water pH 7.2 or distilled water).
• Immediately add an equal volume of Field Stain A and mix.
• Leave stain for 1 minute.
• Rinse the slide with distilled water, drain and dry.
• Examine under oil immersion.

Interpretation of results

In a well stained film, the malarial parasites show deep red chromatin and blue cytoplasm. Leucocytes stain nuclei blue, cytoplasm pale blue. Eosinophils are large and dull red. Neutrophils are small, pale purple. White cells, platelets and malaria pigment can also be seen on a thick film. The malaria pigment will stain brown-black.

Neutrophil:

• Segmented polymorph with purple granules

Eosinophil:

• Polymorph with large orange granules

Basophil:

• Polymorph with large blue granules

Lymphocyte:

• Dark blue nuclei with clear blue cytoplasm

Monocyte:

• Kidney shaped nucleus with smoky blue cytoplasm

Platelet:

• Small pale bluish cells

Red blood cells:

• Pinkish red cells

Malaria Parasites:

• Pale bluish with Red Scifner’s Dot (Trophozoite)
• Cytoplasm of parasite – Blue-mauve
• Chromatin of parasite – Dark Red

Quality
control

Positive: Plasmodium species
Negative: A proven negative smear may be used as the negative control

Limitation of Field’s stain

1. Films for malaria must be made immediately or no longer than 3-4 hours
2. For preparing thick film, allow the film to dry for 30 minutes at least. Ensure film is completely dry before staining. Use a slide warmer to dry the smear to reduce drying time.
3. Absolutely fresh films on the slide can wash off during staining, be aware of this and ensure the film is thoroughly dry.
4. Methanol used as fixative should be completely water free, as little as 1 % water may affect the appearance of the films.
5. The red cells will also be affected by traces of detergent on inadequately washed slides. Sometimes when thick films are stained they become overlaid by residue of stain or spoil by the envelopes of the lysed red cells.

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