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Field’s Stain for Malaria and Helicobacter

Field's Stain

for Malaria and Helicobacter

6
steps
6
materials

This method was originally recommended for staining malaria parasites in thick blood films, but it has also been used as a quick Romanowsky stain for thin blood films and marrow smears. On thin sections of stomach it demonstrates Helicobacter pylori well.

Materials

Solution A

MaterialAmount
Methylene blue0.8g
Azure A0.5g
Di-sodium hydrogen phosphate, anh.5.0g
Potassium di-hydrogen phosphate6.25g
Distilled water500mL

Preparation

  1. Dissolve each phosphate in half the water.
  2. Add one of the dyes to each and dissolve.
  3. The azure may require grinding with a pestle & mortar.
  4. Combine both solutions. Leave overnight and filter.
  5. The solution is stable.

Solution B

MaterialAmount
Eosin Y1.0g
Di-sodium hydrogen phosphate, anh.5.0g
Potassium di-hydrogen phosphate6.25g
Distilled water500mL

Preparation

  1. Dissolve each phosphate in half the water.
  2. Combine both solutions and add the eosin.
  3. Leave overnight and filter.
  4. The solution is stable.

Tissue Sample

Sample preparation

Tissue sections

3µ paraffin sections of formalin fixed tissue are suitable. Many others may be used. Before staining, bring sections to water with xylene and ethanol.

Thick smears

Make a smear by placing 2-3 drops of blood at the center of a slide and spreading it out to an area about 2 cm in diameter with a tooth pick. The smear should be thicker than a regular blood smear, but not so thick that it cracks and peels as it dries. Lay flat and allow to dry. Do not fix as the erythrocytes must be unfixed to be lysed during staining.

Thin smears

Make a standard blood or bone marrow smear and allow to dry. Fix in methanol for a minute or two before staining.

Protocol

  1. Place into solution A for 1-2 seconds.
  2. Rinse with distilled water.
  3. Place into solution B for 1-2 seconds.
  4. Rinse with distilled water.
  5. Air dry.
  6. Coverslip with a resinous medium, or examine without a coverslip.

Expected Results

  • Bacteria, including H.pylori – deep blue
  • Cellular elements – similar to a Romanowsky stain
  • Malaria chromatin – purple red to deep red
  • Malaria cytoplasm – blue

Notes

  • Times in each solution should be adjusted in each laboratory to obtain the colour contrast wanted.
  • Remember that solution B will tend to remove the staining by solution A.
  • For H.pylori, thin sections show the organism best.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Humason, G.L., (1967)
    Animal Tissue Techniques
    W.H. Freeman & Co., San Francisco, CA, USA
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.