Field's Stain
Malaria and Helicobacter

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.

Solutions

Solution A
Methylene blue	0.8	g
Azure A	0.5	g
Di-sodium hydrogen phosphate, anh.	5.0	g
Potassium di-hydrogen phosphate	6.25	g
Distilled water	500	mL
Dissolve each phosphate in half the water. Add one of the dyes to each and dissolve. The azure may require grinding with a pestle & mortar. Combine both solutions. Leave overnight and filter. The solution is stable.

Solution B
Eosin Y	1.0	g
Di-sodium hydrogen phosphate, anh.	5.0	g
Potassium di-hydrogen phosphate	6.25	g
Distilled water	500	mL
Dissolve each phosphate in half the water. Combine both solutions and add the eosin. Leave overnight and filter. The solution is stable.

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.
 
Blood smears
Remember that fresh blood is a health hazard.
 
Thick smears
Make a smear by placing 2-3 drops of blood at the centre 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 blood or bone marrow smear of the usual type and allow to dry. Fix in methanol for a minute or two before staining.

Method

1. Place into solution A for 1-2 second.
2. Rinse with distilled water.
3. Place into solution B for 1-2 second.
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

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

 

Reference
Humason, G.L., (1967)
Animal Tissue Techniques
W.H. Freeman & Co., San Francisco, CA, USA

Drury, R.A.B. and Wallington, E.A., (1980)
Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.