Solutions:- Part 6 – Field Stain A and B, Preparation of the solution

Field Stain A and B

Field stain consists of:

1. Field stain A
2. Field stain B

Thin and thick blood films are made and fixed in methanol for one minute.

Field Stain A (Dark violet color)

Using Commercially Available Powder.

1. Field stain A commercially available powder = 5 grams
2. Distilled water is heated to 80 °C or kept at 60 °C for 30 minutes. = 600 mL.

Procedure to make Field stan A from commercial powder:

1. Field stain A (ready-made powder).
   1. Mix the powder into the water until it is dissolved.
   2. Filter the solution.
   3. Label the date.

Field stain A  from the reagents:

1. Methylene blue (analytical grade) = 1.6 grams.
2. Azur = 1 gram.
3. Disodium dihydrogen phosphate anhydrous = 10 grams.
4. Potassium dihydrogen phosphate anhydrous = 12.5 grams.
5. Distilled water = 1000 mL.

Procedure for Field stain A preparation:

1. Take Methylene blue (analytical grade) = 1.6 grams.
2. Take Azur = 1 gram.
3. Take Disodium dihydrogen phosphate anhydrous = 10 grams.
4. Take Potassium dihydrogen phosphate anhydrous = 12.5 grams.
5. Distilled water = 1000 mL.
6. Mix both salts (3 and 4) in the water.
7. Add 500 ml from above into a bottle containing glass beads.
8. Add the stain powders (Methylene blue and Azur) and mix well.
9. Then add the remainder of the solution.
10. Mix well and filter into another clean bottle.
11. Label the bottle Field stain A and also write the date.

Field Stain B (Orange color)

Using Commercially Available Powder:

1. Field stain B commercially available powder = 4.8 grams.
2. Distilled water is heated to 80 °C or kept at 60 °C for 30 min. = 600 mL.
3. Mix well the field stain B until it is dissolved.
4. Filter and label the date.

Field stain B reagents:

1. Eosin(yellow water soluble) powder = 2.0 grams.
2. Disodium dihydrogen phosphate anhydrous = 10 grams.
3. Potassium dihydrogen phosphate anhydrous = 12.5 grams.
4. Distilled water = 1000 mL.

Procedure for  Field stain B preparation:

1. Mix both salts (2 and 3) in the distle water (1000 mL) to make the solution.
2. Add 500 ml from above into a bottle containing glass beads.
3. Add the stain powders Eosin and mix well.
4. Then add the remainder of the solution.
5. Mix well and filter into another clean bottle.
6. Label the bottle Field stain B, and also write the date.

Staining Procedure for Field Stain A and B:

1. Fill up two Coplin jars or wide-mouth bottles:
   1. Field Stain A (Blue stain).
   2. Field Stain B (Red stain).
2. Make a blood smear on a clean glass slide, and it is dried in the air.
3. Fix in methanol for one minute or get Spray ‘Easyfix’.
4. Dry in the air.
5. Dip fixed smear to Field Stain B (Red Stain) for 5  to  6 seconds.
6. Wash in running tap water.
7. Dip smear into Field Stain A (Blue Stain) for 10 to 30 seconds (adjust it).
8. Wash in running tap water.
9. Dry at air and see under oil immersion objective.
10. The staining time may be adjusted.

Interpretation of the cells in Field stain:

Type of cells	Color appearance
Red blood cells	Pink
Neutrophils granules	Lilac color
Eosinophils	Orange granules
Malarial parasites	Deep red chromatin and pale blue cytoplasm

 

How to prepare thick and thin smears:

1. Thick and thin blood smears can be made into two separate slides.
2. Put two separate drops of the blood. For a thin smear, put a small drop and a large drop for a thick smear.
3. Spread the drops by the cover glass.

Note: The timings can be adjusted by staining a few slides.

Questions and answers:

Q1: What is the purpose of the thin and thick smears.

Q2: Will you fix the thick slides.

Answers:

Q1: Thick smears are made for the identification of malarial parasites. Thin smears are for the appreciation of the peripheral blood cells.

Q2: Thick smear does not need fixation.

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