Principle
Sudan black B is a lipophilic dye that binds irreversibly to an undefined granule component in granulocytes, eosinophils, and some monocytes. It cannot be extracted from the stained granules by organic dye solvents.
Reagents
1) Fixative: 40% formaldehyde solution
2) Stain: SBB 0.3 grams in 100 ml absolute ethanol
3) Phenol buffer: dissolve 16 gm crystalline phenol in 30 ml absolute ethanol. Add 100 ml distilled water in which 0.3 gm Na2HPO4, 12 H2O2 has been dissolved.
4) Working stain solution: add 40 ml buffer to 60 ml SBB solution
5) Counterstain: Giemsa or Leishman stain
Method
1) Fix air-dried smear in formalin vapour. Pour 4-5 drops of 40% formaldehyde on a filter in a petridish. Place the slide in it and cover. Leave for 15 minutes.
2) After 10-15 minutes, remove the slides and stand on end for 15 minutes in air to air wash.
3) Immerse the slides in the working stain solution for 1 hour with a lid on.
4) Transfer the slide to a staining rack and immediately flood with 70% alcohol.
5) After 30 seconds, tip the 70% alcohol off and flood again for 30 seconds.
6) Repeat this step for three times in total.
7) Rinse in gently running tap water and air dry.
8) Counter stain without further fixation with Leishman stain.
Result
The reaction product is black and granular.
Interpretation
1) The most primitive myeloblasts: negative
2) Promyelocytes and myelocytes: most strongly staining cells in the granulocytic series.
3) Metamyelocyte and neutrophils: have progressively fewer positive granules.
4) Eosinophil: granules stained strongly with a clear core.
Image source: Biognost