Sudan Black B Staining

Sudan Black is a slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. An alternative...

Sudan Black B Staining

There are several Sudan dyes such as Sudan II, Sudan III, Sudan IV, Oil Red O, and Sudan Black B. These dyes are used to stain fat and fat droplets. All of these dyes are soluble in lipids and are known as lysochrome dyes.

Sudan dyes also used in coloring textiles, garments, and plastic, that’s why they are known as diazo dyes. This dye is used to stain sudanophilic biological samples such as fats and lipids. It stains fats and lipids blue-black (Sudan Black B stains), the color also varies based on the types of dye.

Sudan II is used to stain nonpolar substances (i.e wax, grease, fats, oils, hydrocarbons, and acrylic emulsions), Sudan III used for staining lipid, Sudan Black B is used to stain Lipids such as phospholipids, strokes, and neutral triglycerides. Sudan Black B is a nonfluorescent and thermally stable dye. 

Sudan Black B is not a specific dye for lipid so it can be used to stain other components such as chromosomes, Golgi bodies, and leukocyte granules.


Sudan Black is a slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. An alternative stain to the Sudan Black B stain.


Use a positive control of a fat smeared slide, and a negative control slide of a paraffin processed tissue, such as lung.


  • Specimen: Frozen sections of the sample
  • Propylene Glycolin two Coplin jars
  • 85% Propylene Glycol (Can be prepared by mixing 85.0 ml Propylene glycol with 15.0 ml Distilled water)
  • Hematoxylin
  • Glycerin Jelly
  • Sudan Black B/Propylene (mixture of Sudan Black B 0.7 gm and Propylene glycol 100.0 ml)
  • Fixative: 10% formalin.

Prepare it by dissolving Sudan Black in propylene glycol, gradually, while stirring. Heat it to 100°C for a few minutes, while stirring continually. Filter the solution using the Whatman 2 filter paper and cool it. Filter the solution again using a frittered glass filter with medium porosity. Store the solution at 60°C in an oven, and the solution can remain stable for 1 year. Solution stable for 1 year.

Sudan Black B Staining Procedure

  • Frozen sections are fixed on a clean glass slide using fresh 10% formalin.
  • Wash the fixed section with distilled water and tap off the excess.
  • Add propylene glycol in two changes, each of 5 minutes.
  • Add Sudan black solution and leave it at 7 minutes for agitation.
  • Add 85% propylene glycol and leave for 3 minutes.
  • Rinse the stain in distilled water.
  • Add nuclear Fast Red and leave for 3 minutes.
  • Wash in tap water, twice, and rinse in distilled.
  • Mount with aqueous mounting jelly, that is glycerin jelly.

Results and Interpretation

  • Fat: Appear in blue-black color.
  • Nuclei: Appear in red color.



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