Sudan Black B Staining – Principle, Procedure, Applications

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.



As SBB is a fat-soluble dye, it discolours lipids, including sterols, neutral fats, and phospholipids. These are present in the azurophilic, secondary, and lysosomal granules of myelocytic and monocytic cells, respectively. As a result of its greater solubility in lipids than in solvent, the dye leaves the solvent during staining. On microscopic analysis, the affirmative reaction reveals variable amounts of black pigments.

The Sudan black technique (modified Chiffelle & Putt) largely use physical processes, as opposed to the majority of staining treatments, which utilise chemical mechanisms. The dye is dissolved in a lipid solvent, which is then used to treat tissue slices. Since the dye is more soluble in the lipid in the tissue section than in the original solvent, the dye will leave the solvent and colour the lipid in the tissue section. In this process, boundary-surface adsorption plays a crucial role. The discoloration is physical in nature. Chemically distinct lipids are indistinguishable with this approach. The best fixatives are formalin with a neutral buffer or formal-calcium. Frozen sections must undergo simple lipid stains that reveal the presence or absence of lipids. The dye solvent is propylene glycol, so it will not dissolve any of the tissue lipids. Because the organic solvents inherent in synthetic resins would dissolve the dye-lipid combination, aqueous mounting medium, such as glycerin jelly, are utilised to mount the final slide.



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


  • Sample: Fresh anticoagulated whole blood or a bone marrow smear can be utilised as a specimen. The slides must be repaired immediately.
  • Fixative: 40% formaldehyde solution vapour
  • Stain: SBB 0.3 g per 100 ml of 100 percent ethanol
  • Phenol buffer: Dissolve 16 g of crystalline phenol in 30 ml of 100% ethanol to create a phenol buffer. Add to 100 ml of dissolved Na2HPO4.12H2O in distilled water.
  • Working SBB stain solution: Add 40 ml of buffer to 60 ml of SBB solution to make a working staining solution. (The composition of the working stain may vary significantly between goods.)
  • Counterstain: May–Grunwald–Giemsa or Leishman counterstain.

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.


Procedure of Sudan Black B Stain

  1. Fix air-dried smears for 10 minutes with formalin vapour, formaldehyde, or formalin-ethanol fixative.
  2. Wash with water for 5 to 10 minutes.
  3. Place the slides in a Coplin jar with a lid and the working stain solution for one hour.
  4. Remove the slides and immerse them in 70% alcohol for 30 seconds. Remove the 70% alcohol and re-inundate. Reiterate three times.
  5. Rinse with running tap water and air dry.
  6. Without further fixing, counterstain with Leishman stain or May–Grunwald–Giemsa stain.
  7. Air-dry and inspect under a microscope.

Results and Interpretation

  • Myelocytic cell azurophilic and secondary granules contain lipids. These are therefore SBB-positive. As myeloblasts evolve into mature forms, the intensity of the staining increases. In general, basophils are negative, although granules may exhibit intense red/purple metachromatic staining.
  • Lysosomal granules of monocytic cells contain lipids. Monocytic cells demonstrate various responses, ranging from negative to weakly positive.
  • Lymphoid cells are negative for SBB. In ALL, however, less than 3% of blast cells exhibit a favourable reaction.

Limitations of Sudan Black B Staining

  1. False Positive Results: Sudan Black B can sometimes bind to lipids that are not part of the lipid droplets, leading to false positive results.
  2. Background Staining: The staining can result in high background staining and low contrast, making it difficult to accurately visualize the lipid droplets.
  3. Non-Specificity: Sudan Black B staining can bind to other biological structures besides lipid droplets, such as lysosomes and melanin, leading to non-specific staining.
  4. Bleaching: Over time, the staining intensity can decrease, which is known as bleaching, making it difficult to accurately quantify lipid droplets.
  5. Difficulty with Automated Image Analysis: Automated image analysis algorithms may have difficulty accurately detecting and quantifying lipid droplets stained with Sudan Black B due to the non-specific staining and high background.

Advantages of Sudan Black B Staining

  1. Simple and Cost-Effective: Sudan Black B staining is a simple and cost-effective method for visualizing lipid droplets in biological samples.
  2. High Sensitivity: The staining has high sensitivity, allowing for the visualization of even small amounts of lipid droplets.
  3. Widely Used: Sudan Black B staining is widely used in both research and diagnostic settings, making it a well-established and well-understood technique.
  4. Multiple Applications: The staining can be applied to a wide range of biological samples, including cells, tissues, and organs, making it a versatile technique.
  5. Easy to Visualize: Sudan Black B staining results in a clear, black staining of lipid droplets that is easy to visualize and quantify under a microscope.
  6. High Stability: The staining has a high stability and does not fade easily, allowing for long-term storage and analysis of stained samples.

Applications of Sudan Black B Staining

  1. Lipid Droplet Analysis: The primary application of Sudan Black B staining is to visualize and quantify lipid droplets in biological samples.
  2. Cell Biology: The staining is widely used in cell biology research to study the distribution, abundance, and dynamics of lipid droplets in cells.
  3. Developmental Biology: Sudan Black B staining can be used to study the development of lipid droplets in organisms and tissues over time.
  4. Obesity Research: The staining is often used in obesity research to study the accumulation of lipid droplets in adipose tissue and other tissues.
  5. Metabolic Disorders: The staining can be used to study lipid droplets in the context of metabolic disorders, such as non-alcoholic fatty liver disease and type 2 diabetes.
  6. Pathology: The staining is also used in pathology to help diagnose diseases associated with lipid droplets, such as Niemann-Pick disease and lipid storage myopathies.
  • It has been used to display triglycerides, lipids, and lipoproteins due to its ability to stain fats.
  • Determine the amount of fat in the faeces in order to diagnose steatorrhea.
  • To dye chromosomes, the Golgi apparatus, and white blood cells.
  • To investigate haematological diseases such as myeloblast deficiencies.
  • It can also detect mitochondrial abnormality-related problems.
  • It is utilised for the staining of granulocytes (eosinophils, mast cells), promyelocytes, and myeloblasts.
  • Oil Red O is used for fingerprinting in forensic pathology.
  • Fabrics and clothing are printed with Sudan staining dyes.

FAQ on Sudan Black B Staining

What is Sudan Black B staining and what is it used for?

Sudan Black B staining is a dye used in histology and pathology to stain lipids and lipoproteins in tissues and cells. It is commonly used for the detection and identification of lipid-containing structures, including fat droplets and lysosomes.

What are the components of Sudan Black B staining solution?

Sudan Black B staining solution typically consists of a mixture of Sudan Black B dye, a solvent such as ethanol or isopropanol, and sometimes a stabilizing agent such as sodium chloride.


How does Sudan Black B staining work?

Sudan Black B dye binds to the lipid and lipoprotein structures in the tissue or cells being stained, producing a dark blue or black stain that can be easily visualized under a microscope.

What is the difference between Sudan Black B staining and other lipid staining methods?

Sudan Black B staining is a selective staining method that specifically binds to lipid-containing structures, while other lipid staining methods, such as Oil Red O or Nile Red, may also stain other cellular components.


How long does Sudan Black B staining take?

The exact time required for Sudan Black B staining can vary depending on the sample and the specific protocol used, but it typically takes anywhere from a few minutes to an hour.

What are the advantages of using Sudan Black B staining?

Sudan Black B staining is a simple, fast, and highly specific method for staining lipids and lipoproteins in tissues and cells. It is also relatively low cost compared to other lipid staining methods.

What are the limitations of using Sudan Black B staining?

Sudan Black B staining can sometimes produce inconsistent results, particularly if the staining solution is not prepared or stored correctly, or if the sample is not prepared properly. Additionally, it may not be suitable for staining all types of lipid-containing structures, particularly if they have a low lipid content.

How should Sudan Black B staining solution be stored?

Sudan Black B staining solution should be stored in a dark, cool place, away from light and heat, to prevent degradation of the dye. It is recommended to store the solution in an airtight container, such as a amber-colored glass bottle, to further protect it from light.

How should Sudan Black B-stained samples be analyzed and visualized?

Sudan Black B-stained samples should be analyzed using a microscope equipped with bright field or phase contrast optics. The images can be captured using a camera or other imaging device, and the data can be analyzed using image analysis software.

Are there any safety precautions to be taken when handling Sudan Black B staining solution?

Sudan Black B dye is classified as a carcinogenic substance, so it is important to take appropriate precautions when handling the staining solution. This may include wearing gloves and eye protection, working in a well-ventilated area, and avoiding skin or eye contact with the dye.



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