Acid Fast Staining

What is Differential Staining?

This staining procedure utilizes more than one stain and differentiates organisms on the basis of the stain that they retain.

The importance of such a staining procedure is that by staining the cells one can differentiate the cells into different types, which is often needed for the identification of the cells.

The commonly used differential staining procedures are Gram staining and acid-fast staining.

What is Gram Staining?

  • Gram staining is a differential staining named after Dr. Christian Gram.
  • It is a simple, widely used staining procedure. The gram character is extremely important in the classification as well as identification of bacteria.
  • The difference between Gram-positive and Gram-negative bacteria appears to reside in their cell wall composition.
  • In Gram-positive cells, the main cell wall component is a mucopeptide which retains the crystal violet-iodine complex.
  • Iodine solution acts as a mordant, which enhances the union between the dye and its substrate.
  • In Gram-negative cells, the mucopeptide makes up only 10% of the cell wall structure and lipids are present in more amounts along with proteins and polysaccharides.
  • Decolorizer dissolves the lipids making the cell wall more porous so that it looses the dye iodine complex.

What Acid fast staining of bacteria?

  • The acid fast staining is a differential staining technique.
  • Acid fast staining technique is also known as the Ziehl-Neelsen staining technique.
  • It was developed by Paul Ehrlich in 1882 which was later on modified by Ziehl-Neelson and is being used by present day microbiologists.
  • Bacteria are classified as acid fast if they retain the primary stain after washing with strong acid and appear red or non-acid-fast if they lose their color on washing with acid and counter stained by the methylene blue.
  • The property of acid fastness appears to be due to the presence of high content of a lipid called mycoloic acid in the cell wall, which makes penetration by stains extremely difficult.
  • Acid fast staining procedure is useful for the identification of members of Mycobacterium.
  • M.smegmatis is a non-pathogenic but normal inhabitant of the genitals and may be mistaken for M. tuberculosis in the urine.

Some Acid Fast Reagents and their colour

Application ofReagentCell color
Acid fastNon-acid fast
Primary dyeCarbol fuchsinRedRed
DecolorizerAcid alcoholRedColorless
Counter stainMethylene blueRedBlue

Requirements for Acid fast staining

  1. Nutrient agar slants or broth cultures of M. smegmatis (48 hrs old),
  2. S. aureus (25 hrs old)

Acid fast staining reagents

  1. Carbol fuchsin
  2. Acid alcohol (3%HCL+95%ALCOHOL)
  3. Methylene blue
  4. Staining rack
  5. Hot plate/water bath
  6. Wash bottle of distilled water
  7. Glass slides

Procedure of Acid Fast Stain

  1. Prepare smears of M. smegmatis & S. aureus on separate slides.
  2. Air dry and heat fix the smears.
  3. Flood smears with carbol fuchsin.
  4. Heat the slides to steaming for 3-5 minutes. From time to time, add more stain to prevent smears form becoming dry.
  5. Cool slides and wash with distilled water.
  6. Decolorize the smear with acid-alcohol for 10-30 seconds or until the smear is a faint pink colour.
  7. Wash the slides with distilled water.
  8. Counter stain smears with methylene blue for 1-2 minutes.
  9. Wash with distilled water.
  10. Blot dry with blotting paper.
  11. Observe under oil immersion objective.
  12. Record the colour of the test organisms and classify the organisms as acid fast/non-acid fast.

Result of Acid Fast Staining technique

Result of Acid Fast Staining, Acid fast staining of bacteria

Bright red or intensive purple: Acid Fast Bacteria

Dark Blue: Non-Acid Fast Bacteria

References and Sources

Further Readings

  1. ABO Blood Group System and Rh system
  2. Algae
  3. Aseptic Transfer Technique
  4. Bacterial Flagella, Fimbriae and Pili
  5. Bacterial Growth and Nutrition
  6. Extremophiles
  7. Fimbriae vs Flagella
  8. Fimbriae vs Pili
  9. Fundamental Microscopy
  10. Growth Curve of Bacteria
  11. Instruments Used in Microbiology Laboratory
  12. MacConkey agar
  13. McFarland Standards
  14. Monochrome Staining
  15. Negative Staining
  16. Nutritional Requirements of Micro-Organisms
  17. Preparation and Sterilization of Culture Media
  18. Serial Dilution in Microbiology
  19. Spread Plate Technique
  20. Sterilization
  21. Streak Plate Technique