07, Jun, 2018
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Acid-Fast Staining: Purpose, Principle, Procedure and Observation

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Acid-Fast Staining: Purpose, Principle, Procedure and Observation Sputum smear stained with Ziehl-Neelsen stain showing acid-fast bacilli

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  • Dayyal Dg.
  • Published: 07, Jun, 2018 | Updated: June 10, 2018
  • Microbiology
Sputum smear stained with Ziehl-Neelsen stain showing acid-fast bacilli

There are certain bacteria which cannot be stained by Gram's method. In 1882, Paul Ehrlich developed a method of staining such type of bacteria. This method was named, and still known as acid-fast staining and the bacteria were named as acid-fast bacteria. In the same year, Ehrlich's method was improved by Zehil and Neelsen. Nowadays, Ziehl-Neelsen method is believed as most important differential staining procedure used for the identification of acid-fast species of Mycobacterium, Actinomyces, and Nocardia. There are many acid-fast bacteria which are pathogenic, such as M. tuberculosis (tuberculosis), A. israelii (actinomycosis), M. leprae (leprosis), and N. asteroides (nocardiosis).

Acid-fast bacteria may be defined as those cells which keep the color of the primary dye (carbol fuchsin) even after the process of decolorization by the acid-alcohol solution. Those bacteria which fail to do so are known as non-acid-fast bacteria.

Acid-fast bacteria are coated with a thick waxy material, mycolic acid, which makes the bacterial cells highly resistant to the penetration of dyes. The penetration of dye is promoted by using heat as mordant. The heat invades the dye through the waxy coat and into the cytoplasm.

PURPOSE

  • Differentiation between acid-fast and non-acid-fast bacteria.
  • Diagnosis of pulmonary tuberculosis from sputum smear. See also: Examination of Sputum

NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

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Reagents

  1. Carbol fuchsin
  2. Acid-alcohol solution
  3. Methylene blue

Equipment

  1. Bunsen burner
  2. Wire loop
  3. Glass Slide
  4. Spirit lamp
  5. Microscope

PROCEDURE

Acid-Fast staining by Ziehl-Neelsen method

  1. Take a clean glass slide and prepare a thin smear from the specimen using sterile technique. The smear should be extremely thin covering a large area of the slide.
  2. Dry the smear is air and then fix the slide by passing the slide through the flame.
  3. Cover the slide with carbol fuchsin. Keep it for 5 minutes over a spirit lamp with constant heating but not boiling. Do not allow the stain to dry over the slide.
  4. When the slide is cooled, wash it with tap water.
  5. Flood the slide with acid-alcohol and leave it for 3 minutes. Wash the slide with tap water and drain.
  6. Counterstain with methylene blue for 2 minutes.
  7. Wash the slide with tap water and keep it for dry.
  8. Observe the slide under oil immersion objective.

OBSERVATION

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved bright red rods. Non-acid-fast cells appear blue.

Acid-Fast Staining by Mobin Method

In 1985, a Pakistani microbiologist, Abdul Mobin Khan developed a method for the staining of acid-fast bacteria. In this method, heating of flooded primary dye on smear is not required. However, initial fixing of the smear over the flame is necessary in order to increase the permeability of the cell wall and promote the newly formulated primary dye to penetrate the cell.

Needs

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

Reagents

  1. Mobin stain
  2. 1% H2SO4
  3. Crystal violet

Equipment

  1. Wire loop
  2. Bunsen burner
  3. Glass slides
  4. Microscope

Procedure

  1. Using sterile technique, prepare a thin smear from the specimen covering a large area of the glass slide.
  2. Dry the smear in the air and then fix it by passing the slide 20 times through the flame.
  3. Place the smear on the staining rack and cover it with Mobin stain. Keep it for 10 minutes.
  4. Pour off the stain and wash the slide with tap water.
  5. Decolorize the smear by 1% H2SO4 solution till it is light pink.
  6. Wash the slide with tap water and keep it for dry.
  7. Observer the slide under oil immersion objective.

Observation

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved red rods while non-acid-fast bacteria as blue.

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Last modified on Sunday, 10 June 2018 09:00
Read 543 times
  • Dayyal Dg.
  • Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

Dayyal Dg.

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Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

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