BACKGROUND
Acid-fast staining is one of the widely used differential staining procedures in bacteriological studies. This stain was first developed in 1882 by Paul Ehrlich. Ziehl and Neelsen independently proposed acid-fast stain in 1882-1883, which is commonly used today. Some types of bacteria resist decolorization by acid as well as alcohol both and hence stated as acid-fast organism.1 In this process carbol fuschin is the primary stain. 3%HCl in 95% alcohol is decolorizer and methylene blue acts as a counter stain.
Most common acid-fast bacteria cause dangerous diseases like TB, and leprosy. Acid-fast bacteria contains large amount of lipid material along with fatty acid and waxes. It has wax-filled cell walls which are impermeable. Due to this waxy cell wall, special precautions are required to stain the cell. Carbol fuschin comprises 5% phenol which plays a role of chemical intensifier and assists in penetration. Heat transfer helps in it and the stain so penetrated is retained by the decoloriser.2
Aim of the experiment is to study the bacterial morphology by acid-fast staining or Ziehl and Neelsen staining method.
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REQUIREMENTS
Apparatus: Microscope
Slide
Staining tray
Inoculating loop
Bunsen burner
Carbol fuschin
Chemicals: Methylene blue
Malachite green
Acid-alcohol (3% HCl in 95% alcohol)
Broth culture (72 h) of M. tuberculosis
Broth culture (24 h) of S. aureus
Procedure of Bacterial Morphology by Acid-fast Staining
Prepare a smear of the given bacterial suspension. Allow the smear to dry in the air and then fix by heating.
Acid fast staining procedure
Take carbol fuschin stain and overflow the smear with it. The heat that slide from below till steam rises, at least for 5 minutes. But do not boil that stain and also ensure that the stain should not be dried out.
Allow the slide to cool approximately for 5 min to protect that slide from breakage in the later step. Wash with tap water. Decolorise the slide by using acid-alcohol until carbol fuschin fails to wash smear.
Wash that slide with tap water and counter stain with one percent aqueous malachite green or methylene blue solution for 1-2 min. Wash the smear, dry in air, and observe the slide under an oil-immersion objective.
RESULTS
Slide-1 shows thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement. Blue-coloured cocci arranged in clusters is found in the second slide.
CONCLUSION
From the thin bright red, slightly curved bacilli with beaded appearance showing palisade arrangement confirms the structure of M. tuberculosis. Blue coloured cocci arranged in clusters hints Staphylococci structure.
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REFERENCES
- Kokare CR. Pharmaceutical microbiology (experiments and techniques]. Career publications, Maharashtra, 2008.
- Gaud RS, Gupta GD. Practical microbiology. Nirali Prakashan, New Delhi, 2006.
