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Acid fast staining

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Z N
STAINING
Presented by
Himalay Baruah
2023/MMB/0006
MMB 1st Semester
CONTENT
 Introduction
 History
 Ziehl-Neelsen staining principle
 Requirements of acid fast stain
 Reagent preparation method
 Procedure of acid-fast stain
 Acid fast bacilli under microscope
 Some ACID FAST ORGANISM
 It is a type of differential staining
 used to differentiate acid fast and non
acid fast bacteria
 used to identify acid-fast organisms such
as members of the genus Mycobacterium.
 also known as Ziehl-Neelsen method
 first introduced by Paul Ehrlich later
modified by two German doctors
bacteriologist Franz Ziehl (1859–1926)
and the pathologist Friedrich Neelsen
(1854-1898).
INTRODUCTION
 In 1882 Robert Koch (German Physician And Microbiologist)
discovered the etiology of tuberculosis.
 Soon after koch's discovery, Paul Ehrlich ( German physician
and pharmacologist ) developed a stain for mycobacterium ,
called the alum(aluminium mordanted) hematoxylin stain.
 Franz ziehl ,in 1882(german bacteriologist) then altered
Ehrlich's staining technique by using carbolic acid (commonly
known as phenol)as the mordant.
 Friedrich neelsen ( german pathologist) kept ziehl's choice of
mordant but changed the primary stain to carbol fuchsin by
adding basic fuschin incredient.
 Ziehl and neelsen's modifications together have developed the
ziehl-neelsen stain.
 Another acid-fast stain was developed by Joseph J Kinyoun
(american physician) by using the ziehl-neelsen staining
 Technique but removing the heating step from the procedure.
 This new stain from kinyoun was named the kinyoun stain
HISTORY
ZIEHL-NEELSEN STAINING
PRINCIPLE
Acid-fast mycobacterium contain a lipoidal
material mycolic acid in their outer membrane,
making the cells waxy and resistant to
staining with aqueous based stains such as
the Gram stain.
The primary stain, carbol fuchsin is applied
to the cells, and heat and phenol are used to
allow the stain to penetrate into the waxy
surface of acid-fast microorganisms.
The excess stain is removed with treatment
by acid alcohol (ethanol and hydrochloric
acid).
A counter stain methylene blue, is then
applied to the cells.
Cell wall structure of acid
fast organism

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himalay baruah acid fast staining.pptx

  • 1. Z N STAINING Presented by Himalay Baruah 2023/MMB/0006 MMB 1st Semester
  • 2. CONTENT  Introduction  History  Ziehl-Neelsen staining principle  Requirements of acid fast stain  Reagent preparation method  Procedure of acid-fast stain  Acid fast bacilli under microscope  Some ACID FAST ORGANISM
  • 3.  It is a type of differential staining  used to differentiate acid fast and non acid fast bacteria  used to identify acid-fast organisms such as members of the genus Mycobacterium.  also known as Ziehl-Neelsen method  first introduced by Paul Ehrlich later modified by two German doctors bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854-1898). INTRODUCTION
  • 4.  In 1882 Robert Koch (German Physician And Microbiologist) discovered the etiology of tuberculosis.  Soon after koch's discovery, Paul Ehrlich ( German physician and pharmacologist ) developed a stain for mycobacterium , called the alum(aluminium mordanted) hematoxylin stain.  Franz ziehl ,in 1882(german bacteriologist) then altered Ehrlich's staining technique by using carbolic acid (commonly known as phenol)as the mordant.  Friedrich neelsen ( german pathologist) kept ziehl's choice of mordant but changed the primary stain to carbol fuchsin by adding basic fuschin incredient.  Ziehl and neelsen's modifications together have developed the ziehl-neelsen stain.  Another acid-fast stain was developed by Joseph J Kinyoun (american physician) by using the ziehl-neelsen staining  Technique but removing the heating step from the procedure.  This new stain from kinyoun was named the kinyoun stain HISTORY
  • 5. ZIEHL-NEELSEN STAINING PRINCIPLE Acid-fast mycobacterium contain a lipoidal material mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the Gram stain. The primary stain, carbol fuchsin is applied to the cells, and heat and phenol are used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is removed with treatment by acid alcohol (ethanol and hydrochloric acid). A counter stain methylene blue, is then applied to the cells.
  • 6. Cell wall structure of acid fast organism
  • 7. Materials  Slides  Sample  Inoculating loop  Bunsen burner  Cotton  Microscope  Acid alcohol Reagents  Primary Stain : Carbol-fuchsin.  Decolorizer : (HCl+Ethanol)  Counterstain : Methylene blue Requirements of acid fast stain
  • 8. Primary Stain: 0.3% Carbol-fuchsin. Dissolve 50 g phenol in 100 mL ethanol (95%) or methanol (95%) then Dissolve 3 g Basic fuchsin in the mixture and add distilled water to bring the volume to 1 L. Decolourizer Solution: Sulfuric Acid (25%) > Slowly add 250 mL sulfuric acid (at least 95%) to 750 mL distilled water. Cool and mix well before using. or 3% acid alcohol : > Add 3% concentrate HCL in 95% ethanaol . Cool and mix well before use. COUNTERSTAIN: 0.3% Methylen blue Dissolve 3 g methylene blue in 1 L distilled water. Reagent preparation method
  • 9. PROCEDURE OF ACID- FAST STAIN BACTERIAL SMEAR PREPARATION: Smear is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope. Method: Sterilize the Slides : In order to have optimal conditions all the slides to be used must be sterilized with a fire source (typically a spirit lamp) applying the heat on both faces for a few seconds From culture sample : -Aseptically a small sample of the culture is spread over a slide surface. -This is then allowed to air dry. -The next step is heat fixation to help the cells adhere to the slide surface. -The smear is now ready for staining
  • 10.  INCASE OF SPUTUM SAMPLE OR OTHER SEMISOLID SAMPLE EXTRACT THE MOST USEFUL PARTS OF THE SPUTUM SAMPLE EXTRACT THE PURULENT AND BLOODY PURULENT PARTS OF THE SAMPLE, SINCE THEY CONTAIN ALL THE BACILLI, AND SPREAD THEM ON THE SLIDE MAKING A THIN UNIFORM BARELY TRANSPARENT SMEAR WITH A WOODEN STICK OR A COTTON HYSSOP. ONCE THIS IS DONE FIX THE SPREAD WITH A LITTLE HEAT FROM THE SPIRIT LAMP APPLIED (ONLY) UNDER THE SLIDE.
  • 11. 1.Pour 0.3% Carbol Fuchsin 2.Gently heat until vapour rises 3.Carbol fuchsin 3-5 mins 4.Rinse with Tap Water & Decolorize with 25% sulphuric acid 5. Let it stand for 15 -30 seconds & rinse gently with tap water 6. Pour 0.3% methylene blue & leave for 1 min to 30 sec
  • 12. Some Acid fast organims under microscope 100x M.leprae M.avium M.tuberculosis