Ziehl-Neelsen staining

or
Acid-Fast Staining
INTRODUCTION
• Differential staining method – Identify Acid Fast Bacilli (AFB).
E.g.: Mycobacterium tuberculosis.
• The acid-fast staining method is a modification of Paul
Ehrlich’s (1882) method.
• Ziehl-Neelsen modified the original method.
• The Ziehl-Neelsen acid-fast staining is most useful for staining
acid-fast bacilli belonging to the genus Mycobacterium
especially Mycobacterium tuberculosis and Mycobacterium
leprae, and also for Nocardia.
PRINCIPLE
•Acid fastness of acid-fast bacilli is attributed to
the presence of large quantities of the
unsaponifiable wax fraction called mycolic
acid in their cell wall and also the intactness of
the cell wall.
•The degree of acid fastness varies in different
bacteria.
PRINCIPLE
• In this staining method, the application of heat
helps the dye (a powerful staining solution
containing carbol fuchsin and phenol) to penetrate
the tubercle bacillus.
• Once stained, the stain cannot be easily removed.
• The tubercle bacilli resist the decolourizing action
of acid-alcohol which confers acid fastness to the
bacteria.
PRINCIPLE
• The other microorganisms, which are easily
decolourised by acid or acid-alcohol, are
considered non-acid fast.
• The non-acid fast bacilli readily absorb the colour
of the counterstain (methylene blue) appearing
blue, while the acid-fast cells retain the red colour
of the primary stain (carbol fuchsin).
REQUIREMENTS
• I Equipments:
• Compound light microscope.
• II Reagents and glass wares:
• Bunsen flame/ torch soaked in methylated spirit, loop wire, glass
slides, slide rack, strong carbol fuchsin, acid/acid-alcohol (25 % H2SO4
or 43 ml HCl + 97 ml ethanol) (decolorizing agent), and Loeffler’s
methylene blue (counter stain).
• III Specimen:
• Sputum smear positive for tubercle bacilli/culture smear of
Mycobacterium species.
PROCEDURE
• 1.Smerar preparation:
• Smear of 2x3 cm is prepared in a new clean grease-free
slide from a purulent portion of the sputum. (thick,
yellow-green, or brownish)
• 2.Heat fixation:
• Fix the smear by passing the slide 2–3 times gently over
the flame with the smear side up.
PROCEDURE
• 3.Primary staining:
• Put the smears on a slide rack and cover the smear with strong
carbol fuchsin. Allow it to stain for 5 minutes.
• During this period, heat the slides from below intermittently
by a Bunsen flame or torch soaked in methylated spirit
without boiling the solution, until the steam rises.
• Heating helps the stain to penetrate.
• Do not allow the stain to dry on the slide, and if necessary add
more carbol fuchsin to cover the smear.
• Rinse the smears gently under tap water.
PROCEDURE
• 4.Decolorization:

• Cover the smear with 20% sulphuric acid for at least 2-4
minutes for decolorization.
• Wash the slides thoroughly with water to remove all
traces of acid and drain the water.
• Clean the back of the slide if needed.
PROCEDURE
• 5.Counterstaining:

• Cover the smear with Loeffler’s methylene blue for 20-

30 seconds.
• Rinse the smears again under tap water and air dry it.
• Observe the smear first under low power (10x)
objective, select a suitable area, and then under oil
immersion (100x) objective.
PROCEDURE
• Examination:
• The smear should be examined following a zig-zag
pattern for at least 100 fields, before declaring the
smear negative.
• Record the observations in the note book.
• Findings are recorded, together with grading of the
positive smear.
30 sec
5 min 25% H2SO4
For 2-4 min
 OBSERVATION

•Presence of 3 x 0.3 µm sized red colored

long slender, straight or slightly curved,
beaded less uniformly stained structures
which are scattered amidst blue-coloured
pus cells and background.
 RESULTS AND INTERPRETATION
•The stained smear contains red-colored acid-
fast bacilli seen among the blue-colored
multilobed pus cells.
•The smear is positive for acid-fast bacilli.
•Probably, the smear contains Mycobacterium
tuberculosis
 RED AFB

RED AFB

c e lls
Bl ue

Blue B.G

Beaded appearance
Counting
List of acid –fast structures
Organisms/Structures and suitable % acid to
stain
Grading (RNTCP or the Revised National
Tuberculosis Control Program)
Kinyoun’s method
• Modified Ziehl-Neelsen staining.
• Procedure is similar to Ziehl-Neelsen stain, but does not
involve heating the slides.
• Kinyoun staining method uses carbol-fuchsin as a primary
stain, followed by decolorization with an acid-alcohol
solution and methylene blue as a counterstain.
• Kinyoun carbol-fuschsin has a greater concentration of
phenol and basic fuchsin and does not require heating in
order to stain properly.
OBSERVATION
Red-colored
ACID-FAST BACILLI

Pus cells

} non AFB