The document summarizes the history and process of acid-fast staining. It describes how in 1882, Robert Koch discovered the tubercle bacillus and developed an early staining method, which was then improved by others including Ziehl and Neelsen, whose combined method became standard. The Ziehl-Neelsen method uses heat to drive stain into acid-fast bacteria's waxy cell walls. It reliably identifies bacteria with mycolic acid in their cell walls, including Mycobacterium tuberculosis. The process involves staining, heating, decolorizing with acid, and counterstaining to identify acid-fast bacteria as red and non-acid fast as blue.

1. Franz Arla O. Palevino
8- Daffodil

2. History of Acid Fast
Staining
In 1882 ROBERT KOCH reported the
discovery of the tubercle bacillus and
described the appearance of the bacilli
resulting from a complex staining
procedure. During the same time period
several other researchers (Ehrlich, Ziehl,
Rindfleisch, and Neelsen), intending to
improve on Koch’s method, introduced
modifications to the reagents and the
procedure.

3. Franz Ziehl was the first to use carbolic
acid (phenol) as the mordant. Friedrich
Neelsen kept Ziehl’s mordant, but
changed the primary stain to the basic
fuchsin . This method became known as
the Ziehl-Neelsen method in the early
to mid 1890s. In this method heat is
used to help drive the primary stain
into the waxy cell walls of these
difficult-to-stain cells. The use of heat
in this method has been the reason that
this technique is called the “hot
staining” method.

4. The Ziehl-Neelsen method has
endured as a reliable and
effective way to demonstrate the
acid-fast bacteria. The acid-fast
stain is performed on samples to
demonstrate the characteristic
of acid fastness in certain
bacteria and the cysts
of Cryptosporidium and Isospora.

5. What is Acid-Fast
Staining
Acid-fast staining, also
known as the Ziehl Neelsen
stain, is used to identify
specialized bacteria that have
waxy mycolic acid in their cell
wall. The presence of mycolic
acid in bacteria is rare, only
found in two genera—

6. —
MYCOBACTERIA and NOCA
RDIA. The bacteria that
possess mycolic acid are
considered “acid fast,”
whereas the vast majority of
bacteria, those that do not
have cell walls containing
mycolic acid, are
considered “non-acid fast”.

7. Due to their waxy cell wall,
these bacteria are
highly resistant to staining
and
treatment. Mycobacterium
tuberculosis , the
causative agent of TB, is one
example of a bacterial
cell with an acid-fast cell wall.

8. Acid-fast organisms include
Mycobacteria, Nocardia,
Cryptosporidium parvum,
isospora, cyclospora
cysts, Legionella micdadei
and the head of a sperm.

9. Gram Staining Vs. AcidFast Staining
TheGram stain is
a technique used to
distinguish between Grampositive cells that have a
thick layer of peptidoglycan
while Acid- fast staining is
used to identify bacteria with
waxy cell walls

10. ACID FAST STAINING
1. Prepare smears of
organisms to be stained.
2. Heat fix the smears.
3. Cut or tear absorbent
paper (bibulous paper) to
fit the slide leaving one
end for handling. Do not
allow the paper to
protrude beyond the
slide, but the smears must
be covered.

11. ACID FAST STAINING
4. Place the slide on
wire gauze on a ring
stand.
5. Saturate the paper
with carbolfuschin.

12. ACID FAST STAINING
6. Heat the slides with a hand-held
bunsen burner until steam can be
seen rising from the
surface. Alternately remove the
burner and reheat the slide to
maintain steaming for 3-5
minutes. As the paper begins to dry
during the staining process add a
drop or two of carbolfuschin to
keep the slide moist. Adding too
much stain will cool the slide (and
drip on the bench). Overheating the
slide or letting it dry will distort
the cells. Under heating the slide
will fail to stain acid-fast cells.

13. ACID FAST STAINING
7. At the end of staining
remove the paper with
tweezers and wash the
slide thoroughly.
8. Drain the slide.
9. Decolorize with acidalcohol for 30 seconds.

14. ACID FAST STAINING
10. Rinse, drain, and
counterstain with methylene
blue for 45 seconds.
11. Rinse, blot, and
examine. First observe each
organism on its separate
smear. Then examine the
mixed smear.
12. Acid-fast organisms will
appear red and non-acid-fast
organisms will be blue.