ACID FAST STAINING

Franz Arla O. Palevino8- Daffodil

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. 

History of Acid Fast Staining

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.

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.

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—

What is Acid-Fast Staining

—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”.

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.

Acid-fast organisms include Mycobacteria, Nocardia,

Cryptosporidium parvum, isospora, cyclospora

cysts, Legionella micdadei and the head of a sperm.

TheGram stain is a technique used to

distinguish between Gram-positive cells that have a

thick layer of peptidoglycan while Acid- fast staining is

used to identify bacteria with waxy cell walls

Gram Staining Vs. 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.

ACID FAST STAINING

4. Place the slide on wire gauze on a ring

stand.

5. Saturate the paper with carbolfuschin.

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.

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 acid-alcohol for 30 seconds.

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.

ACID FAST STAINING