MICROBIOLOGY LABORATORY
Lab Report # 8 (ACID-FAST STAINING)
I. Research and attach the microscopic appearance of acid fast and non-acid fast bacteria and explain
how acid-fast and non-acid fast bacteria will look after Ziehl Neelsen and Kinyoun Staining.
Acid fast bacteria Non-acid fast bacteria
During Ziehl Neelsen staining and Kinyoun, acid-fast bacteria will look red because both of these
staining procedure uses carbolfuchsin as their primary stain. In Ziehl Neelsen, mycobacterium does not
bind readily to simple stains and therefore the use of heat along with carbolfuchsin and phenol allows
penetration through the bacterial cell wall for visualization but in Kinyoun, heating is not included but it
is the duration of how will the sample be exposed. Non-acid fast bacteria in Ziehl Neelsen and Kinyoun
staining will be stained blue because the counterstain used in these procedures is methylene blue.
II. Fill out the table below. EXPECTED RESULT
REAGENT/PROCEDURE FUNCTION ZIEHL NEELSEN KINYOUN
Both acid-fast bacteria Both acid-fast bacteria
Application of Carbolfuchsin will serve and non-acid fast and non-acid fast
Carbolfuchsin as the primary stain bacteria will be stained bacteria will be stained
red. red.
This procedure allows
The red stain will be Not applicable/No heat
penetration of the
Application of heat absorb by the acid-fast is used in Kinyoun
stain through bacterial
bacteria. procedure
cell wall
There is no need for
Bacteria sample will be Allows the stain to The red stain will be
the Ziehl Neelsen
exposed to the stain in penetrate to the absorb by the acid-fast
method to wait 15
15 minutes bacterial cell wall bacteria
minutes
Acid-fast bacteria will Acid-fast bacteria will
To decolorize other retain the red color retain the red color
Application of acid
bacteria that are not while the non-acid fast while the non-acid fast
alcohol
acid-fast bacteria will be bacteria will be
decolorized decolorized
Application of Used as a counterstain Non-acid fast bacteria Non-acid fast bacteria
will have the color of will have the color of
Methylene Blue
blue blue
III. Answer the following questions.
1. What is the principle behind acid-fast staining?
- Acid-fast bacteria have mycolic acid and this layer is very tough because of its high concentration. In
acid-fast staining, the bacterial smear is stained with carbolfuchsin and these substance solubilizes the
lipoidal material present in the Mycobacterial cell wall. In addition heat is added for the stain to be
further penetrated through the cell wall up to the cytoplasm. With these, the mycobacterial can be
stained and observe with a color under the microscope.
2. What is the purpose of heating with application of the primary stain during acid-fast staining?
- The purpose of heating or the use of heat along with carbolfuchsin and phenol allows penetration
through the bacterial cell wall. Heat will loosen up the waxy mycolic acid layer and promotes the entry
of the primary stain inside the cell.
3. What will be the microscopic appearance of Mycobacterium after gram staining? Explain why.
- The microscopic appearance of mycobacterium after gram staining will be uncoloured. It is not colored
or not seen because it has a thick waxy coating on its cell layer and this is called the mycolic acid. This
coating makes the cell unaffected by the stains in the gram staining procedure.
4. What are some diseases caused by acid-fast bacteria?
- According to [Link], there are various disease that is caused by acid-fast bacteria and these
are tuberculosis, leprosy, and Mycobacterium Avium Complex.
5. What makes the acid-fast bacteria more pathogenic than other bacteria?
- Acid-fast bacteria becomes more pathogenic in a way that it is very difficult for our body or natural
immune system to destroy or kill. These mycobacteria is very tough because of the high concentration of
mycolic acids in their cell wall. These mycolic acids form a thick, waxy layer that makes them very
resistant to environmental stressors, like antibiotics.
6. How are the results of sputum smears reported after AFB staining?
- The results of the sputum smears is identified by negative or positive. A normal result for an acid-fast
bacteria smear is negative, meaning no bacteria were found in the sputum sample. A positive result
means that bacteria were found and that you may have an infection.
