Exercise 4

Acid-Fast Staining

1. Look at the picture below and identify the following:

Acid-fast reaction:
Morphology/Shape:
Method used:

Acid-fast reaction:
Morphology/Shape:
Method used:

2. What is responsible for the acid-fastness of these organisms?

- The acid-fast stain is used to identify acid-fast organisms. The high mycolic acid content
in organisms' cell walls is responsible for their acid-fastness, which results in a staining
pattern of poor absorption followed by strong retention.

3. Why is the Ziehl-Neelsen method called the “hot method” while the Kinyoun method the
“cold method?” Explain briefly.
- The fundamental difference between the two carbolfuchsin-based methods is whether
heat is used during the primary staining process. The Ziehl-Neelsen method is also known
as the “hot method” since it uses heat to infuse the carbolfuchsin into the acid-fast cells,
whereas the Kinyoun method is also called as the “cold method” because it does not use
heat.

4. Differentiate the two methods of acid-fasting staining.

Reagent Function Expected Result

Ziehl-Neelsen Kinyoun

Carbon fuchsin It is used as the In the Ziehl-Neelsen The Kinyoun stain is

primary stain dye to stain, a fixed smear modified to make
detect acid-fast covered with carbon heating unnecessary.
bacteria because it is fuchsin is heated, The organisms
more soluble in the rinsed, decolorized appear as slightly
cells' wall lipids than with acid-alcohol, bent, beaded rods 2
in the acid alcohol. and counterstained to 4 µm long and 0.2
with methylene blue. to 5 µm wide.

Acid alcohol It has the ability to After the auramine Acid-alcohol, that's
completely dye has fully stained why they are known
decolorize all non- the smear, a drop of as acid-fast. They
acid-fast organisms, acid alcohol is will keep their red
thus only leaving applied for one to coloration
behind red-colored two minutes to throughout the
acid-fast organisms, decolorize the staining process.
like M. tuberculosis. smear.
The slides are then
stained a second
time with methylene
blue that serves as a
counterstain.

Safranin The safranin stain The modified It gives better

works by binding to safranin technique differentiation
acidic proteoglycans produces a more between oocysts and
in cartilage tissues uniform staining of yeast.
with a high affinity these oocysts.
forming a reddish-
orange complex.