2120 Lab
Week 4
Experiment 6
Today…..
We are going to stain the mycolic acid in the cell walls of certain
bacteria.
What is mycolic acid?
Which bacteria have mycolic acid?
Why are they clinically significant?
Why use Acid fast stain?
Mycolic acid
Mycolic acid is a waxy lipid that is found in the cell walls of some Gram
positive bacteria.
Brown et al. Nature Reviews Microbiology 13, 620–630(2015)
Mycolic acid
The mycolic acid impede the entry of chemicals causing the organisms
to grow slowly.
They are more resistant to chemical agents and lysosomal
components of phagocytes than most bacteria.
These cell walls have fewer porins compared to the Gram-negative cell
wall and the pores are much longer.
This is thought to contribute significantly to the lower permeability of
acid-fast bacteria.
INH (isoniazid) blocks the incorporation of mycolic acid into acid-fast
cell walls thereby inhibiting synthesis of the acid-fast cell wall.
Bacteria that have mycolic acid
Bacteria of the genus Mycobacterium and Nocardia have mycolic acid
in their cell wall.
Note the reddish acid-fast bacilli (Mycobacterium
sp.) among the blue normal flora and white blood
cells in the sputum that are not acid-fast.
Red acid-fast bacilli of Nocardia sp.
www.cdc.gov
Clinical relevance of bacteria that have mycolic acid
Common acid-fast bacteria of medical importance include
Mycobacterium tuberculosis, Mycobacterium leprae,
and Nocardia species.
Mycobacterium tuberculosis
causes TB
https://medlineplus.gov
Mycobacterium leprae
causes leprosy
N Engl J Med 2012; 366:1433
Clinical relevance of bacteria that have mycolic acid
Nocardiosis is a disease caused by Nocardia found in soil and water
affecting the lungs, brain, and skin.
https://openi.nlm.nih.gov/
Why use acid fast stain and not Gram stain?
Acid-fast bacteria stain poorly with the Gram stain procedure,
appearing weakly Gram-positive or Gram-variable.
So they are usually characterized using the Ziehl-Neelsen acid-fast
staining procedure.
Acid fast stain is a differential staining procedure
Acid fast bacteria (with Mycolic acid) are
RED
Non acid fast bacteria (no Mycolic acid) are
BLUE
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.
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.
Method became known as the Ziehl-Neelsen method in the early 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.
Acid fast staining
Acid fast staining
1. Prepare thin smear of organisms to be stained. Heat fix the smears.
2. Cut or tear absorbent paper (bibulous paper) to fit the slide. Do not allow
the paper to protrude beyond the slide, but the smear must be covered.
3. Place the slide over a small beaker containing water. Place the beaker on
the hotplate.
4. Saturate the paper with carbolfuschin. Make sure you do not spill the
stain.
5. Heat the slide over the boiling water in a beaker until steam can be seen
rising from the surface. Maintain steaming for 8 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. Underheating the slide will fail to stain acid-fast cells.
6. At the end of staining remove the paper with tweezers and wash the slide
thoroughly. Drain the slide
7. Decolorize with acid-alcohol for 30 seconds.
8. Rinse, drain, and counterstain with methylene blue for 45 seconds.
9. Rinse, blot, and examine under oil immersion objective.