Hi everyone, and welcome to Biology Professor. Today, we're going to be
talking about the Acid Fast Stain. Now, in the laboratory, when trying to identify
different types of bacteria in a given sample, the most common type of
differential stain used is called the Gram Stain. It differentiates between
gram-positive and gram-negative cells based on their cell wall characteristics.
If you're interested in learning more about that, you can either see my video
on the gram positive versus gram-negative cells or you can also see
my video on the Gram Stain procedure. Now interestingly, there are some types of
bacteria that they just can't be distinguished with the Gram Stain. These
are called gram-indeterminate bacteria. For example, there are some types of
bacteria that they have gram positive cell walls, meaning that they have that
characteristic thick peptidoglycan layer covering a plasma membrane and they lack
gram-negative cell characteristics, for example they don't have the outer
membrane and they don't have any LPS, but they're gram-indeterminate, they just
don't stain well with the Gram Stain. The reason for this is that these bacteria
contain something in their cell wall called mycolic acid
and bacteria that have mycolic acid in their cell wall are referred to as acid
fast bacteria and they can be distinguished from non acid fast
bacteria by the Acid Fast stain. The mycolic acid component is this really
waxy lipid component it is in the cell wall and it's what prevents the Gram
stain reagents from accurately staining these bacteria, however, there is another
dye that can be used with the Acid Fast stain called carbolfuchsin.
Carbolfuchsinis a red dye, it's used first and it will actually dye all of the bacteria
in a sample this red color. The next step is the use of acid alcohol.
Acid alcohol is a decolorizer. For non acid fast bacteria, treatment of acid alcohol will
make the red carbolfuchsin dye come out of the cell wall so that the cell wall
is colorless, however carbolfuchsin is much more soluble in mycolic acid than
it is an acid alcohol. This means that despite the use of acid alcohol, acid
fast bacteria that have all this mycolic acid, they stay red. The final step of the
acid fast stain is to use a counter stain. This is just some stain that will allow
the non acid fast bacteria to be visualized a little better.
The most common counter stain used with acid fast stains is methylene blue.
Methylene blue will stain non acid-fast bacteria blue while the
acid-fast bacteria still have all of these carbolfuchsin dye,
thus they remain red. So now under a microscope, acid-fast bacteria can be
differentiated from non-acid fast bacteria with this stain.
A very common pathogen that is identified by use of the acid fast stain
are species of Mycobacterium. These include Mycobacterium tuberculosis which
causes tuberculosis, also Mycobacterium leprae
which causes leprosy, so this is the main stain for identifying those pathogens
in human samples.