Acid-fast staining works similarly to gram staining, but uses
an acid-alcohol mixture as the decolorizing agent instead of using just alcohol
as in gram staining. The procedure for
preparing a smear is similar to the protocol we used last week for gram staining.
At this point, we are basically masters at this technique. For a quick
refresher, the protocol for smearing includes plating microbes on a slide with
heat fixation; acid-fast staining becomes distinct from gram staining at this
point in the procedure. The slide with the fixed microbe is heated over
steaming water and flooded with dye. The
steam helps drive the dye into the thick cell walls of the microbes. Acid-alcohol
is used to rinse the excess stain, and the slide is then flooded with a
secondary dye that allows for differentiation between acid-fast and
nonacid-fast microbes.
 |
| Our acid-fast soil microbe |
Two genus of bacteria,
Mycobacterium
and
Nocardia, cannot be properly
stained using any technique other than acid-fast staining.
These bacteria are tough to stain because of their
thick, lipid-filled cell walls that are impermeable to many dyes that are
commonly used in staining.
Bacteria with
acid-fast properties require higher concentrations of dyes and heat to
penetrate their cell walls.
The
high-lipid content of their cell walls also makes it difficult to rinse the
excess dye from inside the cell.
Consequently, this characteristic is how these types of bacteria came to
be known as acid-fast, since the cells hold fast to the dyes and require acid
to be removed.
Acid-fast cells retain
the reddish-pink dye, while nonacid-fast cells appear blue due to secondary
staining.
 |
| Nonacid-fast control, M. luteus |
As can be seen in the photos, we determined that our
mystery microbe was, indeed, acid-fast because of its reddish-pink color. It is
visibly distinct compared to the deep indigo color of the nonacid-fast control.
According the dichotomous tree provided to us, the acid-fast nature of our
microbe narrows its possible identity to two genus:
Mycobacterium and
Nocardia.
In order to help us further describe our mystery microbe,
this week’s lab will focus on the organism’s catalase activity and carbohydrate
metabolism. Tune in next week for more info!!!
- Anne and Austin
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