Tripping on Acid-Fast Staining

As we continue to characterize our soil microbe, our sights turned to determining whether our bacteria is acid-fast. This was done using Acid-fast stain, which uses the same process as a Gram stain except for the decolorizing agent used following a primary stain. The name Acid-fast derives from this variation in decolorizing agent: the combination of a mineral acid and organic solvent, such as ethanol, either together or separate.
The decolorizing agent differs from that of the Gram stain due to the physical attributes of bacteria with the acid-fast property. These bacteria all have a cell wall with a high lips content that when grown on agar medium display a wrinkled, dry surface and a pellicle in liquid medium. The lipid saturated cell wall absorbs the primary stain, so that when viewed under a microscope the microbe appears pink. A non acid-fast microbe appears blue due to the methylene blue counter stain. The Ziehl-Neelson Method is the most commonly used procedure, using carbon-fuchsin as a primary stain.
But characterizing a bacteria as acid-fast is only applicable to the genera Norcardia and Mycobacterium. Even though few species are represented, many medically pertinent bacteria display this property, such as Mycobacterium tuberculosis, the tuberculosis causing bacteria.

In order to create a point of reference from which to compare our unknown bacteria, we began by staining a known non acid-fast bacteria, Bacillus megaterium. This acted as our negative control. Below shows a microscope view of our results. The bacteria appears dark purple which indicates that the carbon-fuchsin was not absorbed into the cell wall. They also have a diplobacilus rod-shape.

Bacilus megaterium Acid-Fast Stain

After the acid-fast stain, the soil microbe appeared lighter in color compared to the B. megaterium, as seen below. But it is unclear whether the lighter hue indicates acid-fast properties or a mishap in the experimental procedure. Both cocci and bacilus bacterium are present in the culture.

Unknown Bacteria Acid-Fast Stain

To ascertain the identity of the soil microbe we can use a dichotomous key. It is clear that our microbe is prokaryotic in origin, rather than eukaryotic. We previously established that the organism is gram-positive and rod-shaped, which leads to the question of acid-fast properties. Because the results of the acid-fast stain were unclear, it will be necessary to run more tests, such as determining catalase activity. This will help determine is the bacteria is acid-fast in nature.