Staining techniques for the aerobic actinomycetes (most commonly used for the characterization of the bacteria) utilize:
- Direct smears made from tissue (touch preps)
- Direct smears of pus (obtained from draining areas)
- Smears made from the growth seen on primary culture plates
Gram Stain
In addition to the clinical scenario, a Gram stain helps the microbiologist to characterize bacteria.
- Perform a Gram stain using an appropriate specimen such as sputum, bronchial, exudate, fluid sediment, or tissue. (May also Gram stain growth from agar plates.)
- Use a routine Gram stain procedure.
Acid-Fast Stain
Once a Gram stain has been performed (demonstrating the presence of suspect organisms), a modified acid-fast stain (MAS) should be performed.
Cell walls that contain a high content of a waxy lipid called mycolic acid make staining with acid-fast stains difficult. Organisms such as mycobacteria contain a lot of mycolic acid and do not easily decolorize. (These organisms are referred to as "mycolata".)
Most of the aerobic actinomycetes do not have this mycolic acid in the cell wall, with the exception of Nocardia, Gordonia, Rhodococcus, and Tsukamurella.
- Perform a modified acid-fast stain (MAS).
- The modified technique uses a weak acid decolorizer (1% sulfuric acid - instead of the stronger acid-alcohol used for a traditional acid-fast stain).12
- Procedure:
- Fix the smear
- Flood smear with Kinyoun fuchsin-phenol and allow to stain for 5 minutes
- Rinse with tap water, drain
- Flood the smear with aqueous 1% sulfuric acid and destain for 3 minutes
- Rinse with tap water, drain
- Flood the smear with methylene blue and stain for 1 minute
- Rinse with tap water, drain, then air dry
- Examine under oil immersion at x1000 magnification
12. Leber AL, ed. Clinical microbiology procedures handbook. 4th ed. ASM Press; 2016:Section 6.
