Kemari Simpson

February 20,2019
Title: Acid Fast Stain using Mycobacterium smegmatis and Staphylococcus aureus

Introduction: Many times, to view and identify different types of microorganisms,

Gram stain or simple stains can’t be used because of the characteristics or structures
of the bacterial cells. This lab session sought to explore one differential stain
procedure, the Acid Fast stain, also known as Kinyoun stain (cold acid fast stain) with
the use of Mycobacterium smegmatis and Staphylococcus aureus.

Materials: Mycobacterium smegmatis, Staphylococcus aureus , carbol fuchsin with

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detergent(primary stain with mordant), acid+alcohol(decolorizer), brilliant

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green(counter stain)

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1. Slide was obtained, different bacteria was placed in their respective places, air
dried and heat fixed with a bunsen burner.
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2. Carbol fuchsin with detergent was then applied for 10 minutes and then was
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rinsed gently with water.

3. Decolorizer containing acid and alcohol was then applied for 3 minutes and then
rinsed gently with water
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4. The counter-stain Brilliant Green was applied for 1 minute and then rinsed gently
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with water then blotted dry with bibulous paper

Results: After the appropriate steps were performed, the results were obtained through
microscopy. It was observed that the Staphylococcus aureus was blue green,
indicating that it is acid fast negative as it only retained the color of the counter-stain,
while the Mycobacterium smegmatis appeared to be colorless, but upon closer
inspection, small specks of pink/ red areas were seen under the microscope, indicating
that it was acid fast positive as it retained the color of the primary stain.

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 Staphylococcus aureus Mycobacterium smegmatis
Discussion:
1. Major chemical(structural) differences among bacteria that might explain the

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need to use the acid fast stain include the fact that acid fast positive bacteria

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contain mycolic acid which is a long chain fatty acid found in the cell wall,

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and they form the major component of the cell wall of mycolata species.

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Mycolic acid causes the microorganism to resist the staining of basic
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chemicals.
2. Acid fast staining is not widely used as compared to the normal gram stains
because only a few organisms show the responsiveness towards the Acid fast stain
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inorder to detect the mycolic acid. Acid fast stain is generally more useful than the
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gram stain when the acid fast bacteria like Mycobacterium tuberculosis and M. leprae
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are suspected to exist in a medium. The bacterial genera that are considered to be acid
fast are Mycobacterium and Nocarida.
3. The heating of the bacterial smear during a Ziehl-Neelson stain promote entry of
carbol fuchsin as the temperature allows the mycolic acid or the waxy substance seen
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on the surface to melt, and this allows the carbol fuchsin to penetrate the cell wall
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more easier and in a smaller amount of time. The Kinyoun procedure is referred to as
“Cold Acid Fast Stain” because there is no use of fire to assist in the penetration of the
primary stain into the cell wall, unlike the Ziehl-Neelson procedure, otherwise known
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as the “Hot Acid Fast Stain”. Also, detergent is added to the carbol fuchsin as it helps
to wash away the mycolic acid to speed up the penetration of the dye through the cell
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wall.
4. Acid fast negative bacteria can be stained by carbol fuchsin, but is then removed
with the decolorizer. The Acid Fast Stain is considered a differential stain because it
distinguishes organisms with waxy cell walls that can resist decolorization with acid
alcohol.

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 Bibliography
1. Nataraj, V., Varela, C., Javid, A., Singh, A., Besra, G. S., & Bhatt, A. (2015).
Mycolic acids: deciphering and targeting the Achilles' heel of the tubercle
bacillus. Molecular microbiology, 98(1), 7-16.
2. General Microbiology-Microbiology I (Lab). (2011). Retrieved February 23,
2019,fromhttps://www.koofers.com/flashcards/biol-microbiology-exam-i-la/review
Northwest Missouri State University

3. Leboffe, M., & Pierce, Burton E. (2016). Microbiology : Laboratory theory &
application (BRIEF third ed.).

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