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Acid-Fast Stain: A Technique for Bacterial Differential Staining, Study notes of Bacteriology

The acid-fast stain is a microbiological technique used to differentiate bacteria based on the lipid content of their cell walls. the steps for performing the acid-fast stain using both steam heat and Kinyoun's Acid-Fast Reagent. The process involves heat-fixing and staining cells with carbol fuchsin, decolorizing with acid-alcohol, and counterstaining with methylene blue. The document also includes questions related to the acid-fast stain, such as its similarities to the Gram stain, the role of mycolic acid, and the likelihood of Gram-negative bacteria being acid-fast.

What you will learn

  • How does mycolic acid affect the staining process?
  • What would be the result if acetone-alcohol was used instead of acid-alcohol for decolorization?
  • What is the main difference between the acid-fast and Gram stains?
  • Can Gram-negative bacteria be acid-fast?
  • What color do acid-fast cells appear when stained with the acid-fast stain?

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Exercise 6-C
STAINING OF MICROORGANISMS
ACID-FAST STAIN
Introduction
The acid-fast stain is a differential stain that separates bacteria on the basis of the lipid content of their
cell walls. Bacteria categorized as acid-fast (members of the genus Mycobacterium and some other
Actinobacteria) have Gram-positive walls with thick peptidoglycan containing large quantities of a wax-
like lipid called mycolic acid. These cells are resistant to staining with normal procedures, and require
severe treatment, e.g., heat and potent dyes, in order to be stained. Once stained, their walls do not
readily decolorize, and so can be differentiated from non-acid-fast cells. The acid-fast stain is used
primarily in the diagnosis and study of diseases caused by acid-fast bacteria (Tuberculosis and Leprosy),
however it can also be used on a variety of non-pathogenic organisms. The basic steps in this technique
are as follows:
1. Bacteria cells are heat-fixed and stained with a potent primary staining reagent called carbol fuchsin
while being subjected to steam heat. An alternative procedure uses a different formulation of carbol
fuchsin (“Kinyoun’s Acid-Fast Reagent”) which does not require the application of steam heat.
2. The stained cells are subjected to decolorizing with an acid-alcohol solution; 95% ethyl alcohol
containing 2.5% concentrated nitric or hydrochloric acid (HNO3 or HCl). This step will remove the
primary stain from cells that are not acid-fast, rendering them colorless.
3. Non-acid-fast cells are counterstained with a basic dye of a contrasting color (e.g., methylene blue).
When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the
acid-fast cells will be stained red and the non-acid-fast cells will be stained blue. The presence of
purple-colored cells usually indicates the procedure has been improperly completed; however, some
cells are only partially acid-fast and others may contain acid-fast structures. The endospores formed by
bacteria in the genus Bacillus will often stain red when subjected to the acid-fast stain.
Standard Procedure Using Steam Heat
1. Place about 200 ml of water in a 400 ml beaker on a wire gauze platform over a Bunsen burner
and heat the water to generate steam.
2. At one end of a clean glass slide, prepare a small, mixed smear containing a Mycobacterium
species and Staphylococcus aureus. For best results, thoroughly mix a small quantity of the
Mycobacterium with a loopful of water and break up the cell clusters before adding the
Staphylococcus. At the other end of the slide, prepare a small smear of your morphological
unknown.
3. Allow the smears to air dry and then heat-fix them. It is important to insure that the cells used
are dead, but do not apply excess heat.
4. Place a wire mesh platform over the beaker and place the slide on the platform smear side up.
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Exercise 6-C

STAINING OF MICROORGANISMS

ACID-FAST STAIN

Introduction The acid-fast stain is a differential stain that separates bacteria on the basis of the lipid content of their cell walls. Bacteria categorized as acid-fast (members of the genus Mycobacterium and some other Actinobacteria) have Gram-positive walls with thick peptidoglycan containing large quantities of a wax- like lipid called mycolic acid. These cells are resistant to staining with normal procedures, and require severe treatment, e.g., heat and potent dyes, in order to be stained. Once stained, their walls do not readily decolorize, and so can be differentiated from non-acid-fast cells. The acid-fast stain is used primarily in the diagnosis and study of diseases caused by acid-fast bacteria (Tuberculosis and Leprosy), however it can also be used on a variety of non-pathogenic organisms. The basic steps in this technique are as follows:

  1. Bacteria cells are heat-fixed and stained with a potent primary staining reagent called carbol fuchsin while being subjected to steam heat. An alternative procedure uses a different formulation of carbol fuchsin (“Kinyoun’s Acid-Fast Reagent”) which does not require the application of steam heat.
  2. The stained cells are subjected to decolorizing with an acid-alcohol solution; 95% ethyl alcohol containing 2.5% concentrated nitric or hydrochloric acid (HNO 3 or HCl). This step will remove the primary stain from cells that are not acid-fast, rendering them colorless.
  3. Non-acid-fast cells are counterstained with a basic dye of a contrasting color (e.g., methylene blue). When a mixed culture containing both acid-fast and non-acid-fast cells is subjected to this procedure, the acid-fast cells will be stained red and the non-acid-fast cells will be stained blue. The presence of purple-colored cells usually indicates the procedure has been improperly completed; however, some cells are only partially acid-fast and others may contain acid-fast structures. The endospores formed by bacteria in the genus Bacillus will often stain red when subjected to the acid-fast stain. Standard Procedure Using Steam Heat
    1. Place about 200 ml of water in a 400 ml beaker on a wire gauze platform over a Bunsen burner and heat the water to generate steam.
    2. At one end of a clean glass slide, prepare a small, mixed smear containing a Mycobacterium species and Staphylococcus aureus. For best results, thoroughly mix a small quantity of the Mycobacterium with a loopful of water and break up the cell clusters before adding the Staphylococcus. At the other end of the slide, prepare a small smear of your morphological unknown.
    3. Allow the smears to air dry and then heat-fix them. It is important to insure that the cells used are dead, but do not apply excess heat.
    4. Place a wire mesh platform over the beaker and place the slide on the platform smear side up.
  1. Cover each smear with a SMALL section of paper towel (just cover the smear). Make certain the paper does not extend over the edge of the slide in any direction, as this will allow most of the stain reagent to run off, potentially staining the bench top.
  2. Apply several drops of carbol fuchsin to the smear (thoroughly wetting the paper towel) and allow it to act over steam-heat for 10-15 minutes (apply more reagent as needed). This will color all cells in the smears red. Caution - Do not allow the paper towel to dry out during the staining procedure, and avoid excess flooding.
  3. Remove the slide from the stain rack, hold it in the sink and rinse the smears thoroughly with tap water. The paper towel sections covering the smears will be rinsed off during this process, and can be removed from the sink when staining is completed.
  4. Decolorize each smear with acid-alcohol (95% ethyl alcohol containing 2.5% concentrated hydrochloric acid) by allowing the reagent to flow across the smear. Watch the color run off, and when it stops moving, rinse immediately with water. This decolorizing step will remove the red color from non-acid-fast cells leaving them colorless. Caution – do not over decolorize!
  5. Rinse the smears thoroughly with water to be certain the action of the decolorizing reagent has been stopped.
  6. Counterstain the smears by placing the slide on a stain rack over the sink, applying methylene blue and allowing it to act for 60 seconds without heat. This counterstain will color the non- acid-fast cells blue.
  7. Rinse the slide thoroughly with tap water, remove excess water from the slide bottom and allow the smears to air dry (place near the base of a lit Bunsen burner).
  8. Examine both smears under oil immersion (focus with your 10X objective first) and compare the results. In a properly prepared acid-fast stain, the rod-shaped Mycobacterium cells will appear red and the non-acid-fast Staphylococcus cells will appear blue. Make sketches of the cells present and record their characteristics. Cells from an unknown culture can be expected to appear either red (acid-fast) or blue (non-acid-fast); however, Bacillus cells containing endospores will often appear blue with red spores. Alternative Procedure Using Kinyoun’s Acid Fast Reagent
  9. At one end of a clean glass slide, prepare a small, mixed smear containing a Mycobacterium species and Staphylococcus aureus. For best results, thoroughly mix a small quantity of the Mycobacterium with a loopful of water and break up the cell clusters before adding the Staphylococcus. At the other end of the slide, prepare a small smear of your morphological unknown.
  10. Allow the smears to air dry and then heat-fix them. It is important to insure that the cells used are dead, but do not apply excess heat.
  11. Place the slide on a stain rack and stain each smear by covering it with Kinyoun’s Acid-Fast Reagent. Allow the stain to act for 10 minutes.
  12. While holding the slide in the sink, rinse the smears thoroughly with tap water.

Name ________________________________ Lab Section _______________ WORKSHEET Exercise 6C Staining of Microorganisms: Acid-Fast Stain Goals: __________________________________________________________________________



Materials & Methods: Age of Unknown: ____________ Reagents used: ________________________________________


Data & Results: A) Acid-Fast Positive & Negative Controls B) Morphological unknown Specimen: Total Magnification: Length: units x μm/unit = μm Width: units x μm/unit = μm Notes: Specimen: Total Magnification: Length: units x μm/unit = μm Width: units x μm/unit = μm Notes:

Acid-Fast Results Summary: Specimen Data Result NOTE : If your controls do not appear as expected, SOMETHING IS WRONG. Consult your instructor. Conclusions: Based on your results, what can you conclude about the cell walls of your Morph. Unknown? _____



Does the cell morphology of your Morphological Unknown match what you saw for Exercise 6A (indirect stain) and 6B (Gram stain)? ___________ Additional Comments: ______________________________________________________________