23: ACID FAST STAIN
- Page ID
- 157091
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Explain the purpose of acid-fast staining in microbiology.
- Differentiate between acid-fast and non–acid-fast bacteria based on observed staining results.
BACKGROUND
In microbiology, staining techniques are essential for observing and classifying bacteria based on their structural characteristics. While most bacterial species are effectively stained using the Gram stain, some species possess unique cell wall structures that make them resistant to standard staining techniques. One such group of organisms includes acid-fast bacteria, which are characterized by a thick, waxy cell wall rich in mycolic acids. These waxy lipids create a highly hydrophobic barrier that protects the cell from desiccation, disinfectants, and many antibiotics. This waxy coat also prevents traditional water-based stains from penetrating the cell wall. To visualize these bacteria, microbiologists use acid-fast staining—a differential staining method that distinguishes acid-fast organisms from non–acid-fast ones based on their ability to retain dye. The Kinyoun method, also called the 'cold' acid-fast stain, is a common variation of this technique. It uses a concentrated carbolfuchsin solution along with phenol to allow the dye to enter the waxy cell wall without the need for heat, making it safer and more practical in teaching labs. The Kinyoun stain is particularly important in identifying bacteria from the genus Mycobacterium. Two major species in this genus are Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and Mycobacterium leprae, which causes leprosy (Hansen’s disease). Tuberculosis is a serious infectious disease that primarily affects the lungs but can also spread to other organs. It is spread through airborne droplets when an infected person coughs or sneezes. Symptoms include a chronic cough, weight loss, night sweats, and fever. TB remains a leading cause of death in many parts of the world, especially in immunocompromised individuals such as those with HIV/AIDS. Leprosy, caused by M. leprae, affects the skin, peripheral nerves, mucosal surfaces, and eyes. It is a slow-progressing disease and is transmitted through prolonged close contact, likely via respiratory secretions. Symptoms include skin lesions, numbness, muscle weakness, and deformities if left untreated. Though rare in many countries, leprosy still affects thousands of people globally each year. During the Kinyoun staining process, the slide is first flooded with carbolfuchsin for several minutes. Acid-fast bacteria will retain this red dye even after being washed with acid-alcohol, the decolorizing agent. Acid-alcohol is used in acid-fast staining instead of regular alcohol because it provides the necessary strength to differentiate between acid-fast and non–acid-fast bacteria. The waxy lipids and extremely hydrophobic cell wall of the Acid-fast organisms make the cell wall extremely resistant to the penetration and removal of dyes by mild solvents like regular alcohol.
Acid-alcohol, which contains both alcohol and a small amount of acid (typically hydrochloric or sulfuric acid), is far more effective. The acid component increases the strength of the decolorizer, allowing it to strip the carbolfuchsin dye from non–acid-fast cells while leaving the acid-fast cells stained. A counterstain such as methylene blue is then applied, staining the background and non–acid-fast bacteria a contrasting blue color
• - Acid-fast bacteria → bright red/pink
• - Non–acid-fast bacteria → blue
MATERIALS (Per Group of 2)
1 Acid Fast organism
1 Non-Acid -Fast organism
2 Clean microscope slides
Eye Protection (Goggles)
Kinyoun Staining Reagents
Carbolfuchsin (Kinyoun stain)
Acid-alcohol (decolorizer)
Methylene blue (counterstain)
Distilled water (for rinsing)
METHODS/PROCEDURES
A. Smear Preparation
1. Label your slide *remember to include the organism names.
2. Aseptically transfer a small amount of each bacterial culture onto either side of the slide:
- If using a solid culture, remember to add a loop or two of distilled water before transferring bacteria.
- Mix gently with the loop to create a thin, even smear.
*Remember to draw a slide diagram of a paper towel with three inoculate circles
*Take care to stay within the boundaries of your diagram.
3. In the middle of the slide put one loop of each culture then mix them together.
4. Place the slide on the slide warmer to air dry completely.
5. Heat-fix the slide.
B. Kinyoun Staining Steps
6. Place the heat-fixed slide on a staining tray.
7. Flood the smear with carbolfuchsin. Let it sit for 5–10 minutes.
8. Rinse gently with distilled water.
9. Decolorize with acid-alcohol until the runoff is nearly clear (10–20 seconds).
10. Rinse again with distilled water.
11. Apply methylene blue for 1–2 minutes.
12. Final rinse with distilled water.
13. Place the slide on the slide warmer to air dry completely.
14. Once completely dry use a sharpie to draw landmarks
15. View the cultures under oil immersion
16. Draw the swears in the Results section.
Print a hard copy to bring to lab (PDF).
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Exercise #28 Acid Fast Stain
NAME ______________________
EXPECTATIONS
Which of the two cultures you used today do you anticipate being stained a bright red/pink?
Why is the acid-fast stain considered a differential stain?
RESULTS
Draw what you see on your acid-fast slide under oil immersion. Identify the acid fast and non-acid-fast cells. Remember to use color.
CONCLUSIONS
1. What color and shape were the acid-fast cells?
2. What color would acid-fast cells be if alcohol were used in place of acid alcohol? Explain your response
3. What color and shape were the non-acid-fast cells?
4. What color would non-acid-fast cells be at the end of the staining process if the acid alcohol wash was replaced with alcohol alone? Explain your response
5. Why are acid-fast bacteria often more resistant to disinfectants and antibiotics?