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Microbiology Lab Midterm- Lab 8: Acid-Fast Staining
Terms in this set (31)
What type of stain is the acid-fast stain?
Who discovered that Mycobacterium tuberculosis retained the primary stain even after washing with an acid-alcohol mixture?
Paul Ehrlich (1882)
Which families are acid-fast?
Mycobacteriaceae, Nocardiaceae, Gordoniaceae, Dietziaceae, and Tsukamurellaceae
Which of the families contain pathogenic species?
mycobacterium and nocardia
What can the acid-fast technique be used to diagnose?
tuberculosis and leprosy
What do the cell walls of acid-fast organisms contain that make them impermeable to most stains?
What response does the mycolic acid enhance?
inflammation response initiating the formation of a tubercle, a walled off lesion
What is thought to be the factor in the resistance of Mycobacterium to harsh, dry environments?
What is done to mycobacterium before it is cultured?
a clinical specimen is usually treated with strong sodium hydroxide to remove debris and contaminating bacteria. The mycobacteria are not killed by this procedure.
Who developed the most widely used acid-fast techniques?
Franz Ziehl, Friedrich Neelsen, and Joseph J. Kinyoun
What steps are taken in the Ziehl-Neelsen procedure?
The smear is flooded with carbolfuchsin (a dark red dye containing 5% phenol), which has a high affinity for the mycolic acid in the bacterial cell. The smear is heated to facilitate penetration of the carbolfuchsin stain into the bacteria. The stained smears are washed with an acid-alcohol mixture that easily decolorizes most bacteria except acid-fast microbes. Methylene blue is then used as a counterstain to enable you to observe the nonacid-fast organisms.
What steps are taken in the Kinyoun modification?
It is called a cold stain because the concentrations of phenol and carbolfuchsin are increased so that heating isn't necessary.
What is the mechanism of the acid-fast stain?
The result of the relative solubility of carbolfuchsin and the impermeability of the cell wall. (Fuchsin is more soluble in phenol (carbolic acid) than in water, and phenol solubilizes more easily in lipids than in acid alcohol. Therefore, carbolfuchsin has a higher affinity for lipids than for acid-alcohol and will remain with the cell wall when washed with acid-alcohol.
How do you perform an acid-fast stain?
1.Start with a fixed smear and cover it with carbolfuschin for 5 minutes.
2.Gently wash with off the carbolfuschin with distilled water so it runs through smear.
3.Destaining of the slide: apply acid-alcohol solution on the slide for 1 minute.
a.Acid-fast cells will retain the stain
b.Nonacid-fast cells will release carbolfuchsin (red)
4.Gently rinse the acid-alcohol off the slide with distilled water
5.Counterstain the smear with methylene blue for 1 minute
a.Nonacid-fast cells will become visible- blue color
6.Gently rinse off the methylene blue and place in bibulous paper to dry
7. Ready for viewing under the microscope
a.Acid-fast cells = red stain (carbolfuchsin)
b.Nonacid-fast cells= blue stain (methylene blue)
What changes did we make to the lab?
The only changes we made to the lab were we substituted methylene blue with Brilliant Green K and used Mycobacterium phlei instead of Mycobacterium smegmatis
What color will an acid-fast organism stain in the acid-fast stain?
what color will a non-acid fast cell stain in the acid-fast stain?
Green because we used Brilliant Green K
What bacteria did we use in the acid-fast stain?
Give a description of acid fast Mycobacterium phlei.
Acid-Fast Reaction: Positive
Give a description of acid fast Escherichia Coli.
Color: Brilliant K green
Acid-Fast Reaction: negative
Which of the bacterium was acid-fast?
What is the decolorizing agent in the gram stain?
What is the decolorizing agent in the acid-fast stain?
What diseases are diagnosed using the acid-fast procedure?
tuberculosis, leprosy, nocardiosis, and actinomycete
What is phenol (carbolic acid), and what is its usual application?
Phenol is a disinfectant. It was the disinfectant used by Lister.
Why can heat be eliminated in the Kinyoun modification that you used?
The mycobacterium wax-like lipid is resistant to staining, so heat allowed penetration of the stain. The kinyoun modification does not require heat because a more concentrated fuchsin is used.
How might the acid-fast characteristic of Mycobacterium enhance the organism's ability to cause disease?
The cell wall contains lipids that resist drying and environmental stress so the bacteria can survive harsh conditions, which facilitates their transmission and ability to cause disease.
In 1882, after experimenting with staining Mycobacterium, Paul Ehrlich wrote that only alkaline disinfectants would be effective against Mycobacterium. How did he reach this conclusion without testing the disinfectants?
Mycobacterium are resistant to acid, as demonstrated in the acid-fast stain. The bacteria do no decolorize, even when exposed to acid-alcohol.
Clinical specimens suspected of containing Mycobacterium are digested with sodium hydroxide (NaOH) for 30 minutes prior to staining. Why is this technique used? Why isn't this technique used for staining other bacteria?
Digestion removes unwanted bacteria, sputum, and human cells to facilitate looking for mycobacterium through the microscope. Other bacteria would be killed by the NaOH.
The acid-fast stain is used to detect Cryptosporidium protozoa oocysts in fecal samples. Which of the following would you expect to be a major component off the oocyst wall: carbs, lipids, or proteins?
What disease is caused by Cryptosporidium?
Cryptosporidiosis diarrheal disease
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