Acid Fast Stain 50 FAQs and 30 MCQs:
Acid Fast Stain 50 FAQs:
What is the purpose of the acid-fast stain?
To differentiate acid-fast bacteria (e.g., Mycobacterium) from non-acid-fast bacteria.
Why are acid-fast bacteria difficult to stain?
Due to their waxy cell walls containing mycolic acid, which resists aqueous stains.
What is the key component in acid-fast bacterial cell walls?
Mycolic acid, a lipid that makes them impermeable to standard stains.
Which bacteria are commonly identified using acid-fast staining?
Mycobacterium tuberculosis, M. leprae, and Nocardia (weakly acid-fast).
What diseases can acid-fast staining help diagnose?
Tuberculosis, leprosy, and infections caused by Cryptosporidium or Nocardia.
What is the primary stain in acid-fast staining?
Carbolfuchsin (a lipid-soluble dye with phenol).
Why is phenol used in carbolfuchsin?
It helps the stain penetrate the waxy cell wall.
What is the decolorizer in acid-fast staining?
Acid-alcohol (3% HCl in 95% ethanol).
What is the counterstain in acid-fast staining?
Methylene blue (stains non-acid-fast cells blue).
Why is heat used in the Ziehl-Neelsen method?
To soften the waxy cell wall and allow carbolfuchsin penetration.
How does the Kinyoun method differ from Ziehl-Neelsen?
Kinyoun uses a higher concentration of carbolfuchsin (no heat required).
What is the Truant method?
A fluorescent acid-fast stain using auramine-rhodamine.
How do you prepare a smear for acid-fast staining?
Air-dry and heat-fix the specimen (80°C for 15 min).
Why is a filter paper used in Ziehl-Neelsen staining?
To retain carbolfuchsin and ensure even heating.
How long should carbolfuchsin be applied in Ziehl-Neelsen?
3-5 minutes with continuous heating.
How long should decolorization last?
10-15 seconds (until no more red runs off).
What happens if you over-decolorize?
Acid-fast bacteria may lose the red stain.
How long is the counterstain (methylene blue) applied?
1-2 minutes (Ziehl-Neelsen) or 4 minutes (Kinyoun).
Why must the filter paper stay moist during staining?
Prevents uneven staining and poor dye penetration.
What color are acid-fast bacteria after staining?
Bright red/purple (retain carbolfuchsin).
What color are non-acid-fast bacteria?
Blue (take up methylene blue).
Why might Nocardia appear weakly acid-fast?
Contains partial mycolic acid in its cell wall.
What does a beaded appearance in acid-fast bacteria indicate?
Uneven mycolic acid distribution (common in Mycobacterium).
Can Gram staining identify acid-fast bacteria?
No, they stain poorly (Gram-variable or weakly Gram-positive).
Why do acid-fast bacteria resist decolorization?
Mycolic acid binds carbolfuchsin tightly.
Why might acid-fast staining fail?
Over-decolorization, insufficient heating, or poor smear preparation.
Can blood agar cultures affect acid-fast staining?
Yes, nutrient deprivation may reduce mycolic acid content.
What happens if the slide cracks during heating?
The sample is lost; overheating must be avoided.
Why is tap water avoided in rinsing?
Impurities may interfere with staining (use distilled water).
Can non-bacterial organisms be acid-fast?
Yes (e.g., Cryptosporidium oocysts).
Who developed the Ziehl-Neelsen method?
Franz Ziehl (phenol mordant) & Friedrich Neelsen (basic fuchsin).
What is the “hot stain” method?
Ziehl-Neelsen (uses heat).
What is the “cold stain” method?
Kinyoun (no heat, higher dye concentration).
Which method is faster: Ziehl-Neelsen or Kinyoun?
Kinyoun (no heating step).
Which method is more sensitive for low bacterial loads?
Fluorescent (Truant) method.
What are the layers of the acid-fast cell wall?
Peptidoglycan → Arabinogalactan → Mycolic acids → Surface proteins.
How does mycolic acid contribute to antibiotic resistance?
Blocks drug entry (e.g., in M. tuberculosis).
Do acid-fast bacteria have porins?
Yes, for small molecule transport.
Why are mycobacteria hard to kill with disinfectants?
Waxy cell wall resists chemicals.
How does the acid-fast cell wall resemble Gram-negative bacteria?
Has an outer lipid-rich layer.
Why is phenol hazardous in staining?
Toxic, corrosive, and combustible.
How should phenol waste be disposed of?
As hazardous chemical waste.
What precautions are needed when handling Mycobacterium cultures?
Biosafety Level 2+ practices (aerosol prevention).
Can heat fixation kill all mycobacteria?
No, some survive (follow lab safety protocols).
What sample is used for TB diagnosis?
Sputum (tested for M. tuberculosis).
How many microscope fields should be checked for TB diagnosis?
≥300 fields before declaring negative.
Can acid-fast staining detect dead bacteria?
Yes (retains stain even if non-viable).
Why is fluorescence staining used in large labs?
Higher sensitivity for low bacterial counts.
Can acid-fast staining identify Cryptosporidium?
Yes, its oocysts are acid-fast.
What alternative methods exist for TB detection?
PCR, culture, and interferon-gamma release assays (IGRAs).
Acid Fast Stain 30 MCQs:
- What is the primary purpose of the acid-fast stain?
a) To differentiate Gram-positive and Gram-negative bacteria
b) To identify bacteria with waxy cell walls containing mycolic acid✔
c) To stain bacterial flagella
d) To detect bacterial spores - Which component makes acid-fast bacteria resistant to staining?
a) Peptidoglycan
b) Mycolic acid✔
c) Lipopolysaccharide
d) Teichoic acid - What is the primary stain used in acid-fast staining?
a) Crystal violet
b) Methylene blue
c) Carbolfuchsin✔
d) Safranin - Why is phenol added to carbolfuchsin?
a) To kill bacteria
b) To help the stain penetrate the waxy cell wall✔
c) To act as a decolorizer
d) To enhance fluorescence - What is the decolorizer in the acid-fast stain?
a) Ethanol
b) Acetone
c) Acid-alcohol (HCl + ethanol)✔
d) Iodine - Which counterstain is used in acid-fast staining?
a) Safranin
b) Methylene blue✔
c) Malachite green
d) Nigrosin
- In the Ziehl-Neelsen method, why is heat applied?
a) To kill bacteria
b) To melt the cell wall for better stain penetration✔
c) To speed up drying
d) To fix the smear - How long is carbolfuchsin typically applied in the Ziehl-Neelsen method?
a) 10 seconds
b) 1 minute
c) 3-5 minutes✔
d) 10 minutes - What is the key difference between the Ziehl-Neelsen and Kinyoun methods?
a) Ziehl-Neelsen uses heat, Kinyoun does not✔
b) Kinyoun uses a different counterstain
c) Ziehl-Neelsen is only for fungi
d) Kinyoun requires no decolorizer - What happens if acid-alcohol is applied for too long?
a) Acid-fast bacteria will turn blue
b) Non-acid-fast bacteria will retain red stain
c) The slide will crack
d) Acid-fast bacteria may lose the red color✔ - Which method uses fluorescent dyes for acid-fast staining?
a) Ziehl-Neelsen
b) Kinyoun
c) Truant (Auramine-Rhodamine)✔
d) Gram stain
- What color do acid-fast bacteria appear after staining?
a) Blue
b) Green
c) Red/Purple✔
d) Colorless - Non-acid-fast bacteria appear as which color?
a) Red
b) Blue✔
c) Purple
d) Green - Which bacterium is NOT typically acid-fast?
a) Mycobacterium tuberculosis
b) Nocardia
c) Staphylococcus aureus✔
d) Mycobacterium leprae - Why might Nocardia appear weakly acid-fast?
a) It lacks a cell wall
b) It has partial mycolic acid content✔
c) It is Gram-negative
d) It only stains with methylene blue - What does a “beaded” appearance in acid-fast bacteria indicate?
a) Spore formation
b) Uneven mycolic acid distribution✔
c) Gram-variability
d) Capsule presence
- Which disease is commonly diagnosed using acid-fast staining?
a) Strep throat
b) Tuberculosis✔
c) Urinary tract infection
d) Pneumococcal pneumonia - What sample is typically tested for TB using acid-fast staining?
a) Blood
b) Sputum✔
c) Stool
d) Urine - How many microscope fields should be examined before declaring a TB sample negative?
a) 10
b) 50
c) 100
d) ≥300✔ - Which parasite can be detected with acid-fast staining?
a) Giardia
b) Cryptosporidium✔
c) Plasmodium
d) Entamoeba
- Why might acid-fast staining fail?
a) Over-decolorization
b) Insufficient heating (in Ziehl-Neelsen)
c) Poor smear preparation
d) All of the above✔ - What happens if the filter paper dries during staining?
a) Better decolorization
b) Uneven staining✔
c) Faster results
d) No effect - Why is tap water avoided in rinsing?
a) It contains chlorine
b) Impurities may interfere with staining✔
c) It is too acidic
d) It dissolves the smear
- Who developed the Ziehl-Neelsen method?
a) Robert Koch
b) Louis Pasteur
c) Ziehl & Neelsen✔
d) Joseph Lister - Which method is known as the “cold stain”?
a) Ziehl-Neelsen
b) Kinyoun✔
c) Gram stain
d) Schaeffer-Fulton - What is the advantage of the Truant (fluorochrome) method?
a) No need for a microscope
b) Higher sensitivity for low bacterial loads✔
c) Works without decolorization
d) Only stains live bacteria
- Which layer of the acid-fast cell wall contains mycolic acid?
a) Innermost layer
b) Middle layer
c) Outer membrane✔
d) Capsule - Why are mycobacteria resistant to many antibiotics?
a) They lack DNA
b) Their waxy cell wall blocks drug entry✔
c) They reproduce too fast
d) They produce beta-lactamase - Which staining method is least effective for acid-fast bacteria?
a) Ziehl-Neelsen
b) Gram stain✔
c) Kinyoun
d) Truant - What is the main clinical advantage of acid-fast staining?
a) Detects viral infections
b) Rapid identification of TB and leprosy✔
c) Stains all bacteria equally
d) Replaces PCR testing
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