Acid Fast Stain (Kinyoun-Cold Method) 50 FAQs and 30 MCQs
Acid Fast Stain (Kinyoun-Cold Method) 50 FAQs
What is the purpose of acid-fast staining?
To identify acid-fast bacteria (e.g., Mycobacterium spp.) and parasites like Cryptosporidium and Isospora.
Why are acid-fast bacteria difficult to stain?
Their waxy cell walls (high mycolic acid content) resist aqueous stains.
What are the primary stains used in acid-fast staining?
Carbolfuchsin (Ziehl-Neelsen, Kinyoun) or Auramine-Rhodamine (fluorochrome method).
What is the difference between acid-fast and non-acid-fast bacteria?
Acid-fast bacteria retain carbolfuchsin (pink/red), while non-acid-fast bacteria take up the counterstain (blue).
Which bacteria are commonly detected using acid-fast staining?
Mycobacterium tuberculosis, M. leprae, and Nocardia (weakly acid-fast).
What is the role of phenol in acid-fast staining?
It helps carbolfuchsin penetrate the waxy cell wall.
Why is heat used in the Ziehl-Neelsen method?
Heat drives the stain into the mycobacterial cell wall.
What is the decolorizer used in acid-fast staining?
Acid-alcohol (1–3% HCl in ethanol).
What counterstain is used in acid-fast staining?
Methylene blue or malachite green.
How are results interpreted in acid-fast staining?
Acid-fast organisms: Pink/red; Non-acid-fast: Blue.
What is the Kinyoun-Cold method?
A “cold” acid-fast staining technique (no heat) using concentrated carbolfuchsin.
Why is the Kinyoun method called “cold staining”?
It omits heating, relying on higher phenol and dye concentrations.
How long is carbolfuchsin applied in the Kinyoun method?
3–5 minutes at room temperature.
What are the limitations of the Kinyoun method?
Less sensitive than Ziehl-Neelsen; thick smears may stain poorly.
Can the Kinyoun method detect parasites?
Yes, it can identify Cryptosporidium and Isospora cysts.
What decolorizer is used in Kinyoun staining?
1% sulfuric acid or acid-alcohol.
How is the Kinyoun method different from Ziehl-Neelsen?
No heat, longer staining time, higher dye concentration.
Is the Kinyoun method faster than Ziehl-Neelsen?
Is the Kinyoun method faster than Ziehl-Neelsen?
Can Kinyoun staining be used for sputum samples?
Yes, but Ziehl-Neelsen may be more sensitive.
What is the counterstain used in Kinyoun staining?
Methylene blue (1 minute).
What is the Ziehl-Neelsen method?
A “hot” acid-fast staining technique using heat-fixed carbolfuchsin.
Why is heat used in Ziehl-Neelsen staining?
To melt the waxy cell wall and allow stain penetration.
How long should carbolfuchsin be heated in ZN staining?
5 minutes with steaming (avoid boiling).
What happens if the slide overheats in ZN staining?
The stain may splatter or the slide may crack.
How many fields should be examined before declaring a sample negative?
At least 300 fields under oil immersion.
What is the decolorizer in ZN staining?
Acid-alcohol (3% HCl in ethanol).
How long should decolorization last in ZN staining?
Until the slide runs clear (usually 15–30 seconds).
Can ZN staining be automated?
Manual heating is typically required, but some labs use hot plates.
Why is ZN staining preferred in tuberculosis diagnosis?
High sensitivity and reliability for M. tuberculosis.
What are common errors in ZN staining?
Overheating, insufficient decolorization, or thick smears.
Which is more sensitive: Ziehl-Neelsen or Kinyoun?
Ziehl-Neelsen is generally more sensitive.
Who developed the Ziehl-Neelsen method?
Franz Ziehl (phenol mordant) and Friedrich Neelsen (basic fuchsin).
Who introduced the Kinyoun method?
Joseph Kinyoun (1915) as a cold alternative.
What is the fluorochrome method for acid-fast staining?
Uses Auramine-Rhodamine and UV microscopy (Truant method).
Which method is faster: ZN or Kinyoun?
ZN (5 min with heat) vs. Kinyoun (5–10 min without heat).
Can cold staining replace ZN in resource-limited settings?
Yes, but with slightly reduced sensitivity.
What is the Tan Thia Hok method?
A modified cold staining technique combining Kinyoun and Gabbet’s stains.
Why is acid-fast staining still used today?
Low cost, rapid results, and specificity for mycobacteria.
What are alternatives to acid-fast staining?
PCR, culture, or fluorescent staining (e.g., Auramine-O).
How did Robert Koch contribute to acid-fast staining?
First isolated M. tuberculosis and developed early staining methods.
Why does methylene blue counterstain non-acid-fast cells?
They lose carbolfuchsin during decolorization and take up the blue stain.
What if the smear is too thick in acid-fast staining?
Poor dye penetration, leading to false negatives.
How should smears be prepared for acid-fast staining?
Thin, air-dried, and heat-fixed (80°C for 15 min).
Why is tap water avoided in rinsing?
Minerals may interfere; distilled/deionized water is preferred.
Can acid-fast staining detect dead bacteria?
Yes, if cell wall integrity is maintained.
What causes uneven staining in acid-fast smears?
Inconsistent heating (ZN) or uneven dye application.
Why is phenol used in carbolfuchsin?
Acts as a mordant to enhance dye penetration.
Can acid-fast staining be used for non-microbial cells?
Rarely; some parasite cysts (e.g., Cryptosporidium) are acid-fast.
What safety precautions are needed for acid-fast staining?
Biosafety for Mycobacterium, proper phenol handling.
How can staining quality be improved?
Optimize smear thickness, staining time, and decolorization.
Acid Fast Stain (Kinyoun-Cold Method) 30 MCQs:
- What is the primary purpose of acid-fast staining?
a) To identify Gram-positive bacteria
b) To detect acid-fast bacteria like Mycobacterium✔
c) To stain fungal hyphae
d) To differentiate cocci from bacilli - Which component of acid-fast bacteria makes them resistant to staining?
a) Peptidoglycan
b) Mycolic acid✔
c) Lipopolysaccharide
d) Teichoic acid - Which stain is used as the primary dye in acid-fast staining?
a) Crystal violet
b) Carbolfuchsin✔
c) Methylene blue
d) Safranin - What is the decolorizer used in acid-fast staining?
a) Ethanol alone
b) Acetone
c) Acid-alcohol (HCl + ethanol)✔
d) Iodine solution - After decolorization, acid-fast bacteria appear:
a) Blue
b) Green
c) Pink/red✔
d) Purple - Non-acid-fast bacteria appear _____ after counterstaining.
a) Pink
b) Blue✔
c) Green
d) Purple - Which of the following is NOT an acid-fast bacterium?
a) Mycobacterium tuberculosis
b) Nocardia
c) Escherichia coli✔
d) Mycobacterium leprae - Acid-fast staining is clinically important for diagnosing:
a) Strep throat
b) Tuberculosis✔
c) Urinary tract infections
d) Influenza - Which chemical helps carbolfuchsin penetrate the waxy cell wall?
a) Iodine
b) Phenol✔
c) Ethanol
d) Methylene blue - What is the counterstain used in acid-fast staining?
a) Safranin
b) Malachite green
c) Methylene blue✔
d) Crystal violet
- The Kinyoun method is a _____ acid-fast staining technique.
a) Heat-fixed
b) Cold✔
c) Fluorescent
d) Gram-based - Why is the Kinyoun method called “cold staining”?
a) It uses ice for decolorization
b) It avoids heat by using concentrated carbolfuchsin✔
c) It requires refrigeration
d) It uses a cold counterstain - How long is carbolfuchsin applied in the Kinyoun method?
a) 1 minute
b) 3–5 minutes✔
c) 10–15 minutes
d) 30 seconds - Which decolorizer is used in the Kinyoun method?
a) 95% ethanol
b) 1% sulfuric acid✔
c) Acetone-alcohol
d) Gram’s iodine - The Kinyoun method is less sensitive than:
a) Gram staining
b) Ziehl-Neelsen staining✔
c) Negative staining
d) Capsule staining - Which parasites can be detected using the Kinyoun method?
a) Giardia and Entamoeba
b) Cryptosporidium and Isospora✔
c) Plasmodium and Trypanosoma
d) Trichomonas and Leishmania - The counterstain in Kinyoun staining is applied for:
a) 1 minute✔
b) 4 minutes
c) 10 minutes
d) 30 seconds - A limitation of the Kinyoun method is:
a) It requires a fluorescent microscope
b) It is less sensitive than Ziehl-Neelsen✔
c) It cannot detect Mycobacterium
d) It uses toxic radioactive dyes - Who developed the Kinyoun method?
a) Robert Koch
b) Joseph Kinyoun✔
c) Paul Ehrlich
d) Hans Christian Gram - In Kinyoun staining, acid-fast bacteria appear:
a) Blue
b) Green
c) Pink/red✔
d) Purple
- The Ziehl-Neelsen method uses _____ to facilitate staining.
a) Cold temperatures
b) Heat✔
c) UV light
d) Magnetic fields - How long is the slide heated in ZN staining?
a) 1 minute
b) 5 minutes✔
c) 15 minutes
d) 30 seconds - What is the purpose of heating in ZN staining?
a) To kill bacteria
b) To melt the waxy cell wall for stain penetration✔
c) To evaporate excess dye
d) To fix the smear - Overheating in ZN staining can cause:
a) Smear charring✔
b) False Gram-positive results
c) Rapid decolorization
d) Enhanced fluorescence - How many microscope fields should be examined before reporting a negative result?
a) 50
b) 100
c) 300✔
d) 500 - Which scientist contributed to the ZN method’s development?
a) Alexander Fleming
b) Louis Pasteur
c) Friedrich Neelsen✔
d) Antonie van Leeuwenhoek - ZN staining is the gold standard for diagnosing:
a) Malaria
b) Tuberculosis✔
c) HIV
d) Hepatitis B - The decolorizer in ZN staining is applied until:
a) The slide turns blue
b) The runoff is clear✔
c) The smear dissolves
d) The slide cools - Which staining method is more sensitive: ZN or Kinyoun?
a) ZN✔
b) Kinyoun
c) Both are equal
d) Neither - In ZN staining, non-acid-fast bacteria appear:
a) Pink
b) Blue✔
c) Green
d) Purple
