Auramine- Rhodamine Staining 50 FAQs and 30 MCQs:
Auramine- Rhodamine Staining 50 FAQs:
What is Auramine-Rhodamine staining?
It is a fluorescent staining technique used to detect acid-fast bacilli (AFB) like Mycobacterium tuberculosis under a fluorescent microscope.
Why is it called the Truant method?
It is named after Truant, Brett, and Thomas (1962), who improved the use of fluorescent dyes for AFB detection.
How does it differ from Ziehl-Neelsen (ZN) staining?
It uses fluorescent dyes (auramine-rhodamine), requires no heat fixation, and is more sensitive and faster than ZN staining.
Which bacteria can be detected using this method?
Primarily Mycobacterium spp. (M. tuberculosis, M. leprae) and some sporozoan parasites.
What makes Mycobacterium spp. acid-fast?
Their mycolic acid-rich cell wall retains dyes even after acid-alcohol decolorization.
How does Auramine-Rhodamine stain work?
The fluorochrome dye binds to mycolic acid in the bacterial cell wall, making AFB fluoresce under UV light.
Why is potassium permanganate used as a counterstain?
It reduces background fluorescence by staining non-acid-fast debris black.
Why is heat fixation not required for this stain?
The high affinity of auramine-rhodamine for mycolic acid eliminates the need for heat fixation.
What color do AFB appear under a fluorescent microscope?
Bright yellow, orange, or reddish-yellow against a dark background.
What happens if the counterstain is left too long?
It may quench fluorescence, reducing the visibility of AFB.
What are the main reagents used in this staining?
Primary stain: Auramine-rhodamine
Decolorizer: Acid-alcohol (0.5% HCl in 70% ethanol)
Counterstain: Potassium permanganate (0.5%)
Why is distilled water preferred over tap water?
Chlorine in tap water can interfere with fluorescence.
Is Auramine-Rhodamine carcinogenic?
Yes, both auramine O and rhodamine B are possible carcinogens.
What safety precautions are needed while handling reagents?
Wear gloves, goggles, and a lab coat, work under a fume hood, and avoid inhalation/skin contact.
How long can the prepared stain be stored?
The auramine-rhodamine solution is stable for 1 year if properly stored.
How is the smear prepared for staining?
A thin smear is made, air-dried, and heat-fixed (but no heat is applied during staining).
How long should the primary stain be left on the slide?
15 minutes (without letting it dry).
What is the role of acid-alcohol in this method?
It decolorizes non-acid-fast cells, leaving only AFB stained.
How long should decolorization last?
2-3 minutes (until the slide appears colorless).
Why is potassium permanganate used for only 2 minutes?
Longer exposure reduces fluorescence intensity.
Can a microwave be used for staining?
Yes, microwaving at 80 power for 45 sec is an alternative to conventional heating.
What magnification is used for screening?
400X (high power), with verification under oil immersion (1000X).
How many fields should be examined before reporting a negative result?
At least 30-70 fields, depending on magnification.
Why should stained slides be examined within 24 hours?
Fluorescence fades over time, reducing visibility.
What should be done if the fluorescence is too weak?
Re-stain the slide or confirm with Ziehl-Neelsen staining.
What does a positive result look like?
Bright yellow/orange bacilli against a dark background.
What does a negative result indicate?
No fluorescence or only pale yellow background.
Can rapid-growing Mycobacteria be detected?
No, most rapid growers do not fluoresce.
Why is ZN staining sometimes needed after a negative result?
To confirm the absence of AFB, as fluorescence may fade or be missed.
How are results reported quantitatively?
+ (few), ++ (moderate), +++ (many) based on the number of AFB seen.
What are the advantages over ZN staining?
Faster, more sensitive (~10%), no heat required, easier screening.
What is the major limitation of this method?
Requires a fluorescent microscope, which is expensive and less available in developing countries.
Why is culture confirmation still needed?
Staining gives presumptive results, while culture provides definitive identification.
Can this method give false negatives?
Yes, if smears are too thick/too thin or if fluorescence fades.
Is this method recommended by the CDC?
Yes, CDC recommends fluorochrome staining for primary AFB detection.
Why does the background appear too bright?
Insufficient decolorization or over-counterstaining.
What if the slide shows no fluorescence?
Check microscope filters, reagent quality, or staining procedure.
Why do some AFB appear pale?
Excessive counterstaining or old reagents.
What happens if the smear is too thick?
Decolorization may be incomplete, leading to false positives.
What if the smear is too thin?
Too few AFB may be detected, leading to false negatives.
Is phenol used in this staining?
Yes, it is part of the auramine-rhodamine solution (toxic if absorbed).
What are the hazards of hydrochloric acid in acid-alcohol?
Corrosive to skin, eyes, and respiratory tract.
Can potassium permanganate cause irritation?
Yes, it is a skin and eye irritant and toxic if ingested.
How should waste reagents be disposed of?
Follow hazardous waste disposal protocols (do not pour down the drain).
What PPE is required for this staining?
Gloves, goggles, lab coat, and fume hood use.
Which specimens can be tested with this method?
Sputum, urine sediment, tissue sections.
Can it be used for parasites?
Yes, it can stain sporozoan parasites.
What is an alternative if a fluorescent microscope is unavailable?
Ziehl-Neelsen (hot) or Kinyoun (cold) staining.
Why is this method preferred in high-volume labs?
Faster screening due to lower magnification requirements.
Can this method replace culture for TB diagnosis?
No, culture is still needed for confirmation and drug susceptibility testing.
Auramine- Rhodamine Staining 30 MCQs
1. What is the primary purpose of Auramine-Rhodamine staining?
a) To stain Gram-positive bacteria
b) To detect acid-fast bacilli (AFB)✔
c) To identify fungal hyphae
d) To visualize viral particles
2. Which component of Mycobacterium spp. binds to Auramine-Rhodamine dye?
a) Peptidoglycan
b) Mycolic acid✔
c) Lipopolysaccharide
d) Teichoic acid
3. What type of microscope is required for Auramine-Rhodamine staining?
a) Light microscope
b) Phase-contrast microscope
c) Fluorescent microscope✔
d) Electron microscope
4. Why is Auramine-Rhodamine staining more sensitive than Ziehl-Neelsen (ZN) staining?
a) It uses a higher magnification
b) It does not require heat fixation
c) Fluorescent bacilli are easier to detect
d) Both b and c✔
5. What color do acid-fast bacilli (AFB) appear under fluorescence?
a) Blue
b) Green
c) Bright yellow/orange✔
d) Purple
6. Which reagent acts as the decolorizer in Auramine-Rhodamine staining?
a) Crystal violet
b) Acid-alcohol✔
c) Methylene blue
d) Safranin
7. What is the role of potassium permanganate in this staining?
a) Primary stain
b) Decolorizer
c) Counterstain (reduces background fluorescence)✔
d) Mordant
8. How long should the primary stain (Auramine-Rhodamine) be applied?
a) 1 minute
b) 5 minutes
c) 15 minutes✔
d) 30 minutes
9. Why is distilled water preferred over tap water for rinsing?
a) Tap water is too expensive
b) Chlorine in tap water quenches fluorescence✔
c) Distilled water dries faster
d) None of the above
10. What happens if the counterstain (potassium permanganate) is left too long?
a) AFB fluoresce brighter
b) Background becomes too dark
c) Fluorescence is quenched✔
d) The slide dissolves
11. A positive Auramine-Rhodamine stain shows AFB as:
a) Blue rods
b) Reddish-orange fluorescent rods✔
c) Green cocci
d) Purple spirals
12. How should a negative result be confirmed?
a) Repeat Auramine-Rhodamine staining
b) Use Ziehl-Neelsen staining✔
c) Discard the slide
d) Report as negative without confirmation
13. How many fields should be examined before reporting a negative result at 400X magnification?
a) 10
b) 30
c) 55✔
d) 100
14. What does a pale yellow background indicate?
a) Positive for AFB
b) Negative for AFB✔
c) Contamination
d) Over-decolorization
15. Why must slides be examined within 24 hours?
a) Fluorescence fades over time✔
b) The stain becomes toxic
c) The slide disintegrates
d) None of the above
16. Which of the following is an advantage of Auramine-Rhodamine over ZN staining?
a) Requires oil immersion
b) More sensitive (~10% higher detection)✔
c) Needs a light microscope
d) Takes longer to perform
17. A major limitation of Auramine-Rhodamine staining is:
a) It cannot detect Mycobacterium spp.
b) Requires a fluorescent microscope✔
c) Only works on blood samples
d) It is less sensitive than ZN staining
18. Which organism may NOT fluoresce with this stain?
a) Mycobacterium tuberculosis
b) Rapid-growing Mycobacteria✔
c) Mycobacterium leprae
d) Sporozoan parasites
19. Why is culture still needed after a positive stain?
a) To confirm AFB presence and perform drug susceptibility testing✔
b) Because staining is always wrong
c) Culture is faster
d) None of the above
20. Which organization recommends fluorochrome staining for AFB detection?
a) WHO
b) CDC✔
c) FDA
d) NIH
21. Which reagent in Auramine-Rhodamine staining is a possible carcinogen?
a) Distilled water
b) Auramine O✔
c) Potassium permanganate
d) Glycerol
22. What PPE is required when handling these stains?
a) Gloves and lab coat
b) Goggles and fume hood
c) Both a and b✔
d) No PPE needed
23. What happens if the smear is too thick?
a) AFB fluoresce brighter
b) Decolorization may be incomplete (false positives)✔
c) The slide cracks
d) None of the above
24. If AFB appear faint, what could be the reason?
a) Over-decolorization
b) Insufficient staining time
c) Old reagents
d) All of the above✔
25. How should acid-alcohol waste be disposed of?
a) Pour down the sink
b) Discard in regular trash
c) Treat as hazardous waste✔
d) Reuse for future staining
26. What is the alternative staining method if a fluorescent microscope is unavailable?
a) Gram staining
b) Ziehl-Neelsen staining✔
c) Giemsa staining
d) India ink staining
27. What is the recommended filter combination for fluorescence microscopy?
a) K530 excitation + BG12 barrier✔
b) 488 nm laser + DAPI filter
c) Brightfield condenser
d) Phase-contrast ring
28. Why is heat fixation avoided during staining?
a) It destroys mycolic acid
b) Fluorescent dyes bind strongly without heat✔
c) It makes the slide brittle
d) None of the above
29. Which specimen is NOT suitable for Auramine-Rhodamine staining?
a) Sputum
b) Urine sediment
c) Blood smear✔
d) Tissue sections
30. What is the major drawback of potassium permanganate?
a) It enhances fluorescence
b) It is a skin/eye irritant✔
c) It is non-toxic
d) It stabilizes the stain
Possible References Used
