Acridine Orange Stain 50 FAQs and 30 MCQs
Acridine Orange Stain 50 FAQs
What is Acridine Orange Stain used for?
It is a fluorescent stain used to detect nucleic acids (DNA and RNA) in bacteria, fungi, and host cells in clinical samples.
How does Acridine Orange work?
It intercalates into nucleic acids, causing them to fluoresce orange (RNA) or green (DNA) under UV light.
Is Acridine Orange a Gram stain alternative?
Yes, it is more sensitive than Gram stain for detecting microorganisms in blood cultures and other bodily fluids.
What samples can be stained with Acridine Orange?
Blood cultures, CSF, buffy coat preparations, corneal scrapings (for Acanthamoeba), and Helicobacter pylori gastritis samples.
Can Acridine Orange detect fungi?
Yes, fungal nucleic acids also fluoresce orange.
How do you prepare a slide for Acridine Orange staining?
Fix the smear in methanol and air-dry before staining.
How long should the stain remain on the slide?
2 minutes (without drying).
What is the rinse procedure after staining?
Rinse gently with tap water and air-dry.
What microscope is used for Acridine Orange?
A fluorescent microscope with UV light.
What magnification is recommended?
100x to 1000x.
What color do bacteria fluoresce?
Bright orange.
What color does mammalian DNA fluoresce?
Green.
Why might RNA mask DNA fluorescence?
RNA is more abundant in growing cells, so orange may dominate over green.
Can dead bacteria be detected?
Yes, but debris may distort the image.
What does a dark background indicate?
No nucleic acids present.
What are the limitations of Acridine Orange?
Cellular debris (WBCs, dead bacteria) can interfere with interpretation.
Is Acridine Orange highly sensitive?
Yes, so results should be interpreted cautiously.
Can it detect cell wall-deficient bacteria?
Yes, such as Mycoplasma.
Does it work at high pH?
No, optimal pH is 4.0 ± 0.2.
Can it replace Gram stain completely?
No, but it is more sensitive for low bacterial loads.
What is the pH of Acridine Orange Stain?
4.0 ± 0.2 (acetate buffer).
How should the stain be stored?
Upright at room temperature.
What is the shelf life?
52 weeks from manufacture.
What QC organism is used?
E. coli ATCC 25922 (should fluoresce orange).
What if the stain doesn’t fluoresce properly?
Check pH, storage conditions, and expiration date.
Can Acridine Orange detect Acanthamoeba?
Yes, in corneal scrapings.
Is it useful for Helicobacter pylori diagnosis?
Is it useful for Helicobacter pylori diagnosis?
Can it detect bacteria in CSF?
Yes, more sensitively than Gram stain.
Does it work for buffy coat analysis?
Yes, for detecting bacteremia.
Can it identify yeast infections?
Yes, fungal RNA fluoresces orange.
Why is fluorescence weak?
Old stain, incorrect pH, or insufficient staining time.
What if the background is too bright?
Over-staining or debris interference.
Can autofluorescence be a problem?
Yes, some tissues may fluoresce naturally.
Should the stain be filtered before use?
If particulate matter is present, yes.
What if the slide dries during staining?
Repeat the process; drying affects fluorescence.
Is Acridine Orange better than Gram stain?
More sensitive but less specific.
Can it replace KOH for fungal detection?
It can complement but not fully replace it.
How does it compare to Calcofluor White?
Both detect fungi, but Calcofluor binds to cell walls, while Acridine Orange binds nucleic acids.
Is it used in parasitology?
Mainly for Acanthamoeba, not routine parasites.
Can it detect viral inclusions?
No, viruses lack sufficient nucleic acids for detection.
What is the catalog number for Acridine Orange?
SA16-250 (250 mL bottle).
Is the stain ready-to-use?
Yes, no dilution needed.
Can it be frozen for longer storage?
No, store at room temperature.
Is Acridine Orange light-sensitive?
Yes, protect from prolonged light exposure.
What safety precautions are needed?
Wear gloves; avoid skin/eye contact (potential mutagen).
Why does RNA fluoresce orange and DNA green?
Due to metachromatic properties and binding differences
Can Acridine Orange quantify bacterial load?
No, it’s qualitative, not quantitative.
Does it work for biofilm detection?
Yes, but debris may interfere.
Can it be used in FISH (Fluorescent In Situ Hybridization)?
No, FISH uses specific probes, not intercalating dyes.
Is Acridine Orange used in research beyond clinical labs?
Yes, in cell cycle studies, apoptosis research, and microbiology.
Acridine Orange Stain 30 MCQs
- What type of stain is Acridine Orange?
a) Basic dye
b) Acidic dye
c) Fluorochrome dye
d) Chromogenic dye - What does Acridine Orange primarily stain?
a) Proteins
b) Lipids
c) Nucleic acids (DNA/RNA)
d) Carbohydrates - Under UV light, bacterial nucleic acids fluoresce:
a) Green
b) Blue
c) Orange
d) Red - Mammalian nucleic acids fluoresce as:
a) Green
b) Orange
c) Yellow
d) Red - Why is Acridine Orange more sensitive than Gram stain?
a) It stains only live bacteria
b) It detects low bacterial loads better
c) It differentiates Gram-positive and Gram-negative bacteria
d) It works only at high pH
- How long should Acridine Orange remain on the slide?
a) 10 seconds
b) 2 minutes
c) 10 minutes
d) 1 hour - What is the correct fixative before staining?
a) Formalin
b) Methanol
c) Ethanol
d) Heat fixation - After staining, the slide should be rinsed with:
a) Distilled water
b) Tap water
c) Saline
d) Acetone - Which microscope is used for Acridine Orange?
a) Bright-field
b) Phase-contrast
c) Fluorescent
d) Electron - The optimal pH for Acridine Orange staining is:
a) 7.0
b) 4.0 ± 0.2
c) 9.0
d) 2.5
- Yeast cells stained with Acridine Orange appear:
a) Blue
b) Orange
c) Green
d) Purple - If RNA is abundant in a cell, what color dominates?
a) Green
b) Orange
c) Yellow
d) Red - Dead bacteria may appear as:
a) Bright green
b) Dim or distorted orange
c) Blue
d) Invisible - A dark background in microscopy indicates:
a) No nucleic acids present
b) Over-staining
c) Contamination
d) High bacterial load - Which organism is used for QC in Acridine Orange staining?
a) Staphylococcus aureus
b) Escherichia coli ATCC 25922
c) Candida albicans
d) Mycobacterium tuberculosis
- Acridine Orange is especially useful for detecting:
a) Bacteria in blood cultures
b) Acid-fast bacilli
c) Parasites in stool
d) Viral antigens - Which infection can Acridine Orange help diagnose?
a) Malaria
b) Acanthamoeba keratitis
c) Hepatitis B
d) HIV - In Helicobacter pylori gastritis, Acridine Orange stains:
a) Bacterial nucleic acids
b) Gastric mucus
c) Red blood cells
d) Inflammatory cells - Which sample is NOT suitable for Acridine Orange staining?
a) CSF
b) Buffy coat
c) Urine (without processing)
d) Corneal scrapings - Acridine Orange can detect:
a) Cell wall-deficient bacteria (e.g., Mycoplasma)
b) Only Gram-positive bacteria
c) Only Gram-negative bacteria
d) Viruses
- A major limitation of Acridine Orange is:
a) Cellular debris interference
b) Long staining time
c) High cost
d) Needs refrigeration - How should Acridine Orange be stored?
a) Frozen at -20°C
b) Upright at room temperature
c) In direct sunlight
d) With desiccants - Shelf life of Acridine Orange is:
a) 6 months
b) 52 weeks
c) 2 years
d) Indefinite - Acridine Orange is NOT recommended for:
a) Quantifying bacterial load
b) Detecting fungi
c) Rapid screening of CSF
d) Research applications - Safety precaution when handling Acridine Orange:
a) Wear gloves (potential mutagen)
b) Use in open air
c) No special precautions
d) Store in glass only
- Acridine Orange binds to nucleic acids via:
a) Covalent bonding
b) Intercalation
c) Hydrogen bonding
d) Ionic bonding - Which component is in Acridine Orange’s formulation?
a) Acetate buffer
b) Phosphate buffer
c) TRIS buffer
d) Citrate buffer - At what magnification should slides be examined?
a) 40x
b) 100x–1000x
c) 1500x
d) Only under electron microscope - Acridine Orange is a _____ stain.
a) Metachromatic
b) Simple
c) Negative
d) Differential - In research, Acridine Orange is used to study:
a) Apoptosis
b) Enzyme kinetics
c) Blood typing
d) Antibiotic resistance
