A healthy 47-year-old man has a thick, yellow, crumbling great toenail. Nail clippings are examined with calcofluor white and KOH under fluorescence. The technologist sees several brightly fluorescent strands that are flat, about 15 to 20 µm wide and even in width, twisted at intervals along their length, with no cross walls, no branching and no conidia. They lie on top of the nail fragments, and no fluorescent elements are seen within the keratin itself. Fungal cultures on media with and without cycloheximide have no growth at 4 weeks. The fluorescent strands are best interpreted as:

Cotton fibers, not fungal elements

Candida pseudohyphae invading the nail

Dermatophyte hyphae; culture falsely negative

Broad, sparsely septate Mucorales hyphae

The main purpose of the trichrome-stained permanent smear in an ova and parasite examination is to:

Detect helminth eggs and larvae

Potassium hydroxide (10% to 20%) is added to skin, hair or nail scrapings mainly because it:

Clears keratin so fungal elements stand out

Makes fungal chitin fluoresce under UV light

Stains fungal hyphae blue for easy viewing

Fixes the cells firmly onto the glass slide

Stains: KOH, calcofluor white, trichrome, Giemsa, acridine orange

7 ASCP MLS practice questions on Stains: KOH, calcofluor white, trichrome, Giemsa, acridine orange, part of Microbiology. The first 7 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

Question 1 · Hard
A healthy 47-year-old man has a thick, yellow, crumbling great toenail. Nail clippings are examined with calcofluor white and KOH under fluorescence. The technologist sees several brightly fluorescent strands that are flat, about 15 to 20 µm wide and even in width, twisted at intervals along their length, with no cross walls, no branching and no conidia. They lie on top of the nail fragments, and no fluorescent elements are seen within the keratin itself. Fungal cultures on media with and without cycloheximide have no growth at 4 weeks. The fluorescent strands are best interpreted as:
1. ACandida pseudohyphae invading the nail
2. BCotton fibers, not fungal elements
3. CDermatophyte hyphae; culture falsely negative
4. DBroad, sparsely septate Mucorales hyphae
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Answer: B · Cotton fibers, not fungal elements
Calcofluor white binds beta-linked polysaccharides, chitin and cellulose, so it lights up fungal walls but also cotton, paper and plant fibers. These strands are far too wide for dermatophyte hyphae, which are 2 to 4 µm across, and their even width, flat twisted form and lack of septa or branching are the look of cotton. Dermatophyte hyphae lie within the keratin, are septate and branched, and often break into arthroconidia, while Candida pseudohyphae show constrictions at the septa. Mucorales hyphae vary in width, branch at wide angles, rarely infect nails and would grow within days on media without cycloheximide. The smear is reported as negative for fungal elements, which agrees with the culture.
- A: Pseudohyphae are narrow chains with constrictions at the septa, not 15-20 µm strands.
- C: Dermatophyte hyphae are 2-4 µm wide, septate and branched, and lie within the keratin.
- D: Mucorales hyphae branch and vary in width, and would grow within days on media without cycloheximide.
Question 2 · Medium
The main purpose of the trichrome-stained permanent smear in an ova and parasite examination is to:
1. ADetect helminth eggs and larvae
2. BDetect microsporidial spores
3. CIdentify intestinal protozoa
4. DDetect Cryptosporidium oocysts
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Answer: C · Identify intestinal protozoa
The trichrome stain gives clear nuclear and cytoplasmic detail, which is needed to identify amoebae and flagellates such as Entamoeba histolytica/dispar and Giardia. In a good stain, cytoplasm is blue-green to purple, nuclei and chromatoid bodies are red to purple-red, and the background is green. Helminth eggs and larvae stain poorly and are best found in the concentrate wet mount. Coccidia need a modified acid-fast stain, and microsporidia need a modified trichrome with more chromotrope dye.
- A: Helminth eggs and larvae stain poorly and are found mainly in the concentrate.
- B: Microsporidial spores need a modified trichrome with more chromotrope dye.
- D: Cryptosporidium oocysts are shown with a modified acid-fast stain.
Question 3 · Easy
Calcofluor white makes fungi fluoresce by binding to which component?
1. AErgosterol
2. BMycolic acids
3. CPeptidoglycan
4. DChitin and cellulose
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Answer: D · Chitin and cellulose
Calcofluor white is a fluorescent brightener that binds beta-linked polysaccharides such as chitin and cellulose. Under UV or blue light, fungal walls glow blue-white or apple-green, depending on the filters. It is often mixed with KOH and also stains Pneumocystis cysts and Acanthamoeba cysts. Cotton fibers also fluoresce and must not be mistaken for hyphae.
- A: Ergosterol is a membrane sterol targeted by amphotericin B and azoles.
- B: Mycolic acids are found in mycobacteria and are shown by acid-fast stains.
- C: Peptidoglycan is a bacterial wall polymer that fungi do not have.
Question 4 · Easy
Potassium hydroxide (10% to 20%) is added to skin, hair or nail scrapings mainly because it:
1. AMakes fungal chitin fluoresce under UV light
2. BClears keratin so fungal elements stand out
3. CStains fungal hyphae blue for easy viewing
4. DFixes the cells firmly onto the glass slide
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Answer: B · Clears keratin so fungal elements stand out
KOH digests keratin and host cell material but leaves the chitin-containing fungal walls intact. Hyphae, pseudohyphae and yeast cells then stand out against a clear background. Thick nail material may need gentle warming or more time to clear. KOH itself does not stain fungi, so calcofluor white is often added to make them easier to find.
- A: Fluorescence needs calcofluor white; KOH alone does not fluoresce.
- C: Blue staining comes from lactophenol cotton blue, used for culture mounts.
- D: KOH is a clearing agent, not a fixative.
Question 5 · Hard
A 45-year-old man with HIV (CD4 count 80 cells/µL [0.08 × 10^9/L]) has bilateral pneumonia. BAL results: calcofluor white shows round to cup-shaped cysts about 5 µm across, with no budding; Giemsa shows clusters of small trophic forms with purple-red nuclei. Which organism is most likely?
1. AHistoplasma capsulatum
2. BCryptococcus neoformans
3. CPneumocystis jirovecii
4. DToxoplasma gondii
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Answer: C · Pneumocystis jirovecii
Pneumocystis jirovecii cysts are about 4 to 7 µm, often cup- or crescent-shaped, and do not bud; their walls bind calcofluor white. Giemsa does not stain the cyst wall but shows the trophic forms and intracystic bodies with purple-red nuclei. Seeing both forms in a BAL from a patient with a CD4 count under 200 cells/µL strongly supports Pneumocystis pneumonia. A direct fluorescent antibody test or PCR can confirm it.
- A: Histoplasma forms tiny budding yeasts, usually inside macrophages.
- B: Cryptococcus forms budding yeasts of varying size with a thick capsule.
- D: Toxoplasma tachyzoites have no chitin-like wall, so calcofluor white does not stain them.
Question 6 · Medium
A blood culture bottle from a febrile newborn signals positive, but its Gram stain shows no organisms. Which stain on the broth is most useful next?
1. AKinyoun acid-fast
2. BAcridine orange
3. CCalcofluor white
4. DWheatley trichrome
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Question 7 · Hard
A 24-year-old contact lens wearer who rinses her lenses in tap water has a painful corneal ulcer. Calcofluor white on a corneal scraping shows fluorescent double-walled cysts about 15 µm across, with a wrinkled outer wall and no hyphae. The most likely cause is:
1. ACandida albicans
2. BAcanthamoeba species
3. CFusarium species
4. DPseudomonas aeruginosa
Answer and explanation in LearnFree account

Stains: KOH, calcofluor white, trichrome, Giemsa, acridine orange

The main purpose of the trichrome-stained permanent smear in an ova and parasite examination is to:

Calcofluor white makes fungi fluoresce by binding to which component?

Potassium hydroxide (10% to 20%) is added to skin, hair or nail scrapings mainly because it:

A 45-year-old man with HIV (CD4 count 80 cells/µL [0.08 × 10^9/L]) has bilateral pneumonia. BAL results: calcofluor white shows round to cup-shaped cysts about 5 µm across, with no budding; Giemsa shows clusters of small trophic forms with purple-red nuclei. Which organism is most likely?

A blood culture bottle from a febrile newborn signals positive, but its Gram stain shows no organisms. Which stain on the broth is most useful next?

A 24-year-old contact lens wearer who rinses her lenses in tap water has a painful corneal ulcer. Calcofluor white on a corneal scraping shows fluorescent double-walled cysts about 15 µm across, with a wrinkled outer wall and no hyphae. The most likely cause is: