4.3 KOH, Calcofluor-White, Trichrome, Giemsa, and Acridine Orange

Not every direct smear is a Gram stain. Outline I.C also expects you to choose KOH, calcofluor-white, Wheatley trichrome, Giemsa/Wright-Giemsa, and acridine orange by specimen and target. Each stain answers a different question. Mixing them — using Gram stain to rule out dermatophytes, using trichrome as a blood-parasite stain, or using acridine orange to speciate a GNR — is how M items are missed. Modified trichrome for microsporidia is SM-only; do not study it as an M-tested procedure.

KOH 10–20%: digest keratin, find fungi

Potassium hydroxide 10–20% (sometimes with DMSO to speed clearing) dissolves keratin, mucus, and tissue debris so that fungal elements refract against a digested background. Classic specimens: skin scrapings, hair, nails, and vaginal wet preps for yeast and pseudohyphae. Warm the preparation gently; do not boil it into a crystal garden. Dermatophyte hyphae are septate and refractile; Candida shows budding yeast and pseudohyphae; Malassezia on skin may show spaghetti-and-meatballs (short hyphae plus spores). KOH does not color organisms — it is a clearing agent. A negative KOH does not exclude fungal infection; culture, calcofluor, or tissue PAS/GMS may still be positive. Too much KOH or over-heating dissolves fungi along with keratin. Nail clippings need thinner slices or longer clearing than skin scales. Do not confuse clothing fibers or mosaic artifact (cholesterol/keratin debris that looks like a net) with true hyphae: hyphae cross cell boundaries and have regular septa and cytoplasm.

Calcofluor-white: chitin and cellulose fluorescence

Calcofluor-white is a fluorescent brightener that binds chitin and cellulose. Under UV or blue excitation, fungal walls — and Pneumocystis cysts, plant fibers, and cotton — fluoresce. It is commonly combined with KOH on the same smear so clearing and fluorescence happen together. Advantages over plain KOH: higher sensitivity and easier detection of sparse hyphae and of Pneumocystis cysts (which can show parenthesis-like wall thickening on some calcofluor or GMS preparations). Trap: cotton fibers, plant material, and some artifacts also fluoresce — morphology still matters. Bacteria do not light up as fungal walls. Use calcofluor when the clinical question is fungus or Pneumocystis, not any microbe in a fluid. A fluorescence microscope and a non-quenched slide are required; a bright-field KOH is the fallback if fluorescence is down.

Wheatley trichrome: stool protozoa, cytoplasm versus chromatin

Trichrome (Wheatley) is the permanent stained smear for intestinal protozoa after PVA or similar fixation (or from a concentrated, fixed stool). It is not a bacterial Gram stain and not a fungal wall stain. Cytoplasm of trichrome-stained amebae is typically blue-green; nuclear chromatin and chromatoid bodies stain red-purple. Read nuclear chromatin to separate Entamoeba histolytica/E. dispar from E. coli:

Giardia trophozoites show the face — two nuclei, axonemes, median bodies — on trichrome. Do not use trichrome as the primary stain for Cryptosporidium; those oocysts are shown with modified acid-fast or with EIA/NAAT (see the acid-fast section). Iron-hematoxylin is an alternative nuclear stain; the exam still expects trichrome morphology. Poorly fixed stool (no PVA, delayed preservation) gives pale, distorted nuclei you cannot speciate — reject or re-collect rather than guess E. histolytica.

Giemsa and Wright-Giemsa: blood parasites and inclusions

Giemsa (and Wright or Wright-Giemsa) stains nucleic acids and cytoplasm of blood cells and parasites.

• Thin film: methanol-fixed. Used for species-level Plasmodium morphology (rings, trophozoites, schizonts, gametocytes), parasite size relative to the RBC, Maurer dots, and Schüffner stippling. Also trypomastigotes of Trypanosoma, amastigotes of Leishmania in buffy coat or tissue, and Babesia Maltese-cross tetrads.
• Thick film: RBCs are laked (not methanol-fixed first). Used for screening and quantitation because a thick film examines about 20 times more blood than a thin film. Species morphology is poorer; do not speciate malaria from a thick film alone.

Timing and pH (Giemsa buffer about 6.8–7.2) matter: too acid, everything is too red; too alkaline, too blue, and stippling disappears. Make both films from the same fingerstick or EDTA draw promptly; EDTA distorts parasite morphology if the tube sits. Viral inclusion bodies may be Giemsa-stained in some cytology contexts (historically Tzanck smears), but current M testing emphasizes blood-parasite films more than obscure inclusion trivia. Giemsa is the wrong tool for stool protozoan nuclei (use trichrome) and for dermatophytes (use KOH/calcofluor).

Acridine orange: nucleic-acid fluorochrome for bacteria in fluids

Acridine orange intercalates nucleic acids and fluoresces (typically orange-red for RNA-rich bacteria at acid pH). It is a sensitive screen for bacteria in blood-culture bottles, CSF, or other sterile fluids when Gram stain is negative or the load is low. It does not replace Gram morphology for identification: orange blobs mean organisms present, not GNR versus GPC. Confirm positives with a Gram stain. Host-cell nuclei also fluoresce — know the size difference (bacteria ~1 µm versus leukocyte nuclei many times larger). Acridine orange is not a fungal chitin stain (that is calcofluor) and not a parasite nuclear stain (that is trichrome or Giemsa).

Contrast uses — pick the stain from the question