When infectious agents are suspected on H&E but cannot be definitively identified, special histochemical stains highlight microorganisms in tissue sections. These stains remain essential despite the increasing use of IHC and molecular diagnostics — they are rapid, inexpensive, and provide morphological context.
Tissue Gram Stain
The tissue Gram stain (Brown-Hopps or Brown-Brenn modification) differentiates Gram-positive (blue-purple) from Gram-negative (red-pink) bacteria in paraffin sections. Unlike the bacterial Gram staining technique used in microbiology, which uses fresh culture, the tissue Gram stain works on fixed, processed tissue. Crystal violet stains Gram-positive cell walls; safranin or basic fuchsin counterstains Gram-negative bacteria, nuclei, and background. The tissue Gram stain is useful for identifying bacterial morphology (cocci, rods) and tissue reaction pattern — suppurative inflammation suggests pyogenic bacteria; granulomatous inflammation suggests mycobacteria or fungi.
Ziehl-Neelsen for Acid-Fast Bacilli
Ziehl-Neelsen (ZN) stains mycobacteria (Mycobacterium tuberculosis, M. leprae, non-tuberculous mycobacteria) by exploiting their waxy, mycolic acid-rich cell wall that retains carbolfuchsin dye after acid-alcohol decolorization. Acid-fast bacilli appear bright red against a methylene blue or brilliant green counterstain. ZN sensitivity in tissue is approximately 40-60% compared to culture or PCR — negative ZN does not rule out mycobacterial infection. Modified ZN (Fite-Faraco) uses a milder acid-alcohol decolorizer for M. leprae and Nocardia, which are less acid-fast than M. tuberculosis.
Grocott’s Methenamine Silver (GMS)
GMS is the most sensitive stain for detecting fungi in tissue. Chromic acid oxidizes fungal cell wall polysaccharides to aldehydes; methenamine silver solution reduces silver ions to metallic silver at the aldehyde sites, depositing black silver on fungal cell walls. The background is counterstained with light green.
GMS detects virtually all clinically relevant fungi: Candida (yeast and pseudohyphae), Aspergillus (septate hyphae branching at 45°), Mucor (broad, non-septate hyphae branching at 90°), Cryptococcus (encapsulated yeast, narrow-based budding), Histoplasma (small intracellular yeasts), Blastomyces (broad-based budding yeasts), Coccidioides (spherules containing endospores), and Pneumocystis jirovecii (cysts with characteristic dot-like morphology). GMS also stains elastin, which can serve as an internal positive control.
Periodic Acid-Schiff (PAS) for Fungi
PAS stains fungal cell walls magenta and is complementary to GMS. It is more specific than GMS (fewer false positives from debris) but slightly less sensitive. Many laboratories use both GMS and PAS on each fungal case to maximize detection. PAS with diastase removes glycogen that could be confused with fungi.
Warthin-Starry for Spirochetes
Warthin-Starry silver impregnation stains spirochetes black against a pale yellow-brown background. The technique deposits metallic silver on the microorganisms using a silver nitrate solution reduced by hydroquinone. Warthin-Starry is used to detect Treponema pallidum (syphilis — shows spiral organisms in skin, lymph node, and placenta), Borrelia burgdorferi (Lyme disease), Helicobacter pylori (gastritis), and Bartonella henselae (cat-scratch disease, bacillary angiomatosis). The stain is technically demanding and requires precise temperature and pH control.
Giemsa for Intracellular Microorganisms
Giemsa stain is used for Helicobacter pylori in gastric biopsies (spiral rods on the mucosal surface), Chlamydia (intracytoplasmic inclusions in conjunctival, cervical, or respiratory specimens), Babesia and Plasmodium (intraerythrocytic parasites), and Leishmania (amastigotes in macrophages). Giemsa stains nuclei purple-blue and cytoplasm pink; microorganisms appear as distinct basophilic structures.
Comparison and Algorithm
A diagnostic staining algorithm for suspected infection starts with Gram and GMS for all cases. If Gram-positive rods are seen, add ZN for Nocardia and mycobacteria. If granulomatous inflammation is present without visible organisms, add ZN and GMS. If spirochetes are suspected, add Warthin-Starry. If viral inclusions are present, IHC for specific viruses (CMV, EBV, HSV, HHV8, adenovirus) provides definitive identification. All special stains require positive controls run concurrently, and results must be correlated with culture, serology, and molecular testing for definitive diagnosis.
