Stains, Culture Media, Markers and Subspecialty Techniques

Article initiated by:

Jeremy Kudrna, MD, Stephen C. Dryden, M.D.

All authors and contributors:

Andrew G. Meador, Brian T. Fowler, MD, William Morris, Stephen C. Dryden, M.D., Louise A. Mawn, MD, FACS, Cat Nguyen Burkat, MD FACS

Assigned editor:

Review:

Assigned status Update Pending

 by Louise A. Mawn, MD, FACS on September 13, 2020.

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Microbial Stains

Stain	Uses and Suspected Ophthalmic Microorganisms
Gram stain	Bacteria Fungi Microsporidia
Acid-Fast stain (Ziehl-Neelsen) (Fluorescent Auramine O)	For organisms resistant to gram-staining: Including: Mycobacterium spp. Nocardia spp. Other bacteria with large amount of lipid substances within their cell walls
KOH test	Fungi Candida albicans Dermatophytes Trichophyton spp. Epidermophyton spp. Microsporum spp.
Giemsa stain	Parasites Bacteria Fungi Acanthamoeba spp. Chlamydia spp. (intranuclear inclusion bodies)
Periodic-Acid Schiff (PAS) stain	Stains glycogen Distinguish different types of glycogen storage diseases Stains basement membrane Determining polysaccharide and mucosubstances composition Acanthamoeba spp. corneal cyst wall Fungi Consider PAS staining of eyelashes in chronic anterior blepharitis unresponsive to classic anti-bacterial treatment
Calcofluor white (fluorescent microscopy)	Acanthamoeba spp. Fungi Microsporidia spp.
Grocott-G (Gomori’s) Methenamine-Silver (GMS) stain	Acanthamoeba spp. Fungi (e.g. mucormycosis) Microsporidia spp. Light-green counterstain

Culture Media

Culture Media	Specificity
Blood agar (5-10% sheep or horse blood)	Bacteria Including: Staphylococcus spp. Most aerobic bacteria Most anaerobic bacteria Atypical Mycobacterium Excluding: Haemophilus spp. Neisseria spp. Moraxella spp. Fungi
Blood agar in 5%-10% CO2	Moraxella
Lowenstein-Jensen media (nutrients + bacterial growth inhibitors)	Mycobacteria spp. Nocardia spp.
Chocolate agar (blood agar + cells lysed by heating, contains hemin and nicotinamide adenine dinucleotide [NAD])	Fastidious bacteria Including: Most aerobic bacteria Haemophilus influenzae Neisseria spp. Moraxella spp
Loeffler’s media	Corynebacteria
Sabouraud Dextrose agar (low pH + antibiotic)	Fungi Dermatophytes Trichophyton spp. Epidermophyton spp. Microsporum spp.
Thioglycollate broth	Aerobic bacteria Anaerobic bacteria
Chopped Meat broth	Anaerobic bacteria Propionibacterium acnes Fastidious bacteria
Viral transport	Viruses (e.g. Herpes Simplex Virus) Mycoplasma spp. Ureaplasma spp. Chlamydia spp.
Non-nutrient agar seeded with Escherichia coli (E coli = food source for Acanthamoeba )	Acanthamoeba
Mannitol Salt agar	Differentiating Staphylococcus spp.
Brain-Heart infusion	Difficult-to-culture organisms Including: Streptococci spp. Meningococci spp. Most yeast Most fungi
Potato Dextrose agar	Fungi
Tryptic Soy broth	Aerobic bacteria

Tissue Stains, Cytology and Markers

Tissue Stain	Uses and Specificity
Hematoxylin and eosin (H&E) stain	Hematoxylin: nucleic acids Stains blue Eosin: cytoplasmic organelles Stains pink
Periodic Acid-Schiff (PAS) stain	Basement membranes (e.g. Bruch’s/Descemet’s) Stains magenta Glycogen Fungi Mucopolysaccharides (e.g. goblet cells, mucus)
Masson trichrome stain	Granular corneal dystrophy/Avelino Rhabdomyosarcoma Demonstrates striations (strap cells) Collagen Stains blue/green Hyaline Stains red
Congo red stain	Lattice corneal dystrophy/Avelino Amyloid Stains orange
Crystal violet stain	Lattice corneal dystrophy Amyloid Stains red/purple
Alcian blue stain	Macular corneal dystrophy Mucopolysaccharides (e.g. goblet cells, mucus) Stain blue
Colloidal iron stain	Macular corneal dystrophy Mucopolysaccharide (e.g. goblet cells, mucus) Stains blue
Oil red O stain	Sebaceous cell carcinoma Neutral lipid (fat in tissue) Stains red/orange *Must be fresh tissue Formalin fixation leeches lipid (i.e. fat is removed by alcohol during tissue processing)
Sudan black stain	Optic nerve myelin Phospholipid Stains black
Luxol fast blue stain	Demyelination loses stain Myelin Stains blue
Bielschowsky’s Silver stain	Myelin, neurofibrillary tangles, and senile plaques Stain black
Bodian stain	Nerve fibers Stains black
Mucicarmine stain	Mucus-secreting adenocarcinomas Mucopolysaccharides (e.g. mucus and goblet cells) Stain pink/red Nuclei Stain black
Verhoeff-Van Gieson stain	Elastic degeneration Pterygia Pinguecula Temporal arteritis Skin Elastic tissue Stains black
Alizarin red stain	Band Keratopathy Calcium Stains red/orange
Von Kossa stain	Band Keratopathy Calcium Stains black
Prussian blue stain	Iron Stains blue Iron lines in epithelium Ferry, stocker, etc. Blood breakdown products in cornea or retina
Fontana-Masson stain	Amelanotic melanoma Melanin Stains black
S-100 protein	Heterologous cell lines from neural crest Nevi Melanoma Schwannoma Neurofibroma
Adipophilin monoclonal antibody	Sebaceous carcinoma
Polarizing filters	Regular molecular structure Amyloid Calcium oxalate and other crystals Suture granuloma Vegetable foreign body
Rose Bengal stain	Filamentary keratitis
Giemsa stain	Intracytoplasmic basophilic inclusions Chlamydia
Papanicolaou stain	Intranuclear eosinophilic inclusions Herpes virus (Tzanck smear)

Subspecialty Techniques and Stains

Subspeciality	Application of Techniques and Stains
Cornea	Stromal corneal dystrophies Macular corneal dystrophy Stains with Alcian blue Stains with colloidal iron Granular corneal dystrophy Granular corneal dystrophy type I: Stains red with Masson trichrome stain Granular corneal dystrophy type II: Stains red with Masson trichrome stain Stains with congo red Lattice corneal dystrophy Stains with congo red Stains with crystal violet Familial subepithelial amyloidosis: Stains with congo red Fleck dystrophy: Stains with colloidal iron Stains with Alcian blue
Oculoplastics	Specimen preparation guidelines Michel’s solution for immunofluorescence studies Pemphigus Ocular cicatricial pemphigoid Linear IgA deficiency “Fresh” specimen type Lymphoma (flow cytometry) Sebaceous cells Basics of Mohs micrographic surgery Indicated in areas of the body (eyelids, face) where clear surgical margins must be confirmed with minimal tissue loss to avoid functional and cosmetic deficits Best for removing biopsy proven locally invasive high risk skin cancers Appropriate use Indications for Mohs according to Connolly, et al. 2012 in mask areas of the face, including eyelids: Basal cell carcinoma Squamous cell carcinoma Lentigo Maligna/Melanoma in situ Merkel cell carcinoma Adenocystic carcinoma Adnexal carcinoma Apocrine/Eccrine carcinoma Atypical fibroxanthoma Dermatofibrosarcoma protuberans Extramammary Paget disease Leiomyosarcoma Malignant fibrous histiocytoma Undifferentiated pleomorphic sarcoma Mucinous carcinoma Sebaceous carcinoma
Oncology	Tumors visualized with frozen section Basal Cell Carcinoma Squamous cell carcinoma Tumors suboptimally visualized with frozen section Merkel cell carcinoma Sebaceous cell carcinoma Melanoma

References

1. 1. Trattler B, Friedman NJ, Kaiser PK. Review of Ophthalmology. Louis, Mo. : Elsevier/Saunders, 2018.
2. 2. Bowling B, Kanski J. Kanski’s Clinical Ophthalmology: A Systematic Approach. Edinburgh. : Elsevier/Saunders, 2016.
3. 3. Hogan MJ, Zimmerman LE. Ophthalmic Pathology : An Atlas and Textbook. Philadelphia : Saunders, 1962.
4. 4. BCSC Section 4: Ophthalmic Pathology and Intraocular Tumors. 2017-2018 edition.
5. 5. Mannis, M.J. and Holland, E.J. Cornea: Fundamentals, Diagnosis, and Management. Elsevier, Inc., 2017.
6. 6. Pernick N. Cornea: corneal dystrophy. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/eyecorneadystrophy.html. Accessed May 29th, 2019.
7. 7. Sheehan, D.C., Hrapchak, B.B. Theory and practice of Histotechnology.Columbus, OH. Battelle Memorial Institute, 1987.
8. 8. Ad Hoc Task Force, Connolly SM, et al. AAD/ACMS/ASDSA/ASMS 2012 appropriate use criteria for Mohs micrographic surgery: a report of the American Academy of Dermatology, American College of Mohs Surgery, American Society for Dermatologic Surgery Association, and the American Society for Mohs Surgery. J Am Acad Dermatol. 2012 Oct;67(4):531-50. doi: 10.1016/j.jaad.2012.06.009. Epub 2012 Sep 5. Erratum in: J Am Acad Dermatol. 2015 Apr;72(4):748. PubMed PMID: 22959232.