Stains, Culture Media, Markers and Subspecialty Techniques

From EyeWiki


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.