Figure 4-1 “C” streaks on a chocolate blood agar plate. (Courtesy of James Chodosh, MD.)

Corneal biopsy may be necessary in cases of apparent and significant microbial infection when repeated cultures from corneal scrapings are negative. A small 2- to 3-mm trephine (disposable dermatologic skin punch) can be used to create a partial-thickness incision, and forceps and scissors are used to excise a lamellar flap of cornea. The specimen is generally split into 2 pieces, or separate biopsies are taken so that tissue can be evaluated by both histopathology and microbiology.

In vivo confocal microscopy may be a helpful diagnostic and management tool with reasonable sensitivity and specificity for larger microorganisms such as Acanthamoeba and fungi.

Alexandrakis G, Haimovici R, Miller D, Alfonso EC. Corneal biopsy in the management of progressive microbial keratitis. Am J Ophthalmol. 2000;129(5):571–576.

Kumar RL, Cruzat A, Hamrah P. Current state of in vivo confocal microscopy in management of microbial keratitis. Semin Ophthalmol. 2010;25(5–6):166–170.

Younger JR, Johnson RD, Holland GN, et al. Microbiologic and histopathologic assessment of corneal biopsies in the evaluation of microbial keratitis. Am J Ophthalmol. 2012;154(3):512–519.e2. Epub 2012 May 23.

Isolation techniques

For viral and chlamydial infections, an appropriate tissue-culture cell line is selected for inoculation and examined for the development of cytopathic effects (CPE) and cellular inclusions. For bacterial and fungal infections, directly inoculated blood, chocolate, and Sabouraud’s agar and thioglycollate broth are examined daily to detect visible growth. Microorganisms are studied by chemical staining, chemical reactions, and antimicrobial sensitivity testing. Acanthamoebae may be identified by trophozoite trails on blood agar, but nonnutrient agar with an overlay of killed Enterobacter aerogenes is the optimal isolation medium.

Staining Methods

See Table 4-4 for recommended stains and culture media in the setting of suspected microbial keratitis.

Virology and Viral Infections

Viruses are small (10–400 nm in diameter) infectious units consisting of a singleor double-stranded nucleic acid genome and a protein capsid shell, with or without an external lipid envelope. In