Diagnostic Laboratory Techniques

To minimize contamination of and inhibitory effects on organisms recovered, conjunctival swabbing for microbial specimens can be performed without topical anesthetic. Specimen collection must debride enough surface conjunctival epithelial cells so that intracellular microbes can be seen on chemical stains. Calcium alginate or sterile Dacron swabs slightly moistened with thioglycollate broth are preferable to cotton-tipped swabs because the latter contain fatty acids, which may inhibit bacterial and viral growth. The swabbed material should be plated directly onto warmed solid media (blood, chocolate, and Sabouraud’s). The “nonhandled” distal end of the swab may then be broken off and placed directly into the remaining thioglycollate broth tube. If these media are not available, the specimens should be harvested with any standard culturette tube system that contains appropriate transport media.

When more conjunctival epithelial cells are desired, conjunctival scraping is the preferred method and reduces contamination from debris on the ocular surface. A topical anesthetic is applied to the everted eyelid. Use of proparacaine hydrochloride 0.5% minimizes inhibition of organism recovery. A sterile spatula is scraped firmly across the tarsus; this should cause blanching but minimal bleeding. Alternatively, a cytobrush may be used to rub the conjunctiva, after which it is placed in a buffer solution to release the epithelial cells onto a Millipore filter. The cells are then fixed. Appropriate instrumentation and proper handling are critical for specimens destined for polymerase chain reaction (PCR) testing, because any residual foreign DNA in the specimen may be detected by PCR. Conjunctival biopsy can also be performed to help in the diagnosis of conditions such as Parinaud oculoglandular syndrome, mucous membrane pemphigoid, or human papillomavirus (HPV) infection. See Chapter 14 for a more detailed description of conjunctival biopsy.

Corneal specimens

A corneal culture is indicated for large or sight-threatening ulcers, for ulcers in which an atypical organism is suspected, and for any ulcer that is not responding to therapy. A microbial specimen can be collected from a corneal ulcer by scraping the lesion with any of the following, with similar yields: platinum Kimura spatula, sterile needle, surgical blade, or thioglycollate-moistened calcium alginate or Dacron swab. For larger corneal ulcers (>2 mm), samples should be taken from several regions. A blade or spatula is preferable for preparing smears for chemical staining, but either a spatula or swab is acceptable for inoculation of culture media.

Specimens are best inoculated immediately onto microbiologic media that have been warmed to room temperature in anticipation of the culture procedure; microscopic slides should be prepared for Gram, Giemsa, or other special stains. To avoid contamination and false positives, care must be taken to avoid touching the blade or swab to the eyelids, and a sterile instrument or swab should be used for each row of C-shaped streaks on each agar plate (Fig 4-1) and for each type of broth culture. For a viral culture, a Dacron swab used to obtain viral-infected corneal or conjunctival cells is agitated in a chilled viral transport medium and discarded. Calcium alginate and cotton swabs should be avoided, as both may inhibit viral recovery.