Eyelid specimens

Eyelid vesicles or pustules may be opened with a sharp-pointed surgical blade or small-gauge needle. Material for cytology is smeared onto a glass slide and fixed in methanol or acetone for immunofluorescent staining. Collected vesicular fluid can be inoculated into a chilled viral transport medium for culture isolation in the laboratory. Microbial cultures are obtained by wiping the abnormal area with a broth-moistened swab, followed by direct inoculation of culture media. Culture of epilated eyelashes may be helpful in cases of chronic infection.

Table 9-2 Principal Causes of External Ocular Infections

Table 9-3 Materials for Collecting Eyelid, Conjunctival, and Corneal Specimens for Ocular Microbiology

Table 9-4 Commonly Used Stains and Culture Media for Microbial Keratitis

Conjunctival specimens

To minimize contamination of and inhibitory effects on organisms recovered, conjunctival swabbing for microbial specimens should be performed without topical anesthetic. For specimen collection, the clinician must debride enough 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 dextrose agar). 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 may 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 local anesthetic is applied topically to the everted eyelid. Use of proparacaine hydrochloride 0.5% minimizes inhibition of organism recovery compared with tetracaine. 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 be performed to help in the diagnosis of conditions such as Parinaud oculoglandular syndrome, mucous membrane pemphigoid, and human papillomavirus infection. See Chapter 13 for a more detailed description of conjunctival biopsy.

Corneal specimens

A corneal culture is important to determine the etiology of large or sight-threatening ulcers, ulcers in which an atypical organism is suspected, or 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, fingers, or other nonsterile surface; in addition, a sterile instrument or swab should be used for each separate row of C-shaped streaks on each agar plate (Fig 9-1) and for each type of broth culture. For a viral culture, a Dacron swab is used to obtain virus-infected corneal or conjunctival cells; the swab is agitated in a chilled viral transport medium and then discarded. Calcium alginate and cotton swabs should be avoided, as both may inhibit viral recovery.

Corneal biopsy may be necessary in cases of apparent and significant microbial infection when repeated cultures from corneal scrapings are negative. This is particularly true in cases involving deeper infiltrates. See Chapter 13.

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

Figure 9-1 “C” streaks on a chocolate blood agar plate.

(Courtesy of James Chodosh, MD.)

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.

Isolation techniques

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