Ocular specimen collection

Standard procedures followed for ocular specimen collection, culture isolation and identification of the organisms are described below.

SPECIMEN COLLECTION KIT TO BE KEPT IN THE OPHTHALMOLOGIST OFFICE

The following are the items that must be available for collecting the corneal specimens.

• Culture plates (blood agar, potato dextrose agar)

• Sterile Kimura’s spatula or scraper

• Topical anesthetic agents

• Bunsen burner

• Clean glass slides

• Clean cover slips.

Corneal Scraping

Corneal scraping is performed under aseptic conditions by an ophthalmologist using a sterile Kimura spatula. The procedure is performed under magnification of a slit lamp or binocular loupe following instillation of topical anesthetic agents such as 0.5% proparacaine or 4% lignocaine.

Material obtained from scraping the leading edge and the base of each ulcer specimen are inoculated directly onto sheep blood agar, potato dextrose agar (PDA) and Brain Heart Infusion broth (BHI) without gentamycin sulphate. The material from the corneal scraping is also smeared and labeled onto slides in a thin, even manner to prepare a 10% KOH wet mount and Gram staining. In cases of suspected actinomycetes keratitis Kinyoun’s metho of acid fast staining is performed (Fig. 3.1). Corneal scrapings collected by an opthalmologist by using an ophthalmic microscope in shown in Figures 3.4A to F.

In some of the patients, the ulcer may be predominately in the deeper stromal lesion, with inflammatory outpourings in the anterior chamber. In such a case, a paracentesis is made using a 26 gauge needle mounted on a 2cc plastic disposable tuberculin syringe (Figs 3.2A and B).

Fig. 3.1: Materials used for collecting specimens from corneal ulcers

Figs 3.2A and B: Corneal scraping collected by an ophthalmologist by using slit lamp microscope

Biopsy

Deep stromal lesions and lesion with initially negative culture that may continue to progress clinically may require corneal biopsy. A corneal biopsy in the operation theater. A 3 to 5 mm circular trephine set to a depth of 0.2 to 0.3 mm can be used to outline the are to be biopsied. The edge of the specimen is lifted with a forceps and discussed. The tissue can be bisected and send for both histological analysis and culture. The base of the specimen can also be scrapped for routine culture (Fig 3.3).