Collection of Corneal Tissue

Corneal swabs and scrapings are collected from corneal ulcers for cytologic examination and bacterial or fungal culture and antimicrobial sensitivity testing. Corneal tissue should be examined cytologically when an immediate decision regarding therapy is needed, because cytological examination of corneal tissue often demonstrates the type of organism involved (e.g., gram-negative rods, gram-positive cocci or fungal hyphae). Culture medium inoculated with corneal tissue collected with a spatula often produces more bacterial growth than corneal tissue collected with a swab.

Collection of Corneal Tissue

Indications

• For cytological analysis or bacteriological and fungal culture of a corneal ulcer that appears to be infected, has stromal melting, or is not responding to topical therapy. Clinical signs of infection include:

• Corneal edema surrounding the ulcer
• Miosis, epiphora, photophobia, and blepharospasm
• Conjunctival and episcleral injection
• Corneal neovascularization
• Significant retention of rose bengal stain by the corneal epithelium in the absence of grossly visible corneal lesions or stromal uptake of fluorescein dye
• Purulent ocular discharge

• When neoplastic disease (most commonly, squamous cell carcinoma) of the cornea is suspected.

Materials for Culture and Scraping

• Sedation and a lip twitch to prevent movement of the head
• Topical anesthetic solution, such as 0.5% proparacaine (Ophthaine‚ E.R. Squibb and Sons, Princeton, NJ; Alcaine‚ Alcon Laboratories, Fort Worth, TX) injectable anesthetic agents such as lidocaine HCL or mepivacaine HCL are not advised for topical anesthesia of the cornea, because these drugs are more toxic to corneal epithelium than are topical local anesthetic solutions intended for ophthalmic use. In addition, lidocaine and mepivacaine significantly inhibit bacterial growth, but proparacaine does not.
• Injectable local anesthetic solution (lidocaine HCL or mepivacaine HCL) for anesthesia of the auriculopalpebral nerve
• Sterile, rayon- or cotton-tipped swabs synthetic-tipped swabs are preferred because cotton-tipped swabs contain fatty acids that inhibit bacterial growth.
• Transport medium (e.g., Amies or Stuart’s medium), or thioglycollate broth and Sabouraud dextrose agar for direct culture
• Sterile spatula (Kimura Spatula, Storz Instruments) and an alcohol-type burner for resterilization between multiple scrapings, or a sterile scalpel blade
• Glass microscope slides
• Cold packs for transport

Procedure

• The horse should be adequately sedated and a lip twitch applied to minimize the danger of additional corneal damage during the procedure.
• An auriculopalpebral or palpebral nerve block facilitates the procedure. The auriculopalpebral nerve can be anesthetized with 5mL of local anesthetic solution administered at a depth of 2 cm in a depression palpated at a junction where the dorsal border of the zygomatic process of the temporal bone meets a line drawn along the posterior border of the ramus of the mandible. Or alternatively, one to 2mL of local anesthetic solution is injected subcutaneously over the palpebral branch of the nerve where it can be palpated as it crosses the dorsal aspect of the zygomatic arch halfway between the lateral canthus of the eye and the base of the ear (Fig. 7-1).
• Several drops of a topical anesthetic solution are sprayed on the cornea using a syringe attached to the hub of a small gauge needle that has had its shaft removed (Fig. 7-2). Some ophthalmologists advise that corneal swabs and scrapings intended for microbiological culture be performed without topical anesthesia of the cornea because topical anesthetic solutions interfere with bacterial survival. It may be difficult, however, to collect a corneal swab or scraping from a horse without corneal anesthesia, and proparacaine has minimal deleterious effect on growth of pathogenic bacteria.
• If a culturette swab (Culturette, Marion Scientific, Kansas City, MO) is used, the swab should be moistened with the swab’s transport medium before the swab’s package is opened. The likelihood of culturing an organism is increased if a moist swab is used. Breaking the ampule of transport medium moistens the swab.
• The swab is applied to the edge of the corneal lesion and then inoculated onto thioglycollate broth and Sabouraud dextrose agar for direct microbiological culture, or the swab is placed in a transport medium (e.g., Amies or Stuart’s medium) and placed on a cold pack for transport.
• Edges of the corneal lesion are scraped with a spatula or the blunt end of a scalpel blade (Fig. 7-3 & Fig. 7-4), and the tissue obtained is spread on a glass slide and air-dried. If possible, multiple smears should be prepared. A drop of a 10 to 20% solution of potassium hydroxide (KOH) applied to a fresh smear (i.e., before it dries) facilitates identification of fungi. A minimum staining routine should include Gram’s and Giemsa stains.
• Tissue from the corneal scraping is also placed in transport medium (e.g., Amies or Stuart’s medium) or inoculated into thioglycollate medium and Sabouraud dextrose agar for direct culture.

Figure 7-1. An auriculopalpebral nerve block facilitates the corneal scraping. The nerve can be anesthetized by administering local anesthetic solution in a depression palpated where the dorsal border of the zygomatic process of the temporal bone meets a line drawn along the posterior border of the ramus of the mandible. (A). Or alternatively, local anesthetic solution is injected subcutaneously over the palpebral branch of the nerve where it can be palpated as it crosses the dorsal aspect of the zygomatic arch halfway between the lateral canthus of the eye and the base of the ear (B).

Figure 7-2. Several drops of a topical anesthetic solution are sprayed on the cornea using a syringe attached to the hub of a small gauge needle with the shaft removed.

Figure 7-3. Edges of the corneal lesion are scraped with a spatula or the blunt end of a scalpel blade.

Figure 7-4. Edges of the corneal lesion are scraped with a spatula or the blunt end of a scalpel blade.

Cytological or Microbiologic Findings

Interpretation

• Gram-negative rods seen during cytological examination indicates the possibility of infection with Pseudomonas sp.
• Most fungi are detectable by Gram’s staining.
• A diagnosis of keratomycosis cannot be ruled out by negative cytological or microbiologic findings.

Suggested Readings

Slatter D. Fundamentals of Veterinary Ophthalmology. Philadelphia: WB Saunders Co., 2001, pp101-103. - Available from amazon.com -

Brooks DE. Ophthalmology for the Equine Practitioner (Made Easy Series), Teton NewMedia, Jackson WY, 2002. Order from Teton NewMedia