Taking a corneal scrape and making a diagnosis

Astrid Leck

Astrid Leck
Research fellow, International Centre for Eye Health, London School of Hygiene and Tropical Medicine, Keppel Street, London W1CE 7HT, UK.

	Figure 1. Taking a corneal scrape Photo: J Dart
	Figure 4. Flood the slide with crystal violet Photo: Pak Sang Lee
	Figure 5. Staphylococci sp. Photo: MM Matheson
	Figure 6. Streptococcus pneumoniae Photo: MM Matheson
	Figure 7. Pseudomonas sp. Photo: MM Matheson
	Figure 8. Gram appearance of yeast cells (left) and pseudohyphae (right) Photo: Astrid Leck
	Figure 9. Fungal hyphae visible after Gram stain Photo: Astrid Leck
	Figure 10. Fungal hyphae stained with lactophenol cotton blue Photo: PA Thomas
	Figure 11. Calcofluor white preparation Photo: J Dart
	Figure 12. The trophozoite form of Acanthamoeba Photo: Astrid Leck

Keywords: Eye Infections; Corneal Ulcer; Keratitis; Corneal Diseases / diagnosis;

This article aims to provide a comprehensive guide to taking a corneal scrape and making a diagnosis. However, there are settings in which there are either limited or no laboratory facilities available to the ophthalmologist; for example, at primary level eye care centres in rural locations. In these circumstances, microscopy may still provide valuable information to guide the clinician in their choice of treatment (Figures 5–11 are images of infected corneal tissue as seen by microscopy).

Taking a corneal scrape

What you will need:

21-guage needles or Kimura scalpel
2x clean microscope slides
1x blood agar plate (FBA)
1x Sabouraud glucose agar plate (SGA)
1x brain heart infusion broth (BHI) (for fastidious organisms)
1x cooked meat broth (CMB) (excludes facultative anaerobes)
1x thioglycollate broth (TB)
1x non-nutrient agar (NNA) (if Acanthamoeba sp. is suspected)

In order to have the best possible chance of providing the clinician with an accurate diagnosis, all the media listed are required. In some remote settings, some media may not be available or there may be limitations in the variety of media it is possible to process. For these situations, the minimum requirements are denoted above in bold type, in order of importance. Liquid phase media (broths) must be used when available. If only one liquid phase media is to be used, this should be BHI; it is essential to inoculate more than one bottle. NNA is indicated only if amoebic infection is suspected.

General principles

If possible, withdraw the use of antimicrobial agents for 24 hours prior to sampling. Where this is not possible, the use of liquid phase media, for example BHI, serves as a diluent that reduces the concentration of the drug below the minimum inhibitory concentration (MIC).
Apply anaesthetic drops that do not contain preservative.
Use a different needle to take each specimen or, if using a Kimura scalpel, flame the scalpel between samples.
If fungal or amoebic infection is suspected, it is preferable to sample material from the deeper stromal layer of the cornea.

Order of specimen preparation:

Slide for Gram stain and slide for alternative staining processes
Solid phase media (FBA/HBA, SGA, NNA)
Liquid phase media (BHI, CMB, TB)

If the ulcer is very discrete or only a small amount of corneal material is available, inoculate one solid and one liquid phase medium.

Specimen collection for microscopy

Label slide with patient’s name, date of birth, and hospital number.
Draw/etch a circle on the slide and place specimen within the circle (Figure 2).
Air-dry and cover with a protective slide (tape the ends) or place in a slide transport box.

Figure 2. Slide with label and circle for placing the specimen

Slide with label and circle for placing the specimen

Inoculating culture media

Gently smear material on the surface of agar in C-streaks (Figure 3); taking care not to puncture the surface of the agar.
Sellotape the lid of the plate to the base around the perimeter.
Incubate inoculated culture media as soon as possible. Refrigeration of specimens is to be discouraged and, if not being transported directly to the laboratory, it is preferable to keep samples at room temperature.

Figure 3. Smear the material on the surface of agar in C streaks

Smear the material on the surface of agar in C streaks
Photo: Astrid Leck

Making a diagnosis

Microscopy: the Gram stain

Air-dry and heat-fix specimen using a Bunsen burner or spirit lamp
Allow slide to cool on staining rack
Flood slide with crystal violet; leave for 1 minute (Figure 4)
Rinse slide in clean running water
Flood slide with Gram’s iodine; leave for 1 minute
Rinse slide in clean running water
Apply acetone and rinse immediately under running water (exposure to acetone <2 seconds)
Counter-stain with carbol fuschin for 30 seconds
Rinse in clean running water then dry with blotting paper
View specimen with 10x objective
Place a drop of immersion oil on the slide and view with 100x oil-immersion objective.

Gram positive (+ve) cocci most commonly associated with suppurative keratitis are the Staphylococci (Figure 5) and Streptococci (Figure 6, Streptococcus pneumoniae).
Gram negative (–ve) bacilli, such as Pseudomonas sp. (Figure 7), may be associated with corneal infection.
A definitive diagnosis of Nocardia sp. (Gram variable) may be possible
Yeast cells will stain positively.

Although not the first choice of stains for fungi, yeast cells, pseudohyphae, and fungal hyphae may be visualised in Gram-stained corneal material, typically staining negatively or Gram variable. For microscopy to provide a more definitive diagnostic tool for fungal infection, Gram stain can be destained and restained using a more appropriate stain (Figures 8 and 9).

Microscopy: additional methods

Lactophenol cotton blue (LPCB) or potassium hydroxide (KOH) wet mount preparations are used to visualise fungi (Figure 10).

Add a drop of lactophenol cotton blue mountant to the slide.
Holding the coverslip between your forefinger and thumb, touch one edge of the drop of mountant with the coverslip edge, the lower it gently, avoiding air bubbles. The preparation is now ready.
Initial observation should be made using the low power objective (10x), switching to the higher power (40x) objective for a more detailed examination.
Calcofluor white and Periodic Acid Schiff reaction (PAS) staining may also be used.

Diagnostic criteria

Diagnostic criteria applied to bacterial culture

the same organism growing at the site of inoculation on two or more solid phase cultures, or
growth at site of inoculation on one solid phase media of an organism consistent with microscopy, or
confluent growth on one media.

Diagnostic criteria applied to fungal specimens

fungal hyphae observed in corneal specimen stained on microscopic examination, or
growth at site of inoculation on solid culture media

Amoebic infections

The cyst form of Acanthamoeba sp. can be visualised in corneal material using a direct fluorescent technique such as calcofluor white (Figure 11), haemotoxylin and eosin, LPCB, or PAS. If corneal infection with Acanthamoeba sp. is suspected, inoculate corneal material onto non-nutrient agar in a demarcated area of the plate. In the laboratory, the square of agar where the specimen was inoculated will be excised and inverted onto an NNA plate seeded with a lawn of E.coli. Growth of the trophozoite form is imperative to confirm viability of the organism and thus prove it to be the organism responsible for infection (Figure 12).