This patient-education article is written by the cornea service at Suraj Eye Institute, Nagpur.

Why Microbiology Matters in Keratitis

Several different organisms can produce a corneal ulcer — bacteria, fungi, the protozoan Acanthamoeba and the herpes-family viruses — and each needs a completely different treatment. The clinical appearance often suggests the most likely group, but overlap is common. A corneal scraping taken at the time of diagnosis lets the microbiology laboratory identify the organism and guide treatment with confidence.

Corneal Scraping Workflow

From Slit-Lamp to Diagnosis — Cornea Microbiology Workflow

1. Corneal scrape Kimura spatula or 21-G needle at the slit lamp, under topical anaesthetic

Gram stain Bacteria (cocci, rods); ~30 min

KOH / calcofluor white Fungal hyphae & Acanthamoeba

Culture panel Blood, chocolate, MacConkey, Sab

PCR (selected cases) HSV, Acanthamoeba, ribosomal panels

Same-day result Direct microscopy guides first-line empirical therapy within 1 hour

2–14 day result Culture identifies species and antibiotic sensitivity; tailored therapy

Scraping is taken before starting topical antibiotics if possible — antibiotics suppress culture growth.

Figure 1. The microbiology workflow for keratitis. A corneal scraping is taken at the slit lamp under topical anaesthetic using a Kimura spatula or 21-gauge needle. The sample is divided between direct microscopy (Gram stain for bacteria; KOH or calcofluor white for fungi and Acanthamoeba), inoculation on a panel of culture media, and, in selected cases, PCR for specific organisms. Direct microscopy gives a working diagnosis within an hour; culture confirms the species and antibiotic sensitivities over 2–14 days.

What Each Test Detects

  • Gram stain — bacteria (Gram-positive cocci, Gram-negative rods) within 30 minutes
  • KOH wet-mount — fungal hyphae and yeasts; bedside test, results within minutes
  • Calcofluor white — fungi and Acanthamoeba cysts under fluorescence microscopy
  • Culture media — blood agar (most bacteria), chocolate agar (fastidious organisms), MacConkey agar (Gram-negative rods), Sabouraud dextrose agar (fungi), non-nutrient agar with E. coli overlay (Acanthamoeba)
  • PCR — herpes simplex virus, Acanthamoeba and selected bacterial / fungal panels in difficult or culture-negative cases

What to Expect at the Scrape

The scraping is performed at the slit lamp under topical anaesthetic and takes only a few minutes. Two or three small samples are taken from the edge and base of the ulcer with a Kimura spatula or sterile needle, and the samples are placed directly on glass slides for staining and on culture plates. The procedure is well tolerated; patients describe pressure rather than pain.

Scrape before starting antibiotics where possible. Topical antibiotics started before scraping suppress culture growth and reduce the chance of identifying the organism. In our centre, scraping is part of the initial slit-lamp assessment for any significant suspected microbial keratitis.
✔ Cornea Microbiology at Suraj Eye Institute

We have on-site microbiology with rapid turnaround for KOH wet-mount, Gram stain and direct microscopy, so most acute keratitis cases have a working diagnosis within 30–60 minutes of scraping. Cultures on a panel of media (blood, chocolate, MacConkey, Sabouraud) are inoculated immediately at the slit lamp. PCR is available for selected indications such as Acanthamoeba and herpes simplex.

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Frequently Asked Questions

Will the scraping make my ulcer worse?
No. A few extra microns of epithelium are removed from the edge of an ulcer that already has an open epithelial defect. The clinical course is not affected and the diagnostic information gained is usually decisive for treatment.

How quickly will you know the diagnosis?
Direct microscopy (Gram stain, KOH wet-mount) usually gives a working diagnosis within 30–60 minutes, which is enough to choose first-line treatment. Culture identification of the species takes 2–7 days for bacteria and up to 2 weeks for slow-growing fungi or Acanthamoeba.

If the culture is negative, does that rule out infection?
Not always. A negative culture can occur if antibiotics or antifungals have already been used, if the sample was small, or if the organism is fastidious (slow-growing or difficult to culture). In such cases PCR or empirical treatment based on clinical pattern is used.

Will I need anaesthetic injections for the scrape?
No. The scrape is performed with topical anaesthetic drops only. No needle is used to anaesthetise the eye. The whole procedure takes a few minutes at the slit lamp.

Why do I need PCR if I’ve already had a scrape and culture?
PCR is reserved for specific situations where culture is unreliable — herpes simplex (which does not grow on standard media), Acanthamoeba (slow culture growth), or in any patient already partly treated where culture has come back negative.

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