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 Table of Contents  
Year : 2020  |  Volume : 32  |  Issue : 1  |  Page : 97-101

Scraping in corneal ulcers

Department of Ophthalmology, Giridhar Eye Institute, Kadavanthra, Kerala, India

Date of Submission17-Jan-2020
Date of Acceptance01-Feb-2020
Date of Web Publication17-Apr-2020

Correspondence Address:
Dr. Nadhna Basheer
Chiraparambil House, Kizhuppillikkara (PO), Thrissur - 680 702, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/kjo.kjo_10_20

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Corneal scars resulting from central corneal ulcers are one of the major causes of blindness in developing countries. It is regarded as the second most common cause of preventable monocular blindness in some of the tropical countries. Corneal scraping is the most valuable specimen in cases of corneal ulcer, and its examination is the mainstay in the diagnosis and subsequent management.

Keywords: Cornea ulcer, corneal scraping, microbial keratitis

How to cite this article:
Basheer N. Scraping in corneal ulcers. Kerala J Ophthalmol 2020;32:97-101

How to cite this URL:
Basheer N. Scraping in corneal ulcers. Kerala J Ophthalmol [serial online] 2020 [cited 2020 Aug 12];32:97-101. Available from: http://www.kjophthal.com/text.asp?2020/32/1/97/282646

The cornea is the most exposed surface of the eyes which makes it vulnerable to trauma and infections. It also lends itself to perform diagnostic and therapeutic maneuvers in the clinic itself. Corneal ulcers are usually a result of trauma, especially due to vegetative matter and filamentous fungi accounts for about 67% of ulcers.[1]

Often, the laboratory methods, including microscopy and culture, may be negative despite a clear clinical presentation of suppurative keratitis. As a direct result of this, the treatment is often empirical. This may be due to difficulty in obtaining sufficient corneal material. It is imperative that the quality and quantity of the specimen is optimal for accurate laboratory diagnosis. The administration of antibiotics by patients before seeking medical attention has been thought to affect the recovery of organisms in culture. Even though history and clinical examination may aid in arriving at a probable diagnosis, it is always essential to take a timely as well as an adequate sample for its confirmation.

  Prequisities to Do a Corneal Scraping Top

  • Wire speculum (optional)
  • Proparacaine 0.5%
  • Kimura spatula/Bard parker blade#57-3 No's
  • 3 glass slides

    • Gram stain
    • KOH wet mount

    • Optional glass slide for special stain – AFB, Giemsa, and Calcofluor.

    • Coverslips
    • Culture plates.

  • Blood agar
  • Chocolate agar
  • Sabrodauds dextrose agar

  • Other optional plates include nonnutrient agar with  Escherichia More Details coli overlay, Thayer-Martin medium, and thioglycollate agar.

  Anesthesia Top

Corneal scraping is performed under topical anesthesia preferably instillation of two drops of 0.5% proparacaine in the inferior fornix.[2] Topical proparacaine has the least amount of bactericidal action as compared to other anesthetics agents such as tetracaine and xylocaine. It also provides adequate anesthesia within 1 min of instillation and does not cause intense stinging sensation. General anesthesia and sedation may be required in children, uncooperative adults, or mentally impaired patients.

  Instruments Top

Corneal scraping is obtained using the following instruments:

  • Kimura' s spatula
  • 26 G needle
  • Bard-Parker blade #57 (Becton Dickinson, Franklin Lakes, New Jersey)
  • Hypodermic needle
  • Surgical blade no. 15
  • Calcium alginate swab[2]
  • The platinum spatula (can be rapidly sterilized)
  • A modified platinum spatula is also available with a rounded flexible tip, which is modified with a honing stone to create a narrow tapered roughened edge to enhance the removal of corneal material.

We routinely use surgical blade no. 15 for taking the sample. In case of large ulcers and deep ulcers, extreme caution is advised.

  Technique Top

As before any procedure, we need to explain in detail, the need for the procedure and also request proper cooperation from the patient to get an adequate sample.

Ideally, the scraping is done under the slit-lamp biomicroscope or operating microscope [Figure 1]. The yield of scraping can be improved by taking scraping both from the edge of ulcer where pneumococcus mutiply [Figure 2] and base of ulcer where  Moraxella More Details reside [Figure 3].
Figure 1: Steps and technique in corneal scraping

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Figure 2: Scraping from the edge of the ulcer

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Figure 3: Scraping done from the base of the ulcer

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While performing, the following also keep in mind:

  • To keep the sharp edge of the instrument tangential to the surface to reduce the chance of perforation
  • Move the blade in one direction only
  • Do not contaminate the instrument with lids or lashes while collecting the samples.

  Difficulties in the Collection of Corneal Scrapings Top

There may be various situations where it is difficult to obtain the samples for corneal scraping.

  • In cases of small corneal ulcers, which are less severe
  • In cases of nonsuppurative cases of keratitis, there may be insufficient material to inoculate
  • In advanced keratitis with severe keratolysis and descemetocele
  • In cases of deep stromal keratitis, microsurgical scissors, a no. 11 Bard-Parker blade or a small trephine may be used to obtain an adequate sample
  • In case of intact epithelium, debridement of the epithelium is often required.

  Smear Preparation Top

The ulcer is scraped using the spatula and transferred to a glass slide, and marking is done using a wax pencil or marker to identify the area of smear [Figure 4]. At least two slides are prepared: one for Gram staining and the second for KOH wet preparation. An additional smear may be prepared for special stains such as Giemsa, periodic acid–Schiff, calcofluor, or Gomori modified methenamine-silver stain for which gelatin-coated slide is preferred. Lactophenol cotton blue is usual method to stain fungus, it delineates the chitinous fungal hyphae clearly against a light blue background.[Figure 5].
Figure 4: Slide with a circle for placing the specimen

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Figure 5: Fusarium using lactophenol cotton blue staining

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Negative smears are due to:

  • Prior use of antimicrobial agents
  • Mechanical damage to the cell wall
  • An insufficient sample
  • Poor staining techniques
  • Failure to examine the whole slide
  • Excessive heat fixatione

  Potassium Hydroxide Wet Mount Preparation Top

The smear is spread thinly as possible and 10% KOH is instilled, coverslip is put over the mount and examined under the microscope. KOH loosen the corneal stromal lamellae which help in exposing more fungi and also stain the fungus in a light yellow color.[3]

  Gram Staining Top

Gram staining differentiates the bacteria into Gram-positive and Gram negative bacteria, based on the presence of peptidoglycan layer in the cell wall. Gram-positive bacteria retain the violet color as it resists the decolorization due to thicker peptidoglycan. Gram-negative bacteria are pink in color, which is due to the absence of thin peptidoglycan in their wall which does not retain the crystal violet during decolorization. Various stain for the identification of different organism used in smear is given below [Table 1].
Table 1: Special stains used in identification of pathogens[2]

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Difficulties in gram staining are as follows:

  • When the number of an organism is low
  • Gram-negative organisms are more difficult to identify due to their lighter color
  • Improper decolorization
  • Gram's stain deposits, carbon particles, talcum powder, sodium chloride, crystals, melanin and granules, and precipitated gentian violet may appear as artifacts
  • When the stain is not used frequently, fungus can grow in the stain.

  Culture Examination Top

Culture is the gold standard for the diagnosis of microbial keratitis. The routine used culture media include blood agar, chocolate and Sabourauds dextrose agar, and anaerobic media (if anaerobes are suspected) [Table 2]. The selective media agar plates are inoculated by streaking the platinum spatula lightly over the surface to produce a row of separate inoculation marks in a C-shaped configuration.[2] C pattern streaks differentiate the valid growth from contamination. Fresh samples to be taken for each row of C streak [Figure 6].
Table 2: Different types of culture media for growth of various pathogen[2],[3]

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Figure 6: Blood agar and chocolate agar streaked in C pattern

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Liquid thioglycollate broth is inoculated by transferring the material to cotton-tipped applicator which has been moistened with trypticase soy broth or calcium alginate swab. The swab is inserted to the bottom of the tube to enhance the growth of anaerobic organisms. Different culture media used to grow organism are listed below [Table 2].

Micro-antimicrobial Removal Device is used in cases treated with antibiotics previously to increase the yield of the positive cultures. It is composed of sterile resins (amberlite XAD4 and C-249) which bind antibiotics suspended in sodium chloride.

Duration of the isolation of organism:

  • Aerobic bacteria in keratitis grow within 48 h. In some cases, as early as 12–15 h
  • All plates are examined for any growth using dissecting microscope, and liquid media is evaluated for any turbidity
  • In cases of severe keratitis, the media should be evaluated after 12–18 h of inoculation
  • Growth outside the C streak should be disregarded
  • Indigenous organisms in the tear are distinguished based on their sparse growth, and isolation of the same organism from the ipsilateral lids or the conjunctiva
  • Aerobic cultures should be held for 7 days, anaerobic cultures for 7–14 days, and mycobacterial and fungal cultures for 4–6 weeks before being reported as no growth.

  Interpretation of Culture Results Top

The interpretation of the culture results should be made with regard to the clinical situation, the adequacy of the sample, and the possibility of contamination by the organisms present on the skin, eyelids, and conjunctiva.

  Positive Culture Top

Criteria for a significant positive culture by some investigators include the clinical signs of keratitis plus one of the following:

  1. Growth of the organism in two or more media
  2. Confluent growth of a known ocular pathogen in one solid medium or
  3. Growth in one medium of an organism with positive smear results or growth of the same organism in liquid media.

Jones criteria for positive culture include

  1. Clinical signs of infection plus isolation of bacteria (10 or more colonies) on one solid medium and one additional medium[3]
  2. Isolation of fungi (any detectable growth) on any two media
  3. One medium in the presence of a positive smear.

Liquid media are highly sensitive but less specific in demonstrating the organism, as it is difficult to quantify the growth in broth.

Anaerobic bacteria are suspected in the following situations:

  • Pleomorphic, slender, or fusiform morphology seen on Gram stain
  • Growth in the anaerobic zone of the liquid medium
  • Within the depth of solid agar
  • Production of gas on the liquid media
  • Failure to grow an organism in aerobic media despite organism detection in Gram stain.[3]

Negative cultures reported in

  • In cases of sterile/noninfectious ulcers
  • Prior partial antibiotic therapy
  • Inadequate sampling methods
  • Improper selection of the media and incubation conditions
  • The false interpretation of the data.

When a negative culture is reported, the antibiotics are suspended for at least for 24 h and rescraping is done.

  Antimicrobial Susceptibility Testing Top

The method for testing susceptibility of antibiotics is by the standard disk diffusion and microdilution technique which uses the drug levels in serum rather than concentration in ocular tissues and fluids.

The broth microdilution technique is used to find the minimal inhibitory concentration (MIC) in ocular infections. The bactericidal concentration can be titrated from MIC by subculturing the clear broth in antibiotic-free zone.

  Serological Investigations Top

The serological test may be divided into the following three categories:[3]

  1. Target amplification systems such as polymerase chain reaction (PCR), cell sustaining sequence replication (3 SR), or strand displacement amplification
  2. Probe amplification systems which include ligase chain reaction
  3. Signal amplification in which the signal generated from each probe is increased by using compound probes or branched-chain technology. These techniques detect whether DNA or RNA from a particular organism is present but do not detect the viability of the organism.

Advantages of PCR include greater speed than culture methods (up to 4 h) and disadvantages include cost and false positivity.

16S rDNA typing has been used as rapid detection of the pathogens in patients with bacterial keratitis, whereas the 18S ribosome gene is used to detect fungal keratitis.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Maharana PK, Sharma N, Nagpal R, Jhanji V, Das S, Vajpayee RB. Recent advances in diagnosis and management of mycotic keratitis. Indian J Ophthalmol 2016;64:346-57.   Back to cited text no. 1
[PUBMED]  [Full text]  
Bowling B, Kanski J. Kanski's Clinical Ophthalmology. 8th ed, USA, Elseiver 2016, Chp 6. p. 171-9.   Back to cited text no. 2
Sharma N, Vajpayee RB. Work U of Corneal Ulcer, Corneal Ucers Diagnosis and Management. 1st ed.., Ch. 2. New Delhi: Jaypee Publishers; 2008. p. 35-61.  Back to cited text no. 3


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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Prequisities to ...
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Smear Preparation
Potassium Hydrox...
Gram Staining
Culture Examination
Interpretation o...
Positive Culture
Antimicrobial Su...
Serological Inve...
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