CORNEAL

SCRAPPING,
GRAM STAIN AND
CULTURE
D R . A N J A N A S A P K O TA
2ND YEAR RESIDENT
BPKLCOS
Contents
Corneal Ulcer Introduction
Causative organism Indication
Diagnosis Methodology
Interpretation
Corneal scarping
Introduction Microbial culture and
Indication sensitivity
Methodology Introduction
Interpretation Indication
Methodology
 Culture
 Antibiotic susceptibility testing
Interpretation
Gram staining

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 NORMAL MICROBIAL
FLORA
AEROBIC ANAEROBIC
• Gram‑positive cocci • Propionibacterium sp.
• Staphylococcus epidermidis • Peptostreptococcus
• Staphylococcus aureus • Bacteroides sp.
• Micrococcus • Lactobacillus sp.
• Streptococcus pyogenes • Clostridium sp
• Streptococcus pneumoniae
• Streptococcus viridans
• Gram‑negative cocci
• Moraxella catarrhalis
• Gram‑positive bacilli
• Corynebacterium species
• Gram‑negative bacilli
• Haemophilus influenzae
• Klebsiella sp.
• Escherichia coli
• Pseudomonas aeruginosa
• Moraxella sp.

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CORNEAL ULCER
Corneal epithelial defect associated with necrosis.

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 CAUSATIVE ORGANISMS
EUKAROYTES PROKARYOTES
Protozoa Filamentous bacteria
Sporozoa : Toxoplasma Actinomyces, Nocardia,
Amoebae: Entamoeba, Naegleria, Mycobacterium, Streptomyces
Acanthamoeba
Microsporidia
Fungi True Bacteria
Mould like: Aspergillus Gram-positive Bacilli and Cocci
Yeast like: Candida Gram-negative Bacilli and Cocci
Dimorphic : Histoplasma.
Blastomyces, Coccidioides
True yeasts: Cryptococcus
Spirochaetes
Borrelia, Treponema, Leptospira
Mycoplasma
Rickettsiae and Chlamydia

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DIAGNOSTIC
TECHNIQUES
1. Corneal scrapping
◦ Gram staining, KOH mount
◦ Culture and sensitivity
2. Serological investigations
◦ Polymerase chain reaction
◦ Ligase chain reaction
3. Confocal microscopy
4. Corneal biopsy

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CORNEAL SCRAPPING:
INTRODUCTION
Method to obtain most valuable specimen for
diagnosis of corneal ulcer.

Mainstay in its diagnosis and subsequent

management.

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 As per American Academy of Ophthalmology practice
guidelines initial cultures should be performed for:

◦ Corneal infiltrate is central, large, and/or is associated with

significant stromal involvement or melting.
◦ Infection is chronic or unresponsive to broad spectrum therapy.
◦ Atypical clinical features are present that are suggestive of fungal,
amoebic, mycobacterial keratitis.
◦ Infiltrates are in multiple locations in cornea.

Wilhelmus K, Liesegang TJ, Osato MS, Jones DB. Laboratory Diagnosis of Ocular Infections. Washington,
DC: American Society for Microbiology; 1994.
 CORNEAL SCRAPPING:
METHODOLOGY
ANESTHESIA
◦ Performed under topical anesthesia (after
instillation of two drops of 0.5 percent
proparacaine in the lower fornix of the affected
eye.)
◦ Advantages of proparacaine:
◦ least bactericidal as compared to other anesthetic agents.
◦ provides adequate anesthesia within one minute and does not
cause intense stinging on first installation.
◦ Indication of general anesthesia and sedation:
children, uncooperative adults or mentally
impaired patients.
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INSTRUMENT:
 Obtained using a Kimura’ s spatula.

 Other instruments for corneal scraping:

◦ 26-gauge needle, Bard Parker blade, hypodermic
needle and surgical blade no 15,thioglycolate
moistened calcium alginate
swab(prenriched/premoistened)

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TECHNIQUE:
1. A lid speculum is applied gently to separate the lids.
2. Done under a slit lamp or under an operating
microscope.
3. Under direct illumination, the ulcer is inspected.
(Any mucous or debris on and around the ulcer is
carefully cleaned with a sterile swab stick).

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4. Then, the leading edges and base of the ulcer
scraped.
5. Since material obtained from corneal scraping may
not be adequate, it should be directly inoculated into
the culture media rather than placing it first into the
transport media.
6. Multiple scrapings must be obtained to enhance the
yield of the organisms.

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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).

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CORNEAL SCRAPPING:
INTERPRETATION

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GRAM STAINING:
INTRODUCTION
 Gram’s stain classifies the bacteria into two major groups
based on the cell wall of the bacteria.

 Gram-positive bacteria: retain the gentian violet-iodine complex

and appear blue-purple.

 Gram-negative bacteria: lose their gentian violet-iodine complex

with decolorization step and appear pink when counterstained
with safranin.

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GRAM STAIN:
METHODOLOGY
 Fix smear by: placing in methanol for 5-10 min and allow to air
dry. (preferred method) or passing the slide, through flame two or
three times then allowed to cool.

The slide is flooded with crystal violet stain.

Stain allowed to remain on for 1 minute
Rinsed gently with tap water.
Slide flooded with Gram’s iodine solution. Solution allowed to
remain on for 1 minute.

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Rinsed gently with tap water.
 Decolorized with decolorizer solution until the color stops
running from the smear
Rinsed gently with tap water.
Slide flooded with Safranin stain. Stain allowed to remain on for
30 seconds.
Rinsed gently with tap water.
Allowed to air dry.

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GRAM STAIN:
INTERPRETATION
GRAM POSITIVE GRAM NEGATIVE
APPEARANCE Purple crystal violet Pink
Reason Stain retained by thick layer of Pink safranin
peptidoglycan that forms the outer counterstains the
layer of bacterial cell peptidoglycan layer of
periplasm of the
bacteria that fails to
retain purple crystal
violet stain.

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 KOH MOUNT
 KOH separates the fungal elements from intact cells as it
digests the protein debris and dissolves cement
substances.

 Shining fungal elements can be observed under

microscope.

 Different fungal elements like hyphae, pseudohyphae,

yeast cells, spores, spherules and sclerotic bodies can be
seen clearly in a KOH wet mount.

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ZIEHL-NEELSEN ACID FAST STAIN
Include the use of carbol-fuchsin or Ziehl- Neelsen acid-fast stain
for identification of suspected Mycobacteria, Actinomyces or
Nocardia.

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GIEMSA STAIN
 Consist of compounds made by mixing methylene
blue and eosin.
Differentiates bacteria from fungi, and also identifies
chlamydia inclusion bodies and cysts and trophozoites
of Acanthamoeba species.

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CALCOFLOUR WHITE
Calcofluor white binds to chitin and cellulose

As the cell wall of the yeast and filamentous fungi and
cysts of Acanthamoeba are composed of chitin and
cellulose, these organisms stain bright green with
calcofluor white under epiflorescent microscope

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LACTOPHENOL
COTTON BLUE
A mounting medium and staining agent used in the
preparation of slides for microscopic examination of fungi.
Has three components:
1. Phenol:   kills any live organisms;
2. Lactic acid : It  preserves fungal structures, and
3. Cotton blue : It stains the chitin in the fungal cell walls.
Fungal elements are stained intensely blue.

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MODIFIED GROCOTT-GOMORI METHENAMINE SILVER NITRATE STAIN

More reliable than the Gram, Giemsa, or KOH stain for fungal
infections.
The specimens should be spread onto gelatin-coated slides.

Fungus cell walls and septa appear black and seen against the faint
transparent green background.

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 ACRIDINE ORANGE
 Chemoflorescent dye.

Stains fungi and bacteria yellow-orange against a

green background when the pH is acidic.

 Epiflourescent microscope is used to visualize these

organisms.
 Identifies gram-positive and gram-negative bacteria,
yeast and hyphal forms of fungi and both the
trophozoite and cyst form of acanthamoeba.

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OTHER RELEVEANT
STAINING TECHNIQUES
TECHNIQUE ORGANISM VISUALIZED COLOR OF ORGANISM
Giemsa stain Bacteria Dark blue
Yeast cells and fungal Purple or blue
hyphae
Acridine orange Bacteria Yellow-orange
Fungi, Acanthamoeba Yellow-orange
Calcoflour white Fungi, acanthamoeba Bright green
cyst
Acanthamoeba Reddish-orange
trophozoite
Acid-fast stain (ZN stain) Mycobacterium, Bright red
Actinomyces (variably
stained)

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MICROBIAL CULTURE
AND SENSITIVITY:
INTRODUCTION
•The gold standard for the diagnosis of microbial keratitis.

•The culture media should be stored in a refrigerator.

•The media should be warmed to the room temperature to

prevent lethal cold shock to the organism.

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MICROBIAL CULTURE
MEDIAS
A) Solid medium: Agar is the most commonly used
solidifying agent.

B) Semi solid medium: Such media are soft and are useful
in demonstrating bacterial motility and separating motile
from nonmotile strains .

C) Liquid medium: Are sometimes referred as “ broth “.

Bacteria grow uniformly producing general turbidity eg.
Nutrient broth

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CULTURE MEDIA FOR VARIOUS
ORGANISMS

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CULTURE MEDIA FOR
VARIOUS ORGANISMS

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MICROBIAL CULTURE
AND SENSITIVITY:
METHODOLOGY
o 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.

o Fresh material should be obtained for each row of C

streaks.

o C streak method of inoculation differentiates valid growth

from contamination.

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 o Liquid thioglycolate broth is inoculated by transferring the
material to cotton tipped applicator which has been
moistened with trypticase soy broth or calcium alginate
swab.

o The swab is inserted to the bottom of the tube to enhance

the growth of anaerobic organisms.

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DURATION OF ISOLATION
OF ORGANISM
Most aerobic bacteria are seen on standard culture media
within 48 hours. (may be as early as 12 to 15 hours.)
All plates should be examined daily and liquid media should
be evaluated for the presence of turbidity.
Growth outside the C streak should be disregarded as it
implies contamination.
Aerobic cultures of the corneal specimens should be held
for 7 days, anaerobic cultures for 7 to 14 days and
Mycobacterial and fungal cultures for 4 to 6 weeks before
being reported as no growth.

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INTERPRETATION OF
CULTURE RESULTS

Interpreted based on the clinical findings, the adequacy of

the sample and the possibility of contamination by
organisms present on the skin, eyelids and conjunctiva.

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POSITIVE CULTURE

Criteria for significant positive culture:

Jones Criteria:
Clinical signs of keratitis PLUS Clinical signs of infection PLUS
1. Growth of the organism in two • Isolation of bacteria (10 or more
Or more media OR, colonies) on one solid medium and
one additional medium,
2. Confluent growth of a known ocular • Or isolation of fungi (any
pathogen in one solid medium OR, detectable growth) on any two
media or one medium in the
3. Growth in one medium of an presence of a positive smear.
organism with positive smear results
or growth of same organism in liquid
media.

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Anaerobic bacteria are suspected in following situations:
•Pleomorphic, slender or fusiform morphology seen on gram
stain of corneal smear or culture,
•Growth in the anaerobic zone of liquid medium or within the
depth of solid agar,
•Production of gas on liquid media and failure to grow
organism in aerobic media despite organism detection in
gram stain.

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REASONS FOR NEGATIVE CULTURE

 Sterile/non-infectious ulcers or
 Due to prior partial antibiotic treatment,
 Inadequate sampling methods,
 Improper selection of the media and incubation
Conditions and
 False interpretation of the data.

When the culture results are negative, antibiotic treatment

can be suspended temporarily for 24 hours and rescraping
is done following which repeat cultures are sent and
examined.

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BLOOD AGAR
Enriched media
Helps to isolate the fastidious organisms.
Standard medium for the isolation of aerobic bacteria at 35
c
Helps to support the growth of most saprophytic fungi at
room temperature
Derived from the seaweed, and addition of 5 to 10 percent
red blood cells provides nutrients and an index of
hemolysis.

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CHOCOLATE AGAR
Enriched media.
Prepared by the heat denaturation of blood and provides
hemin (x factor) and diphosphopyridine nucleotide (v factor)
essential for the growth of heamophilus.
Should be incubated at 35°c with 10 percent carbon
dioxide.
Also supports the growth of neisseria and moraxella.

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SABOURAUD’S AGAR
Consists of glucose and peptone agar.
Yeast extract is added to improve nutritional characteristics
and an antibiotic such as gentamicin or chloram-phenicol is
added to inhibit bacterial contamination.

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THIOGLYCOLATE BROTH
Promotes the growth of aerobic bacteria, as well as obligate
and facultative anaerobic organisms at 35 degree Celsius.

Consist of the basic nutrients required to support the growth

of aerobic bacteria and also has sulf-hydryl compound that
act as an oxygen reducing agent to facilitate the recovery of
the anaerobic bacteria.

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ANAEROBICALLY
STERILIZED MEDIA
(PRAS)
Ideal medium for the isolation of the anaerobic bacteria.
A PRAS Brucella blood agar plate enriched with vitamin K
and haemin allows the growth of the anaerobes within 4 to 7
days.
This is also recommended for isolation of nutritional variant
streptococci, which may be a causative organism for
infectious crystalline keratopathy.

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BRAIN HEART INFUSION
(BHI)
Enhances the growth of filamentous fungi and yeasts at
room temperature.

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THAYER MARTIN MEDIA
Enriched chocolate media that suppresses the growth of
the inhibitory components and selectively allows the growth
of the N. Gonorrhoeae.

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LOWENSTEIN-JENSEN
MEDIA
In cases of suspected atypical microorganisms, the cultures
should also be sent on Lowenstein Jensen media.
Allows the growth of mycobacteria.
Contains glycerol and egg mixture which provides fatty
acids and proteins

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 APPEARANCE

Staphylococcus in blood Streptococcus in blood agar

agar Pseudomonas in Yeast
Agar

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Contd…

ASPERGILLUS IN SDA ASPERGILLUS IN BLOOD AGAR

ANTIBIOTIC
SUSCEPTIBILITY TESTING
Preferred methods:
Standard disk diffusion method and
Micro-dilution techniques

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DISC DIFFUSION METHOD
Performed by applying a bacterial inoculum of
approximately 1–2×108cfu/ml to the surface of a large (150
mm diameter) Mueller-Hinton agar plate.
Up to 12 commercially-prepared, fixed concentration,
paper antibiotic disks are placed on the inoculated agar
surface.
Plates are incubated for 16–24 h at 35°c prior to
determination of results.

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The zones of growth The zone diameters of
inhibition around each of each drug are interpreted
the antibiotic disks are using the specified criteria.
measured to the nearest
millimeter.
The diameter of the zone
is related to the
susceptibility of the isolate
and to the diffusion rate of
the drug through the agar
medium.

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Gives “qualitative” results, category of susceptibility (i.e,
susceptible, intermediate, or resistant) is derived from the
test.
Advantages:
 Test simplicity
 Least costly of all susceptibility methods

Disadvantages:

 Not all fastidious or slow growing bacteria can be accurately tested

by this method

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MICRO-DILUTION
TECHNIQUES
Uses small, disposable, plastic
“micro-dilution” trays

Standard trays contain 96 wells,

each containing a volume of 0.1 ml
that allows approximately 12
antibiotics to be tested.

Micro-dilution panels are filled with

broth and supplement of blood.

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Varying concentrations of antibiotics and bacteria to be
tested are then added.

 Plate then placed in a non CO2 incubator and heated at

35 degree Celsius for 16-20 hours

Following incubation, MICs are determined using a manual

or automated viewing device for inspection of each of the
panel wells for growth.

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 Advantages:
The generation of MICs,

Test the susceptibility of

microorganisms to
multiple antibiotics at
once.

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CONFOCAL MICROSCOPY
Fast emerging, noninvasive modality in the rapid diagnosis
of fungal and Acanthamoeba keratitis.

Offers magnifications of up to 200 to 500 with increased

image contrast, and allows details to be visualized even in
hazy corneas.

Aid in the diagnosis, management, and follow-up of cases

of infectious keratitis.

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The fungal filaments: seen as bright, linear, filamentous
structures with bright borders that appear as parallel lines that look
like double-walled structure on confocal microscopy.
Acanthamoeba cysts: presence of oval to round, double-walled,
highly refractile structures with a polygonal inner wall.

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CORNEAL BIOPSY
Indicated if the response to treatment is poor.
 If repeated cultures have been negative and clinical
picture continues to strongly suggest an infectious process.
If the infiltrate is located in the mid or deep stroma with
overlying uninvolved tissue.
Trephine can be used to obtain stromal tissue at the edge
of infiltrate.
The sample obtained is bisected and sent for
histopathology and culture.
Epidemiologic Characteristics, Predisposing Factors,
and Etiologic Diagnosis of Corneal Ulceration in Nepal
Between September 1985 and August 1987
405 patients with corneal ulceration were examined at Tribhuvan University
Teaching Hospital in Kathmandu, Nepal.
RESULT:
 Microorganisms were grown from 324 (80%) of the ulcers.
Pure bacterial cultures were obtained from 256 (63.2%) of the patients, whereas
pure fungal cultures were obtained from 27 (6.7%) of the patients.
 In 41 patients (10.1%), corneal cultures yielded a mixed growth of bacteria and
fungi.
 Of a total of 398 bacterial isolates, 124 (31.1%) were positive for Streptococcus
pneumoniae, the most commonly isolated organism in the series.
 Of 68 positive fungal isolates obtained, 32 (47.0%) were identified
as Aspergillus species. Candida species and Fusarium species were less common.

Madan P. Upadhyay, M.D.1, Purna C.D. Karmacharya, M.D.1, Shasank Koirala, M.D.1, Nhuchhe R. Tuladhar, B.S.1, Larry E. Bryan, M.D.2, Gilbert Smolin, M.D.3, John P. Whitcher,
M.D.3Department of Ophthalmology, Tribhuvan University, Institute of Medicine, Kathmandu, NepalDepartment of Microbiology, University of Calgary, Calgary, CanadaFrancis I.
Proctor Foundation for Research in Ophthalmology, University

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 Microbiological profile of corneal ulcer cases
diagnosed in a tertiary care ophthalmological
institute in Nepal
TIO from April to October 2014
Results:
Out of 101 samples analyzed, 44.6% (45/101) showed positive growth, with
bacterial isolates i.e., 56% (25/45), more prevalent than fungus i.e., 44% (20/45).
Among bacteria Streptococcus pneumoniae (31.1%, N = 14) was isolated in
highest number.
Fusarium (13.4%, N = 6) was the most common fungus species. Pseudomonas
aeruginosa was the only Gram negative bacteria isolated from corneal ulcer
cases.
All bacterial isolates were found to be susceptible to the quinolone group of
antibiotics (Moxifloxacin followed by Ofloxacin and Ciprofloxacin)
Sharmila Suwal, Dinesh Bhandari , Pratigya Thapa, Mohan Krishna Shrestha and Jyoti Amatya

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 REFERENCE
•Ananthanarayan and Paniker’s Textbook of Microbiology, 9th Edition
•CORNEAL ULCERS Diagnosis and Management, Namrata Sharma
•Myron Yanoff; Jay Duker ; Ophthalmology , 2nd Edition
•2016-2017 Basic and Clinical Science Course™, AAO
•Brad Bowling, Frcsed Frcophth FRANZCO; Kanski's Clinical Ophthalmology: A
Systematic Approach, 9th edition
•Role of Confocal Microscopy in the Diagnosis of Fungal and Acanthamoeba
Keratitis
• Pravin K. Vaddavalli, MD,1 Prashant Garg, MD,1 Savitri Sharma, MD,2 Virender S. Sangwan, MD,1 Gullapalli N. Rao, MD,1 Ravi Thomas, MD3

•Epidemiologic Characteristics, Predisposing Factors, and Etiologic Diagnosis of

Corneal Ulceration in Nepal
• Madan P. Upadhyay, M.D.1, Purna C.D. Karmacharya, M.D.1, Shasank Koirala, M.D. 1, Nhuchhe R. Tuladhar, B.S. 1, Larry E. Bryan, M.D.2, Gilbert Smolin, M.D. 3, John P. Whitcher, M.D.3Department of Ophthalmology, Tribhuvan University,
Institute of Medicine, Kathmandu, NepalDepartment of Microbiology, University of Calgary, Calgary, CanadaFrancis I. Proctor Foundation for Research in Ophthalmology, University

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 THANK YOU!!

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