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 Table of Contents  
Year : 2017  |  Volume : 29  |  Issue : 2  |  Page : 72-78

Microbiology for general ophthalmologists

1 Professor in Ophthalmology, Government Medical College, Kozhikode, Kerala, India
2 Department of Ophthalmology, Government Medical College, Kozhikode, Kerala, India

Date of Web Publication10-Aug-2017

Correspondence Address:
V Babitha
Department of Ophthalmology, Government Medical College, Kozhikode - 673 008, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/kjo.kjo_58_17

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Since ocular surface infections are very common in everyday ophthalmic practice, every ophthalmic personnel are familiar with their etiologies and the basic techniques for the diagnosis.

Keywords: Culture and sensitivity, Gram's staining, KOH staining, microbial flora

How to cite this article:
Babitha V, Jyothi P T. Microbiology for general ophthalmologists. Kerala J Ophthalmol 2017;29:72-8

How to cite this URL:
Babitha V, Jyothi P T. Microbiology for general ophthalmologists. Kerala J Ophthalmol [serial online] 2017 [cited 2022 Dec 3];29:72-8. Available from: http://www.kjophthal.com/text.asp?2017/29/2/72/212755

  Introduction Top

Ocular surface infections are very common in everyday ophthalmic practice. Hence, every ophthalmic personnel are familiar with etiologies of these infections and the basic techniques for their diagnosis.[1] Although eyes are relatively impermeable to microorganisms, intraocular infections result from trauma, surgery, or systemic disease.[2] While evaluating a patient with infection, it is of great help to be familiar with the normal microbial flora of the human eye.[1]

  Normal Microbial Flora of Eye Top

Few microorganisms are present normally in the conjunctival sac.[3]


  • Gram-positive cocci

    • Staphylococcus epidermidis
    • Staphylococcus aureus
    • Micrococcus
    • Streptococcus pyogenes
    • 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.


  • Propionibacterium sp.
  • Peptostreptococcus
  • Bacteroides sp.
  • Lactobacillus sp.
  • Clostridium sp.


These are transient and are those found in the environment.

  Microbiology of Ocular Surface Infections Top

To get a better result with microbiologic examination, the ophthalmologist must inform the laboratory about the type of specimen, expected pathogens, and specific antibiotic susceptibilities that must be tested.[4] For diagnosing unusual pathogens, special planning is required.[4]

  Ocular Specimen Collection Top

Peculiarities of ocular specimen:[3]

  1. Small amount of material
  2. Number of organisms very less
  3. Most of the time patients are on topical medication while taking specimen
  4. Specimen may have nonviable organisms
  5. Most of the time liquid media has to be used
  6. Frequently fastidious organisms are encountered; therefore, enriched media are necessary.


Conjunctivitis is usually diagnosed by clinical features.[3]

Conjunctival specimen

Soft-tipped applicators with plastic handles and heads composed of cotton, Dacron, or calcium alginate are used for conjunctival specimen collection.[3],[4] Calcium alginate swabs produce a higher yield of bacterial organisms.[4]


Following are the steps of conjunctival specimen collection [Figure 1].[3]
Figure 1: Conjunctival swab collection

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  • Patient looks up
  • Lower lid pulled down
  • Moistened swab rubbed over conjunctival sac from medial to lateral and then back
  • Swab is directly inoculated onto blood agar (aerobic incubation), chocolate agar, and brucella blood agar.

Conjunctival scraping is rarely taken and used to identify chlamydia and viruses.[3]

  Infective Keratitis (Microbial Keratitis) Top

It is an important cause for corneal blindness. A wide range of microbial agents cause corneal infections. A rapid etiological diagnosis has an important role in initiating an aggressive and targeted treatment. Clinical features of infectious keratitis may help in the initial etiological diagnosis [Table 1].
Table 1: Causative organisms and it's clinical features

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Instruments used for corneal specimen collection are spatulas, Jeweler's forceps, and surgical blades.[3],[4] Cotton-tipped applicators are used in less aggressive infections, and less distinct areas of involvement over a large area require culture.[3],[4]


Corneal specimen is collected as follows [Figure 2].[3]
Figure 2: Corneal scraping

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  • Anesthetize the eye (avoid tetracaine which has antibacterial action), clean the eyelids and conjunctival sac
  • Scraping taken from the edge and the base of the corneal lesion under slit lamp or operating microscope
  • Material is inoculated directly onto various culture media
  • Make smears on glass slides, mark with wax pencil on reverse side, and stain with various stains
  • If any delay for inoculation store the specimen at − 20° C
  • In case of therapeutic keratoplasty, corneal button or corneal biopsy tissues are cut into small bits in sterile conditions and inoculated on various culture media
  • Excessive scraping leads to corneal scarring and long-term poor visual acuity.[6]

Transport media

Direct inoculation of specimen to the culture media is the best but may not be possible always.[4] Many transport systems for supporting bacteria, virus, and chlamydia before direct inoculation on proper isolation media are available.[4] For example, Amies transport medium.[8]

Common stains and tests

Commonly used stains are Gram stain, acridine orange, calcofluor white, and acid-fast, Giemsa stain.[4] All bacteria isolated from the cornea should be considered as pathogens and tested for antibiotic susceptibilities.[4]
Figure 3: Gram staining - Pneumococci

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Figure 4: Gram staining - Candida

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Figure 5: Acid-fast bacilli

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Figure 6: Lactophenol cotton blue staining - Fusarium

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Figure 7: Blood agar - Pneumococcal growth

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Figure 8: Sabouraud dextrose agar - Candida growth

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  Processing and Interpretation of Direct Smears Top

Smears are interpreted by staining methods [Table 2].
Table 2: Staining characteristics and microscopic appearance of causative pathogens

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  Culture of Corneal Specimen Top

Routine media for isolating bacteria from the cornea, conjunctiva, and eyelids are sheep blood agar, chocolate agar, and mannitol salt agar, enriched thioglycollate liquid medium.[4] Inoculated culture media should be kept and examined for at least 5 days [Table 3].[4] The criteria, to consider the growth on culture to be significant, are if growth is confluent (more than 10 colonies) on the site of inoculation on the solid media, or the organism is seen in the smears, or if the same organism is grown in more than one medium.[8]
Table 3: Culture media used for isolating organisms and their appearance

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Corneal biopsy


  • Poor response to treatment
  • Repeated negative culture in the presence of clinical feature strongly suggestive of infectious process
  • Deep stromal infiltrate with uninvolved superficial layers.

Corneal biopsy can be taken under slit lamp or operating microscope. Under topical anesthesia, a trephine or a blade is used to excise a small piece of stromal tissue. Femtosecond laser can also be used as an alternative. A 7-0 or 8-0 vicryl or silk suture can be passed through the abscess without disturbing the overlying epithelium. The organism may attach to the fibers of the suture which can be cultured. Another option is to take biopsy under a lamellar flap.

Uncommon organisms


They are fastidious organisms and require long incubation periods for isolation.[4] Mycobacteria keratitis is due to rapidly growing organisms such as Mycobacterium chelonae and Mycobacterium fortuitum which grows on routine culture media such as blood agar or chocolate agar.[4] Acid-fast staining and inoculation in Lowenstein– Jensen media can be used for mycobacterial isolation.[4]

Nocardia and Actinomyces

These are less frequent ocular pathogens.[4]Nocardia can be cultured on routine bacterial culture media.[4]Actinomyces requires extended growth period, and cytologic examination of corneal specimen by Giemsa stain is diagnostic test (long thin filaments with branching).[4]


The stains commonly used to identify Microsporidia are modified trichrome, Giemsa, Gram, periodic acid-Schiff, Grocott's methenamine silver, calcofluor, and acid-fast methods.[4]


Cell culture isolation, Giemsa stain, and polymerase chain reaction are the methods used to detect chlamydia from ocular samples.[4]

Rare pathogen that cannot be easily cultured need to be tested in special laboratories or at facilities researching the particular disease.[4]

  Antibiotic Susceptibility Testing Top

It is performed on fast-growing bacterial isolates to assess whether an antibiotic will be successful in therapy or not.[4] The disc diffusion method and minimum inhibitory concentration (MIC) methods are used to determine antibiotic susceptibility in vitro. In disc diffusion method, paper disks impregnated with a set amount of antibiotics is placed on a lawn of bacteria and incubate for 24 h, then zones of inhibition are measured and compared to predetermined zone standards [Figure 9].[4] MIC methods include broth dilution, agar dilution, and E-tests.[4] With these methods, MIC of antibiotic required to inhibit bacteria is determined and compared to predetermined standards that represent susceptibility.[4] Ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, oxacillin, cefazolin, vancomycin, gentamicin, and tobramycin are the commonly tested antibiotics in corneal infections.[4]
Figure 9: Minimum inhibitory concentration method for susceptibility

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  Diagnosis of Contact Lens-Associated Keratitis Top

In contact lens-associated keratitis, the lens, container, and the solution have to be examined and cultured.[3],[6] Contact lenses should be removed aseptically, placed in sterile saline, and can be cultured by agar sandwich method.[3] Contact lens solution can be cultured on standard media such as blood agar, MacConkey agar, nonnutrient agar, and Sabouraud dextrose agar.[3] The lens deposits and centrifuged deposit of the lens case solutions can be examined under microscope to detect the organism.[3]

  Serological Methods of Diagnosis Top

The serological tests have little value in the diagnosis of corneal infections.[3]

Newer methods in the diagnosis of infectious keratitis

The newly introduced techniques such as immunochemistry, fluorescent microscopy, enzyme immunoassays, radioimmunoassays, and molecular biologic techniques are highly specific and sensitive [Figure 10].[3] They help to identify the various etiological agents of ocular infections within 1– 6 h.[3]
Figure 10: ELISA test

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Polymerase chain reaction

This test is rapid and requires small sample but expensive and not readily available. It is used for detection of chlamydia trachomatis, slow-growing Mycobacterium, viruses such as herpes simplex virus, varicella zoster virus, adenovirus, Acanthamoeba, and Fungi.[3],[4],[5],[6],[7],[9]

DNA genotyping

This is used for rapid species identification of the organism including fungi.[5]

Confocal microscopy

It is the instant imaging of organisms that are larger than a few micrometer such as Acanthamoeba cysts [7] and fungal hyphae [5],[6]

H1 nuclear magnetic resonance spectroscopy

This test is used to identify Acanthamoeba.[7]

Nucleic acid hybridization technique

This test used to identify both species and strains of the organism.[3]

Smartphone-based digital imaging

This is recently used in the diagnosis and follow-up of keratitis.[3]

Prolonged use of topical antibiotics results in change in normal microbial flora with introduction of fungi and antibiotic-resistant forms.


Special thanks to Dr. Beena Philomina Professor and HOD and Dr. Fairoz CP, Assistant Professor, Department of Microbiology, Government Medical College, Kozhikode, for helping me to get the images.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Levine J, Snyder RW. Practical ophthalmic microbiology. J Ophthalmic Nurs Technol 1999;18:50-9.  Back to cited text no. 1
Armstrong RA. The microbiology of the eye. Ophthalmic Physiol Opt 2000;20:429-41.  Back to cited text no. 2
Therese KL, Madhavan HN. Microbiological procedures for diagnosis of ocular infections. Chennai: L & T Microbiology Research Centre Vision Research Foundation.  Back to cited text no. 3
Krachmer JH, Mannis MJ, Holland E. Cornea, Fundamentals Diagnosis & Management. 3rd ed. London: Mosby/Elsivier; 2011. p.139-46.  Back to cited text no. 4
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. 5
[PUBMED]  [Full text]  
Ansari Z, Miller D, Galor A. Current thoughts in fungal keratitis: Diagnosis and treatment. Curr Fungal Infect Rep 2013;7:209-18.  Back to cited text no. 6
Clarke B, Sinha A, Parmar DN, Sykakis E. Advances in the diagnosis and treatment of Acanthamoeba keratitis. J Ophthalmol 2012;2012:484892.  Back to cited text no. 7
Ramesh S, Ramakrishnan R, Bharathi MJ, Amuthan M, Viswanathan S. Prevalence of bacterial pathogens causing ocular infections in South India. Indian J Pathol Microbiol 2010;53:281-6.  Back to cited text no. 8
[PUBMED]  [Full text]  
Taravati P, Lam D, Van Gelder RN. Role of molecular diagnostics in ocular microbiology. Curr Ophthalmol Rep 2013;1.  Back to cited text no. 9


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]

  [Table 1], [Table 2], [Table 3]


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Normal Microbial...
Microbiology of ...
Ocular Specimen ...
Infective Kerati...
Processing and I...
Culture of Corne...
Antibiotic Susce...
Diagnosis of Con...
Serological Meth...
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