|Year : 2017 | Volume
| Issue : 2 | Page : 72-78
Microbiology for general ophthalmologists
V Babitha1, PT Jyothi2
1 Professor in Ophthalmology, Government Medical College, Kozhikode, Kerala, India
2 Department of Ophthalmology, Government Medical College, Kozhikode, Kerala, India
|Date of Web Publication||10-Aug-2017|
Department of Ophthalmology, Government Medical College, Kozhikode - 673 008, Kerala
Source of Support: None, Conflict of Interest: None
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
| Introduction|| |
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. Although eyes are relatively impermeable to microorganisms, intraocular infections result from trauma, surgery, or systemic disease. While evaluating a patient with infection, it is of great help to be familiar with the normal microbial flora of the human eye.
| Normal Microbial Flora of Eye|| |
Few microorganisms are present normally in the conjunctival sac.
- Gram-positive cocci
- Staphylococcus epidermidis
- Staphylococcus aureus
- Streptococcus pyogenes
- Streptococcus pneumoniae
- Streptococcus viridans
- Gram-negative cocci
- Gram-positive bacilli
- Gram-negative bacilli
- Haemophilus influenzae
- Klebsiella sp.
- Escherichia coli
- Pseudomonas aeruginosa
- Moraxella sp.
- Propionibacterium sp.
- Bacteroides sp.
- Lactobacillus sp.
- Clostridium sp.
These are transient and are those found in the environment.
| Microbiology of Ocular Surface Infections|| |
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. For diagnosing unusual pathogens, special planning is required.
| Ocular Specimen Collection|| |
Peculiarities of ocular specimen:
- Small amount of material
- Number of organisms very less
- Most of the time patients are on topical medication while taking specimen
- Specimen may have nonviable organisms
- Most of the time liquid media has to be used
- Frequently fastidious organisms are encountered; therefore, enriched media are necessary.
Conjunctivitis is usually diagnosed by clinical features.
Soft-tipped applicators with plastic handles and heads composed of cotton, Dacron, or calcium alginate are used for conjunctival specimen collection., Calcium alginate swabs produce a higher yield of bacterial organisms.
Following are the steps of conjunctival specimen collection [Figure 1].
- 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.
| Infective Keratitis (Microbial Keratitis)|| |
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].
Instruments used for corneal specimen collection are spatulas, Jeweler's forceps, and surgical blades., Cotton-tipped applicators are used in less aggressive infections, and less distinct areas of involvement over a large area require culture.,
Corneal specimen is collected as follows [Figure 2].
- 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.
Direct inoculation of specimen to the culture media is the best but may not be possible always. Many transport systems for supporting bacteria, virus, and chlamydia before direct inoculation on proper isolation media are available. For example, Amies transport medium.
Common stains and tests
Commonly used stains are Gram stain, acridine orange, calcofluor white, and acid-fast, Giemsa stain. All bacteria isolated from the cornea should be considered as pathogens and tested for antibiotic susceptibilities.
| Processing and Interpretation of Direct Smears|| |
Smears are interpreted by staining methods [Table 2].
|Table 2: Staining characteristics and microscopic appearance of causative pathogens|
Click here to view
| Culture of Corneal Specimen|| |
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. Inoculated culture media should be kept and examined for at least 5 days [Table 3]. 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.
|Table 3: Culture media used for isolating organisms and their appearance|
Click here to view
- 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.
They are fastidious organisms and require long incubation periods for isolation. 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. Acid-fast staining and inoculation in Lowenstein– Jensen media can be used for mycobacterial isolation.
Nocardia and Actinomyces
These are less frequent ocular pathogens.Nocardia can be cultured on routine bacterial culture media.Actinomyces requires extended growth period, and cytologic examination of corneal specimen by Giemsa stain is diagnostic test (long thin filaments with branching).
The stains commonly used to identify Microsporidia are modified trichrome, Giemsa, Gram, periodic acid-Schiff, Grocott's methenamine silver, calcofluor, and acid-fast methods.
Cell culture isolation, Giemsa stain, and polymerase chain reaction are the methods used to detect chlamydia from ocular samples.
Rare pathogen that cannot be easily cultured need to be tested in special laboratories or at facilities researching the particular disease.
| Antibiotic Susceptibility Testing|| |
It is performed on fast-growing bacterial isolates to assess whether an antibiotic will be successful in therapy or not. 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]. MIC methods include broth dilution, agar dilution, and E-tests. With these methods, MIC of antibiotic required to inhibit bacteria is determined and compared to predetermined standards that represent susceptibility. Ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, oxacillin, cefazolin, vancomycin, gentamicin, and tobramycin are the commonly tested antibiotics in corneal infections.
| Diagnosis of Contact Lens-Associated Keratitis|| |
In contact lens-associated keratitis, the lens, container, and the solution have to be examined and cultured., Contact lenses should be removed aseptically, placed in sterile saline, and can be cultured by agar sandwich method. Contact lens solution can be cultured on standard media such as blood agar, MacConkey agar, nonnutrient agar, and Sabouraud dextrose agar. The lens deposits and centrifuged deposit of the lens case solutions can be examined under microscope to detect the organism.
| Serological Methods of Diagnosis|| |
The serological tests have little value in the diagnosis of corneal infections.
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]. They help to identify the various etiological agents of ocular infections within 1– 6 h.
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.,,,,,
This is used for rapid species identification of the organism including fungi.
It is the instant imaging of organisms that are larger than a few micrometer such as Acanthamoeba cysts  and fungal hyphae ,
H1 nuclear magnetic resonance spectroscopy
This test is used to identify Acanthamoeba.
Nucleic acid hybridization technique
This test used to identify both species and strains of the organism.
Smartphone-based digital imaging
This is recently used in the diagnosis and follow-up of keratitis.
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.
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[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]