Eye Specimens: Collection and Transport (Conjunctival Swab, Corneal Scraping)

There is one instruction in eye microbiology that gets taught over and over and skipped almost as often: swab both eyes, even the one that looks fine. The apparently normal eye is not a waste of a swab. It is the control that tells you what is merely colonizing versus truly infecting, and it is frequently the site of early, subclinical infection, especially in newborns and in viral or chlamydial conjunctivitis. If you take one lesson from this article, take that one.

Eye specimens are collected to diagnose infections ranging from simple conjunctivitis to sight-threatening corneal ulcers and endophthalmitis. The specimen changes as the infection deepens, and the collection is unusually demanding because the material is scanty and the eye is delicate. This article is about getting the right eye specimen to the lab in usable condition.

Both eyes, separate swabs: the rule worth repeating

Collect from both eyes, each with its own swab, each labeled clearly (right and left). Two reasons:

• The uninvolved eye is a built-in control. The conjunctiva has a normal flora. Comparing the two eyes helps the lab tell a true pathogen from a commensal that lives there anyway.
• The second eye is often involved before it looks it. Bilateral or sequential involvement is common in adenoviral conjunctivitis, in chlamydial and gonococcal conjunctivitis, and above all in neonatal ophthalmia, where a missed second eye can mean a missed sight-threatening infection.

Never use one swab on both eyes. That transfers organisms from one to the other and destroys the comparison. One swab, one eye, one label.

The specimen changes with the depth of infection

The site you sample rises in invasiveness as the infection deepens. You should sample as deep as the disease demands, no more.

For conjunctivitis, the swab is the routine specimen. For a corneal ulcer, a surface swab is not enough, the pathogen is in the corneal tissue, so a scraping is taken from the edge of the ulcer. For endophthalmitis, the sample comes from inside the eye. The deeper specimens are collected by an ophthalmologist; the microbiology role is to know which specimen answers the question and how to handle it.

Why eye specimens are inoculated at the bedside

Eye samples are tiny, often just enough to make a visible mark on a plate. A scanty specimen sent across a hospital in a swab tube can dry out and lose its few organisms before it is ever cultured.

So the rule for eye specimens, like the gonococcus rule for genital cultures, is to inoculate the media at the patient's side whenever possible:

• Direct plating onto blood agar and chocolate agar at the bedside (and Sabouraud agar if fungus is suspected) recovers far more than a swab that has to travel.
• Smears for Giemsa and Gram staining are made at the same time.
• If bedside inoculation is not possible, place the swab in Amies transport medium and get it to the lab fast. But bedside plating is better, and for corneal scrapings it is the standard.

The principle: for a scanty, fragile specimen, the shorter the distance between the eye and the culture medium, the better the result.

Timing: before the drops

Collect the specimen before any topical medication goes in. Two reasons:

• Topical antibiotics suppress the very organisms you are trying to grow.
• Topical anesthetics are themselves bacteriostatic and lower the yield, so even the numbing drops matter. Where an anesthetic is unavoidable for a corneal scraping, the least bacteriostatic option is used, and this is an ophthalmologist's judgment.

If a patient is already on eye drops, note it on the request, because a negative culture may simply reflect suppression, not absence of infection.

When to think beyond routine bacteria

Two situations change the specimen and the media requested, and both are worth mentioning on the form so the lab sets up the right cultures.

• Contact lens wearer with keratitis: think fungus and Acanthamoeba. Contact lens use is the major risk factor for Acanthamoeba keratitis and a common one for fungal keratitis. In these cases the contact lens, its case, and the cleaning solution are themselves useful specimens and should be submitted along with the corneal scraping. Acanthamoeba culture uses a special non-nutrient agar seeded with bacteria as a food source, and a Giemsa or wet-mount preparation can show the trophozoites and cysts. Fungal keratitis needs Sabouraud agar and a KOH or calcofluor-white preparation.
• Neonatal conjunctivitis (ophthalmia neonatorum): think gonococcus and chlamydia. In a newborn with purulent conjunctivitis, Neisseria gonorrhoeae and Chlamydia trachomatis are the priority pathogens. Gonococcal specimens follow the fragile-organism rules (bedside inoculation, do not refrigerate), and Giemsa staining is used to look for chlamydial inclusion bodies. Both eyes, as always.

Transport

• Bedside inoculation first. For scanty eye specimens, plating at the point of collection beats any transport.
• If a swab must travel, use Amies transport medium at room temperature, and get it to the lab quickly.
• Do not refrigerate eye specimens intended for culture; fragile organisms such as Neisseria gonorrhoeae and Haemophilus do not tolerate cold.
• Contact lens specimens (lens, case, solution) go in a sterile container and travel with the corneal scraping.

Processing of Eye specimens in Microbiology Lab

As eye specimens are scanty and were usually inoculated onto media at the bedside, the laboratory often receives inoculated plates and prepared smears rather than a swab to process.

Direct examination. Smears made at collection are central here, because a scanty specimen may give more on microscopy than on culture:

• Gram stain for bacteria and yeasts, and for the pus-cell response.
• Giemsa stain, which is important in eye specimens for showing the intracytoplasmic inclusion bodies of Chlamydia trachomatis (inclusion conjunctivitis and neonatal ophthalmia) and for fungal and Acanthamoeba forms.
• A KOH or calcofluor-white preparation and a wet mount when fungal keratitis or Acanthamoeba keratitis is suspected; the wet mount can show Acanthamoeba trophozoites and cysts.

Culture media. Eye specimens are inoculated, ideally at the bedside, onto:

• Blood agar and chocolate agar for common bacteria and fastidious organisms.
• Sabouraud agar when a fungus is suspected.
• Non-nutrient agar seeded with Escherichia coli (or another bacterial lawn) for Acanthamoeba, which feeds on the bacteria; the amoebae leave visible tracks as they migrate.

Incubation. Bacterial plates are incubated at 35 to 37 degrees C, with chocolate agar in 5% CO2, and read over 24 to 48 hours. Fungal and Acanthamoeba cultures are held far longer, because they grow slowly.

Reading and interpretation. The scanty nature of eye specimens changes how growth is judged:

• Because the sample is tiny, growth is often light, so even a small amount of a recognized pathogen, especially if it matches the direct smear, is taken seriously rather than dismissed as light growth.
• The conjunctiva has a sparse normal flora, so the swab taken from the apparently normal eye serves as the comparison: an organism heavy in the infected eye but absent or scant in the normal eye supports a pathogen, while the same light growth in both eyes suggests colonizing flora.
• Corneal scrapings come from a normally sterile site, so any organism recovered from the cornea is potentially significant.
• Confluent growth localized to the site inoculated (for corneal scrapings placed in a "C" pattern) is more convincing than scattered colonies that may reflect handling.

Identification and antimicrobial susceptibility testing

The organisms commonly isolated from eye specimens, and the first approach to each:

• Staphylococcus aureus and coagulase-negative staphylococci: gram-positive cocci in clusters, catalase positive; separated by the coagulase test. Common in conjunctivitis and keratitis. See the Staphylococcus aureus article.
• Streptococcus pneumoniae: gram-positive lanceolate diplococci, alpha-hemolytic; a cause of conjunctivitis and serious keratitis. See the Streptococcus pneumoniae article.
• Pseudomonas aeruginosa: oxidase-positive, non-lactose-fermenting gram-negative rod; the classic cause of contact-lens-associated keratitis and a rapidly destructive corneal pathogen. See the Pseudomonas aeruginosa article.
• Neisseria gonorrhoeae: gram-negative intracellular diplococci; the priority pathogen in neonatal ophthalmia, handled by the fragile-organism rules. See the Neisseria gonorrhoeae article.
• Chlamydia trachomatis: not grown on routine media; shown by Giemsa inclusions or by antigen and molecular tests. See the Chlamydia trachomatis article.
• Fungi (Fusarium, Aspergillus, Candida) and Acanthamoeba: keratitis in lens wearers and after trauma; shown on KOH, calcofluor-white, or wet mount and grown on the appropriate media.

How to Remember

Both eyes, every time. The normal-looking eye is the control and is often subclinically infected. One swab per eye, labeled left and right. This is the point clinicians skip.

Sample as deep as the disease. Conjunctivitis takes a swab, a corneal ulcer takes a scraping, endophthalmitis takes an intraocular aspirate. The surface swab cannot reach a pathogen living in the cornea.

Short trip for a small sample. Eye specimens are scanty, so plate at the bedside. The gonococcus rule applies here too: the less the sample has to travel, the more survives.

Before the drops. Collect before topical antibiotics and even before anesthetic drops, which are bacteriostatic. If drops were already given, mention so on the form.

Contact lens plus ulcer equals think fungus and amoeba. A lens wearer with keratitis raises Acanthamoeba and fungi, so submit the lens, case, and solution, and ask for the special media.

Key exam facts in one table

Where Students Get Confused

"Only one eye is red, so why swab the other?" Because the normal-looking eye is your control for the conjunctiva's normal flora, and it is often infected before it shows symptoms, especially in newborns and in viral or chlamydial conjunctivitis. Swabbing both eyes, with separate swabs, is the rule that is most often taught and most often ignored.

"A conjunctival swab is an eye specimen, so it works for a corneal ulcer too?" No. In keratitis the pathogen is in the corneal tissue, and a surface swab misses it. A corneal scraping from the edge of the ulcer is needed, taken by an ophthalmologist and usually plated at the slit lamp.

"Why plate eye specimens at the bedside instead of sending them like other swabs?" Because the sample is tiny. A scanty specimen dries out and loses its few organisms in transit. Direct inoculation onto media at the point of collection recovers far more, which is why it is standard for corneal scrapings.

"The patient already has antibiotic drops in, does that matter?" Yes. Topical antibiotics suppress growth, and even topical anesthetics are bacteriostatic. A negative culture in a treated eye may reflect suppression, not absence of infection, so always collect before drops and note any prior treatment.

"A contact lens wearer has a corneal ulcer, is that just routine bacterial keratitis?" Not necessarily. Contact lens use is the major risk factor for Acanthamoeba keratitis and a common cause of fungal keratitis. Submit the lens, its case, and the solution, and ask for the special media, or these organisms will be missed.

References and further reading

1. Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
2. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. DOI: 10.1128/9781683670438.CMPH
3. Procop GW, Church DL, Hall GS, et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.