Clinical samples are labeled with the patient's name, type of tissue or fluid, and the date of collection. Universal precautions are followed during collection and processing. Specimens that are shipped elsewhere are put inside double mailing containers with a biohazard label and sent according to current postal regulations.

Submitting specimens and a requisition form to the microbiology laboratory starts a sequence of procedures designed to identify infecting microorganisms (Fig. 1). In the laboratory, smears are stained and examined, other rapid diagnostic tests are performed if possible, and inoculated culture media or cell lines are incubated under specific temperature and environmental conditions to enhance growth.

The laboratory is staffed by accredited personnel who maintain a quality-assurance program with a procedure manual that includes periodic monitoring of equipment and supplies. Communication networks are maintained between office and laboratory personnel to facilitate timely distribution of laboratory test results. Suspected false-negative tests may indicate a need to contact the clinician for additional specimens.⁷ Results are also reported, when indicated, to the appropriate public health agency.

Smearing the specimen in a thin film on a clean glass slide evenly distributes microorganisms. Thick smears cause some stains to clump, whereas excessive spreading fragments cells. Etched circles on the slide or rings made with a wax pencil help the microscopist to find the inoculation site. The examiner must be familiar with the various instruments and methods of microscopy.⁸

WET MOUNT

Scrapings and swabbings of the ocular surface and samples of intraocular fluids can be examined directly by brightfield illumination. Background light is reduced by reducing the microscope's iris diaphragm. Microscopy of fresh, unstained specimens can delineate cells, but differentiation is difficult.

A saline mount is infrequently used in ocular microbiology but can demonstrate large parasites such as mites (Demodex) and lice (Phthirus pubis) from the eyelashes. Onchocercal microfilariae can be directly observed wriggling out of a scraping or biopsy submerged in saline or Hawk's solution.^9,10

Phase-contrast microscopy and Nomarski optics demonstrate the motility of viable organisms (e.g., Acanthamoeba trophozoites).¹¹ Darkfield microscopy increases the visibility of translucent microbes such as spirochetes¹² and spores.

Potassium hydroxide (KOH) 10% is sometimes mixed with the specimen to digest keratinized epithelial cells or thick mucin strands that might be confused with fungal elements. The KOH wet mount can be useful for detecting fungi from ocular specimens.^13,14

Opaque suspensions of India ink or nigrosin help to outline formed elements such as fungi.¹⁵ Lactophenol cotton blue can be used in a wet mount of a clinical specimen to reveal fungi¹⁶ and amebic cysts.¹⁷ This stain is also used to examine yeasts and the spores of filamentous fungi grown on cultures.

FIXATION AND STAINING

A specimen is fixed to a glass slide so staining solutions do not wash it off. Heat fixation by passing the slide through a hot flame is satisfactory, but cytologic details are better preserved by alcohol fixation. The slide is flooded or immersed in a Coplin jar of methanol for 3 to 5 minutes. Methanol is also useful to prepare specimens for immunofluorescent processing.

Staining makes many microorganisms easier to see and helps to classify microbes and human cells.¹⁸ Examination of stained smears begins with scanning the slide under low power. The microscopist looks for inflammatory cells, epithelial cells, other tissue cells, and mucus with an eye to recognize the cytopathologic pattern.¹⁸ Without seeing at least 10 cells in a low-power field, the laboratory will probably question whether sufficient material was obtained. Stained smears are efficient in the evaluation of bacterial and fungal infections¹⁹ and are often the easiest way to confirm parasitic disease.²⁰

Several staining methods are available to evaluate ocular specimens (Table 1). Basic dyes (e.g., hematoxylin) stain acidic structures such as nuclear chromatin. Acidic dyes (e.g., eosin) stain basic substances in the cytoplasm. Stains capable of fluorescence glow under ultraviolet light and make it easier to find microorganisms scattered on a smear. Extra, unstained smears are frozen to prevent breakdown of microbial and viral components. Several stains have high predictive value (Table 2) for confirming chlamydial conjunctivitis,^21–24 bacterial keratitis,^25–27 fungal keratitis,^14,26–30 or endophthalmitis.³¹

TABLE 1. Microscopic Procedures Used in Ocular Microbiology

TABLE 2. Performance of Microscopic ExaminationCompared to Culture for Ocular Infections

LIGHT MICROSCOPY

Gram's Stain

Gram's staining is a standard method in bacteriology laboratories. Crystal violet is applied, then bound to bacterial cells by a weak iodine solution. An acetone-alcohol solvent removes the dye from bacteria whose cell walls lack teichoic acid cross-links that resist decolorization. Dye-retaining, gram-positive bacteria remain dark blue, while gram-negative bacteria are counterstained pink-red with safranin. Color and shape help in presumptive classification. Modifications such as the Brown-Hopps stain are applicable to tissue sections. A fluorescent modification is not as sensitive as the conventional Gram's stain.³²

In microbial keratitis, Gram's staining successfully identifies the responsible organism in many culture-positive cases.³³ During endophthalmitis, the Gram's stain is also useful, although less sensitive, in evaluating vitreous aspirates.³⁴ Gram's staining has some utility for diagnosing bacterial conjunctivitis^35–37 but has high predictive value for gonococcal and meningococcal conjunctivitis. Caution is needed to avoid overinterpreting the Gram's-stained smear. Presumptive identification must be verified by culture isolation.

Giemsa Stain

Methylene blue stains nucleic acids in microorganisms, leukocytes, and tissue cells blue against a light gray background. Giemsa's methylene blue derivative enhances visualization. Modified Giemsa staining highlights DNA in human cell nuclei and cytoplasmic RNA in lymphocytes. Bacteria, intracellular chlamydial elementary bodies, and many viral inclusions stain dark blue. Fungi and parasites are visualized by precipitation of the stain upon their outer walls.

Giemsa staining is used selectively in microbiology laboratories.^21,38–40

Indications are to screen smears for small gram-negative bacteria, such as Haemophilus and Neisseria, that might be missed by Gram's staining; to evaluate inflammatory cells in ocular specimens^21,37,41,42; to detect protozoa such as acanthamoebic cysts, microsporidial spores, and leishmanid amastigotes; and to reveal intracytoplasmic inclusions during chlamydial conjunctivitis.⁴⁰

Giemsa staining is useful for evaluating the cytologic pattern^21,38 and cellular details³⁹ of conjunctival specimens (Table 3). Other Romanowsky stains are Wright's stain, used to evaluate conjunctival cytology, and Field's stain, used to detect protozoa.⁴³ The Tzanck smear of fluid from a herpetic vesicle can be stained with Giemsa or Papanicolaou stain to show multinucleated cells.

TABLE 3. Ocular Cytologic Examination

Acid-Fast Stains

Lipids and waxes of the mycobacterial cell wall resist staining, but once overcome they then prevent decolorization by acid-alcohol solvents. After applying heat (Ziehl-Neelsen technique) or detergents (Kinyoun method), carbolfuchsin binds to long-chain fatty acids in the cell wall. Mycobacteria stain red against a blue-green background.⁴⁴ Other organisms detectable with acid-fast staining are microsporidial spores. A modification helps to visualize Nocardia.

Hematoxylin and Eosin Staining

Hematoxylin and eosin are routinely used to stain tissue sections. Bacteria, fungi, acanthamoebæ,^45,46 and microsporidia stain satisfactorily. This method is most useful for evaluation of inflammatory and tissue cells.

Trichrome Staining

The trichrome stain is useful to detect protozoa such as acanthamoebæ and microsporidia.⁴⁷

Periodic Acid-Schiff Staining

The periodic acid-Schiff (PAS) reaction stains certain carbohydrates. PAS can reveal fungi and amoebæ in tissue sections of ocular infections. PAS staining has been used for cytologic diagnosis of uveitis during Whipple's disease.⁴⁸

Silver Stains

Methenamine-silver nitrate staining is sometimes used in suspected fungal infections. Black fungal cell walls are highlighted against a transparent green background.⁴⁹ Other silver stains are available to detect spirochetes, microsporidia, and protobacteria. The Dieterle stain helps to demonstrate Borrelia burgdorferi during Lyme disease and Treponema pallidum during early syphilis. The Warthin-Starry silver impregnation method is used to identify the organisms of cat-scratch disease in a conjunctival biopsy.⁵⁰

Enzyme-Conjugated Stains

Enzymes, such as horseradish peroxidase and alkaline phosphatase, linked to specific antibodies can catalyze a chromogenic reaction to reveal microbial and viral antigens. Horseradish peroxidase results in an orange-brown precipitate. Immunoperoxidase staining has been used for evaluating epithelial keratitis caused by herpes simplex virus^51,52 or Epstein-Barr virus.⁵³

FLUORESCENCE MICROSCOPY

Acridine Orange

Acridine orange is a useful screening stain because bacteria stain brightly against a dark background and can be detected at relatively low concentration. Cytopathologists use acridine orange at neutral pH to distinguish greenish viable cells from dead cell nuclei that are red-orange. Microbiologists use this chemofluorescent dye at acidic pH to stain bacteria orange against a dark green background containing polymorphonuclear leukocytes with orange nuclei. Nonviable bacteria stain light yellow-green. Atypical mycobacteria are a faint yellow-orange. Yeasts and amebic cysts stain orange. Filamentous fungi and amebic trophozoites remain green.

A fluorescent microscope or a microscope with an ultraviolet light filter is used to examine acridine orange-stained smears. Staining intensity gradually fades, so smears should be examined within 2 hours. Staining artifacts include faint yellow-orange granules from disintegrating leukocytes.

Acridine orange is a useful stain to screen specimens from bacterial,^25,54 fungal,²⁸ and acanthamoebic⁵⁵ ocular infections (Fig 2). Once microorganisms are identified, a Gram's stain can be performed on the same slide.

Calcofluor White

Calcofluor white is a colorless dye used as a fabric brightener because it binds to carbohydrate polymers and reflects short-wavelength visible light. This fluorochrome combines with polysaccharides such as chitin that are present in fungal cell walls and in the exocyst of Acanthamoeba. Evans blue is used as a counterstain to stain cells and tissue debris dark red. Under ultraviolet light, calcofluor white stains yeasts, psuedohyphae, hyphae, and amoebic cysts bright apple-green against a dark background.^56–58 Smears can be subsequently Gram's or silver-stained.

Rhodamine-Auramine

Fluorochrome dyes such as rhodamine and auramine bind directly to mycolic acid in the mycobacterial cell wall and can be detected by fluorescent microscopy. With potassium permanganate as a counterstain, mycobacteria, Nocardia, and microsporidial spores stain yellow against a dark green background when viewed with a fluorescein isothiocyanate light filter. Mycobacteria are usually easier to detect with a fluorochrome stain than with acid-fast staining, but not all atypical mycobacteria stain equally well with rhodamine-auramine. A carbolfuchsin acid-fast stain can be done on the same slide.

Fluorescein-Conjugated Stains

Fluorescein isothiocyanate (FITC) can be bound to proteins known to target particular microbial components, such as antibodies and lectins. Fluorescein-conjugated monoclonal antibodies are available to detect several microorganisms and viruses. Lectins are glycoproteins, such as wheat-germ agglutinin and concanavalin A, that bind to microbial cell-wall polysaccharides.⁵⁹ Optimal use of fluoresceinated lectins is limited by the changing carbohydrate composition of microbial cell walls during different growth phases and under different environmental conditions.

Fluorescent microscopy requires appropriate exciter and barrier filters. Immersion oils, if used, should be low-fluorescing. A high-quality laboratory microscope can be retrofitted with epi-illumination using halogen lamps and interference filters to perform fluorescent microscopy.

ELECTRON MICROSCOPY

The limit of light microscopy is approximately 200 nm. Except for vaccinia and other poxviruses, with diameters of approximately 250 nm, individual viruses cannot be seen with direct microscopic observation using visible light.

Transmission electron microscopy is not generally used for rapid diagnosis except for organisms that cannot be identified by other means. The specimen may be fixed, but better resolution occurs if the fresh specimen is processed immediately. Specific antibody is added for immunoelectron microscopy. Electron-dense substances such as phosphotungstic acid and uranyl acetate negatively stain viruses against a darkly contrasting background.

Presumptive viral identification is based upon the presence or absence of an envelope and the shape and arrangement of the nucleocapsid.^60,61 Transmission electron microscopy is also helpful in the taxonomy of other microorganisms. For example, the spirals of the polar filament of microsporidia aid in classification.

LIMULUS LYSATE ASSAY

Amebocytes are cells circulating in the hemolymph of the horseshoe crab (Limulus polyphemus) that coagulate when exposed to gram-negative bacteria. Standardized lysates of Limulus amebocytes can detect endotoxin. The clinical specimen is mixed in pyrogen-free water, and test reagents are added. Bacterial endotoxin, if present, activates a proclotting enzyme. This reaction is detected by clot formation, turbidity, or a color reaction that gives a yellow solution.

The Limulus lysate test is very sensitive but is specific only for gram-negative bacteria. A positive test indicates the presence and amount of endotoxin but does not assess the viability of organisms in the sample. The assay is applicable to the diagnosis of bacterial keratitis⁶² and to the detection of gram-negative bacterial contaminants in contact lens paraphernalia.⁶³

ENZYME DETECTION

Bacteria and other microorganism produce enzymes. Chromogenic and fluorogenic substrates can detect these enzymes, which permits rapid identification of many bacterial and yeast culture isolates. Automated instrumentation is under development that could apply these laboratory methods of bacterial classification to clinical specimens.

CHROMATOGRAPHY

Gas-liquid chromatography is commonly applied to the identification of anaerobic bacteria by assessing the production of fatty acids in broth culture. This technique can potentially be used to detect microbial cell-wall components of microorganisms in clinical specimens. Among the constituents that could help in preliminary identification are short- and long-chain fatty acids, aliphatic and aromatic amines, isoprenoid quinones, and certain carbohydrates.

TABLE 4. Media Commonly Used for IsolatingMicroorganisms from Ocular Specimens

BACTERIA, FUNGI, AND PROTOZOA

Inoculated media are incubated at appropriate temperatures: 35°C to 37°C for most bacterial cultures, 25°C for fungal cultures, and 30°C to 35°C for amebic cultures. Dehydration is prevented by sealing the plate, using a plastic bag, or placing the plate in a jar to keep humidity at 70% or greater. Many bacteria grow well in an atmosphere of 5% to 10% carbon dioxide (e.g., candle jar or sealed bag with a CO₂ generator).

Early growth is detected on inoculation marks with a dissecting microscope or magnifying lens. Delayed recovery is more likely among patients receiving prior antibiotic therapy.⁶⁸ The characteristics and number of each colony type are noted. Colonies are described according to form, elevation, margination, color, surface, density, consistency, odor, hemolysis, and pigmentation production (Table 5). Representative colonies are picked for staining, biochemical testing (Table 6), and subculture.

TABLE 5. Colony Types of Common Ocular Isolates

TABLE 6. Some Common Biochemical Tests Used in the Initial Identification of Bacteria

Bacterial species identification is based on detecting unique enzymes by chemical indicators and other tests.⁶⁹ Fungi are identified by their macroscopic and microscopic morphology.^70,71 Clonal identity of isolates from two sources can be compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and plasmid DNA analysis.⁷²

VIRUSES AND CHLAMYDIA

Viral and chlamydial specimens are sent to the laboratory for inoculation and incubation as soon as possible. If delay is unavoidable, specimens are refrigerated, but not frozen, in a standard refrigerator. A cell line appropriate to the suspected pathogen is selected.

Most virology laboratories use a diploid cell line, such as human fibroblasts (e.g., MRC-5) or human embryonic kidney, or a heteroploid cell line, such as McCoy (unknown origin), Vero (African green monkey kidney), HeLa (human cervical carcinoma), or HEp-2 (human laryngeal carcinoma). The ophthalmologist should tell the laboratory which virus is suspected because most viruses can be isolated on certain cell lines but not others.

Viral specimens are inoculated onto a cell monolayer and incubated at 37°C. Any residual material can be retained at −70°C. For enhanced isolation, the specimen may be centrifuged onto a cell monolayer in a shell vial or microplate. The centrifugation-shell vial system can culture virus and intracellular bacteria.

Cell cultures are periodically examined with light microscopy for any viral cytopathic effect (CPE). The pattern of CPE helps with provisional identification. Complete identification of viral isolates usually involves immunofluorescence or enzyme immunoassay. Chlamydial inclusion bodies are detected in vitro by iodine staining or direct neutralization and hemagglutination assays. Negative cultures can be repassaged.

Relationships between achievable ocular drug levels and sensitivity concentrations vary for different types of ocular infections and various routes of drug delivery. Thus, the terms resistant and sensitive for ocular isolates may lack accurate clinical correlation for many eye infections.

DISC DIFFUSION

In the Kirby-Bauer method, the microbial inoculum is cultured on Mueller-Hinton agar, and antibiotic-impregnated discs are applied. After incubation, the diameter of the zone of inhibition around each disc gives an approximation of susceptibility (except that oxacillin resistance implies resistance to other β-lactams regardless of other disc readings). Slow-growing bacteria and anaerobes cannot be adequately tested with a disc-diffusion method.

BROTH DILUTION

Each antibiotic is serially diluted in sequential tubes or in wells of a microtiter plate. The MIC is recorded as the lowest concentration with no visible growth. The wells with inhibited growth can be subcultured to determine the minimal lethal (bactericidal or fungicidal) concentration. Checkerboard sensitivity testing determines the combined effect of two antimicrobial agents.⁷³

AGAR DILUTION

An antimicrobial agent can be incorporated into agar plates at a given concentration. Multiple plates are needed to determine the MIC. This method is reserved for testing some anaerobic bacteria.

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