Laboratory diagnosis is made by direct detection of Actinomyces in specimens by microscopy and by isolation of organism by culture.
The specimens include sputum, bronchial secretions and dis-charges, and infected tissues. All these specimens may con-tain large number of sulfur granules. The sulfur granules are also present on the dressings removed from a draining sinus tract. It is essential to transport these specimens immediately to the laboratory for processing, preferably under anaerobic conditions.
Sulfur granules are white to yellow and vary in size from minute specs to large granules. These granules are separated from pus and other specimens and are collected directly from draining sinuses. These are crushed between two slides and are stained by Gram or Ziehl–Neelsen staining method, using 1% sulfuric acid for decol-orization. The stained smears on microscopic examination show Gram-positive hyphal fragments surrounded by peripheral zone of swollen, radiating, club-shaped structures presenting a sun-ray appearance. These club-shaped structures are Gram positive, acid fast, and are believed to be antigen complexes.
Sulfur granules or pus-containing Actinomyces are immediately cultured under anaerobic conditions at 35–37°C for up to 14 days. The specimens are inoculated on blood agar, BHI agar, and into thioglycollate broth and incubated anaerobically at 37°C. A. israeli produces large (0–5 mm in diameter), white, smooth, entire or lobulated colonies resembling molar tooth after 10 days of anaerobic incubation.
Actinomycetes colonies are identified by microscopy, biochem-ical reactions, direct fluorescent antibody test, and gel immu-nodiffusion test. The latter two tests are very useful to differentiate A. israeli from other actinomycotic species and filamentous anaerobes that produce similar type of granules in the tissues.