Semiautomated technique for identification of subgingival isolates

Abstract

A semiautomated approach for the characterization of subgingival bacterial isolates which economizes in media preparation, inoculation, reading, recording, and interpretation of results was tested. Test ingredients were added to a basal medium consisting of Mycoplasma broth supplemented with 5 micrograms of hemin, 0.5 mg of NaHCO3, and 0.5 mg of L-cysteine per ml. Sterile test media were aseptically dispensed into wells of sterile microtiter plates with a MIC 2000 dispenser. Inocula were grown in broth or scraped from agar plates, dispersed, and inoculated with a MIC 2000 inoculator. After 2 to 4 days of incubation, the optical density of growth was determined with an Artek 210 vertical beam reader at 580 nm and stored on a floppy disk. Reagents were added to each well, and the changes in optical density were determined. Thresholds for positive reactions were determined after extensive preliminary studies for each test. The tests were run in duplicate on each plate and interpreted with an Artek vertical beam reader. Tests that were run in this system included: fermentation of carbohydrates, decarboxylase reactions, reduction of nitrate and nitrite, ammonia production, hydrolysis of esculin, growth in the presence of inhibitory or stimulatory substances, and indole production. Approximately 80% of all isolates from subgingival samples could be characterized by this technique. Comparisons were made between the semiautomated and conventional identification techniques. Overall reproducibility of 2,980 strains by the semiautomated and conventional techniques were 95 and 90%, respectively. There was an 86% similarity of results by the semiautomated and conventional methods. The semiautomated technique was more rapid, less expensive, and as reproducible as the conventional method of identification.