Improved isolation of anaerobic bacteria from the human gingival crevice by maintaining continuous anaerobiosis
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Using conventional aerobic and anaerobic culturing techniques only a portion of those microorganisms present in the gingival crevice of man have been cultivated. Since bacteria in this area appear to play an important role in dental and periodontal inflammatory disease, their isolation and characterization are essential. A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms of the gingival crevice area. This technique achieves an anaerobic state by flushing the local environment with oxygen free gas. Once collected the crevicular debris was immediately placed into sterile oxygen free test tubes which were flushed out by the oxygen free gas. In this manner the sample was weighed, dispersed, diluted and "plated" in roll tubes and plates. The media for control (Brewer jar technique) and Hungate techniques was heart infusion agar (Difco)fortified with ten percent defibrinated horse blood. Results indicated that when the Hungate technique was used the recovery of viable bacteria, as a percentage of the total count, was significantly greater than plates incubated aerobically or utilizing the Brewer anaerobic technique. Hungate percent recovery averaged forty-eight percent for ten samples when ninety percent nitrogen, ten percent hydrogen was used; sixty-seven percent for eight samples when eighty-five percent nitrogen, ten percent hydrogen, five percent carbon dioxide was used and sixty-three percent for eight samples when one hundred percent carbon dioxide was the gaseous atmosphere. At no time did the average for the anaerobic plates (Brewer jar) yield more than twenty six percent. This suggests that exclusion of oxygen and the presence of carbon dioxide maximize recovery of gingival crevice material.
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