Detection and quantitation of simulated anaerobic bacteremia by centrifugation and filtration

Fresh human whole blood was inoculated with various anaerobic bacteria or with combinations of anaerobic, facultatively anaerobic, and aerobic microorganisms (3 to 28 microorganisms per ml). The seeded blood was then layered on a reduced Ficoll-Hypaque gradient (density, 1.093 g/ml) and centrifuged (400 X g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered anaerobically through a 0.45-micron membrane filter. The filters were then placed on reduced chocolate Mueller-Hinton agar plates and incubated at 35 degrees C in humidified atmosphere containing 85% N2, 10% H, and 5% CO2 or in air containing 5% CO2. No statistically significant differences were detected between the numbers of microorganisms recovered (alone or in combination) by filtration and by direct culturing of the original inoculum. All organisms were detected within 30 h after filtration. This technique has excellent sensitivity.