Simple method for quantifying viable bacterial numbers in sputum.

Abstract

To establish a simple method of quantitative culture for determining the viable bacterial numbers present in expectorated sputum samples. Sputum samples were homogenised with dithiothreitol, sterile saline or glass beads to determine which method recovered the greatest number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacterial viability. Sputum samples homogenised with dithiothreitol were diluted in sterile saline and sampled using either standard bacteriological loops or a precision pipette to determine which method resulted in the least variation. Homogenisation of sputum using dithiothreitol increased the recovery of viable bacteria compared with sterile glass beads and/or saline, with no apparent effect on bacterial viability when incubated with culture broths. By inoculating agar plates with 10(-3), 10(-4) and 10(-5) dilutions of the homogenised sputum sample, all potential pathogens could easily be identified. A 10 microliter sample volume dispensed by precision pipette and spread with a "hockey stick" resulted in the least variation between plates (less than 16%) and an even distribution of bacterial colonies. Numbers of viable bacteria recovered from different aliquots of individual sputum samples were generally of the same order of magnitude. This method represents a relatively quick and simple technique for accurately quantifying viable bacteria present in sputum samples. The use of a small portion appears to be representative of the sample as a whole.