SIMPLE METHOD FOR QUANTIFYING VIABLE BACTERIAL NUMBERS IN SPUTUM

Aims-To establish a simple method of quantitative culture for determin ing the viable bacterial numbers present in expectorated sputum sample s. Methods-Sputum samples were homogenised with dithiothreitol, steril e saline or glass beads to determine which method recovered the greate st number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacter ial viability. Sputum samples homogenised with dithiothreitol were dil uted in sterile saline and sampled using either standard bacteriologic al loops or a precision pipette to determine which method resulted in the least variation. Results-Homogenisation of sputum using dithiothre itol increased the recovery of viable bacteria compared with sterile g lass beads and/or saline, with no apparent effect on bacterial viabili ty 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 mu l 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 g enerally of the same order of magnitude. Conclusions-This method repre sents a relatively quick and simple technique for accurately quantifyi ng viable bacteria present in sputum samples. The use of a small porti on appears to be representative of the sample as a whole.