2.6. Culture of “Spiked” Clinical Samples

To artificially increase the number of positive samples, 26 samples (18 sputa and 8 cough swabs) were each “spiked” with a unique strain of BCC from the international collection. Each strain was subcultured onto Columbia blood agar and incubated for 24 h at 37 °C. A suspension was then prepared with turbidity equivalent to 0.5 McFarland units using a Densimat. A 1/100 dilution was then performed by adding 10 µL of this suspension to 990 µL of sterile saline (0.85%). Ten microlitres of this diluted suspension were then added to either 1 mL of homogenised sputum or 1 mL of cough swab suspension. A 50 µL aliquot of each “spiked” sample was then cultured exactly as described above. The estimated inoculum of BCC delivered onto each plate was approx. 250–500 CFU.