Cultures were incubated overnight at 37 °C with 5% CO2, and if no growth was present in a culture after this time, it was reincubated for another 24 hours. Researchers used standard techniques (assessment of growth in 0, 1, 2, 3, or 4 quadrants) to measure the presence of pathogenic bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Growth in 3+ and 4+ quadrants was identified as heavy growth.