8 μm filter

The microbial biomass of each sample was concentrated by filtration using the Millipore vacuum/pressure pump (Millipore Corporation, Bedford, Mass.). Each 250-mL sample was mixed with 1/4 volume of saturated sodium chloride solution and incubated at 70°C for 2 min before filtration (Ren et al., 2011 (link)). A 0.22-μm filter (Millipore, USA) was used to collect microbial biomass following an 8-μm filter (Bandao, China). All filters of one sample were collected and stored in a sterilized 10-mL centrifuge tube at −20°C for DNA extraction. The genomic DNA was extracted from the filters using a bead-beating protocol as described previously (Zhang et al., 2007 (link)). The AxyPrep PCR cleanup kit was used to purify genomic DNA, which was stored at −20°C until 16S rRNA gene amplification.

Mtb H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were purchased from American Type Culture Collection (ATCC, Manassas, VA), and the Mtb K strain was obtained from the strain collections at the Korean Institute of Tuberculosis (Korean National Tuberculosis Association, Seoul, Republic of Korea). All strains were cultured in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.02% glycerol and 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC, Becton Dickinson, Sparks, MD) for 28 days at 37°C. Single cell suspensions of each strain were prepared as previously described with slight modifications. Briefly, mycobacterial cells grown in OADC-supplemented 7H9 broth were harvested by centrifugation at 10,000 × g for 20 min and washed three times in phosphate-buffered saline (PBS) (pH 7.2). The pellets were homogenized using an overhead stirrer (Wheaton Instrument, Millville, NJ) for 1 min on ice to minimize bacterial clumping. The homogenized mycobacterial cells were passed through an 8 μm filter (Millipore Corp., Bedford, MA). The predominant presence of single cells in the final preparation was confirmed by acid-fast staining. The seed lots of each strain were stored in small aliquots at -80°C until use. The CFUs per 1 ml of the seed lots were measured by a viable counting assay on 7H10 agar plates.