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This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage conditions, and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29 can kill Mycobacterium tuberculosis, and the phage aerosol can be treated as a potential tool for tuberculosis treatment. The culturabiity of D29 was tested using a test chamber designed for the bioaerosols research against three spray liquids (deionized water, phosphate-buffered saline [PBS], and normal saline), four collection media (suspension medium [SM], nutrient broth, PBS, and deionized water), two sampling systems (the all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20 to 90%). The effect of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. The results proved that viable phage D29 particles generated by deionized water were approximately 30and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or nutrient broth can be maintained up to 12 h irrespective of storage temperature. Relative humidity was found to strongly influence airborne D29 culturability which is 2to 20-fold higher in low humidity (25%) than medium (55%) or high (85%) humidity. This research will help identify the optimal means for the application of D29 aerosol in animal inhalation experiments. [ABSTRACT FROM AUTHOR]