Trypsin edta 1x solution

After incubation, the medium was decanted and the adhered trophozoites were washed three times with freshly prepared PYG medium. Supernatant from the last wash was inoculated on LJ slants for growth of M. ulcerans and also examined for the presence of extracellular M. ulcerans using Ziehl-Neelsen (ZN) acid-fast staining method. Extracellular mycobacteria were not seen in supernatant, neither was M. ulcerans growth observed on LJ after 10 weeks of incubation. The washed trophozoites were finally detached from the walls of the flask using 0.25% Trypsin—EDTA (1X) solution (Gibco, UK) as described above. After centrifugation, the cell pellet was suspended in 5 ml of freshly prepared PYG medium. This was used immediately for the infection experiments. An aliquot of 100μl of the cell suspension was ZN-stained and examined under the microscope (1000X) to confirm the internalization of M. ulcerans in A. polyphaga trophozoites. Additionally, 2 ml aliquot of the cell suspension was treated with 2ml of 0.5% sterile SDS solution for 5 minutes to lyse the trophozoites and then washed three times with sterile distilled water by centrifuging at 3000 rpm for 5 minutes. The lysate was again reconstituted in 2ml of sterile distilled water. An aliquot of 1 ml of the cell lysate was then inoculated on LJ slants and incubated at 32°C to confirm growth of intracellular M. ulcerans.

Mycobacterium ulcerans: A. polyphaga coculture (ratio = 2.96 x 10⁴ CFU/ml: 1.6 x 10⁶ cells/ml) was incubated for 3 days and 42 days without changing growth medium. For the three day culture, the medium was decanted and washed twice (3000 rpm for 5 minutes at 25°C) with freshly prepared PYG medium. The washed trophozoites were finally detached from the walls of the flask using 0.25% Trypsin—EDTA (1X) solution (Gibco, UK). Briefly, 1ml of the Trypsin solution was added to adhered trophozoites in the flask and incubated at 30°C for 5 minutes. Thereafter, 15ml of PYG medium was added and the flask was tapped gently to suspend the cells in solution. The cell suspension was then centrifuged at 3000rpm for 5 minutes at 25°C. The supernatant was decanted and the pelleted cells suspended in 5 ml of freshly prepared PYG medium and then 1 ml aliquots dispensed onto sterile glass cover slides. Slides were incubated for 10 minutes in a humid chamber and thereafter observed through the red channel of a fluorescent microscope (Olympus VX41, Japan). For the cysts, the 42-day old M. ulcerans: A. polyphaga coculture was harvested by centrifuging at 3000 rpm for 5 minutes at 25°C. Cysts were washed twice with sterile distilled water as described and finally centrifuged to concentrate cell pellets. The cells were then applied onto slides and similarly observed under a fluorescent microscope.