Laser intensity and photomultiplier tube gain were kept consistent across all experiments. Image stacks were processed using Imaris 6.3.1 (Bitplane) to generate images for publication. Biovolumes for each image stack were computed using the ‘Surfaces’ feature of the Imaris software with the ‘Absolute Intensity’ setting for background removal. For each co-culutre, 4 replicates comprised of different strain-AFP combinations (to remove any fluorescent intensity bias in the quantification) were used to calculate the mean biovolume. The relative proportion of each strain was calculated compared to the total biovolume. Student’s t-test was used to compare the means of the relative volumes

for Y-27632 supplier each strain pair. Planktonic competition To determine if the WS or SCV had any growth advantage in broth culture competitions were performed with each pair combination. Equal volumes of 16 h cultures of each strain were add

to a total of 150 μL LB media in 96 well plates (30-fold dilution). The plate was incubated at 30℃ with shaking (175 rpm) for 24 h. Prior to incubation samples were removed for determination of Crizotinib mw initial cell numbers. The cultures were serially diluted on LB agar and the number of each colony type were recorded. The SCV and WS could easily be distinguished from the wildtype CHA0 and CHA19 colony types. To control for any phenotypic variation occurring the broth culture the competitions were performed with the strains expressing the fluorescent proteins. Representative plates from each pair combination were imaged with a fluorescent imager (IVIS Imaging System, Caliper LifeSciences) to distinguish the two strains and the numbers were compared to the values obtained when counting based on colony morphology. No phenotypic variation occurred in broth cultures during the time period tested. Fluorescent imaging of the plates was also used to distinguish the CHA0 and CHA19 colonies

as well as CHA0 and CHA19 competed with themselves. The relative fitness [21] of the variant (SCV or WS) compared to wildtype (CHA0 or CHA19) was calculated for each pairwise combination. A relative fitness of 1 indicates that neither strain has a competitive advantage, whereas values higher than 1 indicate that the variant is more fit in the broth culture. A Amino acid one-tailed Student’s t-test was used to determine if the values were significantly greater than 1. P values were adjusted with the Holm-Bonfferoni correction to control for the family-wise error rate [22]. Acknowledgements This work was supported through discovery grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to RJT and HC. NSERC has also provided a Postgraduate Scholarship (Doctoral) to MLW who was additionally supported by a PhD Studentship from the Alberta Heritage Foundation for Medical Research (AHFMR). CLSM was made possible through a Canadian Foundation for Innovation (CFI) Bone and Joint Disease Network grant to HC.