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In the setup ext_modules=cythonize("colonyzer2/maskfill.pyx") must be ext_modules=cythonize("./colonyzer2/maskfill.pyx") for proper path retrieval.

> colonyzer 384 instead of > timeseries 384

Good afternoon,

First of all, thanks for developing this tool, it is being very useful! I found something strange in my analysis, and I wonder if you can help me understand it. I did an experiment with different yeast strains growing in YPD and Calcofluor White (CFW). Surprisingly, I find that some species reach higher growth in CFW than in YPD. This is an example of the output I am getting:

The left plate is rich medium (YPD) and the right is CFW. The growth curves are from 8 technical replicates of Cg-wt1 (Candida glabrata). The images below refer to these 8 replicates in different timepoints. As expected, most yeasts do not grow in CFW. However, Cg-wt1 grows slower, but reaches a higher growth at the end. This is puzzling because we'd expect YPD to be maximum growth. I have seen this in other stressors as well for different yeast species. I wonder if this is i) a true biological observation (i.e. some yeasts grow better on CFW), ii) a biological observation that is an artifact of the plate-based design (i.e. perhaps the higher number of spots growing in the YPD plate generate nutrient deprivation, which results in lower final growth) or iii) a technical artifact of how I measure cell density (or growth). This is not something that I can clarify by looking at the plates, as there is no visual differences between the YPD and CFW growing spots.

For reference, this is how I got this data:

1. I ran colonyzer with colonyzer --lc --diffms --greenlab --plots --remove --initpos --fmt 96

2. I loaded the colonyzer data into R with data_colonyzer = colonyzer.read() from qfa package.

3. I measured growth (Cell Density in the plots) as (data_colonyzer$Trimmed/(data_colonyzer$Tile.Dimensions.X*data_colonyzer$Tile.Dimensions.Y*255)) * 1e7. Note that 1e7 is just a scaling factor to have a value between ~0-1.

I noticed that changing most analysis parameters (i.e. not using --diffms, and/or --greenlab, or using --edgemask in addition to --lc --diffms --greenlab) did not affect this general observation. Similarly, enhancing the contrast of the images also did not change the result. Conversely, I found that reducing --slopefill (i.e. to 0.5 instead of the default 0.9) reduced a bit the differences, but CFW spots had still higher growth.

What would you say is going on? For example, do you think that it is a good idea to use a different --slopefill parameter (I am not sure what it is doing)? I ask because perhaps you also saw something like this in the past.

Thanks for your time!

Best,

Miquel Àngel Schikora Tamarit
Barcelona Supercomputing Center

Good afternoon,

Thanks for developing this tool. I am trying to analyze a 96-well plate of yeasts grown on congo red agar and I am getting misleading results. I get highly underestimated growth / cell density measurements, as compared to the growth in typical YPD plates. For instance, yeasts that appear visually to be growing well in the congo red plate may get growth measurements that are ~10% of their YPD counterparts.

The congo red plate is very red, and I suspect that the lesser contrast between the white yeasts and the red background could be the problem. Have you faced similar issues? Can you think of a simple solution to the problem? I am executing colonyzer --greenlab --lc --diffims --plots --remove --initpos --fmt 96, with version Colonyzer2==1.1.22.

Thanks,

Miquel Àngel Schikora Tamarit
Barcelona Supercomputing Center