One Brett So Many Plates

Monday Observations

I knew I had made up a lot of plates but when it came to critically observing each plate for growth and deciding how I should follow up with the results, it becomes time consuming and taking good notes is critical. It is counter balanced when observing the aroma of the plates though, some are absolutely amazing!

Selective media agars with growth

The Lysine agar media used in this study observed weak growth of colonies. Maybe it works well with bacterias but Brettanomyces yeasts are not a fan. I had read some strains can sustain growth on Lysine and some can’t so figured it was worth observing. I found the other medias to have much better results.This streak had been culturing for five days, no colonies, only stunted growth.

Lysine agar streak
Weak growth after five days

The following MYPG+cycloheximide agar media worked brilliantly, the plates had great uniform growth with the streaks forming solid single colonies. This media looks to be a great selective media.

MYPG w/cycloheximide agar streak
This streak plate had good uniform growth
and single colonies already forming

MYPG w/cycloheximide agar 10^4 serial dilution
10-4 serial dilution

MYPG w/cycloheximide agar 10^6 serial dilution
10-6 serial dilution – Some
of the colonies while uniform
in shape and texture appear
to have a brownish tinge

My absolute favorite plates were the Copper Sulfate agar media. The aroma from these was amazing. Tropical juicy fruit, Hawaiian punch, and some with a slight cocoa buttery aroma. If this is any indication of the aromas produced during fermentation this strain is a winner.

CuSO4 agar streak
A solid streak with uniform observable
growth and morphology.
Good single colonies forming.

CuSO4 agar 10^-2 serial dilution
10-2 dilution – This culture smelled amazing.
The best aromas come from the highest
cell concentrated plates.
Something to keep in mind when
deciding what pitching rates to observe for
optimal fermentations…

CuSO4 agar 10^6 serial dilution
10-6 dilution – Smaller colonies
were observed on CuSO4 agar
then the other medias.

From the 10-6 serial diluted CuSO4 agar plates and the 10-6 serial diluted MYPG+cycloheximide agar plates, single colony tops where looped and streak inoculated onto MYPG agar. Care was taken to only take the top portion of the colony as this assures that pure single cells are looped for streaking in the event that non-cultureable Saccharomyces cells are laying dormant near to a  Brettanomyces colony. The same technique was used to take a colony from each of the streak plates of CuSO4 agar and MYPG+cycloheximide agar.

I’ve deciding to further observe Brettanomyces spp. morphology on another agar medium. I prepared Wallerstein Laboratory Nutrient (WLN) agar plates, which I can further use to observe that Brettanomyces is the only species present during the fermentations as Saccharomyces spp. will form green colored colonies through take up a green pH indicator dye present in the medium, while the Brettanomyces should be observed as white colored colonies.

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