MYPG Compared to Wort as a Growth Substrate

•August 8, 2009 • Comments Off on MYPG Compared to Wort as a Growth Substrate

While most of the batch culture propagations were conducted using wort as the growth substrate, I also looked at how batch culture growth proceeded using MYPG as the growth substrate. This study was performed with each of the three Brettanomyces strains from Wyeast by inoculating them  into 100 ml of MYPG substrate and 100 ml of wort substrate. The substrates were inoculated by looping cells from a single colony grown on MYPG agar. Initial cell growth in both mediums proceeded for 7 days before the propagations were poured aseptically into 500 ml of wort in order to observe any difference the starting medium had over subsequent growth. The second propagation step underwent a further 7 days of cell growth with comparisons made regarding overall cell growth.

Batch Culture Growth Curve Comparison of MYPG and Wort Substrate
Growth curve of three Wyeast strains initially sub-cultured in MYPG substrate
as compared to the same strains initially cultured separately in wort substrate.
Both substrates were further inoculated into 500ml of wort substrate and
grown for 168 hours (7 days) at 28°C with 80-rpm agitation.

Batch Culture Growth Curve Comparison of Collective Means in MYPG and Wort Substrate
Growth curve comparison between the collective means of three Wyeast strains
initially sub-cultured in MYPG substrate as compared to the same strains
initially cultured separately in wort substrate. Both substrates were further
inoculated into 500ml of wort substrate and grown for 168 hours (7 days)
to observe initial impact on cell growth. Mean cell count data was obtained
from all three Wyeast strains grown at 28°C with 80-rpm agitation.

I had previously not had growth this rapid before yielding high cell counts. It would appear that MYPG is a better suited substrate for initial culturing of cells and when looking to propagate up cells for pitching into fermentation. More information on Propagation and Batch Culture Growth can be found by following the link.

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Propagation Data and Graphs

•July 27, 2009 • 3 Comments

I have finished compiling the data on batch culture propagations conducted with five of the Brettanomyces strains. The following two graphs contain data from the standardized propagation method described in the previous post using 12 °Plato (1.048 gravity) wort as the growth medium. Some variation between growth patterns can be seen along with what appears to be step-like growth until the stationary phase is reached after a period of 168 to 192 hours. More information on Propagation and Batch Culture Growth can be found by following the link.

Batch Culture Growth Curve for Brettanomyces Yeasts
Growth curve for five strains of Brettanomyces during semi-aerobic batch culture.
Cultures were grown in 500 ml of wort substrate over a 288-hour
period at 28°C with 80-rpm agitation. Viability was taken into
account to reflect the actual cell number.

Daily Viability of Brettanomyces Yeast During Batch Culture Propagation
Daily percentage of cells which appeared viable after using the
methylene blue staining technique. Cultures were grown in
500ml of wort substrate over a 288-hour period at 28°C
with 80-rpm agitation.

*Methylene Blue staining technique… The use of methylene blue to stain cells in order to determine viability proved difficult with Brettanomyces spp. I found often that cells which were viable would take up the methylene blue into the vacuoles, giving a false positive. Some would take it into the cell cytoplasm but not into the vacuole. The dye would only appear near the tips giving the cells two blue dots at each end. I have spoke with other brewers who have had trouble using methylene blue to yield positive results when doing viability checks on Brettanomyces strains. I would say from the lab experience I had and daily cell counts that an alternative method would be advantageous as I don’t believe methylene blue yields completely accurate results.

Propagations Standardized

•May 15, 2009 • 2 Comments

During the month of April and beginning of May, I worked to standardize batch culture propagation methods for maximizing cell growth. I had previously finished observing colony morphology and growth characteristics on various mediums and chose MYPG and WLN agar as my two mediums of choice. With eight strains of Brettanomyces to use throughout the research and needing to propagate each strain in duplicate (in order to replicate the experiments) I had to use multiple orbital incubators and tied up a lot of the University lab’s resources. The following is the conditions which I found to produce the highest concentration of cells which were physically ready to start fermentation with high vitality and viability.

  • Started with single colonies from MYPG agar, inoculated into 100 ml of wort in a 250 ml flask.
  • All flask pre-filled with the wort solution were autoclaved with a sterile foam bung and aluminum foil loosely around the top.
  • Initial propagation from single colonies was allowed to go for 7 days.
  • The initial propagations were then decanted aseptically into an additional 500 ml of wort in a 1.5 liter flask.
  • This second propagation step went 8 days before it was used for pitching into fermentations.
  • All propagations occurred in orbital incubators rotating at 80 rpm.
  • Temperature was set at 28°C in the incubator.
  • Semi-aerobic conditions were present as a result of using a foam bung in the conical flask tops with aluminum foil covering.

A previous post or two mentions the propagation methods as they were going and some variations in the length of the initial propagation from the colonies. In the end the info above is what I used throughout the rest of the study to propagate yeast cells. Check out the following link to read more about the Propagation and Batch Culture Methods used throughout the dissertation.

Wyeast to the Rescue

•May 6, 2009 • 1 Comment
4/30/09 – Thursday

Over the past month or so I have been e-mailing with Greg Doss Wyeast Brettanomyces packageof Wyeast Labs as previously mentioned and it turns out the gracious people over at Wyeast were able to supply the Brettanomyces Masters Project with three slopes. WY5112 (B. bruxellensis), WY5526 (B. lambicus) and WY5151 (B. claussenii) were shipped over from the USA and have arrived safely. This brings the strain count up to eight different strains that are being used in the research!

The slopes were looped and inoculated into 6 sets of 250 ml conical flasks containing 100 ml of solution for propagating up to pitchable amounts. I will be observing the growth during batch culture in two different mediums this time. Of the six flasks the strains were inoculated into, three contain MYPG solution and three contain wort solution. This is a quick observation to test the difference in growth between the two mediums. Conditions are the same as with all the batch culture propagations: Temperatures of 28°C in the Orbital incubator with agitation around 80 rpm, semi aerobic conditions with foam bung in the conical flask tops and aluminum foil covering.

5/6/09 – Wednesday

What I observed was interesting. I found a slightly higher amount of non-viable cells in all three of the MYPG solution flasks as the over all viability was 96.68%, while after 6 days the cells grown in wort solution had 99.73% viability overall. As for cell growth a different pattern was observed. For B. bruxellensis the wort/mypg cell ratio average per square on the hemocytometer was 228.4/104, for B. lambicus 125.2/76.8 and for B. claussenii 117/134.2. This shows that the yeast have different growth behaviors based on the composition of the media. This could be important in choosing the right wort to use for a 100% Brettanomyces fermentation, or when propagating up cells for pitching into a primary or secondary fermentation. The flasks were then added to 500 ml of sterilized wort solution. As they are grown up into pitchable amounts I will take daily cells counts monitoring the cell growth to see if the initial media continues to have an effect or not. All propagation data from the eight strains will be put into graphs and made available as soon as I get the last of the data.

Wyeast B. bruxellensis on MYPG agar
Wyeast 5112 B. bruxellensis

Wyeast B. lambicus on MYPG agar
Wyeast 5526 B. lambicus

Wyeast B. claussenii on MYPG agar
Wyeast 5151 B. claussenii

Media Cultureablity of Brettanomyces Species

•April 30, 2009 • Comments Off on Media Cultureablity of Brettanomyces Species

Screening of Various Media Agars

I have finished collecting and compiling data on the ability of B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001) to culture on popular brewery related media agar. This is the first part of the research and was carried out to find the relevance of each media, its use throughout the project, and practicability of use in the brewing industry. The observations should be useful for QA/QC labs wanting to culture and maintaining various Brettanomyces strains. The strength of colony growth was judged based on the time it took for single colonies to develop with distinguishable morphological traits and the ease with which each medium could be used to enumerate and identify strains. The medias which colonies were observed on were MYPG, MYPGcc, MYPGch, WLN, WLD, CuSO4, Lysine and UBA agars. To read more about the Evaluation of Yeast Culturability on various media agar check out the link or you can find more information on strain specific Morphological Traits on various media agars.

Media

WLP645

WLP650
WLP653
CMY001
BSI-Drie
MYPG ++ ++ ++ ++ ++
MYPGcc nd + + + +
MYPGch ++ ++ ++ ++ ++
WLN ++ ++ ++ ++ ++
WLD ++ ++ ++ ++ ++
Lysine +/- +/- +/-
CuSO4 ++ ++ ++ ++ ++
UBA + + +/- +/- +

-, No detectable growth; +/-, weak growth; +, moderate growth; ++, strong growth; nd, not determined.

The Many Faces of Brettanomyces

•April 20, 2009 • 2 Comments

Giant Colony Morphologies
When I undertook this project I never imagined the microbiology involved and the hours I would spend in the lab just to get this project off the ground. Three months in the lab culturing organisms so far and I have found that just working with Brettanomyces spp. on various media agars can prove difficult. Each plate takes careful examination to ensure pure cultures are kept and maintained. Overtime this becomes easier and I have started to memorize traits each strain possess on the different media agars. The majority of the past few months have involved working with the Brettanomyces species cultured from Avery’s 15th Anniversary ale and cultures from White Labs yeast company. I have been culturing the Brettanomyces strains onto various medias listed previously and observed the morphologies and growth habits of each strain. The following is photos from the three White Labs Brettanomyces strains and their unique morphologies on various media agars. Along with culturing the different strains on various medias, careful observation under the microscope helps to observe strains general cell morphology but in no way gives a good indication to whether a pure culture is being observed due to the many growth phases and various cell shapes observed by a single culture at one time. I will try to post photos later and detail cell morphology under a microscope but that is too detailed to include in this post. For now here are some photos of the Brettanomyces spp. cultured on the various agar plates as this may be useful for any one trying to culture this incredibly fickle organism. The most difficult part of observing Brettanomyces is that little is written about the behavior and life cycle, if much is even known at all. I have discovered that even when a single colony is taken and streaked onto the agar plates not every resulting colony will grow exactly uniform or have exactly the same color or size. This could be due to a stage of the life cycle when streaked from a single colony or another phenomena, but it seems to be observed in nearly every strain.

Brettanomyces bruxellensis (WLP650) on MYPG agar
Nearly all the colonies form a sand dollar
like pattern on top of the culture
with a round dome in the middle

B. bruxellensis (WLP650) on WLN agar
Nearly all the colonies for a brain
like pattern on top of the culture


Brettanomyces claussenii (WLP645) grown on MYPG agar
White normal round colonies with a rather flat look

B. claussenii (WLP645) grown on MYPG+cycloheximide agar
A distinctive beige color is observable with the cycloheximide

B. claussenii (WLP645) grown on WLN at 4 days since streaking
This species breaks the rules and does not
metabolize the Bromocresol Green
indicator as the literature states
New discovery, look how green the colonies remain

B. claussenii (WLP645) grown on
WLN at 7 days since streaking
Colonies still enlarging

B. claussenii (WLP645) grown on
WLN at 10 days since streaking
Noticeable white dots appear on the top of the colony.


Brettanomyces lambicus (WLP653) on MYPG agar
colonies display a unique curved
ring like top structure

B. lambicus (WLP653) single colony
grown on WLN media with
a ring like top growth
on the colony
The other giant colonies are a Saccharomyes sp.

B. lambicus (WLP653) single colony
on WLN media growing into
the side of a Saccharomyces sp.
I have found Brettanomyces species like to grow on top of and within other yeasts.

Project Updates

•April 12, 2009 • 2 Comments

Over the past couple of weeks I have been in talking with Greg Doss the QC manager at Wyeast labs and they will be helping out the Brettanomyces Masters Project by shipping over slopes of B. lambicus (5526), B. bruxellensis (5112), and the VSS for April to June B. claussenii (5151). Thanks to Greg for his help and I look forward to receiving the cultures once they are shipped and clear customs!

As the project continues, I now have five pure culture strains of Brettanomyces and have started observing cell growth phases during batch culture propagation. All five strains were seperatly inoculated into 250 ml flasks containing 100 ml of wort solution taken from the standard wort produced and being used throughout the entire project. The flasks containing the wort were loop inoculated with cells from the top of a single colony grown on MYPG agar plates. These five strains were then propagated up over a 16 day period and at that time the 100 ml yeast slurry was pitched into 500 ml of new sterile wort solution and grown in a 1.5 litre flask. From here I will again be taking daily cell counts to observe the growth phases for each strain during propagation in order to observe when is best to pitch for fermentation and get a better idea of the ideal propagation methods to be used with Brettanomyces yeast species. Watch for the results in the future and the possibility of further experiments which look to further maximize cell growth during propagation.