13,585
edits
Changes
m
update/typo as per Mark Trent
This page documents an experiment by [https://www.facebook.com/mark.trent.370?fref=nf Mark Trent] to investigate the long term (100 days) survival rates or ''Brettanomyces'' under different media and temperatures. Further work is required to verify these results. Richard Preiss of [[Escarpment Laboratories]] is working on similar research, and Mark Trent intends on evaluating this experiment again.
==Introduction==
Survival of yeast in 4 different liquid media preparations was tested in cold and room temperature conditions. Media were; wort, buffered wort, MYPG, and buffered MYPG. In addition yeast was stored cold on solid media plates and slants containing buffered MYPG or MYPG. For liquid media, the yeast survived better at room temperature conditions compared to refrigerated conditions with the exception of survival in buffered MYPG cold treatment which was not significantly different from survival in buffered wort room or in buffered MYPG room (Fig. 2). Yeast stored on plates were dead on both media as was the yeast stored on MYPG slants while the yeast stored on buffered MYPG "slants" was viable in both replicates (Fig. 34).
==Materials and Methods==
==Discussion==
Previous experiments on survival of ''Brettanomyces'' by Richard Preiss and myself (see [[Brettanomyces_Storage_Survival_Experiment#Preliminary_Work_on_30_Day_Storage|Preliminary Work On 30 Day Storage]]) have shown the yeast survives better in cold temperatures up to 1 month. However, in my previous experiment both isolates seemed to have survived better at room temperature after 6 months although at a very low survival rate. The 6 months evaluation of that experiment had issues and therefore was repeated here. In Richards experiment, evaluation at 6 months revealed mixed results with one strain surviving better in cold temperature and the other better at warm temperature. Therefore, optimum survival temperature may vary by strain. In this experiment only one strain was used but clearly survived better after 100 days at room temperature in unbuffered MYPG. Buffering was deleterious to survival at room temperature in both media, while buffering MYPG for cold temperature was an advantage. It has long been known that ''Brettanomyces'' survives poorly on plates and in my experience poorly on slants. This experiment indicated that Brett survival on slants can be improved by buffering MYPG. Although the survival on slants was only tested as live or dead, the yeast on buffered MYPG grew up nicely after 100 days while the yeast on MYPG was dead. Since the yeast was dead on both buffered and unbuffered plates at 100 days, it is not known if buffering was an advantage for survival on plates.
==Figures==
File:Trent Brett Storage Figure5.jpg|'''Figure 5''' Example of dilution plating, MYPG at room temperature. Dilutions included; left 1:10000, center 1:1000, and right 1:100.
</gallery>
==Preliminary Work on 30 Day Storage==
[[File:Brett viability temp storage.jpg|thumb|300px|[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/ Richard Preiss's results of storing ''Brettanomyces'' at different temperatures for 1 month]]]
[[File:Brett storage MarkTrent.jpg|thumb|300px|[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1145139948847551&offset=0&total_comments=69&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Mark Trent repeated Richard Preiss's results of storing ''Brettanomyces'' at different temperatures, and also in different mediums for 1 month.]]]
Some preliminary work has been done by Mark Trent and Richard Preiss to examine the effects of storing ''Brettanomyces'' in MYPG and wort at refrigeration temperatures versus room temperatures for 30 days. This work suggested that cold storage for as long as one month is better than room temperature storage for the same time. Chad Yakobson also noted that after storing ''Brettanomyces'' in a refrigerated environment (we don't know how Chad was storing the ''Brettanomyces'' cultures when he observed this, for example on agar plates or slants or something else.), after 6 months the ''Brettanomyces'' would die. If ''Brettanomyces'' is stored cold, it will be very sluggish and slow to start fermentation. Non-pure cultures (such as beer bottle dregs with ''Brettanomyces'' in it) should be stored refrigerated. Making a starter is highly recommended if the ''Brettanomyces'' culture has been stored cold <ref>[http://youtu.be/AjVOzBtE27Y?t=43m Yakobson, Chad. Presentation at 2012 Music City Brew Off. At 43:00.]</ref>.
Yakobson's observations were not scientifically quantified and details of his process are lacking (how was the ''Brettanomyces'' stored?), as far as we know. Richard Preiss of [http://www.escarpmentlabs.com/ Escarpment Labs] shared the results of a controlled experiment on MTF that showed that BSI's ''Brettanomyces bruxellensis Drie'' and WLP645 ''B. claussenii'' survived better in low ABV beer when stored at refrigeration temperatures rather than room temperatures for one month. The samples were grown in 1.040 DME wort until typical cell density was reached, and measured for >95% viability after growth with trypan blue stain and microscopy. 10ml samples of each were stored in sterile conical tubes for one month at different temperatures (4°C and 24°C). The samples were burped to avoid having head pressure as a variable. Trypan blue stain and microscopy were used to measure the viability after one month. After one month of storage at 4°C (39.2°F), the viability of ''B. claussenii'' was 92%, and BSI Drie was 72% viability. The samples stored for one month at 24°C (75.2°F) showed a significant drop in viability, with ''B. claussenii'' ending up at 40% and BSI Drie at 6% viability. This experiment also indicates that the viability of ''Brettanomyces'' strains/species after storage is strain/species dependent <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/ Richard Preiss Brett storage experiment results on MTF. 7/24/2015.]</ref>.
Mark Trent repeated Richard Preiss's experiment, but tested different mediums (wort, liquid MYPG, and water). Two ''Brettanomyces'' isolates, one from Orval and one from SARA Bernice, were grown in 10 degree Plato wort and MYPG. The isolates are labeled on the chart as "Brett O (Orval)" and "Brett T (Tim Clifford)" respectively. After growth was complete, 10 mL of aliquots were aseptically transferred to 15 mL centrifuge tubes. In addition, the Orval isolate was grown on a MYPG plate and 3 single colonies for each treatment were transferred to 1 mL of sterile RO water in a 2 mL glass tube. Each treatment was prepared in a duplicate and stored at either 22°C or 1°C. Viability was measured after 31 days. Data shown in the chart to the right. No other statistics were performed (there were no statistically significant differences between the different types of storage mediums at room temperature). All storage mediums shared results similar to Richard's results <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1115768398451373/?comment_id=1145139948847551&offset=0&total_comments=69&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Mark Trent on MTF. 09/10/2015.]</ref>. It appears from these experiments that ''Brettanomyces'' stores better cold when stored for less than a month, however these results need to be verified and more strains should be examined before broad statements can be applied about the effects of temperature when storing ''Brettanomyces'' in wort or MYPG. Interestingly, the current study suggests that '''after''' one month, ''Brettanomyces'' survival rate is generally stronger at room temperatures than at refrigeration temperatures.
==See Also==