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Old 09-03-2014, 03:34 AM   #11
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That was my thought, sorry I realize I didn't clarify.

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Old 09-03-2014, 11:48 AM   #12
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It is very hard to differentiate yeast from pedio/lacto on normal agar plates - the colony morphology is similar, and they are generally unpigmented. An experienced microbiologist may be able to do it, as there are subtle differences, but I know (even having worked in the field for more than a decade) that I couldn't do it.

A microscope is your best bet - yeast can be differentiated from lacto/pedio quite easily this way. Yeast are larger (although Brett can be a lot smaller than Sacc; but still bigger than bacteria). Yeast tend to be round or oval, and sometimes buds can be observed (lactic acid bacteria don't bud, so if you see buds you can be certain you have some sort of yeast).

Lactobacillus tend to be small rod-like bacteria, and depending on the species, tend to be either single cells, two cells connected pole-to-pole, or linear chains with the cells connected pole-to-pole. Pediococcus is also a rod, but the rods are shorter compared to their width than lacto, and under a lower-mag/NA microscope may appear as slightly off-kilter spherical bacteria. Pedio doesn't form chains - instead it forms clumps - usually small groups of cells - typically 2 or 4 together, connected along the long axis of the cell rather than by the poles.

Bryan

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Old 09-03-2014, 12:26 PM   #13
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Quote:
Originally Posted by h-bar View Post
I should be able to pressure cook the plates.
If you can, than I would recommend the following (I'm working on a video showing this process, but it may be a while before it is posted):
  1. Build a stand of some sort that you can use to keep your plates above the water level of the pressure cooker. You need a stand that lets the pressure cooker to be filled at least 1/3rd full.
  2. Prepare your mix of DME and agar; mix it thoroughly but do not boil it. Pour the appropriate amount into each petri dish - swirl constantly to keep the agar evenly suspended (some will stick to the sides; don't worry about this).
  3. Place plates in the pressure cooker, with the petri dish lids on, on the stand. Make sure they are at least 5 cm (~2") above the top of the water level. It is fine to stack the dishes.
  4. Heat, with your pressure cookers pressure release set to the highest pressure setting. Once the cooker comes to full pressure start a timer for 20 min.
  5. After 20 min turn off the stove, but allow the pressure cooker to cool to well below boiling (at least to 80C/175F) before opening
  6. Remove your plates and place in a draft free area to solidify. Gently swirl the plates as you remove them from the cooker to make sure the agar is evenly distributed. It is important to remove them hot, or you may end up with too much condensation inside the plates.
  7. As soon as the agar has gelled, flip the plates. This will limit condensation on the lids.
  8. in a draft-free area, let the plates sit upside-down for ~24 hours (you can place them in a slightly warmed oven if you need this to go faster) - this will let any excess water evaporate.
  9. Place in a zip-lock bag and store in the fridge until needed - again, keeping the plates upside down so any condensation doesn't flow onto the gel.

This process eliminates the risk of infection while making the gel. Your other option is to sterilize empty glass plates on the stand, and to pressure-cook your media (put the bottle/jar into the water - not on the stand), and then to pour the plates as per usual. This later method is closer to what we do in the lab, and is the best way to make a lot of plates, but carries the risk of infection during the pouring process.

Quote:
Originally Posted by h-bar View Post
How will I be able to make sure I'm only collecting yeast from the plate?
I already posted a bit on this in my previous reply. Long story short, its not easy. A few options you may be able to implement at home are:
  1. Add antibiotics (you need something that will kill gram positives - e.g. streptomycin). Always add antibiotics after pressure-cooking, as the heat will destroy them.
  2. Purchase some selective media. This is expensive, but media do exist which allow the growth of yeast but not bacteria.
  3. Use a microscope, as per my last post. You need a microscope that meets certain specifications - I've posted about this before and would recommend you check out that post.
  4. A less certain way is to make your plate using highly hopped wort (60IBU or more). This will inhibit most - but not all - lacto and pedio. It will not inhibit acetobacter or other spoilage organisms.

A fifth method (which is a topic of a video I'm preparing) is to individually test colonies to see if they behave like yeast. To do this:
  1. Prepare a streak plate, as per my video or other instruction you find on the net.
  2. Take a clean plate and draw a grid on the bottom of the plate (roughly 2cmx2cm (1"x1") works well. Draw the grid on the bottom, not on the lid, as lids rotate/etc.
  3. Pick individual colonies and streak each one out on the new plate, keeping each mini-streak inside its own box in the grid. Make sure you are flaming your loop/paper clip between each "pick, and cooling it before "picking", as per my above video.
  4. Let the new plate grow up.
  5. Using a loop or paper clip, inoculate small vials of 1.040-ish wort - 1 colony/tube. Again, flame and cool the loop/paperclip between each transfer.
  6. Monitor the tubes for signs of fermentation - e.g. bubbling, kraussen, yeast-aroma, gravity drops (with a refractometer), etc. After a few weeks you can also taste the wort. Based on this you should be able to ID yeast which will form a kraussen, cause the gravity to drop, smell like yeast, and produce something that tastes non-sour.

Bryan
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Old 09-13-2014, 01:03 AM   #14
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I've let the samples grow for two weeks, and I've tasted them both. Both samples definitely fermented and have white layers of yeast at the bottom.

Jar 1: fruity, solventy, hot. Not very flocculant. If I use any yeast from this jar, I'll try a lower temperature. Hopefully that will reduce the esters a bit.

Jar 2: tastes like a Belgian wit! Very fruity and dry. I have higher hopes for this one. However, the yeast layer at the bottom is less than in jar 1.

I'm going to plate cultures from both next week and will try isolate some strains.

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Old 09-13-2014, 02:53 PM   #15
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Well done! You're lucky to have had such good success; I've found that around here about 1 in 4 cultures is brewable.

Brya

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Old 09-19-2014, 05:09 AM   #16
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I plated the two samples tonight. How soon should I expect to see growth?

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Old 09-19-2014, 11:59 AM   #17
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Sacch should appear in a day or two; lacto and pedio about the same speed. Brett is often a little slower - it may take 3-5 days for those colonies to appear.

Bryan

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Old 09-20-2014, 05:33 AM   #18
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Something is starting to grow in both plates. It's not super visible yet. I'll post a picture once it is. One plate has whitish growth that looks "dry"... That's the best way I can describe it. The other has whitish growth that looks wet and gooey. The one with the "dry" growth came from the jar that had the largest krausen and healthiest looking yeast layer.

Edit: white dry stuff turned out to be mold. It had spread over the entire plate this morning. I chucked it and made a new plate for that sample. I'm so glad I made extra plates!

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Old 09-20-2014, 03:46 PM   #19
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The healthy, non-moldy plate looks like this:

I think I'll scrape off one of those little colonies in the lower corner there. My streaking technique wasn't great, obviously. Hopefully the plate I redid turns out better and doesn't grow mold.

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Old 09-21-2014, 09:01 PM   #20
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I showed that picture to a more experienced brewer (who is also a biochemist) and he said what was on that plate is not yeast. I've retried streaking a couple more times and each time only get mold. I think whatever yeast I have grows too slowly and mold just takes over, or the yeast I collected is no longer viable. I tried to grow more of what I had by adding some fresh wort to the jar, but there has been zero activity. Unfortunately, I think this has a been a failed venture...

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