Novice yeast harvesting thread

[YeastWhisperer]

I would use universal tubes, known as transport tubes in the U.S., if I could not obtain reusable borosilicate glass culture tubes. The only downside to plastic tubes is that one needs to be very careful when flaming the mouth of the tube during aseptic transfers. The upside is that universal tubes are much cheaper than reusable borosilicate glass culture tubes.

[daf]

Here is what's happening so far:


No noticeable bad smells so fingers crossed.

[Jocky]

You can get polypropylene tubes on eBay from places like King Scientific. They are autoclavable and that's where I got mine. If you go to the stickies here, there are some threads on yeast techniques, including making slants (which includes within it a link for stepping up from a slant).

The one thing I have found is that I struggled to find any polypropolene tubes with autoclavable lids. Are these a requirement?

[Goulders]

The ones from king scientific are. Find the vials and if it doesn't say so, just email the seller and ask.

[Kohoutec]

Can I jump in and ask a related question here....I know there are calculators online, but when culturing up yeast from bottles roughly how much yeast would you need for a standard 23l brew length (My OG is usually around 1050)?
I've successfully cultured and pitched Fullers yeast in the past, although the resultant beer did have a fair diacetyl hit. I know the Fullers yeast is known for that but makes me wondered if I drastically underpitched.
My usual process is use the dregs of 3 (or sometimes 4) bottles of whatever "donor" beer I'm using in about 100ml of wort, then step that up to 500ml and then 2l.
I keep reading about stepping up x10 so wonder if I should be starting smaller, say 3 steps of 20ml, 200ml and then 2l...and if I will have enough yeast in my final 2L starter (I drain off the spent wort before pitching)?

[YeastWhisperer]

My usual process is use the dregs of 3 (or sometimes 4) bottles of whatever "donor" beer I'm using in about 100ml of wort, then step that up to 500ml and then 2l. I keep reading about stepping up x10 so wonder if I should be starting smaller, say 3 steps of 20ml, 200ml and then 2l...and if I will have enough yeast in my final 2L starter (I drain off the spent wort before pitching)?

If you are waiting until fermentation is compete to step or pitch your starters, you are waiting too long. A starter is best pitched at high krausen, even if that means pitching the starter wort. Pitching yeast at high krausen is like pitching the yeast equivalent of raring to go military special forces. Waiting until fermentation has completed is like pitching the yeast equivalent of worn-out normal troops. Yeast cells that are pitched at high krausen utilize available dissolved oxygen (O2) better than yeast cells that have reached quiescence at the end of fermentation. This difference has to do with ergosterol and unsaturated fatty acid (UFA) reserves. Yeast cells use dissolved O2 to synthesize ergosterol and UFAs at the beginning of fermentation (a.k.a. the lag phase). Ergosterol and UFAs make a yeast cell's plasma membrane more pliable, which, in turn, makes it easier for the passage of nutrients into the cell and waste products out of the cell. Yeast cells that are pitched at high krausen have greater ergosterol and UFA reserves, and they have not undergone the morphological changes that occur at the end of fermentation in preparation for quiescence. One of these changes is the thickening of the cell wall. This change is a survival mechanism, and it has to be reversed before yeast cells that have reached quiescence can go to work.

With respect to diacetyl, flocculent strains usually have high O2 requirements. Pitching at high krausen will help the culture utilize available O2 better.

With respect to stepping, I would step no more than 5-to-1 with a bottle culture, starting with 30 to 50ml of autoclaved (pressure cooked at 121C at 15 pounds per square inch above normal automospheric pressure for 15 minutes) 1.020 (0.05 grams per milliliter) wort if you have access to a pressure cooker. Bottle cultures are usually not pure, so one wants to start with absolutely sterile wort if at all possible. Boiling only kills vegetative (alive) cells. It does not kill spores. You should also read about how yeast cultures should be handled in a the "Note" section in the following thread: viewtopic.php?f=12&t=70926

[steambrew]

I feel thanks should go to all the people who have made this such a interesting topic

[Kohoutec]

Thanks YW for such a detailed reply. More questions if i may. If I start with say 50ml stepping up x5 I would be going 50, 250, 1250. I assume I want to step up at high krausen each time so would be keeping the wort from each previous step. This means I would actually have a starter size of around 1550ml, do you think this would suffice or would a final step up be advisable (using a 23l batch at 1050 as an example). Appreciate it's difficult to give specifics but just after a general approach.

[YeastWhisperer]

You can step by less than 5. The reason why we step is because it is any opportunity to add O2 as well as a way to ensure that yeast owns the media quickly. You can add media at each step such that the combined volumes is 5x the previous volume because the media is not spent at high krausen. If you pitch a 1L culture at high krausen into 23L of < 1.065 well-aerated wort, I guarantee that it will outperform a 2L starter that is allowed to ferment out. Dissolving additional O2 at every step is critical to yeast health.

As an aside, contrary to what one reads in home brewing texts and on home brewing forums, a stir plate does not provide adequate aeration, even if used at a high speed (which results in shear stress being placed on the cell walls). Stir plates were not designed with yeast starters in mind. They were designed to prevent cells from multi-cellular eukaroyotes (cells that contain a nucleus) from clumping together during a cell culturing technique known as suspension cell culturing. Most brewing strains are NewFlo strain (FLO is the name for the set of genes that control flocculation). NewFlo strains do not flocculate until glucose, mannose, sucrose, maltose, and maltotriose have been reduced to a genetically set level; hence, most brewing strains do not need help remaining in suspension, especially up to the point of high krausen. If you are using a stir plate, please give my well-shaken starter method a shot. I guarantee that you will not go back to using a stir plate and stir bar.

[Jocky]

Does a stir plate not provide additional aeration after the oxygen from the initial shaking is exhausted?

[YeastWhisperer]

No, stir plates do not add additional aeration after the initial O2 that is dissolved by shaking is exhausted. Anyone who has taken physics knows that little to no 02 is enters an Erlenmeyer flask after the culture starts outgassing. Stirring a culture fast enough to dissolve O2 before it starts outgassing results in shear stress being placed on the yeast cell walls.

Stir plates were not designed for growing yeast cultures. They were designed for growing cells from multi-cellular eukaryotes (multi-cellular organisms that have cells that contain a nucleus). Stir plates were introduced to the home brewing community by home brewers who were engaged in cancer research. A technique called suspension cell culturing is used to grow cancer cells in a laboratory. The media has to be kept moving to prevent clumping. That's where a stir plate enters the equation. Yeasts are single-cell eukaryotes. They also do not clump during the biomass growth stage because most brewing yeasts exhibit NewFlo floccultation (FLO is the name for the group of genes that control flocculation). NewFlo strains do not flocculate until glucose, mannose, maltose, sucrose, and maltotriose have reached a genetically set level; therefore, spinning a yeast culture serves no purpose other than to possibly stress the cells. I guarantee that you will never use a stir plate again if try my "Shaken, not Stirred" starter method. It's easier, a lot cheaper, and fermentation performance is better because the cells are not stressed.

[Jocky]

Interesting. Can you comment on why the conventional wisdom that using a stir plate provides a greater yeast growth compared to shaking?

And do you do anything fancy for your method? Just shake it (with foil on the top?) and leave? Do you shake any more after that?

[NotSure]

I use the residual green beer that is left in the primary fermentation vessel to swirl the culture back into suspension, wait a few minutes for the break and dead yeast cells to settle, and carefully decant between 250 and 350ml of thin slurry into a sanitized 500ml Erlenmeyer flask after wiping the pouring lip on the fermentation vessel with 70 to 90% alcohol. The goal here is to leave break, particulate matter, and as many dead cells as possible in the fermentation vessel.

YW, does the same procedure apply to highly flocculent strains, such as WLP002?

[YeastWhisperer]

Interesting. Can you comment on why the conventional wisdom that using a stir plate provides a greater yeast growth compared to shaking?

I have seen no peer-reviewed scientific data that supports the claim that stir plates provide greater yeast growth than shaking. In fact, a stir plate is a poor man's lab shaker in the world of suspension cell culturing. The only thing that a stir plate does is keep the cells in suspension beyond their normal set flocculation levels. However, if one is waiting until the media is exhausted to pitch one's culture, then one is pitching yeast cells that are not in optimum health. A starter should be pitched at high krausen. High krausen is the point where the culture reaches maximum cell density and switches over to replacement-only cellular reproduction.

And do you do anything fancy for your method? Just shake it (with foil on the top?) and leave? Do you shake any more after that?

I believe that you are underestimating how thoroughly the culture is shaken when using my method. There's no way that one could shake a culture thoroughly enough with a foil-covered vessel. The vessel has to have a screw-on cap because it prevents wort from going everywhere during the one minute-plus vigorous shake. One is attempting to turn the starter into as much foam as is humanly possible, which is why the vessel also needs to be at least four times the volume of the starter wort. Foam provides significantly more surface area for the diffusion of oxygen than a liquid.

The only thing that one needs to do after performing the shake is loosen the cap, so that the culture can outgas. Failing to loosen the cap can lead to one having quite a mess on one's hands.

[YeastWhisperer]

YW, does the same procedure apply to highly flocculent strains, such as WLP002?

Yes, even WPL002 will reach maximum cell density before it starts to floc. Remember, we are pitching at high krausen in order to take advantage of healthy, ready to go yeast cells.