From Plate/Slant Propagation Steps(Fold System)

Ingredients
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Hi There,

So im currently research Yeast ranching and gathering equipment.  IVe been doing a lot of research and there seems to be a lot of conflicting information about how many times you should step up from a plate to a viable pitch, and at what volumes.

Assumptions-
Lets assume its a 5G batch of 1.06 Beer.  Mrmalty at the default thickness/trub settings suggests a pitch of 107ML of Slurry.  Without Rinsing and cell counting we are making a big leap, but its theoretical after all.

Braukaiser suggests the following typical steps
10 ml / 22 C / 2 days 80 ml / 22 C / 2 days 300 ml / 10 C / 3 days 2000 ml / 10 C / 4 days 70 ml sediment

The Maltose Falcons website had a wonderful writeup from MB Raines-Casselman, Ph.D.  Doctor Raines-Casselman Suggestions that a 100-Fold system seems reasonable but most brewing colleges advocate 8-10-Fold systems with as small as a 4-Fold System
  4-Fold  10ML ----  40ML ---- 160ML ---- 640ML  -----  2560ML
  8-Fold  10ML ----  80ML ---- 640ML ---- 2560ML
10-Fold 10ML ---- 100ML ---- 1000ML ----
100-Fold 10ML ---- 1000ML

Each step up assumes a 1-3 day propagation time.  Doctor Raines-Casselman suggests that a 100 Fold system is used initially at most breweries, but they go to smaller Folds as the yeast pitch becomes larger so something like
10ML ---- 1000ML ---- 8L ---- 32L ---- 120L

Chris White and Jamil Zainasheff Recommend the first step from a slant/plate to be a 30ml-50ml vial with a 1.020 Medium but does not go farther to suggest the next step.

Wyeast suggestions for larger fermentation’s to use a 10-Fold system assuming you start with a large pitch bought from a Lab.  Something like 2L of slurry into 2 BBLs of Wort, into which you will add an additional 18 BBls of wort once 50-75% of the OG has dropped.

Obviously, there are a lot of Factors that play into this scheme, but im looking for tried and true suggestions from peoples experience.  I cant count cells yet and im trying to get a handle on processes.  Ill be plating Conan onto a plate soon to isolate it and id like to have a clue on how to move forward after the initial 10-30ml step.

Cheers,
Jeff

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For those interested. Here are the links for the sources.  I Found Kaisers website to be the most informative for basic practices and equipment needs.  After that it becomes… Complicated.

Braukaiser - Start here and keep reading.
http://braukaiser.com/wiki/index.php?title=Making_Plates_and_Slants

Docter MB Raines Writeup.  Informative and not super technical.

BKYeasts Lab equipment recommendations, starting at basic and moving towards Advanced

Wyeast’s Info Site.  All kinds fo vague goodies here
http://www.wyeastlab.com/com-propagation.cfm

A Guide to Yeast counting via Serial Dilution from Sean Terrill

Jeff

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I used to propagate from slant using a 10-fold process.

slant → 10mls of autoclaved 5% (1.020) w/v wort (inoculated aseptically) → 100mls of autoclaved 7.5% w/v (1.030) wort → 1L of boiled 10% w/v (1.040) wort → 19L batch

However, over time, I discovered that I could get away with using 30 to 40 milliliters autoclaved 7.5% w/v wort as my initial inoculum and step as high as 20:1 with no ill effects (this discovery was made long before “Yeast” was published).

slant → 30 to 40 milliters of autoclaved 7.5% w/v wort (inoculated aseptically) → 600mls to 1L of 10% w/v wort → 13L to 19L batch

The current preoccupation with calculating the exact number of yeast cells needed to ferment a batch boggles my mind (the overuse of/overdependence on brewing software in amateur brewing also boggles my mind, but that is a completely different topic).  No two yeast cultures behave the same way when pitched into a batch of wort.  Only experience with a culture will let one know if one is underpitching.  Additionally, no yeast culture behaves exactly the same way when used in different breweries because a yeast culture’s phenotype is the result of its genotype being modified by environmental factors.

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Thats essentially what im looking for. im not looking for an exact way to calculate yeast but more a rule of thumb that can then be changed depending on how the strain reacts to my initial trials and batches.

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I believe in the 4 - 7 rule. After propagating yeast from slants for years with out a good idea of the number of cells I was pitching, I finally invested in the equipment to count cells. I was not only surprised by the number of cell being cultured but the difference the fold increase made in the yield factor. Below are the data from an increase from a slant to a 4000ml culture. I began by dilution streaking onto an wort-agar plate and selecting 5 good size colonies to inoculate the 10ml culture. The next steps are outlined below. The volume for each step is a total of the wort plus the inoculum from the previous step. For example the 100ml culture was 90ml of wort with the 10ml culture added. You will notice the first step was 10 fold, the next step was 5 fold, and the final step was 8 fold. Also note the yield factor for each step and how the 5 fold increase achieved the greatest yield factor with only a small drop in the 8 fold increase. You may also conjecture from these data that when making a 2 liter starter from liquid yeast we are making 5 or 6 fold increase as the 500 ml culture produced 1.4 billion cells. Please keep in mind there is an error factor (amount unknown to me) when counting with a hemocytometer. So there may not be a significant difference in say 40.7 million and 37.7 million, thereby the yield factor 30.85 may not be significantly different from 33.60. I would guess the other yield factors are different. Sorry about the table format, I could not make the insert button work. I hope it comes through readable.

Culture  Cells/ml          Total Cells            Cell/ml                  Total Cells            Yield Factor              
(ml)    at Pitch            at Pitch              at Finish                  at Finish

10 10,075,000       100,750,000         144,000,000     1,440,000,000            16.75                                            
100 14,400,000     1,440,000,000         194,000,000     19,400,000,000            22.45
500 40,740,000   20,370,000,000         287,500,000   143,750,000,000          30.85
4000 37,734,375 150,937,500,000         306,500,000 1,226,000,000,000          33.60

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Thanks for the info gents!

I made some plates, slants and stabs today.  When i was first pouring my slants i put them on an angle before autoclaving.  Will they melt back into a liquid as they get autoclaved or will they keep solid since they have already solidified once?  Im unfamiliar with how agar will react.

Cheers,
Jeff

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If it is hot they will be liquid, they have to be on an angle when they are cooling.  If space isnt an issue but time is, I’ll just use chopsticks in my pressure canner and angle my tubes on it through the whole heat and cool process.

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Thats what i did this time.  i laid them at an acceptable angle on my petri dishes because i was unsure.  the canner is still heating up.

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I used to own a proper slant rack before I sold my old brew house.  Now, I just use a couple of bread plates to support my slants while they are cooling.

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Hopefully, you are not autoclaving your plates.  That’s a surefire way to make plates that are mold magnets.

The best way that I have found to make plates is the dry sterilization method.  The petri dishes are dry sterilized in an oven at 177C/350F for 90 minutes and allowed to cool to 66C/150F (dry sterilization kills spores via oxidation).  The media is autoclaved in a separate container with a loosely fitting lid.  The media is allowed to cool to between 49C/120F and 60C/40F before the petri dishes are removed from the oven and poured hot.  It takes a little practice to be able to lift up one side of the cover, pour the media, and quickly place the cover back down in one fluid motion, but most people can master the technique.  If performed correctly, nothing more than a thin layer of condensation should form on the underside of the covers.  This condensation will evaporate during proofing.

Here’s what dry sterilized plates look like shortly after they have been poured:

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I proof my plates with the covers up at room temperature for two to three days.  I also place the piece of aluminum foil that I used to cover my petri dishes while they were in the oven on top of my plates while proofing; however, I do not seal the foil to the pan.  My goal is to prevent dust from settling on my plates while they are being proofed.  Many amateur brewers proof their plates upside down, but proofing plates upside down can result in dust settling between the cover and the bottom of the petri dish.  Any dust picked up during the proofing process can migrate onto the surface of the media when the plate is turned over for streaking.  This problem is compounded when plates are sterilized in an autoclave/ pressure cooker/ pressure canner due to excessive moisture.  I do not invert my plates until the covers have been sealed to the bottoms with Parafilm.  I store my plates upside down in my brewing refrigerator to prevent them from drying out.    I usually store sealed plates in Ziploc bags in a Tupperware container that is dedicated to blank media use.

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Hmm ill have to ponder over dryheating my dishes.  Ive been following the guidelines set down vai Kaiser on Braukaiser.  I Put 2 of my dishes inside the incubator yesterdat afternoon and neither have grown anything that i can see thus far.  I just opened one up.  Altho the surface of the agar is erratic as hell, the whole dish seems clean and sterile(before i opened it anyway)

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Kai is an incredibly smart guy, but his media page is one of the weaker areas of his site.  His plate making technique is completely wrong.  If you Google the phrase “how to pour plates,” you will discover that the basic technique that I outlined above is used to pour plates using dry sterilized glass and pre-sterilized plastic petri dishes.  Condensation is your enemy when it comes to plates.

Kai also uses the wrong piece of glassware for making slants.  The proper piece of glassware for making slants is known as a screw-cap culture tube.  A screw-cap culture tube has a round bottom like a test tube.  A screw-cap culture tube also has a deeper cap that is designed for culturing microbes (compare the screw-cap culture tubes in the photo that I posted above to the vials in used in his photos).  A vial is designed for sample collection.

Kai also seals his plates incorrectly and uses a less than ideal sealant.  A better product to use when sealing media is called Parafilm.  It used to be difficult to acquire, but one can purchase rolls of Parafilm M and Parafilm Grafting Tape on eBay.  Parafilm Grafting Tape is just a modified form of Parafilm M that was designed for grafters who were using Parafilm M to graft plants.  It has a little more olefin and a little less paraffin wax

Here’s a bag of Parafilm sealed cultures in Corning No. 9825 20mm x 125mm culture tubes from my collection:

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Here’s a link to a video that demonstrates how to seal a plate correctly with Parafilm: http://www.youtube.com/watch?v=f47Hq2uiJIs

Here’s a link to one of the better aseptic transfer videos on YouTube: http://www.youtube.com/watch?v=xvgAboJmIsM

The only thing that I would add to the video linked above is that I quickly pass my caps through the flame with the opening down before recapping.  This step is easier to perform using an alcohol lamp than a Bunsen burner.

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Ill check it out when i get some free time.  I know the Vials Kai used are incorrect but ill practice with them ebcause they are cheap.  i also bought a dozen culture tubs but they are smaller and will be hardcer for me to work with.  Parafilm has already been ordered.  Im also thinking about building a hood.  Should be able to do it with MDF fairly cheaply.  probably 2’ wide, 2’ deep and 3’ High.

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A laminar flow hood is a nice to toy to own.

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I think that s alittle beyond my use.  I was more thinking of a hood with a window that will controll up and downdrafts and keep me from breathing on my cultures.