Fractional Sterilization - mushrooms without a pressure cooker.

[BuckarooBanzai]

Cultivating mushrooms in containers on a BRF substrate requires an effective sterilization process to be successful. In order to quickly grow a healthy BRF cake, mushroom mycelia must be the only (significant) fast growing life form in the container/jar. Sterilization of the substrate before spores are introduced is paramount to success. Any bacteria or other microbial life in the jar will compete with the mycelia and slow (or prevent) full colonization.

A pressure cooker can be used to sterilize BRF substrate. Pressure cookers are also bulky, reasonably expensive and very difficult to locate for some (you can’t go pick up a cheap pressure cooker from the local big ‘Mart or thrift store in many other countries).


The thrust of this document is a sterilization process called Fractional Sterilization (hereafter FS). This process is also called Tyndallization (as it was discovered by J. Tyndall in the mid 1800’s). This is not a new idea/process. The Tyndallization process predates home pressure cookers by over 100 years.

The ideas that make FS/Tyndallization work are very easy to understand:

1. Virtually all living contaminants are killed by boiling/steaming temperatures. If it is alive/growing, 60 minutes exposure to steam temperatures will kill it.
2. Most endospores (contaminant “seeds”) are NOT killed by boiling/steaming temperatures. Four hours of steam (at regular pressure) will not kill some very common bacterial endospores.
3. Given time and proper conditions to “hatch” and begin growing, even the toughest endospore becomes a (relatively) delicate living microbe which can be destroyed by steam heat at regular atmospheric pressure.
4. A process that germinates endospores and then destroys the resulting living contaminants can actually be more effective than pressure cooking (some endospores can survive 60 minutes @ 15psi in a pressure cooker).

BRF will contain some endospore contaminants that can not be killed by boiling/steaming. In order to destroy these threats, the high temperature stable endospores must be allowed to germinate (begin growing). Once germinated, the living contaminants are far more sensitive to boiling temperatures and can be destroyed straight away.

Boiling/steaming temperatures can’t kill endospores, but once they germinate/hatch, boiling/steaming temperatures can easily destroy the resulting microbial life.


Please pardon the redundancy of those last two paragraphs, but I want to make sure you understand this process so you can have confidence in it working. Also, please remember, FS is used by professional culture labs when dealing with medias that don’t stand up well to high temp/high pressure sterilization procedures. Boiling is already well established as a preferred method of producing sterile liquid culture media (boiled sugars don’t carmelize and drop out of solution).


The actual process of using FS/Tyndallization to sterilize BRF is:

1. Prepare BRF jars and let them sit at room temperature for 24 hours.
2. Steam the jars for 60 minutes.
3. Wait 24 hours and steam the jars for 60 minutes.
4. Wait 24 hours and steam the jars for 60 minutes.

By the fourth day, all the nasties in the BRF have hatched and will be destroyed by the steam bath. A shorter 3 (or even 2) day process can work, but success rates will probably be lower. Shorter steam cycles (60 or even 30 minutes) can work, but success rates will be lower.


A 4 day process involving three 60 minute steam baths guarantees sterilized jars, even with the lowest quality/dirtiest starter rice. As will be detailed later, it is also possible to use this process (with 90 minute baths) to sterilize popcorn.


It is VERY important not to go longer than 24 hours between steam baths. If you go much longer, the nasties inside will become highly established (and harder to kill). Also, some bacteria will begin producing new endospores at around 30 hours. The 24 hours between steam treatments is critical to success. If you miss the steaming window by more than 4 hours, dump the jars and start again.

[BuckarooBanzai]

Abstract: Use Fractional Sterilization/Tyndallization to sterilize PF recipe BRF jars and popcorn jars. Inoculate half the jars with spores. Incubate all jars at 82F and observe for 10 days.

Test group (20 jars total – 10 BRF, 10 popcorn). Everything was done in ½ pint Ball-Mason jars using Tyvek vented lids and silicone injection ports. All inoculations were done with spore water previously tested and known to be contaminant free. All inoculation work was done in front of a flow hood.

• 5 BRF jars, standard PF 2-1-1 recipe
  1. Jars prepared 8/31/06 and left to sit overnight
  2. 60 minutes steamed 9/1/06
  3. 60 minutes steamed 9/2/06
  4. 60 minutes steamed 9/3/06

• 5 BRF jars, standard PF 2-1-1 recipe
  1. Jars prepared 8/31/06 and left to sit overnight
  2. 60 minutes steamed 9/1/06
  3. 60 minutes steamed 9/2/06
  4. 60 minutes steamed 9/3/06

• 5 popcorn jars, (2 with very little corn for Fahster clone jars)
  1. Popcorn hydrated, drained and put in a plastic bag for 24 hours
  2. 90 minutes steamed 9/2/06
  3. 90 minutes steamed 9/3/06
  4. 90 minutes steamed 9/4/06

• 5 popcorn jars, (2 with very little corn for Fahster clone jars)
  1. Popcorn hydrated, drained and put in a plastic bag for 24 hours
  2. 90 minutes steamed 9/2/06
  3. 90 minutes steamed 9/3/06
  4. 90 minutes steamed 9/4/06

The steaming of jars was done in a large soup tureen. An inverted aluminum pie plate was used to raise the jars off the bottom of the pot, allowing more water to be added and decreasing the chances of cracking a jar. I took pictures of this setup, but since everything is shiny aluminum, the pics aren’t really very much more enlightening than simple text.


The steaming process went as follows:

1. The soup tureen was filled with water (with jars in it), to a point where the water was about ½ way up the sides of the jars.
2. Jars were removed and tureen put on the stove over high heat, until a rolling boil was developed.
3. Heat was reduced to about 1/2, jars were put in and pot was watched until water started to boil again (water was not reheated to a rolling boil).
4. As soon as water started to boil, heat was reduced to a level already known to produce only a gentle simmer. Jars were NOT done over a continuous violent/rolling boil, just a gentle simmer.
5. Lid was placed on pot and timing of the steaming cycle was begun.
6. At the end of the cycle jars were removed (with lifting tongs to avoid burns) and set aside to cool for 24 hours.
7. This exact process was repeated on all jars. The only process change was steaming the popcorn jars for 90 minutes (instead of 60).

So, what were the success rates like you may ask? In a word, they were GREAT! 100% sterilization across the board (even in the popcorn, which was suprising).


Group 1 was 5 BRF jars incubated for 10 days @ 82F without being inoculated. Success rate was 100% with no visible contamination at 10 days. At 10 days, these jars were inoculated and are growing out now.


Group 2 was 5 BRF jars incubated for 10 days @ 82F with spore water inoculation. Success rate was 100% with no visible contamination at 10 days. These jars are very nearly finished.


Group 3 was 5 popcorn jars incubated for 10 days @ 82F without being inoculated. Success rate was 100% with no visible contamination at 10 days. These jars have been inoculated and are growing out.


Group 4 was 5 popcorn jars incubated 10 days @ 82F with spore water inoculation. Success rate was 100% with no visible contamination at 10 days. These jars have already been spawned to a bulk sub!


To push the popcorn test a bit farther, four of the popcorn jars were prepared with only a small amount (perhaps 4 tablespoons) of popcorn. After these jars were fully colonized, 60mL of sterile water was injected, the jars were shaken violently and 55mL-57mL of mycelia slurry was removed (this is Fahtster’s Tek, not mine). Each syringe of slurry was used to inoculate the popcorn layer of a Buckaroo Bulk nugget bag.


All four Bucky Bags colonized their popcorn layer cleanly! All four Fahtster clone jars recovered cleanly (and were used again – Fahtster’s Tek is really great for making mycelia slurry syringes) ! ! !


Not only was there no visible contamination in the popcorn, there were no contaminants at all. Had there been any latent bugs, the speed of mycelia on popcorn might have overtaken them without my notice.

By doing the Fahtster Tek, I am assured that things were clean inside the jar. All that sterile water would have washed any latent nasties off the corn and then deposited them for growth in the bulk nugget bag.

So, not surprisingly, FS proved to be highly effective with BRF substrate.


Somewhat surprisingly, FS proved to also be highly effective with popcorn.


It takes a lot longer and it is a good bit more finicky, but FS/Tyndallization can be used effectively by the home cultivator without access to a pressure cooker.

[roc]

Damn nice work there ole kid!

[Hippie3]

. If you miss the steaming window by more than 4 hours, dump the jars and start again.

how firm is that rule,
what success/fail rate say at 36 hours ?
couldn't one just add an extra episode
to hatch new endospores for another round of FS ?

[BuckarooBanzai]

The primary concern is that at about 28-30 hours, some common nasties are pretty well established and producing new endospores. The newly born spores are less likely to germinate in the next day or two (new endospores have a tendency to go dormant for a short time) and steam is only trustworthy against living contaminants.

At 36 hours, a fast growing bacteria could have done real damage inside the jar and I doubt any amount of regular pressure steam would be 100% effective.

Also, all the research I did on FS was procedurally pretty adamant about the 22-26 hour window.

Then again, only experiments can validate or invalidate a hypothesis. One might well be safe with two 30 minute steams 48 hours apart with a 48 hour pre steam germination period.

This was just an attempt at adapting the existing literature/work (and high success rates) to home cultivation. The fact that the popcorn worked seems like an excellent demonstration of the basic precept of 4 days/3 steams.

I was actually really amazed the popcorn all worked, having seen 60 minutes in the PC leave popcorn dirty before.

[ExecutionStyle]

Buckaroo, this thread is a HUGE help.
Thank you very much.

[golly]

This is good stuff Buck...A common lament for the new guys is the PC hurdle..A big investment for a young person to make when there is no guarantee it'll be fruitfull...
Everything stated,sounds logical ,I imagine that 2 steam cycles is plenty in most cases...
My question is - Did the popcorn show any sighns of mushyness or discolouration after the third steam...?

[BuckarooBanzai]

The "straight" popcorn still looks pretty much the same as when it came out of the double boiler. Still solid and yellow.

Even the jars that had the water squirted in and sucked back out (twice now) are pretty solid looking, though obviously they DO look a tad rough!

[Hippie3]

also see
http://forums.mycotopia.net/showthread.php?t=8227 (No Pressure Cooker Popcorn Success)

[tbonus]

very good thread,always appreciating the knowledge!

[Cryingblackoil]

Very straightforward, excellent writeup as always