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Mycotopia Web Archive Archive Substrate: Shroom Food Grain Teks Karna's Rye Grain Tek Need a pro's help with grain spawn & trich problem Previous Next

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Ryan Waters (Zerogravity)
Posted on Thursday, January 17, 2002 - 08:14 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

We have a FOAF that is looking for someone experienced in rye grain spawn and how to prevent trich (green mold). They have already looked in the archives for answers. Apologies if a question is asked for which an answer has already been posted.

Please excuse the following "book" but if you know all the relevant details then it will be easier for you to isolate their problem(s).

Please note that two previous Karo Tek's were conducted using the same technique as below and no Trich developed.

They knocked up 90 - 1 1/2 litre organic rye grain jars after a successful Karo Tek with B+ & Matias. By successful they mean no contams after 14 days. Within 2-3 days around 80% of the jars contained Trich. In fact there was more Trich than Mycelium.

Here is what they did:
- Soaked grain for 64 hrs; stirred occassionaly with unsterilized hands.

- Rinsed for 1/2 hour. Let stand for a couple of hours then rinsed again in small quantities for 1/2 hour.

- Let stand for 15 min to ensure all water drained

- Put grain in unsterilized 1 1/2 litre jars 2/3 rd's full. This is 1000ML of grain. Used bare unsterilized hands to fill jars. Wrapped tightly with 2 layers of foil. Lids have fiber filled holes stuffed very tight.

- PC'd @ 15lbs for 30 mins. Prior to putting on weighted scale vented for 5 mins.

- Removed jars. Absolutely no clumping. Looked dry but as this is their first time doing this they don't know what it should look like. Placed jars on counter overnight to cool down to room temp. This counter is up against a bed which is assumed to be a haven for Trich. Unfortunately jars were removed with unsterilized oven mits that have never even been cleaned.

- Created a clean room in kitchen. This involved 20% bleach solution on floors, walls, cabinets, switches & ceiling. Thoroughly cleaned range & range hood. Removed everything not bolted down. Enclosed the small kitchen in Poly and let HEPA filter run for 1 hour before they entered. Unplugged fridge as it creates a draft when it runs.

- Physical preparation included full-body disposable suits with hoods. Face mask & rubber gloves. Sprayed themselves down with lysol especially at their feet & head.

- Once in the clean room they turned off HEPA filter to prevent drafts. They moved slowly and were in the room for almost 3 hours. Sprayed the air with water frequently. Unfortunately the lids were sometimes opened too soon and this watered air got in. Once an hour also sprayed the air with a 20% bleach solution. All jars were wipped down with rubbing alcohol and sprayed liberally with lysol. The person actually knocking up the jars did only one task - touched only the needle & the lid on either the innoculation jar or grain jar - nothing else. The other person did everything else. Instead of inserting the needle thru the fiber-filled hole they cracked lid wide open. The needles were sterilized (boiled for 45 mins) and if it touched anything it was re-sterilized with rubbing alcohol. 15CC of innoculant was squirted in across the whole top layer as well as down the sides. The innoculant had been in the fridge in a vacuum sealed bag that was also wrapped in 2 bags. After injecting, the jars were shaked vigorously. Two new layers of foil were applied. Hands were cleaned frequently with rubbing alcohol.

- Jars were placed immediately in incubation chamber and not touched for 3 days. This cabinet has a HEPA filter.

-Once the contam was discovered all grain was discarded, jars sterilized, and the room incubation chamber sterilized.

They were confident they were doing everything right but to have this big a problem in such a short period of time indicates something is grossly wrong.

So what did they do wrong?

Also, they are looking for answers to each of the following questions:
1. Soaking grain. How much time is too long? (In other words can it contam sitting too long?)

2. Soaking grain. Does everything from the soaking bins to the PC need to be sterilized?

3. Rinsing. Can you do too much?

4. Draining. The grain after PC'ing and letting stand overnight looked real dry. They feel too much water is either PC'ing or evaporating out.
Do you want ALL the water out or is some in the jar good? How wet should it look?

5. Is boiling the grain after soaking redundant?

6. PC time. Is 30 min long enough for the large 1 1/2 litre jars? Can it be cooked too long?

7. When knocking up should the HEPA filter be on or should it be off?

8. Bleach residue. If you clean something that was contamed with Trich but don't clean off the bleach residue is the trich still present?

9. Dry grain. Can the grain jars be re-hydrated after being in the incubation chamber for a while if they look too dry?

10. Is Trich killed by PC'ing?

Once again, thanks in advance for your help. They could not have gotten this far without all the help that has already been extended. They hope to return the favor someday when they too are pro's.
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relic (Relic)
Posted on Thursday, January 17, 2002 - 09:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

your problem is very simple, 30 minutes is not nearly long enough to sterilize rye. alot of people say 60 min @ 15 psi, i always do mine for at least 75-90 min. and of course you start timing after the proper pressure is attained.

touching the grain and all that stuff prior to sterilization is absolutely fine.

i preboil/simmer(bring to boil, then lower heat to lowest setting) the grain for no more than 45 min, stirring often and keeping the water level in the pot above the grain by 1/4" or so, adding as needed. then strain the excess water off real good in a collindar and then load the jars 2/3 full. p.c. for 75-90 min. @ 15 psi, then let sit in the p.c. overnight. shake the jars before inoculating in the morning or when it has cooled.

that's a real bummer, 90 jars
i feel for your friend. btw, i wouldn't apply new foil and 1 layer is really enough.

now to answer your questions

1. i don't think you can soak it too long, within reason.

2. no

3. no

4. it shouldn't look too wet after p.c.'ing. if you soaked for 64 hrs i'd say they were fine(keep in mind, i'm not a soaker). the grain can sometimes look too dry when it really isn't.

5. probably yes

6. definately no and yes you could cook it too long though i've known of people who have regularly p.c.'d rye for 2 hrs w/ success. the big factor here is not to let the p.c. go dry. if it does, that's when things go bad.

7. off, unless it's a flowhood you're talking about.

8. no(if it is, it's dead), though bleach residue can not be a good thing to have in your jars wash them babies good w/ dishsoap and rinse them good with hot water before using them.

9. maybe, i dunno. i've never had a grain jar go dry, but then i don't have an incubator either.

10. yes
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Ryan Waters (Zerogravity)
Posted on Thursday, January 17, 2002 - 07:43 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Relic your quick, thorough, & accurate response means a lot. Thanks!

1. So you feel that, even though the large 1.5 litre jars are being used, 90 min in the PC will suffice?

2. Did you recommend not replacing the foil because it is a bad thing to do or merely because it is redundant? The reason new foil is added after knocking up is that it tears when removing it (it was really snug fitting).

3. They are still unclear on how wet the rye grain should look in the jar after PC'ing and while in the incubation chamber. When the jars were opened to innoculate, some of the grains looked very dry. In fact, there was little to no wetness on the jar's walls. It is understood that on the inside of the grain it is fully hydrated. The fiber-filled holes are stuffed tightly and are 5/16 in diameter. Once again they have never seen what it should look like so maybe they are making a mountain out of a molehill. If anyone has any pics that would be extremely helpful. (Too bad they don't have a digital camera so they could post it.)

- What happens if the grain is too wet after PC'ing?

- How fully do you drain?

- What should the glass walls look like after PC'ing?

- What about perlite in the incubation chamber?

- Do you, or anyone else, have any additional thoughts on re-hydrating the grain while it is incubating if required?
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relic (Relic)
Posted on Friday, January 18, 2002 - 04:54 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

yeah, 90 min. will do

after you get them out of the p.c., the foil is basically left on as a second line of defense. to protect agaignst falling particals onto the polyfil. i just think it's redundant to put on two layers then replace them with two new layers after inoc.

i will post some pics below to try and show the grain after p.c.'ing. notice in the pics that there is no condensation in the jars.

too wet will cause poor or incomplete colonization and can also contribute to bacterial contamination.

i put the grain into a screen type strainer and shake it up and down until no water comes out the bottom. i load the jars while the grain is still warm/hot, and when you do this you will see immediate condesation in the jars b/c of the warmth of the grain. this usually goes away. after the jars are p.c.'d and cool down. you may see condensation again when the jars are fully colonized much as you do with pftek brf jars.

depends on the temps in the chamber i guess, prolly can't hurt to have perlite in there. i just put my jars back in the box and put them away at room temps. this, of course, slows things down by a few days.

as far as rehydrating goes, i'd try it on one jar and see how it goes. though it really shouldn't be needed. use sterile water of course.

hope i helped.
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relic (Relic)
Posted on Friday, January 18, 2002 - 05:13 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

ryejar1
few popped grains, oh well

ryejar2
notice, no condensation

ryejar3
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relic (Relic)
Posted on Friday, January 18, 2002 - 05:17 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

ryejar4
same jar as above, other side. cambo cubensis, karo injected 3 days ago.

ryejar5

ryejar6
this jar is prolly ready to be shaken, after that it will completely colonize within a few more days. i usually then shake it up one more time and wait a couple more days.
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Ryan Waters (Zerogravity)
Posted on Sunday, January 27, 2002 - 02:32 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Although I find it hard to believe, my FOAF has again grown Trich. After just 2 days in the incubator green mold is visible in many jars. Too bad Trich doesn't have any market value cause he would be rich by now!

Relic they followed your advice by PC'ing the rye grain in the 1.5 litre jars for 90 mins. (There goes 4 days!) Further, in order to isolate the cause of the original failure, they prepared the second batch exactly the same way as the first batch.

They list the possibly suspicious factors common in both batches as the grain, PC duration, and inoculant. After re-reading the start of this thread, I believe there is nothing they can do to improve their sterile technique. It is for this reason that they do not suspect this. By all means, however, if anyone sees anything that they could do better in terms of sterile technique then they are all ears. Also rulled out is the incubation cabinet because the Trich is uniformly spread out in the jar from top to bottom and the cabinet was sanitized thoroughly just before the jars were placed in it.

They are forced to ask this community questions they feel are ridiculous but they are truly at a dead end street and can go no further without all you guys help.

1. Is Trich visible in liquid inoculant? This is their biggest question. They used the same inoc on both batches. They have studied the liquid as best they can and can see no contams. The myc looks perfect...healthy and lots of it. This inoc is between 3-4 weeks old. That being said, as soon as the first batch of inoc had stopped growing,it was used to knock-up the grain as well as to generate more additional inoculant. They feel that the inoc is the reason for their failure because about 3-4 weeks ago they PC'd some of the same grain for only 30 minutes (albiet it was in only 1/2 pint jars)and didn't inoculate them. Although those two jars have been sitting on the counter all this time there is no contam to report. There is no fiber filled hole, one layer of foil and even that is ripped!

2. Is Trich visible in spore syringes? Having followed everything they have read, they are wondering if the problem started with the original spore syringes.

3. Is it possible that they have "nuclear grade" Trich on the grain that isn't being killed in the 1.5 litre jars after 90 mins in the PC? In other words, are some strains of Trich not destroyed by PC'ing?

4. Are there any other contams out there that are green that this could be?

5. Could there possibly be something wrong with the grain? (Hard to believe this could be the cause when it is considered that those small 1/2 pint jars used the same stuff & have been sitting there without contams for a month now.)

6. Although it has been said that 90 mins is long enough...is it? They ask cause they were told that pc'ing destroys Trich but only when you take into consideration the size of the jar. In other words it is assumed you could PC for less time if the jar was say only a half pint and not 1.5 litres. Please Relic they mean no disrespect by re-asking a question that you were good enough to answer already

Here is what they plan to do.

- Determine if it is the inoc. They are going to use it to knock-up some of the sterilized grain that has been sitting there without contams for 3-4 weeks. If they see Trich within 3 days then that is probably the problem!

- PC some more grain for longer. They are going to PC 4 jars for 2 hours, then 4 jars for 3 hours, then 4 jars for 4 hours. Then they are going to shoot up 6 of them with the inoc. If they still get Trich then it can be assumed it has nothing to do with PC'ing or the grain.

The results of these two tests will be known by this coming Thursday. In the meantime, Hippie, Nan, Relic, Ion, and other pros please read the first post on this thread and see if you can come up with what the cause of this problem could be.

Oh and just in case anyone wants to buy a case load of Trich just let me know...real low price!
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relic (Relic)
Posted on Sunday, January 27, 2002 - 05:05 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

as you said, there are 3 possible problems. i'd get a known "good" syringe of spores, and try to inoc another grain jar prepared in the same manner. if it works, you'll know it was the other inoculant. if it fails you will know it was the grain(assuming it has the proper water content). are you flame sterilizing the needle before incoulating? i get mine to glow then squirt a shot through to cool, then inoc.
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Ryan Waters (Zerogravity)
Posted on Sunday, January 27, 2002 - 06:41 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Hey Relic...good to hear from ya again.

No they do not flame sterilize the needle prior to shooting each jar. The needle never comes into contact with anything but air (which is in an enclosed clean room that has been HEPA filtered. BTW the filter in the unit is brand new as well.) If it does come into contact with anything then it is cleaned with rubbing alcohol.

What are your thoughts...does it need to be flamed before each jar? This will be important to them considering the number of jars.

Thanks again Relic
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hippie3 (Hippie)
Posted on Sunday, January 27, 2002 - 03:23 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

not really, flaming's only needed if the needle touches something dirty.
i'd sat the odds favor it being the inoculant causing problems.
you cannot see trich in syringes.how did you make your syringes ?
you should always test homemade syringes, i use a jar of pf substrate, and test each syringe by firing into 1 hole. if contams grow, i pitch it.
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Ryan Waters (Zerogravity)
Posted on Sunday, January 27, 2002 - 08:44 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Hippie...thanks again for your response.

Your thoughts are mine...they suspect the inoc.

They purchased the syringes from a reputable supplier.

(They would rather not mention them by name because they recognize that whenever there are growing problems between a newbie and a veteran, it is generally the fault of the newbie. The last thing a newbie needs is to point an inexperienced finger only to burn a bridge. If it is discovered to be the fault of the syringe supplier they would only take it up with that individual. They would however, for the benefit of this thread, mention that that was the problem but, unless you feel it wise for mycotopia, not mention the supplier by name.)

Hippie...

1. Is Trich visible in inoc? If so what does it look like? Besides visual clues are there any other ways to detect it? They recognize that any tests would probably render it useless for knocking-up the grain but would provide answers for future reference.

2. Is there such a thing as trich that can not be destroyed by 90 min of PC'ing?

3. Are there other contams that are green besides Trich?

4. Could there be something wrong with the grain?

Please excuse their troubling you with the same questions twice.

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Ryan Waters (Zerogravity)
Posted on Monday, January 28, 2002 - 06:34 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Anyone...

1. Is Trich visible in inoc? If so what does it look like?

2. Is there such a thing as trich that can not be destroyed by 90 min of PC'ing?

3. Are there other contams that are green besides Trich?

4. Could there be something wrong with the grain?
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hippie3 (Hippie)
Posted on Tuesday, January 29, 2002 - 02:35 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

1. no
2. possible, some endospores are very tough.
3. yes
4. yes, and that's the most likely culprit, assuming your syringes were good.
i'd find a new source.
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Eatyualive (Eatyualive)
Posted on Wednesday, January 30, 2002 - 02:16 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Damn this is a detailed post. Many, many questions. but thanks for the good response guys! i always have a bitch of a time with rye. it seems that even when my jars fully colonize ( sometimes less than a week) i always have the mycelia stall out and stop growing near the end after the last shake. i have had success with koh samui. i actually had 5 flushes and then it contamed but it was hell gettin there.