|Anybody have experience with "oven Tek" grain innoculation?
|By onediadem (Onediadem) on Tuesday, February 12, 2002 - 10:51 pm: The Nook
Continued from: Pro's thread on Rye, Contams, Tek, Tips
I would try to construct a glove box. Also, didnt Ion say to shake, then open, and shake again before pc'ing? I also wouldnt put new foil on top of the jars unless it had been pc'd also. Perhaps your hepa isnt working as it should, and allowing the contams to stay in the room. The only contams I have ever had to deal with have been from work done outside of my glovebox. I also spray everything down inside the glovebox till dripping with lysol, and allow things to dry inside the glovebox with a filter patch attached for 24 hrs, then shoot with lids off of everything. Hasnt failed me yet. After pc'ing for 3 and 4 hours, I doubt its the grain, or the karo.
|By Ryan Waters (Zerogravity) on Tuesday, February 12, 2002 - 11:44 pm: The Nook
Thanks for joining Onediadem! I have enjoyed reading your posts in the past.
1. Because it looks like testing will be the norm from now on, they have just commenced work on a glove box! Looks like your thoughts are theirs as well! "Great minds think alike"? They are really getting tired of spending 2 hours cleaning the kitchen, which is the clean room, then waiting 1 1/2 hours to HEPA filter the room when all they want to do is a couple of jars! But they don't think this is the reason cause the room is absolutely sterile (See original post archived at the top of this thread) and while they are working in the room (which is sealed off and all movement is slow to reduce drafts) they are spraying the air with bleach solution every 10 mins. That being said they don't see how a glovebox would be practical considering the number of jars involved.
2. With regard to what Ion said on opening/shaking the inoc jars, they were unclear on that. If one opens/shakes prior to PC'ing then the additional oxygen will boil off while PC'ing...won't it? Someone said that there is a very gentle boil going on but over 45mins it seems as if this is long enough to eat a lot of the extra oxygen. But even if isn't...does anyone see any harm in shaking/opening after PC'ing? After all, the oxygen is being added in a sterile environment and then the jar will be placed in the incubator. Would be nice to have Ion's take on this.
3. Good call on the foil Onediadem! Tough call though. Isn't the foil sterile on the roll? For the first batch of inoc the foil was rolled off while the box was in the Clean Room. The box however had been sprayed with bleach. For the 2nd & 3rd batches of inoc the foil was cut into the pieces prior to the clean room then these pieces were sprayed with bleach on both sides while in the Clean Room. Besides the foil is on top of polyfill which filters between 3-7 microns. They are thinking that to get trich in 100% of the jars on 3 different batches the problem is probably bigger than the foil. At the same time you raise an excellent suggestion that is worth noting. Thanks.
4. The HEPA filters are essentially new so are therefore in great shape.
5. But you got them thinking. When they were emptying the 3 batches of contamed grain the HEPA was on right next to where the work was taking place. This was literally hours. Is it possible that trich is living in the HEPA machine? This, however, would not explain why the first batch of grain failed as no trich had been exposed to the machine. Thanks Onediadem, again you raise a good point. So pros...can trich live in a HEPA machine?
Thanks again Onediadem
|By Karna (Karna) on Wednesday, February 13, 2002 - 12:22 am: The Nook
Here is my 2 cents for what it's worth
Firstly I think you need to cut your batch size by a factor of either 1/10, 1/15 or 1/18 so you have a batch that is not so unwieldy.
Secondly make sure you use ORGANIC rye grain ONLY. Nothing else will work.
Eliminate the Karo step so you don't have to worry about the culture being good or not.
Use the oven tech which in my experience works as well as a glove box w/ the added advantage that you don't have to build it.
Use relic's rye tek or Karna's rye grain tek from the archives to prepare your grain
Keep all your other procedures in place.
From your posts, what I'm getting is either
a) encysted spores which the precooking step should eliminate
b) Karo culture had improper ratio of sugar or was somehow compromised - eliminating the Karo will also of course eliminate this.
c) Spores themselves. But you ordered a new syringe apparently and that SHOULD take care of it although if I were you and I wanted to be 100% sure, I'd order from PF.
d) Improper technique used during inoculation or incubation environment is somehow compromised. The oven tek will take care of the first, just make sure your hands are alcohol doused at all times. I'm sure you can figure a way to get at the second.
If this does not work, my recommendation would be to try a different source for grain, buy a PF amazonian and give it another whirl
I advise you to stick with it though since once you hit it, rye is a great tool to have in your box. I had similar problems when I started out with the stuff. Now I have no problems after I figured my mistakes out a few years ago. Now batches and even exponential expansions in an environment that is not anywhere near as clean as yours using the exact same tek I detailed in the archive, have virtually zero failure rate, so keep it going.
|By Ryan Waters (Zerogravity) on Wednesday, February 13, 2002 - 01:17 am: The Nook
Karna...cool...glad to have your help...your input is worth more than 2 cents to my FOAF! I have enjoyed your previous posts as well.
- You are correct in recomending a smaller batch size for testing purposes. Get it right then make enough to fill the cases. They would have saved many hours and lots of money had they done that. Sounds like experience talking.
- And "Yes", my FOAF has only been using organic rye.
- "Eliminate the Karo step so you don't have to worry about the culture being good or not." Excellent advice for testing purposes. Will do, but need to wait till the spores arrive. Will keep everyone posted as to the results. But when knocking up the grain that will definitely go in the cases my FOAF is thinking that would cost too much to buy a syringe for every jar ($750)
- Your advice to follow the grain teks is wise. To the best of their ability they are indeed using Relic's & your tek re preparing the grain. They have read them many times and learned a lot. As they do not see how they are diviating from these excellent teks this is a mute point. Please inform if they are mistaken.
- "encysted spores which the precooking step should eliminate" They were under the understanding that one either soaked in water for 48 hours or boiled. (PC'ing being the next step & mandatory). With the number of jars involved it is a lot less time & effort to just let them soak. They also read about a lot of people having boiling problems. However it is your tek and you know the most about it, so please set the record straight. If the grain must be preboiled then that is exactly what will happen...just they hope not cause that will add a couple of days to the procedure.
- Your next point is valid and one that they are waiting for a response from Nan on. How could changing the water/dextrose ratio invite Trich? The grow time was much quicker but at the same time newbie's must continually remind themselves to be patient. Note however that even before the Karo Tek was altered Trich reared its ugly head.
- They too feel it is the spores but even this seems to be a long shot.
- Want very much to order from PF, but as my FOAF resides outside of the USA they don't want an envelope reading "mushroom spores" on it to be delivered. Will do it if they have to but will have the spore babies sent to a PO box if Fanaticus doesn't object. (Am currently waiting for a reply to the thread I started re this very point.)
- Karna can you see anything wrong with their sterile technique? You are no doubt very busy but I am thinking that your advice would not only help my FOAF but other newbies as well. I have been extremely thorough in my description of their procedures...probably to the point that some here could say I have "joy fingers"!
- Good call on trying to find another grain supplier. Currently doing so. Relic & Karna..do you know what brand of rye your FOAF is using? My FOAF is using Nunweilers...might try Rogers...those are the only 2 available here.
- Thanks for the encouragement Karna. When I said that my FOAF isn't discouraged...I was lying a little
Wow this is cool...so many veterans helping a newbie!
|By quote: (Quote) on Wednesday, February 13, 2002 - 01:58 am: The Nook
please don't mislead folks by stating that the oven door tek is as good as a glovebox,
it simply is not true,
and causes many newbies grief.
this nonsense should stop here.
oven door tek sux,
it is flawed in theory and reality.
it is totally unsuited for any serious sterile work such as liquid inoculants.
|By Nan (Nanook) on Wednesday, February 13, 2002 - 02:06 am: The Nook
That is the flag I am going to post in the Tek... And I am going to link it to the thread Brettie started on the subject already in the Archive.
Sounds like a plan to me. The box posted by Scottsman even is better than a drafty hot oven... Makes me think some people are just a bit lazy and would rather spend time writing to defend the oven as opposed to the 30 minutes it takes to rig a cheap effective box.
But who am I to say...
|By relic (Relic) on Wednesday, February 13, 2002 - 09:08 am: The Nook
when i asked if you had done the karo tek successfully, i meant had grown out brf or other jars with karo inoculant
you don't really need a glovebox for grain inoculation, only the karo preperation and karo syringe prep.
|By Vitti (Vitticeps) on Wednesday, February 13, 2002 - 09:12 am: The Nook
Then there are those who are/were in the position I was Nan. I had to keep everything secret and hiding the grow chamber and supplies was hard enough, hiding a glovebox too would have been quite difficult for me.
|By Nan (Nanook) on Wednesday, February 13, 2002 - 09:36 am: The Nook
Somebody needs to come up with some EZ plans for a collaspable glove box... Velcro strips on the corners or something... Assemble in 5 min, dissassemble and store under the bed.
|By quote: (Quote) on Wednesday, February 13, 2002 - 02:02 pm: The Nook
has anyone checked out workman's cardboard box-glovebox ?
totally disposable, can be folded down for storage, etc.
it's in the archives....
|By Vitti (Vitticeps) on Wednesday, February 13, 2002 - 07:31 pm: The Nook
A friend of mine who is into paintball stuff once suggested to me that you could use a small, paintball gun style, co2 bottle with the regulator opened slightly to create filtration free positive pressure inside a glovebox. He said they are cheap to refill but didn't define cheap. I really know very little about them but it sounded like it would work well.
On that same note:
Anyone know the effects of compressing air on contaminants in the air? If you compress it to 50 or 60 psi, would that kill any potential contams? I doubt it but it seems that it could, that's an awful lot of pressure for a tiny little organism. Would be nice if you could just use compressed air for positive pressure.
|By onediadem (Onediadem) on Wednesday, February 13, 2002 - 09:29 pm: The Nook
I think people have just been lucky with the oven tek. I used it one time, and was so paranoid about contams, that I built a glovebox, so I knew that the contams couldnt be from enviromental factors. I have never had 1 contam from sterile work done in one. I have however had serious contam issues before I got my computer and found
Nan. My contams before that always came with my procedures after full grain colonizations, mostly mixing grain with other substrates. Since that time, I have only had to toss 2 cakes after 2 flushes from contams. No contams with grain to date. A glovebox, whether it be hard copy or disposable is one item that is indispensable as far as I am concerned. They are extremely cheap compared to costs of tossing green items. one that can be folded would for sure be a cure for those who have security issues.
Just my 2cents for what its worth.
|By Imok Urok2 (Imok) on Thursday, February 14, 2002 - 05:39 am: The Nook
Y'all ever see those cardboard boxes they sell at the office store
for long term financial record storage?
They come in different sizes and can be folded flat after
having been put together and used.
I store personal items that way.
Some of the space in the boxes is wasted that way but I can stack
them vertical and they can go six to eight high, depending on
the weight of the contents.
Saran wrap and glove holes in one of those would probably work.
Then you could fold it back down when you are thru with it.
Cost is less than $15 for three of them.
|By Ryan Waters (Zerogravity) on Thursday, February 14, 2002 - 08:06 pm: The Nook
Wow! Lots of good input here for a thread on gloveboxes! (My FOAF is going to convert an acquarium as foldability is not a prerequisite.)
Sorry to repeat myself but does anyone know if...
Trich can grow in a HEPA machine and infect a clean room?
|By Ryan Waters (Zerogravity) on Thursday, February 14, 2002 - 09:40 pm: The Nook
Quote your thoughts were well written in Hatcher's thread on dextrose. Could my FOAF ask you a question on how to implement your advice? Please do not interpret their question as making light of your advice...they just need help in implementing it. I post this here and not in the original thread as this may or my not be the answer to this thread.
"the problem with quickly venting the steam, aside from the safety issues, is that what happens is-
since the media, be it liquid or solid, is trapped in glass jars, it remains hot longer than the rest of the cooker.
that heat causes the air inside the jars to expand, thus lowering it's density & pressure relative to air outside the jars.
then, as the jars cool, they suck in air from the surroundings, potentialy contaminating.
thus, it's best to leave the pc sealed and minimize the risk until it's all cooled down to room temp."
It is obvious that lifting the weighted scale while sealed inoc jars are under pressure would be dangerous and would also cause the inoc to consume oxygen thereby limiting their life. My FOAF has never done that but knows that they could explode. But this question is not about inoc but about vented rye grain jars.
1.) Do I understand correctly that every poly-fill vented tin foil wrapped rye grain jar must reach room temperature on it's own? Specifically, this would mean not lifting the weighted scale while under pressure, and not removing the PC lid when there is no pressure so that the hot jars could be placed on the counter. Once the jars are on the counter cooling, another batch of jars can commence.
You correctly reason that as the jars cool down they will suck in air. But the vented jars loose pressure at the same rate as the PC. When they are sucking in air it is through the 2 layers of foil and a very tight poly filled 1/4" hole in lid. These holes are supposed to filter between 3-7 microns.
2.)Will this contaminate the rye? Is this the answer to this thread?
To leave the PC alone and let it cool down to room temp would take from beginning to end roughly 14 hours. This PC'ing could only be during the day because of the noise PC's make. At this rate, even with two - 22 qt PC's, it would take 11 days on the stove.
The reason my FOAF choose the rye grain/casing tek was because it is a bulk method.
3.) So how does someone bulk PC rye? Or is the answer that time is the price that needs to be paid? Please note that at no point has my FOAF not applied any of your advice because it was either too much money, time, or effort.
One thing that can be said that out of 190 rye jars placed in the PC and the pressure vented quickly is that not one cracked or exploded. So either my FOAF was lucky or this is not dangerous. When Mirro, the manufacturer of the PC, was called re lifting the weighted scale under pressure, they said it would not hurt the PC but to do it carefully so as to not burn the fingers. They also said pouring cold water on the lid while under pressure would be OK.
My question, however, is whether or not jars will contam by sucking in room air. As I noted somewhere above, as a test they PC'd 16 jars of rye, lifted the weighted scale prematurely, and let them sit on the counter for over a month. None contamed. As you can see my FOAF is so confused. What has all your experience taught you?
In the meantime my FOAF carried out your suggestion on conducting different tests to isolate the cause. As well they are trying some of the above suggestions from posters above. Just waiting for the results.
|By quote: (Quote) on Thursday, February 14, 2002 - 11:14 pm: The Nook
actually, items such as the rye grains trapped inside glass will cool slower than the surrounding pot, as glass is a poor conductor of ir [heat] radiation, while metal [the pot] is not.
as for the polyfil, you are correct in thinking that it should eliminate much of the risk contam-wise, as it should filter any air sucked in.
and properly done polyfil plugs should be fine, but if it leaks any at all, the jar's screwed.
and to go truly bulk,
you want to spawn your rye onto compost/manure/straw, thus multiplying your mass without using more grain spawn, so you need fewer jars to get same yield.
|By Ryan Waters (Zerogravity) on Friday, February 15, 2002 - 12:41 am: The Nook
Thanks for the quick response Quote.
Because my FOAF got 100% failure 3 times, I can now see that a couple of jars with poor polyfilling would not be the answer to this thread.
Not ready to take the plunge into a real bulk tek though. We'll see what happens when they get the tek they are working on now down pat first.
Any thoughts on a HEPA machine spreading Trich in a clean room?
Thanks everyone for bearing with all these questions!
|By Karna (Karna) on Friday, February 15, 2002 - 01:33 am: The Nook
Yes I presimmer for about 50 min to an hour (the water is JUST barely simmering)
Sorry, didn't realize you were outside the states.
I don't see anything wrong with your sterile technique. With large batch sizes, a concern that is always present is that the stuff sits out for a while before getting inoculated which gives more time for contams.
As far as the oven door tek, ya I read that thread that Brettie started. I felt the initial tone discouraged participation. For an intelligent person, PF certainly says some rather silly things like the scientology analogy which was funny, but lame. He makes some good points, but here's the thing, I really doubt he's ever tried it or could even bring himself to surmount his all-encompassing ego and give it a shot. His conclusion is that everybody must be using PF jars because the verm layer prevents contams. This is not so. Rye jars work fine, even rice and brf w/ no verm layer. I don't know if you bother to read them, I certainly would not considering their length, but I did post a tek to draw liquid cultures into syringes using the oven tek. I have used this for almost four years without having a single contam, so it has worked with liquid cultures, consistently. I have also detailed a bulk tek here (in Chronic's thread maybe) that actually involves leaving the grain exposed at numerous points during the process. Once the grain to water ratio was worked out, once again, not a single contam. Before that, only wet spot.
That's not to say, Quote that you are not right, you could be 100% right since I don't use a glovebox. Just our experiences with the oven tek appear to be divergent, that's all. In your experience, sometimes it works, sometimes it doesn't. In mine, it has always worked. Maybe a glovebox is far easier to use and safer and everything else. It does not, however fit into my way of doing things. Too much effort, too indiscrete, too much Lysol inhalation etc. I like the warm air.
As for advising or not advising people to use it, I can only advise people to use what has worked in my attempts. I'm sure the caveat of "this has worked well for me, but hey, it might not work well for you" is an unwritten acknowledgement in all these posts. So while you are obviously free to criticize or recommend against it, I still recommend it.
My sincerest apologies to you Ryan if I exacerbated your already grave situation by recommending you use a tek that always works for me but is actually nonsense, sucks, is flawed inside and out, is totally unbecoming of any self-respecting mycophile, and just no bloody good.
|By quote: (Quote) on Friday, February 15, 2002 - 03:16 am: The Nook
karna, you've been around a long time,
and have been a great member of this community,
so i hope you don't take this offensively.
but surely you must admit that others have failed miserably with the oven door tek,
despite your successes.
you don't see biologists working in front of an oven when they make cultures, right ?
why on earth would you tell people that it's better than a glovebox ?
or are you not claiming that ?
which is better ?
oven or box ?
the vast majority consensus is that while ovens can work for some people some times,
it is better to use a glovebox.
and you must realize my responsibility,
and this board's responsibility,
to steer folks on the path of most likely success.
such should also be your concern,
regardless of the way you personally chose to do things.
you should still recommend the better way,
and not tell people to ignore us when we tell them to build a glovebox, and to use the oven instead like you do.
that is, in my opinion, not in the best interest of either the newbie or the community as a whole.
so if you disagree, that's fine.
but you really should keep it to yourself,
and please don't preach the gospel of the oven door here.
it just confuses the issue as we try to teach them to build gloveboxes and someone else says there's no need, you can be lazy and just use an oven.
|By Imok Urok2 (Imok) on Friday, February 15, 2002 - 04:07 am: The Nook
If your filter is contamed it can spread that bacteria in the
room it is running in.
Same way with cartridge water filters.
You need a way to either replace the HEPA filter or clean it to
prevent cultures from growing in the media.
Also, the particles taken in by the filter clog it up and
reduce its effectiveness.
Hope this helps
|By Nan (Nanook) on Friday, February 15, 2002 - 04:21 am: The Nook
Sigh... The reason Karna gets away with it is the detail of the Tek Karna uses... But there are simply so many variables. Anything that reduces varibles helps the teaching process in this environment.
Gloveboxes are cheap and easy to make. They are reliably effective, even the saggy cardboard ones.
To be honest, I would remove Oven Tek from the archives... But it is entrenched in too many Teks to be omitted. But flag it I will, and the thread Brettie started is already down there. My instincts are dead in line with Quo on this.
We have an obligation to strive for standards of success. My personal mission is to promote Tek that produces the greatest efficiency at the lowest possible cost.
But certain tools are required to be consistantly sucessful at the intermediate and advanced levels. The two most critical are a Pressure Cooker and a Glove Box. That's what separates the independant grower from the wannabes.
|By Ryan Waters (Zerogravity) on Friday, February 15, 2002 - 08:16 pm: The Nook
Imok...thanks for joining! Hope my FOAF can be of assistance to you in the future.
Well Honeywell, the manufacturer of the HEPA machine, says that while some HEPA filters can be washed & reused that is not the type in this machine. (Saw that coming.) Apparently the reusable type are rare and expensive. Probably used for a commercial lab. The reusable type are not made with paper.
Tough decision time because the filters required are $50!
The machine was exposed to Trich for a number of hours when the contamed jars were emptied right next to it.
- Is there anyone else that would like to comment on how great the need to replace is? Someone that has some knowledge of lab procedures would be great!
- What about purchasing the reusable type filter and inserting that into the unit? Where can this be found?
Thanks again Imok
|By Karna (Karna) on Monday, February 18, 2002 - 12:48 am: The Nook
You are right, quo. The gospel of the oven door shall be buried! I didn't think all these people had failed with it. Why is it that it works for me I wonder? As Nan said, maybe it's the way I use it or something. I though that's how everyone used it, obviously not. I should look into this more.
The reason that I recommended it for Ryan was mainly because I didn't see any way 190 jars would be accomodated in a glove box set up. Not that they would fit it the oven door style, but I felt there was more chance of accomodating large batches.
No I don't feel that a glovebox is bad, it's obviously very good. Just my circumstances prevent the building of one appropriate to my needs.
Really, OT's flow hood would be the way to go for me, but for the same reasons, that's out too, so all I have is the bloody oven. I'm quite happy with it, but as you say, those are not the right reasons to be trying to influence others into doing the same.
So for all y'all newbies, listen up, IM A LAZY MOFO. THATS THE TRUTH. ALWAYS HAVE BEEN. SO DON'T DO AS I DO, DO AS HIP AND NAN SAY AND LEARN TO BUILD A GOOD GLOVE BOX, Y"HEAR??!
Peace. And Ryan you green growing goblin you, I hope the trich days are slowly becoming just badly infected memories, yes?