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lastlokean
Stranger

Registered: 07/30/07
Posts: 106
Last seen: 11 years, 10 months
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LC always fails, agar to grain works fine WTF?
#13017272 - 08/07/10 01:51 PM (14 years, 5 months ago) |
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So... awhile back the green meanies hit hard, failing multiple monotubs!
Sense then the decision to return to a martha for smaller controlled cakes was made. Sense then success has been limited, with a seemingly endless trich problem.
Currently spores -> Agar -> Agar -> Agar -> LC -> Grain -> Bulk is being utilized to get clean healthy culture. (In theory)
With about a 90% contaminate rate using good sterile procedures in a glove box. Originally the blame was cast onto sterile process. And now it is way overkill, and only FRESH brand new syringes jars and all equipment are used. (Just out of extreme paranoia)
After an entire month of failures a change was made to straight agar transfers to grain. This has found a much higher success rate close to 100%.
Whats interesting is the LC would look fine, test fine on agar and look fine when colonized on grain about half the time and green the other half. Any of the grain that looked fine would contam instantly when spawned to bulk.
Years prior have had no problems with LC and I can't seem to figure out whats wrong with the LC.
Using sugar based LCs at anywhere from 2-4% solution of turbinado or honey using distilled water. They are PC'd for half an hour to 45 minutes at 15 psi. Always wrapped in foil, sometimes removed while still warm for filter to dry fast, sometimes let cool in the PC for 24 hours. (Not sure which is 'correct')
For a LC lid two holes are drilled. One for 2 pieces of micropore tape and one for a silicon injection port. The holes are always sanded down very well till smooth.
Anyone figure why the LCs would consistently FAIL?
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Recipe:
How a Lokean makes chocolates
For a fully enclosed gh with a coolmist piped in, one may find that adding one of these to the opposite end as the intake will seriously help reduce pressure buildup and strain on the poor weak coolmist motor, while also dramatically improving fae without cutting polyfill stuffed slits all over the damn thing. (That looks suspicious )
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,387
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Re: LC always fails, agar to grain works fine WTF? [Re: lastlokean]
#13017873 - 08/07/10 03:59 PM (14 years, 5 months ago) |
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You could try PCing for less time. 20 minutes is fine at 15 psi. You dont ever want to exceed 15 psi even by a little with an LC.
Micropore tape is a poor choice in filter material for LCs. You would be better off with one of those rigs with the flexible tubing stuffed with polyfil in the lid and a single rtv port. This way you can pinch the tube off with a clip or something and shake the jar very well every day or two to speed up growth and get a dense culture, which you cant do with a micropore taped up hole. Plus micropore tape has got to have some huge micron spaces. I would just wildly guess by eybelalling it that it has to be in the 50-100 micron range or even higher. You can literally see gaps you know.
Are you trying Agar to LC Or spore to LC?
All in all LC is an art form, kinda like being a good chef. It takes some practice for some situations and some people to excel at it.
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,387
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Re: LC always fails, agar to grain works fine WTF? [Re: sandman420]
#13017889 - 08/07/10 04:02 PM (14 years, 5 months ago) |
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or you could just do what youre doing, and then use the grain to make a "Grain LC" by injecting sterile water and shaking the jar up.
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MrPinkFloyd


Registered: 07/13/09
Posts: 683
Loc: United States
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Re: LC always fails, agar to grain works fine WTF? [Re: sandman420]
#13018171 - 08/07/10 05:13 PM (14 years, 5 months ago) |
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I think it's the micropore tape smuggling in the nasties.
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lastlokean
Stranger

Registered: 07/30/07
Posts: 106
Last seen: 11 years, 10 months
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Re: LC always fails, agar to grain works fine WTF? [Re: MrPinkFloyd]
#13018269 - 08/07/10 05:40 PM (14 years, 5 months ago) |
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Thanks for the insight, will give it a fresh try with a more proper filter!
--------------------
Recipe:
How a Lokean makes chocolates
For a fully enclosed gh with a coolmist piped in, one may find that adding one of these to the opposite end as the intake will seriously help reduce pressure buildup and strain on the poor weak coolmist motor, while also dramatically improving fae without cutting polyfill stuffed slits all over the damn thing. (That looks suspicious )
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Javadog
Continuing along



Registered: 05/03/10
Posts: 7,385
Loc: USA
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Re: LC always fails, agar to grain works fine WTF? [Re: lastlokean]
#13018617 - 08/07/10 07:06 PM (14 years, 5 months ago) |
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Quote:
lastlokean said: So... awhile back the green meanies hit hard, failing multiple monotubs!
Sense then the decision to return to a martha for smaller controlled cakes was made. Sense then success has been limited, with a seemingly endless trich problem.
Currently spores -> Agar -> Agar -> Agar -> LC -> Grain -> Bulk is being utilized to get clean healthy culture. (In theory)
With about a 90% contaminate rate using good sterile procedures in a glove box. Originally the blame was cast onto sterile process. And now it is way overkill, and only FRESH brand new syringes jars and all equipment are used. (Just out of extreme paranoia)
After an entire month of failures a change was made to straight agar transfers to grain. This has found a much higher success rate close to 100%.
Whats interesting is the LC would look fine, test fine on agar and look fine when colonized on grain about half the time and green the other half. Any of the grain that looked fine would contam instantly when spawned to bulk.
Years prior have had no problems with LC and I can't seem to figure out whats wrong with the LC.
Using sugar based LCs at anywhere from 2-4% solution of turbinado or honey using distilled water. They are PC'd for half an hour to 45 minutes at 15 psi. Always wrapped in foil, sometimes removed while still warm for filter to dry fast, sometimes let cool in the PC for 24 hours. (Not sure which is 'correct')
For a LC lid two holes are drilled. One for 2 pieces of micropore tape and one for a silicon injection port. The holes are always sanded down very well till smooth.
Anyone figure why the LCs would consistently FAIL?
I have posted about sudden and consistent problems in making LCs for three new stain syringes.
I think that I may have figured out why by testing on agar though, as I am seeing a weak bacterial infection in each syringes petri dish.
Good luck!
JD
-------------------- Boyd Rice told my brother that life is a corny pack of freesakes
Myco-tek.org
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lastlokean
Stranger

Registered: 07/30/07
Posts: 106
Last seen: 11 years, 10 months
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Re: LC always fails, agar to grain works fine WTF? [Re: Javadog]
#13018714 - 08/07/10 07:34 PM (14 years, 5 months ago) |
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I'm getting trich. not a weak bacterial infection...
--------------------
Recipe:
How a Lokean makes chocolates
For a fully enclosed gh with a coolmist piped in, one may find that adding one of these to the opposite end as the intake will seriously help reduce pressure buildup and strain on the poor weak coolmist motor, while also dramatically improving fae without cutting polyfill stuffed slits all over the damn thing. (That looks suspicious )
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Doc_T
Random Dude




Registered: 03/06/09
Posts: 42,395
Loc: Colorado
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Re: LC always fails, agar to grain works fine WTF? [Re: lastlokean]
#13018807 - 08/07/10 07:56 PM (14 years, 5 months ago) |
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LC w/ hidden trich. Happens. If you look carefully, there's probably a green grain in each fully white jar.
-------------------- You make it all possible. Doesn't it feel good?
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lastlokean
Stranger

Registered: 07/30/07
Posts: 106
Last seen: 11 years, 10 months
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Re: LC always fails, agar to grain works fine WTF? [Re: Doc_T]
#13019176 - 08/07/10 09:26 PM (14 years, 5 months ago) |
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But hidden green shouldn't happen to 30+ LC in a row made from different strains. I've decided not to continue trusting micropore tape on the LC lids anymore... Instead gonna use the auto tubing + poly-fill. Hopefully this fixes the problem!
--------------------
Recipe:
How a Lokean makes chocolates
For a fully enclosed gh with a coolmist piped in, one may find that adding one of these to the opposite end as the intake will seriously help reduce pressure buildup and strain on the poor weak coolmist motor, while also dramatically improving fae without cutting polyfill stuffed slits all over the damn thing. (That looks suspicious )
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sandman420
Saint PP



Registered: 06/17/04
Posts: 5,387
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Re: LC always fails, agar to grain works fine WTF? [Re: lastlokean]
#13019184 - 08/07/10 09:28 PM (14 years, 5 months ago) |
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I think that will do you good my friend.
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