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Peabody


Registered: 07/14/09
Posts: 123
Last seen: 13 years, 4 months
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Help me figure out where I went wrong? -Bacterial contam/wet rot
#10990237 - 09/03/09 12:15 AM (15 years, 4 months ago) |
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I've made a diagram below that I think should be easy enough to read. (Easier to read than what I'm about to write below)

So a week or so ago I inoculated lots of WBS grain jars, and some of them showed zero growth and signs of bacterial contamination or wet rot. I've been very frustrated with this. This has thrown my schedule off and I want to prevent it from happening in the future.
Here are all the clues, please help me piece it together to determine where the contamination likely came from.
1. Received SA and Z-strain spore syringes in the mail from Ralph. 2. Prepared lots of WBS using FooMan's tek. This has not failed me before. Sterilized jars in PC. (Quart jars, lids are agar's method with tyvek and paper tape) 2. Inoculated a couple jars straight from the SA syringe. Made LCs using brown rice syrup from SA and Z-strain. 3. Inoculated more previously PC'd jars with spores from new LCs. Made more WBS jars, inoculated those too. 4. Inoculated some jars with old but fresh LC from previous grow, Argentinas. 5. Noticed very slow growth in SA syringe-only jars, so added 2ccs of LC. 6. All of these jars, except for the jars inoculated only with straight spores from the SA syringe, did not grow at all or growth disappeared. Wet rot suspected. 7. New LCs opened and investigated. SA LC smells like cheese. Z-strain LC smells and looks fine. Both thrown away. 8. Jars opened and investigated, smell like seeds. Slightly sweet smelling. Ever so slightly mushy looking. Also thrown away. 9. Jars bleached, washed, PC'd, starting again.
Sterility of ... WBS: PC'd @ 15psi for 60 min LC: PC'd @ 15psi for 20 min Syringes: used right from mailed package Needles: brand new, alcohol rubbed, did not flame, used separate new-in-wrapper needles per LC Myself: hot shower, gloves, surgical face mask, shower cap, clean clothes Inoc environment: very clean unused guest bathroom, bleached, lysoled, doors closed, all tile
Inoculation technique: -Unwrap needle, attach to syringe. Remove cap, swab needle and jar lid with alcohol. Insert needle into LC, suck up enough, insert needle through paper tape into jar lid. Deposit 2cc per quart jar, 1/2cc in each corner of jar. Remove needle from jar, detach from syringe, place both aside. New paper tape on lids of jars, new coffee filter, rubberband. Repeat.
It seems not to be the... - spore syringes, because they work (at least for SA) when used alone - WBS preparation, because some of those worked too, and have worked very well in the past.
As you can see in the diagram I have been successful at preparing WBS and LCs. At what point do you think the bacteria got in?
Thank you so much, and I will do my best to answer any questions that come up. I just don't want to fail like this again. 12 jars in all wasted.
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Triptonic



Registered: 04/23/08
Posts: 3,707
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Re: Help me figure out where I went wrong? -Bacterial contam/wet rot [Re: Peabody]
#10990494 - 09/03/09 01:16 AM (15 years, 4 months ago) |
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The only thing I can think of is its probably your WBS and the pressure cooking.....I'm not sure about foomans technique but does he soak and simmer or just soak or just simmer? I usually do both. Maybe try PCing for 90 mins instead of 60. Do you shake your jars after you see growth? Maybe your grains are to wet.
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solumvita
Q.B.E.


Registered: 02/12/08
Posts: 2,061
Loc: South Africa
Last seen: 1 year, 7 months
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Re: Help me figure out where I went wrong? -Bacterial contam/wet rot [Re: Triptonic]
#10992682 - 09/03/09 01:48 PM (15 years, 4 months ago) |
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You say that your SA-LC smelt bad but the Z-strain was fine? yet you threw both away?
Seems like from the diagram that your LC technique was not good on the day as the previous two of the B and Argentina strain seemed to work fine. You make no mention of a glove box, perhaps you should consider using one.
I like your picture much better as it explains far better than the text can.
I reckon the bacteria got in when you were inserting the needle when injecting the LC.
-------------------- One of these days all the answers will be revealed until then we learn from each other!
www.mushrush.co.za
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Brennus
Student of Life



Registered: 05/31/08
Posts: 3,297
Loc: SE United States
Last seen: 6 years, 10 months
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Re: Help me figure out where I went wrong? -Bacterial contam/wet rot [Re: solumvita]
#10992793 - 09/03/09 02:13 PM (15 years, 4 months ago) |
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You didn't mention using a glove box. You also always need to flame sterilize syringes (or any other tools, such as scalpels and inoculation loops).
What temperature did you colonize your jars at? Did you use an incubator?
Also, I always sterilize grains for 90 minutes @ 15 PSI. 60 minutes may or may not work. One more stage to botch things up at, though.
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Peabody


Registered: 07/14/09
Posts: 123
Last seen: 13 years, 4 months
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Re: Help me figure out where I went wrong? -Bacterial contam/wet rot [Re: Brennus]
#10993203 - 09/03/09 03:38 PM (15 years, 4 months ago) |
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Quote:
You say that your SA-LC smelt bad but the Z-strain was fine? yet you threw both away?
I had more spores in the original syringes so I made new LCS, they are in progress but don't really look right. :\ Too much sediment at the bottom. I may try one more time with honey instead of BRS.
Quote:
You didn't mention using a glove box. You also always need to flame sterilize syringes (or any other tools, such as scalpels and inoculation loops).
I don't have a glove box, yet. I was hoping my guest bathroom would be sterile enough because it is never used, small, and all tile. I think you two are right and my next step will be constructing a glove box.
Do you need to flame sterilize the needles if they are straight out of the sterile medical packaging, used once, then discarded? I have lots of needles. I will absolutely flame them from now if it is still necessary.
Quote:
What temperature did you colonize your jars at? Did you use an incubator?
I did not use an incubator, I had them in an otherwise empty plastic tub in my closet with the lid off. My house stays at around 78-80F.
For the previous jars, the successful ones, I made a habit of turning off the A/C when I left the house and letting my house get up to around 85F for most of the day, (Florida in the summer) then back down to 78F when I got home. But now I have four monotubs going so I try to keep it around 78F all day.
Quote:
Also, I always sterilize grains for 90 minutes @ 15 PSI. 60 minutes may or may not work. One more stage to botch things up at, though.
I was thinking that 90 minutes would mush up the seeds too much, but I have not tried it. Next time I sterilize grains I will use 90 minutes and see how it goes. Also I have switched from Kaytee to Pennington brand WBS, which may make a difference.
Thank you so much for your help!
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