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OfflineNikoyo
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Eliminating possible sources of contam/bac
    #24726930 - 10/21/17 02:08 PM (3 years, 6 months ago)

Hi all, I apologize in advance as this will be a rather long post, but bear with me! As the title states I have been having a difficult time creating clean WBS spawn. After a few rather successful BRF cake grows, I made the transition to using agar and trying bulk. While I did have some initial success taking clones to agar from the cake grows and agar to WBS it seemed for months I struggled from there, ie lots of metabolites, mono's triching out before the first flush etc etc. It got to the point that I gave up as I just couldn't seem to achieve clean spawn.
 
    Fast forward some half a year later; after a hiatus but continuing to read up on info, checking the site almost religiously, trying to gain more knowledge about cleaning up agar, learning what piggybacking bac looked like etc etc I decided to give it another go. This time starting via spore prints on agar and trying to narrow down possible sources of contam/bac I had previously been dealing with.
 
    While I have definitely improved my sterile technique all around I am still having issues with creating clean spawn. I am currently working with a TC and fiji culture each some six to eight transfer in. Here's a few examples of what my plates are looking like.

(as you can see the first one is an earlier transfer on grain soak agar while the others are a BRF recipe.)
   
    While they are definitely looking better than my previous attempts to clean up cultures, I am still having contam issues in my quart WBS jars.  Heres a couple that I recently tossed and one example of how my other jars are looking.

      As you can see Ive been dealing with the greens ie penicillum, aspergillus and "trich". The 2nd pic looks like "cobweb", the 3rd pic is a day after shaking at 30%, hard to see but next to my thumb there is overlay from trich starting to appear. While all my jars are not affected (a few have finished colonizing 100% and are currently being used on small "test" trays 1:1 ratio cvg to see if my agar indeed has piggybacking bac). 3 trays were made up two days ago so we shall see what happens with them.

    Just to cover the bases here's a few things I have done to narrow down and eliminate possible sources of bac/contams. Upped WBS pc times from an hour to two hours. Worked to improved my overall sterile technique while working in SAB ie making smooth but quick transfers, using less iso and other disinfectants as a crutch, making more transfers to clean up agar prior to innocing grains, switched up agar recipes to get cultures to spread quicker, loading up WBS on the drier side prior to pcing ; likewise I have been cutting out the middle of my plates thus not using the section where the donor piece was transferred to ie donut shape.
   
    While these changes have helped eliminate possible sources there are still a few things to narrow down and rule out. some are the following.
-Lids- Need to get SFD's as Ive been using polly stuffed in GE holes.

-try cleaning work area more; Due to the amount of tub failures I had previously Im sure the spore count is extremely high; I also grow herb so Im sure there is a large amount of trich in the house. 
- Moving all myco work to a backyard shed and out of the house.
     
    I know all of these could be affecting my results to a degree so I am going to continue trying to narrow it down. I'm sure people will ask specifically about my agar and innoc/ grain prep procedures which I will gladly explain; tried not to make this post a novel. None the less if anyone else has had similar problems what changes did you make and how did it help? Any advice would be greatly appreciated; I enjoy this hobby a lot but the amount of failure definitively is draining and takes the fun out of it.


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OnlinemushboyM
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Re: Eliminating possible sources of contam/bac [Re: Nikoyo]
    #24726937 - 10/21/17 02:11 PM (3 years, 6 months ago)

cutting out the agar doughnut is too much time spent with your hand/blade hovering above open media/cultures.

and from the pics your wbs prep looks rather weak. soak that shit 24hrs drain and load. pc 2hrs minimum.


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OfflineNikoyo
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Re: Eliminating possible sources of contam/bac [Re: mushboy]
    #24727007 - 10/21/17 02:56 PM (3 years, 6 months ago)

Quote:

mushboy said:
and from the pics your wbs prep looks rather weak. soak that shit 24hrs drain and load. pc 2hrs minimum.




Have been doing 24 hour soak, strain, putting some on paper towel to see how wet before loading up. I will admit these jars were on the drier side, been trying to find that healthy medium as previously was loading up when wetter which lead to lots of slime.  Good call on the "donut shape" transfer, was previously working strictly with pastey plates and tiger dropping so its been a bit of playing catch up getting used to working with plates. Will definately keep it in mind on the next set of jars. Im going to get some sfd's for my lids and see if the above help before I go about making my shed a more permanant lab space.


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