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 bbox244
Legally Insane
Registered: 03/17/11
Posts: 205
Loc: USA 
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 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine 
#14712567 - 07/03/11 09:55 PM (1 year, 11 months ago) |
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I had 2 bacteria resistant mono-cultures which survived bacteria invasion on MEA agar, then survived invasion on WBS, but when spawned to bulk via:
--From Log--
1/4 cup lime
1 cup coffee grounds unspent
1/2 cap kelp extract
3 qts verm
4 qts + 800ml h20
1 qt worm castings
1 cup gypsum
boiled lime/gypsum/coffee/kelp extract/h20/
buffered ph of liquid to 6.6
--End Log--
The added bacteria destroyed both mono-cultures. **Bulk sub was Sterilized** No fungi/spores present. Only added bacteria (endospores).
Background:
I have a lot of both working and hands on knowledge working with WBS, and Agar. My bulk substrate knowledge is high when it comes to forum/book knowledge, but low when it comes to hands on. I've been working on producing these 2 mono-cultures for awhile now. It's taken a long time to find these 2 mono-cultures which were both resistant on both agar and grains.
Question:
I've noticed mycelium has greater resistance on certain mediums than others (Agar/Grains). Is this true for bulk substrates as well? In the advanced mycologist's opinion, is the problem I experienced due to my lack in working hands on with bulk substrates (hopefully)? Or is it due to mycelium having different strengths against bacteria in different mediums? What do you think happened? I'm really hoping it was purely ineptitude on my part. I know everyone will first blame the coffee, but, this is the same formula I used successfully in my past 3 bulk grows without issue. Naturally with those, I was simply growing and wasn't adding bacteria cultures to destroy mycelium.
You are far more experienced than am I with the behavior of mycelium on different substrates, and even "conditioning" the mycelium to behave differently on different substrates. If you think the culture were not properly conditioned to work on the bulk substrate, please let me know, and I can prepare some experiments to better condition the mycelium for transfer to bulk. Or, if there genuinely is a difference between the behavior of mycelium in terms of the "foothold" it had on different substrates, is there a better medium I can grow on which will more universally show it's behavior, perhaps a different agar, or something which will show better how the mycelium will react on multiple substrates.
Right now, I just test on MEA, then test on WBS, and so far I've found 2 resistant mono-cultures after all this time, and when I took them to bulk, they started colonizing fine, until the bacteria was added via injection and then they got wiped out. I can still see the grains bare, without mycelium anymore. The grains literally were stripped of the mycelium and destroyed. These same mono-cultures destroyed bacteria on both MEA and WBS. Any ideas are greatly appreciated, I've been really scratching my head on this, and the only thing I can think of is that the mycelium didn't have as great of "foothold" (for lack of a better terminology), on bulk substrate as it did on Agar and WBS. Did the sterilization give the bacteria a stronger presence on the bulk sub? Is mycelium inherently weaker on bulk subs?
If there is a better medium to colonize and test against instead of MEA and WBS, and if it's strain dependent, please let me know, this will greatly reduce the time I've spent on this. Thank you,
One more thing, I did 2 trays of different MS WBS grains, same stains as the 2 mono's, as a control group with the same bulk sub, those are almost fully colonized, no bacteria was added. All trays are kept in a totally sterile environment with HEPA filtered air for GE. **Sterilization** was done to ensure I had total control over bacteria/fungi in the substrates. I'm aware of beneficial bacteria/fungi in past. process, etc. These 2 trays flourished in sterile sub under totally sterile conditions. They are both cubes, as are the mono's. Would manually adding actino's, microbes, and other beneficial thermophilic bacteria help to simulate a traditional bulk grow better? Could this be the reason the mycelium was destroyed?
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cc2
Mush
Registered: 05/15/10
Posts: 2,106
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine [Re: bbox244]
#14714216 - 07/04/11 06:09 AM (1 year, 11 months ago) |
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Quote:
bbox244 said:
I had 2 bacteria resistant mono-cultures which survived bacteria invasion on MEA agar, then survived invasion on WBS, but when spawned to bulk via:
The added bacteria destroyed both mono-cultures. **Bulk sub was Sterilized** No fungi/spores present. Only added bacteria (endospores).
sorry but by bacteria resistant I think they should be immune, mycelium winning contams on agar isn't bacteria resistant.
you probably had healthy strong mycelium growing, which managed to survive moderate attacks but then kept going worse. endospores are a tricky thing to kill.
since you were doing a controlled experiment, which kind of bacteria did you introduce? or was it just random contams?
let us know 
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mycoelf
Agent Of Chaos
Registered: 06/26/09
Posts: 553
Loc: hyperspace
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine [Re: cc2]
#14715007 - 07/04/11 12:20 PM (1 year, 11 months ago) |
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So the culture you started with was clean? You got to be clean on the petri before you can move further. If U are having trouble acquiring a clean isolate you might look into an antibiotic agar, U can also peroxidate your agar, and if all else fails look up agar sandwich TEK by RR. If your mother culture is not absolutely clean contamination comes from within the spawn run, and anything U do to expend the myc you will expand the contams 10,000 times first,
Maybe I don't understand what U are asking, but if your culture was clean to start and you sub con-tamed maybe you have a sterilization protocol issue. I was confused by you r phrase "resistant" myc can defend themselves sure, in a naturalized environment, but they selectively nuke those critters they do not like, and live with many other organisms in the soil/ wood sub. When you are in captive conditions the rules change because the theater of action has so drastically changed,so the presence of bacteria in the first instance is totally unlike a bacteria in the second  and please continue to clarify what you are looking for, We hope to help U.
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Mycoelf
Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
    
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 ozzysmygod
Late Night PC'ing Enthusiast
Registered: 11/26/08
Posts: 835
Last seen: 2 months, 23 days
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine [Re: mycoelf]
#14715044 - 07/04/11 12:30 PM (1 year, 11 months ago) |
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Hello Bbox! Having read your thread, and your pm's I have an understanding of what your trying to achieve. Im new to agar work, only 150 plates under my belt, and no isolates as of yet, still working on them. So dont take what I say as fact.
I have seen a thread where RR suggests, if your trying to clean up a plate, to use agar with less nutrients than normal. This allows the mycelium to have a head start over the contaminates, so it can be transfered to a new dish.
Maybe your bulk substrate has too many nutrients, allowing the contamination to take over, even though it works fine on your agar plates.
This is only a hunch, your doing some pretty advanced work!
I don't really know what to suggest. Maybe try your agar work with slightly MORE nutriants so it better replicates a bulk substrate? Although this might slow down growth.. I'm sure there's a happy medium though.
Good luck!
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SKrink
KING MOB
Registered: 01/29/11
Posts: 1,042
Last seen: 4 months, 2 hours
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine [Re: ozzysmygod]
#14716427 - 07/04/11 05:54 PM (1 year, 11 months ago) |
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Probably not my place to jump in with no hands on experience, but your work sounds fascinating. I would like to learn too, and I have a couple questions:
For MEA, WBS, and Bulk Sub, at which point ( X% colonization of each) do you introduce the bacteria?
Is there an optimal point for each, and are they all the same or different?
What if the FORM of the media has something to do with it?
2D surface of agar and individual seeds of WBS , VS. the indistinguishable mass of bulk substrate material.
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RogerRabbit
Bans for Pleasure
Registered: 03/26/03
Posts: 39,374
Loc: USA Mountain Northwest
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control grp fine [Re: bbox244]
#14719458 - 07/05/11 10:41 AM (1 year, 11 months ago) |
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Quote:
bbox244 said:
I had 2 bacteria resistant mono-cultures which survived bacteria invasion on MEA agar, then survived invasion on WBS, but when spawned to bulk via:
--From Log--
1/4 cup lime
1 cup coffee grounds unspent
1/2 cap kelp extract
3 qts verm
4 qts + 800ml h20
1 qt worm castings
1 cup gypsum
Try it again without the lime, and always brew coffee before using the spent grinds. Don't use unspent coffee grinds in bulk substrates. Kelp is good plant food, but I don't know what fungi would do with it. Also, worm castings make a poor substrate, so your mycelium didn't have a chance from the start.
Also, there's nothing in that mix which would harbor bacterial endospores.
RR
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bbox244
Legally Insane
Registered: 03/17/11
Posts: 205
Loc: USA
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control group fine [Re: RogerRabbit]
#14726857 - 07/06/11 06:08 PM (1 year, 11 months ago) |
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Thanks for the replies! First off, resistant is a bad word, it was used because of the type of assay I'm doing. I can measure the rate at which mycelium is penetrated, therefore, I can measure the mycelium's resistance to said bacteria. With an assay, if only a minuscule amount of mycelium is penetrated, it is resistant, or even perhaps immune to invasion from that particular bacteria. I cannot say it's entirely immune because it's a visual method. My methods only allow me to tell the rate of invasion. I would need an electron microscope to see if something was 100% immune. However, I can, by comparison, tell which mono-cultures are immune. The 2 mono-cultures I used were annihilated on a bulk substrate whereas they were both totally immune on agar and WBS, determined by reference.
Basically, this all started from a thread from a couple months back where I was asking about bacteria and mushroom mycelium, and I did some research and found that mushroom mycelium very commonly carries genetic traits which allow for it's use in the production of antibiotics, as well as many other medicinal uses. So, I decided to try and make bacterial immune mono-cultures from the strains I presently have. After a couple months of work, and A LOT of plates, I have 2 mono-cultures which showed no cellular penetration from visual inspection via an assay. So, I thought I had hit the jackpot, but, when I put the cultures to bulk substrates and prepared a solution from several bacteria samples I've cultured from both agar contamination and SMS, and injected it into the bulk tray, about 1 week later, the mycelium slowly disappeared.
At first, it was growing well, excitement was high and then, total disappointment. Also, I colonized 2 other trays with MS versions of the 2 different strains, and the exact same bulk substrate and they both are almost done colonizing. No signs of any contamination at all, but none was introduced. This was to test the bulk substrate to make sure it wasn't contaminated and make sure it was sound for the experiment.
With regards to comments/questions so far:
For the bulk sub: The only thing on the use of the coffee, was that by boiling it for an hour or so in h20 first, I ended up with spent coffee grounds + coffee. This may be no different from adding unspent grounds and may be a contributing factor, if it is the same thing as adding unspent grounds (which I am sort of thinking it would be now), then I will remove that and the castings and give it another go. The lime was only a PH buffer, so I'm fine without it, I don't mind going with a lower PH, and it won't really matter without the coffee anyway. So, I can ditch the castings/coffee/lime and just do verm/gypsum/coir, since nothing germinates on coir.
For bacterium introduction: On MEA, 4 different cultures of common bacteria was grown and fixated on a single plate for a couple days before introduction. The mono-cultures to be tested were about 1/3 the size of a single plate. The bacteria was introduced at this point in solution and the plate was incubated for a couple hours.
With WBS, no assay was performed, just inoculation from a resistant mono-culture and if partial colonization was successful after 2-3 days, a grown bacterium culture solution was produced and injected. If the mono-culture survived on WBS (many didn't), then it was tested for fruiting by just casing the WBS and watching for primordia.
For Bulk, I only have 2 mono-cultures which made it this far, and I added 4 cc's of bacterium solution after preparation of the bulk substrate and within a week the mycelium was gone.
Quote:
Mycoelf: When you are in captive conditions the rules change because the theater of action has so drastically changed
This is kind of what I'm after here. I've only been working with mushrooms for a year, and I had no experience other than basic fungi work in college. In my work, I don't work with fungi either, so my experience is limited, but thankfully, a lot of the same procedures I've used in the past work in mycology as well. So, after some work with a lot of plates and WBS, I'm really starting to find that the mycelium from mushrooms have a different "foot-hold" for (lack of a better term) on different substrates. It seems that even if a mono-culture is immune on agar, it will sometimes get destroyed on WBS. However, the 2 mono-cultures I have now are fine with 3 different of temperature conditions for colonization, as well as 3 different WBS recipe's. So, I'm pretty sure I've got something here, but it all goes back to the bulk sub.
I'm realizing that mycelium behaves differently in different mediums. Is there a better medium I can grow mycelium in that will better simulate how it will grow on WBS/bulk substrates?
I will make another bulk attempt using just coir/gypsum/verm. I'm not ready to give up on these 2 yet. I've had so many failures, that I really want them to work. So I'm thinking it has to do with my technique with bulk substrates. Just a bit frustrating because, the other MS WBS spawns are doing great on the exact same bulk sub. I appreciate the answers so far, and if there is a better medium to test on, that would be great. Perhaps PDA would better represent alternate medium growth? I don't know the answer with mycelium. Thats where your expertise comes into play, you have far more experience working with bulk substrates and working with different mediums for mycology. It would be impossible for me to have this knowledge unless I went to Mushroom U , which I did not. Thanks again,
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.... Always curious, always looking for better ways, mind always wonders ....
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bbox244
Legally Insane
Registered: 03/17/11
Posts: 205
Loc: USA
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control group fine [Re: bbox244]
#14731365 - 07/07/11 03:59 PM (1 year, 11 months ago) |
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Ozzy, I can't believe I missed your post! Sorry bro! Your point on nutrients is very good. Perhaps the nutrients in the substrate is what affects the myceliums "foot-hood" (Still don't have a term for this), that makes a lot of sense. I am using "light" Agar presently. So, this is perhaps why so many of my mono-cultures fail when put to grains. So, the nutrients give the bacteria a stronger foot-hold. It makes sense that the different types of nutrients would play a factor on whether or not the mycelium or bacteria would have a stronger position. This is interesting. I'm willing to bet that cellular wall strength of hyphae varies between mediums as well. Thats something I know is tied to genetics as well however. I'm going to start testing on different nutrient agar's to see if results change. Which ever agar I use will be fine for the staining, so I can change it up a bit. Ok, I'm gonna first study up a bit on specific nutrients for mycelium and proceed from there. I'd be happy to have something immune on specific mediums at this point. This has taken up a lot of my time, and I'm really starting to feel it mentally/physically.
I really need to try getting access to an electron microscope. I know a lot of people at a near by university and they have a mass spec, electron microscope, chromatograph, everything I would ever need to actually "see" whats going on here. Well, I just need the EM, but the other equipment would be very useful for other things. The problem with microscopy assays is that you can just get a visual chromatic deterministic evaluation, it's not a 100% accurate method to work with, it's an old tech, but it's still used a lot, since you can do a lot "visually". But at some point, you really do need to look at a sample in pm's instead of nm's. Thanks again everyone,
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.... Always curious, always looking for better ways, mind always wonders ....
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tanman1990
Intermediate Mycologist
Registered: 02/28/11
Posts: 217
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Re: 2 bacteria resistant mono-cultures destroyed on bulk substrate, control group fine [Re: bbox244]
#14757944 - 07/12/11 06:18 PM (1 year, 11 months ago) |
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Wow, this is some hardcore research you have going here. I'm very interested in the outcome and impressed by the depths you have gone to. I'm fairly new to mycology so this thread blew my mind a bit haha. I hope you have success, keep us posted!
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