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I'm back in the saddle again....... (w/ questions)


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#1 shoomer

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Posted 02 March 2017 - 05:30 PM

[Skip near the bottom for questions]

 

So after I found out that my drying technique is not crisp enough to keep stored boomers from getting the "blues" in the freezer, I whipped out the fixins and started a new run about 1 1/2 mo. ago.

 

1st 2 runs were a mix of 1-1/4,15 - 1/2,  and 3 pt. jar BRF's, 12 or so of which are sitting on CD's in a bubbling tub of perlite (other methods may be better/more efficient, but this one works for me as until I get some uninfected LC syringes, Martha type bulk FC is not my aim) w/ 5 more 1/2 pts from the 2nd run waiting to mature to "rattle" age for easy removal.

 

Only 2 (1-1/4 pt. and 1- 1/2 pt.) got trichy from lackadaisical tape application.

I'm going to keep them in vitro to see if the cake or trich wins as it's still too cold out to throw them in the compost pile.

 


 

Anyway, things are going according to plan except during that 2nd run I made up some new LC's (baby bottle tek) and had some questions about the activity (or lack thereof) of the LC:

 

1.) First LC run was tried and true B+, Pink Buffalo, and (new for me) South American.

     2nd LC run was King Oyster, Brown Oyster, portabella, Hawaiian PE, and Amazonian PE.

    

     a.) 1st run had 3 LC failures due to sterility (some nipples need new silicon) of 2 different

           types.

           1 was cloudy, 3 were a bit cloudy, but started to devour all the air in the sealed

           bottles and the sides of the bottle started to pucker in. These were easy for me to 

           identify because of previous experience but I wanted to ask about the "air eating" 

           infection.

           Anyone had a similar experience?

 

     b.) 2nd LC run was a BB each of the 5 strains listed above and here's the results

            I.) Brown oyster = cloudy after 1st day [LC syringe]

           II.) Portabella     = cloudy after 1st day [LC syringe]

          III.) King oyster    = crystal clear (or as crystal as ME LC gets) 4 days in [LC syringe]

          IV.) Am. PE         = cloudy after 2nd day [spore syringe]

           V.) H. PE            = crystal clear [spore syringe]

 

These syringes are over 1.5 years old and I know that ANY LC syringe should be well dead by this time but the spore syringes (even clumped as they are) should be good.

 

2.) The cloudy boomer LC's are toast I know, but I've never had a good run of edible LC and wonder if ANY edible LC gets cloudy (instead of ropey boomer myc goodness) and is still uninfected?

These are all 2X failures IME and after 2X's I deem the cloudy toast and the clear just a dead LC syringe.

 

3.) The cloudy PE is toast, but the clear one seems fine except for no myc. The clump of spores in both PE syringes is so dark and cohesive I thought the plunger rubber was starting to disintegrate.

Do you think the clear PE LC syringe had a clump contained ALL the spores, so much so that if  that clump was not in the 'noc squirt, it was like injecting sterile water?

 

4.) What's your favored way of breaking up spore clumps?

 

Just curious folks. :wink:


Edited by shoomer, 02 March 2017 - 05:41 PM.


#2 panaho

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Posted 02 March 2017 - 09:53 PM

Of the spore syringes I have used I drew back so a bubble is there and just shook as much as possible to break things up. Four days now and no growth. I saw somewhere where the water was as dark as coffee so I am wondering. I never had too

long a wait if I was using spore prints- my favorite method by the way.


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#3 The1unknown

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Posted 03 March 2017 - 11:00 AM

Of the spore syringes I have used I drew back so a bubble is there and just shook as much as possible to break things up. Four days now and no growth. I saw somewhere where the water was as dark as coffee so I am wondering. I never had too
long a wait if I was using spore prints- my favorite method by the way.

Give them more time some take longer than others. Also what temps are your jars sitting in? Colder temps will slow things down. You want to shoot for 70-75 °F. Also important to note if you pull air into your syringes to do it over a flame so you're drawing in cleaner air.
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#4 The1unknown

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Posted 03 March 2017 - 11:08 AM

I'm not experienced with LC but I understood that spores will germinate in LC so you should have mycelium not spores (please someone correct me if I'm wrong) The cloudiness in the LC could be from dying mycelium perhaps? As far as the viability of the spore syringes being viable that'll have allot to do with genetics and/or how they were stored. High temps =dead spores.

#5 The1unknown

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Posted 03 March 2017 - 11:24 AM

As far as air eating. Mycelium are living creatures and consume oxygen and give off carbon dioxide. Idk if they give off equal amounts of carbon as they take (oxygen) but I find it strange that it was "puckering in" that would indicate low/negative pressure inside compared to outside. Where did you get these LC's from?

#6 Myc

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Posted 03 March 2017 - 02:45 PM

I'm not experienced with LC but I understood that spores will germinate in LC so you should have mycelium not spores (please someone correct me if I'm wrong) The cloudiness in the LC could be from dying mycelium perhaps? 

 

You're correct. If you have a perfectly clean print and can pull-off a perfectly clean transfer, spores will germinate in LC.

This is like picking the trifecta at the race track. It can be and has been done. 

LC is highly available nutrition and bacteria is your most likely competitor to establish dominance. They are far faster than fungi and exist almost everywhere.

 

 

Your best bet is to do live tissue transfer to LC - Agar wedge, slurry preparation, proven LC, etc.


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#7 The1unknown

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Posted 03 March 2017 - 05:45 PM

Wonder if the cloudiness is dead bacteria? Maybe nothing germinated the clear ones and all the spores died?
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#8 shoomer

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Posted 03 March 2017 - 08:51 PM

Thank you all for the replies!

 

 

 

Of the spore syringes I have used I drew back so a bubble is there and just shook as much as possible to break things up. Four days now and no growth. I saw somewhere where the water was as dark as coffee so I am wondering. I never had too
long a wait if I was using spore prints- my favorite method by the way.

Give them more time some take longer than others. Also what temps are your jars sitting in? Colder temps will slow things down. You want to shoot for 70-75 °F. Also important to note if you pull air into your syringes to do it over a flame so you're drawing in cleaner air.

 

 

The1unknow - jar temps are @ 70F (kept in the warmest room of the house),

 

I'm not experienced with LC but I understood that spores will germinate in LC so you should have mycelium not spores (please someone correct me if I'm wrong) The cloudiness in the LC could be from dying mycelium perhaps? As far as the viability of the spore syringes being viable that'll have allot to do with genetics and/or how they were stored. High temps =dead spores.

 

If it died, it's died faster than anything I've seen. I like LC because 1cc of MS syringe = 150cc of LC

All syringes kept in the fridge.

 

As far as air eating. Mycelium are living creatures and consume oxygen and give off carbon dioxide. Idk if they give off equal amounts of carbon as they take (oxygen) but I find it strange that it was "puckering in" that would indicate low/negative pressure inside compared to outside. Where did you get these LC's from?

Exactly, It's not air exchange (the LC baby bottles don't "breath").

2 LC's (BO and Port) from ebay vendors (both are trash IMHO) and the KO and all MS syringes (10 for $60) from thesporedepot<dot>com.

I am happy w/ the purchase from the depot as the 2 that I have tried have preformed flawlessly and even though the KO LC syringe is dead, it was sterile enough not to grow anything in the ME LC so far.

 

 

 

I'm not experienced with LC but I understood that spores will germinate in LC so you should have mycelium not spores (please someone correct me if I'm wrong) The cloudiness in the LC could be from dying mycelium perhaps? 

 

You're correct. If you have a perfectly clean print and can pull-off a perfectly clean transfer, spores will germinate in LC.

This is like picking the trifecta at the race track. It can be and has been done. 

LC is highly available nutrition and bacteria is your most likely competitor to establish dominance. They are far faster than fungi and exist almost everywhere.

 

 

Your best bet is to do live tissue transfer to LC - Agar wedge, slurry preparation, proven LC, etc.

 

I've done LC before and I've had a 5 of 8 survival rate (I like multiple as not everyone will fail).

I tried some store bought port flesh to LC  in the 2nd run but ended up cloudy for all 3 (soaked in provodine, ripped open in SAB, scraped flesh into just opened LC holding lid ajar).

I'll try again, but it'll have to wait until I get VIP status and can purchase (beg, borrow, or steal) a clean LC of edibles.

I _will_ take spores too. ;)

 

Wonder if the cloudiness is dead bacteria? Maybe nothing germinated the clear ones and all the spores died?

I'm thinking my sterility technique probably could use a little tweaking and you're probably right as all mushies I know of transpire (O2/CO2) and do not eat the atmosphere in the BB.

 

Again, thank you one and all for your replies!



#9 shoomer

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Posted 03 March 2017 - 10:05 PM

I've been thinking that in my short span here @ 'Topia I've pretty much asked questions about problems.

 

I did want to show that not everything is FUBAR'd here and I do have a handle on clean technique (I used to brew beer for a living and never had a batch go bad!).

 

The proof is in the pudding so here's my proof (and future "pudding pops"):

 

BRFs.jpg

 

the 1st & 2nd BRF's

 

Excess.jpg

 

The mature ones from last pic getting ready to pin in the FC.

 

Just didn't want folks to think I was completely inept. :-)


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#10 Myc

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Posted 04 March 2017 - 10:30 AM

 

Just didn't want folks to think I was completely inept. :-)

 

Nothing of the sort. We're all here to learn and improve.

I only weighed in because this seemed like a good discussion which presented an interested problem - i.e. the implosion of your culture vessel. I've always used the Airport Tek for this type of procedure - knowing that growing mycelium requires gas exchange. The implosion is a sign of a plugged vent or other issue.

 

Liquid Culture seems to be regularly misunderstood. Many folks mistake LC as a substitute for agar as far as propagation of spores. I see it more as a media for rapid expansion once a clean isolate has been established using traditional agar techniques. Otherwise, there is no advantage of one over the other in regards to inoculation/colonization. I've tested both (1cc of LC vs. 1cm^2 of colonized agar each in a jar of grass seed) and the results are identical. 

There are, however, enormous benefits of agar over LC when one considers that with agar one can rescue contaminated samples. When it comes to the time invested in learning advanced aseptic technique I think agar is the best place to begin. Once mastered, everything else just becomes simple. 

 

This guy had a problem similar to the one you've described:

https://mycotopia.ne...rt-tek-mistake/

 

Just my .02


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#11 Seeker2be

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Posted 04 March 2017 - 10:40 AM

Agar is the window to the soul of mycelium.  L/C is the unknown metropolis.


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#12 shoomer

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Posted 04 March 2017 - 03:24 PM

The amount of good non-condescending advice on 'Topia has me thinking it's an anomaly in the world of interweb forums. :D

<snip>
Liquid Culture seems to be regularly misunderstood. Many folks mistake LC as a substitute for agar as far as propagation of spores. I see it more as a media for rapid expansion once a clean isolate has been established using traditional agar techniques. Otherwise, there is no advantage of one over the other in regards to inoculation/colonization. I've tested both (1cc of LC vs. 1cm^2 of colonized agar each in a jar of grass seed) and the results are identical.
There are, however, enormous benefits of agar over LC when one considers that with agar one can rescue contaminated samples. When it comes to the time invested in learning advanced aseptic technique I think agar is the best place to begin. Once mastered, everything else just becomes simple.

This guy had a problem similar to the one you've described:
https://mycotopia.ne...rt-tek-mistake/

Just my .02


I've only ever done LC.

Agar scares me since if I'm having these types of issues (even though the greater majority are successful) with LC means I'm not consistently "clean" enough even w/ my handy dandy SAB.

I want to do agar work, I just think it's a waste until i build a flow table as I want my success average to stay the same instead of declining and using the right tool for the job is sometimes a better answer than using the one that "works most of the time".

I hear you about the LC vs. agar, but I like making more LC than any syringe could hold as high numbers of substrates still keeps the average high and ME is )relatively) cheap. The other reason (although I didn't do it last year) is I have 20 acr. of untouched woods I'd REALLY to get some pre-staged oyster/port/shitake/miatake/lionsmane/reishi wild stands growing in and that will take a LOT of substrate and/or LC.

If it wasn't against the rules I'd challenge you to send me an edible LC or spore on the promise that I'd only use it on agar to cut my teeth. ;-)

The LC tek I use is non-vented Evenflo autoclave-safe baby bottle (and some jars w/ tyvek breath ports this time) w/ RTV sealed nipples (not inverted).
I filled them to 150ml this time (1/2 full) to give the LC some more breathing room and allow better growth before they either drown in their own CO2 or get stalled in the fridge. They do not breath which is why I dropped the LC amount to 150ml instead of 200ml as I did last time (although those had a 3-4 wk. life before asphyxiation).

Last time I had the same type of failure and was just curious if a myc/bacteriologist knew of what wild 'tam eats air since they're sealed and cannot breath.

I'm round-filing eBay LC's ('tam'd/dead) and the depot (dead) LC syringes, but I'll recycle the works.
I'll try the air bubble in the MS syringes during needle flame and see if that helps break up the spore clumps as I'd really like to get some PE going.

Thanks for your suggestions though! Every little bit helps. :D

Edited by shoomer, 04 March 2017 - 03:24 PM.


#13 peacefrog

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Posted 05 March 2017 - 09:10 AM

I have never owned a flow hood, although I do want one, and have only used an SAB. I use agar for every grow with great results personally. Do I see contamination from time to time? Of course. No one can grow at 100% contamination free rate. I don't care what anyone says, it just happens sometimes.

I do read a lot of people say they are afraid to try agar out of fear of contamination issues. But I advise, that's the wrong way to look at it. Agar is there as a tool to easily differentiate between clean and dirty growth then easily clean it up.

Once you have a clean culture on agar, you know it's clean and you can expand to more agar, LC, LI or grain and know that what you put in either of those medias is clean. If anything comes up dirty after inoculating, it may either the receiving media or your sterile tek to blame. But you know your agar plates are clean.

With spore syringes, unless you make them yourself under very sterile procedures, they are a crap shoot. You have no idea how clean or dirty they are unless you have a microscope and the knowledge to identify all.

My thoughts lean toward your success rates astronomically improving if using clean agar to inoculate LCs with.

My 2 pennies.
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#14 Arathu

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Posted 05 March 2017 - 11:10 AM

I concur with peacefrog et. al. and add.................IMHO agar IS a tool allowing us to view and leads to the path getting away (assuming everything else is in order of course) from contamination, minimally before wasting a bunch of raw materials but also allowing us to choose and isolate mycelium that demonstrates characteristics we are interested in. (keep notes and lab books damn it........... :biggrin: )

 

.........nur meine zwei pfennige..........

 

A


Edited by Arathu, 05 March 2017 - 11:13 AM.

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#15 shoomer

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Posted 06 March 2017 - 08:32 PM

I appreciate the advice on agar, but remember I've just gotten back into the saddle.

​The plan has been (this time) to do MS into LC, 'noc BRF, pick the choicest of the fruits, do flesh to LC to isolate biggest/best pure strain and go from there.

Agar work is in the plans just not immediately.

 

I have (plastic) plates so they are a "once and done".

 

[Although...back in the day Ron Popiel of Ronco fame had this handy dandy machine that would make glasses out of bottles which would be stellar for making heavy duty petris out of liquor bottles, if reverse engineered (a couple of jigs and a glass cutter). Then all you need is finding complimentary sizes.]

The agar/scare was a little extreme, but I HATE wasting resources if not necessary (I blame engineers in my family blood line) and I like having better than 80% odds w/ once and done.

​Don't hate me 'cause I'm anal. :wink:

​I'll get there....promise! But it's probably going to wait until I have edibles to propagate.

​I'd try this week but only have the supermarket ports (and lately shitake) for material that are reportedly to have "end of strain" characteristics and the last try didn't do that well.

​The season here isn't trustworthy for postal edible LC yet as I hear it can survive a light freeze but not all the way solid.



#16 The1unknown

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Posted 07 March 2017 - 12:31 PM

Imo you've already wasted resources with the LC as they didn't work out. If you would have went to agar and cleaned up your cultures then used those to inoculate your LC then that would greatly increase your chances for success. LC is good for fast colonization not for increased success.

#17 shoomer

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Posted 08 March 2017 - 01:20 AM

Imo you've already wasted resources with the LC as they didn't work out. <snip>

 

​No offense intended, but I'll take losing a bit of time and ME over losing a petri dish forever as I have a finite amount presently and a water heater that finally ate itself a week ago means hobby cash is tight for a bit as I'm going tankless.

 

​The good news is while my LC average was 65/35 success which could be me or the syringe, every BRF (20) I have done this time from accepted LC is clean and some are already fruiting (my "trich" infections were really bruising instead).

 

I think I might have a good enough handle on sterile procedure finally to do agar and a grocery store run for some shitake/ports (and in a month or so, get some Oyster LC via post).

​It would be nice to be able to clone some woodland species this spring as I've got lots of interesting fungi that pops up in my woods (even if morels don't always),

​If I get a mind for it, I'll take my camera w/ me morel hunting this spring as last time I did I got some great shots (it was also an AWESOME year for morels).

​Thanks all!

​You'll see me again when I have problems w/ agar. :wink:


Edited by shoomer, 08 March 2017 - 01:24 AM.


#18 shoomer

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Posted 09 March 2017 - 06:43 PM

It's taken about a week, but the FC cakes are proving to be worth the time.

lil_fuggers.jpg

 

I forgot that I had examples of the "air eaters" too.

 

All BB's filled to 150ml and if you can't see the sunken sides that easily, check out the fluid levels.>vacuum.jpg


Edited by shoomer, 09 March 2017 - 07:27 PM.


#19 shoomer

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Posted 11 March 2017 - 05:42 PM

Just a small update as it might as well be a grow log too.

 

Just hours ago.......

 

top.jpg

 

and a close up of the top producers so far.

 

top_close.jpg

 

Seems my luck is running towards the good. I hope it carries over to edibles THIS time. ;)


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#20 fahtster

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Posted 12 March 2017 - 02:02 AM

Looking good! That's one way to use a cd nowadays. Lol :thumbs_up:

Faht
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