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WelkomAanTranskei
!xi

Registered: 07/26/15
Posts: 5
Last seen: 6 years, 9 months
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Maize to 5050 "experiment"
#22007973 - 07/28/15 04:05 AM (8 years, 6 months ago) |
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Good Day, Folks.
Firstly, I have obtained a wealth of information from this site so thank you to everyone who shares their experience here, it does not go unappreciated.
Secondly, hi, I am from the southern hemisphere.
On to things then...
I am hoping to do two things here: share what I learn and thereby hopefully assist others, and secondly, I hope to gain further insight into this fun little hobby by examining my own short-comings.
So, to begin. Preparations.
I have a room dedicated to plant cultures, in which I tinker with plant tissue culture. I have the basic equipment - a flow hood, an autoclave, glassware, incubator etc etc. Things like the incubator and flow hood I built myself some years ago.
Mushrooms interest me for both the meticulous care required to grow them - along with the multitude of different methods which one may employ, and of course I am as fond as the next person in taking a moment to see the world differently.
Day 0. *All working surfaces were sterilsed with a mixture of bleach and isopropyl alcohol prior to work and whenever new instruments were introduced. I took a shower prior to starting and wore face mask, hairnet and gloves, all of which were sterile.
I received two spore prints: B+ and Golden Teacher. B+ I considered contaminated off the bat as I was given only a piece of tinfoil folded over twice with no regard for sterility. I used a flame sterilised inoculation loop to scrape these spored into a liquid culture of honey and distilled water (because it sounded so interesting), which I autoclaved for 45 minutes at 21psi. A simple glass jar was used with two self sealing injection ports, one of which is to be used as a filtered pressure balance port when the mycelium is to be extracted. As I type this I am on Day 11 with healthy mycelium growth although contamination may still be present; one simply cannot tell.
With the GT print, sterility looked more promising. Using a freshly flamed inoculation loop, spores were scraped into a sterile beaker of distilled water under the flow hood and stirred. two, new, sterile syringes were used to suck up the mixture and stored in a spare gamma irradiated (overpriced) box meant for explants, in the fridge.
Now is where I make the first of my mistakes.
Day 0. +4 hours
I soaked the required amount of maize kernels for four hours in warm water in the sun. I ignorantly assumed the soaking procedure was required for the grain to absorb moisture... After the soaking I placed the grain in a massive aluminium pot on the gas and brought it to a boil. I then washed the grain under cold water for about 5 minutes and left it in hot water while I prepared 16 jars.
...second and third and fourth mistakes incoming...
Despite searching rather extensively I could only acquire 500ml jars which were appropriate. I knocked two holes in the lid of each jar. As I could only fit 8 jars at a time in my autoclave I would have to do the sterilisation in batches. I topped up all the jars with the prepared maize kernels (without care for moisture content - no toweling etc... they were sopping wet and dripping). I autoclaved the first batch for 60 minutes at 21psi... and this is where I may have redeemed myself with half the jars.... and then I had to go to the pub! So I left the 8 jars in the autoclave to cool down and I resumed the next day.
Day 1.
I checked on the jars I autoclaved the night before to inspect the maize to see what had happened to it physically. Then I autoclaved the jars a second time. Then I autoclaved the second batch of jars after transferring the first set to my incubator mycology box thingy...
***mycology box thingy***
This thing is an experiment in itself. It is controlled by an Arduino Mega and has 4 programmed settings. One for spawn, one for transfer/primordia, one for pre-pinning and one for fruiting.
The box itself is 500mm x 1000mm x 500mm and has an lcd read out for temperature, relative humidity, the current program, and space for other sensor read outs like CO2, O2, pH, lumens, which I haven't used.... There are four 30mm holes on the sides, 135mm from the bottom, which are connected to 400mm long, vertical filters I built. They are as efficient as a 400mm long tube filled with polyfil... that is what they are. On top there is a 55mm hole over which there is a second small box in which there is an HEPA filter and an extraction fan - all this is for Fresh Air Exchange, automated and Arduino controlled of course. In addition to this there is also heating, dehumidifier and humidifier set up and controlled by the Arduino to keep the required environmental conditions depending on the program running.
Its possibly needless but it was fun to build and has other uses.
*************************
Day 1 +6 hours
So with 8 jars autoclaved once, left over night, and autoclaved again, and 8 autoclaved only once, the jars were inoculated using the syringes prepared the day before. Fresh needles were used, inoculation was done in the flowhood, in the same room as the (sterilised head-to-toe with iso) myco box thingy. I thoroughly screwed up on measuring the correct amount between the jars as the needle had a habit of sticking into a maize kernel and then suddenly erupting with force and a few extra cc's of inoculant with the first few jars. After each jar was inoculated, I wiped the top with an iso solution and blocked off the holes with 3M's falsely named Micropore tape... I did two layers of tape because, lets face it; it ain't micro anything. All the jars were sealed in the myco box thingy and set to spawn, which would maintain a temperature between 26 degrees Celsius and 27.8 degrees Celsius. Being sealed in jars I didn't think that the rest was required. A blanket was put over the box, not to keep the dark out (its mohair and about as micro as the micropore tape) but just to keep up the thermal efficiency.
Day 2 - 5
Things were slow and I didn't actually check on anything until day 5. When I did there was a little mycelium growth.
Day 11. Today.
This morning after I showered, I went to check the jars. There is a lot of growth in most jars, some with sparse but healthy growth and some with good growth but suspected contaminants. These contaminants, if I assume correctly can most likely come grom the maize - having not soaked it long enough to allow foreign spores to germinate before dry steam sterilisation, right? I also have a hopeful theory in my heart that they may simply be oxidisation of either metal flakes from the lids or plant matter.
I'll put some pictures up, any input would be graciously received:
The first image is what 12 of the jars look like.

second image is evidence of pooling water due to previously mentioned ignorance as well as that discoloration. Bacteria? there is minimal growth in this one. Either way, this jar has an X and will have the contents buried in the field.

Third image is a black spot. The black spot. Two jars have this, one, which you will see now, looks very ominous, this one looks like it could be a flake of metal from the hole in the lid...? Right? Hey guys? Its just rust, right?

Fourth is lots of black spots on the other black spot jar... nooooo! It looks very ominous indeed. Both these black spot jars have been marked with a K and have not had their fate in an hole in the field sealed yet.

Final image is just interesting growth, notice the really high water content? Is this growth caused by this?

Thank you folks, if you read this entire lot, I applaud you and any input and questions and critiques you may have.
As stated in the title I plan on transferring this lot, all that are healthy that is, to a coco-verm mix, pasturised of course, over the weekend. It won't be a lot but it will be fun to observe the outcome and I shall document it here.
Now I know I have done things all over the place and didn't follow any specific instructions. I have done the PF tek some years ago in varsity and it was fun and easy and rewarding. With this I just wanted to have some fun and see what I could do. I have nothing else on the go at the moment so I have all the space available to play mycology so why not. If anything, all my mistakes may provide some guidance to another.
Peace!
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insanemike

Registered: 02/23/14
Posts: 4,272
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I'm sorry to have to tell you this op but your jars are infected with a bad case of bacteria. From reading your well thought out procedure, I think you may have over heated those jars. If you can maintain a room temperature between 65-75* F, you can leave them on a shelf in the open air. If the jars begin to reach temperatures above 80*F internally, it will begin to slow mushroom mycelium growh and will begin to promote more bacterial growth. The best thing you can do is have bad short term memory, meaning cut your losses and start over again. Good luck and don't be afraid to ask us questions, we love to help.
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swatsqad
What title? Where? whatt.



Registered: 07/09/15
Posts: 194
Loc: EU
Last seen: 2 years, 11 months
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Re: Maize to 5050 "experiment" [Re: insanemike]
#22008013 - 07/28/15 04:42 AM (8 years, 6 months ago) |
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Black spots look like spore lumps.. As for the last image, is that a maggot in the lower left??
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WelkomAanTranskei
!xi

Registered: 07/26/15
Posts: 5
Last seen: 6 years, 9 months
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Re: Maize to 5050 "experiment" [Re: insanemike]
#22008024 - 07/28/15 04:51 AM (8 years, 6 months ago) |
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Thanks for the reply, it really does help.
So, first off, my temperature settings may not actually be correct? I should be aiming for 19 - 23 degrees Celsius? In my area that is usually the temperature in winter, at night. Should I use cooling to maintain temperatures or is there some other way to curb the growth of bacteria?
In addition to this, how did you spot the bacterial infection, was it the discolouration of the liquid content?
I do not plan on salvaging anything and will commit the contents to holes in a field but apart from the incorrect temperatures and high water content could you perhaps offer any advice as to where I may improve? I know larger jars would be a start but in addition to that?
Considering I am starting over I will examine the black spots, discoloured liquid and myceliuym itself under microscope. If I can find a way to get decent pictures I will post them for interest sake...
And swatsquad, that definately is NOT a maggot! I did just check. After boiling the maize and having it cool some of the kernels cracked and left their insides puffing out, for lack of a better description... they also excreted an off colour liquid. All of this is, I assume, due to overcooking the maize...?
And guys, what cause those rhizomorphs?
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swatsqad
What title? Where? whatt.



Registered: 07/09/15
Posts: 194
Loc: EU
Last seen: 2 years, 11 months
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Well Rhizomorphic growth is AFAIK normal for cobes. As for contaminations, I should suggest you go to the contams subforum
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SteveRogers
gandy dancer



Registered: 10/24/06
Posts: 3,450
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OP next time you get a print like that you would save yourself a lot of potential headaches by putting that inoculation loop to agar first and not LC. That print was probably not clean as you said so yourself. LC can be risky even when done properly, but going straight from a dirty print is very risky.
Mike shot you straight and I would go with his advice as well.
-------------------- "General, I am loyal to nothing......except The Dream"
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Maize to 5050 "experiment" [Re: SteveRogers]
#22008157 - 07/28/15 06:21 AM (8 years, 6 months ago) |
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Quote:
SteveRogers said: OP next time you get a print like that you would save yourself a lot of potential headaches by putting that inoculation loop to agar first and not LC. That print was probably not clean as you said so yourself. LC can be risky even when done properly, but going straight from a dirty print is very risky.
Mike shot you straight and I would go with his advice as well.

Sorry to say it but ya need to pitch that mess and start over. Use agar since yer already rocking a flowhood.
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WelkomAanTranskei
!xi

Registered: 07/26/15
Posts: 5
Last seen: 6 years, 9 months
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22008235 - 07/28/15 07:11 AM (8 years, 6 months ago) |
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So far you folks have been a great help. Thank you.
I had considered doing agar but I just loved the sound of a liquid culture with just honey and water. I mean, come on, I've done liquid cultures before and they are hellishly complicated. These seemed crazy simple. I just had to. And anyhow, I can transfer the mycelium to a bunch of agar plates and pick out the best ones, cut the contams out and move on, surely? An interesting exercise at the least.
So I dissected all but 9 of the jars I had, taking samples of all the nasty looking stuff, the black spots, the mycelium and anything that looked rotten and I hauled out the old (and I mean old) microscope. Here are some pics purely out of interest. I buried all the rest of it. It actually all smelled rather pleasant, nowhere near the horrid smell I was expecting.
On to the pics... I had to hold my cellphone camera against the objective and it took some practice so the pics get better as I go. Also, excuse the artifacts... my objectives and lenses had serious fungal damage when I was given the microscope, this is as good as I can get it.
First one here is of those mysterious black spots... or not so mysterious as swatsqaud correctly identified, spores. Here we see the spores mixed in with the mycelium.

And here is a closer look:

OK, so then I went looking for some evidence of bacteria. Most of what I found was just epidermal cells from the maize mixed with mycelium but then as I was moving across a slide I found lots of these little guys (the brownish discoloration in the original image #2)I circled them:

Clicking up to a higher zoom we can see that this little cell has a nucleus, a cell wall and some endoplasm I guess. So it is not fungi and not plant. So my guess would be bacteria considering the size.

Yeah, so I just figured it would be nice for people to look at.
I am going to try and culture some of that mycelium in the LC onto agar plates, with the idea of simply cutting out the good myc to use, unless I receive a resounding no...
And as for the jars, I am going to keep at least 3 of them at the same elevated temperatures I have been using to see what happens and what more progresses - we learn through observation after all. After opening up all those other jars I, as inexperienced in mycology as I am, admittedly, I cannot see how you guys can tell it is so thoroughly contaminated? There certainly is no fungal contamination but there is bacterial, as I have seen myself, although of what I have observed I do not see any such discolouration on the other jars.
Either way, I am taking sage advice when it comes my way and rebooting, but until a new print comes my way (about 10 days or so), I am going to flog this mess to death and see what I can learn from it, if only just identifying mess!
Edited by WelkomAanTranskei (07/28/15 07:12 AM)
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Galba Cubensis
azur's handdoll



Registered: 06/16/15
Posts: 173
Loc: Norway
Last seen: 8 years, 5 months
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Nǃxau is that you bro? I thought you were dead Next time ask for the shrooms directly instead of waiting around for the gods to send you spores
-------------------- RETARDS! I'LL IGNORE ALL OF YOUR ADVICE! BUT TELL ME WHAT TO DO STILL!
Edited by Galba Cubensis (07/28/15 07:35 AM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Liquid culture is fine if inoculated with a clean wedge. Spores are almost never clean. Go ahead and use a simple sugar LC. But use a clean inoculation source like an agar wedge. I like to take a wedge with a really aggressive clone and use that for my LC. Then your playing with power. Spores to LC is like playing with a time bomb.
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swatsqad
What title? Where? whatt.



Registered: 07/09/15
Posts: 194
Loc: EU
Last seen: 2 years, 11 months
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22008358 - 07/28/15 08:14 AM (8 years, 6 months ago) |
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nice pics! The black spore spots did confuse me at first, as I had them in my forst grow. that is currently still growing. http://www.shroomery.org/forums/showflat.php/Number/21985284/fpart/1/vc/1 as you can see in the first post, I had a severe case of spore lumps
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insanemike

Registered: 02/23/14
Posts: 4,272
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22008362 - 07/28/15 08:15 AM (8 years, 6 months ago) |
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I think most of us (atleast for me) started with a ms spore syringe to a lc after realizing how long spores take to germinate on grain. For some reason when I began, I was so reluctant to really get into agar but ever since I have, I've never looked back. Agar is easy and the most reliable way to get clean cultures, nothing else even comes close.
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WelkomAanTranskei
!xi

Registered: 07/26/15
Posts: 5
Last seen: 6 years, 9 months
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Re: Maize to 5050 "experiment" [Re: insanemike]
#22008448 - 07/28/15 08:43 AM (8 years, 6 months ago) |
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That is pretty incredible swatsquad. The speed at which you reached full colonisation is unbelievable. I find it incredible just how many individual spores are in one miniscule black dot. I was not even able to view the whole dot under the microscope, yet it was barely visible under the naked eye.
I'm definately going to try and culture some of the good mycelium. I just need to get some agar - all I have on hand is mixed with hormones and whatnot for plants. Have any of you had good results with the food grade agar that you can get from the store? I assume there shouldn't be any difference in this case? I will have to wait like 2 weeks for lab grade agar.
As an interesting side, has anyone experimented with gelatin supplemented with the right additives as an alternative to agar?
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insanemike

Registered: 02/23/14
Posts: 4,272
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Re: Maize to 5050 "experiment" [Re: insanemike]
#22008604 - 07/28/15 09:27 AM (8 years, 6 months ago) |
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Food grade agar is fine, just make sure that its not got sugar added. Telephone agar is pretty good. Also speed is worthless if you don't end up with a harvest at the end. LC is a powerful tool but when it fails, the fail is often huge. Proper inoculation and testing is key to success with LC.
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Galba Cubensis
azur's handdoll



Registered: 06/16/15
Posts: 173
Loc: Norway
Last seen: 8 years, 5 months
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22009262 - 07/28/15 12:12 PM (8 years, 6 months ago) |
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Pro question of the evening: Everyone says that you have to test run your lc with one jar first too check for contams since "you can never know whats in there" (thats a quote). If you have to test run it you "lose time", compare that to the "time you gain" by doing lc since its faster. What is fastest between that (lc+test run before inoc of "true jar") and just doing it 'yi ol way'(inoc of true jar with simple agar, dont count the days it takes for it to grow on the agar since thats the same for lc)? Whats the difference in time week(s), couple of days?
-------------------- RETARDS! I'LL IGNORE ALL OF YOUR ADVICE! BUT TELL ME WHAT TO DO STILL!
Edited by Galba Cubensis (07/28/15 12:17 PM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Testing does take some time but not enough to make it a drawback. If you test it on agar you can see a lot faster if its clean which helps. Usually takes 2 - 3 days to verify its 100% good. I never test LC on grains or cakes. IMO LC isn't so much about speed as it is the ease of inoculation. The speed is simply a bonus.
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Galba Cubensis
azur's handdoll



Registered: 06/16/15
Posts: 173
Loc: Norway
Last seen: 8 years, 5 months
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22009355 - 07/28/15 12:27 PM (8 years, 6 months ago) |
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Yeah Ive never tryed out lc before actually, doing it right now while waiting for some 0.2 syringe ports that should be here any day now. Too bad I already started the cultures, itll have to be the GDSAB all over again this time too, unless one counts it as a win if they get contaminated? Then I could to it over again just for the satisfaction of using the ports (was actually planning on doing it on the balcony in a dry storm can you believe? (you shouldnt(thats a parantese withing a parantese, and yes this is the third one)))even with the ports, now go back and read that sentece again cause I know you didnt make sense of it the first time
-------------------- RETARDS! I'LL IGNORE ALL OF YOUR ADVICE! BUT TELL ME WHAT TO DO STILL!
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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I read it three times. Its still gibberish
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maddchef
Vaginal escape artist



Registered: 09/04/09
Posts: 5,602
Loc: Your mom's vag
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Re: Maize to 5050 "experiment" [Re: Pastywhyte]
#22009893 - 07/28/15 02:09 PM (8 years, 6 months ago) |
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You can either spend 4 days testing lc or you can not test it and lose the hours you spent prepping and cooking grain, the electricity/gas it takes to cook it, the money it took to buy grain, and if you grow edibles the lost income.
Your choice
-------------------- In the land of the blind, the one eyed man is king. All mushrooms are edible, but some only once..... Easier than cakes I do science and shit.
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