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Mikeallojee
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transfering agar cultures
#12209763 - 03/15/10 10:52 PM (14 years, 2 months ago) |
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http:// 
The original cultures are one and three. I used a quarter of a print to make a syringe. Zero contamination found. I did two transfers from each multispore dish making two and three from one and five and six from three. I hope this doesn't scramble anyones mind if so far, sorry if it does:)
This is the first time I have done this type of transfer wanting to select the best sections to fruit. I have done clones but the multispore is kinda intimidating. I wish to transfer the best looking mycelium. So far what I have read says to select the fastest growing edges. But thats about it. I appologize about the duplicates of one, I couldn't get the pics off once I posted. I have no idea why. myc
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wisp
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Re: transfering agar cultures [Re: Mikeallojee]
#12210483 - 03/16/10 12:52 AM (14 years, 2 months ago) |
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Pick the rhizomorphic sectors, like those on the bottom of plate 5.
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Mikeallojee
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Re: transfering agar cultures [Re: wisp]
#12210673 - 03/16/10 01:38 AM (14 years, 2 months ago) |
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How much longer should I allow them to grow?
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wisp
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Re: transfering agar cultures [Re: Mikeallojee]
#12210691 - 03/16/10 01:45 AM (14 years, 2 months ago) |
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Transfer now.
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Mikeallojee
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Re: transfering agar cultures [Re: wisp]
#12212680 - 03/16/10 01:20 PM (14 years, 2 months ago) |
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Should I transfer the Rhyzomorphic sectors and just toss the remainder dishes, or should I keep them for additional transfers?
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mycoelf
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Re: transfering agar cultures [Re: Mikeallojee]
#12213096 - 03/16/10 02:34 PM (14 years, 2 months ago) |
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Good effort, I would say that your plates look good and agree that the bottom of pl 5 is strong, also the upper left in the third pic looks good. I always keep MSG plates for "observation" You never know how a culture will work out at the time of transfer. I designate the mother culture as "M1" for mother, 1st generation.
I am assuming that we are looking at P cube, every species has a top form in 2D, even each strain, you will know these when you see them-intuition is paramount to judging which sectors to culture from. Always looking for strong rizomorphic fans showing multiple cross-linking, these nodes are the genesis of fruit formation and therefore are an indicator that shows strain strength.
Since you are troubling your self to do the work of MSG, remember to take the final step of inoculating at least 5 slants of each sub strain you isolate. Mini trials conducted through time will show the best performing sub-strains that then be cultured into many F2 slants, thereby allowing you to tap this investment in work for many years to come.
Congratulations
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12214724 - 03/16/10 06:48 PM (14 years, 2 months ago) |
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The variety is Ps. Cyanescens from northern Washington. I appreciate your commentary. I will do as you say by labeling M1 and so forth. I have a few test tubes to make slants but those buggers are expensive. I will have to invest in some more.
Mycoelf it sounds like you have been doing this for a long time. Thank you for your input. As I said above I am new to this hobby, and don't really care so much about the ingestion, its become more about the success of the operation. I would love to hear more if you care to share about the intuition piece of picking the best growth. myc
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wisp
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Re: transferring agar cultures [Re: Mikeallojee]
#12215274 - 03/16/10 08:19 PM (14 years, 2 months ago) |
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Slants are overkill for species such as Panaeolus cyanescens in my opinion. You can easily get very high yields with little or no isolation work. Take a look a Blue Helix's grow logs for example of what I mean.
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Mikeallojee
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Re: transferring agar cultures [Re: wisp]
#12218864 - 03/17/10 01:47 PM (14 years, 2 months ago) |
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Panaeolus cyanescens??? I am doing Ps. Cyanescens, is your opinion still the same?
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wisp
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Re: transferring agar cultures [Re: Mikeallojee]
#12219730 - 03/17/10 04:23 PM (14 years, 2 months ago) |
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Oops, misread. I thought that looked a little too rhizomorphic for Panaeolus cyanescens.
In that case, isolation work may be worth the effort. However, seeing as grows with woodlovers are generally dependent on seasons, isolating a single sector, fruiting it and comparing it to other single sectors is going to take a long time. Personally, I think you should just isolate several rhizomorphic sectors and combine them to make one aggressive culture.
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RogerRabbit
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Re: transferring agar cultures [Re: wisp]
#12221824 - 03/17/10 11:32 PM (14 years, 2 months ago) |
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Quote:
tripsis said: Slants are overkill for species such as Panaeolus cyanescens in my opinion. You can easily get very high yields with little or no isolation work.
Slants are for culture storage, not isolation. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Mikeallojee
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Re: transferring agar cultures [Re: RogerRabbit]
#12222077 - 03/18/10 12:18 AM (14 years, 2 months ago) |
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Whats the best way to combine the sectors. Should I combine two at a time and then add additional at subsequent transfers? Or is there a more efficient way to do this?
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wisp
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Re: transferring agar cultures [Re: RogerRabbit]
#12222501 - 03/18/10 03:12 AM (14 years, 2 months ago) |
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Quote:
RogerRabbit said:
Quote:
tripsis said: Slants are overkill for species such as Panaeolus cyanescens in my opinion. You can easily get very high yields with little or no isolation work.
Slants are for culture storage, not isolation. RR
Correct. I was not implying that they were. I was saying that you don't need slants for Panaeolus cyanescens, because there's no need to store an isolated culture, as yields are good from multispore.
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wisp
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Re: transferring agar cultures [Re: wisp]
#12222505 - 03/18/10 03:13 AM (14 years, 2 months ago) |
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Quote:
Whats the best way to combine the sectors. Should I combine two at a time and then add additional at subsequent transfers? Or is there a more efficient way to do this?
If you're making an liquid culture, just throw them all into the same jar, if not, just put them into the same grain jars, or even put them into individual jars and mix them together when spawning. I really don't think the method should matter too much.
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Mikeallojee
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Re: transferring agar cultures [Re: wisp]
#12225367 - 03/18/10 03:28 PM (14 years, 2 months ago) |
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I guess I could just cut up the agar and drop it into a grain jar. Thanks for the info.
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12248601 - 03/22/10 12:27 PM (14 years, 1 month ago) |
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Hello,
As Rodger pointed out, slants are for storage. At this time your fuzz is at a peak of genetic vitality, which should be preserved in cold storage. As the cultivator continues to transfer from petri to petri, the mycelium ages, as it does it looses the aggressive capture quality, which for obvious reasons is un-desirable. Sub-culturing into in-vitro is optimally achieved at F1, but is acceptable at f10. more than ten transfers from origins risk (in my opinion) selecting a sub-strain or causing a strain to mutate due to a breakdown in genetic vitality due to the aging process
If you are totally broke on slants the same end can be accomplished in baby food jars. Both jar and lid can be PC'd and be rigged in front of a flow bench to "slant" huge mother cultures can be inoculated from your first clean isolated plate grown out and stored either under water or under mineral oil for a great many years. When you determine that a series of descending transfers are showing signs of petering out, or if the archetypal pattern of growth changes, it is time to tap cold storage, subculture onto another plate and re start production runs. I would recommend para-filming the top of any in-vitro and the in sterile arena re wrapping in a sandwich baggie as a second barer to contamination. I also have a thermos that the finished slants go into at room temperature, providing for a slow cool down, in 24 hours the cool down is complete, I transfer my cultures into a small cooler inside the fridge so to prevent fluctuation due to fridge use or power failure. I rigged a small remote digital thermometer to the cooler so I can monitor every time I walk by. I maintain at 42 deg Fahrenheit.
It is hard to comment on the process of intuition. It comes to mind that in a MSG that I did a couple of years ago, on an Indian strain of PC, there was many sites of white fuzz on a very large 100mm plate, maybe 75 to 100, there was one particular site that had not only germinated but run a maze that looked like a game of tetris and had squeezed thru bottlenecks created by other points of origination, but by far had captured more of that plate than any other contender. After deciding where start and finish was on this particular fuzz, I sub-cultured the leading edge of this isolate and it became one of my best performing strains of PC ever. I suppose intuition is a feeling and a connection with the fuzz and deciding where on the table you are going to place your bet, as I see every plate as investment in resources, time, money, and most importantly you cant run out and investigate every culture so bottom line, the cultivator picks based on appearance so many strains and the success or failure of those impending runs is based on those picks, AND intuition is an integral part of this selection.
I concentrate very hard on strain work, as it would seem to be the base of successful cultivation, if you are working towards the end of small production, i.e., for home consumption multispore inoculation will work and the spores store infinitely in a very convenient package. It is my observation and is also reflected in the literature, that multi strain cultivation will produce results, but not the same as an isolated strain given the same conditions. Some strains would rather vegetativly run the substrate a.d. infinum, never moving towards fruiting, while others originating from the same MSG will perform notably better. I maintain actively around 20 species, and have many strains of isolate for each species that I maintain. I make no less than 5 slants for each isolate, which adds up to many hundred slants. A great many of my strains come from wild clones, which always amaze me at the demonstration of vigor and aggressive domination of niche'.
http://www.emsdiasum.com/microscopy/products/preparation/vials.aspx
This is a website that I really like for finding the perfect container for sterile work.
Keep up the good work, I am glad that you are working so hard to understand a very difficult process, but on which has many rewards to those who achieve mastery.
Blessings of the mycelium be upon you.
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12251214 - 03/22/10 08:13 PM (14 years, 1 month ago) |
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Wow thanks mycoelf for taking the time to help me along. I understand what you are saying and it is very encouraging. I fell in love with mushrooms a long time ago, I now have fallen in love with mycology. I enjoy watching the mycelium run across the agar.
If I understand correctly the white fuzzy mycelium is the most aggressive and is the best to store on a slant. I planned on taking two slants from one or two of the petris but was not sure which would be the best to do so from. It probably doesn't make a difference.
Will the white fuzzy mycelium turn rhizomorphic at some point or will it continue to be white fuzz?
It sounds like I have a ton of transfers to do. The bear of it is that unlike cubes the cyan's take a longtime to fruit so a year has to go by to know if you did it right. I plan on doing several patches. I think I will do a few multispore to ensure success and a couple isolates just to play and go from there. Thanks again for the replay mycoelf and the insightful tutoring. -myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12252273 - 03/22/10 11:10 PM (14 years, 1 month ago) |
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Basically I sub the most promising sectors, and feel that you have to challenge the myc nutritionally by changing up the agar, especially the protein base. At some point you will get a fan or sector that is showing strong rizomorphic growth, that is the section you want to sub from. I use a liquid process called myc fermentation that is detailed to some extent in Stametes growing gourmet and medicinal mushrooms. The mother culture must be in no less than prime condition at the inception of the spawn run as the few micro grams of myc on the petri can easily give descent to many potential tons of captured substrate.
I think it is ideal to slant culture in prime condition, but if you are approaching generation f10 or greater it will be better to slant young mycelium than to chase an ideal that you may not achieve easily or soon.
With PC it has always been easy to get the culture into shape just by selection and changing the nutrient medium from transfer to transfer. for spawn runs I sub from a prime culture to generate F2 that then mother liquid culture, from there I can create 7 to 60 1 quart grain masters that can each transfer to as many as ten 5 lb bags of substrate when you put it into that perspective, why would a cultivator use less than a prime origin??
I don't know what the archetype for p cyan is as I have never kept it, but I would love to find out some day. I have always dreamed of taking a road trip to the Columbia river head waters to collect and possibly field clone a wild specimen, as those strains closest to nature are usually more vigorous yet less tame than a strain that has been through many generations in captivity.
Light and peace
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12258099 - 03/23/10 08:37 PM (14 years, 1 month ago) |
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I found a log this week with some nasty looking oysters on it. I plan on going back hear soon to see if any new ones have come out so I can clone them. I also have plans to visit point disapointment this fall and do the same...collect some wild speciems. I was thinking of cardboard and some agar dishes Azures too. But for now I only have a print that someone here gave me out of the kindness of their harts. myc
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Mikeallojee
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Re: transferring agar cultures [Re: Mikeallojee]
#12258167 - 03/23/10 08:49 PM (14 years, 1 month ago) |
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I had no idea that changing agar from transfer to transfer made a difference. Is there a proper order of sequence for transfers as it applies to expansions and which type of agar to use when? myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12265896 - 03/24/10 08:59 PM (14 years, 1 month ago) |
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Mycelium develops substrate specific digestive enzymes in order to digest what it is on, If you culture only on MEA, eventually just like us the myc gets "bored" with the same old same. If I know I am doing a specific grain master the agar for the mother plates on the spawn run gets a pinch of every thing that the myc will encounter in that planned expansion. If the final sub is wood, I obtain a sample of the intended wood and a pinch goes in the pot. This may be over kill in some viewpoint, But I have demonstrated to myself and it is also reflected in the literature that "leap off" time, or the time between inoculation and the time aggressive capture starts can be delayed by the myc having to stop and develop digestive enzymes for a sub that it is unfamiliar. keeping the base grain and the source of protein changing challenges the myc and encourages archetypal growth.
To me this is why LC and specifically fermentation is so important in a big production run, also in other instance, a questionable sterile arena, this enzyme preparedness may be the difference between a more rapid colonization that overwhelms minor contams preventing failure, by not allowing 24 to 72 hr for the contam to get a leg up. Nature is all about niche and dominance, when we create a sterile vacuum for a myc to colonize it sometimes is that a perfect job is not done, in small percentage I have seen a tiny spot of trich or asperilligus get overwhelmed and killed by a rapidly colonizing myc.
In reference to your aspirations of bulk outdoor substate left untreated, this could make a difference that would matter.
I keep a grain storage facility for makeup in a special bin, some of my favorite grains for strain work are,
Rice white millet red millet quinowa wheat
My favorite protein sources,
High quality dog food spirulina peptone oats soybean meal Salmon/Sardenes (believe it or not)
in every solution I add an extra light Malt Extract as a basic source of easy calories, The ratio of the other grains is never meant to be specifically nutritive, only to flavor the agar so the myc is ready when I open the gate for a rapid expansion.
When I am mixing for slants I go with a strictly MEA with a double agar content, In that instance the medium is to no other end than grow out and storage. The double agar ensures a firm sub to work on in tight quarters and that it will stay conformed to the container.
I also activate a specific wine making yeast manufactured by Lalvin 71b 1122.
I use this strain becuase it nutritional requirements are low and it is easily activated on ME The culture is inactive saccharomyces cerevisiae, I add a generous amount of yeast, let to activate on the surface before agitating for 15 min or more on a stir table. This activates the yeast and then the autoclave kills them. I believe that higher fungi benefit from the fact the the yeast mineralize nutrition from the sub then making it more available to the myc. I also believe that the myc will digest said yeast both living and dead on a sub.
And as always (I seem to say this allot) refer to chapter 12 of Stametes Growing gourmet and medicinal mushrooms, GGMM for further reference
And don't forget the hotly debated peroxide- it makes an unforgiving substrate a little more doable for the common man. See Rush Wayne or Kerry Orgame for formula.
Fun fun fun with agar
Blessings and light
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12279877 - 03/26/10 10:26 PM (14 years, 1 month ago) |
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Its taken me a while to digest what you wrote. I now understand that I don't know shit yet and have a lot of learning ahead of me.
I think I understand some of what you wrote. Fermentation? Is something I don't understand. I will also refer to chapter 12. Thanks again for the schooling. -myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12284363 - 03/27/10 06:05 PM (14 years, 1 month ago) |
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knowing that you don't know is the point of the wheel where knowledge becomes wisdom, and is the first step onto a new path. The rewards of knowing where this one leads are infinite.
I am a Stametesian, I don't worship the man, but subscribe to his school of thought regarding mushrooms and their cultivation. He is an excellent teacher in his many books and does not tell you what to think but merely suggest which questions to ask. To me this is the core of the scientific process.
The process of mycelial fermentation is one of a myriad of forms that Liquid culture can take. The man who tutored me in mycology taught me a process called myc fragmentation. In short you take a kick ass culture in petri, transfer it as a whole into a sterile blender like device and chop it to bits using a very sharp high speed blade. There are special blenders that are sold for this that are fully autoclavable and very expensive. I am a retired metalworker and manufactured mine. The result of this is a liquid solution that contains huge quanitys of slightly damaged hyphae in solution. This solution can be applied directly to a grain substrate for further colonization. The advantage there is that one 100mm plate fragmented into 600 ml or so can inoculate 60 or so 1 quart grain masters each at a substrate weight of @ 200 g. Each of these grain masters could the innoculte from 1 to 10 5lb bags of end substrate or to create second generation grainmasters, wich then could inoculate a bulk substrate at @ 20% of volume.
The point of this really is to take one really kick ass culture in run out to quite potentially hundreds or thousands of pounds of spawn. Just depends on how you design the spawn run which moves you make.
To generate equivalent spawn using traditional technique 2 wedges become 1 master, then in turn becomes 1 to 10 bags. grow out is slower as the sub is not as uniformly inoculated, more leap off points, quicker colonization. you also have to generate 1 plate for every six grain masters. LC gives a tenfold advantage, and also allowing the cultivator to create large spawn runs using only the cultures in most premium form.
When I cultivate a poly pore I use the traditional technique. My Reishi strains grow so aggressively a master inoculated w/ 2 wedges grows out in 4 days with one shaking, overgrows in less than 7. No real need to ramp up spawn there, as the old way suffices.
But take a stropharia like Stropheria Rugusoannulata, the red wine stropheria, (also a close relative of PC) grows very, almost painstakingly slow on agar the strain that I have takes 2 to 3 weeks to make a plate, consequently the cultivator has to make many many plates to achieve enough agar to make 60 GMs, but with liquid spawn, one plate could make 60 GMs' which could fold out according to the above scenario. using traditional technique, a GM1 might take 3 or 4 weeks to grow out. Even longer in a 5 lb bag.
But fragmentation is only half of the story, you will remember that I said that the fragmentation process slightly damages the hyphae. Fermentation is the process of adding your above described LC into a sterile vessel that is being agitated by a stir plate. The traditional vessel is a Erlenmeyer Flask, I have done it in a ball jar. The recipe for the media is allot like that for agar, except there is no agar, obviously because you don't want it to solidify.
After a 24 to 72 hours on the table the myc has a chance to heal severed ends. every where it has healed a bud forms in the segment previous to the cut. each bud is a branch that gives rise to aggressively running myc that conquers territory after innoc so fast that thermo-genisis becomes an issue during incubation. If cultures are stored too closely the thermal insulative property of wood can actually retain so much heat that the core temp an exced 109 deg F killing the spawn.
Fermentation also gives the cultivator to add a representative sample of all the substrate along the path of the planned Myc expansion. In the time of fermentation the myc has a chance to not only heal from the frag, but also to develop digestive enzymes for a specific sub that will be encountered, thereby reducing the leap off time to near zero. The remaining nutrition inside the fermented culture serves a a nutritive base for the myc to consume during the process of leap off onto the grain. I use whole grain and it takes some effort to penetrate that PC'd hull and start to gain a foothold.
I am sure that there are forum members out there that frown on the process. There are really only two draw backs, the first is the danger of thermo genisis, the solution there is proper placement of cultures in a properly regulated environment. The other drawback is what intimidates most cultivators.
This is an all or nothing technique!!!
If you fuck up the setup for the fermentation by allowing a contam into the bottle, it multiples with even a more exponential force than using the traditional technique. when I was pioneering this for myself I once accidentally included a scarid fly into the broth. the result was the most uniformly contaminated grain masters I had ever generated. Biggest mess that I ever had to remediate also. It took a week of cleaning before I was at ground zero.
But you shouldn't let that scare you, for this one fuck up, I have pulled off so many successful operations it is beyond count in my measure.
This pic is Two cultures sitting side by side, They are of the same strain of a Strophariaicae. The bottle on the left is 72 hours ahead of the other, which was freshly inoculated in the pic. note the dramatic color change This is due to a massive folding out of the myc while in the bottle, due to the fact that it is in a perfect short term environment.

This pic shows a prototype of the breathing apparatus that I have developed to supply sterile gas exchange to the culture. I found that passive Gas exchange was insufficient for the needs of the culture. I went thru allot of trial and error to perfect this technique as there are allot of delivery and transfer obstructions to pull off in sterile arena. I worked R+D on this for about two years before finally settling into a routine that works as good as I know how to make it.

Blessings and Light find you
Mycoelf
-------------------- 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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RogerRabbit
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Re: transferring agar cultures [Re: mycoelf]
#12285138 - 03/27/10 08:49 PM (14 years, 1 month ago) |
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Quote:
mycoelf said: Mycelium develops substrate specific digestive enzymes in order to digest what it is on, If you culture only on MEA, eventually just like us the myc gets "bored" with the same old same.
Prove it besides reading about it in GGMM.
I also took his seminars, including the masters seminar ten years or so ago when he still did that one. However, I've seen no evidence of this 'memory' or 'boredom'. I use MEA exclusively and have for three decades. I've kept master slants on MEA for many years. Most of my strains are ten or more years old and have been on MEA the entire time and they still perform as well as the day they were made.
I've also not seen one whit of evidence that adding some of the final substrate to the agar makes the slightest difference. The mycelium jumps off the agar to the grains and jumps from the grains to the sawdust or compost just as fast as ever. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Mikeallojee
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Re: transferring agar cultures [Re: RogerRabbit]
#12285700 - 03/27/10 11:32 PM (14 years, 1 month ago) |
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I hadn't thought of liquifying a full agar dish. Mycoelf you said that you will take a 100mm dish and turn it into a 600ml LC. In order to do this do you have to place the liquified agar to the flask on the stir plate? Also do you add sterile water to dilute the solution so it isn't so thick?
I thought that I had read that when making agar you want it less nutritous than the spawn so when it is introduced it will grow faster because of the nutritional boost.
I didn't know that mycelium has to create a new enzyme each time it encounters a new substrate. It sounds like RR has never encounter evidence of mycelium memory. I will be honest this is the first time I have heard about it. It sounds like a neat idea and plausable if in fact that mycelium has to develop unique digestion enzymes depending on the food source. Mycoelf have you noticed a signifigant difference it leap time?
I have been growing reishi and grey oysters on cardboard and I have learned that not all cardboard is not the same. Nike shoe boxes work well, the cup holders from fast food restraunts easily attract mycelium. Cerial boxes don't seem to work very well though. I was thinking if I was to soak some of the cardboard in water and then use the water to make the agar or maybe soak the grain in the cardboard water with some gypsum and coffee. What do you think?
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RogerRabbit
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Re: transferring agar cultures [Re: Mikeallojee]
#12286549 - 03/28/10 08:17 AM (14 years, 1 month ago) |
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Quote:
I thought that I had read that when making agar you want it less nutritous than the spawn so when it is introduced it will grow faster because of the nutritional boost.
Actually, it's the opposite. Mycelium grows fastest on a less nutritious substrate. That's why a 40 pound straw log can colonize faster than a 3 pound manure substrate, even though the manure is inoculated with a spawn rate of 1:3 and the straw inoculated at 1:20.
Mycelium doesn't have to evolve to grow on various substrates. That seems to be one of paul's pet theories, but I've never seen it in action.
With shoe and cereal boxes, etc., that's chipboard, not cardboard. It's the final recycling process and can't be recycled again. I roll them up into logs and use them for heat in my wood stove. They burn almost as long as wood logs. Most of them will support mushrooms though, especially oysters which aren't picky at all about substrate. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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badman


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Re: transferring agar cultures [Re: RogerRabbit]
#12286566 - 03/28/10 08:26 AM (14 years, 1 month ago) |
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To be fair to Paul his theory is not that far fetched, its similar to operons.
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RogerRabbit
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Re: transferring agar cultures [Re: badman] 1
#12286575 - 03/28/10 08:31 AM (14 years, 1 month ago) |
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Paul's a long-time friend. I wasn't attacking him, just disagree with that theory based on experience. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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badman


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Re: transferring agar cultures [Re: RogerRabbit]
#12286771 - 03/28/10 09:28 AM (14 years, 1 month ago) |
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I know you werent trying to buckle his self esteem. I havent noticed any difference either by adding sawdust or horse shit. But if this theory is in fact true then I wouldnt be that surprised. I can see why Paul thinks this.
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Mikeallojee
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Re: transferring agar cultures [Re: badman]
#12289777 - 03/28/10 09:11 PM (14 years, 1 month ago) |
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When using the chip board for oysters do you roll it up like a log or just tear it up? The first few bags Ive done have smelled really bad after a few days is this normal?
I noticed that the mycelium both from Ps. Cyan has begun to grow over each other kin a layering form on the agar. I had hypothesized that they would meet being from the same print and just merge creating dikaryon. How off am I? Are there spores from the same print that are not compatible for mating? -myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12295314 - 03/29/10 08:43 PM (14 years, 1 month ago) |
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Quote:
Quote: mycoelf said: Mycelium develops substrate specific digestive enzymes in order to digest what it is on, If you culture only on MEA, eventually just like us the myc gets "bored" with the same old same.
Prove it besides reading about it in GGMM.
I can't say that I am prepared to prove my observations in an empirical sense, I would have to set up some serious control, work over hundreds perhaps thousands of generations of transfers, develop strict protocol for appearances, then go and analyze which enzymes were in every sample.
I wish I could prove it, if i was a giant corp or the gov, Id' would just cut a huge check and wait for the report..
Respectfully I can say that I can only attest to my own experiential observation.
The man who was my first teacher was basically a hobbyist who worked quite prolifically with one particular strain of PC, a Golden teacher. He was working with a hood on agar and was fragmenting cultures and inoculating PF style jars with the slurry. His sense of genetic management was a continuous descent of transfers onto the same store bought agar. He made no note of the P value system, but made a new generation every couple of weeks. He said that he noticed no problems or changes with the myc over that period of time. His technique was to specifically innoc each dish with as small a fragment as possible. The myc responded from this position with four perfect fans on quarters of direction, corresponding to the four sides of his agar cut. his solution for the inevitable genetic crash was to refer back to a spore print. That worked for him, but was not satisfying enough to me, so I hit the books and the main bible of study was of course, GGMM.
Well, the prover proves what the thinker thinks. Having taken late gen cultures from the aforementioned strain I was convinced that the behavior that I was noticing (a degradation in to cottony sectors) was a result of poor nutrition. I was incorrect, but as the part of me that thought this wanted approval from the part of my mind that validates, I issued a vain attempt to "save" the strain. I started manipulating agar in make up searching for the one magic ingredient that would make my sacred strain live again. In parallel transfers from the same mother cultures, splitting the best fans on edges into multiple plates, I observed that where I loaded protein (quinowa) and High quality DFA (sardine based) the myc jumped and for a few transfers tried to resume archetypal growth.
In essence it would be my experiential conclusion that diverse and challenging substrate stimulates the myc to try harder even with limited genetic resource.
Point is that the same old IS the same old. What is true in the lab may not be so in the field, and struggle in the context of natural conditions ALWAYS involved diverse multi part nutrition, some of which may have been encountered previously further up the trail. It is Myc Vs. Myc out there and waiting around to find the exact combination to a nutritional lock is precious time lost. When you go out into the field looking for cool mushies to clone you are always looking for diverse intermediary habitats- places where flood, land slide, fire, cattle, whatever has created small scale disaster. Those environs are loaded with diverse origin of debris, The myc runs between points of best nutrition looking to capture the most squares first- competing in niche' with other higher fungus, tho that gets the most wins and there are acrobatics in between, gaps to jump, areas of inorganic material etc. Nature is rarely homogeneous.
Also noting the mycellium is very old, as old as the first life to come up onto land, a true survivor. In that time in it's descent from pre history I am going to say that there were times in earth history that were more than challenging. I would argue that adversity is a positive pressure towards retaining the advantage of the retention of a nutritional memory. I do think that this memory fades past a factor of myc expansion.
You may say with an empirical club that I can't "prove" such things , but to me all of those factors given interplay seem reasonable to allow the jump to infer that mycellium has developed a few tricks up its Sleeve. NOT having a memory for specific sub that was encountered within a perimeter of expansion would seem to me to be a distinct disadvantage, we know that Myc can run sub specific maze (i.e.mycellium running) Why does this seem so unreasonable???
Besides the observations above my INTUITION is ok with the idea of that being true. Alternative mycology, which this I presume this forum is, has got to leave room for those mechanisms that serve us where the "as taught" scientific method fails us. I am fascinated by sterile culture and have spent hours looking and meditating on specific strains. They are like little pets, an entity that is more than just an embodiment of running mycellium. They have a awareness that extend outside the petri- connected on the cosmic level. I feel as if they communicate and teach in a very silent and passive way, the cultivator who looks at a culture for ten seconds before conducting transfers and running off mentally to all else that must be accomplished in his day misses the connection- that hidden message that is there to guide us. A principal that I find valuable to be sensitive to and remains part of the myco world view that I pass on to others. All Ideas that I submit in this forum are for the stimulation and entertainment of you all. None of my views represent the be all or end all of all discussion. When I leave the established track of what the empirical book tells us it is not in spite of the established institution, but only to honor and seek out the chapters of those books that are still blank as they are unwritten.
All I ask of those of you, my esteemed fellows, is that the more skeptical of you agree that a belief and the suspension of dis-belief are two separate modes of looking a singular object. You don't have to believe anything because I do, I am not asking you to by perpetuating my feelings on any given subject, but, Staying open to the suggestions of other may lead you down paths of investigation that may lead somewhere we would all like to go...
The blessings of the mycellium find you,
Mycoelf
Ps I am enclosing a pic OF a PC Super KOH Sammui that I manipulated by placing a sterile Phalaris Arundinecia grass seed, A grass that in Nature is known to have a symbiotic relationship with some Psilo Spec. Not only did I get an amazing archetypal myc pattern, but also multiple fruiting, It is important to note that this is a 60mm plate and the mother mushroom in the middle is almost 2" long, note the spore print on upper lid!!!!
-------------------- 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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mycoelf
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Re: transferring agar cultures [Re: mycoelf]
#12295477 - 03/29/10 09:10 PM (14 years, 1 month ago) |
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Quote:
I hadn't thought of liquefying a full agar dish. Mycoelf you said that you will take a 100mm dish and turn it into a 600ml LC. In order to do this do you have to place the liquefied agar to the flask on the stir plate? Also do you add sterile water to dilute the solution so it isn't so thick?
I have tec that is still in rough draft that I wanted to publish on shroomery, there are pics, but for now I will offer a brief over view.
The flask with the nutrient solution are autoclaved for 45 min at exactly 15 psi. I made a container that is a stainless steel cup that has a tight fitting lid whose center accepts a modified cutting blade I adapted from a "smoothie" blender, the type of hand held blender made to liquefy ingredients for the drink, I replaced the bearings and modified the blade. The cup is pc'd with 150 ml of water. after cooling to below 90 F I open a 100mm dish, cut into sections, both concentrically and radially, and use the scalpel to transfer the sections into the cup. 3 one second burst followed by 1 one second burst at high speed grinds the culture into little bits and liberates thousands of hyphae into solution. The medium is then transferred into the flask via sterile Pyrex funnel, the breathing apparatus is para filmed on, then 72 hours later is delivered to whatever the next gen substrate is.. I peroxidate the culture before inoculation, and transfer via syringe, free pour, peristaltic pump etc.
In answer to your second question regarding leap off, yes I have noticed a dramatic difference. Same strain, sister dish, fermentation vs fragmentation alone time difference for PC captureing a PF style jar goes from 23 to capture, over 30 to fruit, to capture in 14, fruit in 17 days.
I am not a math guy but just by looks somewhere around a 50% improvement.
Peace
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12315964 - 04/02/10 02:32 AM (14 years, 1 month ago) |
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The petri dish looks cool. You added a grain of rice to the dish and a couple mushrooms grew? Did you expose to fruiting conditions or just let it sit in closet? myc
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RogerRabbit
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Re: transferring agar cultures [Re: Mikeallojee]
#12319330 - 04/02/10 05:36 PM (14 years, 1 month ago) |
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Quote:
The flask with the nutrient solution are autoclaved for 45 min at exactly 15 psi. I made a container that is a stainless steel cup that has a tight fitting lid whose center accepts a modified cutting blade I adapted from a "smoothie" blender, the type of hand held blender made to liquefy ingredients for the drink, I replaced the bearings and modified the blade. The cup is pc'd with 150 ml of water. after cooling to below 90 F I open a 100mm dish, cut into sections, both concentrically and radially, and use the scalpel to transfer the sections into the cup. 3 one second burst followed by 1 one second burst at high speed grinds the culture into little bits and liberates thousands of hyphae into solution. The medium is then transferred into the flask via sterile Pyrex funnel, the breathing apparatus is para filmed on, then 72 hours later is delivered to whatever the next gen substrate is.. I peroxidate the culture before inoculation, and transfer via syringe, free pour, peristaltic pump etc.
With the exception of peroxide, that sounds like exactly the method Paul teaches at his seminars. I take it you attended one? RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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mycoelf
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Re: transferring agar cultures [Re: RogerRabbit]
#12320678 - 04/02/10 10:18 PM (14 years, 1 month ago) |
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No, But thanks for the complement, it is on my wish list to do so. I know that I would benefit with a more experienced person looking over my shoulder, critiquing hand motions and technique. I would also love to qualify for the advanced class so that I could hear more theory-
I do one thing differently than Paul does, when I first tried this fermentation technique, I misread the information and added LIVE yeast, as opposed to yeast extract. I know that sounds crazy, and I am not sure what is happening on a micro level, but I experimented with the addition of a specific wine making yeast. It took a few failures to figure out proportion, but as soon as the flask cools I add @ 2g of the inactive yeast in sterile arena, and give them 15 min to float and do their thing. I usually run my fragmentation protocol while the yeast is activating, after adding the mother culture I start the spin, so I am running both cultures PARALLEL, then at the end I peroxidate and the yeast are destroyed. My running theory is that the myc is grabbing the yeast as well as the ME while they are also healing from the frag and training enzymes for the next substrate.
I have also experimented with not killing the yeast, and injecting the parallel culture into PF style jars, PC takes forever to fruit but captures in record time. The fruits are unusually dense, have a yeast tinge where they should be white and are very heavy. I would say experientially that evidence points to an unusually active flush considering I ran same strains parallel and did a taste test side by side on week after another.
 I would really like to talk more about this, but the lady of the house is baking choc chip cookies just in time for munchies--
who-hoo, munchies... Mycoelf
-------------------- 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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mycoelf
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Re: transferring agar cultures [Re: mycoelf]
#12320736 - 04/02/10 10:30 PM (14 years, 1 month ago) |
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Quote:
Did you expose to fruiting conditions or just let it sit in closet? myc
I have a special incubator that uses a 15W full spectrum compact fluorescent as a heat source. The bulb is wired to a thermostat. intermittently the bulb fires and gives the light exposure required to initiate pinning on petri's, for the purpose of evaluating a strains willingness to pin. I sterilized the RCG and dropped it at the perimeter of a new but established culture. The section in the center got real thick and did not colonize further, then it exploded radially with the thinner "running" myc, and when it hit the boundary, it fruited. If you look in the pic there are many "nodules" that would give rise to many fruits under different circumstance. I.E a spawn run.
what amazed me is the spores that it dropped in culture.
I had never seen a sporulateing pin on a dish before that, This strain originate from a clone that had been spawned using the fermentation method. I don't know if it means anything, but i could hardly coax the myc to colonize much less run.
lATER
mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12326257 - 04/03/10 10:10 PM (14 years, 1 month ago) |
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It is clear that you have a lab to work in unlike most of us who use our kitchen sink and counter. How big is your lab mycoelf?
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12327832 - 04/04/10 08:44 AM (14 years, 1 month ago) |
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I have a space that is 18X 18, and 2 stories. This space is divided into a clean room that is 12X12, a spawn storage room, that is 8x8 and a fruit room that is 8X11. Th remaining room is 8x 10 and is a makeup, extraction lab, and is where I autoclave. It is my 3rd lab. My first was a spare bedroom, my second was built into a auto garage and was 11X15. I had every operation except autoclave in that room, and boy let me tell you it was tight.
My current lab is currently under construction. I finally bought property and it had the above mentioned space already built and I spent the winter framing out. I still have a bit of wiring and sheathing to do and I will be ready to work again. I have not worked in sterile culture since November and boy do I miss it, The last time spent in the old lab was a mad dash to get all of my new clones for 09' into In-Vitro, And I am happy to say that they are all looking good and happily chilling out.
I am including a pic my last work space- just for prosperity.
Happy belated Osatra,
Mycoelf

-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12331941 - 04/04/10 08:42 PM (14 years, 1 month ago) |
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11x15 would be a dream at this time of my life. The kitchen blows to say the least. You have flasks and a flow hood yes definitely a lab and a nice one at that. We have an offer on new house with plenty of space so hopefully our offer will be accepted and my lab will be in the works in the next few months.
Back to agar...
I have been using fungi perfecti's antibiotic agar and am just about out. I have some agar that I bought at an Asian mart and have some potato flakes. I was reading about making ones own agar but I have not done it yet and was wondering if you have any pointers or should I just purchase my agar of the next meaning the kind that you just add water and autoclave. myc
Ps I did the transfers you suggested and they are taking off and looking great. I took to slants and now am soaking my wood chips that I plan to inoculate her in the next day or two.
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12332391 - 04/04/10 09:57 PM (14 years, 1 month ago) |
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I started with FP agar- and high quality agar it is. when I first started, I was afraid to make my own because I was afraid that I would mess something up and my cultures would suffer. I was also convinced that I could not live with out the antibiotic gentymiacin sulfate. I was just expressing insecurities about my noobieness. I used AGAR AGAR from a health food store, it is with the bread making stuff for an additive to make bread moist, and went from there. I use the kelp base agar, I don't know why. One of my strains is P carulecens, incidentally I found in ancient archival material that this mushie HATES rye and will not grow on any medium containing rye. I don't remember if I called FP and asked or it was on the label, but I believe that his base is wheat and rye, explaining my poor success with that strain on FP AGAR, live and learn. FP claims that their AGAR is a superior lab grade, and I believe it to be true, but you can buy the AGAR alone outside of the premix. I found that the FP AGAR slices and dices nicely under scalpel, but resist dissolving in fermented culture. The Health food AGAR dose not pour as clear but dissolves nicely in Fermentation. The reason that this is important is if your method of delivery involves passage through a fine port, such as a needle on a syringe or a peristaltic pump. I would encourage you to experiment with your own make up and record your base ingredients and note in your system somehow which agar is which, I always pour new plates before I run out and sometimes it is helpful to have different kinds depending on the type of work that you are doing. I have a clean cooler in a sterile enviorn that I keep blank AGAR culture in so they are all in one spot when I go to work, and I avoid useing a blank the same day it is poured so I know that it is a clean plate when I start. I also use sandwich baggies to store my blanks so that nothing comes in from the outside before use. I wipe them down outside and place 2 100x15 in each bag and use as needed. I don't usually keep them in the bag after the innoc, but sometimes do as an extra measure of quarantine if I have made a clone or i am not sure of a culture's purity. I also parafilm every inoculated culture
I know that someone on this list poo-pooed peroxide in agar, but I told my mouse story previously in a recent post here it is copied
If you are poring from a flask remember that the #1 vector is contact. I have a couple of silicone pot holders that I pc with the agar, one to provide grip and thermal protection from the hot flask and also to allow a firm grip lower from the lip, the other to set the flask on if for some reason I must set down he flask. This prevents rapid cooling when doing a large run. If your clean alcohol-ed gloved hand comes closer than 1 inch to the top you will probably contaminate. I set up an agar run so that once the flasks picked up it does not leave my hand until I am done. I use the stack method in GGMM, while intimidating to the novice is easy once you achieve practice.
A steady hand is also important, I usually smoke a bowl while I wait for the PC to achieve Zero psi.
Last year I had shut down working over the winter a couple of months and a mouse moved in in my absence. First thing after an opening deep clean I poured peroxidated agar and had fumbled the lid on one plate, so I left it out on my table overnight for "observation" The next day I came in to find the lid off the plate and the agar had bee munched by the starving mouse. Deeply curious as to the real power of peroxide I wrapped the plate and quarantined it in an incubator, expecting to find contam inside 72 hours.
six weeks of observation yielded no contam whatsoever. I was astonished as I discarded the plate
end quote
This is my testament to the power of peroxide in AGAR. It does not survive the PC but I prepare a 10 ML graduated cylinder in foil and peroxidate 8ml in 600 ml of agar. I pour and swish to distribute throughout. I keep my peroxide in the fridge and I usually forget to let it warm up before hand so it usually goes in cold. I know that Logic says that it should breakdown but I have proved to myself that it is still active not only by the above story but also years of observation.
I also noted that it seems as if you are going to inoculate your wood from AGAR?? This probably wont work if that is the case. The point of the intermediate grain master is to not only provide many points of inoculation but also to provide a nutritive base for the myc as it gains a foothold in the bulk sub. 10 to20 % by volume is a good base. Less than 10% and you run the risk of the other critters overrunning the sub or an extremely slow/partial capture.
You might review GGMM for the model onto a bulk sub using grain masters, You might also check out Myc Running, there is a tec in there on how to do a dirty clone on cardboard and then a strategy for laying out outdoor beds, believe it or not this tec features the very same mushie that you are attempting to cultivate.
You are looking for a 38 to 50% moisture content in your wood for colonization. I usually do this by "feel" but you could weigh a representative sample in a container at 100g, then bake at 350 for three hours and reweigh. Don't forget to tare the container. the second weight is the percentage of dry mass minus the water, then you can do the math.
I could not decrypt the PM you sent me as I am not yet a proper member, could you resend your reply with no code?
Good luck with your agar.
Mycoelf
-------------------- 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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iluvfungi


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Re: transferring agar cultures [Re: mycoelf]
#12332771 - 04/04/10 11:24 PM (14 years, 1 month ago) |
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Unless I missed something, if all you are doing is keeping cultures, isolating cultures, growing fungi; it ain't that complicated.
Perhaps some people like Paul, who are much more intellectual then the simple hobby of growing fungi, blow it up just a little bit too much.
It only gets complicated if you are going into the genetic level of fungi, then it is very complex. But still, with time even something that is complex is no longer complex.
This just goes to show the sad state of the philosophy of science; I see something, it happens a few times, I call it factual knowledge. Given that people work as one, and everyone experiences group think, it makes one question any and all scientific knowledge.
Back on topic; transferring agar cultures, working with agar was the simplest thing I have ever learned in my entire life. It is just so natural. If you have a sterile environment, contamination is almost like a blessing to do more work, since it happens so infrequently.
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PrimalSoup
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Re: transferring agar cultures [Re: iluvfungi]
#12338922 - 04/05/10 10:10 PM (14 years, 1 month ago) |
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Quote:
it ain't that complicated.

Nah, you're right. The simply doing part isn't all that complicated once you get it sussed.
But the "now why the hell did that happen?" part is something else entirely. And tends to be far more interesting as well...
BTW this is a great thread. 
Peace -PS
--------------------
if you stand too close to the machine it'll start to eat youPrimal's simple tested teks and projects: Wheat Prep 2.0 Acidic Tea Tek Potency Project!
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Mikeallojee
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Re: transferring agar cultures [Re: PrimalSoup]
#12340192 - 04/06/10 02:08 AM (14 years, 1 month ago) |
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Thank you for all the information on agar, you have saved me many years of trial and error. I read that you do some deep archival research, you must have been doing this for several years?
I will definitely do the agar to grain master. Today I PC three myco bags of wood chips I will spawn with Ps. Cyan. I read Faukface's tek on out door growing. He wrote he doesn't have problem with contamination with the woodchips and outdoor grows. That cyan mycelium must be some aggressive shit to move through and take over the way it does. Mother nature is awsome.
The problem I have is that I am in a rental and will be moving soon so I want to take my chips with me. Plus I want to be successful so I sterilized the chips. Prior I soaked in a coffee/gypsum soup with lots of water. I wasn't sure if I should add coffee or gypsum so I said what the heck in the name of science why not. I was planning on adding one quart of grain to each bag I'm not sure if that will be too much or not. I plan on placing the inoculated chips into some of my planter pots and some of my bonsai pots. I have no idea if this will work but hopefully the chips will stay conlonized so when I get someplaced more perminent I can just transplant. Not sure if I have a chance in hell but you never know unless you try or you get a better idea to try something different.
Hopefully I will get a chance to make up some agar this weekend, I want to expand the specimen on agar to begin isolation. It sounds like a kick to do it. I really need to get a flow hood so I can have some free movement. Working in a dead air box is so confining it seems like I am always fumbling around in it. Again thank you for your years of advice. myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12340956 - 04/06/10 08:42 AM (14 years, 1 month ago) |
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It is my pleasure to serve. That is part of my personal purpose being involved, to find those who want to know and open the doors that are available to me to open. My years in are but a mere Decade, But my background as a student goes back a life time. When I got interested in mushies I sought out all experience in the friend collective, many of which are Old hippy types, they were glad to offer what information they had, part of which was the original Oss and Oeric book written by the late great Terrance Makenna, and his bro under pen names. As a side note It stated that fact about P. Car, that I have not seen restated anywhere. I think that is why there is so little interest In this species, it is listed as mildly active in the lit, but the strain that I have is Var. Mazatecorum, which all of my freinds agree is the most mind blowing mushie that I have.
Good luck with your grow, I love your potted plant idea, maybe a mulch to preserve moisture would help, I think it will work. You never know you may stumble upon some unknown symbiosis.
My teacher told me that when he started his first flow hood was made from a HEPA furnace filter, that is only 1" thick and considerably cheaper. If you ae going to build a nice box plan one to accept a proper hepa, but a cheap hepa may get you by in the meantime. I think that the duct tape approach has a value to serve the short term goals and adds greatly to long term understanding. Stametes wrote that failure is a profound teacher, and I believe that to be true
Gypsum is definitely beneficial, it helps in many ways but lacks the ability to buffer PH. Coffee grounds I would guess are acidic, so watch your PH at make up. You can buffer your mix with aglime or oyster shells, the latter of which will only buffer when acids are present, the former of which should be added in at around 2-4% by mass.
Peace Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12343741 - 04/06/10 05:25 PM (14 years, 1 month ago) |
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I appreciate your help and you taking the time to answer my noob questions. If you don't mind I would like to continue picking your mind as time goes on and I continue my work. I am unfamilure with Var. Mazatecorum. You said mind blowing Im not sure what you mean by this? I don't dabble in psych too much so they all blow my mind. I have noticed in myself that the PE gives me better visuals than the other strain I have going. The other strain is a mystery to me. I bought some mushies at a phish show a while back and broke open a closed cap and placed one of the gills on agar and it grew very well. I had no contamiation which still astonishes me. Something odd happend when I had grown it out three times, I had a white colored mushies pop up. I expected it to turn to a darker color but it stayed white. I decided to clone it to see if I can get more white ones. I have no idea at this point if there is any active properties but I am hopeful. myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12349143 - 04/07/10 02:24 PM (14 years, 1 month ago) |
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Hello,
It is my pleasure and my service to continue to correspond. Part of a Mushroom inspired mission to be a point of inoculum, also this is how I can give back to the community folks who put on the site, I could keep my viewpoint to myself, but a seed that is not planted does not grow.
By mind blowing I mean the general feed back that I get is that the strain that came to me from and old seasoned hippie "mazatec" I believe to fit the profile of P Caerulescens, physiologically, I have not had the resources to do any microscopy, some day I will and know forever for certain. There is a Var called Mazetecorum, that I feel that this strain is, This is the shroom in my signature pic. If anyone else has a more educated opinion I am dying to hear it, but by mind blowing I mean that it lends its experience towards the spiritual mystical realm, not really a party trip, but putting one in a state of in tune with the green force of nature, everything has a purpose, and is Karmicly bound to serve that purpose, any entity not serving its purpose risk extinction, The purpose of Psilocybes in my meager opinion is to serve the interface between humans and the rest of life on earth/ cosmos, why else would they produce those psi compounds that appear to have no other purpose?? That is their purpose, to speed our evolution so that we can become tech monkeys and carry life as a whole to other planets in space!!
But I digress, back on thread,
I would love it if you would share a pic or two of your fruiting cultures. I have extracted genetic material from the center of such sterile fruit and generated strains that were legendary and memorable to my cultivation experience.
Mycolight find you,
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12351288 - 04/07/10 07:52 PM (14 years, 1 month ago) |
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http:// http:// http:// http:// http://
Here is that weird white one that popped up. I have no idea if it will continue to produce white fruit but it was cloned. I did three transfers and all look okay as of a few days ago. Once I get a grain transfer and fruit it I will know more. Hopefully I can get it to spore out white fruit after a few hundred cycles of isolation. We will see.
The majority of my other strains are not actives. I have a bunch of prints that I have traded for but as I said before I don't trip much so I use what I have and give most of them away or just throw them out with the trash. Some may feel this is a waist but for me its more about the growing than then consuming. Consuming is a nice bye product of a kick ass hobby.
I will be honest, I got into cultivation last fall because I got so frustrated not being able to find any cyan's. I live in the PNW about an hour from Astoria and couldn't find shit. It pissed me off so bad that i became motivated. I think I spent over a hundred hours shrooming last fall. I found tons of cool mushrooms and one azure.The other problem was that there were state troopers all over out at fort stevens and that really sketched me out. The kids are getting good at finding them and we work on identifying as many as we can. Its funny because we go down by the river all of us in our rain gear tromping through the trees and brush.
The other reason I got into cultivation is that I love to grow things and typically have a garden and bonsai but there isn't much to do with either in the winter and the house plants I have are not that entertaining so I jumped on the ban wagon of cultivation and now it has become a lifestyle. I love this website and the trades that take place...it is cool as hell. I am trying to build a decent stock of prints and trade or purchase what I can from who ever is interested. I won a contest a while back for ATL #7 I plan on getting them going here in the next few weeks. Have you grown any of these?
I am off now to search for an agar recipe...I am still intimidated on creating it from scratch but that is the only way one learns. myc
If you would like I could post more of my non active pics. myc
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Professor Frink
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Re: transferring agar cultures [Re: Mikeallojee]
#12351445 - 04/07/10 08:18 PM (14 years, 1 month ago) |
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What the hell is that? That's a cube right? The gray color is confusing the hell outta me, never seen one like that....
You need to clone that and for sure get a damn spore print
-------------------- Culture trade list Wanted Culture list: Panellus stipticus Yellow Morel
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PrimalSoup
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Re: transferring agar cultures [Re: mycoelf]
#12351610 - 04/07/10 08:52 PM (14 years, 1 month ago) |
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Quote:
That is their purpose, to speed our evolution so that we can become tech monkeys and carry life as a whole to other planets in space!!
Including spores in space - the master plan.  
@Mikeallojee: That white fruit is a nice sport. Be interesting to see how the cloning turns out!
Peace -PS
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if you stand too close to the machine it'll start to eat youPrimal's simple tested teks and projects: Wheat Prep 2.0 Acidic Tea Tek Potency Project!
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Mikeallojee
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Re: transferring agar cultures [Re: PrimalSoup]
#12352141 - 04/07/10 10:29 PM (14 years, 1 month ago) |
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http:// http:// http://
Here are three samples taken off the grey mushroom. Notice that one looks kinda normal with a little contamination. The other two look odd to me. They started out with standy looking hairs from the sample. All clone pieces were taken from various spots of the stipe. Low, mid and high. Ideas of what may be happening??? myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12354212 - 04/08/10 09:58 AM (14 years, 1 month ago) |
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3127110 looks fab, there is some kind of bacterial contam going on in the lower edge of the plate, contams on the edge means your fingers got too close to the edge, something hopped the wall. This type of contam is easy to clean, a couple of drops of peroxide via syringe will react on the contam direct, your best fans is lower left, I would sample at 3 or 4 o'clock, invert the inoculum so that the myc makes contact with a peroxidated agar and your culture should be clean. Just based on looks and intuition I would pursue this substrain on a small scale, just to see what will come of the mutant mushroom. I also noticed that there is a real nice blueing going on where the clone flesh was inoculated-cool, this means that this particular strain is going well and getting everything it needs for active compound production.
I have grown PC parallel with yeast culture and got similar flat looking distorted color, I really do wonder what is going on with the one shroom, where the rest is normal looking.
The mushies in the tray look to be cub's ???? I thought that you were growing cyan, Are we looking at a wood sub in these trays???
I have had success growing on wood indoors myself-- intrigued as I have always wondered if P cyan would fruit on wood in a pod setup?????
Congratulations, you are more accomplished then you give yourself credit for- you obviously have a grasp of basics- now it is just a matter of time.
I also cultivate a large variety of edible and medicinal. MY best crops are reishi and Shitake. I am glad that you have a broader interest, I started out with cubes like everyone else, found out how deep the rabbit hole really goes,
Welcome to brotherhood
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12354974 - 04/08/10 12:22 PM (14 years, 1 month ago) |
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Thank you for the compliment. I certainly don't feel accomplished. Yes they are cubes...I thought I would start out with something easy to grow first and from my studying cubes and oysters seemed to be the best way to start i figured that once I had some success I would branch out. I have several mushrooms grows happening all at once. I have reishi growing too. I had some kick ass antlers but I bumped the tub and the suckers just fell off...big bummer. I am growing them for friend who is Dx with terminal cancer. I dried the antlers boiled them up making a tea and gave the tea to him. I hope it helps. Since the first reishi grow which I began on alder wood dust and chip I have moved on to paper/cardboard. I noticed that the cardboard that I soaked in a coffee/gypsum mixture is taking off. Where as the ones with cardboard just soaked in water have been moving slowly.
I also have two strains of shiitake. One from FP and one 75 from noobieshroomy. The blocks I have going now are clones from a block I bought for a friend who was Dx with prostrate cancer a few years ago which is in remission. I read that shiitake help reduce the return of some cancers so I decided to buy him the block with the plan of cloning and continuing the cycle so he can always have fresh ones for his salad or what not. I think I placed too much bran in the block because all I have growing now are big blobs. RR said to pick them off which I did. I now have four petri dishes with 75 on it that need to be placed in some grain jars. Just getting around to is seems to be the biggest struggle.
Thanks for the advice on the petri dish. Do you have any idea as to why the other two dishes grew the way they did with the strandy looking mycelium and the other dish performed much better? I do plan on doing some small scale experimenting on the the white cube just for fun. The anomaly has really caught my interest.
I had no idea that placing my figures too close would cause a contamination to jump the wall. It isn't the first time this has happened I just wrote it off as, "oh well it happens." Peroxide for the contamination what great idea. I will have to do this. I kinda like watching competing fungus compete to see what will happen.
The sub in the trays is CCV with no casing layer. It occurred on the third flush.
On your signature is that a PE shroom? Did you grow it in a bag? myc
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mycoelf
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Re: transferring agar cultures [Re: Mikeallojee]
#12355872 - 04/08/10 02:26 PM (14 years, 1 month ago) |
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Hello,
My first strain of cultivation was PC, var golden teacher. I believe that there are some mushrooms that are so damn hardy as to allow you to make all of the mistakes in a run and still they are so forgiving/ hardy they still pay you with a moderated success. Cubies are one, oysters are another. The tag of "golden teacher" took on a new meaning as I first tuned into what was going on on the dish with them. I was growing cub's and studying my ass off, while one day I was sitting outside in to forest reading about mushrooms and Lo and behold I found a wild Reishi. I preformed a low impact tissue sample/ clone and met with perfect success this was the pinnacle of my then growing love of the myc. when I came to understand what power the Reishi had I realized that ALL mushrooms have something to offer, and the world was bigger than just growing to trip w/ my friends. I have been taking reishi for years now and wouldn't do with out it.
As for the differentiation in your clone, your guess is as good as any. For Stopharia type's I usually start with a longitudinal dissection, and examine a the interior. I am a believer in a quiet mind while in sterile arena and I wait for a chunk of fuzz to call to me for transfer. This is the intuitive process that I have referred to in the past, with cubes the long ropey myc in the core seems to be the winner. I have cloned the majority of my strains from the wild and have spent many hours pondering the subject. It may just boil down to getting lucky, I keep a 10x 60 mm plate for cloning and strain work They are so cheap, like 500 for 80$ that I pour special runs just for this purpose. If I really want a clone I do 10 plates, if it is precious I do 20, if it is just another strain of oyster I may do a few as 3. I have found that the myc that leaps off from the wild clone is as aggressive as it gets, I try to go in-vitro within 2 transfers of this clone. This is when the myc is in top genetic form and should be preserved thusly.
There is an excellent discussion on myc archetypes in GGMM, you might check it out, and while there look at the section (so dry) on hand position protocol while handling plates in sterile arena, it takes a bit of practice but once you get what he teaches there you wont commit the same mistakes over and over, train you hand how to make those motions and when habit is burned in you won't have to give it another thought. You will also save yourself some work and expense by not having to re-mediate cultures.
my sig pic is the "Mazetec" strain that I mentioned earlier in this thread, it weighed in at 155g wet and was part of an experiment fruiting on an enriched wood substrate spawned in a 5lb SAB, run flat with a vermic casing layer in an aspirated mono tub type setup. I don't know what you mean by "PE"
time to go feed the critters.
Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12358298 - 04/08/10 09:27 PM (14 years, 1 month ago) |
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"try to go in-vitro within 2 transfers of this clone" What is meant by invitro?
PE=penis envy. I saw another post where the person had grown them in a bag and they were as big as the one you have there. Its obvious that I still have a lot of learning to do as it goes for identification. I will go to the GGMM and look for the hands technique and strain archetype. I had never thought of taking 20 cultures from one mushroom, that's one sleeve that I am using. But $80 for 500 sounds like a good deal. Where do you get your dishes from?
I just poured 30 agar dishes today, I plan on expanding the white clone in about five days. As well as the shiitake 75 I have. I want to wait to make sure there is no contamination before I do though.
Reishi? what do you use for your sub? Do you have a specific type of agar you use specifically for reishi? You also said that you found a wild specimen of reishi that would be a great find. I talked to the wife about spending our summer vacation out on the Washington peninsula. I was thinking that I might get lucky and find some wild samples out there. I also go hiking alot in the cascades but until about three months ago I didn't know what reishi was. Reading a thread on a guy who made a reishi monotub that kicked ass. I want to try one of those as well but am afraid that it will fail miserably. Since I have other people counting on me to provide, I want to make sure I am successful so I will continue with the wood blocks and paper/cardboard. I would love some advice on reishi as well. Tips are always appreciated. myc
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badman


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Re: transferring agar cultures [Re: Mikeallojee]
#12358437 - 04/08/10 09:50 PM (14 years, 1 month ago) |
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In vitro means behind glass, with shrooms...I like these pics
   
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mycoelf
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Re: transferring agar cultures [Re: badman]
#12363944 - 04/09/10 07:37 PM (14 years, 1 month ago) |
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Hello,
As badman astutely pointed out, the literal definition, I was referring to storage slants. These are test tubes that are autoclaved then "slanted' to cool causing great surface area for colonization. I have been getting the 20x 100 that FP offers, I am looking further into lab supply when I next order a collection. They have screw on lids and are easier to handle while inoculating. Once grown out they are kept in the fridge. You can then revive restart strains from these masters when needed. I would think the standard is 10 transfers from origins would be considered prime, but we all know that a good Myc will go for many more transfers than that before collapse.
I have always referred to stock cultures as IN-vitro. Perhaps this is wrong, I scanned GGMM this morning and did not see the term associated with the appropriate section. I suppose I shall have to adjust my language to fit the definition closer.
http://www.tritechresearch.com/T3308.html
These are the plates that I was referring to. They seem to have dropped in price since last year when I bought last. They also have a vented version, but I would say since you are working at home you may prefer the ones that I am accustomed to.
where Reishi are concerned I have two strains that I cloned from the wild. I have found them very easy to clone once you adapt to the polypore physiology, and learn to clone at the base of the shelf anywhere above the pores in cross section. I really like polypores, they are SO aggressive in culture, I don't make any special provisions, they are fed what ever agar that I have made for my other work, I can innoc a 100 mm plate and get a full colony in 5-7 days. Polypores then to thicken as they mature on a plate and can be difficult to cut with a razor sharp scalpel. I have never expanded them to senescence, as it is bad practice, I suspect that they would go even further than your standard cube before failing.
As for substrate they are as easy to please as on a plate. I get sawdust from a stone industry palate maker, mixed hard wood, oak and poplar, even with some assorted pine shavings and they eat it all. Stametes states that augmenting sawdust with grain at greater than 10% can actually reduce yield. I like to use rice or WBS or red and white millet for grain masters, and I usually match the enrichment of the sawdust with the same grain as so they are prepared enzymaticly for colonization of the bag. They grow so fast when you set them up right that thermo-genesis is the biggest danger.
Out where you are there is the "Oregoneese" variety that should be easy enough to find for a rare mushroom, don't bother looking until summer solstice as their fruiting time is from about June to September. They like the warmth of those months.
My cloning technique is similar to MR Stametes as it was inspired by him. I have a 5/16 stainless steel tube that I sharpened in a lathe. I keep it in my "field collection kit" that is a converted camera case. I alcohol the tube while holding the other end with tweezers, flame it with my bic, then holding the un-sharp end with gloved hands I insert the tube for the underside with a twisting motion cutting a core as I go. when I sense that I am well above the pores I change the angle of the tube thereby,severing the end of the core, once I am sure that I have liberated the core, I withdraw the instrument and quickly drop into a baggie, All this with gloves and alcohol on the hands.
Upon returning to the lab I prepare sterile arena, and use a sterile solid round stainless steel that fits in the handle end of the tube to push it out onto a prepared stainless dissection tray. I usually drop some peroxide onto the specimen and wait for the reaction to stop at @15 min. Then U use a pick to secure one end and slice away the pores and move them down stream from the remaining clone. The remainder minus the field end wich is also removed and discarded is sliced into thin sections like cookie dough, speared with a scalpel and neatly each "cookie" section is placed flat into its own dish.
Cloning wild specimen's is always a crap shoot. That is why I like to use the 60mm plates, they are cheap and I pour no less than 80 at a time. Sometimes I get 7/10 clean then pick the best looking plates for further work. sometimes I get 3/ 10 clean and it is good to have a clean plate to start with then to try to separate using subculture technique.
Where fruiting Reishi is concerned, once they colonize and start to form "snakeheads" I strip or roll the bag down and place them out side. They grow kind of dry so You don't worry about watering them. They form a "skin" around the sawdust block and do quite well retaining moisture. I have run them both flat and as a block and the fruits on the flat seem to be more numerous but smaller. I have seen no evidence that a casing layer in any way helps. When I have run side by side comparison, I would say that casing a culture of Reishi is actually detrimental

This is the first one that I found and cloned in-situ as to allow the mother mushie continue to sporulate. If not "Rare" they are definitely not common. I feel as if I should let those spores do their thing in the wild. I have attempted to collect Reishi spores and failed in the past.

This is the same mushroom showing two wounds where I sampled tissue. I monitored this mushroom until the end of the year and the sampling appeared to have no negative effects.

These are resulting outdoor fruiting colonies from the above mother fungi.

This is a Reishi that I found last fall just before I closed my last lab and moved. It was found in an old growth forest on the remains of a huge tree that was more than likely to have been virgin timber. I made several successful clones and many successful culture slants. I have yet to give this one a trial. The mushroom in this pic weighed 2 oz dried, and was 6 inches across. I have high hopes for this strain.
May the myc be with you all, Mycoelf
-------------------- 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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Mikeallojee
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Re: transferring agar cultures [Re: mycoelf]
#12365320 - 04/10/10 12:01 AM (14 years, 1 month ago) |
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Those are beutiful reishi. I hope the paper works, I was kinda sketchy about using it but part of this hobby is expereimentation plus I have seen other people on here being successful. Thank you for the wild culture extraction technique. I can't imagine how bad I might have mangled the first wild reishi I found with out your instructions. The tube idea is good. I need to learn more about poly pores there seeme to be alot around the area. I think I will buy some of those petri dishes 500 for 49 bucks is a good deal. mcy
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