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Mikeallojee
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Registered: 10/02/09
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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
Registered: 04/13/08
Posts: 5,304
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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
Registered: 04/13/08
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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
Registered: 04/13/08
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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
Registered: 04/13/08
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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
Registered: 04/13/08
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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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Registered: 06/26/09
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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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Registered: 10/02/09
Posts: 897
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Last seen: 11 years, 3 months
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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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Registered: 10/02/09
Posts: 897
Loc: SW WA
Last seen: 11 years, 3 months
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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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Registered: 10/02/09
Posts: 897
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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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