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 SwampTromper
Registered: 02/02/11
Posts: 31
Loc: Florida Swamp 
Last seen: 12 years, 11 months
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 Senescence in Isolation 
#13944338 - 02/11/11 09:17 AM (13 years, 2 months ago) |
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I've researched and experimented but still do'nt fully understand. When i isolate a multispore on agar and it takes six transfers to get an isolate, am i not left with senescence in my master?
It seems that its almost counter productive as i'm isolating good genes while making the good cells multiply over and over which in turn causes them to degrade?
I could be wrong in my thinking, my mind is so full of information. How about some clarification?
Why can i do six agar transfers but six g2g tranfers leaves me with poor fruiting?
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 cyanara
jedi in training
Registered: 12/22/09
Posts: 1,205
Loc: your grow closet
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well first off because the number of cell divisions are greater in a jar than are on a petri plate.. second you are constantly changing your media and this seems to be what helps slow how fast senescence takes place. I think though that domesticated varieties are more likely to senescence because of the limited gene expression, just my thought though, and please if anybody has anything that could clarify by all means please do.
Actualy I was going to make a post cause I'm having problems with my petri culture and it's showing senescence expression.
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Pinback
Stranger
Registered: 07/20/02
Posts: 836
Loc: Europe
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Re: Senescence in Isolation [Re: cyanara]
#13944955 - 02/11/11 11:21 AM (13 years, 2 months ago) |
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There isn't much information around on senescence in higher fungi. Most of what you read is hearsay and assumptions.
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cyanara
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Registered: 12/22/09
Posts: 1,205
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Re: Senescence in Isolation [Re: Pinback]
#13945004 - 02/11/11 11:28 AM (13 years, 2 months ago) |
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so could you explain to me why a healthy culture, was brought out of the fridge, placed on MYEA and some was used for grain masters and the rest was transfered to home made PDYA where now it's expressing some degree of sectoring and the rhizo's are not as nice?
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Pinback
Stranger
Registered: 07/20/02
Posts: 836
Loc: Europe
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Re: Senescence in Isolation [Re: cyanara]
#13945330 - 02/11/11 12:33 PM (13 years, 2 months ago) |
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Quote:
cyanara said:
so could you explain to me why a healthy culture, was brought out of the fridge, placed on MYEA and some was used for grain masters and the rest was transfered to home made PDYA where now it's expressing some degree of sectoring and the rhizo's are not as nice?
Do you consider this indicative of senescence, and if so, why?
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mycoelf
Agent Of Chaos
Registered: 06/26/09
Posts: 555
Loc: hyperspace
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Re: Senescence in Isolation [Re: Pinback]
#13946036 - 02/11/11 02:42 PM (13 years, 2 months ago) |
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If you are starting your spores on agar and it is taking 6 transfers to get an isolate, might I suggest you run a larger dilution rate for the spores over the plate? the points of origin will be more distinct and the resulting sectoring will be easier to spot. I usually achieve an isolate within two transfers if not the first using this approach.
secondly cyanara is correct in that the lines of cells divide further in G2G then when it is successive transfers on agar. This expansion on agar can be further minimized by choosing the dominate or better looking sector and sub-cultureing from the leading edge as soon as it has leaped off the transfer I do a Multi spore germination and usually by the first or second transfer there is a clear dominate isolate, and more often than not it goes to stock culture slant as an F1 or F2 max.
SO what are we talking here? cubes? if so there are many commercial strains that have been selected so many times as cyanara said they are compromised when U get them, their genetic expression is already narrow, or it could be said that their gene pool is shallow. This very thing is the reason why the prophet Paul is so adamant about continuous efforts to collect and preserve the genetic integrity of the wild strains. They are always strong and the cultivator that is Nature does a fine job of keeping the strains viable by continuously mixing genes from different sources, something that rarely happens in the laboratory.
My culture library is dominated by the wild strains that I have cloned. Good genetics could still be strong at 150 generations out from the original material, if U are really experiencing senescence at gen 6, consider the source.
U might also try to run some variations in you agar formula. I have also found that sometimes PH will play a role in the poor performance of a transfer, have U checked the PH of your agar after makeup? PH is so easy to overlook and your source water will largely determine your final PH, depending if U are adding any buffers or additives
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Mycoelf
Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
    
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cyanara
jedi in training
Registered: 12/22/09
Posts: 1,205
Loc: your grow closet
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Re: Senescence in Isolation [Re: mycoelf]
#13947882 - 02/11/11 08:10 PM (13 years, 2 months ago) |
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no buffers or additives, and I use distilled water. what are your suggestions to change PH and how do you messure after makeup?
Also explain to me how you delute down? and are you talking delution to the point of monokariots..?
Please teach me mycoelf, it seems that you have alot of knowledge and are patient and willing to share with those who are ready to learn.
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SwampTromper
Registered: 02/02/11
Posts: 31
Loc: Florida Swamp
Last seen: 12 years, 11 months
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Re: Senescence in Isolation [Re: mycoelf]
#13970706 - 02/15/11 09:41 PM (13 years, 2 months ago) |
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I'm talking oysters, and to re-state it i mean - If i do an oyster multi-spore on pda and it takes 6 tranfers to get an isolate. Then i do a few g2g's from the isolate to get enough mycelium to make some straw bags.
Is that going backwards in terms of senesence as compared to making a multispore lc to grain, then straw, then making a clone of a strong fruit and growing from that clone?
I've done small experiments with both and do'nt really see where the isolate is outdoing the clone. Maybe i just need to do more isolations or more experiments to get a better picture?
The way i am seeing it, its more like volume creates more division and increases signs of senesence. So back to the main thought, are 6 agar transfers similar in cell division to about one small g2g transfer?
Edited by SwampTromper (02/15/11 09:55 PM)
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PrimalSoup
hyperspatial illuminations
Registered: 11/17/09
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Re: Senescence in Isolation [Re: cyanara]
#13970865 - 02/15/11 10:02 PM (13 years, 2 months ago) |
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Quote:
so could you explain to me why a healthy culture, was brought out of the fridge, placed on MYEA and some was used for grain masters and the rest was transfered to home made PDYA where now it's expressing some degree of sectoring and the rhizo's are not as nice?
Because you changed the substrate and it takes a while to readapt. I see this a lot when I'm using PDY plates made from "scratch" (boiling a potato, mixing up batches with my scale). In other words, ran out of Mycomedia MEA again. 
Little variations in plate prep lead to lengthy lag times when transferring cultures off one batch and onto another. But they do sort it out and get it together again. The same thing will happen going onto grain, the myc always has to adapt to the new substrate. It's not senescence.
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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mycoelf
Agent Of Chaos
Registered: 06/26/09
Posts: 555
Loc: hyperspace
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Re: Senescence in Isolation [Re: cyanara]
#13974482 - 02/16/11 04:17 PM (13 years, 2 months ago) |
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Sorry for the late reply, I have been traveling and have not had net access. My technique for dilution is to sterilize 10ml distilled water in a test tube, and heat a innoc loop until color in the metal is achieved, then I quickly touch the loop to the center of an agar plate, liquefying the agar on the loop, which should immediately cool., then from the plate I go to the spore print and pick up spores on the innoc loop and insert into the test tube followed by several vigorous seconds of motion inside the tube. The spores then move into the water,(spores are hydrophillic) by the millions, and there you have a inoculation solution. Sometimes if the print is old I will sit the test tube in storage and wait until I see hydrated and sometimes monocaryotic germination in the tube, which goes no where and therefore is time sensitive, because the solution is non nutritive. At this point maybe I would put a drop of solution in the center of a plate and wait for the myc to take hold. If I want to spread out the points of origin I would roll the plate back and fourth so the water rolls around on the agar, leaving behind many spores/monocaryotic germination points, that then run the agar as separate points of origin. If I was doing something like oysters that are notorious for the high density of the spores, I might sterilize about 100 ml in a 300 ml beaker with a stir bar and add the solution in the tube to the beaker giving a 10 to 1 dilution, while stirring I would draw for the beaker with a syringe and then use the resulting solution to do 1 drop per plate. At the advanced diluted rate there would be no need to roll, but it is optional depending on what will work for U. As a rule of thumb I pour two size plates. 60mm and 100mm. I use the 60mm for "strain work" which can be anything from cloning to isolation to MSG work. If something is precious I might do 10 60mm plates, each with one drop. When the plates start to show I watch every day and when the strongest origins take more territory than their counterparts, those are the points that I subculture into about ten plates, subsequently labeled A,B,C etc. Mini trials are in order to see what if any strain has superior characteristics, in the case of oysters I would say a nice trait would be to not over run a g1 before you have time to prepare substrate, and of course willingness to fruit, overall size and quality of yield etc.
when it is all over the strains that are kept go into stock culture slants ideally within a couple of transfers of origins. Those then are put in the fridge to lay dormant and occasionally sub-cultured from to start new plates, I may run a F1 from slant through about ten generations of subculture or a season, whichever comes first. I always cycle my strain bank over the cold months because it s easier during the low spore time of winter to make totally clean slants, and most of us are not actively running spawn during that time, and you should always keep working to keep in practiced form.
Distilled water is always best for agar work just cause it is PH neutral. After I moved last year my first batch in a hurry of agar was done with tap with out considering this, and The myc did not like the 8.5 PH agar, except the poly pores that did not seem to care. I salvaged those plates by using them for the polypore runs and things have been fine since. My point of mentioning Ph is that it is really easy to forget about and when facing less than optimal results I suggest that the issue of PH be considered as a possible solution/culprit
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Mycoelf
Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
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cyanara
jedi in training
Registered: 12/22/09
Posts: 1,205
Loc: your grow closet
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Re: Senescence in Isolation [Re: mycoelf]
#13974653 - 02/16/11 04:46 PM (13 years, 2 months ago) |
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thank you so much mycoelf... do you use a digital device to messure out PH or do you use PH paper?
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mycoelf
Agent Of Chaos
Registered: 06/26/09
Posts: 555
Loc: hyperspace
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Re: Senescence in Isolation [Re: cyanara]
#13977860 - 02/17/11 07:55 AM (13 years, 2 months ago) |
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I used to have a fancy 400$ ph pen and it was very accurate but eventually it died for the sensor stopped working. These days I use a high range PH test kit from the aquarium store, that has its limitations but I find I can get a good idea of what is going on PH wise, and for our purposes PH is a shotgun, not a rifle
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Mycoelf
Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
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RogerRabbit
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Registered: 03/26/03
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Quote:
SwampTromper said:
So back to the main thought, are 6 agar transfers similar in cell division to about one small g2g transfer?
No, not even close. The flat plane of a petri dish holds very little mycelium compared to the three dimensional space of a jar full of grains. In addition, when we're isolating strains, we never let the dishes grow fully out. In fact, I make transfers when the culture is no more than 1cm to 2 cm in diameter. This is not going to result in senescence even after 100 petri dishes.
As for grain to grain transfers, I use large spawn bags with six quarts of rye in each one. I make five G2G transfers before spawning to bulk, and senescence isn't a problem.
However, doing a series of grain to grain transfers before cloning the fruit you want, gets you down that road, and isn't recommended. When you isolate strains, always save the master cultures in refrigeration, and have them well marked. Once you see which strains fruit best, you don't clone fruits, but rather pull the original cultures out, and you now have P5 to P6 to grow essentially forever if you store it properly on master slants.
RR
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Chad1
Stranger
Registered: 12/02/10
Posts: 279
Loc: USA
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Sorry for going off topic, but how much grain do you use for each g2g transfer into the 6 quart grain bags?
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RogerRabbit
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Re: Senescence in Isolation [Re: Chad1]
#13982299 - 02/17/11 11:39 PM (13 years, 1 month ago) |
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My sterilizers hold 8 bags, so I do it at 1:8 for convenience, but it could easily be 1:10 or less.
RR
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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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mycocozm
Registered: 05/13/10
Posts: 181
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Not to mention, Swamp (I don't think anyone has hit on this yet), if my understanding is correct, another reason why senescence is exponentially greater in g2g compared to dishes, beside the substrate and the sheer volume differences, is that while you are waiting for a monoculture isolate to emerge there are various living strains that you are filtering through. Its not until two compatible strains fuse (and become rhizomorphic, depending on species) that you come to the end of the sexual reproduction phase and the beginning of your isolated strain's longevity. In other words, you aren't loosing vitality until you start expanding the mycelium of your final isolate. All the ones that came before it don't count, they were just trying to make your isolate because they want inside you!
Can I get a fact check? I know I will
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cyanara
jedi in training
Registered: 12/22/09
Posts: 1,205
Loc: your grow closet
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Re: Senescence in Isolation [Re: mycocozm]
#14013038 - 02/23/11 11:33 AM (13 years, 1 month ago) |
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ya I would have to agree with mycocozm. I was thinking about this last night and it seems pretty logical. if there are multiple strains in a petri or jar they are constantly rerouting and charing new genetic information. and although clones are a quick way to get a guarenteed fruiter it seems as though they are the quickest to senescence, prob due to have already past there sexual reproduction phase
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shroomie_glen
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Re: Senescence in Isolation [Re: cyanara]
#14052013 - 03/01/11 11:15 PM (13 years, 1 month ago) |
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I haven't had problems with senescence in a few years since I have adopted the practice of constantly testing genetic expression and trashing my masters after viability has been pushed.
Keep those myc. running and begging for more.
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No. No, man. Shit, no man. I believe you'd get your ass kicked sayin' somethin' like that man.
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SpawnRun
Morchella esculenta
Registered: 11/12/10
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^^Indeed.
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Clayton Wiseman
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SwampTromper
Registered: 02/02/11
Posts: 31
Loc: Florida Swamp
Last seen: 12 years, 11 months
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Re: Senescence in Isolation [Re: SpawnRun]
#14090718 - 03/09/11 02:28 AM (13 years, 1 month ago) |
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I tried creating senescence by doing a bunch of g2g transfers then fruiting... Fruited great. Makes me feel better about running into problems. I guess myceliumm is not as sensitive as i thought.
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RogerRabbit
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Another misunderstood aspect is chronological time, not just the amount of cell divisions. A single kernel of colonized grain can be expanded to fill a large truck of grains without senescence if one works fast. However,if you took each jar of grains and let it sit for a month before making the transfer, you wouldn't get nearly as far, even if they were refrigerated. This is why we keep the masters on test tubes and don't begin the expansion until the time comes.
RR
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"I've never had a failed experiment. I've only discovered 10,000 methods which do not work."
Thomas Edison
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cyanara
jedi in training
Registered: 12/22/09
Posts: 1,205
Loc: your grow closet
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so when you pull a test tube and make a new plate, will the test tube that was pulled be shot?
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RogerRabbit
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Re: Senescence in Isolation [Re: cyanara]
#14093217 - 03/09/11 03:40 PM (13 years, 1 month ago) |
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No. I keep them for years and years. I always pull cultures from my oldest source, which will deliver the youngest cell lines. If you'll put wood in your culture slants, they'll last up to a decade if opened once a year in front of a flowhood to let in some fresh air.
RR
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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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mycocozm
Registered: 05/13/10
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Last seen: 3 years, 3 months
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Quote:
RogerRabbit said:
if you took each jar of grains and let it sit for a month before making the transfer, you wouldn't get nearly as far, even if they were refrigerated. This is why we keep the masters on test tubes and don't begin the expansion until the time comes.
RR
You are saying that you have to transfer the mycelium while its in its most vigorous stage of growth?
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RogerRabbit
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Re: Senescence in Isolation [Re: mycocozm]
#14094267 - 03/09/11 06:44 PM (13 years, 1 month ago) |
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Yup, that is if you want to make half a dozen or more grain to grain transfers at a 1 to 20 ratio.
RR
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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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SwampTromper
Registered: 02/02/11
Posts: 31
Loc: Florida Swamp
Last seen: 12 years, 11 months
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You go 1:20 g2g? Is your succeess rate still comparable to a lower ratio or is it compromised? I guess i'll just have to experiment and find out although i never go over 1:10.
Then again i havent been doing g2gs for twenty years. What would you reccomend? I have a pretty low contam rate.
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