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Offlinejuturna
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senescence, degradation question
    #10070551 - 03/29/09 10:13 PM (15 years, 3 days ago)

okay lets say you have a master slant,lets say its an original {T1}. and down the line a couple months you do a transfer for making an identical master slant to keep the process flowing.
so now you have a couple of your {T2} master slants right? now for senecense pusposes i know RR likes to keep track of how many days its been since the earliest cell divisions like an inoc. from a multispore correct? {minus time in the refrigerator, because not much cell division takes place}. now for the {T2} master would you add the days that the {T1} had already had on it? to keep track of cell division, or does it start over  (ex. master slant was dated 04/01/09 P.Cub.-EQ from petri dish number lets say A1A1A2B.thats how i label them. and the other part of the label i put {T1}for first gen. and i put the days since germination lets say it took me 15 days to isolate that strain. ok so when you make your {T2} slant would it start at 15 days since earliest cell division or does it start back at 0 cause its a second generation..... i know its tough to follow but ikm real pressed on this issue any help would be greatly appreciated, theres a a couple free prints in it for anyone who can explain this to me thanks..


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Invisiblegreys
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Re: senescence, degradation question [Re: juturna]
    #10070706 - 03/29/09 10:36 PM (15 years, 3 days ago)

wouldnt the amount of distance it travels on a petri dish determine the number of cell divisions?

if its a 90mm dish and you transfer tissue to the center...let it grow to the edge and then transfer from the leading edge near the petri dish side...isnt that a measurable amount of cell divisions?


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Offlinepotatonet
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Re: senescence, degradation question [Re: greys]
    #10070894 - 03/29/09 11:03 PM (15 years, 3 days ago)

I believe once you have an isolate as the strain sectors out in the agar plate and remove a piece from the leading edge you can start that at day 0, so the 15 days to isolate does not come into effect because it is a small amount of time when considering the life of the slant.

you could keep the 15 days if you want but as RR says, he has some slants that are 10 yrs old... 15 days is .5% of the life of a slant that old.

If it were me, once I got an isolate and slanted it with wood, I would call it day 0 the day I slanted it. keeps the math simple.


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In the Quechua language of Peru there are over 1000 words for "potato"

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Offlinejuturna
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Re: senescence, degradation question [Re: greys]
    #10070941 - 03/29/09 11:09 PM (15 years, 3 days ago)

yes but i dont think there is a way to count the cell division on a single plate. and from what i understand you want the least amount of cell divisions so letting it colonize the whole dish defeats the purpose. also the edge of the petri dish is prime real estate for contaminates. so my question still remains..


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Offlinepotatonet
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Re: senescence, degradation question [Re: juturna]
    #10070992 - 03/29/09 11:16 PM (15 years, 3 days ago)

um.... I think I just explained it...

to quote RR:

" no piece of the tissue is 100% sterile, the smaller the piece of tissue you transfer the less chance of there being contaminants onboard"


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In the Quechua language of Peru there are over 1000 words for "potato"

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Offlinepotatonet
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Re: senescence, degradation question [Re: potatonet]
    #10071027 - 03/29/09 11:23 PM (15 years, 3 days ago)

" If your intention is to simply make another test tube slant to return to the refrigerator, let it grow out on a petri dish for about a week or so, as soon as you get some good healthy growth, at that point, go ahead and transfer to a fresh test tube, don't allow it to grow any longer than necessary."

he says to label with date, species,and strain when you make the petri dish from the initial slant.

you probably do the same thing when making the new slant, so the new slant starts the day its made.


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In the Quechua language of Peru there are over 1000 words for "potato"

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Offlinejuturna
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Re: senescence, degradation question [Re: potatonet]
    #10071059 - 03/29/09 11:29 PM (15 years, 3 days ago)

i know you did  that wasnt directed towards you potatonet and thanks for the help bro. but i wasnt asking about the days for isolating.. read it again i was talkin about the time between the T1 slant and the creation of your T2 slant wich is second gen. correct? read the top again bra.


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Offlinepotatonet
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Re: senescence, degradation question [Re: juturna]
    #10071096 - 03/29/09 11:33 PM (15 years, 3 days ago)

If you make a slant and keep it refrigerated, then make a petri dish to make a continiuation of the slant (because you pulled the slant to do some plates and/or jars) then the date would be from the initial slant.

make multiple test tubes of the same slant every 6 months to a year after growing on agar for a week for each new slant group.

you're question just asked about continuation of the slant


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In the Quechua language of Peru there are over 1000 words for "potato"

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Invisiblegreys
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Re: senescence, degradation question [Re: juturna]
    #10071103 - 03/29/09 11:34 PM (15 years, 3 days ago)

i was just providing a hypothetical example, not saying you would want to transfer from th edge of the dish...i suppose you could put a tissue sample under a microscope and measure cells in microns and figure out how many cells are in a linear cm of tissue..is there some particular reason you need to be so accurate with your records of cell lines?


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Offlinejuturna
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Re: senescence, degradation question [Re: greys]
    #10071154 - 03/29/09 11:41 PM (15 years, 3 days ago)

thank you so much potatonet i understand now.. just make the T2 slant the same date as the T1 slant cause T1 is the earliest cell divisions..thanks bro.. if you wanna trade some prints in the future hit me up dog. no reason greysRDbest i just like making notes and being very thourough thats all. im sure you understand. but thanks for your input...hit me up if you wanna trade prints some time... and thanks again guys..


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Invisiblefastfred
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Re: senescence, degradation question [Re: juturna]
    #10082068 - 03/31/09 05:41 PM (15 years, 1 day ago)

>  just make the T2 slant the same date as the T1 slant cause T1 is the earliest cell divisions..

The date doesn't matter really in comparison to cell divisions.  You also don't want to label things like T1 -> T2.  Label them like T1 -> T1-A or T1-2.  Your first number needs to indicate what isolate it is.  Other numbers can indicate which transfer, etc. it is.  Besides labeling them like this you also should date all of them.  The date should be the date you made THAT tube, slant, petri, etc..


-FF

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Offlinepotatonet
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Re: senescence, degradation question [Re: fastfred]
    #10083644 - 03/31/09 09:19 PM (15 years, 1 day ago)

but its a continuance of cell divisions from an original slant, the cell divisions still occured.

yes you should date the day you made the slant but the day of the original slant should be on there as well and should be considered in the age of the slant.  otherwise people would just split slants all day long starting them at 0 and senescence would still occur.


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In the Quechua language of Peru there are over 1000 words for "potato"

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Offlinejuturna
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Re: senescence, degradation question [Re: potatonet]
    #10083999 - 03/31/09 10:10 PM (15 years, 1 day ago)

:thumbup:good info potato...makin this thread mean something


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Invisiblefastfred
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Re: senescence, degradation question [Re: potatonet]
    #10099984 - 04/03/09 11:41 AM (14 years, 11 months ago)

Quote:

potatonet said:
but its a continuance of cell divisions from an original slant, the cell divisions still occurred.

yes you should date the day you made the slant but the day of the original slant should be on there as well and should be considered in the age of the slant.  otherwise people would just split slants all day long starting them at 0 and senescence would still occur.




No.  The date is just for checking your lab notes.  The date does not indicate the number of cell divisions in any way.  You can figure the cell divisions or a p-value type score based on how many transfers made since you'll usually be growing it out about the same from each transfer to the next.

Suppose you have this label: LCDF3-T3 4-1-02
That tells me that it is the third LCDF (LC Determined Fruiter) isolate and that it is on it's third transfer.  I then look back in my notes to 4-1-02 and can see whatever I wrote down about it at the time.

If I transfer it again it becomes LCDF3-T4 4-3-09 and I make a note of it in my lab book.  Then when I look in my notes on 4-3-09 I will read my notes on it which will refer me back to 4-1-02 to see notes on the original isolate.

No lab work you are going to do is going to lead you to senescence problems.  We only try to keep the cells young because it's good lab practice and young cells are often better at surviving various lab procedures.

There are way to many posts worrying about senescence when it's not really a problem except in rare circumstances.  The only time you're going to run into problems is after many repeated G2G transfers when you expect the myc to continue dividing/colonizing a lot more.  That is the equivalent of hundreds, if not thousands, of petri dish transfers.

There really should be a guide to keeping lab notes, but traditionally each scientist comes up with their own methods through trial and error, and they don't always want others to be able to follow their lab notes easily.

I used to write a lot of cryptic abbreviations and tried to index and compile things rather than go by date.  After awhile I realized that after a few months I usually had no idea what I was talking about.  I used to leave space for updates to cultures but that space would run out or I couldn't locate it again, or for some other reason the data always got fragmented and became somewhat useless.

So always date everything and run from date to date in your lab book.  That way every culture is tied back to the original entry and you can easily find all your info.  In the example above for the T4 I would simply look up the date, which would refer me back to the T3 culture date.  I would also put something like Ref: T4 4-3-09 on the page with the initial entry.  In that way if you look up the 4-1-02 date you will find all the initial info plus you'll see a date leading you to the T4 entry.

Using that system you always can look something up, find the initial entry, then the initial entry will also lead you to other transfers made from it.

Hope that helps.  There are plenty of other systems out there, but that's what works for me.


-FF

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Offlinepotatonet
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Re: senescence, degradation question [Re: fastfred]
    #10102142 - 04/03/09 07:07 PM (14 years, 11 months ago)

gotcha

good info


--------------------
In the Quechua language of Peru there are over 1000 words for "potato"

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Offlinemetalhead
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Re: senescence, degradation question [Re: potatonet]
    #10102325 - 04/03/09 07:36 PM (14 years, 11 months ago)

on the topic of strin degeneration.. is there a way to prevent the degeneration of morel myc. and it's sclerotia forming ability? cuz it's a real bitch to keep starting fresh cultures only to have them crap out a little while into the experiments. especialy if you get a good culture going with great sclerotia forming tendencys.


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Offlinepotatonet
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Re: senescence, degradation question [Re: metalhead]
    #10102413 - 04/03/09 07:58 PM (14 years, 11 months ago)

excellent question!


--------------------
In the Quechua language of Peru there are over 1000 words for "potato"

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OfflineRogerRabbitM
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Re: senescence, degradation question [Re: potatonet]
    #10103089 - 04/03/09 11:03 PM (14 years, 11 months ago)

Morel mycelium shouldn't crap out after a few plates.  Some of my morel cultures are more than ten years old.  Perhaps it doesn't like your agar.  I use MEA, and never change it, despite advice to the contrary.  I just stick to MEA with a hardwood stick in each slant, and cultures just keep right on going.
RR


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Offlinemetalhead
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Re: senescence, degradation question [Re: RogerRabbit]
    #10105928 - 04/04/09 03:59 PM (14 years, 11 months ago)

i'll try that what kind of wood? alder? it's usually around 5-6 transfers that it starts. i remember stamets mentioning it in his gourmet and medicinals book.


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OfflineRogerRabbitM
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Re: senescence, degradation question [Re: metalhead]
    #10106039 - 04/04/09 04:27 PM (14 years, 11 months ago)

I use medical tongue depressors bought from a medical supply house. It doesn't say what type of wood it is, but from the color I suspect elm, birch or maple.
RR


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