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OfflinePOWAtrippinDiscord
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First attempt at strain isolation
    #9116642 - 10/22/08 04:12 PM (15 years, 5 months ago)

I have recently been interested in creating isolates of strains as I cant see how I couldnt benefit from this.  I have a syringe at the ready but I also have a bunch of dif colonized subs going right now as well as fruiting casings.  What would be the best means for a nub to innoc these plates in your opinion?

I know that if I use tissue the process will be probably shorter cause there will be fewer to choose from but at the same time would it be better to have more to choose from to get the best?  Should I just innoc one plate with three drops in three areas or just use tissue that probably has multiple genetics?

Also contamination here isnt really an issue as soon as you isolate to another plate correct?  I have heard this mentioned briefly but never heard it flat out.


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OfflineRogerRabbitM
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Re: First attempt at strain isolation [Re: POWAtrippin]
    #9116798 - 10/22/08 04:34 PM (15 years, 5 months ago)

Contamination is always an issue, and every time you open a dish, you risk contamination.  Follow the most anal sterile procedure at all times.

It's best to start from a spore print, and isolate as many strains as you can, then carefully label and fruit each one.  When you find the best strains, simply go back to your well labeled petri dishes to find the original culture that matches the fruits you prefer, and there you go.  Save it on master culture slants for future use, and grow only that/those isolates from now on.
RR


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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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Offlineveda_sticks
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Re: First attempt at strain isolation [Re: RogerRabbit]
    #9116833 - 10/22/08 04:37 PM (15 years, 5 months ago)

Buy RR's dvd, it will show in much much more detail than is on the website.


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PF TEK - writeup by EvilMushroom666
Lets Grow Mushrooms - RogerRabbit & RoadKills website with sample videos plus the full PF TEK video series. Alot of great information - BUY THE DVD
Cakes can and will pin! - So you think cakes suck for pins. Your wrong
Franks Simple Coir/Verm Tek
Franks Proper Pasturisation Tek
Franks Spawning To Bulk - Monotub
Professor Pinheads RTV Injection Port Tek
Foo Mans No Soak WBS Prep Tek

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OfflinePOWAtrippinDiscord
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Re: First attempt at strain isolation [Re: veda_sticks]
    #9117013 - 10/22/08 05:04 PM (15 years, 5 months ago)

So it would be worth it to start from spores for the mass of genetics that would happen.  Being sterile isn't to big of an issue I have worked in labs and understand how to be clean.  I was more refering to if god forbid there is a contam if I isolate it away from the contam it should resolve the problem correct?  Or if a plate contams should I chuck it.  Thats all I was asking.

On a side note I don't have to wait any ammount of time to iso to a new plate or is there a set ammount of time I should wait between isolations?

Also Agar mediums (currently usind PDA) are there any that are best and any that just suck or are they all really about the same?  I was trying to get light malt agar extract but failed as my home brew supply in town went belly up.  I was a little peeved but I am not sure if it makes any difference.  I have looked at vast lists of recipes but haven't really seen any info about the pros and cons of each.


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OfflineRJ Tubs 202
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Re: First attempt at strain isolation [Re: POWAtrippin]
    #9117041 - 10/22/08 05:08 PM (15 years, 5 months ago)

Quote:

GreedAndVanity said:
I know that if I use tissue the process will be probably shorter cause there will be fewer to choose from but at the same time would it be better to have more to choose from to get the best? 



I'm not sure what you mean by the above . . .

Cloning and selecting isolates from multi-spore cultures are two different methods.

This is one method, of many good ones:

1) Do the PF tech of the types of your choice.
2) Clone the first 3 or 6 in-vitro primordia that emerge.
  (easy to do since they are sterile)
  Clone to LC (or directly to grain or other spawn)
3) Fruit
4) Evaluate
5) Select the best clone(s) for the traits you desire

There are many methods that work well.
I like this one.
One nice thing about it, is that you can go directly from 1/2 pint jars into LC and skip the whole Petri plate routine.

PS
I probably don't need to say this, but if anyone wishes to try this approach, be sure to make at least 2 cultures of each clone, so you can fruit one, and save at least one...so that in the end, after you select your winner(s), you can do a grain to grain or back to a plate for storage and propagation....bla bla bla....OK, I'll shut up now...


Julius Richard Petri (1852–1921) was a German bacteriologist who is generally credited with inventing the Petri dish while working as assistant to Robert Koch.

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OfflinePOWAtrippinDiscord
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Re: First attempt at strain isolation [Re: RJ Tubs 202]
    #9117086 - 10/22/08 05:17 PM (15 years, 5 months ago)

I am talking about taking a piece of mycelium from a ms substrate. Say you take one piece of colonized grain and put it on an agar plate, that piece may have two or more different substrains on there and then you should be able to choose the healthiest.  This would be apparent due to sectoring, I have seen sectoring agar plates that were from substrate you would just have a lot less sectoring and would have to go through less isolations to get one refined strain with no sectoring.  I was just wondering if it would be better to go through a ms inocculation of 1000s of competeing DNA types to find the best one or choose from fewer to begin with.


--------------------
Don't believe everything you think.  TRADE LIST

‹Sell Your Soul› You know this place is owned and operated by the Illuminati, right?
‹lsdwithme› i possibly just smoked a rat turd :facepalm:

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OfflineRogerRabbitM
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Re: First attempt at strain isolation [Re: POWAtrippin]
    #9117968 - 10/22/08 08:22 PM (15 years, 5 months ago)

You don't have to go through thousands.  Let them grow for a few days, and many of those sectors will combine together via anastomosis.  At that point, begin your transfers.  If you'll isolate a dozen strains from a swipe of spores on agar, you'll get a few good ones at least.
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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OfflinePOWAtrippinDiscord
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Re: First attempt at strain isolation [Re: RogerRabbit]
    #9118449 - 10/22/08 09:41 PM (15 years, 5 months ago)

How many different samples should I try to choose from in your opinon RR?  The more the better or is ten enough?


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Don't believe everything you think.  TRADE LIST

‹Sell Your Soul› You know this place is owned and operated by the Illuminati, right?
‹lsdwithme› i possibly just smoked a rat turd :facepalm:

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OfflineRRVideoV
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Re: First attempt at strain isolation [Re: POWAtrippin]
    #9118654 - 10/22/08 10:13 PM (15 years, 5 months ago)

Ten is plenty.  Otherwise, you can easily get overwhelmed trying to save and fruit them all.  For the short time it takes a quart of rye berries to colonize, you can put the petri dish in the refrigerator after using 1/4 or less of it to inoculate a quart jar.  Be sure to properly label each jar of grains the same as the petri dish that inoculated it.  Then, label the bulk substrate to match also.  This way, when you harvest, you know exactly what petri dish to go back to in order to capture that strain and save it in master culture slants.
RR


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OfflinePOWAtrippinDiscord
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Re: First attempt at strain isolation [Re: RRVideo]
    #9118690 - 10/22/08 10:17 PM (15 years, 5 months ago)

YES SIR!  I will try to update with pictures to let you know how I am doing!


--------------------
Don't believe everything you think.  TRADE LIST

‹Sell Your Soul› You know this place is owned and operated by the Illuminati, right?
‹lsdwithme› i possibly just smoked a rat turd :facepalm:

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