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krypto2000
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Registered: 12/05/06
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Last seen: 4 years, 3 months
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Does my procedure sound... sound for serial dilution? MS -> Agar
#23848525 - 11/19/16 12:55 PM (7 years, 2 months ago) |
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I'm trying to start some new cultures as my old ones are.. well getting old. I normally do MS -> agar -> fruit -> clone and then I have a good culture. I'm thinking instead of just scraping some spores onto some agar though I'm better off diluting it to minimize both the amount of strains and contaminants that are on each dish. I'll go over what I'm thinking and let me know if I should modify anything.
I'm thinking of taking a ~100ml beaker, 50ml of dh20 with a 1/100th of a drop of dish soap (tween substitute) and scraping a small amount of spores in there. Maybe stir it up for a bit to make sure they're evenly dispersed. From there take an inoculation loop dipped in the solution and streak the plate.
If I take the smallest amount of spores scraped I physically can does this seem like an appropriate amount of dilution? I have not done this before to know. I will do this in front of a flow hood however none of the equipment will be sterile, is this a big deal? I figure the spore print itself is not sterile so does it really matter if I'm adding in a few more contaminants? Maybe I should sterilize it just to minimize bacteria?
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enlightenment
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Registered: 08/09/09
Posts: 1,647
Loc: Europe
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: krypto2000]
#23848631 - 11/19/16 01:21 PM (7 years, 2 months ago) |
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It should work. But I would make a spore syringe with a very low spore amount. This syringe can be further diluted. I think it is not that beneficial trying to inoculate with a very diluted spore solution.
The classic method does not need to be "improved" IMO. Inoculate a dish and transfer a healthy piece away. What don't you like about MS->agar->fruit->clone? Do you have that much contaminations? You just need one to two transfers from the initial spore dish to get a nice MS grow and a clone. If the spore print is VERY dirty you can try the method you thought about. 
I had only a few dirty prints that were hard to clean on agar.
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krypto2000
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Registered: 12/05/06
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: enlightenment]
#23848677 - 11/19/16 01:32 PM (7 years, 2 months ago) |
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Well MS -> agar is not *bad*, but I have a couple issues that I was hoping to clear up. For one all of my spore prints are old and have been sitting in a drawer that I rumage through a lot, I also have gotten them from trades so I'm going to assume they are not clean at all and thus I'd like to minimize my chances of contamination.
The second reason is that when I do MS -> Agar even after a few transfers it seems to me like a total gamble whether I have a clean culture or not. You just can't tell by looking at it unless you spend a couple of months and 5-10 transfers more to narrow it down.
What I don't like about MS -> Syringe -> agar is that even when using the tiniest of drops the liquid will run all over the agar and thus I can't really isolate anything, the whole dish is either growing everywhere or if there's bacteria in it then growing nowhere because it's full of bacteria.
edit: I'm currently PCing two things of water. I figure I better take this extra step to minimize contamination, I hate when nothing germinates bc it's overtaken by bacteria. I plan to scrape spores in one, stir it up, then dump a small bit of that in the other. This way I'll have two spectrums of dilution. I'll scrape ~2 plates of each and that will hopefully give me a good starting point.
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enlightenment
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Posts: 1,647
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: krypto2000]
#23848696 - 11/19/16 01:39 PM (7 years, 2 months ago) |
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Quote:
krypto2000 said: What I don't like about MS -> Syringe -> agar is that even when using the tiniest of drops the liquid will run all over the agar and thus I can't really isolate anything, the whole dish is either growing everywhere or if there's bacteria in it then growing nowhere because it's full of bacteria.
second transfer:

first transfer:

I use a syringes especially for old spores to rehydrate them. One drop in the center of the dish and carefully place the dish on a shelf without spreading the solution.
If the print is dirty i place one drop on the dish and swipe it several times with an inoculation loop to "separate" healthy parts and contaminated parts.
I recommend to use low nutritious agar. It is easier to notice sectors IME.
I love to use MEA.
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krypto2000
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Registered: 12/05/06
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: enlightenment]
#23848727 - 11/19/16 01:47 PM (7 years, 2 months ago) |
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Hmm.. well you've proved me wrong, it must be my technique then. Or maybe it's just how dilute your syringe was too I guess.
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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
Last seen: 8 months, 21 days
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: krypto2000]
#23848737 - 11/19/16 01:51 PM (7 years, 2 months ago) |
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Low nutritious MEA recipe I use:
15 g Malt extract 20 g Agar 1 L Distilled water
Give it a try 
How I inoculate if the spore print is very polluted:
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krypto2000
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Registered: 12/05/06
Posts: 11,579
Last seen: 4 years, 3 months
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Re: Does my procedure sound... sound for serial dilution? MS -> Agar [Re: enlightenment]
#23848757 - 11/19/16 01:59 PM (7 years, 2 months ago) |
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Wow, that's a great idea, very obvious in retrospect!
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