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Offlinecyankid
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Registered: 10/20/00
Posts: 49
Last seen: 5 years, 11 months
Agar/liquid Q's
    #1567179 - 05/21/03 10:03 PM (13 years, 6 months ago)

Hey all. Pretty well rounded when it comes to mycology, but I'm about to try this liquid innoculant thing. Anybody have any ideas for a port on the lid that would facilitate easy/clean dropping of an agar wedge, as well as clean closure? I was thinking some sort of compression fitting attached to the lid. Also, what's best? I have access to malt extract of all types, dextrose, yeast nutrient, PDY etc... I've heard dextrose is cool because you can see anything it it, but the nutes suck. Also, what about adding sulphites to protect against bacteria like they do in wine? Or what about hops? Both are used to protect liquids from bacterial contamination, only the sulphites affect yeast.

Also, I recently made some PDY agar, and I'm having bad results. Lots of contams and slow growth. Is it a pretty broad based substrate? I would assume that's why I'm seeing so many contams. Back to MEA for me. Been doing all my work in my dirty ass kitchen in my dirty ass apartment, and surprisingly, until now, I've had great results. Go figure.

Thanks for any help/insight. Just trying to beat this contam war.



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Invisiblemicro
bunbun has a gungun
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Registered: 05/09/03
Posts: 7,532
Loc: Brick City
Re: Agar/liquid Q's [Re: cyankid]
    #1571073 - 05/23/03 02:31 AM (13 years, 6 months ago)

Don't know about an actual agar wedge; this really needs to be done under a flow-hood/glovebox, however, you could use a sterile innoculating loop to transfer from petrie -> liq. media (broth.)

As for a seal on the jar with the broth, a septum plug (like on blood vials) should work well if it's air-tight (it's rubber so all you need is a bit smaller but almost perfectly round hole in the jar lid.)  I don't know who came up with this idea; I think someone on Mycotopia.  HINT, HINT -- however, if you could come up with a seringe full of sterile innoculant you could alcohol-swipe the rubber septum plug and just innoc.  Petries are the hardest, IMO, because you have to open them up, but in a glove-box you could always inject water/scrape/draw back; this is a lot better than waving an innoculating loop all over the place in unsterile air.  This brings in the possibility for a vector of contamination from the sides of the petrie, however.  It's sooooo much easier under good laminar flow, (make sure the cabinet is empty and not full of air obstructions) however.

As for PDY -- yes, it does get contaminated pretty easily.  Just a problem with making media.  The slow growth can be a result of a lot of things; most likely carmelized sugars (from over sterilizing -- only use 15 psi for 15-20 min at full pressure,) insufficient nutrient base (I add a .5X non-essential amino acid mix to promote fast healthy rhizomorphic growth) or too thick of a media if you are not diluting it at the end (1L water/300 g sliced potatoes should boil down to less than 1L, and much water is absorbed by the potatoes,) pH (unlikely), insufficient moisture (there should be a good amount of condensation on the tops if incubated right-side up.  I just put the petries back in the sleeve to incubate to help this a bit and make life a lot easier,) weak strain or contaminatio.

As for the decision of what type of media, nutrient broths (ie PDY or malt extract) are far better at growing mycelium, especially under submerged culture (shaking constantly) than a simple d-glucose solution.  IMO PDY broth is the best, but malt xtrct works fine, too.  The only problem is that these are nutrient broths and thus get contaminated mad easily, so you must be 100% sterile.  Malt extract broth is given as:

Per liter:

Malt extract 6g
Maltose 1.8g
Dextrose 6g
Yeast Extract 1.2g

Ster @ 121C/250F (~15 psi) for 15 min

Sulphite is not a good idea; its main purpose is to inhibit yeasts and moulds and it is very unspecific for what type of fungi it attacks.  It has many functions, but mainly disrupts metabolism (I think the electron transport chain in particular, but I could be wrong.)  If you are worried about bacteria, a bacteriostatic would be more affective, i.e. gentamycin or penicillum/streptomycin, but when it comes to the fungi there is not much you can do except be sterile, but yes, there are some reagents (i.e. peroxide) that will kill the weaker faster-growing actinomycetes, etc. and not readily kill the basidios (stronger) at similar concentrations.  It's up to you what to do :wink:

--
Micro 


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OfflineBenFungTorez
Los Rancheros!

Registered: 04/06/03
Posts: 38
Loc: Way up there...
Last seen: 12 years, 3 months
Re: Agar/liquid Q's [Re: micro]
    #1576268 - 05/25/03 01:57 PM (13 years, 6 months ago)

hey dudes,
i work if a food pathology lab and prepare agar plates for culturing fungus all the time. most often we use PDA. I've recently tried culturing ecuadorians and mexicana on plain PDA plates, the mexis are taking slowly but the EQs are not taking yet.....i think my spores are a bit off. anyways, to my point.
if you have access to antibiotics try using PDA with 100ppm of streptomycin. many antibiotics stop only bacterial growth while allowing fungus to still grow. just make sure you let the agar cool a little before you add the antibiotics or they will be inactivated.


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Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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