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InvisibleAtheist
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Agar with Dried Tissue
    #5332848 - 02/23/06 07:53 PM (17 years, 11 months ago)

Does the tissue have to be fresh or can it be from the inside of a dried mushroom?


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InvisibleFooMan
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Re: Agar with Dried Tissue [Re: Atheist]
    #5332865 - 02/23/06 08:00 PM (17 years, 11 months ago)



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InvisibleAtheist
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Re: Agar with Dried Tissue [Re: FooMan]
    #5332880 - 02/23/06 08:06 PM (17 years, 11 months ago)

Wow that is a huge thread. Thanks foo


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OfflineSnaggletooth
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Re: Agar with Dried Tissue [Re: FooMan]
    #5332881 - 02/23/06 08:06 PM (17 years, 11 months ago)

When reading that post pay attention to Roger Rabbit  :thumbup:


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InvisibleJaeger
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Re: Agar with Dried Tissue [Re: Snaggletooth]
    #5332943 - 02/23/06 08:22 PM (17 years, 11 months ago)

yea, thanks for linking that foo :smile:


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InvisibleFooMan
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Re: Agar with Dried Tissue [Re: Atheist]
    #5332959 - 02/23/06 08:25 PM (17 years, 11 months ago)

No problem fellas. Took me awhile to dig that one up :grin:


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InvisibleDIRTYMAN
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Re: Agar with Dried Tissue [Re: FooMan]
    #5333827 - 02/24/06 12:52 AM (17 years, 11 months ago)

I could imagine that Foo's thread could be used a bit with this journal entry http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5236115/an/0/page/0
Note in Foo's link RR states contams will most surely happen from the dirty hippie not being clean in his drying procedure (why would he?) and transfering to the agar will most certainly contam...hence Agar Saver Tek. Could be transfered to other substrates too I guess... Acquire an eigth and grow!!!:headbanger:


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OfflineRogerRabbitM
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Re: Agar with Dried Tissue [Re: DIRTYMAN]
    #5334210 - 02/24/06 07:15 AM (17 years, 11 months ago)

Yes, you must assume that contamination will develop from a dried fruitbody. That's the beauty of antibiotic agar. Another tip is to use a very small piece of tissue for the transfer. Remember, the larger the piece you use, the more contaminant spores will be on it. You can also soak the dry tissue in water for fifteen minutes with bleach added at the rate of two tablespoons per gallon. This will help to neutralize the bacteria and fungi spores that may be present, while still allowing the mycelium to recover. Just remember, the VERY first mycelium you see must be transferred to a new petri dish, so have some made up and ready. The trick is to make a series of transfers until the mycelium has outran the contaminants.
RR


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InvisibleAtheist
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Re: Agar with Dried Tissue [Re: RogerRabbit]
    #5334214 - 02/24/06 07:19 AM (17 years, 11 months ago)

Awesome RR, I'll definately try this in the future.


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Offlinemogur
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Re: Agar with Dried Tissue [Re: RogerRabbit]
    #5336897 - 02/25/06 01:46 AM (17 years, 11 months ago)

Speaking of decontamination, RR, I found these neat little tricks awhile ago during some research, so this seems like an appropriate place to post them--- (check the tips in bold font below, very clever.)

Novel Method for Selective Isolation of Actinomycetes- Hirsch/Christenson
A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.

Decontamination- Malloch
The simplest means of separating [fungi] is to spread their apart far enough that the colonies resulting from their germination will not grow together for a time. A common method is to place a spore mixture on the agar on one side of a Petri dish and streak it up and down that side until the spores are evenly spread along a line. The streaking is done with an inoculating wire having its tip bent into a loop so that it will not cut the surface of the medium. Sterilize the loop and do a second streak at right angles to the first along another side of the dish. This results in a second line bearing far fewer spores than the first. Repeat this a third time to yield another line. If it seems necessary, do it a fourth time, but be careful not to streak into the first line. After two or more days one of the streaks will be seen to contain several separate colonies that can be used for transferring. The previous streak will be too crowded and the subsequent one too sparse. (Figure 1)


A similar result can be obtained by using dilutions. It will prove more useful than the streaking technique when the [fungi] that is wanted is greatly outnumbered. Just take a mixture of spores and stir it up in about 10 ml of sterile water. Dilute this down several times and plate out each dilution s
eparately. The ideal dilution will produce a plate containing about 60 colonies, of which at least some will be the desired [fungi].

When a [fungi] is contaminated with yeasts or bacteria, dilution techniques usually work well. At times, though, yeast or bacterial cells so outnumber the spores of the desired fungus that dilutions are useless. Then it may be helpful to take advantage of the tunnelling or bridging abilities of fungus filaments. A technique sometimes used is to embed glass rings in the medium and surround the contaminated colony. A sterile glass ring, about 10 mm high and 15 mm in diameter, with three "feet" 2-3 mm in diameter glued or welded to the rim on the bottom, is placed on the bottom of a Petri dish. Sterile medium is then poured in to the usual depth, being careful not to overtop or wet the glass ring. After the medium has solidified, the mixed colony is inoculated into the glass ring. The [fungi] filaments will be able to tunnel down under the ring and back up to the surface, while the bacteria or yeast, lacking the forward pressure of hyphae, will be confined to the ring.

The apical growth of [fungi] hyphae can be used to bridge gaps in the medium insurmountable to bacteria and yeasts. A good method to follow is to make a small moist chamber by putting a few layers of wet filter-paper in a Petri plate. Then flame-sterilize a microscope slide, put a square of agar medium near the edge of it, inoculate the agar with the mixed culture, and put the whole thing in the moist chamber. After a few days the hyphae of the desired [fungi] may extend out into the air from the agar block. If they do, place another agar block on a second sterile slide and move it close enough to the first block to contact the aerial hyphae but not the block itself. If one is successful, the hyphae will grow into the new block, leaving the yeast or bacteria behind.

In some cases, mechanical purification techniques just don't seem to work. If they don't one can take advantage of the chemical or nutritional differences between micro-organisms. The most basic technique is to use antibiotics to discourage bacteria. Most bacteria are sensitive to antibiotics, although no antibiotic will work for all of them. In routine isolation work we use a mixture of antibiotics, most often streptomycin and chlortetracycline at a concentration of 30 and 2 mg/l respectively. Few bacteria are able to grow in such a medium, while most fungi are unaffected. In fact, fungi are so tolerant of these substances that we sometimes just sprinkle a little of the powdered antibiotic right on the agar surface in a Petri plate.

Most [fungi] have at least some nutritional preferences that distinguish them from other moulds. These preferences can be used in the purification of cultures if the physiology of the organisms involved is known. Zygomycetes, for example, grow rapidly on glucose or maltose media such as Leonian's and will overgrow everything, but cannot grow on some complex compounds like cellulose. Thus a medium such as Czapek's, with cellulose substituted for sucrose, will put zygomycetes at a distinct disadvantage.


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InvisibleMyconut
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Re: Agar with Dried Tissue [Re: mogur]
    #7777757 - 12/19/07 01:53 PM (16 years, 1 month ago)

such nice information!

EDIT: didnt see the date on this, found it by chance... my bad.


Edited by Myconut (12/19/07 01:54 PM)


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