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OfflineDistrict
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tetracycline concentration on agar?
    #23018103 - 03/17/16 08:33 PM (8 years, 9 months ago)

What's a good concentration on adding tetracycline to agar? I have 500mg pills.

I found in the forum:

Quote:

The ones related to pennicillin, like ampicillin , use at 50mg/liter and the ones related to
tetracycline, use at 10mg/liter.




Quote:

Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml.  Mark the plates with a single black line on the side.




And do be clear that ml means agar solution in liquid form right?

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Re: tetracycline concentration on agar? [Re: District]
    #23019176 - 03/18/16 01:52 AM (8 years, 9 months ago)

anyone?

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Re: tetracycline concentration on agar? [Re: District]
    #23019275 - 03/18/16 02:37 AM (8 years, 9 months ago)

Ummm idk about this dude. Those 500mg pills probably have another half gram or so of additives, fillers, and binders, that you probably don't want in your agar. As far as the math goes it says to put 10-15mg of tetracycline per liter of agar solution, and unless you have a milligram scale you can't reliably eye ball 15mg trust me! All in all I would highly recommend not doing this, and potentially wasting a whole litre of agar. I'm no expert on agar but any time I've seen or read about agar added to culture it was gentamicin and it was in pure form. Never have I read anything about people crushing up prescription antibiotics. If you want culture antibiotics check out mycopath they're a sponsor and that's where I got mine when I tried to germinate some dirty prints.

Hope this helps!
Peace

Edited by St.Owned (03/18/16 02:42 AM)

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Re: tetracycline concentration on agar? [Re: St.Owned]
    #23019286 - 03/18/16 02:42 AM (8 years, 9 months ago)

Gentamicin, not available, in pure form where I am. Hence, tetra. Fish antibiotic. A liter of agar is cheap here.

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Re: tetracycline concentration on agar? [Re: District]
    #23019313 - 03/18/16 03:06 AM (8 years, 9 months ago)

Gentamicin is very common in feed and farm supply stores in rural areas. I found some once as an anti parasitic injection for pigs. Why do you need antibiotics in your agar so badly though? Are you trying to use dirty spore prints from wild mushrooms?


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Re: tetracycline concentration on agar? [Re: St.Owned]
    #23019405 - 03/18/16 04:41 AM (8 years, 9 months ago)

Thanks for the info, Tetra is what I have in this environment. Not interested in gentamicin.

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InvisiblebodhisattaMDiscordReddit
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Re: tetracycline concentration on agar? [Re: District]
    #23019521 - 03/18/16 06:58 AM (8 years, 9 months ago)

Chances are you only think you need antibiotics because you're a noob.
:shrug:

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Re: tetracycline concentration on agar? [Re: bodhisatta]
    #23019554 - 03/18/16 07:13 AM (8 years, 9 months ago)

Forget complicated extra steps and just get better at the simple ones. I suck at agar transfers and am having wild print problems but the only way it's going to get better is practice.

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Re: tetracycline concentration on agar? [Re: Adden]
    #23019680 - 03/18/16 08:19 AM (8 years, 9 months ago)

I too reccomend to you to steer clear of antibiotic agar. Just yesterday or the day before there was a thread made by someone who uses it exclusively. The result was that bacteria was being suppressed to a point where he could not see it on his plates. Thinking he has a perfectly healthy plate he used wedges on jars and spawned to a monotub. Obviously from the day he dropped that wedge in his jar the antibiotic agar was useless.

If you really have bacteria problems, it's definitely nothing the sandwich tek can't fix.

Grab a fresh plate and your bacteria contaminated plate after prepping your work area, wiping plates etc etc. Open the new plate and dig a small hole with your scalper in the middle of it. Then cut a small wedge from the contaminated plate and put that in the hole of the fresh plate (try to cut the wedge small enough for it to fit in the hole). When that is done cut a square of agar from the fresh plate and put that on your contaminated wedge. Wait until the mycelium grows on the surface of the patch you put on your wedge and scrape some mycelium to a new plate. This will, without a doubt have no bacteria in it unless you fucked up on sterile technique. Bacteria cannot grow through agar like mycelium does and the barrier of agar makes sure that the mycelium cannot drag any bacteria with it.

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Re: tetracycline concentration on agar? [Re: Supalemonhaze]
    #23022273 - 03/19/16 02:32 AM (8 years, 9 months ago)

Well, guys, after months and months of trying to cultivate grocery bought shrooms, I finally have stopped bacteria.

I'm like, "anti-bacteria, where have you been all my life?"

Seriously, if you are a noob, and don't have access to clean spores, syringes, whatevers from the sponsors. Give yourself some anti-bacteria. Now I can finally proceed to sectoring and other advanced shit.

It's worth it.

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Re: tetracycline concentration on agar? [Re: Supalemonhaze]
    #23022304 - 03/19/16 02:50 AM (8 years, 9 months ago)

Quote:

Supalemonhaze said:
I too reccomend to you to steer clear of antibiotic agar. Just yesterday or the day before there was a thread made by someone who uses it exclusively. The result was that bacteria was being suppressed to a point where he could not see it on his plates. Thinking he has a perfectly healthy plate he used wedges on jars and spawned to a monotub. Obviously from the day he dropped that wedge in his jar the antibiotic agar was useless.

If you really have bacteria problems, it's definitely nothing the sandwich tek can't fix.

Grab a fresh plate and your bacteria contaminated plate after prepping your work area, wiping plates etc etc. Open the new plate and dig a small hole with your scalper in the middle of it. Then cut a small wedge from the contaminated plate and put that in the hole of the fresh plate (try to cut the wedge small enough for it to fit in the hole). When that is done cut a square of agar from the fresh plate and put that on your contaminated wedge. Wait until the mycelium grows on the surface of the patch you put on your wedge and scrape some mycelium to a new plate. This will, without a doubt have no bacteria in it unless you fucked up on sterile technique. Bacteria cannot grow through agar like mycelium does and the barrier of agar makes sure that the mycelium cannot drag any bacteria with it.





This is good stuff. I might do it to make sure. Right now, it's nice to not see bacterial blotches after 24 hours, and nicer to see actual mycellium blossoming.

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InvisibleSupalemonhaze
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Re: tetracycline concentration on agar? [Re: District]
    #23022342 - 03/19/16 03:23 AM (8 years, 9 months ago)

Quote:

District said:
Quote:

Supalemonhaze said:
I too reccomend to you to steer clear of antibiotic agar. Just yesterday or the day before there was a thread made by someone who uses it exclusively. The result was that bacteria was being suppressed to a point where he could not see it on his plates. Thinking he has a perfectly healthy plate he used wedges on jars and spawned to a monotub. Obviously from the day he dropped that wedge in his jar the antibiotic agar was useless.

If you really have bacteria problems, it's definitely nothing the sandwich tek can't fix.

Grab a fresh plate and your bacteria contaminated plate after prepping your work area, wiping plates etc etc. Open the new plate and dig a small hole with your scalper in the middle of it. Then cut a small wedge from the contaminated plate and put that in the hole of the fresh plate (try to cut the wedge small enough for it to fit in the hole). When that is done cut a square of agar from the fresh plate and put that on your contaminated wedge. Wait until the mycelium grows on the surface of the patch you put on your wedge and scrape some mycelium to a new plate. This will, without a doubt have no bacteria in it unless you fucked up on sterile technique. Bacteria cannot grow through agar like mycelium does and the barrier of agar makes sure that the mycelium cannot drag any bacteria with it.





This is good stuff. I might do it to make sure. Right now, it's nice to not see bacterial blotches after 24 hours, and nicer to see actual mycellium blossoming.




Be careful with antibacterial agar, as I've said before that there is always a possibility that the bacteria is able to survive the antibiotic but is too weak to grow enough to be seen.


Here is a pic of a culture that like you, I just couldn't clean up. It's an oyster culture which was started with a piece of oyster tissue bought from a store. After about 3 failed transfers I found the sandwich tek and did it. bam, BAM! Clean as a whistle:


Notice how clean it looks. Digging a hole in the middle to put your wedge in ensures that the patch you put on it fully covers the contam and it has nowhere to go. I could have easily taken a wedge from elsewhere in the plate because of this but I went for the safest option, which is the top of the patch.


See the 2 scratches on the patch? This is where you take your piece from because it's impossible for bacteria to go up there whereas the condensation water inside the plate can easily distribute the contamination on the lower level. Notice that I didn't take wedges, I took scrapings because if I cut a wedge like normal, my scalpel would have come in contact with the bacteria.



Now I'm waiting for this plate to grow so I can inoculate a couple master jars and some extra plates.

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Re: tetracycline concentration on agar? [Re: Supalemonhaze]
    #23022508 - 03/19/16 06:20 AM (8 years, 9 months ago)

On same agar plate where mycellium runs. Dig hole in middle. Cut wedge size of hole. Invert. Take 'scratches' at top of inverted wedge. Transfer.

Did i get that?

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Re: tetracycline concentration on agar? [Re: District]
    #23022519 - 03/19/16 06:38 AM (8 years, 9 months ago)

Actually, no. I see now. That pic is fully colonized from a small contam'd wedge covered in that trapezoid shaped patch. The mycellium punctured through that agar patch, which you then scraped.

I'll do this next.

Edited by District (03/19/16 06:40 AM)

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Re: tetracycline concentration on agar? [Re: District]
    #23022546 - 03/19/16 07:06 AM (8 years, 9 months ago)

Exactly. Now since I made a hole to start with the patch sat flat on the rest of the agar, completely trapping the contaminated wedge in there so I probably could have used the rest of the plate for wedges but if you want to play it safe, the top of the patch is the way to go.

When I didn't do the hole the patch became like a small hill which could slide all around the plate easily. It can also break in two or completely fall off the wedge. The hole is something I added and it keeps everything in place.

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Re: tetracycline concentration on agar? [Re: District]
    #23023860 - 03/19/16 04:46 PM (8 years, 9 months ago)

.

Edited by Mandarinfish (07/17/20 09:25 PM)

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Re: tetracycline concentration on agar? [Re: Supalemonhaze]
    #23024122 - 03/19/16 06:15 PM (8 years, 9 months ago)

Quote:

Supalemonhaze said:
Exactly. Now since I made a hole to start with the patch sat flat on the rest of the agar, completely trapping the contaminated wedge in there so I probably could have used the rest of the plate for wedges but if you want to play it safe, the top of the patch is the way to go.

When I didn't do the hole the patch became like a small hill which could slide all around the plate easily. It can also break in two or completely fall off the wedge. The hole is something I added and it keeps everything in place.




There are two types of antibiotics: bacteriostatic (prevents the bacteria from growing) and bactericidal (kills the bacteria).  Here's a list of bactericidal antibiotics:

https://en.wikipedia.org/wiki/Bactericide

You can often get antibiotics without a prescription by ordering stuff for aquariums.

I don't see why using bacteriostatic antibiotics would be that bad, either.  Isn't most of growing mushrooms all about giving the fungi a "head start" on the contams?

Edited by mushroomnoob1981 (03/19/16 06:16 PM)

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Re: tetracycline concentration on agar? [Re: mushroomnoob1981]
    #23024140 - 03/19/16 06:19 PM (8 years, 9 months ago)

Plus, doxycycline is stable-ish for about 90 days at 40°C.  Even though it is a bacteriostatic, it should last long enough for your grow. 


http://chromsci.oxfordjournals.org/content/45/9/623.full.pdf

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Re: tetracycline concentration on agar? [Re: mushroomnoob1981]
    #23024528 - 03/19/16 08:17 PM (8 years, 9 months ago)

Quote:

mushroomnoob1981 said:
Quote:

Supalemonhaze said:
Exactly. Now since I made a hole to start with the patch sat flat on the rest of the agar, completely trapping the contaminated wedge in there so I probably could have used the rest of the plate for wedges but if you want to play it safe, the top of the patch is the way to go.

When I didn't do the hole the patch became like a small hill which could slide all around the plate easily. It can also break in two or completely fall off the wedge. The hole is something I added and it keeps everything in place.




There are two types of antibiotics: bacteriostatic (prevents the bacteria from growing) and bactericidal (kills the bacteria).  Here's a list of bactericidal antibiotics:

https://en.wikipedia.org/wiki/Bactericide

You can often get antibiotics without a prescription by ordering stuff for aquariums.

I don't see why using bacteriostatic antibiotics would be that bad, either.  Isn't most of growing mushrooms all about giving the fungi a "head start" on the contams?




Just because it says it kills bacteria it doesn't mean it's effective on all of them, especially if you use it constantly which can give bacteria a resistance to that specific antibiotic.

My first premade agar was antibacterial but I was still getting bacteria contams, when I asked what this is about I was told that different bacteria needs different antibiotics, which makes perfect sense.

And yes you are correct we are giving our fungi a head start on other organisms but if you have a supressed bacteria contamination on a plate and you use that plate to inoculate jars, the head start is gone and the bacteria is free to grow on your grains, rapidly multiplying in numbers. After g2g or spawning it will be hard for mycelium to keep up with the bacteria because of the sheer number of it. Some bacteria grow at a phenomenal rate especially in higher temps.

Bacteria is very easy to get rid of completely with normal transferring so that is why experienced mycologists refrain from using it. That and the reason mentioned above makes most of us rely on our skills more than a medicine that might or might not work.

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Re: tetracycline concentration on agar? [Re: District]
    #23379604 - 06/25/16 04:00 AM (8 years, 6 months ago)

Bumping this.


Most research recommend a 'pinch' of tetracycline. I want to share my experience with concentration.

I usually run about 250ml agar solution. With a 'pinch' I found that bacteria still blooms. So, I would say, 1 tsp per 250ml is a safe amount based on my experience.

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Re: tetracycline concentration on agar? [Re: District]
    #23768941 - 10/25/16 03:57 AM (8 years, 2 months ago)

Adding more info.
1 mycelium colonizes slower in tetra-anti bac agar
2 one pack = one use. the tetra powder itself will be contamed, so you only get to use a pack once, once you open it.

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