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Invisiblebbox244
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Gentamicin Preparation in solution suspension with additives
    #14539219 - 05/31/11 04:25 AM (12 years, 9 months ago)

Hello everyone, as a newbie mycologist (only about 9 months), I've learned a lot, but I've been an amateur chemist since college and have done a lot of chemical work, both organic and inorganic.  Anyway, I had an agar bacteria issue, and someone mentioned using an antibiotic, specifically gentamicin.  While this acts on both gram positive and negative bacterias, it makes it tough to see what kind of bacteria you're dealing with, but anyway, I've decide to prep some in an inexpensive way, I've sourced some good medical quality powder with no additives for around $40/gram.  Since the solution I will be making is going to be 100mg/ml I'm wondering if anyone here knows the amount of water necessary to achieve that "density" of Gentamicin solution in terms of percentage.  I could always get out the graduated cylinder and guess it, but I figured someone may know the exact percentage based on the ion separation, etc.  Plus, has anyone here tried adding an excipient, if so, what was used?  There are several I've considered already, but I'm curious if there are any other people who have had success with suspension and shelf life increase in solution with an excipient?  Maybe pe-glycol for starters.  I thought about posting this in the chemistry section, but I figured people here would have more experience with Gentamicin powder.  If nobody knows a density calc, thats cool, I can calculate in manually when it arrives, but I was thinking of getting the formulation done first since I want to try a few excipients have been thinking about that may prolong the life for more than 4 years, also, when working with the powder, do you use the batch analysis to calculate how much you use, i.e. my batch is 678mcg/mg. So, that means a lot was lost during the methly drying, so I have to take that into consideration.  Also, the mol weight for all three components, I'm showing 477mol for the C2 component correct?  The greater being used in the average mol weight?  I appreciate any help, I just want to come up with a really good additive and I will gladly share whatever formulation I come up with on my own, or with the help of others.  Basically, the idea is to make sure it distributes evenly, maintains good suspension in solution, try to extend the actives shelf life as much as possible, and to make sure it'll stay stable during PC and evenly distribute in Agar solution.  Also, there are a ton of other gram +/- anti's out there, some with much more effectiveness against bacteria in terms of outright bacterial destruction, and I was wondering if anyone has experimented with any of the others or more recent ones available.  I have a decent setup, and with the elimination of basically every cool chemical by certain Nazi organizations, I'm stuck with playing with polymers and other hobby shit.  So I'm pretty excited to do something organic again.  I'm definitely going to get some different additives to identify whatever bacteria is attacking my plates.  Sometimes I get a small spot or two doing indoor stuff, and I've never even thought of a chemical solution until someone brought it up yesterday.  Ever since then I've been looking at all sorts of chem related stuff with mycology.  Much thanks,


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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14539529 - 05/31/11 06:56 AM (12 years, 8 months ago)

The most common types of  bacteria in agar work come from the breath and skin of the cultivator. I quit using Gentamicin sulfate in agar a few years ago because it's completely unnecessary. Rapid transfers can isolate the mycelium from bacteria.  I noticed the antibiotic would often knock the bacteria back, but it would show up later in the grains.

The USDA organic standard allows antibiotics in agar for organic mushroom farms, but I'd like to see that changed.

It's common to add about .1 gram of gentamicin sulfate per liter of media prior to sterilization.
RR


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Invisiblebbox244
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Re: Gentamicin Preparation in solution suspension with additives [Re: RogerRabbit]
    #14539974 - 05/31/11 09:56 AM (12 years, 8 months ago)

1g per liter?  It's method of action and penetration abilities in study have shown effectiveness with 8-10mg/1000ml.  Granted, this would be the minimum, I was thinking of starting with 20mg/liter.  My reasoning is 2 fold.  One, since I did some searching on the boards, a lot of people use it for outdoor isolation, so I figured I would refine a process for making it, specifically with longevity and equilibrium in solution in mind and share this technology for those interested.  Personally, what I want to try is doing a cellular assay with it.  I know chromatic assays are more effective, but, even though I am still new to mycology and do not fully understand the genetics involved in the different types of mushrooms, I do know that they are cellular growth fungii.  Stating that, I would imagine that some genes present in different species/strains are more resistant to bacterial infection.  That is what I'm really after.  First, stabilize it, making a tek for procuring and producing a perfect solution of Gentamicin.  Then personally start performing different assays on isolates.  I am curious to see if there are mushroom genetics which may/may not be more susceptible to bacteria.  You probably already know the answer, but, I don't, which is why the thought of mushroom antibiotics intrigued me so much the other day.  Yesterday I think, I work too much, time flies, not sure when I read the post exactly, but it was several hours ago.  Besides formulating a really good method of solution, do you think there is value in isolating genes which are less susceptible to bacteria?  Especially for new growers, or for people who don't have their sterile techniques down quite right, and for risks in inoculation, etc.  From what I recently experienced, some of my spore syringes got infected with bacteria and they never germinated on agar, just a fast form of bacteria grew, indicating to me that the spores themselves had been compromised by bacteria.  If we could isolate genes making mushrooms much more resistant to common forms of bacteria, I believe this would be a positive thing on multiple levels.  Granted, all of this is just 24-36 hours of thought, so I may be way way out in the desert on this one.  But, the idea hit me hard the second that poster mentioned mushroom antibiotics.  I did a lot of R&D in college with microbiology.  Thus, I would love to try genetic isolation via assays, not just with Gentamicin, but some other micin derived antibiotics as well.  Even some other -+gram biotics as well.  I just think there may be some genetic predisposition traits present, and it may be worth while to take a look.  I'm sure this has already been done and researched, but, the idea came to me a little while ago and thought I'd run with it.  Combining my new hobby with my love of chemistry and long long ago love of microbiology feels right. 

Also, I see that most people actually add GM to their growth media.  I was thinking of using it as a coating to medium instead, perhaps 1.5mm thick.  Just enough to protect for a streak.  I think adding it to the entire medium would be a waste, unless you're going to re-use the agar, or something like that.  I was planning on doing a second pour right after my first, with the second pour containing the antibiotic.  Directly over the still warm first pour.  Just enough to coat for a streak or tissue clone.  I also had a dish get contaminated when my parfilm tore because I stretched it a bit too much.  Even though the tear was barely visible, I ended up getting a couple bacteria spots on the plate section exposed.


You are correct about it "knocking back bacteria" but, if penetration has taken place, there is nothing you can do, the bacterial will definitely re-appear unless further suppressed in the growth medium.  Just from what I've read in the past few hours, I bet that if there is an infection, no amount of GM will fix it.  Perhaps a leading edge sector might not be infected, but, I think even that is doubtful.  Once bacteria penetrates a cell, depending on type, it is usually inter-cellular, meaning it will move from cell to cell as growth of the underlying takes place, i.e. it'll spread with the material as it grows once the material has been infected.   

Also, you said you wish the FDA would regulate the usage of GM for Organic grows?  I'm curious why?  GM is a byproduct of a G+ natural bacteria itself, therefore, it is organic.  I don't know anything about it's synthesis, but I do know it comes from a naturally occurring strain of bacteria.  Just curious why you are adverse to it's usage on agar.  Wouldn't it make plates less susceptible to accidental infection?

Again, please comment on these ideas, this is from a brain storm I had from someones post and my curiosity is overwhelmed at the moment.  Thanks to this community as always.  bbox


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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14539983 - 05/31/11 10:01 AM (12 years, 8 months ago)

Too dense, couldn't read.
I'm curious as to the goal here- are you putting gentamicin into agar dishes or LC?
And why? Bad spores or what?


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Re: Gentamicin Preparation in solution suspension with additives [Re: Doc_T]
    #14540113 - 05/31/11 10:43 AM (12 years, 8 months ago)

Quote:

It's common to add about .1 gram of gentamicin sulfate per liter of media prior to sterilization.
RR




As in 0.1 grams/L. If you have sterile technique down this is really a non issue. We've been inoculating agar in the oven or above the gas stove and are still able to get clean cultures.

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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14541043 - 05/31/11 02:54 PM (12 years, 8 months ago)

I don't like antibiotics for the reason they tend to knock the bacteria back, but then it re-appears in slightly more antibiotic resistant form later. If one continues to use Gentamicin sulfate routinely, he's going to breed more and more antibiotic resistant bacteria, and the rods are going to be attached to the mushroom mycelium, growing with it. 

I also disapprove of using pyrethrin as an insecticide in organic agriculture, even though it's 'organic'.  After all, so are poison ivy and arsenic, but I don't use them in food preparation.
RR



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Invisiblebbox244
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Re: Gentamicin Preparation in solution suspension with additives [Re: RogerRabbit]
    #14544982 - 06/01/11 09:31 AM (12 years, 8 months ago)

Sorry, had to finally sleep, was up all night on another project, got some mold in my ... plants...  Anyway, just got back from my "farm", and I see by your post RR, that the bacteria is becoming more resistant to gentamicin.  Which makes perfect sense.  Basically, what I was interested in doing is called a gentamicin assay.  What we do here is culture bacteria via samples from around areas in a common household/and or mushroom patch for indoor/outdoor growers.  Then, we culture and get plates ready for these bacterias.  Once we have plate sections which are growing active mycelium, we go ahead and infect the mycelium.  The purpose for this is to do an exact timed infection, say 2-4 hours and see how the cells of the mycelium react to the infection.  Shortly thereafter, we add gentamicin and see exactly how effective it is in killing the bacteria which did not breech the cells of myc.  Now, in the case of gentamicin, we can see it kills all or most of the bacteria which did not infect the myc. and gauge the rate of infection for the cells which did get infected.  Basically, gauge how effective the infection rate is for a specific bacteria.  Some are more aggressive than others.  By doing this, we find the most aggressive ones, and work with them for awhile doing a count after gentamicin cleanup.  So, we go ahead and repeat the same test using different species/strains of mushrooms and the same strains of aggressive bacteria.  Now, after time, if we do a culture of an isolate, and infect it with an aggressive strain of bacteria, we can then add the gentamicin and voila, we find that most of the bacteria was killed and many less myc cells were infected by microscopic analysis.  This indicates we have myc cells which are resistant to the aggressive strain of bacteria.  Thus, we keep this strain/species and perform further work on it, trying to isolate the genetically bacteria resistant isolates.  After that we can try cross breeding, combination, etc, to get the genetically bacteria resistant culture to bind with other isolates in the same species/stain which have positive traits we look for in growing.  If all goes well, we end up with material which has the positive growing genetics combined with additional genes which make it less susceptible to bacterial infection.  I know this has probably been done a thousand times, since it's basic bacterial microb., but, I think it may have a good application here. 

With what you said about creating "super bugs", the science is the same in people.  Like I was saying before, once cellular penetration takes place, it's pretty much impossible to get rid of.  If you take those cells and try transfers, etc, more than likely, the bacteria which did penetrate will travel right along with the culture via cellular division, therefore, there isn't really anything you can do to get rid of it unless you want to risk making a sub-culture of the isolate which has permeable cell walls to allow for bacterial biocide penetration.  But, while this is common research in humans, I don't think it will apply here.  If we try to "fix" an isolate which is infected, it would take hundreds/thousands of plates and some really harsh and dangerous methods to degrade the cellular structure to allow for penetration, then, once the bacteria is killed, we would have to rebuild the cellular wall strength and keep going, in my opinion it's not worth it. 

In fact, I will bet anything that there are strains out there right now which people grow and use everyday which have some type of bacterial infection present within the cells of the mushroom from it's inception as spores.  These mild bacteria become part of the cellular makeup of the mushroom, thus will always be there, perhaps dormant, but, will always be present under microscopic analysis.  Even with h202, or tri-oxide, unless the cells are destroyed the bacteria or even perhaps a virus for that matter, will always be present in the mushroom, and it's spores.  Just a theory,

To answer another poster, my purposes for using a gentamicin coat are to "create a bacterial resistant skin on my plates", for cloning tissue, spores, etc.  So I don't have to worry about small amounts of bacteria that sometime will move in and start a culture.  In the process of gentamicin solution prep, I plan on making a perfect tek for people who want to use it to keep their plates sterile, etc, for a long time and reduce the risk for accidental contams. i.e. a small parafilm tear, which happened to me.  With that I want the tek to use molar properties, excipients, etc to make the perfect gentamicin solution for use in microscopy.

My other purpose is above, to do assays using gentamicin to find isolates which are resistant to bacterial penetration.  I just want to see if the assays work, and offer up some slants to the community if I am successful.  I've already begun some basic bacteria study in my totally isolated lab I filmed up and sealed a few days ago.  This is where I'll be conducting my experiments since, bacteria, and especially mold, can really mess up a grow op.  I grow purely as a hobby, and now that I've come up with a perfect fruiting chamber based on RR's design, for rapid temp changes, I have a lot of fruits, so now, I want to spend a few months collecting species/strains, and doing some isolate work to find bacterial resistant traits.  Also, my agar techniques need further refinement and practice, and I am perfectly content spending the next few months just working on again with occasionally fruiting a jar or two with some genetically promising isolates, especially if I can discover some bacterial resistant traits to try combining with positive growth genetics, to create a bacterial resistant, genetically stable and abundant mushroom,

bbox

I just updated this post to make up for all the pre-coffee mistakes this morning.  Also, I want to add that since this all started from a post in regards to 3 infected spore syringes I had, I've now taken those syringes and analyzed them under an ms non-stereo, I wish I had one of those, but, anyway, sure enough, the bacteria has broken through the walls of the spores.  I'm almost positive this is the case.  Need a few chems to be sure, and will be ordering them shortly, but, I bet none of these spores will germinate if left in solution forever.  On another note, using my last bag print which I dried and added Silica Gel too, I made several tri-section plates streaked with those spores, and I actually have a full section growing myc with zero bacterial contamination.  The other 8 sections are contaminated, but one of them isn't.  So perhaps the bacteria I'm dealing with isn't aerotolerant after all.  I decided to keep the syringes and will make another plate in a few weeks.  Anyway, I just would like to thank the community again for all the wonderful help.  Thank you RR for your guidance and replies.  If I didn't come across your videos and purchase them, I would have never discovered this absolutely fascinating hobby.  I totally understand now why you do it full-time.  It's very very addictive.  More than smoking in my opinion.  :smile:


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Edited by bbox244 (06/01/11 01:52 PM)

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OfflineRogerRabbitM
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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14546779 - 06/01/11 04:52 PM (12 years, 8 months ago)

You might want to confine your thoughts to one or two points per post.  If you write a book, it's too much to read in one post.  I've never seen bacteria inside a mushroom mycelium cell (yet).  I've seen lots of bacteria on the surface, but mushroom mycelium is only one cell in thickness.  I'm not sure where it would hide, but at any rate, I've not ever seen it.
RR


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Invisiblebbox244
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Re: Gentamicin Preparation in solution suspension with additives [Re: RogerRabbit]
    #14546994 - 06/01/11 05:39 PM (12 years, 8 months ago)

Yeah, it pretty much impossible without an electron microscope to see it, but, the indications are there.  I'm seeing what I think are penetrated cell residuals.  Anyhow, I am going to get some chromatic media and some chems to tell for sure.  This is the only way you will actually get a for sure visual method of seeing it under a scope.  Thats how I'm going to do the assays as well, without the proper chems there is no way to isolate the infected cells from the non infected ones.  I'm pretty sure what I saw is some evidence of penetration though.  Like I said, no way to be sure without chems and no I don't have an electron mic :wink:  It would be a cake walk with one of those.  Those buggers are really really small, way beyond my magnification.  With the right chems you get phosphorescence of the cells in play, so you can get a good count based on surface area.  This will tell you the rate of infection when timed.  It's pretty much how I plan on doing what I said in my other post.  It's not as perfect as a good escope, but, since I'm just doing counts, it'll work fine.  I'm just waiting for that large surface area to glow once I add the gentamicin and it does it's job, or whatever antibiotic I use, then I'll know for sure there is bacteria resistant genetics in mushroom tissue I'm testing.  But, that could take awhile.  I've seen a lot of bacteria in my lifetime, but always with an escope, I've seen cultures under ocular magnification, but, never individual cells without using "particles". 

On a side note, I actually knew someone in college who build an escope, it was insane, I actually built a mass spec with my TA in grad physics, and believe me, that was totally insane.  It worked like a charm though.  We we're taking apart bee pollen for the CRI (Cancer Research Institute), and analyzing the components and creating sample matrixes for them to test.

On another note, I totally agree, nobody wants to read a book, it's an ADD thing combined with too much coffee.  I am a total coffee addict, and my mind wanders, etc.  But I will make an effort to stay on point.  Thanks,
bbox


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Edited by bbox244 (06/01/11 05:43 PM)

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Invisiblebbox244
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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14549300 - 06/02/11 03:25 AM (12 years, 8 months ago)

I took some time tonight to do some research into the genetic attributes of mushrooms.  The implications of genetic research into mushrooms is staggering.  In my previous post, I was talking about an idea with which one could isolate bacterial resistant species/strains of mushrooms using gentamicin assays as a starting point.  Well, I have to share this.  After my research tonight and a couple phone calls,

I believe mushrooms can kill cancer. 

I had no idea, no clue.  I am truly amazed at the limitless genetic potential of mushrooms at this point.  Just from looking into it for a few hours to prep for my experiment, I've not only discovered that finding bacteria resistant genetics is not only possible, but is highly probable.  Mushrooms have more beneficial attributes to negative organisms and in-organics than anything I ever studied at the CRI or anything I studied during all my years working in microbiology.  I feel really cheated that I was never given the opportunity to work with mushrooms in my research and feel like sending some questioning emails to some of my former research professors for being so unknowledgable regarding research into mushroom biology.  I'm definitely going to do some assays as a starter, and will keep logs for anyone interested, but I will try to stay within the confines of isolating genetics for assisting in growth of mycelium and mushroom fruits, since that is what this community is primarily about.  However, I must say again, that I am truly shocked by the endless possibilities of the mushroom.  I had no idea, I feel like years of my life went by and they were totally wasted.  Why on earth are people not researching mushrooms more?  After my findings tonight, I would think they would be the organic of choice for an incredibly wide spectrum of microbiological research.  I will start with spore research primarily at first, then into mycelium, which is where the magic really happens.  Mycelium has endless possibilities in research.  I just had no clue, so for those of you who knew all of this already, thanks.  Actually, I'm just in a state of amazement now, my mind is overloaded, and thats all for tonight.  Thanks to the community, and I will hopefully scratch the surface of this soon.

So, there will be 2 things, a Gentamicin/Antibiotic TEK, and perhaps some species/strain genetics which will have good resistance properties against infection as well as being good candidates for cross breeding into good growth/fruiting genetics.  Good night all.
bbox


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Re: Gentamicin Preparation in solution suspension with additives [Re: bbox244]
    #14549739 - 06/02/11 07:18 AM (12 years, 8 months ago)

Many species of fungi have very powerful anti-tumor properties.  This is all well documented and why we have a gourmet and medicinal mushroom forum here.
RR


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Re: Gentamicin Preparation in solution suspension with additives [Re: RogerRabbit]
    #14549921 - 06/02/11 08:22 AM (12 years, 8 months ago)

by my memory stametes states in GGMM the gentamicin sulfate is adde to agar at 1/20th g per 1 L of agar solution.

I stir my agar thoroughly before PCing in order to make sure all ingredients are homogenous. Gentamicin is one of the few broad spectrum antibiotics that will survive PCing, making it a popular additive. It is also one of the few that is not controlled by the NAZI science institutions.

I agree with RR in the opinion that it is entirely unnecessary if you have your sterile technique down. I pour straight agar, no additives, in my new clean room (specially built) and have a ZERO contam rate. :dancer:

In my last lab I routinely peroxided my agar. There are others here who have theorized that peroxide instantly boils off. IMHO I can not agree. I had a plate that I accidentally touched that I made using this method, so I left it out in front of my flow bench overnight. It was a new winter season and unknown to me a mouse moved into my lab and starving attacked the agar. There were paw  prints all over the agar and several large chunks missing. as per usual with mice feasting there was several mouse pellets by the dish, in addition to the fact that the lid was knocked off for several hours after the feast in open air.

Realizing this was the ultimate test of peroxided agar. I para filmed and quarantined this plate after putting the lid on and scraping off the mouse poo.


Several days later there was ZERO contams. I observed that plate for a month and there was never a bacteria or a mold.

Either that mouse washed his body and sterilized his hands with my alcohol and brought tiny little gloves to work with.........

I am thinking that no matter what the experts say that peroxide will work against bacteria equally as mold spores.

I cant remember the exact rate of peroxide I used but I think that it was something like 16 ML per L. I added after I PC in a PC'd 10 ml graduated cylinder. I added and stirred while on the hot side of molten to ensure consistent distribution.

On a final note The "Agar sandwich" method has worked very well for me against isolating myc from bacteria. The bacteria simply do not have the ability to move through a solid media. The myc will survive the end heat of almost not liquid agar, and will emerge shortly after immersion. A tiny sample from the emergence on a new plate and Viola :goodluck:


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Re: Gentamicin Preparation in solution suspension with additives [Re: mycoelf]
    #14554357 - 06/03/11 06:03 AM (12 years, 8 months ago)

You had a brilliant mouse :wink:  Peroxide will survive higher temps than people think.  The oxidation rate has many dependencies, air pressure being one of them, temp, etc.  No doubt it will survive if you add to cooler agar.  This can easily be proven as well.  A simple steel wool test would prove it.  Anyway, I really dig the sandwich idea.  That is genius.  It still depends on the type of bacteria, but, it will work for a lot of them.  If it's aerotolerant, then it will survive no matter what you do unless you use something biocidic to that specific type of bacteria.  The problem is in the Hyphae themselves, if infected, the bacteria will move between the "dividers", in the cells, I think they're made of Centin or something like that, totally different from cellulose found in plant cell walls. So even if the mycelium was sandwiched, depending on the bacteria, it could in theory move along the hyphae as it is forming, one cell at a time.  I'm definitely going to try it out though, once my chems get here.  Since I will be inducing bacteria to phosphoresce, I'll be able to see if the mycelium which grows to the surface in an agar sandwich remains infected if there is infection in the underlying sandwiched mycelium.
Like you said, sterile technique is everything, I just opened up a sleeve I poured a week ago that was sitting on my shelf, and zero contams.  I'm still using a GB, I am working on a really wild flow hood using a 2'x3'x7" H13 filter for $40.  Long story, it's laser scanned, H13, 99.995%@>=0.3um, etc, whole other post on that deal when it gets here from Europe.  Once I have that I will definitely have an advantage, plus, I hate my GB, it's such a PIA, sweating my ass off in my murder suit, it blows.  50mg in the Agar will work.  So for h202, you used 16ml at 3%, I have 97% in the lab.  So, I'll break that down.  I like the peroxide idea, I think it would last for a few days at least.  I just read my first article on Paul S. tonight, regarding some of his research.  Fascinating man.  He found a 2000 yo mushroom in Oregon which covers like 20,000 acres or something insane like that.  Anyway, thanks for your help, I cant wait to get started on the bacteria stuff.  I've already been swabbing away :wink:  Have a great one!  Thanks!


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