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OfflineABC
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Agar before PC'ing
    #9959441 - 03/12/09 04:52 PM (15 years, 10 days ago)

Hello, not quite sure if I did it correctly. I don't have a scale

500 ml water
1 heaping tbsp agar powder
1 heaping tbsp yellow corn starch
5 g turbinado sugar
1 tsp honey

This is my first attempt at agar (the recipe is kindof thrown together, but I thought it might be 'close enough'). I'm currently PC'ing some bags, so I just let my mixture sit. After an hour it's still liquid. I've been agitating it every so often, but it seems that a bunch of it settles to the bottom, which indicates that there is too much solute to be suspended in the water.

So why is it still liquidy? Does agar need to be heated before it can solidify at room temperature?

There are tiny spots of solid where it stuck to the glass from agitating, but it's mostly liquid

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Re: Agar before PC'ing [Re: ABC]
    #9959907 - 03/12/09 06:03 PM (15 years, 10 days ago)

This shouldn't be in advanced mycology...

Your on the right track.

A level tablespoon of agar is 7g, so a heaping tablespoon is probably about 10g which is what you need for 500ml of water.

I like to heat my agar before I PC it just to make sure that it all goes into solution, it sucks when you wait 3 hours for the PC to cool and then your agar doesn't harden. What I do is bring it to a boil, turn off the burner, stir it, then bring it to a boil again, put it into the container I'm gonna PC in and PC for 30-45min. After waiting for the PC to cool down, take the agar out and swirl it to mix it all up again, while in the PC often some stuff will settle towards the bottom and you want everything to be in suspension.

Agar won't get solid unless its heated.


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Re: Agar before PC'ing [Re: PitcherCrab]
    #9964700 - 03/13/09 12:56 PM (15 years, 9 days ago)

Hi: So lets make your question fit into advanced mycology. Your water needs to be heated to dissolve the agar, other wise it will just settle out of solution. The trick is to just heat the solution enough to dissolve the agar. Never let it come up to a simmer or worse a boil as this will start to coagulate the starches and the sugars to start caramelizing that's in the agar solution. This can reduce the jell strength of the agar so it will not set up right. Plus binding up some of the nutrients in the finished agar so they are unusable by the fungi you're trying to grow.

Heat the solution you're dissolving the agar in a double boiler or what is called a water bath in a lab. Many homes have one to make candy or you can just take a large pan with some water in it and placing a smaller pan that your going to use for the agar solution into it. The hot water will heat the inner pan that holds your agar solution helping you to not over cooking it. Just heat and stir the agar solution tell the agar dissolves and then remove from the big pan and pour into the bottles you're going to sterilize it in. This is an old lab trick that even the big boy's use like BBL/DEFCO.

Agar is what is called a mucofiber compound (because they look some what like mucous when dissolved in water) that is made up of a mix of low viscosity soluble microfibers. Carrageen, carob gum, guar gum and xanthan gum are other common mucofiber compounds. What makes agar unique is that it will jell because it has just the right mix of these soluble microfibers. When you over cook your agar and some of the starches and sugar start to coagulate they act like microfibers and upset the balance of the microfibers naturally found in the agar, braking its jell strength. Things that have a lot of soluble fiber in them like oat meal can do the same thing; that's way oat meal agar can have a real soft set.  Good luck; Hipster

Edited by CheeWiz (03/13/09 01:40 PM)

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Re: Agar before PC'ing [Re: CheeWiz]
    #9964936 - 03/13/09 01:39 PM (15 years, 9 days ago)

Quote:

The trick is to just heat the solution enough to dissolve the agar. Never let it come up to a simmer or worse a boil as this will start to coagulate the starches and caramelize the sugar in the agar solution. This can reduce the jell strength of the agar so it will not set up right. Plus binding up some of the nutrients in it so they are unusable by the fungi you're trying to grow.




I don't know where you're getting that from.  Standard procedure is to boil to dissolve the agar.  Besides you're going to be heating it well above boiling during sterilization.  I've never had any problems with carmelization.  It's very rarely a problem and then only with certain formulations at higher than normal temperatures and sterilization times.  It's not something to worry about.  Certainly no need for double boilers or waterbaths, and no need to waste energy and overcomplicate things.

I'm not sure WTF the OP is trying to make with starch, sugar, and honey.  But here is the proper preparation of what he should be making...

Quote:

Potato infusion 200 g 
Dextrose 20 g 
Agar 20 g 
Distilled water 1 liter

To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form).

Mix in other ingredients and boil to dissolve. Autoclave 15 min at 121°C. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 ± 0.2.

Source: Bacteriological Analytical Manual Online (January 2001)
FDA M127 Potato Dextrose Agar




One thing that RR has suggested is that for MEA you add the malt extract to cold water, which allows it to have time to dissolve without clumping.  That seems like a reasonable idea even though you'd be deviating from FDA specs a bit.

The FDA/BAM has this to say about malt extract agar:
Quote:

Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color. Autoclave 15 min at 121°C.




Here is what the FDA suggests for starch agar...
Quote:

Heat to dissolve agar in 500 ml water. Dissolve starch in 250 ml water. Combine and dilute to 1 liter. Autoclave 15 min at 121°C.




Not sure why the OP wants to use starch, but as suggested you can add it after boiling the agar in a separate solution or just by itself.  I doubt it will be any sort of problem.  If you're unlucky you might discolor your media a bit, but this is hardly a problem.  I would simply boil to dissolve ingredients and see if it's a problem first.  Usually most discoloration results from oxidation rather than carmelization (add a tiny pinch of ascorbic acid if you want PDA and others to look lighter colored).


-FF

Edited by fastfred (03/13/09 01:53 PM)

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Re: Agar before PC'ing [Re: fastfred]
    #9965085 - 03/13/09 02:03 PM (15 years, 9 days ago)

The pre-mixed MEA from fungi perfecti can be mixed more easily with cold water.  I usually mix the desired amount with about 1/4 cup of cold water until I have a paste, and then add the rest of the water.  The paste then mixes right in.  I've noticed when I use agar agar flakes from the oriental market along with malt extract, I need to heat to boiling or nearly so, to dissolve the flakes.  Perhaps Paul is using lab grade agar, or perhaps it's just because it's ground to a fine flour.

I've never experienced carmalization at 15 psi.  I think it needs to be quite a bit hotter than that.
RR


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Re: Agar before PC'ing [Re: RogerRabbit]
    #9965206 - 03/13/09 02:25 PM (15 years, 9 days ago)

Quote:

I've never experienced carmalization at 15 psi.  I think it needs to be quite a bit hotter than that.
RR




Carmelization is usually said to occur at 320F (160C).

Here's a table:
Fructose 110°C, 230°F
Galactose 160°C, 320°F
Glucose 160°C, 320°F
Sucrose 160°C, 320°F
Maltose 180°C, 356°F

The term "carmelization" doesn't refer to a specific reaction.  It generally means polymerization and/or oxidation of sugars, but any time you heat a sugar and it darkens "carmelization" has occurred.

As I mentioned earlier you can just add a pinch of ascorbic acid to the mix and it will prevent or even reverse any oxidation and return it to a lighter color.  It may lower your pH a bit, so don't go overboard.


-FF

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Re: Agar before PC'ing [Re: fastfred]
    #9965468 - 03/13/09 03:04 PM (15 years, 9 days ago)

Hi: The not letting your agar boil come from some 20 years of going into university and research labs plus BBL where they prepare agar all day because that's what they make there and talking to the people that prepare the agar while I'm there working on their sterilizers. They consider it poor technique to let agar solution to come up to a simmer and will dump any thing that been aloud to boil as part of their quality control. Plus it can be found in a number of books on preparing agar's; DEFCO Handbook and the BBL Manual warn not to over cook medium by boiling as this can led to the sugars to caramelize. Caramelize in this case is the point where starches and sugars start coagulate into fiber like compounds and not the candy makers caramel and hard set points.

Myself over the years I've seen so many things used in preparing agar mediums that I reserve judgment as to what some one might want to use in preparing their own medium, different folks different strokes.

I've boiled it down two, DME or Oat Meal agar to work with for myself and I use room temperature Di water to start with. Adding most every thing to this. I've found for myself that they just dissolve better in room temperature water or in the case of oat meal that needs to sock over night or 12 hours before being simmered and filtered and then the other things added. I add the agar when heating on the water bath before sterilizing. I hope this will clarify any thing I've may have written. Hipster

PS a finial note: I agree with RR that the DME medium mix from Fugi Perfecti will mix right into room temperature water and this is true with most medium mixes from DEFCO and other major manufactures. I now use only agar powder from Fungi Perfecti because it's so fine I don't have to heat it as long as most others before I can pour it into smaller bottles and sterilize. The only reason I go though all the trouble is because I'm finding it next to imposable to find an oat meal medium mix from some one that doesn't keep a lot a records of who they sold things to. There is some one on E-bay who for about seven dollars plus post will sell you enough DME medium mix to make around 335ml of medium. That's enough to pour a sleeve of 20, 100mm plates. I've been trying to get him to stock oat meal medium mix.

Edited by CheeWiz (03/13/09 09:36 PM)

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Re: Agar before PC'ing [Re: CheeWiz]
    #9965583 - 03/13/09 03:28 PM (15 years, 9 days ago)

> DEFCO Handbook and the BBL Manual warn not to over cook medium by boiling as this can led to the sugars to caramelize.

Sure, you don't want to overcook by boiling.  So don't boil it for an hour or something absurd like that.  I doubt they mean don't boil it.  The boiling is just to dissolve the ingredients.  Once they dissolve you remove the heat, most of the time you won't need to actually bring it to a boil.

Boiling is far too low temperature to cause carmelization, and remember you're going to be heating it to well above boiling during sterilization anyway, so I don't really see why you would be worried about boiling it.

I guess if you're not in research you can do whatever you want.  But if you're doing research and preparing your own media you're going to have to follow procedure or list it as "modified XXX media" and it's going to look pretty silly when you call it "modified FDA/BAM M127 PDA" and then list that the "modification" was that you just didn't boil it.  Otherwise you better "Boil to dissolve ingredients", however you interpret that I guess.

Most people are pretty serious in research and if the FDA spec says "15 shakes" then you better shake it exactly 15 times with an arm motion at the elbow from horizontal to 90 degrees.

I encourage people to do whatever works for them.  But there's no reason not to start with standard methods and work from there.  Otherwise we get this "Oh no! It'll carmelize if you don't stroke it gently!" impression just because that's the way you learned it.  If that goes on long enough then we end up with dogma instead of science.


-FF

P.S.  No offense to you, you just learned a non-standard method and so believe that there's a reason for it, which there probably is for certain types of media.  It's always best to use the least heat necessary, I just think you took it a little too far.

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Re: Agar before PC'ing [Re: fastfred]
    #9966181 - 03/13/09 05:52 PM (15 years, 9 days ago)

Hi: FF we will have to agree to disagree on some points. We are not that fare a part, just seeing things in a different way. No I don't want people to be afraid of what there doing, but at the same time I want people to responsible in what they are doing and that means staying with what there doing and only heating tell every things gone into solution. Any thing longer is not needed and can degrade the medium.

I'm 56 now and one thing I've learned over the years is that when you say you can do some thing that means to the max for most people. Many people will put some thing on the burner and go to 7-11 for some cigs & beer thinking every thing is fine when they get back an hour later because it's just boiling. Your just a little more trusting in what people will and won't do. But again if you've been around so of the people I've been around you would understand my cynicism.

Plus I've read here on this site where people have started to PC some thing, walked away and come back two hours later to find the safety plug from their PC blown out and wondering why and some one tells them that you can fix that with two fender washers, rubber washers, a bolt and nut. It does make one think about what you tell others that they can or should do some thing. Hipster

Edited by CheeWiz (03/14/09 12:50 AM)

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Re: Agar before PC'ing [Re: ABC]
    #9968267 - 03/14/09 01:45 AM (15 years, 8 days ago)

:nothingtoadd:


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Re: Agar before PC'ing [Re: deladude]
    #9969582 - 03/14/09 10:44 AM (15 years, 8 days ago)

Quote:

safety plug from their PC blown out and wondering why and some one tells them that you can fix that with two fender washers, rubber washers, a bolt and nut.




I think Mr. Darwin covered that well.  Those who would do such a thing probably won't be around to do much breeding.
RR


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Re: Agar before PC'ing [Re: RogerRabbit]
    #9969693 - 03/14/09 11:09 AM (15 years, 8 days ago)

Quick question, the FDA recommends PC'ing agar for 15min, FP says 45min on the side of the jar.  Is 15min fine for sterilizing? I only ask because my agar turned out darker than I was expecting last time and I fear it may have been due to the longer PC cycle.

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Re: Agar before PC'ing [Re: RogerRabbit]
    #9969787 - 03/14/09 11:26 AM (15 years, 8 days ago)

Quote:

RogerRabbit said:
Quote:

safety plug from their PC blown out and wondering why and some one tells them that you can fix that with two fender washers, rubber washers, a bolt and nut.




I think Mr. Darwin covered that well.  Those who would do such a thing probably won't be around to do much breeding.
RR




I must say to the credit of this site, RR and the other mediators here that when some one does come up with pearls of wisdom like fixing a PC in a most questionable way that they are corrected about their ill fated ways in a short time.

It was an over sight on my part not to state this in my original post. You guys & gals here do a very good job of staying on top of things! Thank You; Hipster

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Re: Agar before PC'ing [Re: CheeWiz]
    #9969998 - 03/14/09 12:00 PM (15 years, 8 days ago)

Quote:

I'm 56 now and one thing I've learned over the years is that when you say you can do some thing that means to the max for most people. Many people will put some thing on the burner and go to 7-11 for some cigs & beer thinking every thing is fine when they get back an hour later because it's just boiling.




You're probably right.  I guess I'm giving people too much credit when I expect the instructions "boil to dissolve" and the advice "use the least heat necessary" to be actually understood.  Seems pretty self-explanatory to me, but come to think of it I've seen a lot of stupid stuff recounted here.

Quote:

Quick question, the FDA recommends PC'ing agar for 15min, FP says 45min on the side of the jar.  Is 15min fine for sterilizing? I only ask because my agar turned out darker than I was expecting last time and I fear it may have been due to the longer PC cycle.




15 minutes is technically the time it takes to sterilize something at 121C.  If all parts of the media reach 121C and hold there for 15 minutes the FDA declares that sterilized.

Remember that this is with a lab autoclave though.  Most of them have temperature readings and they don't start the cycle timer until they actually reach the set temperature.  It's also based on Erlenmeyer flasks filled no more than 1/2 full or less.

Personally I always go at least 20 minutes, that is a more standard sterilization time.  If you don't have a good setup that you are experienced with (e.g. if you have contam problems you will know where they are coming from) then I would go 25-30 minutes.  45 minutes is WAY overcooking your media.  A damn PF jar only takes 30 min!  And that's a non-liquid with poor heat transfer through the substrate.

One note... Media with yeast or yeast extract sometimes needs longer.  I've had some stubborn yeast refuse to sterilize in a reasonable time.  Boiling it separately for a bit or pre-sterilizing it seems to work pretty well.


-FF

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Re: Agar before PC'ing [Re: Seroind]
    #9970071 - 03/14/09 12:15 PM (15 years, 8 days ago)

Hi: This could be what is called caramelization of your medium. This can start with over cooking your medium and the Sterilizing is the last nail in its coffin or just sterilizing to long. 45min is a long time to PC a medium, it should be around 20min or so for a liter flask. That time is after the PC seals it self and comes to 15psi!

Water is a great conductor of heat and it is heat that kills off every thing so that it is sterile. It only takes about 5min at 250*f for the heat to kill off every thing the rest of the time is for the heat to conduct though the solution so that you have uniform and complete heating. Air on the other hand is a relativity a poor conductor of heat! When sterilizing things that have air trapped in them like grain jars, ex; there has to be extra time add to compensate for the fact that air is a poor conductor of heat so that there is uniform and complete heating to attain sterilization. Sterile is the complete lack of any thing living. I hope this helps; Hipster

Edited by CheeWiz (03/14/09 12:25 PM)

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Re: Agar before PC'ing [Re: CheeWiz]
    #9972814 - 03/14/09 08:17 PM (15 years, 8 days ago)

I use the MEA from stamets and always sterilize for 45 minutes after fifteen PSI is reached.  So far, it's never caramelized.  I realize many folks use less time, but I'm a firm believer in following label instructions, especially if I have reason to trust whomever made the label.
RR


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Re: Agar before PC'ing [Re: RogerRabbit]
    #9982290 - 03/16/09 02:44 PM (15 years, 6 days ago)

45 minutes is way too long.  There is no reason why a liquid would take that long to sterilize.

It's easy enough to verify if you are getting sterilization.  You can use an autoclave sticker or just check to see if you're getting strange contamination.  If it's sterilized, it's sterilized and cooking it longer can only waste energy and degrade your media.

I almost always use 25 min.  I figure that's 15 min sterilization time + 5 min allowance for heat transfer and circulation + 5 min (25%) safety margin.

30 minutes is fine if your setup isn't the best or you haven't used it enough to get the hang of it.  But 45 minutes is double what you need and seems way overkill to me.

BTW difco and some other suppliers specify a longer time, so for research purposes you are either going with the FDA prep or following the manufacturer directions unless otherwise stated.


-FF


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Re: Agar before PC'ing [Re: fastfred]
    #9986319 - 03/16/09 11:55 PM (15 years, 5 days ago)

Wouldn't the FDA be more concerned with food safety?  I'm sure fifteen minutes at 121C would kill any organisms that could harm our health, but with agar that's been supplemented with grain products, especially for culture slants that will be stored for months or years, it's important to nuke the endospores.  At any rate, once the sterilizer has come up to pressure, that extra fifteen minutes gives me peace of mind and doesn't use much energy since the stove is on its lowest setting by then anyway.  It also doesn't harm the product, so I'll stick with it.  I'm just hard headed that way I suppose.
RR


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Re: Agar before PC'ing [Re: RogerRabbit]
    #9987670 - 03/17/09 10:03 AM (15 years, 5 days ago)

I didn't mean to sound like I was criticizing you for following the directions, sometimes you have to just to say that you did.

I have no idea if endospores survive the spray drying process used for making malt extract.  I would suspect that they don't since beer brewers only boil their mash and then ferment for a long period and don't usually have any contamination problems.

I've never had any contamination problems using malt extract sterilized for 30 min.


-FF

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Re: Agar before PC'ing [Re: fastfred]
    #9991167 - 03/17/09 07:48 PM (15 years, 5 days ago)

Could it have something to do with the water also?
I use DI water which I would assume to have no endospores, whereas tap water would have alot.

I get away with 22 min at 13.5-14.5psi (all my cheap PC does without modifying) for MEA, using a pyrex flask.

It always has worked for me, and I am now digging in to my 2nd box of 100 pre-sterilized dishes.

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