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What's up with my agar?? :(

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Bancopuma

Bancopuma

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I'd really appreciate some mycological input with what's going on with my agar, it keeps getting this film on it. I have a fair bit of experience growing cubensis and Pan cyans and haven't encountered this before, although it has claimed all my agar jars the last two attempts at inoculation. I want to get a Hawaiian Pan cyan grow on and this is preventing things from moving forward.

I can't think why this is happening. I think my pressure cooker and agar source may have changed since my last grows that never had this problem but that is all that has changed. So does this look like mold, a weird emulsion or something else? The only thing I can think of is that the pressure cooker isn't doing its job somehow.

Would appreciate any input...image is attached (not the best quality, hope it's ok).
 

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Was this agar made in that container and opened later, or transferred ? If it just came from the PC and hasn't been tampered with then all I can think of is that it somehow wasn't sterilized to begin with.
 
Hey Orion, so the agar was indeed made in this jar and opened only very briefly in a pretty sterile environment for inoculation with Pan cyan spores. In past grows hygiene has never been an issue, but the last two times the agar has produced this weird film, and this time it only took half a week or less to appear. I've had bacterial molds before and they didn't look like this. Maybe the pressure cooker isn't doing its job? It's a larger model than used with previous grows for convenience but I'm not sure I have 100% faith in it any more.
 
That agar looks severely contaminated. Definitely not a mold but the colonies looks similar to yeast plaques. It reminds me of Candida spp grown on agar, but since colony appearance and expansion can change dramatically in different agar formulations, it could be something else, maybe even Saccharomyces spp.? How does that smell? Smell can be useful indicator if you are trying to find out what that is.

One thing is for sure - since the colonies all look the same, your contamination issue is likely caused by a single microorganism.
 
Hey dude thanks for the input. Just had a sniff and it definitely pongs alright. Hard to describe really but pungent, sour, acrid smell. It definitely doesn't resemble a mold from one's I've encountered before. Can yeast survive boiling, let alone pressure cooking?? I thought they were more fragile than bacteria.

Not sure what is going on here, may change agar source and inoculation tank cleaning fluid. I doubt very much it's the spore prints as this hasn't ever happened before bar the last two grow attempts. My main suspect is the pressure cooker...when doing its thing it never seems as potentially (and reliably) dangerous as previous models, when it moves it hisses ever so slightly, unlike the others. Right now I think I may have a large cooking pot that happens to have a lid on it. Otherwise I'm a bit stumped. But yes this contam is what has affected all batches for the last few grow attempts which itself is a bit weird...could it be somehow in the agar powder itself?
 
It could very well be the agar, but I doubt it; what is the recipe for your agar medium? If it contains yeast extract then this might be a source of contamination but I doubt it since yeast extracts are not only pretty much dead, but if it were something from yeast extract (which is most commonly made of saccharomyces cerevisiae), you would have picked a pleasant, bready yeasty smell, not as you describe.

From your overall description it is possible that sterilisation is the culprit, especially if you use the same previously contaminated jars to pour your agar. Have you done a test of sterility? i.e. to pressure cook agar in a jar, then leave it as is and see if anything grows. If no, PC is fine - if yes, the PC is the culprit.
 
Appreciate the input. For the agar I use water, agar agar powder, a few organic chicken manure pellets (the Pan cyans need the N to thrive) and some dark barley malt extract, it's the stuff in the jar like treacle as oppose to the powder. Have only used the current jar of this for the last few grows actually, but have only ever used this stuff for all previous grows without problems. But yeah smell is acrid and sour and in no way wholesome.

I actually used different jars this most recent grow attempt bar one. But thanks for this suggestion it makes sense, I'll cook up some jars this eve as a control and see what happens. My hunch right now is the pressure cooker, weird though how it seems to be the same bug that is dominating all jars, and whatever it is seems to be fast growing.
 
So I made up 3 jars, pressure cooked them and then left them in the incubation chamber without having opened them at any point, and 2/3 jars still got contaminated by the same thing. So I must have a faulty pressure cooker that ain't doing its job for some reason. At least I know the problem now and can move on, thanks for the input.
 
If contams seem to be plaguing you, I would recommend taking the jars and pressure cooker and cleaning out really good with a bleach water solution and maybe afterwards rub down with IPA to be extra sure.

Unless the pressure cooker is visibly leaking pressure from a point other than the steam release, I think you're okay there. As I'm sure you're aware, the temps in there get upwards to about 250* F and will kill off any nasties and bugs, so realistically, everything in there should be sterile, but it wouldn't hurt to wash this with bleach water solution just to be sure.

Contams suck. :(
 
Hi Ancotar, thanks for the input. I always clean my jars before use and clean and dry the pressure cooker after use. I strongly suspect this is a pressure cooker issue, prior to this model I had very few contam incidents, now it is a little shy of 100%. Surely if there happened to be any microbes in the pressure cooker or in the jars, if the pressure cooker was doing its job it wouldn't make any difference? My last model was much more feisty, and it was obvious it was doing its job, if it got moved a bit it would hiss etc. This one doesn't really, I just get the impression not much in the way of pressure is going on inside, so think I need to look into getting a new one. I bought this one I think because it was larger and could hold more jars than my previous one which I never had issues with.
 
Hi Bancopuma,

I agree that dish is 100% contaminated. When you opened the jar briefly were you working in a still air box or was it in open air?

I suggest buying disposable plastic petri dishes. Place them in a clean still air box still in the original plastic sleeve.
Prepare your agar in a glass liquor bottle with a cap that has a hole with some kind of filter.

Let the agar cool enough to be able to handle it, wipe it down with alcohol and place it in the still air box.

Slip on some latex gloves and wipe them with alcohol too. Keep an alcohol soaked paper towel inside the box and wipe the bottle and your hands once your hands are inside. pour your dishes in the box stacking them as they are poured then slip the plastic sleeve they came in back on them while they cool.

Just my two cents.
Whatever you decide to do I hope you figure it out and get back on track.

Good luck to you!
 
Bancopuma said:
[...] I bought this one I think because it was larger and could hold more jars than my previous one which I never had issues with.
Click to expand...
Clearly, the changed variable is the pressure cooker. How much bigger is it? Does it need a larger heat source perhaps? There are ways of increasing the pressure in a PC so that you might not need to buy a new one.
 
Hi peeps thanks for the input. I inoculate in an aquarium sealed with bubble wrap and two holes for the arms. So not airtight but has been absolutely fine until now. Unlike the last pressure cooker, this one doesn't seem to have an obvious pressure valve or weights. The heat source should be fine given its size. Will have a look for the previous model for now.
 
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