My first yeast slant, why does it have so low viscosity?

I tried making my first batch of yeast slants yesterday but noticed the viscosity of the slants did not seem to match what I was following in this thread. After using 35g of DME, 400ml of distilled H2O, and 2.5g of Agar-Agar, and mixing it all together in a sauce pan, then pressure cooking the slants for 15 minutes at 12PSI would give me what I need.

Here is a video I uploaded on youtube showing how the slants came out.



Any suggestions on what I did wrong, and how I can fix this? Can I reuse the slant solution that is currently in the vials to make the correct solution I need?

I don't see a video and I don't use agar agar, I use gelatin, but it sounds like the agar didn't set. I use 250 ml water, 25g DME and 7g of gelatin (1 packet) and never have a problem.

I've never used agar-agar; I use Difco Bacto-agar, but it's probably more or less the same.
The media formulations I've seen are 1.5 to 2 grams of agar per 100 mL, which would be 6 to 8 grams for 400 mL. Even so, your slant looked pretty runny. Is the agar-agar completely dissolved? It (agar) can form semi-transparent little snot balls that keep it from completely dissolving easily.

I made a new batch last night, and finished around 5AM this morning. I will post a picture later in the day, but this time I used approx 1 tea spoon of agar agar, 2 tea spoons of DME, and 1 cup of distilled water, I just looked at the slants and they have seemed to slanted properly, meaning their the viscosity is what it is suppose to be . I will store them away around 5AM. I used little amounts because I wasn't sure if it was going to work or not, but I have 8 slants in the box right now.

Has anyone had issues with their slants lifting from the bottom during incubation periods? If so, could such a phenomenon be avoided by adding more agar agar to the solution?

ipatch said:

I made a new batch last night, and finished around 5AM this morning. I will post a picture later in the day, but this time I used approx 1 tea spoon of agar agar, 2 tea spoons of DME, and 1 cup of distilled water, I just looked at the slants and they have seemed to slanted properly, meaning their the viscosity is what it is suppose to be . I will store them away around 5AM. I used little amounts because I wasn't sure if it was going to work or not, but I have 8 slants in the box right now.

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I find that I have to vary the amount of agar from batch to batch. I use actual beer wort at 1.030 rather than DME/LME. I've done a little experimenting with the use of acids and find that acid degrades the agar during pressure cooking. So depending on the acidity of my wort I can have different outcomes. My findings indicate that the lower the ph the more agar I need for the media to set at room temperature.

What I do is mix up the media in a sauce pan and boil until all the agar is disolved. Then I take a tablespoon of media and put it in the freezer until it cools and sets. Leave the sauce pan on the stove with a lid on to keep it hot. When the media has solidified I basically play with it a little bit at room temperature (poke it, squeeze it, prod it). If I'm satisfied I add yeast nutrients, pour the slants and pressure cook. If it's too solid I add more wort if too soft I add more agar and boil until it all disolves and repeat the test.

ipatch said:

After using 35g of DME, 400ml of distilled H2O, and 2.5g of Agar-Agar, ...
Any suggestions on what I did wrong, and how I can fix this?

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Various references I found say to use 1.5% by weight of agar - you are using less than half that, which is probably why it's not setting well.
The powdered agar-agar I get from the Asian supermarket works fine at 1.5-1.6% and I've never had any problems, so I'd suggest you double the amount of agar and you should find it sets without problem.

Wolfy, you're right on about the 1.5% (1.5 grams per 100 ml) for standard plating media, but I also think spareparts is right about the acidity.
I've had to use 0.7% "top agar" to plate lambda phage in the past. It is made from bacterial media components, so pH isn't much of an issue, but it sets up OK. I wouldn't want to streak on it, as it's pretty thin/soft, but it gels OK.
I'd bet the 1.5% would make up for a fair bit of acidity.

Hey JohnMc, most likely pH could be an issue (and I'd suggest you know much more about it than I do) but we're dealing with a Home Brew type situation, and what looks like a pressure cooker, not something concocted in a biomedical science lab.

So as far as the OP is concerned, simply double the amount of agar, as he did later (1tsp Agar in 1cup water) and the problem is solved without having to over analyze and complicate things to the nth degree as happens on the forums sometimes. Maybe when he's done it 100 or so times the OP can check the pH and adjust the Agar if required, however in a home situation just adding more agar works as well.

Hey JohnMc, most likely pH could be an issue (and I'd suggest you know much more about it than I do)

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Actually I don't, because I've been spoiled by lab stuff. I don't remember the last time I checked bacterial media pH, it just works out OK.
I guess I was trying to say it's not the percent of agar-agar that's the problem in and of itself, instead, there must be something compounding it, which I think spareparts had hit upon.

So as far as the OP is concerned, simply double the amount of agar, as he did later (1tsp Agar in 1cup water) and the problem is solved without having to over analyze and complicate things to the nth degree as happens on the forums sometimes.

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Guilty as charged! Plus, when I read his retest the first time, I misread it, thinking it was still runny.

Well, I got back up on the horse, and decided to make another batch of yeast slants, and this batch came out a lot better meaning it is not liquid. *yay*