Agar Amount in Yeast Slants

I mixed the agar DME solution up per the following proportions:

500ml water
1.5% agar powder (7.5g)
7% DME (35g)
1% yeast nutrient (5g)

The above proportions and my process were from this thread (the yeast slant sticky). https://www.homebrewtalk.com/f163/slanting-yeast-133103/

However, I have a problem. My slants never set up. In doing some research I have discovered that the percent agar that folks are recommending is all over the place. It is obvious to me that people just kind of get to an amount of agar that will allow the substance to solidify just enough to stay together and not move around (I would say a consistency of almost candle wax or perhaps a little thinner). That is, the amount of agar is not critical.

I also believe that there are different percentages of agar based on the type of agar you have. So far I have heard of the block form, the flaked form, and two type of powdered forms (the nutritional kind and a laboratory grade). They may all require a different amount of agar to get the proper consistency in the slant. This is my perception from reading about it in multiple places. Thoughts?

My agar is from Now Nutrition and says 100% agar. Here's my procedure:
I boiled my DME and yeast nutrient for around 1 minute. Let cool for a few minutes then dumped in the agar powder and stirred. Then used a syringe to measure out for my vials. I pressure cooked as instructed, let cool 1 hour, tightened caps and taped.

I also read something about getting the agar mix up to boiling temps to avoid separation while filling your vials. I think this is the cause of that white looking stuff at the bottom of the vials. Perhaps if I would have boiled it all together than the amount would have been fine?????


Additionally I am suspicious of is my yeast nutrient. I used Fermax yeast nutrient. After boiling my wort, I got a strong ammonia smell. Perhaps Fermax is not the same concentration as the wyeast nutrient mentioned in the yeast slant sticky?


So any input would be appreciated.

Bullgator,

I am in the exact same situation. I used the same recipe from the same thread with the same agar and I'm finding that the agar mixture is too "juicy". The yeast grows well enough in it but spreads down the sides of the vial so CO2 gets generated underneath the plug of Agar and it gets pushed to the top of the vial. When I open a vial the entire plug gets pushed out!

I found your thread while looking for a solution. Did you arrive at a recipe that is working better for you?

MichaelBrock said:

Bullgator,

I am in the exact same situation. I used the same recipe from the same thread with the same agar and I'm finding that the agar mixture is too "juicy". The yeast grows well enough in it but spreads down the sides of the vial so CO2 gets generated underneath the plug of Agar and it gets pushed to the top of the vial. When I open a vial the entire plug gets pushed out!

I found your thread while looking for a solution. Did you arrive at a recipe that is working better for you?

Click to expand...

I went to a friends house and remade them. We used the same amount but we did boil the agar/dme solution for a few minutes. I think this helped prevent the agar from separating from the solution and settling at the bottom. Now I did have a problem with moisture causing my yeast to slide down but I think it was mainly because we cooled the vials in the freezer which may have not let moisture escape the vial as easily. Next time, I am going to let them cool naturally.

One thing I definitely will do is put less agar/dme in per vial and tilt them to allow the slanted surface to expose some of the vial bottom. I read this on the yeast slant sticky. It is a great idea. This will make the possibility of the agar/dme from popping out impossible. I didn't get many pop out but around 10% did and it was very fustrating.

I hope this helps. I am no expert but let me know if I can share anything else.

I work in a lab. I make bacterial growth medium all the time. Cut down on the water! Instead of 500 mL, try cutting that by 5-10%. (450 - 475 mL). Should make the media less watery and less likely to slide around on you. Also, are you sure the white precipitate isn't hot break or something? Just wondering.

" I pressure cooked as instructed, let cool 1 hour, tightened caps and taped." After autoclaving (pressure cooking), be sure to cool to about 56 C. Then, swirl the vials to get the agar mixed in. It looks like you failed to mix the molten agar. It simply is denser on the bottom and solidified there. You'll be fine with the recipe you have.

StusBrew said:

I work in a lab. I make bacterial growth medium all the time. Cut down on the water! Instead of 500 mL, try cutting that by 5-10%. (450 - 475 mL). Should make the media less watery and less likely to slide around on you. Also, are you sure the white precipitate isn't hot break or something? Just wondering.

Click to expand...

Thanks for the response. Won't that increase my gravity? I have read you want your gravity lower to control your yeast growth on the slant. I am sure it was agar on the bottom because when I cleaned out all my vials there was a thin 'puck' of hard agar/dme at the bottom.

Tiltie: I think you may be on to something. I will try that next time for sure.

Thanks!

From the Yeast book by Chris white, states quantities etc, one problem I've had is that residual moisture in the slant causes a carpet if yeast to grow over whole slant and down sides. I avoid this now, just before I streak by opening the slant over a flame and flaming the lid and vial for just a second to dry out. Works great if its been stored upside down

I think my agar is pretty well distributed. I don't have a different color band at the bottom. I do however, have a pocket of yeast nutrient. I used Wyeast nutrient and it doesn't seem to have dissolved very well into the agar mixture. I'm having the same problem as Larso, there is moisture in the vial and the yeast is spreading down the sides of the agar to the bottom. As it reproduces the CO2 is pushing up the agar plug. My technique was the same as BullGator's and I did not let the vials dry out before taping them. I have loosened the lid on a few in a warm room to see if I can get them to dry out a bit.

Oh yeah tiltie. It's one of those things you do, but don't think about. In most recipes making bacterial media. It calls for mixing the water and the ingredients and then adding to a hot water bath and heat "till clear". While heating you periodically swirl the mixture. We use an autoclave at work for like 2 minutes to heat till clear. It comes out of autoclave. Gets a swirl. Then back into autclave for 15 minutes. If it's going to used for making plates!

I have the Yeast book. but I just went here. Saw someone using 35 g DME and 2.5 g agar and 400 mL water. It made 10 plates for me. No problems. Swirling, heating, autoclaving. I did it at work while making other media. lol