Slanting yeast

[TimWeber]

I've used a simular process in the past. One problem I keep having, is after inoculating the slants and the yeast start to grow they produce a lot of C02. I vent the tubes but am always worried about contamination. When activity starts to die down (less gas to vent) I place the tubes in the fridge. But they still ferment a bit, and when I go to use the slant, I get a large puff of C02 and the agar actually starts to bubble up from the base of the tube and push its self slowly out of the tube.

Do you vent your culture tubes?

Have you ever had this problem?

Maybe I just used the wrong mix of agar/wort.

 

[Saccharomyces]

No I incubate the tubes in a sanitized beaker covered with foil with the lids slightly loose so they can offgas. After 10-14 days I tighten them up and into the fridge. I have had one vial give off a slight puff of CO2 so far when opening it but it was very slight.

Yeast/bacteria/mold can only get in through dust which can't get through the threads under the cap, but moisture and CO2 can escape.

 

[mordantly]

i tested that theory, i sterilized (15 psi for 30 min and cooldown time) 500ml wort. left the flask for a week with an shimmed, inverted beaker.. to leave plenty of gap for airflow. no mold or anything visibly colonized it. so i agree. airborn bad guys cant fly up.

 

[JKoravos]

There is enough yeast that a 250mL starter is actively fermenting on the stir plate after 24 hours. Assuming a growth rate of one generation every two hours, and a maximum yeast concentration of 80M cells per mL, there would have to be at least 5M cells in the slant. So a conservative estimate would be somewhere around 1M ~ 10M cells... but it's just a guess.

This is the best online resource IMO regarding yeast cell concentration and growth rates in wort:

http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices

If you read, you will see why I use a stir plate AND sterile wort for the first step; the method he gives you can use non-sterile wort safely but it requires adding 10mL of wort (which you could add directly to the slant vial) and wait 2-3 days before pitching the 250mL starter. Since I use sterile wort and am very very careful with my sanitation in the first step, I go straight from the slant into 250mL without any problems.

I'm just getting started in slanting, but I've been going from slant to ~200mL for the first step, then from there to ~1000mL, then higher if necessary. It's working fine so far.

Also, 'he is a 'she'. The author of that Maltose Falcons article is Maribeth Raines.


Here's a question i don't think I've seen an answer to...What makes a slant 'go bad'?

 

[TimWeber]

A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.

Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.

 

[Bru]

To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?

 

[dooksh]

i got 12 ml vials . a little small. do you think i could work with those?

 

[Saccharomyces]

A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.

I have only had one slant 'go bad', it had visible green mold growth from a single spore that managed to sneak into the slant vial while I had the cap off.

Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.

I haven't been that concerned about it, doesn't seem to effect anything.

To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?

Washing yeast the viability drops off quickly so you have to re-use the yeast relatively soon, or re-wash and propagate the yeast prior to pitching. I have also had a few infection problems with washed yeast, whereas building up from a slant everything is sterile (you are always working with yeast which came from a pure culture rather than from a fermenter).

i got 12 ml vials . a little small. do you think i could work with those?

Most folks use the 12ml vials for yeast banking. I went with the bigger ones because I think they are easier to handle.

 

[dooksh]

thanks for all the information Saccharomyces .

 

[mordantly]

i can only find gelatin in 1oz packaging. will regular gello work? thicker of course.

it looks like you penetrated the surface of the medium at innoculation time vs swiping the surface?

 

[Saccharomyces]

i can only find gelatin in 1oz packaging. will regular gello work? thicker of course.

I have heard it will work although I have not tried it.

it looks like you penetrated the surface of the medium at innoculation time vs swiping the surface?

Yeah I just poke it 5-6 times.

 

[mordantly]

11-8-09

made the following "jiggler" ratio. (jell-o reports 76g sugar in 3oz):

1 3oz package lemon jell-o
1 tbsp dme
5oz water

got 5 tubes each of ~20ml firming in fridge. gonna try to put some yeasties in them tomorrow using my homemade nichrome wire loop.


11-09-09

it seems if you put this "slant" in the fridge to set, once at room temp there is moisture/softening of the media. resting at room temp till nearly firm so far has absorbed the moisture.

 

[dooksh]

i made some slants yesterday .most of the liquid slanted but there is still a small amount of liquid that wont slant.
do you think i should start over?

 

[Saccharomyces]

i made some slants yesterday .most of the liquid slanted but there is still a small amount of liquid that wont slant.
do you think i should start over?

You really want solid media for storage, if the slants aren't solid you need to use more agar and try again. I have some slants that got watery after storage, they still seem to be building up OK but they were solid when I cultured the yeast, for those I will be tossing them pretty soon because I don't expect them to be viable nearly as long.

 

[mordantly]

how will yeast respond if taken from a washed starter @ 40F and innoculate a slant at 65F?

 

[Saccharomyces]

how will yeast respond if taken from a washed starter @ 40F and innoculate a slant at 65F?

Don't think it would be a problem, but I wouldn't try it unless the yeast are first generation or you streak a plate to make sure it isn't contaminated before using the slants.

 

[cmcnair1]

Would a pressure cooker that only does 10 psi work sufficiently? It seems like a lot of the new cheaper models don't go to 15psi.

 

[Saccharomyces]

Would a pressure cooker that only does 10 psi work sufficiently? It seems like a lot of the new cheaper models don't go to 15psi.

You need at least 12 PSI above ambient pressure to reach the recommended 240*F for canning. You could probably get away with it... but I wouldn't try.

 

[Hugh_Jass]

Sacc,
I've heard of people dumping some boiled/cooled starter wort into a couple of cultured slants to begin their starter. They would use a sanitized loop to mix the yeast and wort and dump that solution into their flask and place on a stir plate. They would re-cap the emptied tube and allow the culture to re-grow on the same agar/slant.

Does this sound o.k.?

 

[Saccharomyces]

Sacc,
I've heard of people dumping some boiled/cooled starter wort into a couple of cultured slants to begin their starter. They would use a sanitized loop to mix the yeast and wort and dump that solution into their flask and place on a stir plate. They would re-cap the emptied tube and allow the culture to re-grow on the same agar/slant.

Yep, you should do this if you don't have canned wort. Reason is, if the wort isn't absolutely sterile, you could get hosed by bacteria in the slant to 250mL step because it's a large one and the lag time is long (especially if you don't have a stir plate).

 

[Hugh_Jass]

Thanks Sacc,

Is using multiple slants to increase the initial cell count?

I have 5 slants of a few different varieties. Each have a good size culture. Should I use all 5 when beginning a starter or just a few?

I have been using only one slant. I would scrape the slant with my loop and inoculate the starter wort with the single culture.

 

[Saccharomyces]

I have been using only one slant. I would scrape the slant with my loop and inoculate the starter wort with the single culture.

I only use one slant. Making slants is a PITA, I wouldn't want to use them all up at once.

 

[Hugh_Jass]

I'm sorry. I wasn't clear on my initial post.

If I dump sterile wort into a few inoculated slants, scrape the cultured yeast off the agar to get it into solution and pour that solution back into a sanitized Erlenmeyer flask, can I re-use the slant. According to another person who slants, the culture will re-grow onto the same agar. In your opinion this correct method or no?

 

[Saccharomyces]

I'm sorry. I wasn't clear on my initial post.

If I dump sterile wort into a few inoculated slants, scrape the cultured yeast off the agar to get it into solution and pour that solution back into a sanitized Erlenmeyer flask, can I re-use the slant. According to another person who slants, the culture will re-grow onto the same agar. In your opinion this correct method or no?

Oh, I suppose you could do that, though usually the slant falls apart when I add wort to it.

 

[Hugh_Jass]

I'll try it next time and let you know if it re-grows a clean culture.

This thread has been very helpful. Thanks

 

[Saccharomyces]

I'll try it next time and let you know if it re-grows a clean culture.

I don't see why it wouldn't as long as everything is sterile... provided the slant doesn't fall apart.

 

[mordantly]

the candle creates carbon deposits on the nichrome. maybe i need an alcohol lamp?

 

[Bokonon]

the candle creates carbon deposits on the nichrome. maybe i need an alcohol lamp?

Until recently I was just using a bic lighter to flame my loop and other anything else I needed. It worked fine just required a bit more planning

 

[mordantly]

im sad to report, on day 5 or 6, no visible life of any kind on my three test-slants of the previously mentioned jell-o media. how long should i wait at 65-70F before i scrap the test? i think the stuff is too dry for growth.

 

[Saccharomyces]

Might as well wait a few more days. But if it's too dry, it's too dry.