Making yeast slants?

[floydmeddler]

Have just finished preparing my slants and they are 'setting' now. Will inoculate two tomorrow -

I leave my newly made slants in a warm place for about 3 days just to be sure they are 'clean'.

Have read about this too and gave it a thought. However, my vials spent 15 mins in a pressure cooker with full heat then I left it to cool naturally. I then opened it up and immediately tightened the caps so I'm 99.9% sure they are sterile.

Plan is to finally start collecting yeast from pubs. Was worried in the past about nasties but now I'll be able to pick out healthy colonies... I think! Still learning!

Streaking onto petri-dishes would be best for that - as long as the yeast you are collecting is a single strain type.

Yes, this makes more sense actually but will hopefully be able to get good results from inoculating a few slants. Have pretty good surface area to work with as they are quite broad. Intend to buy me a petri dish though.

[floydmeddler]

Well, here are my babies:



1.0: WLP005 - Source - WLP005 Vial.
2.0: Duvel - Source - From small 100ml starter I created during the week.

Fingers crossed these will take off!

The rest are waiting to be inoculated with whatever comes along.

[Wolfy]

Well, here are my babies.

As you start collecting more yeast you might find it useful to make at least 3-4 slants for each strain (I'd love to be able to culture some real-yeasts directly from the pub!)
That way if you get an infection in one it does not deplete your entire stock of that particular strain, and it also makes it easier to archive each strain without having to re-slant every time you re-culture one.
I find that when I get down to only one slant for a particular yeast strain it's a good reminder that I need to make some more.

[floydmeddler]

Good idea. Went to a pub in town last night and got a pint of Harveys Best pulled into a bottle. Added 3/4 tsp of sugar and am now waiting for some yeast to form. Will then use this to inoculate a few slants as you suggest (and maybe a petri dish) then isolate if need be. Very excited at the prospect of having Harveys yeast!

[mitch]

got a pint of Harveys Best pulled into a bottle. Added 3/4 tsp of sugar and am now waiting for some yeast to form.

You don't need to use a whole pint. I take a clean slope and a sterile 1mL syringe to the pub, take my freshly pulled pint to a quiet/dark corner and transfer ~0.1mL of beer to the slope (just enough to wet the surface of the slope). A couple of days later I select a few good looking colonies and streak them out onto plates (to get them away from any mould) before selecting colonies and storing them on slopes.

I always perform a small trial fermentation with cask isolated yeast before using it on a full batch. If I am not happy with the attenuation, flocculation or flavour of the trial beer then the yeast is binned.

[floydmeddler]

got a pint of Harveys Best pulled into a bottle. Added 3/4 tsp of sugar and am now waiting for some yeast to form.

You don't need to use a whole pint. I take a clean slope and a sterile 1mL syringe to the pub, take my freshly pulled pint to a quiet/dark corner and transfer ~0.1mL of beer to the slope (just enough to wet the surface of the slope). A couple of days later I select a few good looking colonies and streak them out onto plates (to get them away from any mould) before selecting colonies and storing them on slopes.

I always perform a small trial fermentation with cask isolated yeast before using it on a full batch. If I am not happy with the attenuation, flocculation or flavour of the trial beer then the yeast is binned.

Seriously? That's amazing!! Will try this right now with the bottle I have. What yeasts have you taken from pubs? Need some recommendations!

[floydmeddler]

OK, so I dropped around 0.2ml of Harveys Best onto the yeast slant. How long does it usually take before I notice any signs on the slant?

Cheers

[mitch]

How long does it usually take before I notice any signs on the slant?

With a bright light shinning almost parallel to the surface of the agar I can usually make out tiny specks after about a day.

It will be at least two days before you can start poking around with your loop, the actual time depends on your incubation temperature. You want enough growth to be able to identify and avoid contaminants, but not so much that the contaminants start to merge with the yeast.

[floydmeddler]

How long does it usually take before I notice any signs on the slant?

With a bright light shinning almost parallel to the surface of the agar I can usually make out tiny specks after about a day.

It will be at least two days before you can start poking around with your loop, the actual time depends on your incubation temperature. You want enough growth to be able to identify and avoid contaminants, but not so much that the contaminants start to merge with the yeast.

The specks are there today. So, here's my plan:

When it starts to establish itself, rub my needle (don't have a loop) over the small healthy white colonies then use this needle to inoculate a fresh slant. I'm presuming this tiny quantity on the new slant will spread out as it grows over a week then I'll put it in the fridge.

Then either:

When I want to create a starter, take my slant out of the fridge the night before, rub my sterile needle over the yeast then dip this needle into a mini wort. Put slant back in fridge until needed again. Build up mini wort to pitching quantity.

or

Take my sterile needle, rub over yeast on slant then use it to inoculate a fresh slant. This slant can then develop at room temp for a week before being put in the fridge. With original slant, add a tiny amount of wort, swirl around then add to mini wort. Build up to pitching quantity from there.

What do you think?

Cheers

[mitch]

The first option sounds better, it involves fewer transfers (particularly of the 'mini wort') and you are not serially propagating your master yeast.

I would also do a trial fermentation with about a pint of 'proper' wort from your next brew before deciding whether to use the yeast on a full batch.

[Fuse]

You say "rub my needle over the small healthy white colonies then use this needle to inoculate a fresh slant" - ideally you want to pick from a Single colony rather than multipe that way you know you will have a pure strain as opposed to potentially multiple. Its not the best practice to take a colony and streak it directly onto another slant or plate, ideally you would want to grow up a small starter culture and then plate from that - so you are playing from a healthy "log phase" growing culture so it can crowd out anything else that may be about.

For making my pitching culture i usualy grow a single colony in 10mL media and then up to 100mL when its looking quite cloudy then up to the 1L 36/48 hours before pitching.

[floydmeddler]

The first option sounds better, it involves fewer transfers (particularly of the 'mini wort') and you are not serially propagating your master yeast.

I would also do a trial fermentation with about a pint of 'proper' wort from your next brew before deciding whether to use the yeast on a full batch.

Makes sense. Cheers Mitch.

[floydmeddler]

You say "rub my needle over the small healthy white colonies then use this needle to inoculate a fresh slant" - ideally you want to pick from a Single colony rather than multipe that way you know you will have a pure strain as opposed to potentially multiple. Its not the best practice to take a colony and streak it directly onto another slant or plate, ideally you would want to grow up a small starter culture and then plate from that - so you are playing from a healthy "log phase" growing culture so it can crowd out anything else that may be about.

For making my pitching culture i usualy grow a single colony in 10mL media and then up to 100mL when its looking quite cloudy then up to the 1L 36/48 hours before pitching.

Thanks for this. I'll try to select a colony and add to 10mls of wort. Have a few mini starter vials already and they are around 7ml. Should do the trick! What size should a single colony be in mm? Cheers.

[Fuse]

they will be anything from 1mm to 5mm (and bigger) depending on the number and density of the cells you plated, but more likeley to be at the lower end of that scale - you may even end up with a confluent slant so that individuals colonies are not visible. They should start appearing as little dots, if you hold the agar slant up to your eyes and look accross it you should start to see litle bumps, ill take a pic of some plates i have tomorrow so you can see.

[floydmeddler]

Here's a pic of mine.




I'm assuming these individual specks are individual colonies. So, can I use one of these tiny 1mm colonies to kick off 7ml of mini wort?

Many thanks