# Calling All Yeast Platers!



## dreamboat (15/4/05)

I have just commenced my first foray into yeast plating in order to resurrect a wheat yeast which I obtained a small sample of over a year ago. The yeast paste had gone somewhat brown, so i figured that plating was the best way to go to check viability, and to get get a nice healthy population.

I made a pair of agar / dme plates in the steamer at home, and generally followed the instructions available around the web to streak the plates. Now, 60 hours later, I have some life showing on the plates, with some distinctive white blobs, and nothing else to be seen... hopefully a good indication for both my sanitation methods and the viability of the yeasties.

The question is - how big should I let the blobs get before collecting them (one?)and pitching to a small starter? pictures attached.


----------



## Gulf Brewery (15/4/05)

dreamboat said:


> The question is - how big should I let the blobs get before collecting them (one?)and pitching to a small starter? pictures attached.
> [post="54571"][/post]​



Dreamboat

I normally get the equivalent of 2 match heads in size and put that in 50ml of starter, then step it up to 100ml, 500ml, then starter size. You can do it from less, but more is better.

Cheers
Pedro


----------



## Steve Lacey (15/4/05)

Yes, you could probably do it now if you were in a hurry, using say three colonies. But you can let them go a bit longer if you like. I put my plates in a ziplock placky bag and then inside a biscuit tin. They last for ages like this and the colonies can get quite big.

steve


----------



## dreamboat (15/4/05)

Thanks Steve/Pedro.
I am in no rush at all - really a couple of weeks before I need a pitchable quantity. My concern is as to whether I need to prevent the colony growing to the size where the individual colonies start to merge, or whether I am better of to take a smaller quantity of yeast, but from a single colony.
Since I have two plate to play with, I might look at making a starter from one colony from one plate this weekend, and let the other go until maybe next weekend.... by which time I will know how the first has performed. I will post a picture of my setup - but I can run a nice sporadic aeration of my starter, so should be able to get up to big numbers in pretty quick time.




dreamboat


----------



## Kai (15/4/05)

I would say that you can collect the blobs when they're big enough to tell whether they're yeast colonies or not. Or, leave longer if that floats your boat.


----------



## pint of lager (15/4/05)

Welcome dreamboat, to the wonderful world of petri dishes.



> have just commenced my first foray into yeast plating in order to resurrect a wheat yeast which I obtained a small sample of over a year ago.



Plating is definitely the way to go from a small sample. 

Because the sample is old, try and make sure that you select your colonies to make your starter for uniform size and appearance. After a year in less than ideal conditions, the chances are that there will be more mutations in your sample. 

Let the plate grow for at least a week at about 20 deg C, so the overall macro appearance of a colony is obvious to the naked eye.

The colonies will self limit in size, they are not going to grow and take over the whole plate.

I also tried steaming plates, but found that 3 out of 10 plates were infected. Then I went to using a pressure cooker.

I collect about 2 matchheads worth of yeast and pitch to 50ml of sterilized wort, then when that is active continue stepping up.


----------



## Steve Lacey (15/4/05)

pint of lager said:


> I also tried steaming plates, but found that 3 out of 10 plates were infected. Then I went to using a pressure cooker.
> 
> [post="54668"][/post]​



I now have a pressure cooker too. BUT, I never got an infection using steaming. However, I added the step of doing the initial boil-up of the media in a tough glass wide-mouth bottle (fruit juice) in the microwave. You have to watch it carefully or it tries to boil right over the top. I wouldn't boil this for long, only a couple of minutes, and it is very on/off because of said boilover problems. Sitting the bottle in a shallow water bath reduces the boilover effect but takes a bit longer. Anyway, I then poured that into a triple cleaned petri dish (soaked in bleach, then boiled). Then I would give that a blast for 20 minutes or so in the steamer. A lot of frigging around that is mostly eliminated by a pressure cooker, but it has always worked for me. 

One tip: The media in the juice bottle can be made up to more than you need, say 100 mL. The excess is just stored in the fridge and you zap it with the microwave whenever you need some more. Works a treat.

Steve


----------



## sluggerdog (15/4/05)

I have no idea what this is all about.

Is this growing yeast from scratch or something?


----------



## Gulf Brewery (15/4/05)

Sluggerdog

What we are talking about is a way of storing yeast on plates. Have a read on this article on Craftbrewers that may help explain it. 

Cheers
Pedro


----------



## Kai (15/4/05)

I don't think any method short of a pressure cooker is fully sufficient for sterilising plates, but if it works then it works.

Do you platers use selective media for your yeasties?



pint of lager said:


> Because the sample is old, try and make sure that you select your colonies to make your starter for uniform size and appearance. After a year in less than ideal conditions, the chances are that there will be more mutations in your sample.



That's a better way of putting it... big enough so you can see what the colonies look like too.

Look at the pattern on the edge, the colour, the sheen and the three-dimensional shape (is it a bubble? flat? a crater? a nipple(i forget the technical term)?). Then make sure you choose several colonies, as you can't pick up any metabolic mutations with the naked eye. Choosing several kind of averages this out.


----------



## sluggerdog (15/4/05)

Gulf Brewery said:


> Sluggerdog
> 
> What we are talking about is a way of storing yeast on plates. Have a read on this article on Craftbrewers that may help explain it.
> 
> ...




Thanks GB/ Pedro, Will read up on it.

Cheers!


----------



## pint of lager (18/4/05)

Dreamboat, you may want to streak some yeast from a more recent sample, this is more likely to grow into an easier plate to work with.

For a first yeast petri dish project, using a fresh source of yeast will give you an easier introduction to the world of streaking.


----------



## kungy (18/4/05)

Kai said:


> I don't think any method short of a pressure cooker is fully sufficient for sterilising plates, but if it works then it works.



By sterilising plate, are you referring to both the yeast growing media and petri dish? Or are you referring just to the petri dish?

Cheers

Will


----------



## Gout (18/4/05)

where does everyone get their agar? i have looked around and found it very expencive 

mind you in 500g or 5Kg (got upto $1800) I gather i only need about 50-100g to last a long time


----------



## pint of lager (18/4/05)

Have purchased it from both Asian foodstores and a health food store.

You want the fine powder, not the stuff that resembles noodles.

And yes, 25 gms will last you a long time. My recipe is 100ml water, 12 gms DME, 1.5 gms agar.

Boil the water in a beaker, remove from stove, add DME and agar, stir, reboil till agar is dissolved, pour into glass plates and pressure cook.

If you don't dissolve the agar, some plates end up with not enough and do not set.


----------



## Airgead (18/4/05)

Gout said:


> where does everyone get their agar? i have looked around and found it very expencive
> 
> mind you in 500g or 5Kg (got upto $1800) I gather i only need about 50-100g to last a long time
> [post="55057"][/post]​



Local chinese supermarket. Food grade agar agar powder. About $2 for a 25g packet. Its used a lot in asian deserts and things.


----------



## bradmcm (18/4/05)

I've got a 25g packet here of "Best Quality Agar-Agar Powder" (must be good then  )
that set me back all of $1.55 from a grocer in Adelaide's Central Market.
Seeing Gout is in Melbourne, there are no shortage of Asian groceries
for him to choose from!


----------



## Steve Lacey (18/4/05)

kungy said:


> By sterilising plate, are you referring to both the yeast growing media and petri dish? Or are you referring just to the petri dish?[post="55047"][/post]​



He was talking about sterilising the plate with media already poured into it.


----------



## Gout (19/4/05)

thanks people i wll try to find some then, i didnot know the supadupa markets would have it, thought it was more a science supply thing and hence the huge quantities and price i was finding


----------



## kungy (19/4/05)

In this talk of sterlisation, Does anyone sterilise there plates via tyndallisation?

Will


----------



## Gulf Brewery (19/4/05)

kungy said:


> In this talk of sterlisation, Does anyone sterilise there plates via tyndallisation?
> 
> Will
> [post="55255"][/post]​



Will
Would you like to tell us WTF tyndallisation is?

Cheers
Pedro


----------



## kungy (19/4/05)

Taken from the Maltose Falcons website by MB Raines. Tyndallisation is 

"This is done by boiling the media for 15 minutes every other day for a week. Note that this is similar to canning where the media is immersed in a pot of boiling water and boiled. At least two to three successive boilings are necessary for complete sterilization. "

What your essentially doing, is heating it up, to sanitise. Any bacteria, spores etc that remain, germinate, then you heat the media up again and repeat a number of times. So each time the spores use up their energy to germinate until they reach a point when they can't germinate. (Correct me if i'm wrong microbiologists). This process is the next best thing to pressure cooking.

Will


----------



## pint of lager (19/4/05)

> Autoclaving may be too harsh a process to sterilise certain of the growth media used in diagnostic microbiology. To overcome this, Tyndallisation may be used. The medium is boiled on the first day, and held for ten minutes and then is allowed to cool. This kills the vegetative microbes in the medium but permits spores to survive and then to germinate. On the second day of the process the medium is once again heated for ten minutes, killing any microbes that have germinated. The process is completed with a second overnight incubation and heating.



From some dental sterilization notes found using google.

This is what I did for my first batch of plates. And 3 out of 10 sprouted bacterial growth. If you have heaps of plates and no pressure cooker, then, by all means follow this path and also follow Steve Lacey's suggestions on cleanliness earlier in the thread. You could use any heatproof container, such as baby food jars, or any shallow jar that you can get a streaking probe into.


----------



## Airgead (19/4/05)

kungy said:


> What your essentially doing, is heating it up, to sanitise. Any bacteria, spores etc that remain, germinate, then you heat the media up again and repeat a number of times. So each time the spores use up their energy to germinate until they reach a point when they can't germinate. (Correct me if i'm wrong microbiologists). This process is the next best thing to pressure cooking.
> 
> Will
> [post="55262"][/post]​



I'm no microbiologist (but my old man is). From my understanding the process you described is indeed what happens. Eventually the spores can not germinate and the media is sterile. However, some bacterial spores are very hardy and the repeated boiling is not enough to completely eliminate them. 

As far as the old man is concerned, it is the next best thing but it runs a very poor second. In practice though it *should* be sufficient for homebrewing. You wouldn't use it to sterilise surgical equipment or anything like that.

Cheers
Dave


----------



## RobW (19/4/05)

IIRC (and it was a long time ago) tyndallisation is performed using dry heat (in an oven). The theory is as described in that you encourage the spores to germinate while killing off all the vegetative forms over 3 cycles but I seem to remember it is less efficient than autoclaving (pressure cooking) & not widely used.


----------



## Plastic Man (19/4/05)

The things you learn on this web site !!.

My only hope now is that tyndallisation comes up in Trivial Pursuit this weekend. I'll have a winner....


----------



## Borret (12/5/05)

Hey Folks,

I am looking at doing some slants for something to do and have obtained some small vials to have a crack at it with. They are unused blood vials and sterile but unfortunately they are internall sprayed with silica so I will have to rinse that out to start with. (Vials free of course with a nurse in the family)
So that is not the issue here.
Has anyone tried sterilising in the microwave sterilisers that you use for baby bottles? They may sound dodgy but they work on the principal of a semi sealed environment which does hold some preesure and you put a small amount of water in the bottom, put an insert in to it to sit your bottles (or vials) on and then whack on the lid. Put in the microwave on high for a about 6 mins and 'bob' is an uncle of yours. My only fear is if I am sterilising the agar solution in the tubes in the micrwave then I may have a boiling stuff crawling out the top type problem. But is this the same in the pressure cooker?

Also on streaking tools. Are they just a really fine wire. Could I substitue this with a really fine piece of stainles wire ( eg a single strand out of a multistrand fishing line trace) and alcowipe and flame it before use to the same effect?

Regards

Borret


----------



## Gout (12/5/05)

I use some stainless wire

also the pressure cooker holds a pressure and hence up's the boiling temps, so the agar does not boil out of the tubes, yet is at a high enough temp to kill germs etc

if yours hold the pressure to prevent a boil over then ya ok i guess... giv it a try??

also note, i used my wire in a starter thinking ahhh not "that" much yeast will make it onto the plate (petri dish) but there is a white line where ever the wire was placed, i gather yeast coming up all over the place

i think you are ment to have a tiny amount of yeast in water so its just cloudy and then you limit the yeast wiped on the agar...

Lastly i used some tubes like you have and after pressure cooking them the agar is clean after a week at room temp so it must be ok!


----------



## RobW (12/5/05)

Borret said:


> Hey Folks,
> 
> I am looking at doing some slants for something to do and have obtained some small vials to have a crack at it with. They are unused blood vials and sterile but unfortunately they are internall sprayed with silica so I will have to rinse that out to start with. (Vials free of course with a nurse in the family)
> So that is not the issue here.
> ...



Borret

Stainless is excellent for that. What you may find useful is to make a loop rather than a straight wire by turning the middle of the wire around a knitting needle or similiar to make a loop of 3 or 4 mm then twist the ends together all the way to the bottom. You can put a handle on too. The loop makes it easier to streak colonies lift them off the agar & transfer them. Sterilise it in the gas flame on the stove (or your burner).


----------



## Borret (12/5/05)

Well. Troubles a bruin,

I aquired some agar for cheap at the chinese grocer today for $1:10 a pack and also 1/2 kilo of dried orange peel for under $10 and a few packs of sugar beet based rock candi for very little while I was at it, so the plans were running smoothly.

However this evening I put the above described vials to the test in the bottle steriliser and disaster struck. I guess the steriliser does get pretty damn hot and that the tubes are not suitable. This is what happened. So now I'm back to square one on the tubes :angry: 

Pic shows the steriliser in the background for those who were wondeing, the before and after blood vials at the front.





Borret :blink:


----------



## Gout (12/5/05)

i used similar and the cap (plastic part) melted, but the rubber bung and the tube was ok.... must be a different type etc


----------



## sosman (12/5/05)

My first DIY slant:





Mouldy petri:





More info at http://brewiki.org/Yeast/Culturing


----------



## NRB (12/5/05)

You finally grew that mould you were expecting...

Are your petri's 2 piece glass numbers, or a single plate sealed with plastic film? I've only used complete glass jobbies.


----------



## pint of lager (13/5/05)

Sos, that plate looked good, you had two sorts of mould growing. You could have used some of the yeast that was away from the mouldy bits and restreaked a fresh plate. This is how you can get around infected samples.

There is quite a bit of mould near one edge of the plate, this may or may not be due to handling and your aseptic procedures.

Borret was asking about streaking probe metal. You want a metal that will take many cycles of heating and cooling without flaking too much. And of course, it mustn't melt when heated in a flame. SS is good, so is nichrome wire as used in heating elements.


----------



## Steve Lacey (13/5/05)

sosman said:


> Mouldy petri:
> More info at http://brewiki.org/Yeast/Culturing
> [post="58866"][/post]​



Sos, good info as usual.

Coupla tips for reducing contamination. When you do the quadrant method, don't reflame the loop. Just work your way around quickly from one dip into the yeast source. In other words: flame loop, dip loop, pick up plate out of lid (it is upside down), turn and work above a low gas flame now if possible and/or hold breath, streak one, rotate plate 90 deg, streak two, rotate, streak three, rotate, streak four, replace plate into lid. Exhale. From picking up dish to replacing it should take all of 10 to 20 sec at most.

I then pop the plate into a ziplock bag and then that into a biscuit tin. Actually, it goes into the ziplock bag after pressure cooking and during the day or two wait before streaking for the condensation to bugger off.

Wow, that yeast supplies place in Colorado has some stuff. I'd like to know how to use all those viability indicators and things...and I wonder what the tubing is for? Shame they seem to only have disposable loops. I aquired my loop from a mycologist friend, so I don't know what to tell people about getting their own.

Steve

P.S. Cagney and Lacey indeed!! :lol:


----------



## Borret (13/5/05)

POL- Yep got some nichrome wire as well. Use it for hot wire cutter for foam on my model planes. 

Gout- how do you sterilise. I am wondering if it was actually the microwave that did that to the plastic as the bottom bit that may have sat in the water was not as bad

Borret


----------



## Gout (13/5/05)

pressure cooker from Aldi $69... works a treat!

i have left the agar out for over a week now (in sealed test tubes) with zero growth so it must be rather sterile i would think...


----------



## sosman (13/5/05)

NRB said:


> You finally grew that mould you were expecting...
> 
> Are your petri's 2 piece glass numbers, or a single plate sealed with plastic film? I've only used complete glass jobbies.
> [post="58888"][/post]​


They are two piece glass. I only acquired the Parafilm later so they were unsealed for a few days after I streaked them.


----------



## sosman (13/5/05)

From the science supply australia catalog I believe the nichrome wire they use for loops is 22 gauge. I think that is about 0.6mm. I happened upon a stash of SS wire at work which is 0.7mm and works fine. If it is too thick then making a small enough loop could be a problem.

PS if anyone is around vermont and wants some wire to make a loop - get in touch.


----------



## sosman (13/5/05)

pint of lager said:


> Sos, that plate looked good, you had two sorts of mould growing. You could have used some of the yeast that was away from the mouldy bits and restreaked a fresh plate. This is how you can get around infected samples.
> [post="58964"][/post]​


POL I was tempted to do that but this was yeast I already have a fair bit of. Interestingly this yeast is a couple of generations old and apart from the mould which I am guessing it has come from the atmosphere, there doesn't seem to be much else growing with the yeast.


----------



## Gout (13/5/05)

Ok i finally got off my bum and took a pic

the left is the little blood tube i was talking about

the mid is the 20Ml tubes that are great

and right is the streaked plate, i now know i had to much yeast on the wire and will do it better next time, however there is some single cells


----------



## pint of lager (13/5/05)

If you can see visible yeast on your probe, you have too much. As Kai said, you need a plate the size of a cricket pitch to effectively streak.


----------



## Gout (13/5/05)

i plucked two small dots off this dish and placed onto a slant (dont plan to use) This is all practice so if/when i stuff it up, its only a few cents wasted... once i have it down pat i will then move to my yeast bank and start moving to this method....

first time is always blind to a point... but we will all get there


----------



## pint of lager (13/5/05)

Gout, practise is good. 

Sos, moulds are common on plates. This is why when you streak a plate up, don't just do one, do a couple. Hopefully one will be mould free.

To inhibit mould growth, you need some sort of bacteria inhibitor in the agar solution. A small bit of antibiotic tablet as per GLS's notes works. Or some sodium or calcium propionate.


----------



## sosman (14/5/05)

pint of lager said:


> Sos, moulds are common on plates. This is why when you streak a plate up, don't just do one, do a couple. Hopefully one will be mould free.
> 
> To inhibit mould growth, you need some sort of bacteria inhibitor in the agar solution. A small bit of antibiotic tablet as per GLS's notes works. Or some sodium or calcium propionate.
> [post="59020"][/post]​


Good tips. There is someone at work who spent a year in a yeast lab so she has given me some good tips too. I have some scrap perspex and I might try to make a miniature culturing hood. I also scored one of those 254nm wavelength UV tubes which I might rig up inside the hood to sanitize everything before I get going. Another brewing project.


----------



## sosman (14/5/05)

Steve Lacey said:


> sosman said:
> 
> 
> > Mouldy petri:
> ...


Good tips, I will try that. I was under the impression that leaving the dish for more like a week was needed to see if it had something nasty growing on it. Having said that, I probably infected mine during the streaking part because they were clear after a week.

Oh yes, my homemade innoculating loop:


----------



## ausdb (14/5/05)

I found this site last night it has excellent info on yeast culturing. Actually the whole website is pretty good, check out their club brew system!!

http://www.maltosefalcons.com/tech/MB_Rain...t_Culturing.php

Cheers


----------



## sosman (14/5/05)

ausdb said:


> I found this site last night it has excellent info on yeast culturing. Actually the whole website is pretty good, check out their club brew system!!
> 
> http://www.maltosefalcons.com/tech/MB_Rain...t_Culturing.php
> [post="59046"][/post]​


That page inspired me to make a stir plate and I referred to it lots on yeast plating stuff. That link and a few other informative ones are collected at http://brewiki.org/Yeast/Culturing


----------



## sosman (1/6/05)

Steve Lacey said:


> Coupla tips for reducing contamination. When you do the quadrant method, don't reflame the loop. Just work your way around quickly from one dip into the yeast source.
> [post="58976"][/post]​


I went ahead with your tip Steve and voila, no mould (2 out of 2). I guess I did improve my other techniques also.


----------



## Borret (1/6/05)

Congrats SOS, it's looking good. Hope to up to that some time. 
And I see Post 1000 is about to pop up for you- Wuhoo. Millenium poster.

Cheers
Borret


----------



## Hoops (29/6/05)

OK here's a few photos of my first attempts at streaking.
Comments?


----------



## Ross (29/6/05)

Hoops said:


> OK here's a few photos of my first attempts at streaking.
> Comments?
> View attachment 3017
> 
> ...



Looking pretty impressive hoops - no sign of rouge bacteria...


----------



## sosman (29/6/05)

Hoops said:


> OK here's a few photos of my first attempts at streaking.
> Comments?
> [post="65390"][/post]​


:beer:
Give me the streaker from the 1982 grand final any day.


----------



## TidalPete (17/10/05)

Airgead said:


> Gout said:
> 
> 
> > where does everyone get their agar? i have looked around and found it very expencive
> ...



I received my first slants the other week & had no option but to make up an inoculation loop, sterilise three 10ml starters (just in case of contamination), & have a go building a starter from a slant. I am happy to say that all three starters are alive & well. It was a lot easier than I expected so I contacted a mate in Brisbane to get me some agar to continue my yeast culturing experiences. As I did not specifically mention agar powder I finished up with the agar in the pic. I don't suppose it really matters as it should still dissolve with the DME in the pot. Please tell me if I'm wrong here.

:beer: 

Edit ---- My first attempt at posting a pic didn't turn out as expected  . Will try again when I resize it. 
Edit 2 ---- Please bear with me. I have never used a digital camera before. This should work.


----------



## TidalPete (17/10/05)

Tidalpete said:


> Airgead said:
> 
> 
> > Gout said:
> ...



Success  My first & last stuff-up with piccies (hopefully) :beerbang: .


----------



## NRB (17/10/05)

You'll be able to use that agar no worries. It liquefies at around 85C but solidifies somewhere in the 30-40C mark.


----------



## TidalPete (17/10/05)

NRB said:


> You'll be able to use that agar no worries. It liquefies at around 85C but solidifies somewhere in the 30-40C mark.
> [post="83497"][/post]​



Thanks for your response NRB. Just playing it safe. I am typing this out of the corner of my right eye whilst admiring your lovely avatar.  

:beer:


----------

