Calling All Yeast Platers!

[Gulf Brewery]

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]

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]

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]

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]

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]

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]

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]

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]

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

[post="58774"][/post]​

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]

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]

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]

My first DIY slant:

Mouldy petri:

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

 

[NRB]

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]

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]

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]

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]

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]

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]

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]

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.