Making Agar Slants (Slopes) - In Pictures

[Wolfy]

Q. Are polystyrene test tubes able to withstand 121C?

No, but you should be good with polypropylene, however if you are buying from Ebay (or similar) ensure they say 'suitable for autoclave' or similar, because some polypropylene tubes have polystyrene lids and stuff like t hat.

[greenxpaddy]

Finally my shipment from Asia has arrived. I'll have a go at making slants tonight.

On another note, and without trying to bore you to death, you will remember the Mild I brewed recently with the minorly infected wort that I reboiled for 20 mins for the starter. I thought it was ok because there were no off flavours in the beer...then we had that mysterious looking layer in the yeast rinse...

Well it still tastes absolutely fine. However after having a couple of bottles yesterday lets say had an interesting belly for the following few hours, a lot more turbulent than normal effects of yeastiness activity. Not saying it was violent tummy upset or anything but made me think. Coincidentally I did a trial pressure cook yesterday and when it was finished was looking for somewhere to store the sterile jars. I thought that Wilkos storage box/FV was ideal. I had washed that out the same as all my other FVs after that brew. However when I opened it there was a bad amount of black mould growing on the bottom of the rubber grommit for the airlock and grey mould growing round the tap screw collar. Maybe I didn't clean it will enough - but still the amount of mould would indicate there was a high level of spores in the immediate FV environment. When I used the little bottler to fill from the FV I recall it being very clear except for the odd what I thought were clumps of flocculent yeast running through every now and then initially. Putting two and two together, maybe they were clumps of something else? So here's the plan....I am going to take two samples - one from the rinsed yeast from that brew and one sample from the bottom of a bottle of the bottled mild and streak them each on a petri dish of agar wort. Lets see what grows. Interestingly I would be keen to hear if anyone tests their beer anyway in this way to see how sterile there set up really is. If anyone has any photos of cultures which show very good sanitising and poor sanitising as comparisons that would be useful. As I am guessing no matter how hard you try there will always be a very small number of invaders....which is not an issue. Its all matters of degree. In the meantime I won't be drinking any more of that one. Lets hope its completely unrelated and the suspicious elements are just coincidences and misinterpreted.

[gregorach]

So here's the plan....I am going to take two samples - one from the rinsed yeast from that brew and one sample from the bottom of a bottle of the bottled mild and streak them each on a petri dish of agar wort. Lets see what grows. Interestingly I would be keen to hear if anyone tests their beer anyway in this way to see how sterile there set up really is.

No, I've never tried streaking the dregs... Problem there is that it's probably going to be hard to see any contaminants through all the yeast. The professional way to do the job would be to membrane filter the beer and then plate it on one or more selective media - which kill the yeast and favour other organisms - but that's not the sort of thing you can easily get into at home.

Still, give it a shot and see how it goes... Do bear in mind that producing and streaking clean plates takes a bit of practice though!

[greenxpaddy]

Thanks. I cooked my 10 slants and poured my 6 petri dishes this morning. I'll leave them for a week to see if they are a success. For info I got the same hot break as you. I'm guessing if you want them to look all perfect it would be best to boil your DME first and cool once, decant into a new jug before then adding agar and reheating.

That's a point about the number of yeast cells in the dregs. May just streak the beer itself.....Interestingly the rinsed yeast in the 50ml pot appears to be developing isolated black spots in the refrigerator, not on the surface - within the yeast cake. I'll keep you posted and load up some pics.

Lastly - on petri dishes topic i bought plastic ones. They say these are sterile in the main pack. they're not individually hermetically sealed though. I am guessing they won't go in the pressure cooker - think they are normally polystyrene. Does appear to make it harder to prepare with no contamination risk as they need to be opened to pour in the sterile agar solution.

[gregorach]

Lastly - on petri dishes topic i bought plastic ones. They say these are sterile in the main pack. they're not individually hermetically sealed though. I am guessing they won't go in the pressure cooker - think they are normally polystyrene. Does appear to make it harder to prepare with no contamination risk as they need to be opened to pour in the sterile agar solution.

Normally when you pour plates you'd autoclave the petri dishes (if using glass ones) and the medium separately, and then pour them once the medium has cooled a bit - you want it to be as cool as possible whilst still hot enough to pour smoothly, which happens to be about the temperature at which the flask can be handled without it hurting too much. It's not really practical to load dishes of medium into the autoclave - even if you manage to avoid spilling them, you'll end up with excessive amounts of condensation inside them when they cool. Pre-sterilised plastic dishes don't get autoclaved, so that saves you some messing around.

When pouring plates, you need to work near an open flame, such as a bunsen burner or alcohol lamp - I use a little butane torch. I also wear nitrile gloves (sanitised with hospital-grade alcohol gel) and a disposable surgical mask. Have your foil-covered flask of still-liquid medium to one side (the side of your dominant hand) and an empty petri dish on the other. Pick up the flask of medium, remove the foil cover next to the flame, pass the mouth of the flask through the flame, lift the lid of the dish just enough to get the flask under it (keeping it over the dish), pour 10-20ml of the medium into the dish, replace the lid, flame the mouth of the flask again, flame the foil cover and replace it, put the flask down, and move the dish somewhere to cool. Repeat.

There's a lot to be said for keeping your plates (a "petri dish" is the empty container, once its got medium in it it's called a "plate") in a closed, sanitised container - I use a large sandwich box. Also, you need to be very careful to avoid getting any of the medium on your hands or the outside of any your dishes - it's incredibly sticky, almost impossible to clean off, and guaranteed to go horribly mouldy. If you do get any on your hands, stop and change your gloves immediately or you'll end up getting it everywhere. Be aware that there will probably be a drip on the lip of the flask, so be careful how you handle it and where you put it down.

Once your plates have set, invert them. Once most of the condensation has dissipated (usually takes a couple of days) they're ready to use. You can stack them up, wrap a rubber band around them and store them in the fridge until needed.

One other thing to watch - when taking a petri dish or plate from the top of a stack, slide it off sideways rather than picking it straight up. If they're properly clean, they tend to stick together just because of air pressure, so you can end up picking up half of the one below as well without realising it.

[greenxpaddy]

Here's the results of my plates

the first pic shows after 3 days. The streak of beer is on the left -nothing yet- and the streak of slurry from the rinsed yeast on the right - took straight away.



For along while it looked like there was nothing at all on the left plate however after 7 days we have the following. Just to confuse you they are the other way round in this photo.



The slurry streak is not growing much more currently. We have no evident foreign bodies on the agar. The beer streak was clearly too runny and i used too much on the streak it would appear. Maybe there was too much condensation. Although the pattern is vastly different the growth suggests only yeast.

There is no mould evident ... yet. If there is any bacteria its not surfaced yet.

[Wolfy]

The slurry streak is not growing much more currently. We have no evident foreign bodies on the agar. The beer streak was clearly too runny and i used too much on the streak it would appear.

The main idea behind streaking yeast onto plates is so that you can isolate individual cells that then grow up into colonies (which you can then use to grow a 'pure' culture from).

To do this you'd take a (very very) small sample and run the loop backwards and forwards in one direction in one sector of the plate, before turning the plate at about 45degrees and then streaking over the same 'lines' into the next sector of the plate. When you repeat that process 2 or 3 times (if done correctly) you should have only individual cells deposited, which grow up into small round colonies.
It does take some practice and there are some online tutorials that will explain in pictures better than I can in words.

However, I'm not sure that your experiment has shown much at all other than I don't think I'd trust whatever is growing on the 'beer streak plate'.

[greenxpaddy]

Thanks Wolfy - its interesting you think that the growth on the beer plate is suspect. At first glance the bumpiness of the growth looks very similar. Initially I thought that the yeast cells were deposited like a blanket over the surface in very low density when the streak ran. and that explianed the randomness.

Do you have any links to streaking tutorials

[Wolfy]

Here is one: http://www.alsand.com/beer/yeast/index_E.html
The animation shows what I was trying to explain, and the BW pictures show how individual colonies have been isolated.

[greenxpaddy]

Nice one. That's really helpful. Now to do a repeat!

[gregorach]

Right, first off, if nobody's said this yet: you clearly need the yeast book.

This is (more or less) what a streaked plate is supposed to look like:



It's not perfect, because the colonies from the last set of streaks aren't as well isolated as I would like, but it's not bad. i'm not getting any of the really good ones out long enough to photograph...

I've never had very good results with the streaking patterns usually recommended - rather than using a zig-zag pattern, I streak a number of not-quite-parallel lines at each step. Hopefully you can see the pattern in the photo. One thing to remember is that you need to flame the loop between each "set" of streaks.

1: Take a tiny sample from your yeast source, pick up the plate, and run the loop back and forward several times in a short line near the edge of the plate - you need to be able to track where this is, so I do it next to my left thumb when holding the plate. Put the plate back down and rinse your loop. (I like to rinse at this stage so that you don't end up with crud burnt onto the loop.) Rotate the plate clockwise through about 45 degrees.
2. Flame the loop. Pick up the plate (keeping it inverted for now), cool the loop by touching it to an area of the plate you're not going to be working near (if you've picked the plate up in your left hand, this is likely to be somewhere around the edge of your palm on the far side from your thumb. You can see the distorted area where I cooled my loop on this plate near the bottom of the photo.) Turn the plate over and quickly draw a set of streaks across the line you did in step 1. Put the plate back down and rotate it clockwise again.
3. Using the same technique, do another two sets of streaks, flaming the loop each time.

Incubate for two or three days, and you should have a lovely plate like mine.

[greenxpaddy]

OK, I though I could do it without reading Yeast but maybe i should buy it...onto Amazon now

[floydmeddler]

You can do it without the book... Most of us on here have no doubt. Plenty of stuff online.

[Wolfy]

Right, first off, if nobody's said this yet: you clearly need the yeast book.

I suggested it would be a good idea before spending lots of money on yeast-propagation-hardware, farming yeast is not for everyone, nor is it cheap or quick.

You can do it without the book... Most of us on here have no doubt. Plenty of stuff online.

Very true, but its also a useful resource and has all the info in one location, when I was looking for streaking examples just before, about 1/2 of my 'reference bookmarks' were no longer valid.
I might have to try the Way-back-machine and retrieve the info.

[gregorach]

Oh, I forgot to mention - the entire streaking procedure should be carried out within 10cm of an open flame.