Pouring Plates

So this is the cloning mode where we do all of our work. That sterols is a sterile wood before using it. We should wipe this down percent ethanol. You never know who the last person wants to use it, but this is a nice spray, so it will spray down before we even begin. So we know that it's clean because there are media spots on here. You can see that haven't quite been cleaned off, but if at least the surfaces and sterilized that represents particularly eco life may be in that media and may contaminate our plates, and we don't want that to happen. So how to save as draft instagram want to keep everything as clean as possible. Now we have, this is some media that is does not have auger in it. You need to make immediate as auger in it so that it's, it will form bacteria Petri plates bacterial plates or our culture plates are stored in this fridge, and we want to generate something like this. It'S a hard auger that we can grow out our ecoli on. So now I have 10 Petri plates here. This is a sleep of Petri. Plates 10 have been used already, it's been using correctly and I'm showing you a bad example here. What happened? Was someone ripped open, the top, which is fine? You got to get out of somehow right, but they ripped it all. The way down to here problem is is that the 10 that were used originally that's fun that we're fine, but then these have sat up on the counter, for I don't know how many weeks right and they could have been contaminated because it was closed often so, Ideally, you'll open a slit here at the top and then squeeze out and like a tube of toothpaste right and you squeeze out the ones the number of plates you need from the bottom, and sometimes you use a hole leak. A hole leader will put more all the plates in one sleeve, we only a half a liter, so we have about half a sleeve here now. The other thing about this, let's get these out, is with the plates, our first they're upside down and that's good. It'S just a way that normally handle bacteria when you, when we incubate it with the plates upside down. We'Ll also do all of our writing and annotation on the bottom. Sometimes it's cut nicely, then you can reuse the sleeve as a container and tape the top. Now, every one of these plates that I'm poori, I need to put my name on them: the class or project that I'm doing it and also the date, and so those three things are important to have on every plate. Every so often we go through the fridge and we'll throw a place that are not labeled and plates that are expired more than you know, six weeks or so, which is a bacteria and really aren't that good anymore anyway, and usually people go up what they need. After time, so the other thing to check is that we need to pour the media when it's not too hot or not too cold, and right now we do not have. We only have the media in here. It'S starting to set the way to check the temperature. Is with our little digital kilometer now this is a laser thermometer show be careful not because it will bounce the laser bounces off these stainless steel sides to try optics shine it in your eyes, but it does a fairly good job. So this is now at 46 degrees Celsius right right at about 30 or so it'll start to set up. So I need to get my iptg x-gal and my ampicillin in there before it gets too late now's a good time to do that. It'S at 43. It'S going to start setting up soon, so we better put the ampicillin in it now. This is this, I know, is half a liter, it's 500 milliliters. So for half a liter we need to put in half a mil half a milliliter of ampicillin. The ampicillin is at ten milligrams per mil milliliters swirl that around and get well mixed. Okay, generally, what we're going to do as a class is put the ampicillin in now, let it cool it's hardened a little bit and then we'll put the IPTV and x-gal on top of that and spread it. So we're just surface treating the plate with those two reagents for the screening process. Now, if you look closely here, the plates are up upside down in general. You want to have about twenty milliliters per plate and fill it up on being generous here, because I know we've got more media than than we need. Okay, it's better to start on the bottom, so I'm just looking at the side of the plate, and I want to have it about halfway full and you notice that I have put bubbles in this. As I was shaking it, those bubbles can be confused for colonies later on. So it's nice not to have bubbles in the media when you're pouring it and I'll show you a trick how to get rid of it doesn't have to be this full, although you do run into problems if the media is too thin on the plate, you want To make sure you've got ample media in there. What happens is it ends up drying out too quickly in the refrigerator, and you don't have good bacterial growth. So one thing you can do to get the bubbles away. We'Re starting this lighter here, what you can do occasionally is if you can have a long enough hose, you can run the flame this on medium and it will pop all those bubbles and then you won't, then you'll have a smooth surface, so you don't have the Bubbles confused with e.coli, ideally because I want them to cool I'm spreading them out a little bit. I just have to be careful, otherwise just end up making a mess, but I'm going to put them two to three deep and we'll. Let them sit for 20. 15 minutes 20 minutes. Let them let them cool like this, and then they'll harden a little bit and we'll put on the X gala night PTT. The bacteria will grow best when the media is when the plates are pre-warmed, so we're going to stick them back after they've hardened, we'll stick them in the growth in the incubator, for a little bit before. We put this in actually put the cells on there and it won't shock the bacteria if you take plates cold plates right out of fridge, put bacteria on it. He'Ll shock the cells and you won't have very good growth results coming out of it. So