now cover culturing um techniques um that we typically use in laboratory settings and so these are going to be useful for individuals that are looking to grow up bacteria in order to study them and one of the main challenges about studying any organism is culturing it in a laboratory setting outside of its natural environment and so in order to grow a microbian in a laboratory setting you really need to have a good idea of what nutrients that microbe needs and for some microbes that is pretty easy they you know aren't that picky and other microbes are fastidious or difficult to culture and that's because they might be requiring a certain vitamin or trace element in order to function and if you're not providing it in your culturing conditions that microbe is not going to grow so the culture medium is the the food that we are providing for the microbe and this medium can is going to have to contain all of the nutrients required for the growth of that desired organism and it can be solid in the solid form or a liquid form an important aspect of this medium is that it must be sterile and this is again think about it in a laboratory setting if you're interested in growing one specific species of microbes or one specific species of bacteria you need there to be no other living organisms on that bacterium or in that growth medium and there are different ways of sterilizing culture medium we can filter them to remove all microbes or we can autoclave or pasteurize or heat to remove them and the the [Music] process of introducing microbes into a growth medium is inoculation so the microbes that you introduce are the inoculum and there are there are two types of mediums that we can use to grow microbes and here we have two examples um the first one is a diff is a defined medium and it's defined because you know exactly what is in that medium so you know exact composition right and here's an example of a defined medium that we use to grow e coli it's got glucose it's got several salts that provide trace elements and and trace minerals that are needed for that microbe to grow some microbes that is not sufficient and you need to provide other nutrients that um in order for them to grow and if that's the case you're probably going to have to use a complex media and a complex media you know exactly what goes into it but you don't know the exact quantities and amounts of every single nutrient that is found inside of it and most often these complex medias use extracts from either animals so beef extract you can also use uh yeast extract and so so forth you can also use plant extract and all sorts of things you can also find a common ingredient that you can also find in complex media is peptone peptone is simply just protein mixtures that have been partially digested and that is because some microbes cannot [Music] bring in right transport large protein molecules if you recall the cell wall of microbes there's a reason for that and so in order for the nutrients to be bio active or available for the microbes they need to be broken down into smaller pieces or these peptides which are just smaller protein chunks right and that's what peptones are in addition to other meat uh nutrients for which you know the exact quantities so you can see that for some in this example we know the exact amount of uh sodium chloride but in the beef extract we know we're going to find some lipids uh right some fat from the beef we're going to find some proteins from the beef we're going to find some amino acids from the beef but we don't know the exact quantity of any particular nutrient it's a mixture and that's what makes it complex the media can also then be differential and selective and this is something that we've covered in the lab portion of the course so hopefully this is uh going to be just review but to reiterate a selective media is going to inhibit the growth of certain microbes and support the growth of others whereas the differential media is going to help you distinguish between different microbes depending on whether they can or cannot consume a certain nutrient and usually it's based on appearance so it's pretty easy to tell it's a color change in the colonies or something like that and so here's an example of cultural mediums that are selective where you initially inoculate a and over time you see that this colony or the these bacteria did not grow whereas these grew quite well and it looks like maybe these also didn't grow too well so what we're seeing is that these are dying off they're being selected against and these microbes are growing just fine so they're so they're being positively selected for we then have differential media so here we've inoculated different microbes and you're seeing that there's a color change where in these two inoculums right these two bacteria we see a pink color compared to the other two so that's differential so we're able to see so in this case this microbe might be able to use a certain nutrient that's present there because it's able to carry out um fermentation it's a specific uh form of fermentation whereas this one is not and in addition indeed we're seeing that not only are we seeing a color difference which means that this medium is differential but we're seeing that some microbes are not growing and that would make it selective so they are dying off so in this kind of in this example it would be both differential and selective and here's another table of different types of uh growing media and we'll now talk about uh reducing media which is going to be used to culture anaerobes and so anaerobes again are organisms that do not tolerate oxygen and that is because they can't neutralize the reactive species generated by the presence of oxygen and so they would need a reducing media to be able to grow one or at least one of the ways to grow anaerobic microbes is by using reducing media the this media is going to contain chemicals that help to neutralize and sorry are you going to use to chelate or take out you can also use as your book describe uh a candle to help burn off oxygen um in in a jar and then once you seal the jar with the burning candle the burning candle continues to consume the oxygen until it consumes so much of the oxygen that there's not enough oxygen to continue the flame and it dies out and in that case you would have depleted oxygen levels which would facilitate the growth the growth of anaerobic organisms you also then have organisms that require co2 carbon dioxide levels to be higher than ambient atmosphere and these organisms are referred to as capnophiles and there are different ways of culturing these microbes sorry i guess the candle jar would be used for canophiles uh not so much for anaerobes because there would still be oxygen even though small amounts of oxygen if you just use the candle so the candle would be used more for the cabinophiles and you would need to remove all oxygen if you want to grow anaerobes and next we'll talk about ways of plating bacteria there are several ways these are two that are sometimes used and really this is probably the better way of plating bacteria the first way is where you take uh a stock solution of bacteria and you inoculate the clean petri dish so you spread some bacteria on an empty petri dish and then you pour some liquid liquid agarose the liquid agarose has to be hot enough that it's it's been liquefied but not still hot that it's going to kill your microbes and so you just need to let the aggress cool a little bit but not too much because it'll stop to solidify and that's when you pour it in you then swirl around your plate to help diffuse the bacteria and to disperse them and hopefully if you've done it well you'll end up with colonies and again what you're striving for is individual colonies where each colony starts from one cell and for that you cannot have colonies that are growing in close proximity to one another because then you can't assure that each colony came from one cell so the better technique to do is to do a spread plate where you have a petri dish that already has the the media and it's usually solidified using agros and that's what you would have received in your kit all of your petri plates are agros based media and you would take seminar some bacteria inoculate and you would use a spreader to help spread the bacteria and ideally again you want to spread such that you end up with bacterias so far apart from each other that every colony that you observe is from one bacterial cell and again there's no way to definitively know but you can look at the size of the colony to help gauge uh if most of them are the you know the relatively the same size but then you've got a monster colony where that's you know to twice the size of all the other colonies well probably that colony didn't start off with you know one bacteria and maybe two or so and that's why it's much bigger there's that many cells um and another challenge in growing microbes is that you often want to study and isolate a single bacterium to then uh closely expand and work off of just to reduce genetic diversity here for a number of other reasons so often we want to have a pure culture and this is also something that you would have encountered if you've done the if you kind of take bacteria from an environment it's going to be a mixture of bacteria but if you just want to grow out one species you would need to be able to obtain one single cell and then grow it and in order to do that we would do um a streak a streak plate and what that means is you heat your your rod um to sterilize it you then inoculate it by dipping it into your stock solution of bacterium whether it's a mixed population or all the same species but you just want to isolate one single bacterium you then take your petri dish and you pass it and go back and forth and this will deposit the microbes from the inoculating loop onto the agar you then reheat your uh inoculating loop to clean clean it from or get rid of all the microbes that are still there so you typically dip it in alcohol first and then flame it to help burn off the alcohol and get rid of bacteria you then use that sterile loop to barely touch your first set of streaks which are here right now using the sterile um what you may call it uh loop you're going to touch the original streak a little bit and move up and down and touch maybe once or twice and spread those microbes and so hopefully what you're doing is taking a few of the microbes that were here and spreading them as you move up and down on this side of the petri dish you then sterilize your loop again to make sure you get rid of any microbes that were present from when you did this streak and so after sterilizing it you then barely touch the second streak that you've made and in a single motion you zigzag to help spread the microbes and essentially what you're doing is you're diluting the microbes as you do these various streaks and again you can sterilize your loop and you do your final fourth streak where you barely touch your third streak and spread that around and what you should hopefully get is something like this where your first streak is very um overgrown a lot of microbes a lot of bacteria then fewer and fewer and you start to see some colonies and there are various sizes and again they're various sizes because a different number of microbes that were there and eventually in your fourth or fifth streak you should be able to see individual colonies and then you can be more or less assured that by collecting this colony you're getting a colony that came from just one single bacterium