Welcome to the bumbling biochemist’s bacterial pancake bakery! One of my guilty pleasures in the lab is the smell of LB – so I slyly suggested we teach our new grad student how to pour agar plates! Bacteria love these sugary treats. And biochemists love that we can use agar to make gel matrixes filled with Lysogeny Broth to feed bacteria in exchange for them doing favors for us like copying DNA. So let’s take a look at the ingredients and baking process.
When it comes to growing bacterial colonies, LB-agar steps up to the plate – but first you have to get it into a gel state (a situation to which its friend agarose can surely relate!) “-ose” is an ending that’s usually used to indicate something’s a sugar – and agarose is a sugar – but so is agar! So what’s the difference? They’re related, but they differ by more than just a few letters and those differences make them useful for making gel matrices for different tasks – agarose gels for separating DNA fragments by size and agar gel plates for growing bacteria)
A gar is a fish-like thing and AGAR (aka agar-agar (seriously!)) is a mixture of 2 sugars – agaropectin & the one we’re more familiar with, agarose. So you get agarose by purifying agar. And to understand why you’d go through that trouble sometimes, but not other times, it helps to know a bit more about what these sugars are.
Agarose is a polysaccharide (“poly” means many & saccharide’s sugar, so a polysaccharide is a long chain of repeating sugar subunits joined together). It’s an example of a polymer. Polymers are long chains of repeating subunits. Different polymers have subunits of different types (e.g. nucleotide subunits link up to give you the polymers we call DNA or RNA; amino acids join to form proteins; and monosaccharide sugars chain-ify to give make complex carbs (like agarose!))
Sugars have lots of hydroxyl (-OH) groups (which water happily sticks to, helping you form a gel – an “infinitely” interconnected (like 7° of Kevin bacon) polymer mesh containing water. (more to come)). Individual sugar units (monosaccharides) often adopt ring structures (as is the case in agarose) in which the -OH groups stick out like legs. Sugars can have the same “linkage” but have the linked groups sticking out in different ways & we use “L” and “D” to refer to which direction in space the legs point. More on such stereochemistry here: bit.ly/2Q8Dnax
Monosaccharides can use their -OH’s to link together. Linking 2 gives you a disaccharide . Add a few more and you get oligosaccharides (oligo means few). Link lots and you get a polysaccharide (poly meaning many).
And speaking of many, the multiple -OHs mean there are multiple potential linkage sites (which we specify by which number “addresses” on the sugar rings are joined). You can get “branching,” but in agarose, you have linear chains (of about 400 subunits). (Although branches can be introduced using “crosslinkers” to strengthen agarose so that it can do things like make size exclusion chromatography (“gel filtration”) resin which can withstand the high pressures generated in FPLC (Fast performance liquid chromatography)).
In agarose, the repeating subunit is a sugar duo (disaccharide) of galactose (D-galactose to be precise) linked to a modified galactose monosaccharide, 3,6-anhydro-L-galactose. We call this duo AGAROBIOSE (Linking a galactose to a glucose gives you lactose, a disaccharide you might be more familiar with).
In galactose, the rings have 6 sides with 4 -OH legs, 5 -H legs, & 1 methylhydroxyl (-CH₂OH) leg. We often don’t draw the “-H” legs because they hide the more interesting legs that are capable of reacting. In agarose’s modified galactose, 2 of the -OH groups have linked up & kicked out a hydrogen (“anhydro”) so that the methyl hydroxyl arm is bridged to an -OH arm forming a “bridge” over the ring.
About 2/3 of agar is agarose but, agar also contains AGAROPECTIN. It’s really similar (it has alternating D & L galactoses) BUT many of those galactoses have modifications. There are several different modifications including adding sulfate(s), pyruvic acid, or methyl groups, or sneaking in one of those linked-leg versions like’s in agarose.
Unlike the consistent repeating nature of AGAROSE, it’s only the alternating D- & L- galactose “skeleton” that’s consistent in agaropectin. Its modifications are scattered throughout the chain (for instance, ~every 10th is attached to a sulfate through an -O-sulfate linkage, but that’s just on average, and it’s not likely they’re precisely, evenly, distributed). And, while the chains tend to be shorter than the agarose chains, their length is also variable, so, agaropectin’s really quite a mix & you never know quite what you’re gonna get!
Why use one over the other?
DNA has a lot of negatively-charged phosphate groups (phosphorus surrounded by 4 oxygens). This will serve the basis for them moving through the gel towards the positive end. So we need the gel to be neutral.
Agar has a lot of sulphate groups (sulfur surrounded by oxygens). These are also negatively-charged, so they can interfere with how the DNA moves through the gel. So it would make a bad matrix for electrophoresis.
BUT agarose is neutral, making a good matrix for electrophoresis.
BUT agarose is also more expensive because it has to be purified. So if you don’t need to worry about the physical charge issue, might as well use something that has a lower *monetary* charge! Because agar requires less processing, its cheaper & perfect for use as a matrix to hold bacteria food!
In agar plates, it’s not the agar itself that’s providing nutrients. That’s one of the great things about agar – *most* bacteria can’t eat it. Instead, the agar just provides “scaffolding” to house the nutrients the bacteria need. And often those nutrients are provided through a liquid bacterial food “growth media” called LB, which, no matter what your textbooks might say, (originally at least) stands for Lysogeny Broth. Sometimes initials for Luria, Lennox, or Luria & Bertani get credit for the name, but it really stands for LYSOGENY BROTH and its recipe was first published (by Giuseppe Bertani) in 1951. He was using it when studying lysogeny (a process where a bacteria-infecting virus called a bacteriophage (“phage”) inserts its own DNA into a bacteria’s DNA & bides its time until conditions are right for entering the lytic phage where it cuts itself out, makes lots of copies and bursts open the cell) http://bit.ly/2HLuB1
The point of LB is basically to give the bacteria what they need to grow, divide, and do what we want (like make copies of DNA we put in them and/or make copies of proteins using instructions from DNA we put in them). And to do it cheaply.
Note: For the protein-making and large-scale DNA-making, we grow bacteria in straight-up LB liquid – “in suspension” with shaking – but when we need to isolate specific groups of bacteria that are all derived from the same “parent cell” and thus genetically-identical, we embed that LB into a gel so that different genetically-distinct “colonies” don’t intermingle, but instead grow as gloopy dots. If you want to learn more about various media for the suspension stuff check out this post http://bit.ly/bacterialmedia but today I’m going to focus on the gel-trapped form.
We don’t give the bacteria 5-star cuisine. Instead, we want to spend as little money as possible while still giving them the nutrients they need. At a minimum, we need to give them a source of energy, something they can break down (catabolize) to make ATP – such things can be sugars, proteins, fats. In addition to breaking things down, they need to be able to make things like proteins and DNA (do the anabolic part of metabolism). This requires nutrients that provide the elements needed like carbon and nitrogen, which thankfully you can get with a simple recipe that’s sufficient for lots of bacteria.
There are 3 main components (though 2 of those components themselves have a lot of components.
- TRYPTONE -> this is a mix of peptides formed by the digesting a protein called casein with pancreatic enzyme -> this provides amino acids the bacteria can use to make new proteins
- YEAST EXTRACT -> this “autolysate” of yeast is basically just whatever happened to be in yeast (organic compounds including vitamins, trace elements, etc.) – and if it was good enough for the yeast…
- SODIUM CHLORIDE (NaCl)(table salt) -> allows for osmotic balance, transport, etc.
A few of the major LB formulations are the “Miller,” “Lennox,” & “Luria” versions & they differ in the amount of salt they have. Miller & Bertani drown the bacteria in NaCl (10g/L) whereas Lennox just uses 5g/L and Luria just 0.5g/L -> such low salt recipes are good if you’re using a salt-sensitive antibiotic. in the original paper, Bertani also added glucose, but most later recipes leave it out.
And speaking of leaving things out, we need to make sure we “leave out” bacteria we don’t want, which we can do by “selecting for” the bacteria we do want using selection media, which contain things like antibiotics etc. that suppress the growth of things you don’t want to grow. For example, when we put genes into bacteria, we normally do it in the form of circular pieces of DNA called plasmids. We design those plasmids to also have an antibiotic resistance gene, so we can spike the food with that antibiotic and it can still grow, but other stuff can’t http://bit.ly/2tcW4ky
There’s also differential media – this allows for “screening” as opposed to “selection” – you don’t keep things from growing, but you change how they appear – for example, we use X-gal for blue-white screening http://bit.ly/2MxNPs2
After I put a plasmid with my gene into bacteria and get colonies, I pick a few of those colonies and put them in liquid LB (with antibiotic) to grow overnight to make lots of copies of the plasmid, then I can purify out those copies and send them for sequencing to check for typos before l enter the “expression prep” part.
Regardless of what media you use, you need it to be sterile. So you autoclave it (stick it in a really hot, high pressure dishwasher) -> make sure the bottles aren’t sealed tight or they’ll explode (thankfully I haven’t made this mistake) – and don’t re-tighten the lids until the bottles have cooled of or the lids will get stuck (I *have* made this one). Another mistake not to make -> don’t add antibiotics before autoclaving, or you’ll inactivate them. We usually don’t add it until right before we’re ready to use it. More on autoclaves: https://bit.ly/autoclavessteam
In undergrad, I made all my own media, but here, we use so much of it, we have a “media-maker” lab technician who’s amazing and makes & sterilizes our bacterial growth media. After making the LB-agar mix, she sticks it in the cold room where it happily gel-ifies. In the bottle. Then, when we want it she (or we if she’s not around or if we just want to show someone how such as today) stick it in the autoclave. With the lids NOT tight. You want a bit of water in the tray to cover the bottom of the bottles (I think to try to disperse extra heat).
Speaking of heat, you know something’s been through an autoclave if the lines on its autoclave tape are black – the tape is temperature-sensitive so it color-changes when it gets really hot. I really want to design a line of joke and/or trivia messaged autoclave tape – so if the whole teaching thing doesn’t work out, I guess I have a back up!
After the autoclave runs, the bottle’s really hot. Way too hot to pour and too hot to stick antibiotics into without them falling apart. So first you stick it in a warm water bath to cool down to the point where they’re warm enough to stay pourable but cool enough to not kill your antibiotics or melt your dishes (they’re just plastic) or burn your hands (speaking of which, be sure to wear insulated gloves when taking the bottle from the autoclave!
After an hour or so, it’s good to go. But when you take it out, the race begins. We stick it on a stir plate at 37C and add the antibiotic(s) we select for our selection. Conveniently, our media maker leaves a stir bar in the bottle so it’s already in there – and sterile.
Then it’s batter pouring time. It’s good to do this next to a flame from a Bunsen burner. And if you get bubbles in your plates that flame is handy to give them a quick zap to burst those bubbles. But don’t overflame or you’ll inactivate the antibiotic.
After pouring, you leave the plates to set. You’ll know they’re ready because you can see them change color and look denser. Then you can stick them in the plastic sleeve the dishes come with and stick them in the cold room or fridge or whatever. And when you do this you want to invert them so the gel side is up and the lid is on the bottom. This way, condensation that forms on the gel drips down onto the lid and the lid doesn’t drip on the gel.
Depending on the antibiotic, the plates should be good for a pretty long time – but keep a lookout for mold or fungi growing or cracks forming.
When you’re ready, add bacteria with the matching antibiotic resistance gene and it’s bon appetit! The bacteria will divide where they sit, so you get goopy dots called “colonies” of a bunch of bacteria that are genetically identical.
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0⠀