Since joining the Biological Oceanography Group at Lamont, I’ve been (understandably) learning a lot more about biology and classic microbiological techniques. In my previous degrees, I focused on geology/biogeochemistry and then theoretical ecology, while also pursuing research experience in environmental microbiology. In terms of studying microbes, I’d used lipid fragments, DNA, RNA, iron assays, and plenty of other tools. Yet, one basic skill that I somehow missed was pouring a petri plate. When I announced that I actually didn’t know how to do that, I was met with disbelief (and what seemed like disappointment). That feeling inspired this blog post, which will hopefully help someone else avoid the same sticky situation!
The first thing to know about petri plates is that they are essentially sterile petri dishes filled with selective media on which you can culture particular bacteria. For my work on pathogens in urban water, I make highly selective media, with different chemicals that make it easy to distinguish the colonies. For example, the salmonella media I use has ferrous citrate and sodium thiosulfate, so when hydrogen sulfide production occurs during the growth of salmonella, a black colony forms. That same media also selects for shigella, which display creamy white colonies, while general lactose fermenting bacteria turn pink (check out the picture of bacteria cultured from Piermont Pier in the Hudson River).
When you’re culturing bacteria in a petri dish, think of it like Goldilocks – you want to enough colonies that you can see them, but not so many that you can’t accurately count them. For some microbes in water samples (like the pathogens I study), you often use filtration, instead of simply spreading an aliquot of water over the plate (usually about 100µL is the right amount to cover the agar surface, but not drip).
This is because the concentration of these pathogens is lower (below a few hundred colony
forming units [cfus] per 100mL) than some other microbes, like general heterotrophs (over 10,000 cfus per 100 mL). To filter water samples, you attach a vacuum pump to a tube that’s holding up glassware, which will allow you to pump water through a filter (I use a 0.2 µm gridded filter to capture my bacteria). The amount of colonies you grow from a filter incubated in a petri dish relates directly to the amount of water you pass through the filter. Think of it like an air filter: the longer it’s there (which means the more air passing through it), the more dirt/dust it collects. Sometimes, you’ll be able to find information on the optimal amount of water to filter to grow different bacteria by searching through the literature (like by searching keywords on google scholar and then following up with relevant cited papers). Other times, bacteria quantities will be so variable, like with Enterococcus in the Hudson River, that you’ll have to do volumes of 1, 10, and 100 mL, with the goal of having 2 of the plates be countable.
Filling Your Plate
Pour Plate Method
The process of pouring petri plates is quite simple, which is why many microbiologists might assume that you know how to do it already. You create your media according to a recipe, autoclave it (to dissolve the agar and sterilize) and then place it in a water bath to slowly cool it to within the range of 45-60°C (check your recipe for the exact temperature), but to still keep it warm enough to remain a liquid. I tend to let my cool for 1-2 hours. After it is safe to handle, return to your plating area (ideally a fume hood or laminar flow hood, but you can also use a biosafety cabinet if you skip the flame sterilization). Using pre-labeled plates (suggested to put the date and media type – plates do expire!), pour your agar solution off the center and fill the dish until a portion on the opposite side is still showing (a half moon-like shape, see picture). Gently swirl your plate in a circle, spreading the agar to each edge of the plate. Once done, replace the top of the petri dish and move it to the back of your workspace to cool. Continuing filling your other petri dishes. When the agar has sufficiently solidified (depends on your recipe and size of plate, but usually within 5 minutes), invert the petri dishes, so condensation on the top of the dishes won’t drip onto the agar surface. If you make 500 mL of agar, you’ll have enough to do this method for 50, 100 x 15 mm plates.
Pipette Plate Method
If you’re making smaller plates (like 47 mm), which you’ll tend to make when you filter water for culturing (why waste good agar?), you won’t be able to simple pour the petri dishes, which is a pain. Instead, you’ll use a squeeze bulb pipette and a sterile tip (I use 1-10 mL tips). Take up about 5 mL of agar from your bottle and empty your pipette in the petri dish. Gently swirl the plate around to cover the entire plate evenly. Because agar will very quickly solidify in your pipette tip (and on the plate), thereby making it impossible to keep preparing plates, this process has to be done with care and speed. Some of my tips for success include:
- Before beginning, line the petri dishes up with the tops angled open (though still covered), so you can quickly open them and pipette in your agar.
- Only empty the pipette using the down arrow part of the bulb. The last ejection push (by the base of the bulb) should be over a paper towel or another catchment item. This last ejection will create bubble surfaces in your agar, which will complicate any later colony counting.
- Pre-label everything, so you only have to worry about making sure the agar gets to every plate quickly. This will also prevent lumpy media surfaces.
The smaller plates are much more difficult to prepare, so I would recommend that you don’t worry if things go wrong. To first learn the technique for smaller plates, I started with only 250 mL of agar, which filled 2 sleeves of 25, 47 mm petri dishes.
Spreading Your Samples
This last technique is for when you’re using larger petri dishes and dilution is adequate for attaining countable samples. When I do my dilutions, I do the full suite of 1:1, 1:10, and 1:100 (or 1:30, depending on environmental abundance), where the dilution liquid is PBS buffer or autoclaved, 0.2 um filtered water from the sample location. I recommend making the dilutions in sterile tubes of the appropriate size (for my experiments, I use 15 mL vials). To mix the sample, I tend to pipette up and down in the vial or cap the tube and shake gently. If you’re short on pipette tips (or want to generate less waste), I’d recommend first mixing and plating the most dilute sample tube and then proceeding to more concentrated samples. This is because any crossover will be minimal compared to the higher concentration samples.
Then, pipette 100uL of sample onto the surface of your plate, which should be on top of a “turntable” (left). Before using the spreader, you should dip the end in isopropyl and then flame (using a Bunsen burner or butane torch). You want to use this combination so that the spreader is sterile, but not hot enough to kill the bacteria that you’re about to plate. To test your technique, you could clean the spreader and then touch it to your gloved hand. It should be completely comfortable to touch (temperature-wise). While gently spinning the petri dish on the turntable, run the spreader over the surface of the agar to create an even layer of the culture. This will make it easier to count the colonies when they come out from incubation. It’s not a bad idea to wipe your spreader down with a Kimtech wipe afterwards, so you don’t have any agar that will get burned during sterilization (and then transferred to the next plate in line).
Some General Tips & Tricks
Now that you can handle plating a multitude of petri plates, here are a few tips that will make you even more efficient and effective!
- Keep your water bath on while you’re plating, so you can put the agar back in if you notice it starting to solidify again. Also, before you autoclave, be sure to turn your water bath on, so it has the chance to heat up.
- If you have to inoculate aliquots of agar with your sample, put your agar in tubes (I use 50 mL tubes) that you immediately put back into the water bath until you are ready to use them. This will prevent them from solidifying.
- Add your sample (and any other solution) to those 50 mL tubes and then pipet up and down or shake to fully mix them.
- Immediately plate this layer before it solidifies and creates an uneven surface for future counting.
- When adding a chemical after the autoclave process of media (agar) making, it’s often a good idea to filter sterilize the solution. Autoclaving kills all bacteria, making the media sterile; however, the additional chemicals could be carrying bacteria.
- A way to do this is to mix the chemical will MilliQ (or DI) water and taking it up in a syringe. Pass the fluid through a 0.2µm filter (which removes bacteria) into a sterile tube for storage.
- To make sure you get all of the chemical through the filter is to fill the syringe with MilliQ water and push down on the plunger until the first bead of water forms on the opposite side of the filter. Dispose of the remaining MilliQ water and proceed with sterilizing the chemical solution.
- Store your plates in a cool, dark place. I use a fridge incubator set at 4°C, though most plates suggest a range of between 2°C and 8°C.