How to Make the Perfect Agar Plate Every Time
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Making agar plates, whether they contain LB, M9, blood agar, or any other growth media, is a simple procedure. But there are a few finer points that will kill your experiment, make a mess, or just cause you inconvenience if you get them wrong.
So let’s put on the record exactly how to make the perfect agar plate for those of you who are new to the world of working with bacteria.
Follow these steps, and you’ll be on your way to making agar plates that are perfect—with no lumps, bubbles, or excess moisture—every time.
8 Tips for Pouring Perfect Agar Plates Every Time
1. Use a Recipe
Make up the medium according to the recipe, then add the desired amount of agar powder (normally about 1% w/v) and stir. If you autoclave without stirring, with the agarose still floating on top of the liquid, you get an agarose cake in the medium. Interesting, but useless.
When making up the agar, only use 3/4 of the volume of the bottle. This allows space for bubbles to rise while the agar is melting in the microwave (and saves you cleaning up overflowing agar from the microwave!).
2. Autoclave
Autoclave your medium for 25 minutes. After autoclaving, you can, of course, store the medium-agar mix in a toughened glass bottle then melt it in a microwave or water bath when needed. Make sure you use toughened glass bottles, or disaster (see #2) can strike.
Remember to use heat-resistant gloves when handling hot medium straight out of the autoclave.
3. Cool It!
Cool the medium-agar mix to 55°C. For routinely consistent results, do the cooling for a couple of hours in a 55°C water bath. Agar starts to solidify at about 50°C.
Using the water bath when making agar plates means you can consistently cool the mixture to just above the solidification temperature.
Before I used a water bath, I used to just cool it in the air, but would inevitably forget about it and come back to find solidification had already started—lumpy plates are no good for spreading!
4. Supplement It
You can now add any antibiotics or supplements, and be confident that the agar is at a suitable temperature because you have cooled it in the water bath.
5. Pour the Plates
Use about 30 mL of the agar-medium mix to create each plate when using 100-mm diameter dishes. The less agar-medium mix in each plate, the more easily they will dry out.
30 mL is a good amount for long-term storage, 10–20 mL is fine if you are going to use the plates relatively soon.
For consistency when making agar plates, I’d recommend using a serological pipette. Suck up 2–3 mL more than you need to minimize blowing bubbles into the plate.
Make sure you’re careful not to contaminate the media and plates when pouring—use clean gloves (such as nitrile gloves) when handling the dishes, and don’t forget your sterile technique. Use an open flame (e.g., bunsen burner) to help keep the area sterile when preparing plates.
6. Let It Set
If there are any bubbles in the plates, briefly pass the flame over to pop them. Classic error: trying to move the plates before they’ve set and cooled completely is just asking for trouble. Just leave them alone (and maybe admire your perfect agar plates while you wait)!
7. Get Dry
Dry the plates in the laminar flow hood with the lid slightly off for 30 minutes (or in a 37°C incubator for 2–3 hours, or room temperature for 2–3 days). Drying the plate is very important for storing the plates and growing colonies on them.
If you don’t dry the plates, the moisture will evaporate and condense on the lid during storage or incubation and give you horrible wet plates. At worst, the moisture can affect the plating of your cells.
Use a timer to remind you when the 30 minutes are up as—in my experience—it is very easy to forget about your plates and come back to find your plates have turned into agar crisps/chips. Tasty.
8. Use It or Store It
Once you’ve poured your perfect agar plates, you can use them immediately or seal them for later use. You can use Parafilm, or stack them and store them upside down in the plastic bag or plastic sleeve that the plates came in for easy storage.
Store the plates in the refrigerator or cold room at 4°C. Guidelines suggest using agar plates within approximately 2 to 4 weeks.
Depending on the additives you have included, the shelf life of the prepared plates might be shorter—make sure you check this before you start so you don’t end up wasting your time (and resources) making too many plates or end up with contamination on your plates!
A quick way to label your plates is to have a color code for each antibiotic and medium type you tend to use (e.g. red for ampicillin, black for kanamycin, green for LB, blue for M9).
Stack the plates and use the appropriately colored lab marker to draw a line down the whole stack. Make sure you keep the color code to hand, though.
Now you should have no issues making agar plates that are perfect every time. If you’ve got any further ideas or additions to this protocol, please leave a comment below.
Originally published July 5, 2011. Reviewed and updated February 2021.
I am about to start a lab based project at my university where i will need to prepare my own agar. This project will span a couple of weeks and i am unsure of how often i would need to prepare new agar plates to prevent contamination
Hello!
Agar plates stored at 4°C are usually used within one or two weeks. But if your plates are not contaminated or dehydrated you can use them for longer periods.
Hello!
I had prepared agar plates last week and stored them without inoculation. I checked the plates daily to ensure zero contamination. 2 days ago, (i.e. about 5 days were over) the plates were still not contaminated. But when I checked today the plates are contaminated.
What do you suggest?
Where did you store the plates? How did you store them (e.g., wrapped in foil or in a bag or loose)? I hate to ask so many questions, but the answers will help me troubleshoot! 🙂
There are a few ways you can stack your plates to get them to cool/dry faster, although you need good sterile technique to avoid compromising your agar. E.g. for round plates you can make an ‘agar pyramid’:
Pour the first plate, and leave it to cool with the lid half-off (i.e. covering only half the plate). Pour the next plate and put it next to the first one. Now remove the lid and put it between the two plates, so it sits on top of both, covering each by about half.
There are four points of contact, so you can put another Petri dish directly on top of that lid, and so on: each plate is supported by the two underneath it. You can go for three- or four-level towers if you’re feeling brave! Convection with the (sterile) air in the flow hood will make your plates cool more quickly.
I pour a lot of square plates, and you can rotate the lids by about forty-five degrees to leave the agar exposed to air while you put the next plate on top. Again, with the caveat that your technique needs to be good – don’t stick your fingers in the agar, and so on.
I keep a small batch of sterile, pH-adjusted liquid media in the cold room, so that if I need plates in a pinch, I can just add agar and autoclave. It needs to be changed fairly regularly though!
Hope this helps,
Peter
hey nick i tried this and did not work. How much time is needed for bacteria to grow in the petri dishes with agar??????
I’m not Nick, but generally you need about 12 hours at a minimum.
Proper drying is important too. After the plates have set, I turn them over ( they are typically in a stack of 10-20 plates) and leave them on the lab bench overnight (or longer). This results in excess moisture absorbing back into the agar, not condensing on the lid. NB I mostly pour yeast agar media, which has 2% agar.