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How to Make the Perfect Agar Plate Every Time

gloved hands holding up an open perfect agar plate

Making agar plates, whether they contain LB, M9, or any other medium, 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 get great, or even perfect, agar plates – 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 (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.

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 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 for each plate when using a 100 mm diameter plate. 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, I’d recommend using a serological pipette. Suck up 2–3 mL more than you need to minimize blowing bubbles into the plate.

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 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 pop them in the bag that the plates came in for easy storage. Store the plates 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.

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 perfect agar plates every time. If you’ve got any further ideas or additions to this protocol, please leave a comment.

Originally published July 5, 2011. Reviewed and updated February 2021.

26 Comments

  1. David Gilmore on July 24, 2016 at 7:38 pm

    1. Agar is normally 1.5% and will wet and settle to the bottom with no problems. Swirling after autoclaving mixes efficiently.
    2. Large volumes (like a liter) take 30 minutes, but smaller volumes (200 ml) are easily sterilized in 15,
    3. Water transfers heat effectively, again depending on volume. 200 ml is cool enough to use within 30 minutes easily.
    5. As previously commented, 30 ml is a huge volume. I doubt anything less than 15 ml will cover a plate, though. 20-25 ml is standard.
    8. I have my own reasons for disliking refrigerator storage, but that’s personal preference.
    I like all the rest of the advice though.



  2. labman on May 28, 2012 at 12:16 pm

    hey,
    has anyone experience in adding antibiotics to an already poured plate?
    (e.g. if you are in a hurry)
    thanks…



    • ali250719 on June 7, 2012 at 4:26 am

      Paper discs impregnated with antibiotics are routinely used for antimicrobial susceptibility testing. The discs are placed right on top of the agar and antibiotic then flows into the surrounding agar resulting in clear zones of inhibition.

      Similarly, it is indeed possible to spread the antibiotics on the surface of the agar plates prior to adding bacteria.

      Ali



    • Jen on February 12, 2016 at 2:33 pm

      I have done this on occasion. I normally have 1000x stocks, which would mean I’d have to spread 20 uL on a 20 mL plate, so I usually will dilute the stock 1:10 and then plate 200 uL antibiotic, letting it dry before spreading cells.



  3. awiklendt on July 6, 2011 at 10:42 pm

    Once I became accustomed to the correct volume of agar required in each plate, I now pour my plates in a stack – that is, I pick up my empty stack from the bottom lid, exposing the last base, pour, replace stack, grab next lid up, pour, replace stack… etc.

    This makes pouring many plates (hundreds) at a time very easy, as moving them becomes a simple matter of slowly sliding the stack to the back of the bench to make room for more pouring close to the bunsen (i.e., in the sterile field). *Keep in mind stacked plates take longer to set.*

    Also, if you pout in a laminar flow, they set and dry faster, and you don’t need a bunsen in the flow.

    Hope that helps.



    • Eric on March 23, 2020 at 2:52 am

      Suggestions for finding used laminar flow hoods? Tryin to hold two families together and work and be daddy on a budget. Lol. But im crushin it.



  4. Senthil Gandi on July 6, 2011 at 12:55 pm

    A cooler agar bottle is also much easier to hold while pouring it out on plates, also very hot agar will deform the lids of your petri dish.

    When making up agar, only make up to 3/4 of the volume of the bottle. This allows space for rising bubbles while melting the agar in a microwave. Saves time and effort of having to clean the insides of the microwave.



    • Nick on July 6, 2011 at 2:25 pm

      Good stuff Gandi, thanks!



  5. Chen Guttman on July 5, 2011 at 1:17 pm

    Hey Nick,
    Just one comment – Laying 30ml of LB on a 100mm plate is quite a lot. In our lab we routinely pour between 10-20ml (max!) – this saves on media + you prepare much more plates/flask.
    Chen



    • Nick on July 5, 2011 at 3:16 pm

      Thanks Chen — I was meaning that 30 mL is better for longer term storage since the plates don’t dry out easily but you are right – you can get away with 10-20ml. I’ve changed the text accordingly!



      • Donald on January 19, 2018 at 9:26 pm

        30 mL is excessive. 1 month of storage at 4 C will result in degradation of ampicillin by about half. Even 20 mL is enough for plates to last for many months or possibly years, but then you start having to wonder about antibiotic degradation.



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