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How to Make the Perfect Agar Plate Every Time

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.

Follow this and you’ll get great plates – with no lumps, bubbles or excess moisture – every time.

1. 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 argarose still floating on top of the liquid, you get an agarose cake in the medium. Interesting, but useless

2. Autoclave 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 this (see #2) can happen

3. 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 waterbath, I used to just cool it in air but would inevitably forget about it and come back to find solidification had already started — lumpy plates are no good for spreading!

4. Add any antibiotics or supplements
You can 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 for each plate in a 100mm 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 mLs more than you need to minimise blowing bubbles into the plate.

6. Allow the plates to 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 the set is just asking for trouble. Just leave them alone!

7. 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 RT for 2-3 days).
Drying the plate is very important for storing the plates and growing colonies on them. If you don’t dry them, the moisture will evaporate and condense on the lid during storage or during incubation and give you horrible wet plates to work with. At worst the moisture can affect the plating of your cells. Use a timer to remind you when the 30 minutes is 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 the plates immediately or seal them. You can use Parafilm, or in the bag that the plates came in. Store the plates at 4°C
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 etc). 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 plates every time. If you’ve got any further ideas or additions to this protocol, please leave a comment.

22 Comments

  1. Sush on May 30, 2019 at 8:32 am

    What is the exact amount of agar that should be added in 100 ml of media?

  2. Catherine E Davis-Sparks on September 27, 2018 at 5:44 am

    Hi, I made NGM agar with the powder and water, added the dextrose, autoclaved it, then plated it. I found out today that I missed out on 3 ingredients. Can I save the current media by heating the plates and returning the liquid to the jar I made it in, and add the required ingredients and autoclave again? I have never made it before, and I made 1000ml of it at my professor’s request. I don’t want to waste it if I can fix my mistake.

    • bernard_muthuku on March 10, 2019 at 7:58 pm

      you can re-use it

  3. Olu on March 14, 2018 at 5:05 pm

    What happens when inoculated plates are incubated beyond the prescribed periods?

  4. CuriousUniStudent on December 4, 2017 at 12:18 am

    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

    • lucas.souza on December 26, 2017 at 4:36 pm

      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.

  5. Jayateerth S Bhavikatti on July 17, 2017 at 9:19 am

    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?

    • Dr Amanda Welch on July 21, 2017 at 2:50 pm

      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! 🙂

  6. Peter on January 31, 2017 at 6:20 pm

    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

  7. Andrea Garcia on January 16, 2017 at 2:05 pm

    hey nick i tried this and did not work. How much time is needed for bacteria to grow in the petri dishes with agar??????

    • Dr Amanda Welch on January 17, 2017 at 9:01 pm

      I’m not Nick, but generally you need about 12 hours at a minimum.

  8. Labrat on September 30, 2016 at 11:31 pm

    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.

  9. 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.

  10. 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.

  11. 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.

  12. 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!

  13. 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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