Petri dishes are where much microbiological work occurs. They are used to isolate, grow, and propagate cultures of micro-organisms. They are also used to “see what’s there” when one wants to analyze which micro-organisms might be present on a surface or inside of a solution.
But, petri dishes are just glass or plastic. Hence we need something for micro-organisms to grow on within them. That something is agar.
What is agar?
Agar (or agar-agar) is a powdered substance that is used to create a nutritious media upon which micro-organisms can grow. It is a long-chain carbohydrate (polysaccharide) typically extracted from red algae. When boiled and then cooled it forms a gelatin. Because of this it is used in the culinary world and is most easily found in stores being sold as a vegan gelatin substitute.
A petri dish will almost always contain a base of solidified agar, but it’s less frequent for this media to contain only agar. Like any other organism, micro-organisms can have specific nutritional requirements in order to thrive. Therefore it is most common to use a mixed agar media, or recipe, which contains agar plus other additives. These additives can serve many purposes: enhancing growth, inhibiting growth, identification, or coloring.
With a real laboratory supplier you can simply buy these other mediums as a pre-mixed powder. We will need to create these mediums ourself, but fortunately for growing most yeast this is quite easy and cheap to do. For our purposes there is not a large advantage using expensive commercial mediums.
Materials
You will need the following items to make an agar-based petri dish, regardless of the specific media you with additives you intend on using.
- Petri dishes
- Flask (or something similar) to prepare your media in
- Alcohol lamp or bunsen burner (and something to light it)
- Agar
- Water
- Scale
- Stove or hot plate
- Aluminum foil
Preparing agar
First consider how many petri dishes you intend to pour. For a standard sized petri dish (90mm-100mm) about 25-30ml of agar media is required. You will need 2% agar powder by weight to the quantity of water. This means that if you’re using 100ml of water you will use 2g of agar powder.
It is entirely fine use regular tap water. If you’re concerned about contamination or the minerals that may be present within it you can purchase distilled water and use that instead. In either case, the water must be brought to a high temperature so that the agar can dissolve within it.
Once the water has been brought to a high temperature, slowly add your agar and stir or swirl to prevent clumping. Ensure that it is all dissolved. A stirring hot plate makes this simple task simpler, but it remains a simple task without it.
Once all of the agar has been dissolved, cover the mouth of the flask with a piece of foil to prevent environmental contamination from floating into it. Pressure cook this solution for 15 minutes at a high temperature and high pressure, with as much water surrounding the flask as possible, to sterilize it.
The pressure cooking is a bit unnecessary for our purposes. You can sanitize the media sufficiently enough by boiling it for 5 minutes. This will kill the bacteria we’re concerned about, leaving only anaerobic organisms that can survive high temperatures but won’t grow with the oxygen they’ll be exposed to. Please see the Sterilizing vs Sanitizing page for more information.
Pouring agar
Let your media cool until it’s between 48-65° C/120-140° F. The precise temperature is not so important; we just want it to be cool enough that it does not crack our glass petri dish or melt our plastic dish, but not so hot that it solidifies in our flask or becomes too thick to pour out.
This can be measured with a thermometer or laser reader directly, but a good heuristic to go by is that the flask should be almost too hot to hold (a bit uncomfortable). Imagine the temperature you wait for your tea or coffee to get to before drinking it.
It’s important to use asceptic technique when pouring agar. Your alcohol lamp or bunsen burner should be lit, and you should keep the plate as close to it as possible so that it remains within the updraft current created by the heat to preventing environmental contamination from floating into it.
- Ensure the petri dish is oriented with the lid up so that it can be quickly removed and replaced
- Remove the foil lid from the flask
- Flame the lip of the flask with the alcohol lamp to sterilize it
- Lift the lid of the petri dish as little as possible to pour the media in
- Slowly pour approximately 20-25 ml, trying to prevent air bubbles
- Quickly re-cover the lids on both the petri dish and the flask
- If any air bubbles are present, give the petri dish some small swirls to try and free them
Looking at the petri dish from the side, the top of the media should roughly align with where the bottom of the lid sits when the dish is covered. It should take about 30% of the volume of the dish.
Now, let the dish sit for about 20 minutes until the agar solidifies. This cooling process will cause condensation on the top of the lid, which could eventually drop onto the plate and displace our cultures or otherwise ruin them. Therefore, once the agar has solidified it is best to keep the plate upside-down.
It is okay to leave the lid of the dish slightly open to allow the moisture to escape as the agar solidifies so long as it’s near the burning alcohol lamp. Be mindful of the contamination you could cause with your breath!
Potato Dextrose Agar (PDA)
Potato Dextrose Agar is a classic microbiological media. It is often used for growing bacteria like Lactobacillus as well as to grow fungi, the latter of course being inclusive of yeast. This media is extremely cheap and easy to make: you only need potato and corn sugar as additives to our agar. Dextrose is a fairly generic sugar that can be found in grocery or health food stores. Here in Norway it is most easily found as druesukker. It can be substituted with regular table sugar if necessary.
First, determine how many plates you wish to pour and calculate how much water that requires. You will need 50% potato weight by water. This means if you need 100ml of media, you will need 50g of potato. The following recipe uses 100ml as an example. Note that it’s adequate to use even less potato than this, as low as 20% weight by volume of water.
- Shred 50g potato (yes, a regular chunk of russet potato)
- Simmer it in 100ml of water for 25-30 minutes
- Strain the potato shreddings out with a fine-meshed wire strainer
- Stir and dissolve 2% by weight of dextrose (in this case 2g)
- Slowly pour, stir, and dissolve 2% by weight agar, being careful to avoid clumps
- Cover the mouth of the flask with foil
- Sterilize in a pressure cooker, or bring to a boil for 5 minutes to sanitize
- Pour the media into your plates as described above