Eureka, I guess this might be an interesting post for some of you. I will mention some details about my technique about agar plates. But I will not get into much details, just a brief overview. I learned the techniques in the lab, so the following techniques are very basic microbiological methods.
About the media, I normally use Sabouraud agar. This is a media for growing yeasts and some other bacteria. The agar consists of the following components [g L-1] (when used in a concentration of 65 g of the powder to 1 Liter of water):
10 g Meat- and casein protein, 40 g glucose, 15 g agar, pH around 5.6 +/- 0.2 (according to the label). Produced by Carl Roth GmbH, Karlsruhe, Art. Nr. X932.1, Sabouraud – 4% – Glucose Agar, Ph.Eur. f.d. Mikrobiologie, Agarmedium C.
Why Sabouraud? I first tried to make my own malt agar with dried malt extract and agar but the firmness of the agar turned out to be very inconsistent. And I used Sabouraud before in the lab for culturing yeasts.
It begins by adding 26 g of the Sabouraud mixture to 400 mL of tap water. I use a 500 mL Schott bottle and a pressure cooking pot and boil the whole thing for approximately 15 minutes to sterilize the media. I then let it cool down in the pot until the pressure is back to normal, take the hot bottle out and let it cool.
When the agar is approximately 50°C (122°F), I pour about 20 mL of the media in a petri dish (I use 94 mm Greiner plastic petri dishes with vents) and let it cool down. 400 mL should be enough for 20 plates but I always have less than that. Thats all about the procedure to get a petri dish with agar ready to plate some bugs.
I plate with an inoculating loop which I heat up in a gas burner until it is red, let it cool down until the red color vanishes and stick the loop into the agar to cool it down. I then pick up a colony, some liquid or whatever I want to plate and plate it. I normally use a dilution streak technique to have single colonies later on. This makes it easier to determine the morphology of a colony and distinguish between different microorganisms (see picture below).
And I do not have a fancy flow bench. I just use a piece of glass as a base which I can clean with some alcohol. The most important part, in a flow bench as well, is to work fast. Just open the dish for a very short time for streaking and close it again. This technique works for me, the plates with molds and other contaminations are very limited.
After a few days of incubation at room temperature, the plate might look as the on the left. As already mentioned, the advantage of the dilution streak is that you get single colonies. A colony is a single spot. This is easy to observe on the picture on the left. The single colonies are the ones on the upper right corner. A typical yeast colony looks like (in my experience): Circular, white- to off-white color, even, convex, a bit glossy and a diameter of about 2 mm. About the terminology:
- Circular: Defines the form of the colony. Colonies might as well have an irregular shape.
- Even: Defines the margins of the colony. Some colonies might be wavy, filamentous etc.
- Convex: Describes the elevation of the colony. Some colonies are flat, some convex, hemispheric, raised etc.
And thats how I prepare my Sabouraud plates and use them for growing and screening for different yeasts and some bacteria.