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.
Wow those are some pretty steamed up plates! I wait for them to cool before putting lids on to prevent that…
You can leave them on the bench and let air dry over a few days. Agar won’t dry out because of all that moisture.
Thank you for your comment. The photo was taken right after pouring. The plates were still hot.
I already tried your technique way back and all the plates were infected with molds… I am therefore very uncomfortable letting the plates dry without the lid on. So, I let the plates dry with the lid on for some hours and pack them and off they go in the refrigerator for later use. The best way would be to let the plates dry in a flow bench. Unfortunately, I do not have a flow bench at home… If I do plates in the lab, I pour them in a flow bench and leave them there overnight to cool and dry.
Yes good. If they’re too steamed up, just letting them sit for a day or two does the trick. I think I’ll also start closing them right away because even losing a couple plates out of a batch is unpleasant.
You mentioned in your post about potato agar that you had problems with molds. I guess this had something to do with cooling without the lid. But that’s just a guess of mine. As long as the amount of contaminated plates are acceptable, keep your technique as it is.
I have enjoyed reading your posts so far, I have some micro testing experience at my current job (chemicals etc), but will be taking on a position for QC testing and “yeast management” at a brewery very soon. I will have to propagate and maintain yeast for batches, some starters as high as 33M cells per mL of wort. For micro testing etc the concepts are the same but the organisms are a little different (“good” vs “bad”) as are some of the techniques. I dont think we will have an autoclave to sterilize things in at first so I am wondering about your pressure cooker idea. You fit the pyrex bottles inside the pressure cooker with the lid on? I dont know why I am confused about this. Seems to me I remember my mom’s pressure cooker being quite small….Anyways I am sure I will have more questions to come as I read through your site and will make good use of the information!
I have to tilt the Pyrex bottles (with the lids on) to fit them into my pressure cooker. But indeed there is not much of extra space. It just happens that my pressure cooker has enough space for two 500 mL Pyrex bottles. Thanks for commenting, happy reading and good luck with your new job. Cheers, Sam
Hi, I was wondering if the Sabouraud agar would not do some selective pressure on yeast and allow it to get used only to glucose instead of to more complex sugars like maltose or even maltotriose. Regards from Mexico City
Hi, to me such an event seems very unlikely. Simply because the time the yeasts spend growing on a plate is too short to get any evolution/adaptation going. Cheers, Sam
Hi, this might be a silly question but I was wondering, is it safe to consume wild yeast specifically saccharomyces and brettanomyces after growing them on sab plates?
Hi, I don’t see any problems here. To stay on the save side, go with home-made dry malt extract agar instead. Cheers
I see you’re using this agar because you had difficulties formulating your own malt agar using DME. I find that using Malt Extract Agar (Difco), pH adjusted 5.4, works quite well and has a consistent firmness when used at the recommended 33.6 g/L. Given that Sabouraud agar doesn’t have a maltose component are you at all worried about this media’s impact on the wort utilization potential of the strains you store on it?
I initially encountered problems in formulating my own agar due to the agar agar I initially used. I now use DIY malt agar as well.
> Given that Sabouraud agar doesn’t have a maltose component are you at all worried about this media’s impact on the wort utilization potential of the strains you store on it?
Good point. I would be worried if I store my yeasts on these plates. I however use Sab only for isolating purposes and yeasts spend a rather short amount of time on these media. And I highly doubt they rapidly adapt to the non-maltose conditions withing the given time frame.
you should really use YAPD agar…
Can you elaborate on that?