I would like some advice on the preparation of the media used to screen for killer enzyme activity in yeasts. I prepared yeast extract - malt extract - methylene blue agar (YM-MB agar) containing 0.3% yeast extract, 0.3 % malt extract, 0.5% peptone, 1.0% glucose, and 2.0% agar supplemented with 0.003 % methylene blue, buffered with 0.05 M citrate buffer to pH 4.2 according to Young (1987). The issue I am having is the media in the plates is soft and does not set properly, which makes spreading the yeast of interest on the plate difficult. I imagine it is the combination of the dye, low pH and possibly more agar that may be the issue. I would also like to know if the dye should be added to the media prior to, or after, autoclaving the media. Alternatively, should the dye be filter sterilized and added afterwards or can the dye be added as is without sterilizing it. I am going to try and make the changes to the pH and add more agar like I mentioned, but if anyone has dealt with this issue already and has advice on how to sort this issue out right away I would greatly appreciate it.
Do the following:
mix the components without agar and dissolve with a stirring (make 2x concentration containing 0.6% yeast extract 0.6% malt extract 1% peptone (make sure you leave out enough water volume to add the citrate and MB and glucose to a final concentration of 2x then filter); heat together with stirring after cooling add MB and citrate and glucose (all filtered or can be filtered after addition) to a final concentration of 0.006% and 0.1M and 2% respectively. for 1 L media autoclave water + agar (you might want to increase the final agar concentration for a 3 % final concentration your can add 30g of agar to 500ml of water for example). Once the agar has cooled add 500ml of your 2x mix and pour the plates. allow to set overnight in the fridge. You are right the low pH causes the plates to be soft also autoclaved YM media usually turns soft if heated too much. Be careful though about adjusting the pH some killer toxins have a pH dependent stability/activity/production and you may not see activity if you raise the pH depending on your toxin.
Hanna Alalam thank you very much for the assistance, I appreciate it. I tried adding more agar to 3% (or 30 g in 1 litre of media) but the plates are still soft. I will try and make the media as you suggested it. If I may ask, is adding the rest of the media components to the agar only once the agar has cooled imperative for the media to work? I did autoclave the agar and other media components separately but I added the media components to the agar immediately after autoclaving the two separately.
Not likely, however, I do it to make sure there is not reaction/degradation between the added components but for growth it does not matter that much unless an essential substance you are adding is very heat labile. I think incubating in the cold should help with the softness.
Hanna Alalam okay great thank you very much, I really appreciate your help with this I will give it a try then right away.
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