I have isolated a plant protease, for which I have to perform Casein agar plate assay. I am unable to sort out the following trouble faced during the preparation of the same.
Preparation protocol that I followed:
1. 1 g of Casein in 75 ml of distilled water, adjusted the pH to 7.4 , heated and stirred untill the casein is completely dissolved.
2. 1 g of Agar to the above solution ,heated for 20 mins to dissolve it and then the final volume is made upto 100 ml.
3. Lastly, I sterilized the Casein agar solution by autoclaving.
Problem faced:
Casein and agar was completely dissolved before sterilization. Once the sterilization was over, I found precipitated substances in the flask.
What is the reason behind the precipitation? Can anyone recommend me any other best ways to prepare casein agar ?
Thanks.
3/9/22
Dear Jegadheeshwari,
I think the reason your casein agar looks the way it does is that you combined the casein with the agar, then autoclaved them together.
You can try an alternative approach. Add 2 g agar to 90 mL of your buffered solution (pH 7.4) in a 250 mL Erlenmeyer flask. Heat with stirring to melt/dissolve the agar. Cover the mouth of the flask w/ a foam plug. Also dispense 10 of skim milk into a 50 mL Erlenmeyer flask. Cover the flask mouth as above. Autoclave the agar and skim milk separately for 20 min. After that, allow the milk and agar to cool to ~55-60C in a water bath. The agar will remain molten at this temperature. Next, aseptically add the skim milk to the agar and mix by swirling the flask. When the solution is uniform, pour it into sterile petri dishes (~25 mL/dish) and allow the skim-milk agar to harden. The major protein in milk is casein, so using skim milk is easier to use rather than trying to dissolve casein powder. Using the above formula, your plates will have 2% agar, which will make the agar "stiffer", and usually easier to work with. (If you prefer, you can use 1.5% agar.) This should work for your protease assay.
Since your protease solution will probably not be sterile, after adding it to the agar, you may get microbial growth on the plate, depending on how long you incubate, and at what temperature. You can prevent this by adding an antibiotic (e.g., penicillin) to the agar after the autoclaving step and just before pouring the plates. If you don't have an antibiotic, you can also use sodium nitrite (NaN3). This is an effect antimicrobial and works at concentrations of ~0.02-0.04% (0.2-0.4 mG/mL).
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa USA [email protected]
Thank you for your brief explanation. Will definitely try this and update you @W.J. Colonna
3/12/22
Dear Jegadheeshwari,
In my original e-mail, I suggested using NaN3 as an antimicrobial. I mistakenly identified this as sodium nitrite. It is actually sodium azide. I apologize for any confusion.
Bill C.
Respected dear Colonna, much obliged towards your time and thoughts to share. Thank you!
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