I want to perform MICs for staphylococci with D-Aspartic Acid (soluble in 1M NaOH 50mg/mL) and Ala-Phe (soluble in 80% acetic acid). However, when using these diluents it seems that it is the diluents that kill the staphylococci and not the reagents. How can I dilute D-Aspartic Acid and Ala-Phe in order to perform MICs?
Thank you in advance.
Virginia
Hi there,
For the D-Asp solution, neutralization of the 2 carboxylic functions should be enough to solubilize it. So what I suggest is to add NaOH pellets (one by one) or drops of concentrated NaOH until full solubilization is observed, at this point the pH should be theoritically close to neutrality. With 1M NaOH, there is an excess of NaOH towards the acidic content of the solution (50mg/mL Asp is 0.375M and therefore 0.75M in terms of carboxylic acid content). About the dipeptide, it's a bit different as there are hydrophobic side chains. Did you try 5% or 10% DMSO instead of acetic acid?
After making a stock solution in the said diluent, you can make your working solution using buffer such as PBS to minimize the effect of the diluent/s. Also, if you doubt if the killing effect is because of the diluent/s, you should confirm with treatment using NaOH or acetic acid only (same volume as what you add on your concentration).
Try dissolving and diluting Ala-Phe in 100% DMSO, which the bacteria should be able to tolerate at 2% by volume. Dilute the Ala-Phe solutions1:50 into the bacteria suspension for broth MICs.
I agree with Dominique Liger that you should be able to make a concentrated stock solution of D-Asp by titrating it with NaOH just until it reaches a neutral pH. You could also try dissolving it in 100% DMSO. However, to avoid acidifying the broth, you should probably use the monosodium salt to make the DMSO solution.
Thank you all very much for your suggestions.
Ala-Phe was not soluble in DMSO, but I dissolved it in 20mM HCl (50mg/mL concentration). I also dissolved D-Aspartic acid in 0.1M NaOH (12.5mg/mL concentration).
Thank you again for your answers.
All the best,
Virginia
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