You can prepare intermediate solutions to reduce the DMSO concentration to less than 1%. Some articles mention that concentrations lower than 5% have no antimicrobial effect depending on the strain used. Use a DMSO control before using the extract.

Also, the extract did not completely dissolve in any solvent other than DMSO, and when I prepare 1 % DMSO it separated into two layers

Have you tried dissolving it in water or growth medium? Some of the extract may dissolve. The only problem with that is maintaining sterility. You can pass the aqueous supernatant through a sterile membrane syringe filter to sterilize it.

Yes, I tried to dissolve it in water but It didn't dissolve the only solvent is DMSO which is completely soluble @ Adam B Shapiro

There are alternatives to DMSO, but as far as I know, all of them show antibacterial activity (DMF, Acetonitrile, Ethanol, etc.). I have observed some extracts dissolve in buffers like PBS. You can search for relative research articles for the same. Lastly, you can check for its miscibility in ionic liquids; although they have antibacterial properties, not all. Maybe plan an experiment where you can test Ionic liquids against your bacteria to see if this idea works. There could be a concentration at which your extract dissolves, yet, it is non-lethal for your bacteria.

If the purpose of your experiment is to identify antimicrobial compounds in plant extract, you can dissolve it in various solvents (e.g. water, DMSO, ethanol/methanol) and use those for your antimicrobial activity determination. Of course, you need solvent-only control to see, whether the effect you observe is due to the solvent or your extract.

This will also serve as first separation step.

You don't have to use 100% solvents either. If you use e.g. 50% organic solvent to dissolve your extract, you will reach lower concentration in your antimicrobial activity determination.

Also, how did you prepare your extract? If it was once dissolved, it should get dissolved again.

Solvent-only controls are commonly used for extract testing but are pretty lame. Synergy is not addressed and esp. for ethanol, low levels ineffective in testing are known to synergize water soluble antimicrobials.

Prob. more important - an extract that requires DMSO solution for efficacy is a useless lab experiment.

What other solvent than DMSO and Water can I use to dissolve an aqueous extract (lyophilized extract) to evaluate the antimicrobial activity?

My extract is not easy to dissolve. For example, I need a stock concentration of 500 mg/ml. I have used DMSO but I can only dissolve for 250mg/ml(thick or viscous) and in water at 125mg/ml(it is very thick or viscous). The extract is very voluminous and light that it absorbs all the liquid and if I add more solvent the concentration is reduced.

Adam B Shapiro, Gloria Arely Guillén-MeléndezAditya Trivedi

Tatiana Shiguango

Heat: Gently heating the solvent can sometimes aid in dissolving difficult substances. Be cautious not to overheat, as this can degrade your extract or solvent.

Solvent Choice: Experiment with different solvents or solvent mixtures. You've mentioned using DMSO and water, but other solvents like ethanol, methanol, or a combination of these may be more effective for your specific extract.

Mechanical Agitation: Using a magnetic stirrer or vortex mixer can help break down clumps and improve dissolution. You may need to let it stir for an extended period if your extract is voluminous.

Ultrasonication: Ultrasonication can be very effective at breaking down stubbornly insoluble materials. Ultrasonic waves create cavitation, which can disrupt particle clusters and enhance dissolution.

Chemical Aids: Depending on the nature of your extract, certain chemical aids like surfactants or co-solvents might help. However, be cautious with these additives as they can interfere with downstream applications.

I hope this helps. All the best.