Serial dilutions, plating several replicas, count isolated colonies then calculate amount of UFCs per volume.

isolated it in colonies ,then make serial dilution , or make gram stain . its already used for used biofilms by crystal violet staining then determined the safranine in photometer.

Honestly, what I would do is dilution plating with selective agar. Something like a mannitol-salt agar would inhibit growth of P. aeruginosa but allow quantification of S. aureus. I'm not aware of a selective agar for Pseudomonas that inhibits the growth of staphylococci, although MacConkey's is close due to the crystal violet and bile salts (not sure how inhibitory that is to staph in the real world but they sure won't be happy). An alternative/complementary strategy is to incorporate vancomycin in the media which would inhibit gram positive organisms but not Pseudomonas. Simply plating serial dilutions of your sample to both types of media and back-calculating CFU/mL in your original sample should solve your problem. Good luck!

You can use a real-time fluorescent quantitative PCR reaction with specific primers of S. aureus and P. aeruginosa.

You should streak the culture for isolated colonies on Nutrient agar, both the bacteria will be form two different types of colonies easy to distinguish, if any problem do oxidase test for the colonies, oxidase negative are staph and +ve are Pseudomonas.

Thank you so much all for your generous help. 

I wish you all the best.

Legesse