The use of supplemented Minimal Salts media to grow pseudomonas aeruginosa in 96 - (Jul/28/2013 )

Hello

My research revolves around studying the ability of plant extracts to act as antibacterial and antibiofilm agents. I am testing this compounds against pseudomonas aeruginosa and use six different strains, PAO1, ATTC 25619, and four clinical isolates. My MIC and growth curve experiments were preformed in Mueller-Hinton 2 broth. I then used this media to preform a crystal violet attachment assay in 96 well plates. In this assay the bacteria are seeded into the plate from an overnight culture along with media(supplemented with a test compound or not) and then allowed to adhere to the plate during a 24hr incubation at 37C with 25rpm shaking. After which the plates are washed and stained with crystal violet, which is then solubilized with ethanol and the absorbance of the CV in the attached bacteria is read.The bacteria were seeded at a final concentration of 1x10^6 CFU/mL. Using the Mueller-Hinton 2 broth, my bacteria  were actually over growing and there was too much background adherence. For this reason I have switched to a Minimal salts media supplemented with thiamine and casamino acids. The following is my recipe for 1L of media:

200mL 5x M9 premade stock from sigma Aldrich (contains in g/L: 33.9 disodium phosphate, 15 monopotassium phosphate, 2.5 sodium chloride, and 5 ammonium chloride)

20mL 10% glucose

20mL 10% casamino acids

2mL 1M MgSO4

1mL 1M CaCl2

1mL 0.5% thiamine

ddH2O to 1L

My issue is that the ATTC strain will not grow in this media at all, even with the casamino acid supplement. All of my clinical isolates will grow on plates of this media with 1.5% agar, as well as broth media incubated at 37C and 200rpm, however when I plate the bacteria into the 96 well plates with only 25rpm shaking and 37C for 24hrs, I get practically no growth. Furthermore, If I assess the viability of this bacteria using a Celltiter-Blue assay dye (reading results of absorbance not fluorescence) there is practically no viability of what bacteria has grown by visual assesment grown. PAO1 still manages to grow so I have ruled out the possibility of the volume being too small or my stocks being unviable (since I plated bacteria from them). I just don't know what else I could look at or change in order to increase the growth to preform my assay properly.

I greatly appreciate any input, Thank you for your time.

Suggest you use diluted Mueller Hinton broth (10% - maybe titrate to greater %'s if necessary) .  This will also bring more practical relevance to your experiments.  Think the crystal violet assay quantifies biofilm - not just cells ( http://www.ajtmh.org/content/71/5/687.full )

Also it's ATCC - American Type Culture collection - not "ATTC."