Whole Cell Phosphate Uptake Assay

Reference:

Purchase ³²P, phosphoric acid in HCl 2.0 mCi/ml from NEN (NEX-054).
Set up 10-25 ml overnight cultures in low phosphate media (see recipe file for Pseudomonas aeruginosa and E. coli low phosphate Hepes buffered minimal media). Harvest cells by filtration with Nalgene 0.45 m m filter units. wash cells by filtration 3X with 10 ms of Hepes buffered minimal media WITHOUT phosphate.
Resuspend washed bacteria in the same phosphate-free media to a final OD₆₀₀ = 0.2-0.3. Store on ice until needed. Cells are good for about 3 hours.
Place ³²P (2.5 m M = 1.25 m l of source) in a 10 ml diposable tube.
Warm bacterial suspension approximately 10 minutes at 37^oC before using in assay. Take one ml sample of bacterial suspension and add to the ³²P. Immediately start timing, vortex to insure good mixing. Remove 200 m l samples at the desired time intervals and filter using 0.45 nitrocellulose filters. (The old manifold filtration apparatus with filter cups from Amicon is useful for this.) Immediately wash the filter with Hepes buffered moinimal media WITH 1 mM Phosphate (from a squirt bottle for fast washing). For a good time course try 15, 30, 45, 60 seconds after mixing cells with ³²P.
Remove filters , dry and count in 5 mls of scintillation cocktail.
Duplicate experiments should be done on two different days to verify results.
Controls: 200 m l of Hepes buffered media alone to get background counts and 1.25 m l of ³²P in 1 ml buffer, no cells, to get maximum counts.