Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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MATERIALS:
Soil samples (agriculture or forest)
Two 2.0-ml Eppendorf microcentrifuge tubes
P-nitrophenyl-phosphate solution (100 mM), SIGMA= LOT 054H6178, FW = 371.1
g/l = 1M, 0.7422 g / 20 ml = 100 mM
CaCl 2 solution (0.5M)
NaOH solution (0.5M)
P-nitrophenol standard stock solution (100 µM), PNP standard: PNP FW = 139.11 g,
0.013911 g / 100 ml = 1mM solution, to make 100 ml of 1 µM, take 10 ml of 1mM
solution + 90 ml water. Use the 1 µM solution to make five standard solutions
between 0 and 10 µM (10, 5, 2.5, 1.25, and 0).
Microplate reader (vertical path length photometer with 405 nm interference filter)
Two flat bottom 300-µl wells
96-well microplate reader rack
Aluminum foil
Micropipettes and micropipette tips
Spatula, marking Pen, and tape
METHOD:
Label the microcentrifuge tubes as enzyme assay treatment and control.
Wrap each microcentrifuge with aluminum foil
Weigh out 0.25 g soil (dry mass basis).
Add 1 ml dH 2 O to the soil and swirl gently
Initiate the reaction by adding 0.25 ml of 100 mM p-nitrophenyl-phosphate to the
treatment microcentrifuge tube but NOT to the control
Place microcentrifuge tubes in rack and shake at 175 rpm for 30 min.
NOTE: The algae MPN assay should be started during this incubation time.
After the incubation period, add 0.25 ml of the p-nitrophenyl-phosphate solution to
the control.
Terminate the reaction by adding 0.25 ml of each 0.5M CaCl 2 and 0.5M NaOH to all
tubes.