ESTCP Project #1011, Rhizosphere
3.26 g BH medium
1.0 L deionized H2O
(one drop cyclohexanol (Aldrich#10,589-9) added to each of 40 test tube
containing 4.5 mL BH medium/tube)
Materials (for one soil sample)
test tubes (6-10mL)
test tube racks
test tube caps
sterile tips for multi-channel pipetter
sterile multi-channel pipetter basins
aluminum weigh dish
mL BH negative control medium
Dextrose positive control medium
Motor Oil medium
Vegetable Oil medium
Sodium Benzoate medium
95 mL MPP dilution bottle (10-1 dilution)
90 mL MPP dilution bottle (10-2 dilution)
45 mL MPP dilution bottles (10-3 through 10-9 dilutions)
(total of 9 MPP dilution bottles per sample)
stoppers for dilution bottles
wide tip 10-mL sterile pipette
5-mL sterile pipettes
MPN Media Preparation per Sample
1. Prepare the MPP buffer using direction from Appendix B. You will need 9 dilution
bottles and 9 stoppers per soil sample.
2. BH negative control: Prepare medium using recipe on media page, add 4.7 mL of
medium to each of 5 test tubes. To allow for volume loss during autoclaving, initial tube
volumes should be measured to 4.7 mL to achieve final volumes of 4.5 mL of medium.
The 10-2 dilution will be the only dilution inoculated for BH negative control medium.
Cover test tubes with foil and autoclave @ 121C for 15 min.