ESTCP Project #1011, Rhizosphere
Final Report
3. Dextrose positive control: Prepare medium using recipe on media page, add 4.7 mL of
medium to each of 5 test tubes. The 10-3 dilution will be the only dilution inoculated for
Dextrose positive control medium. Cover test tubes with foil and autoclave @ 121C for
15 min.
4. Motor oil medium: Prepare medium using recipe on media page, add 4.7 mL of medium
to each of 40 test tubes. After the medium has been added to the tubes, add one drop of
30W non-detergent motor oil to each test tube. Cover test tubes with foil and autoclave
@ 121C for 15 min.
5. Vegetable oil medium: Prepare medium using recipe on media page, add 4.7 mL of
medium to each of 40 test tubes. After the medium has been added to the tubes, add one
drop of Wesson vegetable oil to each test tube. Cover test tubes with foil and autoclave
@ 121C for 15 min.
6. Sodium Benzoate medium: Prepare medium using recipe on media page, add 4.7 mL of
medium to each of 40 test tubes. Cover test tubes with foil and autoclave @ 121C for 15
min.
7. Cyclohexanol medium: Prepare media using recipe on media page, add 4.7 mL of
medium to each of 40 test tubes. After medium has been added to test tubes, add one drop
of cyclohexanol to each test tube. Cover test tubes with foil and autoclave @ 121C for
15 min.
8. Autoclave 170 test tube caps @ 121C for 15 min.
Procedure
Preparation of MPN Dilutions
See Appendix B
Inoculation of Media
1. Motor oil, Vegetable oil, Sodium Benzoate, and Cyclohexanol media: 0.5 mL of
each dilution (10-2 - 10-9) will be added respectively to each of 5 test tubes. Begin by
shaking the dilution bottle to ensure even dispersion. Then pour an appropriate
amount of the dilution into a sterile pipetter basin. Add 0.5 mL of the dilution to the
media. (It is best to add, for example, the 10-2 dilution to all of the media and then
add the 10-3 dilution and so on. It is quicker and uses fewer pipette tips). Cover tubes
with sterile caps after adding inoculant.
2. BH: 0.5 mL of the 10-2 dilution will be added to each of five test tubes. To save
space, you can put the neg. control tubes in the same test tube rack with the dextrose
samples. Cover tubes with sterile caps after adding inoculant.
3. Dextrose: 0.5 mL of the 10-3 dilution will be added to each of five test tubes. Cover
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