ESTCP Project #1011, Rhizosphere
Final Report
tubes with sterile caps after adding inoculant.
Reading Tubes
1. Incubate in the dark at room temperature (25C).
2. Read the MPN tubes on the following schedule:
Medium
Weeks of incubation
BH - Negative
5
Dextrose
5
Cyclohexanol
5
Benzoate
6
Motor Oil
8
Vegetable Oil
8
3. Microbial growth or a positive reading is indicated by turbidity in the tube(s).
Vortexing the tubes is helpful to discern microbial growth. When positive tubes are
determined, the MPN value can be determined using appropriate MPN tables
(Woomer, 1994).
Reference: Woomer, P.L. 1994. Most probable number counts. p. 59-79. In R.W. Weaver
(ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA
Book Ser. 5. SSSA, Madison, WI.
Clean-up Procedures
See Appendix B.
Note: The MPN determination method appears suitable for adaptation to microtiter plate
methodology. References:
Haines, J.R., B.A. Wrenn, E.L. Holder, K.L. Strohmeier, R.T. Herrington, and A.D. Venosa.
1996. Measurement of hydrocarbon-degrading microbial populations by a 96-well plate
most probable number procedure. J. Indust. Microbiol. 16:36-41.
Wrenn, B.A., and A.D. Venosa. 1996. Selective enumeration of aromatic and aliphatic
hydrocarbon degrading bacteria by a most-probable-number procedure. Can. J. Microbiol.
42:252-258.
To determine temperature of media after autoclaving, use stick on thermometers. DO NOT
1
autoclave thermometer.
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