(isolated by electroelution from 0.7 % agarose gels as described by Maniatis et ai, 1982)
contained in 0. 1 M Tris buffer, pH 7.2, and held for 10 min at 0°C. The cell suspension was
subjected to the following heat treatment : incubation for 10 min at room temperature,
followed by 2 min at 37°C plus 10 min at room temperature. One milliliter of UBYE broth
was added to the cells and the suspension incubated at 37°C for 20 min. Following this
incubation, 2.5 ml of ESW soft agar at 47°C was added to each tube of cell suspension.
The contents were mixed and poured onto ESWA plates overlaid with phenanthrene.
Radiolabeled (
14 C) -phenanthrene metabolic experiments
Five microliters of (9 -
14 C) -phenanthrene (Amersham Corp., Arlington Heights, Illinois)
in hexane were added to 1/4 inch diameter dry sterile blank paper discs (BBL Microbiology Systems, Cockeysville, MD) in a glass petri dish, yielding a nominal concentration of
41 ppb of phenanthrene and a count of 11,000 dpm. The paper discs were allowed to stand
for 10 min for the hexane to evaporate. One 14 C -phenanthrene-impregnated disc was
introduced into a Durham tube. To the Durham tube containing the disc was added 0.3
ml of minimal salts broth. Each Durham tube was introduced into a larger vial containing
2 ml of preblend 3a80 scintillation cocktail/carbon dioxide absorbent (Research Products International, Elk Grove Village, Illinois) to drap the 14 CO
2
evolved. To each
Durham tube was added 0.1 ml of culture. Two replicate batches of vials similarly
prepared were capped and incubated at 25°C. The two triplicate batches of vials in the
experiment included : 1) uninoculated controls, 2) E. coli HB101, 3) Flavobacterium sp.
SB23 (pG05), and 4) cured derivative of strain SB23. At the end of the incubation period,
the discs were removed. One batch of three replicates was counted after 2 days and the
other batch after 7 days, using the Beckman LS 7500 microprocessor-controlled liquid
scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). Percent of total
above uninoculated control was determined using 11,000 counts as initial disintegrations
per minute (dpm) per vial, i.e,
dpm x — dpm c
% of total above control =
x 100
11,000
where the subscrips, x = test vial, and c = uninoculated control.
Hydrocarbon adherence/emulsification tests
Adherence to, and emulsification of, selected liquid hydrocarbons was investigated using
the method of Rosenberg and Rosenberg (1981) with some modifications. The hydrocarbons tested were n-octane, n-hexadecane, cyclohexylbenzene, and 1,2, 3,4, -tetrahydronaphthalene (tetralin) all of which were obtained from Chemical Samples Co., Columbus,
Ohio. Cells harvested at stationary phase (48 hr) from 50 ml of UBYE broth culture grown
at 30°C with shaking, at 120 rev/min were washed twice with PUM buffer (K 2 HOP 4 .
3H 2 O,22.2g ; KH 2 PO 4 ,7.26g ; urea, 1.8g ; MgSO 4 .7H 2 O, 0,2 g ; deionized water, 1 liter;
pH, 7.1). Washed cells were then resuspended in PUM buffer to an absorbance of 1.5 at
400 nm (Gilford spectrophotometer model 2600, Corning Glass Works, Overlin, Ohio).
To round bottom acid-washed test tubes (10 mm diameter) containing 1.5 ml of washed
cell suspension were added various volumes (0.1, 0.2, 0.3, and 0.4 ml) of the test
hydrocarbons. Other steps in the procedure were according to Rosenberg and Rosenberg
(1981).
604
contained in 0. 1 M Tris buffer, pH 7.2, and held for 10 min at 0°C. The cell suspension was
subjected to the following heat treatment : incubation for 10 min at room temperature,
followed by 2 min at 37°C plus 10 min at room temperature. One milliliter of UBYE broth
was added to the cells and the suspension incubated at 37°C for 20 min. Following this
incubation, 2.5 ml of ESW soft agar at 47°C was added to each tube of cell suspension.
The contents were mixed and poured onto ESWA plates overlaid with phenanthrene.
Radiolabeled (
14 C) -phenanthrene metabolic experiments
Five microliters of (9 -
14 C) -phenanthrene (Amersham Corp., Arlington Heights, Illinois)
in hexane were added to 1/4 inch diameter dry sterile blank paper discs (BBL Microbiology Systems, Cockeysville, MD) in a glass petri dish, yielding a nominal concentration of
41 ppb of phenanthrene and a count of 11,000 dpm. The paper discs were allowed to stand
for 10 min for the hexane to evaporate. One 14 C -phenanthrene-impregnated disc was
introduced into a Durham tube. To the Durham tube containing the disc was added 0.3
ml of minimal salts broth. Each Durham tube was introduced into a larger vial containing
2 ml of preblend 3a80 scintillation cocktail/carbon dioxide absorbent (Research Products International, Elk Grove Village, Illinois) to drap the 14 CO
2
evolved. To each
Durham tube was added 0.1 ml of culture. Two replicate batches of vials similarly
prepared were capped and incubated at 25°C. The two triplicate batches of vials in the
experiment included : 1) uninoculated controls, 2) E. coli HB101, 3) Flavobacterium sp.
SB23 (pG05), and 4) cured derivative of strain SB23. At the end of the incubation period,
the discs were removed. One batch of three replicates was counted after 2 days and the
other batch after 7 days, using the Beckman LS 7500 microprocessor-controlled liquid
scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). Percent of total
above uninoculated control was determined using 11,000 counts as initial disintegrations
per minute (dpm) per vial, i.e,
dpm x — dpm c
% of total above control =
x 100
11,000
where the subscrips, x = test vial, and c = uninoculated control.
Hydrocarbon adherence/emulsification tests
Adherence to, and emulsification of, selected liquid hydrocarbons was investigated using
the method of Rosenberg and Rosenberg (1981) with some modifications. The hydrocarbons tested were n-octane, n-hexadecane, cyclohexylbenzene, and 1,2, 3,4, -tetrahydronaphthalene (tetralin) all of which were obtained from Chemical Samples Co., Columbus,
Ohio. Cells harvested at stationary phase (48 hr) from 50 ml of UBYE broth culture grown
at 30°C with shaking, at 120 rev/min were washed twice with PUM buffer (K 2 HOP 4 .
3H 2 O,22.2g ; KH 2 PO 4 ,7.26g ; urea, 1.8g ; MgSO 4 .7H 2 O, 0,2 g ; deionized water, 1 liter;
pH, 7.1). Washed cells were then resuspended in PUM buffer to an absorbance of 1.5 at
400 nm (Gilford spectrophotometer model 2600, Corning Glass Works, Overlin, Ohio).
To round bottom acid-washed test tubes (10 mm diameter) containing 1.5 ml of washed
cell suspension were added various volumes (0.1, 0.2, 0.3, and 0.4 ml) of the test
hydrocarbons. Other steps in the procedure were according to Rosenberg and Rosenberg
(1981).
604