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This may be due to the fact that cell hydrophobicity was obtained using the washed cells while demulsification tests were carried out on cell-free products of these strains.
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That is, unless you are using the washed plaster sand backfill.
An established procedure [ 90] was used for lipid extraction from the washed cells and from the cultural medium combined with the two washes.
Then, 1 mL of 1 M NaOH was used for the lysis of the washed cells.
The YPA culture was centrifuged in a Jouan CT 4 22 in a swing out rotor at 4000 rpm for 5 min at 4°C, the cell pellet was washed with 200 ml dH2O, and the washed cells were used to inoculate 400 ml of 0.3% potassium acetate sporulation medium.
The washing step was repeated one time more and the washed cells resuspended at 107 cells mL−1 in ice-cold PBE buffer.
The washing step was repeated one time more and the washed cells were resuspended at 5 × 106 cells mL−1 in ice-cold CL buffer.
The fixed cells were washed with 60% (v/v) isopropyl alcohol (Sigma-Aldrich), and the washed cells were completely dried.
Wash the cells by centrifugation again and suspend the washed cells in 20mL of PBS/BSA.
The same amount of heat inactivated bacteria as used in the co-culture with live bacteria was suspended in 5 ml of respective medium and added to the washed cells.
The membrane filters with the washed cells were transferred to 50-mL centrifuge tubes and then frozen in liquid nitrogen.
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