Experimental Study of Biodegradation of Ethanol and Toluene Vapors

Appendix B. Calibration Procedure and Calibration Curves
Appendix B-1. The Bacteria
Preparation Scanning-Beam Electron Microscopy (SEM)
Bacteria were cultured in benzyl alcohol medium solution (See Section 3.2.3).
Normal morphology was defined as that cell form exhibited during free exponential
growth. The organisms were harvested by centrifugation at 2,000 rpm for 5 minutes and
were washed 3 times in 5-10 ml distilled water; mixed and re-centrifuged for 5 minutes.
The organisms were then fixed in buffered 2% (vol%) glutaraldehyde (pH 7.2) for 2
hours at room temperature. The organisms were again centrifuged at 2,000 rpm for 5
minutes, and were then washed 3 times and re-suspended in 5 ml of sterile, distilled,
demineralized water. A drop of the suspension was placed on 5-mm disks of heavy-duty
aluminum foil and allowed to dry at room temperature (29.0 o C). The disks were then
attached to specimen stubs. Specimens were coated with pure gold on a rotary turntable
high-vacuum coating unit to obtain a uniform coating.
SEM
A SEM 505 Philips (Holland) in the Department of Biology, University of
Saskatchewan, was employed. Specimens were examined and photographed at
accelerating voltage of 30 kv. Figure B-1 (9,600 X) and B-2 (4,780 X) show the SEM
graph of Pseudomonas putida (ATCC23973) used in this study.
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