Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1985, p /85/ $02.00/0 Copyright 1985, American Society for Microbiology Vol. 49, No. 2 Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy S. M. KUTZ,' D. L. BENTLEY,2 AND N. A. SINCLAIR'* Department of Microbiology and Immunologyl and Electron Microscope Facility, College of Agriculture,2 University of Arizona, Tucson, Arizona Received 6 August 1984/Accepted 15 November 1984 Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide. Scanning electron microscopy (SEM) is a valuable tool for evaluating biofilm formation and biodegradation of celluloseacetate (CA) reverse-osmosis membranes used in water and advanced wastewater treatment. SEM has been used in previous studies to determine biofilm thickness and composition as well as to reveal chlorine-induced lesions in CA membranes (8, 9). In addition, SEM allows visualization of the microenvironment within reverse-osmosis units and provides information on the characteristics of bacteria present. Glycocalyx networks and bacterial stalks or appendages are indirect evidence of bacterial colonization and adherence. SEM sample preparation often involves the use of glutaraldehyde or glutaraldehyde followed by osmium tetroxide fixation (2). The material is then dehydrated in an ethanol series, critical point dried, and coated with gold or a goldpalladium alloy. CA membranes processed in this way are readily damaged by an electron beam. Destruction of the CA occurs even at low accelerating voltages (5 to 10 kv) and low magnifications (<5,000x) (8). Optimum focusing and correction for astigmatism are severely limited. Details of bacterial fine structure and the overall physical condition of the CA membrane surface cannot be discerned at high magnifications (>5,000x) because of electron beam lability. The application of additional coatings of a gold-palladium alloy to CA membranes does not alleviate beam damage. The purpose of this research was to develop a method for the fixation and preservation of CA membrane integrity for SEM analysis. Membranes fixed with glutaraldehyde and treated successively with ruthenium tetroxide, thiocarbohydrazide, osmium tetroxide, thiocarbohydrazide, and osmium tetroxide (RTOTO) were scanned at high and low magnifications at accelerating potentials of 10 to 30 kv. For comparative purposes, CA membranes were fixed with glutaraldehyde and treated with either ruthenium tetroxide or osmium tetroxide. The electron beam damage to CA membranes and attached microorganisms was assessed. Three strips (2 by 5 cm) of a low-pressure CA membrane (degree of substitution, 2.67) were used as the sole carbon source for naturally occurring well water bacteria inoculated into flasks of 0.1% Bacto-Peptone (Difco Laboratories, Detroit, Mich.). Cultures were incubated for 2 weeks at 25 C. The CA strips were then fixed overnight in 4% glutaraldehyde (Electron Microscopy Sciences, Ft. Wash- * Corresponding author. ington, Pa.) in 0.1 M Millonig buffer (5) at ph 7.2. Samples were rinsed three times for 5 minutes each time in Millonig buffer, followed by three five-minute washes in high-pressure-liquid-chromatography (HPLC)-grade water. The first CA strip was postfixed for 30 min in 2% osmium tetroxide (EMS, Ft. Washington, Pa.) and then rinsed nine times in HPLC-grade water. The second CA strip was postfixed for 30 min in 1% ruthenium tetroxide (Polysciences, St. Louis, Mo.) and rinsed nine times in HPLC-grade water. The third CA strip was treated like the second but in addition was incubated for 30 min in a saturated thiocarbohydrazide solution (Sigma Chemical Co., St. Louis, Mo.). Immediately before use, thiocarbohydrazide was suspended in water and allowed to stand for 1 h at 50 C. The liquid was decanted, and the procedure was repeated until the resulting saturated solution was a clear straw color. The solution was allowed to cool to room temperature before use (6). After being treated with thiocarbohydrazide, the CA strip was washed nine times in HPLC-grade water, incubated in 2% osmium tetroxide for 30 min, and then washed again. Treatments in the thiocarbohydrazide and osmium tetroxide solutions were repeated once (4). All three samples were dehydrated through an ethanol series (30 to 100%) and critical point dried with CO2 (1). The samples were sputter coated with 30 nm of gold-palladium alloy (target composition, 60% Au-40% Pd) with a magnetron sputtering device. Samples were observed with an International Scientific Instruments DS-130 scanning electron microscope. Caution must be used when handling osmium tetroxide and ruthenium tetroxide. Only HPLC-grade water should be used in the preparation of aqueous solutions, and glassware must be free from organic compounds to avoid rapid reduction of ruthenium tetroxide. CA membranes conventionally fixed in glutaraldehydeosmium tetroxide (Fig. 1) were labile, as evidenced by rapid electron beam damage and tearing of the membranes at 20 kv. Beam damage to samples occurred in less than 1 min at magnifications greater than 2,000x and limited the amount of detail visible. Use of a lower accelerating voltage (10 kv) improved the situation somewhat; however, moderate beam damage still occurred in less than 1 min (Fig. 2). Although postfixation with ruthenium tetroxide increased sample stability, beam damage was nevertheless encountered (Fig. 3 and 4). Photographs at magnifications near 5,000x could be attained only by rapid focusing on an adjacent area. Recoat- 446

2 VOL. 49, 1985 NOTES 447 FIG. 1. Scanning electron micrograph of a CA membrane fixed with 4% glutaraldehyde and postfixed with 2% osmium tetroxide. Accelerating voltage, 20 kv. FIG. 2. Scanning electron micrograph of a CA membrane fixed with 4% glutaraldehyde and postfixed with 2% osmium tetroxide. Accelerating voltage, 10 kv.

3 448 NOTES APPL. ENVIRON. MICROBIOL. FIG. 3. Scanning electron micrograph of a CA membrane fixed with 4% glutaraldehyde and postfixed with 1% ruthenium tetroxide. Accelerating voltage, 20 kv. FIG. 4. Scanning electron micrograph of a CA membrane fixed with 4% glutaraldehyde and postfixed with 1% ruthenium tetroxide. Accelerating voltage, 10 kv.

4 VOL. 49, 1985 NOTES 449 FIG. 5. Scanning electron micrograph of a CA membrane fixed with 4% glutaraldehyde and postfixed by the RTOTO method (see text for details). Accelerating voltage, 10 kv. FIG. 6. Scanning electron micrograph of a CA membrane showing attached microorganisms. The sample was fixed with 4% glutaraldehyde and postfixed by the RTOTO method (see text for details). Accelerating voltage, 20 kv.

5 450 NOTES ing the samples with gold-palladium did not improve the stability. In contrast, CA membranes treated with RTOTO tolerated accelerating potentials from 10 kv (Fig. 5) to 30 kv at magnifications exceeding 30,OOOx for periods of 5 min and longer with no detectable signs of beam damage (Fig. 6). As shown, microbial fine structure was preserved. As no discernable electron beam damage was produced, RTOTO samples could be examined for future reference if desired. Once damaged, areas on conventionally prepared membrane samples cannot be reevaluated. The RTOTO method markedly enhanced the stability of CA membranes. The use of osmium tetroxide for postfixation of electron microscopic samples is well documented; however, osmium tetroxide does not react chemically with most saturated and nonsaturated polymers and polysaccharides, including CA (3). Ruthenium tetroxide, on the other hand, reacts strongly with these compounds, as well as with polar lipids, glycogen, and monosaccharides (3). Rapid darkening of CA membranes as the ruthenium tetroxide is reduced during postfixation is evidence of the binding of the heavy metal ruthenium to the sample (6). However, like osmium tetroxide, ruthenium tetroxide alone cannot effectively reduce electron beam damage of CA membranes. For this reason, thiocarbohydrazide was used as a mordant or ligand for successive postfixation with a combination of ruthenium tetroxide and osmium tetroxide treatments. Its use with osmium tetroxide to produce conductive coatings is well documented (2, 4, 6, 7). However, its use with ruthenium tetroxide to stabilize otherwise electron-beam-labile material is not evident from the literature. Our results show that RTOTO postfixation markedly improved CA membrane stability and preserved microbial fine structure. LITERATURE CITED APPL. ENVIRON. MICROBIOL. 1. Anderson, T. F Techniques for preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. 13: Hayat, M. A Introduction to biological scanning electron microscopy, p University Park Press, Baltimore. 3. Hayat, M. A Fixation for electron microscopy. Academic Press, Inc., New York. 4. Kelley, R. O., A. F. Dekker, and J. G. Bluemink Thiocarbohydrazide mediated osmium binding: a technique for protecting soft biological specimens in the scanning electron microscope, p In M. A. Hayat (ed.), Principles and techniques of scanning electron microscopy, vol. 4. Van Nostrand Reinhold Co., New York. 5. Millonig, G Laboratory manual of biological electron microscopy. Mario Saviolo, Vercelli, Italy. 6. Murphy, J. A Non-coating techniques to render biological specimens conductive, p In R. P. Becker and 0. Johari (ed.), Scanning electron microscopy 1978: international review of advances in techniques and applications of the scanning electron microscope, 1978, part II. Scanning Electron Microscopy, Inc., Chicago, Ill. 7. Murphy, J. A Non-coating techniques to render biological specimens conductive/1980 update, p In 0. Johari (ed.), Scanning electron microscopy 1980, part I. Scanning Electron Microscopy, Inc., Chicago, Ill. 8. Ridgway, H. F., C. A. Justice, C. Whittaker, D. G. Argo, and B. H. Olson Biofilm fouling of RO membranes-its nature and effect on treatment of water for reuse. Am. Water Works Assoc. J. 76: Ridgway, H. F., A. Kelly, C. Justice, and B. H. Olson Microbial fouling of reverse-osmosis membranes used in advanced wastewater treatment technology: chemical, bacteriological, and ultrastructural analyses. Appl. Environ. Microbiol. 45: