Microb Ecol (1983) 9:307-315

MICROBML ECOLOGV @ 1983 Springer-Verlag

Surface Attachment of a Sediment Isolate of Enterobacter cloacae S. D. Salas and G. G. Geesey Department of Microbiology,California State University, Long Beach, California 90840, USA Abstract. Enterobacter cloacae was recovered from surface sediments of a flood control channel in an area where freshwater runoff mixed with coastal seawater. Cells o f this bacterium elaborated an extensive capsule when cultured under laboratory conditions designed to promote extracellular polysaccharide production. Colonization of glass surfaces by cells was similar under aerobic and anaerobic conditions. Temperature exerted little effect on m a x i m u m adherent cell density in the range of 15-25~ The availability o f organic nutrients also had little influence on the tendency of cells to adhere to surfaces. M axi m um adherent cell densities decreased (76%) as salinity increased from 0 to 12cY~.The results suggest that cells of E. cloacae are suitably adapted to maintain a sessile existence in brackish water sediments o f temperate coastal areas.

Introduction

It is well known that microorganisms exhibit the capacity to adhere to and colonize surfaces under both natural and laboratory conditions. Submerged surfaces in aquatic environments are rapidly colonized by a variety of bacteria, algae, and protozoa. Bottom sediments, in particular, provide an abundance o f surfaces for microbial colonization and enable bacteria to achieve densities many orders o f magnitude greater than that which exists in the overlying water [281. Although the advantages of surface attachment to microbial survival and growth in aquatic habitats has been recognized for many years [29, 35], the role o f adhesion in specific biochemical transformations has just recently been demonstrated. Nealson and Ford [30] found that manganese oxidizing activity o f a marine bacterium was enhanced when surfaces were provided for colonization. Likewise, it has been shown in several aquatic habitats that heterotrophic activities o f particle-associated bacteria are greater than those of free cells [23]. Although it is not yet known to what extent adhesion affects sediment microbial processes, the rates of many in situ biochemical transformations are likely to be influenced by the density o f the particle-associated bacteria present. Numerous studies have determined the effects o f various factors on bacterial attachment to surfaces. Surface characteristics [1, 7, 10], as well as pH and ionic strength o f the aqueous medium [27, 34], appear to be important in this

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regard. In aquatic sediments, there are additional factors which may influence bacterial attachment such as nutrient availability, oxygen concentration, and temperature. Little is known, however, of the influence these parameters have on sediment bacterial adhesion. A study was therefore undertaken to determine t h e i r e f f e c t s o n s u r f a c e a t t a c h m e n t o f a r e c e n t s e d i m e n t i s o l a t e o f Enterobacter

cloacae.

Methods

Sampling Sediment cores were collected manually with a Plexiglas coring tube and immediately transferred to the laboratory. Portions of sediment (approximately 0.2 g) were aseptically recovered from the top centimeter in the center of cores, serially diluted in d-H20, and plated on an agar medium which promoted extracellular polysaccharide production during growth of freshwater sediment bacteria based on the India ink staining method of Duguid and Wilkinson [8]. The medium consisted of a 50 m M phosphate buffer solution (pH 7.9) containing per liter sucrose, 5 g; yeast extract, 0.25 g; sodium succinate, 0.2 g; sodium citrate, 0.5 g; sodium nitrate, 0.2 g; magnesium sulfate, 0.1 g; calcium chloride, 0.03 g; ferrous sulfate, 0.1 mg; agar, 15 g.

Sampling Site Sediment cores were collected from Cerritos Channel in Long Beach, California in an area where intruding coastal seawater mixes with freshwater runoff. Preliminary studies indicated that the salinity and temperature of the water in the sampling area varied seasonally from 0 to 12%o and 15 to 22~ respectively.

Characterization of Isolated Bacteria Bacteria obtained from the most frequently encountered colonies on isolation medium were stained with India ink and examined microscopically for capsule production. An encapsulated bacterium, frequently recovered from mucoid colonies, was subsequently identified as E. cloacae using an API 20E kit.

Surface Colonization A sediment isolate of E. cloacaewas cultured in liquid broth (seed culture) at 25~ for 20 hours in a gyratory shaker at 100 rpm. The broth consisted of isolation medium without agar. When desired, anaerobic conditions were achieved by purging culture vessels with N2 before and after inoculation, placing the vessels in vented GasPak 100 jars (BBL) containing a GasPak anaerobic generator, then purging the jar with N2 prior to incubation. Colonization studies were performed in presterilized 1 liter flasks containing 300 ml liquid medium and 12 acid-washed glass cover slips (Coming # 1.5, 18 m m 2) supported in Teflon retainers. Experiments were initiated by inoculating the flasks with ceils from 20-hour cultures. Cells that formed a "collar" on the inner wall of the seed culture vessel served as the inoculum. When desired, anaerobic conditions were established by purging the flasks with N2 and incubating in GasPakjars, as described above. All flasks were agitated on a gyratory shaker (100 rpm) during incubation. At intervals, 2 cover slips were aseptically removed from the Teflon holders and dipped in sterile

Sediment Isolate of E. cloacae

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0.05 M phosphate buffer (pH 7.9) to remove any cells that were not firmly attached to the glass surface. In anaerobic incubations, the culture flask and the anaerobic jar were purged with N2 during cover slip removal and a fresh GasPak anaerobic generator added to the jar prior to its replacement on the shaker. The cover slips were subsequently processed for determination of bacterial densities, as described below.

Starvation Studies Bacterial ceils adhering to the vessel wall of an anaerobic seed culture were transferred to a mineral salts medium and stored in a 1 ml plastic syringe for 3t/2 weeks at 25~ The mineral salts medium consisted of broth medium without the organic constituents. Following incubation, the contents of the syringe were injected into nitrogen-purged flasks containing the glass cover slips submerged in complete broth medium. The flasks were incubated anaerobically at either 15~ or 25~ and cover slips removed at intervals for determination of bacterial densities. Ceils obtained from the collar of anaerobic seed cultures also were washed in nitrogen-purged, sterile mineral salts medium and inoculated into nitrogen-purged 1 liter flasks containing 300 ml mineral salts medium and 12 acid-washed glass cover slips in Teflon retainers. The flasks were incubated anaerobically at 25~ Cover slips were removed periodically and the adherent bacterial densities determined, as described below.

Bacterial E n u m e r a t i o n Washed cover slips containing adherent bacteria were transferred to 10 ml beakers containing filter-sterilized acridine orange solution (0.1 mg/ml 0.05 M phosphate buffer, pH 7.9), stained for 3 min, rinsed in sterile phosphate buffer to remove excess fluorochrome, mounted on microscope slides, and air dried. The cover slips were examined under a Zeiss epifluorescence microscope containing a filter set optimized for detection of acridine orange fluorescence [ 12]. Adherent bacterial densities were determined by enumerating fluorescent cells that appeared within an area delineated by an eyepiece gradicule. Cell densities were calculated from the mean number of ceils obtained from an examination of 20 different areas of the cover slip. Bacterial densities in the liquid phase were determined by blending 10 ml volumes in a microcontainer (Eberbach Corp.) to disperse cell aggregates, followed by serial dilution in phosphate buffer, addition of acridine orange to achieve the same final concentrations in each dilution tube as above, filtering portions of the appropriate dilution through a Nuclepore membrane (0.2 #m pore size, 25 m m diameter), mounting the membrane in immersion oil between a clean microscope slide and cover slip, and enumerating fluorescent cells, as described above.

Results Enterobacter cloacae w a s o n e o f t h e d o m i n a n t

bacteria recovered from a sediment sample when an isolation medium was used which exhibited a high carbon : nitrogen ratio (approximately 70:1) and contained sucrose as the major carbon source. Fresh isolates of this bacterium produced large capsules and formed a visible collar of growth on the inner wall of swirling culture flasks at the air-water interface. When clean glass microscope cover slips were introduced into freshly inoculated media, they became rapidly colonized by the encaps u l a t e d cells. T h e c e l l s c o l o n i z e d t h e s u r f a c e s i n a u n i f o r m m a n n e r a l t h o u g h clumps of 2-5 cells were occasionally observed. The standard deviation for counts of cells attached to 20 different areas of the same cover slip was 21%

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S . D . Salas and G. G. Geesey

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Fig. 1. Colonization of glass cover slips submerged in broth medium by E. cloacae under aerobic (11) and anaerobic (0) conditions at 25~ Suspended cell concentration under aerobic (n) and anaerobic (O) conditions was determined at the time maximum adherent cell densities were established. Vertical bars in this and subsequent figures represent +_1 SD of cell densities obtained from 2 cover slips. Fig. 2. Anaerobic colonization of glass cover slips submerged in broth medium at 15"C (ll) and 25"C ((3) by cells (a) obtained from a fresh collar population and (b) incubated for 31/2 weeks in mineral salts medium.

o f the m e a n density. T h e standard d e v i a t i o n o b t a i n e d f r o m counts o f cells attached to different c o v e r slips in the s a m e colonization flask was 7% o f the m e a n density. Microscopic e x a m i n a t i o n o f the bacteria suspended in the liquid p h a s e revealed m a n y cell aggregates which often c o n t a i n e d m o r e than 50 cells per aggregate. Surface colonization by E. cloacae was unaffected b y a variety o f e n v i r o n m e n t a l factors. A d h e s i o n to glass c o v e r slips was similar u n d e r b o t h aerobic and anaerobic conditions (Fig. 1). In the presence or absence ofO2, a m a x i m u m a d h e r e n t cell density o f a p p r o x i m a t e l y 3 X 105 c e l l s / m m ~ was achieved 14 hours after inoculation w h e n cultures were incubated at 25~ Immediately thereafter, the a d h e r e n t cell density decreased to 5 0 - 7 5 % o f the m a x i m u m value and r e m a i n e d relatively constant for at least 86 hours. T h e concentration o f cells in suspension at the t i m e the m a x i m u m a d h e r e n t cell density was achieved was not significantly different in aerobic a n d anaerobic cultures (Fig. 1).

Surface colonization o f E. cloacae at 15~ was similar to that at 25~ (Fig. 2a). D e t a c h m e n t o f cells f r o m the c o v e r slips occurred m o r e rapidly, however, following e s t a b l i s h m e n t o f m a x i m u m a d h e r e n t cell densities at 15~ t h a n at 25~ T h e culture m e d i u m also c o n t a i n e d m o r e visible c l u m p s o f cells when incubated at the f o r m e r t e m p e r a t u r e . I n c u b a t i o n o f E. cloacae for periods o f at least 31/2 weeks in m i n e r a l salts m e d i u m at 25~ h a d little effect on subsequent colonization o f c o v e r slips

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311

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Fig. 3. Anaerobic colonization o f glass cover slips submerged in broth medium (0) and mineral salts medium (11) at 25~ The cells were washed in mineral salts medium prior to their inoculation into the colonization flasks. Fig. 4. Anaerobic colonization of glass cover slips submerged in broth medium containing no NaC1 (O) and 15% NaCI (m) at 25~

submerged in growth-promoting medium at that temperature (Fig. 2b). This was also the case when cells were incubated in growth-promoting medium at 15~ following maintenance at 25~ for 31/2 weeks in mineral salts medium (Fig. 2b). The longer lag period exhibited by the culture inoculated with cells previously maintained in mineral salts medium was likely due to uncontrollable differences in the number of viable cells used in the inoculum. Colonization also occurred when the growth-promoting organic constituents were deleted from the suspending medium. Fresh, washed cells, suspended in mineral salts medium at an initial concentration of 2.7 • 107 cells/ml, achieved a maximum adherent cell density of 8.4 • 103 cells/mm 2 after 120 hours of incubation under anaerobic conditions (Fig. 3). Although the rate of colonization was slower in mineral salts medium, the ratio of attached cells to suspended cells at the time maximum adherent cell densities were established was similar to that of cultures containing quantities of organic nutrients which supported growth and capsule production (Table 1). However, adherent cells maintained in mineral salts medium were noticeably smaller (3 um 3) than those suspended in growth-promoting medium (5 #m3). Cells of E. cloacae were able to grow and adhere to the glass cover slips in saltwater media. The maximum adherent cell density, though, was considerably less in the presence of 12% NaC1 than in the absence of this salt (Fig. 4).

Discussion

Most sessile aquatic bacteria adhere to surfaces with threadlike fibers composed primarily of polysaccharides [5, 6, 13]. At the ultrastructural level, these fibers resemble those encapsulating bacterial cells which produce " s m o o t h " or "mucoid" colonies on solid laboratory culture media. Encapsulated bacterial cells

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Table 1, Ratios of attached to freely suspended cells in laboratory culture studies Suspended ceils (bacteria/ml)

Attached cells (bacteria/ mm 2)

Ratio (attached/ suspended)

Present study from data Fig. 1 Anaerobic conditions Aerobic conditions

5.6 X 108 8.4 X 108

3.4 X 105 3.0 X 105

6.0 X 10-4 3.6 X 10 4

From data Fig. 3 Growth media Salts media

1.0 X 109 3.0 X 107

3.2 • 105 8.4 • 103

3.2 X 10-4 2.8 X 10-4

Floodgatea [11]

1.0 X 104

2.3 X 103

5.0 X 10-1

MarshalP et al. [28]

1.5 X 108

4.5 x 104

3.0 x 10-4

Hendricks c [ 16] Heterotrophs Enterics Total direct counts

1.0 X 106 1.0 x 10~ 1.0 X 10s

3.2 x 103 3.0 X 103 5.3 • 105

3.2 • 10-3 3.0 X 10-s 5.3 X 10-3

Fletcher~ [9]

3.7 X 109

4.0 X 105

1.1 X 10-4

Reference

a Suspending medium was composed of artificial seawater and the surfaces for attachment consisted of glass cover slips b Suspending medium was composed of artificial seawater and the surfaces for attachment consisted of glass slides Suspending medium was composed of autoclaved river water and surfaces for attachment consisted of glass cover slips. Ratios of attached to suspended cell density were derived from values obtained after 1 day of incubation d Suspending medium was composed of sterile-filtered seawater and surfaces for attachment consisted of polystyrene Petri dishes. The ratio of attached to suspended cell density was derived from values obtained under conditions yielding the maximum adherent cell density after 2 hours incubation of cells from logarithmic phase cultures

f r o m t h e s e t y p e s o f c o l o n i e s , i n c l u d i n g t h o s e o f E. cloacae, h a v e a t e n d e n c y to a d h e r e t e n a c i o u s l y to e a c h o t h e r a n d to i n e r t s u r f a c e s [4, 22]. S i n c e s m o o t h , e n c a p s u l a t e d s t r a i n s o f E. cloacae w e r e a m o n g t h o s e b a c t e r i a r e c o v e r e d in t h e g r e a t e s t n u m b e r , it a p p e a r s t h a t t h e i s o l a t i o n t e c h n i q u e u s e d h e r e w as s u c c e s s f u l in s e l e c t i n g f o r a d h e r e n t b a c t e r i a in s e d i m e n t s . A l t h o u g h E. cloacae is c o n s i d e r e d a n e n t e r i c b a c t e r i u m o f w a r m - b l o o d e d a n i m a l s a n d a n " o p p o r t u n i s t i c " p a t h o g e n o f m a n , it is w i d e l y d i s t r i b u t e d in f r e s h w a t e r h a b i t a t s [2, 20, 24]. D e m o n s t r a t i o n o f s e r u m a n t i b o d i e s a g a i n s t E. cloacae in n a t u r a l p o p u l a t i o n s o f b o t t o m - f e e d i n g cat f i sh l e d T r o a s t [32] to suggest t h a t t h e fish c o n t a c t e d t h e b a c t e r i u m in r i v e r s e d i m e n t s . A l t h o u g h t h e purpose o f the present study was not to d e t e r m i n e the distribution and density o f E. cloacae in t h e a q u a t i c e n v i r o n m e n t , t h e r esu l t s d e m o n s t r a t e t h a t this b a c t e r i u m i n d e e d exists in s e d i m e n t s a n d t h a t its r a n g e e x t e n d s i n t o b o t t o m deposits of brackish water habitats. T h e s i m i l a r a d h e r e n t cell d e n s i t i e s o b t a i n e d u n d e r a n a e r o b i c a n d a e r o b i c conditions contradicts the general belief that reduced oxygen concentration i m p e d e s bacterial a t t a c h m e n t to surfaces and p r o m o t e s d e t a c h m e n t of previ-

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ously established adherent cell populations [ 19, 25]. In this regard, E. cloacae may represent the adherent bacterial population responsible for the continued biological activity in the bottom layers of fixed-film reactors following the development of anaerobic conditions [19]. Though there is presently no information relating natural densities of these or other adherent bacteria to oxygen concentration in the environment, the data indicate that bacteria such as E. cloacae will continue to colonize and remain associated with surfaces as conditions become anaerobic, provided there are sufficient surface binding sites available. In this regard, a recent study revealed a significant negative correlation between coliforms and dissolved oxygen concentration in very eutrophic northern brackish waters [18]. The ratios of attached to suspended cells at the time maximum adherent cell densities were established in cultures of E. cloacae were similar to those calculated from data obtained from several planktonic bacterial isolates (Table 1). The broad range of ratios likely reflected differences in experimental design. Surface orientation, vessel configuration, and solution agitation rate all influence the ratio of attached to freely suspended cells. The fact that the ratios obtained for E. cloacae were within the range of values calculated from the other studies suggests that the capacity of this sediment bacterial isolate to irreversibly sorb to surfaces is not substantially different from that of planktonic bacteria. Zobell [35] initially suggested that low nutrient conditions stimulated attachment of marine bacteria to surfaces. Since then, several studies have demonstrated that carbon limitation promotes attachment of various planktonic bacteria to surfaces [3, 26, 27]. In the present investigation, the similar ratios of attached to freely suspended cells in both mineral salts and growth-promoting media indicate that the concentration of organic nutrients has little influence on the tendency of E. cloacae to attach to surfaces. Organic nutrient limitation is not as prevalent in bottom sediments as in overlying water [14, 15, 17]. Thus, the nutrient-responsive attachment that appears to promote the survival of planktonic bacteria may not be an important selective pressure for those bacteria residing in sediments. The temperature response of E. cloacae is consistent with the results of Fletcher [9] which indicated that low temperatures exert little influence on maximum adherent cell densities. Reduction of the incubation temperature from 25~ to 15~ in the present study did result in increased clumping of cells in broth cultures, however. This phenomenon has been observed in cultures of Corynebacterium xerosis [31]. Why these temperature-induced changes in cell surface properties did not affect adherent cell densities on the submerged glass surfaces remains to be determined. The reduced maximum adherent cell density in the presence of 12~ NaCl compared with that observed in the absence of this salt is consistent with other studies indicating that adsorption efficiency is greater in water of lower salinity [21]. However, Wardell et al. [33] found that although cell densities decreased with increased NaC1 concentration, Enterobacter spp. remained the dominant adherent bacteria in river water enrichments in which the salinites ranged from 0-13%0. The response of E. cloacae to oxygen and organic nutrient availability, sa-

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linity, and temperature suggests that cells of this bacterium are well adapted to colonize bottom sediments in areas where the overlying water varies from brackish to freshwater. Acknowledgment. This study was supported by the Space Technology and Research Program, California State University, Long Beach. It is contribution no. 32 of the Southern California Ocean Studies Consortium.

References 1. Baler RE, Shafrin EG, Zisman WA (1968) Adhesion--mechanisms that assist or impede it. Science 162:1360-1368 2. Bergey DH (1957) Bergey's manual of determinative microbiology. 7th ed, Williams & Wilkins, Baltimore 3. Brown CM, Ellwood DC, Hunter J R (1977) Growth of bacteria at surfaces. Influence of nutrient limitation. FEMS Microbiol Lett 1:163-166 4. Costerton JW, Geesey G G (1979a) Which populations should we enumerate? In: Colwell RR, Costerton JW (eds) Native aquatic bacteria: enumeration, activity, and ecology. ASTM Press, Philadelphia, pp 7-18 5. Costerton JW, Geesey G G (1979b) Microbial contamination of surfaces. In: Mittal K (ed) Surface contamination. Plenum Press, New York, pp 117-127 6. Costerton JW, Irwin RT, Cheng K-J (1981) The bacterial glycocalyx in nature and disease. Ann Rev Microbiol 35:299-324 7. Dexter SC, Sullivan JD, Williams J, Watson SW (1975) Influence of substrate wettability on the attachment of marine bacteria to various surfaces. Appl Microbiol 30:298-308 8. Duguid JP, Wilkinson JF (1953) The influence of cultural conditions on polysaccharide production by Aerobacter aerogenes. J Gen Microbiol 9:174-189 9. Fletcher M (1977) The effects of culture concentration and age, time and temperature on bacterial attachment to 9olystyrene. Can J Microbiol 23:1-6 10. Fletcher M, Loeb GI (1979) Influence of substratum characteristics on the attachment of a marine pseudomonad to solid surfaces. Appl Environ Microbiol 37:67-72 11. Floodgate G D (1965) Factors affecting the settlement of a marine bacterium. Veroeff Inst Meeresforsch Bremerhaven 2:265-270 12. Geesey GG, Mutch R, Costerton JW, Green RB (1978) Sessile bacteria: an important component of the microbial population in small mountain streams. Limnol Oceanogr 23:12141223 13. Geesey GG, Richardson WT, Yeomans HG, Irvin RT, Costerton JW (1977) Microscopic examination of natural sessile bacterial populations in an alpine stream. Can J Microbiol 23: 1733-1736 14. Gerba CP, McLeod JS (l 976) Effect of sediment on the survival o f Escherichia coli in marine waters. Appl Environ Microbiol 32:114-120 15. Hendricks CW (1971) Enteric bacterial metabolism of stream sediment eluates. Can J Microbiol 17:551-556 16. Hendricks CW (1974) Sorption of heterotrophic and enteric bacteria to glass surfaces in the continuous culture of river water. Appl Microbiol 28:572-578 17. Hendricks CW, Morrison SM (1967) Multiplication and growth of selected enteric bacteria in clear mountain stream water. Water Res 1:567-576 18. H i m J, Viljamaa H, Raevuori M (1980) The effect ofphysiochemical, phytoplankton, and seasonal factors on faecal indicator bacteria in northern brackish water. Water Res 14: 279-285 19. Hoehn RC, Ray AD (1973) Effects of thickness on bacterial film. J Water Poll Contr Fed 45:2302-2320

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20. Hofie M G (1979) Effects of sudden temperature shifts on pure cultures of 4 strains of freshwater bacteria. Microb Ecol 5:17-26 21. Kaneko T, Colwell R R (1975) Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Appl Microbiol 29:269-274 22. Kauffman F (1966) The bacteriology of Enterobacteriaceae. Williams & Wilkins, Baltimore 23. Kirchman D, Mitchell R (1982) Contribution of particle-bound bacteria to total microheterotrophic activity in five ponds and two marshes. Appl Environ Microbiol 43:200-209 24. Kisilev BS, Dusmyhamedov NS, Golubeva YV, Tashpulatov RU, Bondarensko VM (1978) Isolation of bacteria of the Klebsietla genus in diarrhea in polar researchers. Zh Mikrobiol Epidemiol Immunobiol 12:49-52 25. Mack WN, Mack IP, Ackerson AO (1975) Microbial film development in a trickling filter. Microb Ecol 2:215-226 26. Marshall KC (1980) Bacterial adhesion in natural environments. In: Berkeley RCW (ed) Microbial adhesion to surfaces. Ellis Horwood Ltd, Chichester, Great Britain, pp 187-196 27. Marshall KC, Stout R, Mitchell R (1971) Mechanism o f the initial events in the sorption o f marine bacteria to surfaces. J Gen Microbiol 68:337-348 28. Meyer-Reil LA, Dawson R, Liebezeit G, Tiedge H (1978) Fluctuations and interactions of bacterial activity in sandy beach sediments and overlying water. Mar Biol 48:161-171 29. Munison RJ, Bridges BA (1964) "'Take-over"--an unusual selection process in steady-state cultures of Escherichia colt. J Gen Microbiol 37:411-418 30. Nealson KH, Ford J (1980) Surface enhancement of bacterial manganese oxidation: implications for aquatic environments. Geomicrobiol J 2:21-37 31. Stanley SO, Rose AH (1967) On the clumping of Corynebacterium xerosis as affected by temperature. J Gen Microbiol 48:9-23 32. Troast JL (1975) Antibodies against enteric bacteria in brown bullhead catfish (lctalurus nebutosus, LeSueur) inhabiting contaminated waters. Appl Microbiol 30:189-192 33. WardelI JN, Brown CM, Ellwood DC (1980) The use of continuous culture to study bacterial attachment. In: Berkeley RCW (ed) Microbial adhesion to surfaces. Ellis Horwood Ltd, Chichester, Great Britain, pp 221-230 34. Weiss L (1970) Adhesion in biological systems. Academic Press, New York 35. Zobell CE (1943) The effect of solid surfaces upon bacterial activity. J Bacteriol 46:39-56