Modification of the Interaction Between Escherichia Coli and Bacteriophage in Saline Sediment

MARGARET M. ROPER AND K. C. MARSHALL Department of Agricultural Science, University of Tasmania, Hobart, Tasmania, Australia

Abstract The effects of sorption phenomena on the interaction between a parasite and its host bacterium have been investigated using an Escherichia coil-bacteriophage-saline sediment system. The sediment contained organic matter and a high proportion of clay, predominantly montmoriUonoid. Both E. coli and phage remained firmly sorbed to saline sediments or montmorillonite, but were rapidly desorbed following dilution of the electrolyte below a critical concentration. This desorption coincided with the dispersal of sediment colloids. Escherichia coli was protected from phage attack by the presence of sediment, montmorillonite, or organic matter at salinity levels both above and below this critical point for dispersal and desorption. Evidence is presented indicating that E. coli is protected from phage attack at low electrolyte concentrations by an envelope of sorbed colloidal materials around the cell, whereas at high electrolyte concentrations protection results both from the colloid envelope around the cells as well as from the sorption of cells and phages to solid particles. The protection of E. coli and possibly other fecal bacteria may result in their accumulation in saline sediments, producing a possible health hazard in estuaries and lagoons if the bacteria are desorbed following dilution as a result of heavy rainfall.

Introduction
Van Donsel and Geldreich [26] and Hendricks [7] have detected higher numbers of fecal coliforms in marine bottom sediments near sewage outfalls than in the overlying waters. They have suggested that sedimentation of the bacteria prolongs their survival. Fecal coliforms normally are rapidly eliminated from seawater by a variety of natural predators and parasites [19 - 21]. It is feasible that sorption of the fecal bacteria to particulate materials in sediments may protect the bacteria from parasites and possibly predators. Marshall [14] has emphasized the distinction between the sorption of bacteria to particles of bacterial size and larger [ 18, 23, 24] and the sorption of colloidal materials to the surface of bacterial cells [10 - 13]. The purpose of the present investigation was to demonstrate the possible involvement of both sorption mechanisms in the modification by sediments of the interaction between coliform bacteria and a natural parasite. Escherichia coli and its specific bacteriophage were used as a model host1 MICROBIAL ECOLOGY, Vol. 1, 1-13 (1974) @1974 by Springer-VerlagNew York Inc.

M.M. Roper and K.C. Marshall

parasite s y s t e m , since b a c t e r i o p h a g e s also sorb t o p a r t i c u l a t e m a t t e r w h e n t h e e l e c t r o l y t e c o n c e n t r a t i o n is suitable [4, 6, 8, 2 5 ] . This host-parasite c o m b i n a t i o n provides a c o n v e n i e n t s y s t e m for s t u d y despite t h e suggestion b y Carlucci a n d P r a m e r [3] t h a t phages are n o t a significant f a c t o r in t h e c o n t r o l o f E . coli in seawater.

Materials a n d M e t h o d s

Organisms. Escherichia coli strain M13 was obtained as culture No. 200 from the Microbiology Department, University of Melbourne, Australia. A bacteriophage capable of lysing M13 was isolated from water near a sewage outlet in Orielton Lagoon, a saline lagoon located near Hobart. Sediment Sampling. Sediment samples were obtained by means of a Peterson dredge [27] from Orielton Lagoon. This lagoon is cut off from freely circulating seawater by a causeway, although salt water occasionally enters the lagoon over two small weirs at high tide. The input of freshwater from Orielton Rivulet is intermittent and, consequently, the salinity of the lagoon water generally is equal to or greater than that of seawater except flood conditions. PartiaUy treated sewage is discharged into the lagoon and, because of the lack of any tidal scouring action, it must contribute to the sediment in this eutrophic lagoon. Analysis of Sediment. Particle-size analysis of sediment samples was performed by a modification of the hydrometer method of Buoyouces [ 1 ]. Sediment samples and the clay fraction obtained above were subjected to X-ray diffraction analysis [22]. The clayfraction was washed three times with the appropriate normal chloride salt to obtain clays homoionic to Ca and K. All samples were air-dried and then a sample of K-saturated clay was heated to 550~ prior to analysis. Bacterial and Phage Counts. Counts of E. coli were obtained on MacConkey agar (Difco) after incubation at 37~ for 18 hr, while counts of indigenous bacteria from sediments were obtained on nutrient agar (Difco) containing 2.5% NaC1 after incubation at 28~ for 5 days. A double-layer plating technique was employed for phage counts, the number of plague-forming units being determined after incubation at 28~ for 24 hr. Stepwise Desorption of Bacteria or Phage from Sediment or Montmorillonite, Samples of Orielton sediment or crude montmoritlonite (Wyoming bentonite) were made saline by suspension in an excess of seawater. The samples were centrifuged at 1935g for 10 min, and 20g (wet weight) resuspended in 9 ml of distilled water prior to the addition of 1.0 rrd of a suspension ofE. coli or phage. After standing for 10 min, the samples were centrifuged at 480g for 5 rain, the supernatant was decanted, and its volume recorded. In subsequent washings, the sediment or montmorillonite samples in the centrifuge tubes were mixed with 10 ml of distilled water, allowed to stand for 10 min, and then centrifuged as before. Numbers of E. coli and plaque-forming units, specific conductance (Ls in mho/cm), and absorbance (at 420 nm) were determined for all supernatant samples. For convenience in plotting results, conductivities were expressed as the reciprocal form (1/Ls). In most experiments, the procedure was repeated until after the dispersion of the colloidal materials in the sediments or clay. This corresponded to values of 1/Ls of 1500 to 2000.
The desorption of indigenous bacteria from fresh Orielton sediment samples was carded out by the same stepwise washing procedure.

Escherichia-phage Interaction in Sediment

Effect o f Salt Concentration on E. coli or Phage. Suspensions of E. coli or phage were added to various dilutions of seawater in distilled water chosen to cover the range of specific conductivities encountered in resorption experiments. Numbers of E. coli or phage were determined after 2 hr of exposure at room temperature to the various seawater concentrations. Effects o f Sediment, Montmorillonite, or Organic Matter on the Survival o f E. coli in the Presence and Absence o f Phage. The survival of E. coli over a range of salinites was determined in the presence and absence of different sediment components in the following treatments: (a) E. coli + spent nutrient broth, (b) E. coli + phage in spent nutrient broth, (c) as in (a) + sediment (or montmorillonite or organic matter), and (d) as in (b) + sediment (or montmorillonite or organic matter). Spent nutrient broth was used in treatments (a) and (c) to ensure that nutrient additions were the same as in the phage treatments. A colloidal montmorillonite sample was prepared by suspending the bentonite in distilled water, separating the coarse fraction by centrifugation at 12,000g for 20 rain, and finally concentrating the fine fraction by further centrifugation at 23,000g for 45 m i n [ 10]. Organic matter was extracted from Orielton sediment by the pyrophosphate method [5 ], The salinity of the organic matter was adjusted by dialysis with different dilutions of seawater, whereas the salinity of the sediment and crude montmoriUonite samples was adjusted by suspending these materials in seawater diluted to the required salinity. All treatments were incubated at 28~ for 24 hr and those containing sediment or crude montmorillonite were centrifuged at 480g for 5 min. Numbers of E. coli in these supernatants and in the controls (a and b) were determined. Numbers of E. coil sorbed onto sediment or crude montmorillonite were determined following desorption by dilution of the samples to a constant conductivity (1/Ls = 1440) regardless of the initial salinity. The numbers were adjusted for the volume of diluent employed. Samples containing organic matter or colloidal montmorillonite were diluted directly to 1/Ls = 1440 without prior centrifugation and the numbers of E. coli in the liquid were determined. Electron Microscopy. Suspensions of E. coli and phage in ammonium acetate (0.1 M) were dried on grids and negatively stained with potassium phosphotungstate (2% solution containing 0.05% bovine serum albumin). To observe the sorptive interactions between E. coli, phage, and montmorillonite, the suspensions on grids were freeze-dried as recommended by Kittrick [9]. These suspensions contained various combinations of montmorillonite (100/~g/ml), E. coli (107 ceUs/ml), and phage (108 plaque-forming units/ml). Freezing prior to drying of the suspensions prevents artefacts resulting from surface tension effects. The material on the grids was either negatively stained or shadowed with gold-palladium (60% gold - 40% palladium) prior to examination under an A.E.I.-E.M.6 microscope at 60 kV or a Hitachi HU-12 microscope at 70 kV.

Results
A n a l y s i s o f S e d i m e n t . T h e particle-size d i s t r i b u t i o n o f t h e O r i e l t o n sedim e n t s u s e d in these investigations was 3 4 . 5 % coarse sand, 4.5% silt, a n d 4 9 . 3 % clay, t h e r e m a i n d e r b e i n g organic m a t t e r . Since t h e r e was a n a b u n d a n c e o f clay p r e s e n t , t h e n a t u r e o f t h e clay m i n e r a l s was investigated f u r t h e r . X - R a y diffract i o n analysis o f t h e fresh s e d i m e n t gave n o evidence o f t h e p r e s e n c e o f clay (Fig. l a ) , p r e s u m a b l y due to t h e i n t e r s t r a t i f i c a t i o n o f organic m a t t e r b e t w e e n t h e crystal layers o f the clay. Analysis a f t e r t h e removal o f t h e organic m a t t e r b y H 2 0 2 t r e a t m e n t a n d s a t u r a t i o n o f the clay f r a c t i o n with Ca ions revealed

M.M. Roper and K.C. Marshall

(Fig. ib) that significant amounts of chlorite, vermiculite, or montmorillonoid (d = 15.2 A,) and small amounts of kaolinite (d = 7.25 A) were present [2]. Similarly, analysis of a potassium-saturated clay fraction (Fig. lc) revealed the presence of chlorite, vermiculite, or montmorillonoid (d = 14.24 A), possibly small amounts of illite (d = 10.04 A,), and a peak at 7.13 A.. Heat treatment of the potassium-saturated clay fraction caused a collapse of the crystal layers from a "d" spacing of 14.24 to 9.8 A (Fig. ld), indicating that this peak probably was a montmorillonoid clay and not chlorite (does not collapse) nor vermiculite (collapses to a lesser extent). The peak observed at 7.13 k in the potassiumsaturated clay fraction disappeared in the heated sample, an observation that is consistent with the behavior of kaolinite.

!~

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~--3.35

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~ . . . . '- s - - - " ' ~ 3.35 ~ d

! ~" 4.27 ~
~
9

~____.4.27
f

7.13 ,~

Fig. 1. X-ray diffraction patterns for: (a) air-dried sediment sample from Orielton Lagoon; (b) air-dried calcium saturated clay fraction; (c) air-dried potassium saturated clay fraction; (d) air-dried heat treated (550~ potassiumsaturated clay fraction. These clay fractions were prepared from Orielton sediment by treatment with H202 followed by dispersion with hexametaphosphate [ 1].

Stepwise Desorption of Bacteria or Phage from Sediment or Montmorillonite. Although high numbers of E. coli were added to Orielton sediment, viable counts of the organism in the supernatants of successive washing of the sediment revealed that few bacteria were released at high salinites (Fig. 2a). Absorbance readings of the successive supernatants were not continued throughout the experiment, but desorption of the bacteria coincided approximately with the dispersion of colloidal material in the sediment as the electrolyte concentra. tion was lowered. The results obtained for the desorption of E. coli from montmoriUonite (Fig. 2b) demonstrated that desorption was dependent on the attainment of a critical electrolyte concentration. The maximum number of cells were desorbed at an approximate value of 1/Ls of 450. The numbers of bacteria desorbed declined rapidly in later sediment washings, suggesting that

Escherichia-phage Interaction in Sediment

most viable bacteria were desorbed in earlier washings. As before, the increase in bacterial desorption closely followed the increase in the dispersion of the clay. The desorption of phage (Fig. 2c) from sediment showed a slightly different pattern from that observed with E. colL Moderate numbers of phage were desorbed at the highest salinities. With decreasing salinity, the number of phages desorbed decreased initially, but this was followed by a rapid increase which coincided with the dispersal of sediment colloidal material. The desorption of indigenous bacteria from a fresh sediment sample (Fig. 2d) resulted in a pattern similar to that observed for E. colL

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Fig. 2. Numbers of bacteria or bacteriophage desorbed from sediment or crude montmorillonite as a function of decreasing salinity in successive washings of the sample: (a) E. coli + sediment (input = 1.0 X 107, total recovery in all washings = 5.98 X 106); (b) E. coli + montmorillonite (input = 2.0 X 109, total recovery in all washings = 2.1 X 109); (c) phage + sediment (input = 5.0 X 107, total recovery in all washings = 1.3 X 10s); (d) indigenous bacteria from sediment. Numbers desorbed (0), and absorbance at 420 nm (zx)

M.M. Roper and K.C. Marshall

The Effect o f Salt Concentration on E. coli or Phage. No significant effect on E. coli or phage numbers after exposure for 2 hr to varying concentrations of seawater was observed (Figs. 3a and 3b). These results show that the large differences in numbers observed in the desorption experiments were not the result of salt effects on E. coli or phage.

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Z o 0 --I

"

"

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/ 0

20

30

40

50

60

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80

90

100

Percentage Seawater

Fig. 3. The effect of salt concentration on numbers of (a) E. coli and (b) phage following exposure for 2 hr.

Effects o f Sediment, Montmorillonite, or Organic Matter on the Interaction Between E. coli and Phage. Since the degree of salinity affects the desorption of E. coli and phage from the sediment materials, the effect of such materials on bacterium-phage interactions was examined over a range of different salinities. The results in Fig. 4a give the ratios of the input of E. coli to the total output of this organism from the Orielton sediment samples following dilution of all samples to a constant conductivity (1/Ls = 1440)to desorb the surviving bacteria. In the controls, a marked reduction in the recovery ofE. coli was observed in the presence of phage at all but the highest salinity level. The addition of sediment affected recovery of E. coli particularly at the higher salinity levels in the absence of phage, but the sediment apparently prevented lysis ofE. coli by phage at all salinity levels.

Escherichia-phage Interaction in Sediment

The addition of montmorillonite to cultures (Fig. 4b) showed a similar effect to that found with the Orielton sediment, except that restriction of growth of E. coli was more evident at the lower salinity levels in the presence of the clay. Phage attack of E. coli was prevented where montmorillonite was present. Organic matter extracted from Orielton sediment also protected E. coli from phage attack at all salinity levels (Fig. 4c). By comparison with the E. coli control, the organic matter stimulated growth of E. coil at all but the highest salinity level.
Effect o f Low Concentrations o f Colloidal Montmorillonite on E. coliPhage Interation. The protection of E. coli from phage attack by sediment

materials at low electrolyte concentrations in which the sediment colloids were in a dispersed state suggested that the fine colloidal materials were sorbed to the surface of the bacterial cells to form a protective envelope. This effect was tested using a very low concentration of montmorillonite (150 ~tg/ml)at two different electrolyte concentrations. The results in Table I show that most cells ofE. coli were protected from phage attack by the clay at both electrolyte levels. This result was supported by electron microscope observations of bacteriumphage-clay interactions. The phage was found to sorb to montmorillonite either

Table I
Survival o f Escherichia coli in the Presence and Absence o f Phage and~or Colloidal Montmorillonite at Two Different Electrolyte Concentrations

Treatment
E. E. E. E. E. E. E. E. coli coli coli coli coli coli coli coli

No. of 1/Ls E. coli (X 10-7) 125.3 4.0 108.2 0.015 139.9 92.6 123.8 76.5

% Survivala 3.19 0.014 66.16 61.81

Output/input b 8.89 0.28 7.67 0.001 9.92 6.57 8,78 5.43

72 72 719 + phage 719 + clay 72 + phage + clay 72 +clay 719 + phage + clay 719
+ phage

aPercent survival = (No. of E. coli recovered in phage treatment/No, recovered in control treatment) X 100. bOutput/input = total No. of E. coli recovered following dilution to achieve desorption/initial inoculum ( 14.10 X 107).

M.M. Roper and K.C. Marshall

-1

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--o

-1
o

~>
/

-2

~3

-1

-2]
L L ~ i i

200

400 1/Ls

600

800

1000

Fig. 4. The effect of different adsorbents on the survival o f E . coli in the presence or absence of phage: (a) sediment; (b) crude m o n t m o r i l l o n i t e ; (c) organic matter. E. coli (m); E. c o l i + phage fo); E. coli + adsorbent (~); and E. coil + adsorbent + phage (0). O u t p u t / i n p u t -- total n u m b e r of E. coli recovered following dilution to achieve desorption/initial inoculum ( 10 - 20 X 107).

Escherichia-phage Interaction in Sediment

Fig. 5. Bacteriophage associated with colloidal montrnorillonite particles. The preparation was negatively stained (PTA). The phage particles were orientated in two directions on the clay particles: (i) face-to-face (i.e., lying across the clay platelet) as shown by (1 ~ ); (ii) tail-to-face as shown by (2 -~ ).

in a face-to-face or a tail-to-face orientation (Fig. 5). The formation of an envelope of montmorillonite around an E. coli cell is shown in Fig. 6. The exclusion of phage from the surface of the bacterium by the clay envelop (Fig. 7) demonstrates the probable mode of protection of the bacterium.
Discussion

Biological parasitism and predation probably are the major factors responsible for the destruction of large numbers of fecal microorganisms entering aquatic environments in untreated sewage [19]. Any interference with mechanisms maintaining normal microbial balance may favor the survival of potentially pathogenic microorganisms in the aquatic ecosystem. The results presented here show that in the presence of sediment material E. coli is protected, at least on a short-term basis, from the lytic action of bacteriophage. The protection pro-

10

M.M. Roper and K.C. Marshall

Fig. 6. A shadowed preparation showing E. coli coated with colloidal montmorillonite. Note the relative absence of clay platelets away from the bacterial cell. vided to the bacteria by sediment materials from phage attack and possibly other forms of parasitism and predation may provide an explanation for the reported accumulation of fecal microorganisms in saline sediments [7, 26]. The interaction between E. coli and sediment components may be considered from two viewpoints: (a) the sorption of bacteria to larger sediment particles or aggregates at relatively high electrolyte concentrations, and (b) the sorption of colloidal clays or organic matter to the surface of bacterial cells at both high and low electrolyte concentrations. The factors involved in the sorption of a negatively charged bacterium to a negatively charged surface have been considered in detail by Marshall et al. [ 17]. A reversible attraction of a bacterium to a surface occurs when the electrolyte concentration is high enough to compress the diffuse electrical double layers at the interacting surfaces to a point where van der Waats attraction energies become effective. Conversely, on lowering the electrolyte concentration beyond a critical point the double-layer repulsion energies become more effective at a

Escherichia-phage Interaction in Sediment

I1

Fig. 7. A shadowed preparation showing E. coli coated with colloidal montmorillonite and phage particles excluded from the bacterial surface (indicated by white arrows). greater interparticle distance and result in a repulsion of the bacteria from the surface. The same mechanism is involved in the dispersion of the colloidal materials of the sediment. An irreversible sorption of bacteria to surfaces involves firm adhesion resulting from polymeric bridging between the bacteria and a surface [ 15 - 17]. Since E. coli is readily desorbed from the sediment components on reduction of the electrolyte concentration, it is unlikely that such firm adhesion is involved. The phage apparently behaves in the same manner. Evidence has been presented indicating that colloidal clays are sorbed to the surface of bacterial cells by means of electrostatic attraction [ 10- 13]. The surface of a bacterial cell is saturated by clay platelets in suspensions of about 100 pg of colloidal montmorillonite per milliliter. This envelopment of the bacterium by colloidal clay is seen in the electron micrographs presented in this paper. The Orielton sediment, montmorillonite, and organic matter all provide similar protection to E. coli from phage at all salinity levels. This suggests that the protective mechanism is a physical one and not associated with particular

12

M.M. Roper and K.C. Marshall

chemical properties of any o f the individual sediment components. At high electrolyte concentrations, protection probably results from the sorption of b o t h the host bacteria and the phages to particulate matter in the sediment, and from the formation of a colloidal envelope around the surface o f the bacterial ceils, preventing attachment o f phage to its host. At low electrolyte concentrations, the bacteria and phages are desorbed from the particulate material, but the bacteria are still protected from phage b y the envelope o f colloidal material remaining sorbed to the cell surfaces. In view o f the results obtained in this investigation, it is interesting to speculate on the possibility o f an accumulation o f viable fecal microorganisms in the sediments of Orielton Lagoon as reported in other saline sediments [7, 2 6 ] . This lagoon is subjected to occasional flooding with freshwater from the surrounding catchment area. It is feasible that under such circumstances the salinity of the sediments may be reduced to a level at which large numbers of fecal microorganisms may be desorbed from the particulate sediments, but would still be protected from phage attack by the colloidal envelope remaining around the bacterial surface.

Acknowledgments This investigation was supported by grants from the University of Tasmania and the Australian Research Grants Committee. The authors wish to thank R. J. Ford and Dr. J. A. Beattie for assistance with the X-ray diffraction analyses.

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Kittrick, J. A. 1965. Electron Microscope Techniques. In: Methods of SoilAnalysis, Part 1. C. A. Black, editor, Americal Society of Agronomy Monograph No. 9, Madison, pp. 632-652. Lehav, N. 1962. Adsorption of sodium bentonite particles on Bacillus subtilis. Plant Soil 17: 191-208. Marshall, K. C. 1968. Interaction between colloidal montmorillonite and cells of Rhizobium species with different ionogenic surfaces. Biochim. Biophys. Acta 156: 179-186. Marshall, K. C. 1969. Studies by microelectrophoretic and microscopic techniques of the sorption of illite and montmoriUonite to rhizobia. J. Gen. Microbiol. 56: 301-306. Marshall, K. C. 1969. Orientation of clay particles sorbed on bacteria possessing different ionogenic surfaces. Biochim. Biophys. Acta 193: 472-474. Marshall, K. C. 1971. Sorptive interaction between soil particles and microorganisms. In: Soil Biochemistry. A. D. McLaren and J. J. Skujins, editors. Vol. 2. Marcel Dekker Inc., New York. pp. 409-445. Marshall, K. C. 1973. Mechanisms of adhesion of marine bacteria to surfaces. Proceedings of the Third International Congress on Marine Corrosion and Fouling. United States National Bureau of Standards Special Publication (in press). Marshall, K. C., and Cruickshank, R. H. 1973. Cell surface hydrophobicity and the orientation of certain bacteria at interfaces. Arch. MikrobioL 91: 29-40. Marshall K. C., Stout, R., and Mitchell, R. 1971. Mechanism of the initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68: 338-348. Meadows P. S. 1965. Attachment of marine and freshwater bacteria to surfaces. Nature (London) 207:1108. Mitchell R. 1968. Factors affecting the decline of non-marine microorganisms in seawater. Water Res. 2: 535-543. Mitchell R. 1971. Role of predators in the reversal of imbalances in microbial ecosystems. Nature (London) 230: 257-258. Mitchell R., Yankofsky, S., and Jannasch, H. W. 1967. Lysis of Escherichia coli by marine microorganisms. Nature (Londonj 215: 891-893. Nuffield E. W. 1966. X-ray diffraction Methods. John Wiley and Sons, Inc., New York. Peele, T. C. 1936. Adsorption of Bacteria by Soils. Cornell University Agricultural Experiment Station, Memoir No. 197. Santoro, T. and Stotzky, G. 1967. Effect of electrolyte composition and pH on the particle size distribution of microorganisms and clay minerals as determined by the electrical sensing zone method. Arch. Biochem. Biophys. 122: 664669. Sproul, O. J., Warner, M., La Rochelle, L. R., and Brunner, D. R. 1969. Virus removal by adsorption in waste water treatment processes. In: Advances in Water Pollution Research. S. H. Jenkins, editor. Fourth International Conference, Prague. Pergamon Press, London. pp. 541-547. Van Donsel, D. J., and Geldreich, E. E. 1971. Relationships of salmonellae to fecal coliforms in bottom sediments. Water Res. 5: 1079-1087. Welch, P. S. 1948. Limnological Methods. McGraw-Hill, New York.

10. 11.

12.

13. 14.

15.

16. 17. 18. 19. 20. 21. 22. 23. 24.

25.

26. 27.