WIHHIHIIHHIWII|IIIWHIIWIIllNHHHHHleI meets i Q .1 '- — View.“ ‘1’ ‘ J " 245» l ‘ " .' 3" 47? fl? :5“ «a ~ \ I.“ ‘4." .115 a ‘3. t .. . = v a 5 ~23 l a, _ ,.., v ._ ’59 :rj .1 l 4 o ‘L‘ ’ This is to certify that the dissertation entitled THE EFFECT OF SOIL MOISTURE ON AMOEBA POPULATION presented by NAKISAH MAT AMIN has been accepted towards fulfillment of the requirements for Mas ter' S degree in Zoology Wwflé Major professor Date 12/22/83 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 )V1531_J RETURNING MATERIALS: Piace in book drop to LJBRARJES remove this checkout from —_ your record. FINES will be charged if book is returned after the date stamped beiow. . . ,, ..' Jr I‘ ' ‘ f. 4 'I -. '-. I" ." ‘ .- I JJ 1 u up ' ' ‘ r , .5 s ‘ ‘. 4, x r . E I 3 "o 1‘ . .,~_ - ‘. . . ' , , P- : . . ~- ‘0‘ » ~ ~- ' r-I THE EFFECT OF SOIL MOISTURE ON AMOEBA POPULATION by Nakisah Mat Amin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1984 ABSTRACT THE EFFECT OF SOIL MOISTURE ON AMOEBA POPULATION by Nakisah Mat Amin A pressure membrane extractor was used to measure the relationship between soil moisture and soil suction for a sandy loam soil. The lower and upper limit of available water for this soil was 25.4696 and 3.79%. Diameters of soil pores which were emptied at corresponding suction levels were calculated. Soil pores larger than 7.5 u m effectively maintained the most water which was required for amoeba growth. A peak population density of amoebae in soil was achieved at soil moisture of 3096. Soil moisture levels higher or lower than 3096 negatively affected the growth of amoebae. A comparison of the species composition of amoeba populations at soil mositures of 1096 and #096 revealed no difference at these two moisture levels; however, the total number of amoebae was higher at a soil moisture of 4096 (p 0.01). Acanthamoeba polyphaga was the most abundant of the various species identified. 1;. c_oli was used to feed soil amoebae and the survival of this bacterium in soil showed that its numbers decreased over time. The factors which lead to this decrease, including predation by amoebae in soil, are discussed. DE DICATIO N To my parents ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation and thanks to Dr. Neal R. Band, my major advisor, for his patience and valued guidance during the course of this study. My thanks are also due to Dr. Richard J. Snider and Dr. Michael J. Klug for their constructive comments and helpful suggestions in preparing and writing this thesis. I also wish to express my appreciation to Mr. Allen K. Sample, Dr. Sterling Thompson, and Nouri for giving me assistance in doing my research. I would like to thank Kay for editing this thesis and Barbara for her typing. To my close friends, I also acknowledge their help and support while finishing my study at this university. Finally, a special thank you is extended to Abdullah for his encouragement and help which sustained me throughout my preparing this thesis. iii TABLE OF CONTENTS List of Tables List of Figures INTRODUCTION LITERATURE REVIEW Occurrence of Amoebae in Soil Soil Water Effects of Soil Properties on Amoebae Escherichia coli MATERIALS AND METHODS Soil Sampling and Treatments Determination of Soil Moisture at Various Suction Levels Enumeration of Amoebae in Soil Soil Dilution Series Amoeba Counting Species Identification Experiments with Escherichia coli Culture of Bacteria Cell Counting Biochemical Tests RESULTS Soil Moisture Characteristic Curve Effects of Soil Moisture on Amoeba Populations Amoeba Species at 1096 and 40% of Soil Moistures Survival of Escherichia coli in Soil DISCUSSION Soil Moisture Characteristic Curve Effects of Soil Moisture on Amoeba Population Amoeba Species at 1096 and 40% of Soil Moisture Survival of Escherichia CL“. in Soil Appendices Literature Cited iv vi — \Iauw 10 ll 11 12 12 13 M 15 15 15 l7 l7 I9 20 20 22 22 2Q 28 3O #5 LIST OF TABLES Soil Moisture Characteristic Curve for Sandy Loam Soil Effect of Soil Moisture on Amoeba POpulation in Natural Soil Amoeba Species at 1096 and 4096 of Soil Moistures Survival of E. gall in Natural Soil Biochemical Tests of Escherichia coli 18 26 27 28 31 Ia lb 2a 2b LIST OF FIGURES Soil moisture characteristic curve for sandy loam soil Soil moisture characteristic curve in relation to diameter of soil pores Effect of soil moisture on amoeba population Log plot of amoeba papulation at various soil moisture levels Survival of E. coli in natural soil vi I9 21 23 2Q 30 IN TROD UCTION Naked amoebae are the most abundant among soil protozoa (Cutler, I923). The genera Hartmannella Alexeieff, Naegleria Alexeieff, Vahlkampfia Chatton and Lalung-Bonnaire and Acanthamoeba Volkonsky are common naked soil amoeba (Crump, I950; Geltzer, I963). The cells of these amoebae are highly flexible, making it easy for them to move around in the soil microenvironment (Sleigh, I973). Tropic forms consume other microorganisms such as bacteria and yeasts, demonstrating that their presence in soil is important as micropredators (Nikoljuk, I969; Sleigh, 1973; Clarholm, 1981). To grow soil amoebae and other protozoa in a culture medium (not axenic culture), edible bacteria such as Escherichia coli (Migula) are used as food (Singh, 1946). There is a close relationship between soil properties and amoeba life. Soil moisture is the most important factor for the growth of amoebae in certain soils (Stout 6c Heal, 1967). Water is held by adhesive and cohesive forces to soil particles and between water molecules, and it completely occupies soil pores when saturated (Foth, 1978). External forces are required to drain water from soil pores (Childs, 1940). Although measurement of soil moisture does not indicate potential energy of soil water, it gives an estimate of the air-filled pores (Griffin, 1963). A soil moisture characteristic curve is used to estimate soil pore sizes (Childs, 191:0). Objectives of the present study were to (a) evaluate the relationship between habitable soil pores and the amoeba population at various soil moisture levels; (b) determine relative species composition of amoebae at soil moistures of 1096 and I40%; and (c) since no previous studies have reported Exili as food for amoebae in soil, another objective was to measure the survival of E.coli in soil. LITERATURE REVIEW Occurrence of Amoebae in Soil The protozoan fauna are similar in all soils (Dixon, 1936), and their numbers are roughly related to bacterial numbers (Sandon, 1928). Of these soil protozoa, naked amoebae are the most abundant (Cutler, 1923). Their numbers increase in cultivated soil and the amoebae cause a 60% decrease in bacteria added to soil (Clarholm, 1981). The accumulation of protozoa in a rhizosphere can be traced to greater activity and multiplication of bacteria, which can serve as food for amoebae in that region (Linford, 191:2). In soil, most of the protozoa present are heterotrophic. The trophic forms of amoebae ingest other soil microorganisms such as bacteria or yeasts. Acanthamoeba castellanii (Douglas, 1930) consumes both unicellular and filamentous forms of cyanobacteria (Wright et al., 1981), and it has surface binding sites for flagella of seveal bacteria (Preston at King, 1982). Various authors have reported on the selectivity of soil protozoa in consuming different bacteria (Singh, 191:6; Nikoljuk, 1969). Although naked amoebae present in soil are free-living and feed on other soil microorganisms, a few of them are potentially pathogenic to laboratory mice (Culbertson, 1971). Naked amoebae commonly found in soil include the genera Hartmannella Alexeieff, Naegleria Alexeieff, Vahlkampfia Chatton and Lalung-Bonnaire and Acanthamoeba Volkonsky (Crump, 1950; Geltzer, 1963). Fellers and Allison (1920) conducted a quantitative survey of protozoa in New Jersey soil and found Naegleria gruberi (Schardinger, 1899) to be the most abundant soil amoeba. The latter was subsequently re-identified by Sandon (1928) as Hartmannella halLina (Dangeard). In New Brunswick soil, Sandon (1928) found that the predominant species of amoebae was very similar to Hartmannella M. Dixon (1936) discovered 25 species of Rhizopoda in Russian soil, of which the three most common were Hartmannella hyalina, m s2 5 (Sandon, 1928), and Amoeba diplodea Hartmann and Nagler. Rhizopoda testacea were poorly represented. Although numbers and species of protozoa did not show a significant relationship with soil depth, more species and numbers of individuals were found in the top layer (Dixon, 1936). One factor found important to the occurrence of varied species of amoebae in soil was the number of bacteria present as food supply (Sandon, 1927). Trophic forms of amoebae are frequently present in soil (Fellers & Allison, 1920); in its microenvironment, such organisms live on soil particles (Russel 6: Appleyard, 1915) and move about to graze on bacteria (Sleigh, 1973). When the soil microenvironment is inhospitable, amoebae will be encysting (Stout, 1973). Encystation can be induced by moistening the cysts or stimulation with compounds excreted by living bacteria (Crump, 1950; Drozanski, 1963). Soil Water Soil texture refers to the relative size of soil particles and aggregation of these particles into groups is called soil structure (Buckman 6: Brady, 1969). The United States Department of Agriculture categorizes soil particles as very coarse sand, coarse sand, medium sand, fine sand, very fine sand, silt, and clay. The proportion of all these particles defines a textural soil class by using ”The Soil Textural Triangle" (Foth, 1978). Since soil particles are not spheres, their arrangement or aggregation forms pore spaces of complex interconnected shapes (Collis-George, 1959). The number and nature of pore spaces vary in an individual soil and are determined by size, shape, and arrangement of soil particles (Darbyshire, 1975). These pore spaces are either filled with gases or various concentrations of gasses and water (Foth, 1978). To remove water from these pore spaces, the water-air interface has to be able to pass through the pore necks; this can be done only if the force applied is greater than the equivalent surface tension force (Collis-George, 1959). In unsaturated soil, water is held to soil particles by adhesive forces and between water molecules by cohesive forces, which leaves some space for air- filled pores (F oth, 1978). At saturation, all of the pores are filled with water and there is a continuity or conductivity of water flow in soil. If it is not saturated, some of the pores become air-filled and water conductivity will decrease. When pressure (or suction) is applied to soil, the largest pores, which are most conductive according to Poiseuille's law (the rate of water flow is proportional to the fourth power of the pore radius), will be emptied first so the water only flows in smaller pores (Hillel, 1980). Soil moisture content (or soil moisture) is a term used to express the ratio of water to soil, per mass, based on dry soil. Generally, this ratio is multiplied by 100 to express it in terms of percentage (Buckman 6: Brady, 1969). Soil water has two forms of energy, kinetic and potential. The movement of water in soil is very slow so that its kinetic energy is not significant compared with its potential energy, which becomes the major factor in determining the state and movement of water in soil. In the field, soil water is subjected to many forces. These forces may be combined to derive the total potential of soil water as is explained below. Pt = $9 + ¢p + ¢o where 45 = total potential 1: Pg = gravitational potential ¢p = pressure or matric potential ¢O = osmotic potential The ellipses indicate the possible additional terms which can be included in this equation. Among various potentials, pressure or matric potential describes the tenacity of soil water to soil particles (Hillel, 1980). Measurements of soil moisture do not express the potential energy of soil water, but give estimates of air-filled pores (Griffin, 1963). The relationship between soil moisture and corresponding soil suction is graphically expressed by a soil moisture characteristic curve (Childs, 19140) which is strongly affected by soil texture and structure (Hillel, 1980). Soil water is available for plants in the range between field capacity (FC) and permanent wilting point (PWP). PC is the upper and PWP the lower limit of available water in soil (Buckman 6t Brady, I969). Available-water capacity (AWC) depends on texture and structure of soil; it correlates negatively with the percentage of coarse sand and positively with the percentage of fine sand (Salter 6: Williams, 1965; Salter et al., 1966). Field capacity is commonly defined as moisture retained in soil two days after the soil was partially wet and represents the maximum storage capacity (Sykes 6c Loomis, 1967). In most soils, PC is equivalent to 113 bar of soil moisture suction (Baver et al., 1972). PWP is the percentage of moisture left in a soil that can cause permanent wilting in plants (Sykes 6t Loomis, 1967) and normally taken to be at 15 atm (bars) (Salter 6: Williams, 1965). As the water capacity of soil is affected by its texture and structure, these properties and AWC at varied levels, affect the numbers and Species of amoebae. Effects of Soil Properties on Amoebae Soil structure and texture influence the distribution of soil microorganisms in soil (Heal, 1962). The number of protozoa retained in fine or coarse sand is much larger than those retained in silt or clay (Cutler, 1919). The diameter of soil spaces is also found to greatly influence the distribution of various testate amoebae (Heal, 1962). Amoebae with larger shells (or tests) are found to inhabit larger soil spaces within the organic soil horizon; amoebae with smaller tests inhabit smaller soil spaces in mineral layers. Mahler and Wollum (1981) reported that populations of Rhizobium s2 were lowest in sandy and clay loam soils. The low occurrence of this bacterium in these soils limits the number of amoebae present. Generally, pore space sizes decrease with increasing depth with a corresponding decrease in the vertical direction of small cavities available for soil inhabitants (Wallwork, 1970). The amount of water present in soil plays an important role in influencing soil atmosphere, soil temperature, and. freedom of animal movement (Collis- George, 1959). Water volume is not constant, but varies with time. Water conduction is determined by soil particle size (Donahue et al., I977). Sandy soil is more permeable than clay and water capillary action increases as particle size decreases (Collis-George, 1959). The last remnants of water in capillaries is held at the corner of the soil spaces due to surface tension and serves as habitat for the smallest soil inhabitants like protozoa and bacteria in dry soil. Alexander and Jackson (1954) observed soil bacteria in the film of colloidal material covering soil particles. To get water, soil microorganisms have to perform work equal to the energy expended in retaining water in soil (Collis-George, 1959). Soil animals have to maintain the osmotic pressure of their body fluids equal to the energy of water in soil. If not, water will diffuse out from their bodies, eSpecially in dry soil. Normally these orgnisms form some impermeable outer coverings (cysts) to prevent water loss; others tolerate extreme environmental conditions or shift to a new environment (Collis-George, 1959). Moisture is the most significant property for the growth of amoebae in certain soils (Stout 6c Heal, 1967). This includes the alternative processes of wetting and drying of soil, movement of water to narrow pores, association of water film with mineral and organic colloids and variations in osmotic tension. Cutler and Dixon (1927) showed that reproduction of protozoa falls when the moisture content of soil drOps below half of field capacity. The growing period of the ciliate Colpoda steini Maupas, 1883 decreased in a soil sample subjeted to increased suction (Darbyshire, 1976). The large amount of water lost from soil pores under this suction affects the growth of this species (Darbyshire, 1976). While no direct relationship has been found between bacterial number and soil moisture content (Waksman, 1916), Clark (1967) reported that at a moisture tension of three bars or higher, bacterial activities are reduced, especially at 15 bars of mositure tension (at PWP). Although previous studies have not focused on the effect of soil aeration on amoebae, its effects on other aerobic soil microorganisms show that the presence of air (i.e., oxygen) in soil pores promotes their growth. The occurrence of aerobic and anaerobic bacteria in soil depends on pore space air. Filling up these pores with water reduces the availability of oxygen and affects aerobic bacterial growth (Clark, 1967). A study done by Winogradsky (19215), and cited by Skinner (1975), shows that the presence of Azotobacter (aerobic bacteria) and Clostridium (anaerobic bacteria) at various soil depths depends on water content which directly affects soil aeration. Azotobacter grow throughout the soil column at low soil water content (e.g., 1596 or lower). Soil water content higher than 2096 favors the growth of Clostridium. In general, lack of aeration slows down biological activities in soil. Decomposition and mineralization rates of organic materials in soil will also be reduced (Baver et al., 1972). Aeration is the most effective factor influencing fungal activity in soil compared with moisture content, soil texture, and structure (Griffin, 1963). Williams et a1. (1972) found that Streptomycetes grow best in pore spaces which are humid and air-filled, rather than water-logged. Soil atmosphere is not static, but changes over time because of several on- going processes (Burges, 1967). Living organisms use oxygen from air-filled pores and produce carbon dioxide as a byproduct of respiration. The latter gas diffuses to the external environment and, likewise, fresh oxygen will diffuse down from the above ground atmosphere. The partial pressure of both gasses in the soil environment is controlled by the diffusion process. The COZ/oz ratio depends on soil pore sizes and pathways (ColIis-George, 1959). If pathways are straight and pore sizes large, this ratio will decrease. The diffusion coefficient for various water-air ratios is a linear function of air porosity (Penman, 1940). Carbon dioxide formed in soil pores is increased with depth (Kuhnelt, 1961). The response of soil organisms to this gas is variable (Burges dc Fenton, 1953). Saprophytic soil protozoa show a positive chemotaxis to carbon dioxide at lower concentrations and show a reverse reaction at higher concentrations (Kuhnelt, 1961). Such other soil properties as pH, salinity, and temperature also influence the life of soil amoebae. Soil water (with dissolved nutrients) tends to move from warmer to cooler areas, indirectly affecting microorganisms distribution (Cary at Maryland, 1972). A significant change in soil pH will affect its ionic condition (Buckman & Brady, 1969). Microorganisms are affected directly by the change in H-ion concentration which indirectly influences nutrient elements. Fluctuations in the reaction of soil solution also affect soil organisms. Bacteria and actinomycetes grow in mineral soils at relatively high pH (Warcup, 1951, cited by Warcup, 1967), but the tolerance of individual species of soil protozoa to 10 soil pH is largely undetermined (Heal, 1967). The presence of protozoa in various soils at different pH levels, however, gives an impression that they can grow within a relatively wide range of pH (Stout, 1956). Barnes and Ali (1917) showed that an increase of soil salinity will decrease the number of microorganisms, as well as inhibit some of their activities. Increasing salinities may trigger the encystment of soil protozoa (Band, 1963). The transformation of soil amoebo- flagellates, Naegleria M is determined by the concentration of cations or anions in soil solution (Willmer in Stout & Heal, 1967). Fluctuations in soil aeration, pH, and temperature affect the growth and survival of amoebae and _E. cili. It is useful to be aware, at least, of all these effects in examining the treatment effcts that are the focus of this study. Escherichia coli Escherichia 9g is a gram-negative bacillus, indigenous to the gastrointestinal tract. I; 99g is not a soil inhabitant, so its presence in soil is used as an indicator of faecal contamination and its density is proportional to the degree of faecal contamination (Geldreich et al., 1962). Survival of E; cLli in soil is very low due to the low availability of its Specific required nutrients in soil (Waksman 6c Starkey, 1923; Waksman & Woodruff, 19110). Additional factors antagonistic to the success of _E_L_c_ofl in soil are the type of bacteria added, treatment, and temperature incubation of the soil. Waksman and Woodruff (1940) suggested bacteriostatic and bactericidal substances might be produced by soil actinomycetes. MATERIALS AND METHODS Soil Samplingand Treatments Soil samples were taken in September 1981 and 1982 at the Rose Lake Wildlife Research Area (see Appendix A for sampling sites). These two soil samples were then used as sources for soil subsamples throughout the experiments. The area of each sample taken was approximately 18 x 18 square cm, using a shovel. To obtain homogeneous soil samples, grass Agropygon Qpens (Linneus) was discarded and the soil was passed through a 2.0 mm sieve and mixed thoroughly by hand before it was stored. Samples were kept in covered buckets and stored at 14C. Generally accepted numbers of amoeba species present in soil were not affected by storage (Sandon, 1927), especially when soil samples were taken in a dry, hot season with comparatively low moisture (Dixon, 1936). Soil texture and pH were determined by the Soil Testing Laboratory, Michigan State University. The soil used in this study was a sandy loam with pH 7.3 for the 1981 sample and pH 6.6 for the 1982 sample. Soil moisture was 3.9096 and 8.6396 (g water] g dry soil x 100) respectively, for the two soil samples. Experiments with soil suction and the effect of soil moisture on amoeba population were done using the first (1981) soil sample. The second (1982) soil sample was used for determination of amoeba species‘ composition and the survival of I; cLli. The first and second soil samples are in the same soil textural class (sandy loam) so results obtained from using the two samples were correlated. ll 12 Determination of Soil Moisture at Various Suction Levels A pressure membrane extractor (Soilmoisture Equipment Corporation) was used to determine the soil moisture (for moisture content) at various suction levels. After subsamples were subjected to each pressure, they were put into an oven at 105C for about 214 hours until their constant weight were attained to determine their moisture content (gravimetric method), which was expressed as g water] g dry soil x 100. The suction level used ranged from 0.3 bar to 15 bars, which is the range of available water for plants in soil. The results, soil moisutre and soil suction (expressed in bars), were used to plot a standard soil moisture characteristic curve for the soil under study. A one-way analysis of variance to find the variation of soil moisture under various soil suctions was done. Enumeration of Amoebae in Soil Enumeration of amoebae at various soil moisture levels in this study was done by Singh's (1946) method. The soil samples were moistened with distilled water to get variations in soil moisture levels and incubated before their numbers were counted. Details of these stepes added to Singh's method are explained below. Variations in soil moisture levels were artifically made by adding known amounts of water to the soil subsamples. Knowing the soil moisture, the desired moisture level was achieved by adding an appropriate amount of water to the soil. Additional water was also used to make a suspension of g, 2911 as food for amoebae. A spatula was used to mix the water and soil before the soil was incubated. The purpose of soil incubation was to measure the effect of additional water on the amoeba population within the incubation time. Ten g of moist soil was put in a 50ml sterile culture flask for enumeration of amoebae. A 13 suspension of F: _c_ofl (0.03g packed cell volume with 6996 viable cells) was added to the soil in aqueous suspension. Soil with added bacteria was incubated at 23C for 48 hours before counting. The following steps were carried out as described by Singh (1946). Deviations from his method are specified. Soil Dilution Series In the amoeba counting, incubated soil was diluted with 50ml Low Salt Solution (LSS) (Band 6: Mohrlok, 1969) instead of the normal salt solution used by Singh containing 2.92g of NaCl, 1.33g of MgSOqJHzO and 0.04g of CaClz in 1000ml of distilled water to give the initial dilutin of 1:5. A set of 15 two-fold dilution series, ranging from U5 to 1/81, 902, was prepared. From each of such dilutions of a series, eight 0.5ml aliquots of the suspension were distributed to 8 wells of a 24-multiwell plate containing sterile Dilute Stock Agar Glucose (DSAG) containing 2.13g McC12.6Hzo, 0.136g KHZPOI” 0.568g NazHP04, 1.03 Trypticase, 1.0g Yeast Extract, 1.0g Glucose, 15g Bacto-Agar and 1000ml distilled water (Band, personal communication). In this experiment, DSAG was used to replace 1% agar containing only NaCl. Multiwell plates were used instead of glass rings and petri dishes to facilitate replication of the experiment. To support the growth of amoebae in multiwell plates, a drop of £2921; suspension was added to each well. The plates were incubated at 23C for four days before observation. Both vegetative forms and cysts were found healthy at that time, making species identification and clonal isolation relatively easy. Amoeba Counting The examination of each series of plates was done by direct observation under an inverted microscope. The density of the population of amoeba in various moisture-induced soil samples was calculated from the number of 14 negative wells in a given series. Using the table in the appendix presented by Singh (1946), the density of amoebae per g. dry soil was determined. Determination of positive wells was based on the occurrence of cysts and vegetative forms. Two replications of each treatment were performed. A one- way analysis of variance of log-transformed number of amoebae was done to determine soil moisture effects. A comparison of amoeba number at 1096 and 4096 of soil moisture levels was done using a student t-test which detailed species compositions examined in this study. Species Identification A method similar to the enumeration of amoebae in soil described above was used to identify amoebae at two soil moisture extremes. Soil moistures at 1096 and 4096 (g water/ g dry weight soil x 100) were used for this purpose. The 1982 soil sample was used for species identification so the minimum soil moisture used was 1096. Species identification was based on morphology for both vegetative forms and cysts: locomotion for vegetative only, using descriptions by Page (1967a, 1967b, 1976). A phase contrast microsc0pe was used to observe amoebae in wet-mount preparations and sizes were measured by using a calibrated ocular micrometer. A flagellation test was done to identify soil amoebo-flagellates by suspending amoeba cells in LSS, in wet amount preparations and sizes were measured by using a calibrated ocular micrometer. A flagellation test was done to identify soil amoeba-flagellates by suspending amoeba cells in LSS, in wet-mount preparation, without E: % (Fulton dc Dingle, 1967). The slides were left at room temperature at least for seven hours for transformation. Low Salt Agar (LSA) containing 196 Bacto-agar (Band 6: Mohrlok, 1969) was used as a medium in colonal isolation for further species identification. 15 Observations and identification were made based on two replicates of each treatment of soil moisture levels. Experiments with Escherichia coli Culture of Bacteria Escherichia coli strain K12 was obtained from Dr. Robert Brubaker, Department of Microbiology and Public Health, Michigan State University. The cultures were transferred weekly and maintained in Tryptone-Agar Slants (0.5g NaCl, 1.0g Bacto-Tryptone and 1.0g Bacto-Agar in 100 ml of distilled water). Before it was used as food for amoebae, E: c_ofl was grown in Tryptone Broth (0.5gm NaCl and 1.0g Bacto-peptone in 100 ml distilled water) for 24 hours at 35C. The cells were then centrifuged at 3020 x g, at 4C for 10 minutes and washed three times in LSS before they were weighed and diluted in distilled water. A similar amount of the E; CL“. in a method similar to enumeration of amoebae in soil (0.03g packed cells volume with 6996 of viable cells) was used to examine the survival of this bacterium in soil (10g dry weight). Cell Counting For total plate counting with the surface plate technique, the initial dilution series was done by adding 50ml LSS to incubated soil. A decimal dilution series using 9ml of 0.196 Peptone Solution (lg Bacto-Peptone in 1000 m1 of distilled water) was prepared as described by Deibel and Lindquist (1981). The diluent (0.1m1) at each dilution level that can give the colonies numbers between 30 and 300 was spread on the Levine Eosin Methylene Blue Agar plate (BBL) using a "hockey stick" glass rod. Triplicate sets of EMB plates were used for each dilution due to their success in producing _E_z ggli colonies, green black, metallic sheen in color (Konenman et al., 1979). All plates were incubated in an l6 inverted position at 35C for 24 hours before counting. Experiments with -_E_: cLli were carried out at 2596 soil moisture. Biochemical Tests A series of biochemical tests were conducted to identify colonies counted on EMB agar as E; cili. Colonies which were randomly taken from EMB plates were grown on Tryptone-Agar slants for 24 hours before identification. Tests for Indole, Nitrate, Triple Sugar Iron Agar (TSA), Citrate utilization, Methyl Red Voger Proskaur (MR-VP) and motility were utilized for this purpose. The tests and media followd standard procedures described by Koneman et a1. (1979) and the DIFCO Manual (1977). RESULTS Soil Moisture Characteristic Curve The soil moisture characteristic curve for the soil investigated (using the 1981 soil sample) is shown in Figure 1a. The curve shows a relationship between soil moisture and soil suction within the range 0.3 bar to 15 bars which is the range of available water for plants in soil. Results from a one-way analysis of variance showed that the amount of water loss varied at different soil suctions. F calculated was 192.730 which was highly significant compared with F(g’27,o.01) which was 3.26 (refer to Appendix B; for detailed ANOVA table). Variations in each replicates were presented as standard deviation of each mean (see Table 1). According to this curve, a soil suction of 0.3 bar corresponds to 25.46% (3 water/ g dry soil x 100) of soil moisture and a suction of 15 bars corresponds to soil moisture of 3.7996 (Table 1). The soil moisture shows an exponential decrease at soil suctions greater than 0.3 bar until a soil suction of 5 bars is reached (Figure la). Beyond this soil suction, the amount of water drained from soil pores is almost constant; this is demonstrated by the horizontal part of the curve. There is little water left (3.9796 of soil moisture) in the system after 5 bars of soil suction. The soil moisture at 014 bar was estimated from this curve at $2 1096. Figure lb describes the distribution of various sizes of soil pores. The diameter of soil pores is calculated by using the formula of Baver et a1. (1972). _ 2V Cosd> pgr h 17 18 .MH wusmflm 600m owumeflumw mm: USHm> mane; 60.0.“ 60.6 00.6 06.6 66.6 00.0 00.0 0.60 06.0.“ 06.0 66.6 66.6 66.6 60.0 06.0 0.6 00.0.“ 06.6 06.0 06.6 60.0 60.0 06.0 0.6 60.0.“ 60.6 06.0 00.6 00.6 06.6 60.0 0.0 00.0.0 66.0 60.0 06.0 60.0 06.6 0.0 0.0 66.0.“ 06.0 06.6 00.0 00.6 00.6 6.0 0.0 06.0.“ 06.6 06.0 06.6 66.6 06.6 0.0 0.0 66.0.“ 66.0 06.0 66.0 60.6 06.0 0.6 6.0 600.00 IIIIIIIIIIIIIIIIIIII 6.0 6.0 66.0 H 60.60 00.00 66.00 06.60 60.60 0.00 6.0 06 H 082 z 2 0a 00 E 3 6.83 mwumuflammm mmNHm cofluusm 6000 0006 0000 x 0006 606 0\06063 00 musumwoz afiom 000m 6600 wccmm 00m w>usu Uflumfluwuumumno wusumfloz Hfiom 0 60660 19 m0 ‘P 'T .0 manna cu co>0w nuns .aeofl soon no Aomv nodue0>uv vuevceua usu nuceuouauu unwoa gone mo pun .m:00ua>uunno c we owauu>e ozu aouu :uxeu an: ucqoa scam .000. Inca avcea new o>uau uauuuuUuueuenu uuaueuoa 000m .60 .wuu Aauenv nouuuau Hwom 00 6 Q 0 ---o—- --------- 0---- coc1 o u-A I O N (00; x 1108 fizp 8/13395 8) axnastou {tog 20 where h : height of water rise in a capillary tube with radius r Y : water surface tension 0 : water density 9 : acceleration due to gravity 45 : the contact angle between water and soil pore (assumed to be zero) which is then simplified as d = 3/H where d: diameter of the soil pores in mm H: the amount of tension which the water in the pore is in equilibrium. One bar is approximately 100cm of water suction (Griffin, 1963). The relationship between soil moisture and the diameter of soil pores is shown in Figure lb and Table 1. At saturation (_cg. 4896 of soil moisture), soil suction was equal to 0 bar, all soil pores were saturated. When pressure was applied to the soil, a certain size of pore was emptied. At 0.3 bar, soil pores with diameters of 10 um or more were emptied, followed by other smaller pores as suction increased. Many pores were considered emptied when pressures higher than 5 bars were applied, as shown by a small amount of water loss beyond this suction (Figure 1a and Figure lb). Pores emptying (a) and pores empty (b) in this system are shown in Figure lb. At 0.3 bar of soil suction, 25.46% of soil water remained in the system occupying soil pores with diameters of less than 10 um. At a soil suction of 0.5 bar, remaining water in the soil (7.8496 of soil moisture) was present in pores with diameters smaller than 6.0um. The amounts of water left in various sizes of pores for the rest of the data in Table l are explained in a similar manner. When soil suction exceeded 5 bars, all pores available in the soil were empty. In 21 «.0 m0 .3 an huan ounce can a an cacao cue unahunao euuom .60005 “006 we wonozeqc ou couueaeu =0 u>u=u caucuHOUUeuezu ousuuwofi Hmom .n0 .wam Aanv mouoa H006 mo nauQEUan n.w o.o mu.% 6.“ m cm 11‘- AmuuLv couuu:e chm wow s (001 x {108 51p fi/zaaea 3) aznnsxom ITOS v'-"" I $0 ON .on Ten INN rem oo~ saaod {108 go aSeauaazad 22 this context, soil pores are said to be emptied when no water can be extracted as suction increases, even at 15 bars (see Figure lb). Assuming the relationship between soil moisture and soil pore percentage is a straight line-that pores were empty at "b" and water-filled at zero bar—the percentage of soil pores with various diameters can be estimated (Wallace, 1958). So the percentage of soil pores with diameters of 0.611 m and 1011 m for the soil investigated were about 2496. The rest of the pores in the system had diameters larger than 1011 m. Since most water was lost between 0.3 bar and 5 bars of soil suctions, the soil pores which effectively contained water in the soil investigated (within the range of available water in soil) were the soil pores with diameters of 0.6um - 10 pm. At 15 bars soil suction, water retained in soil was 3.79% of soil moisture. This water occupies soil pores with diameters between 0.211 m and 0.611 m. Effects of Soil Moisture on Amoeba Populations Amoebae need a critical amount of water for growth as shown in Figure 2a. The maximum population of amoebae in the soil investigated was found at 3096 soil moisture. Populations of these organisms decreased at soil moistures higher or lower than this critical point. A poor growth of amoebae was apparent between soil moistures of 596 and 2096, resulting in a low number of amoebae at these moisture levels. A dramatic increase in amoeba number was observed starting at a soil moisture of 2596. The amoeba numbers decrease again after a peak population is attained (i.e., at 3596 and 4096 of soil moistures). The effect of soil moisture on amoeba population was significant (p 0.01). This was shown by high value of F obtained which was 110.3442 from a one-way analysis of variance, as compared with F(7,g,o,oj) which was 6.18 (see Appendix 82 for detailed ANOVA table). When the log-transformed number of amoebae was plotted against soil moisture, the population incrased in a straight line as soil Moisture increased (Figure 2b). The linear relationship between amoeba Number of amoebae (x 10‘) 35 30 25 20 15 10 23 U U l 10 - 20 3° ‘0 g0 Soil moisture (a water/g drv soil x 100) Fig. 2a. Effect of soil moisture on amoeba population. Each point is the average of two counts. Bar of each point represents the range of amoeba population counted. The amoeba number at 52 of soil moisture was 0.1 x 10‘ (see Table 2). Number of amoebae (log. no./g dry soil) 5.5 T 5.0 ‘ 3.5 - 3.0 241 fl T T ‘ 20 3O 40 50 Soil Ioisture (g water/g dry soil x 100) Fig. 2b. Log plot of amoeba population at various soil moisture levels Each point was taken from the average of two observations. Bar of each point indicates the standard deviation (SD) of each mean. 25 population and soil moisture was obtained at soil moisture levels between 596 and 3096. A regression line of this relationship is T = 2.57 + 0.099x (Figure 2b). The estimate value of 8 is 0.099 1 0.01 log (no.)/ soil moisture (p 0.01). The value of t for testing 8 = 0 against 8 > 0 was 4.5249. The value of t¢,o.oj was 3.747. A regression test for the soil moisture levels of higher than 3096 was not done, since only two data points were observed. Amoeba Species at 1096 and 4096 of Soil Moistures Amoeba species found at 1096 and 4096 soil moisture, with measurements of their cysts and vegetative forms are listed in Table 3. Occurrence of each amoeba species at these two moisture levels is roughly presented by frequency found, not absolute numbers. The total pOpulation of amoeba species at these two moisture levels was listed in Table 2. Data presented in Table 3 were for the second (1982) sample of the soil investigated based on two replications of the experiment. There was no difference in species composition at 1096 and 4096 soil moisture, but generally the number of an individual species was increased at 4096 of soil moisture (Table 3). The difference between the number of amoebae present at 4096 and 1096 of soil moisture levels was significant at p 0.01, using a student t-test (Table 2). The number of species of Vahlkampfia juggg Page, 1967 was found to decrease at a soil moisture of 4096. Another species that was identified as a member of family Vahlkampfiidae was rarely observed at either soil moisture levels. Acanthamoeba glyphaga (Puschkarew, 1913) was commonly observed at soil moisture levels of 1096 and 4096. Survival of Escherichia coli in Soil Escherichia coli counts, using Eosine Methylene Blue plates (EMB) and the spread technique, are shown in Table 4. The survival of this bacterium in natural 26 .ummulu unmosum o 0:06: .moch0M0cm0m mo 00>00 *0 0 pm .600 um some on» 5000 6000000 ousum0oe 0006 no wow 00 c005 0:900 .06060 .000060 0m0.0 00 0:000M0Cm0m no: 0003 00506005 0006 mo 00>00 sumo um c000603m0m 630050 G0 6:00900Ho>0 mm0.0.H 0000.m mm00.m vm00.m 000.0 000.0 mmmr m «0mm0.0.fl mmvo.m momm.m mm0>.m 066.0 000.m 00m.m 00 000.0.H 0000.0 0000.0 Nehm.v 006.00 00N.00 000.00 60 000.0.H 00mm.0 000m.v 0000.0 00m.00 006.0N 006.00 om 600.0.H mmvN.m mvmm.m 0000.6 000.060 000.00m 00.m00 mm 000.0.H vmmo.m 00mm.m Nomv.m ooo.MHm ooo.v¢m ooo.mmm 0m 000.0.H 0mmm.m mmmm.m mmmv.m 006.060 000.m00 000.00m mm «0mmo.o.fl NNON.m v0®0.m 0mvm.m 000.000 ooo.mva ooo.m00 co 06 H 0002 00 0.. :00: 00 00 000 6000000000 «6000000000 x 00006 >00 0\Hmuns 00 0006 600 0\ 0000024 60 .02 000 0006 000 0x0000os0 60 000502 00906002 000m 000m Housuoz :0 :00000500& mnoOE¢ :0 00506002 000m «0 uommmm N OHQMB 27 .:O006>00600 no 6006000000 030 :0 00660 0003 6>Ono 000:0600m 6060 00¢ 0060 + 0:000 600:030000 6600 ++ 0:000 >0u:0svm0m +++ 0:000 >0u:0swo0u 0608 0:0 ++++ 0.0 u 6.0 00 I m +++ ++ 06660 .060:00060060 0000500 60000mwoz .0 60 I 60 + + 0060 .000000 0060000500000> "506 .6 6.6 I 6.6 00 I 0 +++ ++ 0660 .0600 6006000 000005050006 .6 6.6 I 0.0 60 I 6 +++ ++ 0660 .0600 6050000500> 000055050006 .0 6.6 I 6.0 00 I 00 + ++ 0660 .0600 06060000060500000> .6 0.00 I 0.60 I ++ + 00660 .60060000 00000000600 000050000006 .0 0.60 I 6.6 00 I 00 ++++ ++++ 06060 .30000006000 060006006 000050000006 .0 05:0 05:0 00 00 00008600 :000060000 0000 x 0006 >00 0\ 06>U :0 500:00 00063 my 0000600: 0006 600006002 0006 00 wow 0:6 600 06 6000006 mnoofia 6 00000 28 v0m.0.H 0N0m.m 000v.m m0om.m 000.0 000.0 moo.0 Noo.0 00.0 m 0m0.0.H 0000.0 000m.0 0000.0 0m00.0 m0.0 No.0 m0.0 00.0 0 0mm.0.H 0600.0 000m.0 0000.0 0000.0 00.0 No.0 00.0 00.0 0 mmm.o.H 000N.0 0000.0 0mvw.0 mvmm.0 vm.0 00.0 00.0 00.0 m mmm.0.H m00m.0 0000.0 000m.0 00mm.0 00.0 0m.0 mm.0 00.0 v 0m0.0.H m000.0 0060.0 0000.0 m0mm.0 00.0 60.0 00.0 m0.0 m 000.0.H 00N0.0 m0m0.m m00m.m 0000.0 0m.0 00.0 00.0 00.0 m 000.0.H mmov.m 0000.0 v0vm.m Nomm.m mm.m 00.m NN.N ov.m 0 000.0 + m0m0.0 m00m.0 Nmm0.0 m000.m mm.v 00.m 00.0 00.0 0 06 0 0000 6* N0 00 0000 60 2 00 00600: 6000000000 6000000000 0809 0006 000 0\ 60:500 0000 000 0000 x0 0006 000 0\ 60:500 0000 :0000050:H 0006 0005002 :0 0000 um 00 00>0>056 v 00006 29 soil in this study was examined at a soil moisture of 2596 for a period of eight days only, using the 1982 soil sample. The number of E _c_o_l_i decreased over time in a linear form (Figure 3). The best fitting of the straight line is given by the equation {I = 8.8 - 0.357x. The estimated value of B is -o.357 1 0.03 log (no.)l day (p < 0.01). The value of t for testing 8 = 0 against B + + + + com dunno: + + + + maoccH v: mw .I m# an mononumnzm mmflcoHou waoo .m mo mmumofiammm no mamas flHoo mazofiumcomm Mo mumme HMUMEonoon m mfinma DISCUSSION Soil Moisture Characteristic Curve The soil moisture characteristic curve for a sandy loam soil (Figure la) graphically demonstrates the differing levels of water retained in soil at various soil suctions. The shape of this curve is determined by the soil texture and structure (Hillel, 1980) and may also be influenced by the history of soil usage of drying and wetting (Griffin, 1963). The curve shows that at 0.3 bar of soil suction, the corresponding soil moisture was 25.14696 which was close to the estimated mean value for a sandy loam of 2696 which was derived from the regression relating percentage of different particle size fractions and soil moisture content described by Salter and Williams (1969). The lower limit of available water in the soil investigated was 3.79% which corresponded to 15 bars of soil suction. The estimated value of soil moisture at this pressure was 996. The slight difference between these two values might have been due to the nature of the soil used for this investigation. The value prOposed by Salter and Williams (1969) was taken from an average of several soil samples in their study. The water retained in the soil in the present study at both PC and PWP was comparatively low (the corresponding soil moistures were 25.4696 and 3.7996) and it increases when the soil texture becomes finer (Salter 6: Williams, 1965). The maximum water that could be retained by this soil at saturation was g lI896 (g water] g dry soil x 100), and all pores in the system were considered to be saturated. When suction developed, some of this water was drawn out of the pores. The pores which were emptied at corresponding suction levels are shown 32 33 in Table l and Figure lb. Bigger pores were emptied first because they were most conductive according to Poiseuille's law (Hillel, 1980). At field capacity of 0.3 bar, only 25.06% water was left in the soil, which was the upper limit of available water for the soil investigated. As soil suction increased, the amount of water dropped gradually until it reached a soil suction of 5bars (Figure la). The amount of water loss was almost constant beyond this suction, even at 15 bars (i.e., at PWP). The water left in the soil at PWP was bound to soil particles by adhesive and cohesive forces, cannot be used, causing permanent wilting in plants (Foth, 1978; Sykes 6c Loomis, 1967), and this may affect the availability of water for soil microorganisms too. As seen in Figure lb, about 2#% of the pores in the soil investigated had diameters between 0.6um and 10 um; the rest of soil pores were larger than 10 um. This means that this sandy loam soil contained a number of large pores which could retain a considerable amount of water when saturated or at field capacity. This would influence the nature of microflora present (Bhaumik 6: Clark, 1907). When the suction was higher than 0.3 bar, these large pores were emptied and less water retained in the small pores for which higher soil suction was required. The results obtained in this study agreed to previous findings (Hillel, 1980). The application of pressure to the soil caused the pores filled with the air that replaced the water in the system. In soil, air and water wre inversely related: when water fills the pores, only a little air is present in the soil; and when the water is drawn out of the pores, the pores fill with air (Buckman & Brady, 1969). At higher pressure, only a very little water retained in the soil and, tightly bound to soil particles, it could not be drained, even at 15 bars of soil suction. However, this water could be drawn out of the soil if it was put in an oven at 105C for 24 hours at which the equivalent pressure was 10,000 bars (Foth, 1978). 34 Effects of Soil Moisture on Amoeba Population The peak population of amoebae in this study was achieved when the soil moisture was at 3096 (Figure 2a). The amoeba populations decreased at soil moistures below or above this moisture level. The population decreased at soil moisture below 2596 were also observed by Cutler and Dixon (1927) for protozoan populations in their study. When the numbers of amoebae were log-transformed and plotted against soil moistures, they showed a linear increase with increasing soil moisture until that moisture reached 3096 (Figure 2b). The smaller amount of amoeba growth at various soil moisture levels below 2596 was shown as short lines connected to each point within this range (Figure 2a). This study showed that soil moistures ranging from 2596 to 3096 represented Optimum water availability for the rapid growth of amoebae. Active amoebae need a moist environment for feeding, as well as oxygen for respiration (Sleigh, 1973). At these moisture levels, an equilibrium exists among the solids, liquids, and gasses of the soil system, providing optimum soil conditions for microorganisms. Above the optimum soil moisture, at 3596, there was a slight decrease in amoeba number and it dropped further, below the number of amoeba at field capacity, when the soil moisture was at #096. Filling the large soil pores with water created an anaerobic environment, preventing amoebae from growing. Although the effects of anaerobic conditions on amoeba populations in soil were not addressed in this study, a study done on bacteia showed that the growth of aerobic bacteria was affected by filling the soil pores with water, due to the lack of oxygen in soil (Clark, 1967). The effect on amoebae would be similar, either directly because amoebae are aerobic organisms, or indirectly because amoebae are bacterial consumers in soil. Another factor which can explain the decrease in amoeba numbers at higher levels of soil moisture is the production of organic substances in flooded 35 soil which can be toxic to microorganisms in soil. Osa-Afiana and Alexander (1979) found that sulfides and organic acids were produced in flooded soils and one of these substances, butyric acid, caused the death of root-nodule bacteria in their investigation. Wolin (1979) found the organic acids' toxicity for soil microorganisms incrased as soil pH decreased. The pH of the sandy loam soil in the present study was relatively high (pH 7.3 for the 1981 soil sample) so the toxicity of these acids was relatively low. If the acids existed in high concentration, however, they would cause injury to these organisms and have a significant effect on trophic interactions of soil microecology (Osa-Afiana & Alexander, 1979). If water is added to the soil, according to Rost and Fieger (1923), it will lower the soil pH. This phenomenon may contribute to many aspects of the relationship between soil moisture and other soil properties and merits future study. At the lower ranges of soil moisture (e.g., at 2096 or less), amoebae grow poorly (Figure 2a and Table 1), but their increase in number is almost linear with increasing soil moisture within this range. The small amount of water present in this low range favors the multiplication of amoebae although at very slow rates. Even though 20% or lower soil moisture will allow much air to be present in soil (too many pores are air-filled), this is not the only factor that is required for the growth of amoebae. A moist environment is important for amoebae to move, feed, and multiply. The size of amoeba population increased at soil moistures above 2596 proved that water was important for their growth. Protozoa failed to multiply at the 1096 moisture level due to the inability of these organisms to move to their prey (Osa-Afiana 6: Alexander, 1979) and Losina-Losinsky and Martinov (1930) noticed that amoebae moved very slowly in loam soil at 1596 to 2096 of soil moisture. Bryant et al. (1982) discovered that in dry soil, no bacteria were ingested by amoebae because the amoebae were in an inactive state. 36 When the environment is unfavorable, particularly when the soil becomes too dry, the movement of amoebae will be limited, the organisms will be inactive, and normally they will encyst. If sufficient water is added to the soil, it will induce excystation (Bryant et al., 1982). The additon of water to dry soil can cause an increase in protozoan numbers within two to four days (Osa-Afiana 6: Alexander, 1979), due to excystation when the soil is wet. In this study, the additon of water to the soil produced favorable conditions for amoebae to excyst and reproduce in a relatively short time (within #8 hours of incubation). This phenomenon is similar to what happens in nature as unfavorable conditions for cell divison are relieved (Stout, 1973). The numbe of these organisms shown in Figure 2a and Table 2 is the result of this relatively swift multiplication in soil within #8 hours of incubation. Each point in Figure 2a was calculated from the average of two total counts (each count represents each replication of each treatment), that is, the counts which included both vegetative forms and cysts. These two counts were not significantly different because they did not exceed eight negative wells in their original counts (p 0.05) (Singh, 19%). In nature both vegetative forms and cysts are commonly present in soil even though only the former state play an important role as micropredators. In this study, differentiation in the number of cysts from vegetative forms, by treatment with 296 HCl to kill the vegetative forms (Cutler, 1920), was not done. However, Alexander (1961) found that at low soil moisture, the cystic stage of amoebae were predominantly present in the soil. The occurrence of cysts and vegatative forms in the environment always fluctuates, so the total count of both of these forms, rather than only one, better represents the normal pOpulation. Based on Figure 1b, the 0.3 bar suction, the pores that were effectively emptied were the pores which had diameters bigger than 10IJ m. 50 the ga_._ 25% 37 water retained in soil at this suction was found in soil pores with diameters less than 10 um. Amoebae in soil use oxygen from air-filled pores and move into the moist environemnt between pore spaces with diameters of less than 10 u m. Amoebae can move into soil pores which are smaller than themselves because they can change the shape of their bodies easily (Elliott at al., 1980). The water remaining in pores after draining, however, will restrict their movement in soil (Darbyshire, 1976). The results of the present study explained this phenomenon, and this is why the amoeba number varied at different soil moistures. When the suction increased, the smaller pores were emptied and the amoebae encysted because they were not able to move to a new environment. If the soil water in this study had not restricted the movement of these organisms, they might have shifted to a new environment, as was seen when amoebae entered smaller soil pores in the presence of nematodes, i.e., the phenomenon of escapism from predators (Elliott et al., 1980). In this study amoebae were found to inhabit the soil pores which had diameters larger than 10 u m. Amoebae show good growth at soil moistures between 2596 and l5096 (Figure 2a); and even at l4096 soil moisture, when the soil environment is considered anaerobic for amoebae, the population was higher than the population at 2096 or less. Therefore, a moisture level of 2596 to #096 is needed for rapid growth; the oxygen loss at these levels is less important. Referring to Figure lb, the range of soil moisture that promoted good growth of amoebae was when soil suction was at 0.3 bar or less, which corresponded to effectively drained pores of size 10 um or larger. As the soil suction increased (e.g., at 3 bars), (5.11396 (Ea: 596, Table l) of soil water was left in the soil. This water occupied the pores with diameters of less than 1.0um, and amoeba number was low at this soil moisture level (Figure 2a and Table 2). This meant that the absence of water in larger pores (with a diameter bigger than 10 u m) which 38 amoebae typically inhabit, affected their growth and movement. The soil organisms moved easily in coarse soil, compared with fine soil, because the former had a greater number of larger water-filled soil pores (Elliott et al., 1980). The number of protozoa in soil is always related to the bacterial number present (Sandon, 1928; Chlarholm, 1981), so any unfavorable physical conditions which affect the bacterial population will also affect an amoeba population. Too much or too little water present in soil limits the activity of bacteria and may slow their rate of multiplication, thus limiting the supply of food for amoebae. Amoeba Species at 1096 and #096 of Soil Moisture The amoeba species found in sandy loam soil with a pH of 6.6, in artifically induced conditions of 1096 and #096 of soil moisture, are shown in Table 3. The ratio of amoeba species present at these soil moisture levels was similar, but the number of each species was increased at #096. This explained why the total number of amoebae was higher at #096, as compared with 1096 (p 0.01) (see results in Table 2). Since the soil moisture levels used were 1096 and #096, the amoeba species identified in this study occupied the soil pores with diameters bigger than 7.511 m (Table l). The number of species found at these two levels of soil moisture failed to show any differences because the soil samples used in this experiment were taken from the same bucket which was stored at #C. The addition of water induced the excystation of those species already present in the soil; thus it incrased the number in each species, but not the number of species. A difference in number of species present at 1096 and #096 of soil moisture might be expected if the soil samples were taken from two sites in the field, with moisture content for each site was at 1096 and #096. Although the types of soil 39 protozoa present in soil vary according to soil moisture (Bamforth, 1969), other factors such as soil texture, fertility, pH, and temperature are also important in ifluencing these organisms to be varied in certain soil environment. In this study, Acanthamoeba alyphaga was found to predominate in soil at both soil moisture levels. Even though the number of all species seemed to be higher at #096 than 1096, Vahlkampfia jggfi was rarely found at #096. This can probably be explained by the sensitivity of this particular species to the presence of oxygen in soil. At #096 of soil moisture the soil pores are water-filled, less oxygen is present in soil, and this may have limited the ability of this amoeba to multiply. The number of this species was found to be higher at 2596 of soil moisture (data are not shown) than at 1096 and at #096. This signified that at 2596, the soil microenviornment was optimum for this amoeba to grow and multiply. At #096 of soil moisture, not only were other amoebae numbers increased, but the number of ciliates such as M s2 also increased. A crowded environment may increase competition for soil spaces and available food (LeFevre et al., 1952, cited by Bamforth, 1963). One species that was found to belong to family Vahlkampfiidae, based on its morphology, locomotion, and negative results with a flagellation test was very rare at both moisture levels in this study. The cyst of this species could not be investigated, as attempts to isolate its cyst were unsuccessful because of its sparceness in the soil. The identification of Acanthamoeba castellanii was based only on the morphology of its cyst, rather than its vegetative form. In this experiment, DSAG was used as a medium for isolating amoebae from soil and LSA was used to make clonal isolation. This use of different media may explain why the sizes of L Ego—sat and i M were smaller than Page's descriptions (19067a). Another reason may be that different strains of these #0 ameobae were present in the soil investigated. The morphology and size of amoebae are not always constant, so it was difficult to measure them accurately (Adam, l96#). Size and morphology vary depending on the specimen's growth conditions, culture age, and method of preparation (Culbertson, 1971), as well as structure, composition, and fertility in nature (Reinhard et al., 1969). The confirmation of these two species was completed after doing a flagellation test which took about seven hours for E. M to give a positive result at room temperature. it juggsg was recognized by observing at its locomotion which was erruptive, characteristic of family Vahlkampfiidae, and also by the morphology of its cysts and negative results on a flagellation test. The identification of amoebae in this study was done based on their morphology and locomotion of trophic forms, cyst morphology as described by Page (1967a, 1967b, 1976), and flagellation tests (Fulton 6c Dingle, 1967). Due to time limitations, identification based on nuclear structure and patterns of mitosis and division were not done, although at present they are considered the most reliable techniques for identifying and classifying the amoebae (Pussard, 1973; Singh 6: Humaiah, 1976). However, the detailed explanations by Page were helpful for identification of these species. Survival of Escherichia coli in Soil The presence of Escherichia coli in soil was found to decrease linearly over an eight day incubation time (Figure 3). Klein and Casida (1967) made a similar observation after 2# days incubation although the decrease was not linear. In their study the decrease of the _E_._ ggli population was less abrupt when sugar was added to the soil or when one percent of autoclave-sterilized soil was added to normal soil. The decrease in E coli numbers in the non-nutrient-amended soil in #1 the present study was due to a dying out of cells because of a lack of the food necessary to support the growth of a species nonindigeneous to soil. McGrew and Mallette (1965) found that viable cells of g, 993 decreased with time in normal soil but showed a stable growth (represented by a straight horizontal line) when fed with glucose. This phenomenon signified that glucose was used for cell maintenance, but not for growth. While starving due to the lack of food in the environment, the endogenous metabolism of E ggl_i was increased and cellular glycogen was used very rapidly (Dawes 6c Ribbons, 1965). Cell "aging" may also cause a decrease in g, Q numbers with incubation time in soil. As the age of cells increases, their respiration rates will decrease, and the cells eventually will die out. During the aging process, the cellular content (i.e., ribonucleic acid) was found to be oxidizing, resulting in an accumulation of uracil in the suspension medium (Clifton, 1966). Waksman and Woodruff (l9#0) discovered that by adding E w to the soil, the total soil population was increased, due to the rapid multiplication of antagonistic bacteria, particularly spore-forming bacteria, to exclude this bacteria from the soil. This factor causes _E_. _co_1i to die out when added to the soil. The addition of one percent of autoclave-sterilized soil, which killed some of the antagonists, probably helped the l; 5&11 to live longer in normal soil. However, Klein and Casida (1967) claimed that the decrase of _E_._ gli in soil is not caused by toxic compounds produced by indigeneous microorganisms, but by its inability to slow down its metabolic rate in keeping with the low availability of usable organic carbon in soil. There is an inverse relationship between numbers of bacteria and numbers of active amoebae in soil (Cutler, 1923; Cutler dc Crump, 1920) and the decrease in _E_._ 9211 population in this study was also due to predation by amoebae. In fact, this was the main reason why E coli were added to the soil; that is, to serve as #2 food for amoebae. Even though some indigenous bacteria present in soil can provide natural food for amoebae, the additional food was needed to support the normal growth of amoebae in moister soils. It is assumed that in this experiment, E c_o_li_ that were added to the soil could occupy the soil spaces or the water films that were accessible to amoebae. The decrease of the _E_. _c_o_1_i population because of predation (the term partial predation may be appropriate here as E _c_o_li was not the only food availale in soil) will never be total because the bacteria will not be totally consumed by amoebae (Habte 6: Alexander, 1975). Bdellovibrio bacteriovorus Stopl and Starr, a parasite of gram-negative bacteria (Starr 6r Baigent, 1966) which is present in any American soil can also contribute to limiting of the establishment of E: egg in soil (Klein 6: Casida, 1967). The decrease of the E; 9211 population in the soil in this study was less abrupt over time because the pH of the sandy loam soil used was relatively high (i.e., 6.6. for 1982 sample). 1:; c_ol_i_ can survive longer in alkaline soils than acid soils (Cuthbert et al., 1955); this may be comparable to the actual pH environment of E go_li_ in the gastrointestinal tract. Since E in can survive even after #8 hours of inoculation in soil, it can serve as food for amoebae as long as the amoebae only consume its vegetative forms, not its dormant form or dead cells as observed by Darbyshire (1976) on Colfla steini with Azotobacter. APPENDIX A .Awnmfiv woa>nmm coaum>uwmaou Hfiom «usuH50fiuw< mo unmEuumnwn mmumum vmufias an xwaaaoo umhom cam EMOH hwam “whom we umnwuummv mm3 HHom mnH .mmcfiaaamm Haom new dawn wsu moumofivcfi aouum n< .mmud sopmmmmm omaavafi3 oxma omom no one < n < xfivamnm< It. I. u nail 2E