RELEASE AND RECOVERY OF RHIZOBIUM FROM TROPICAL SOILS FOR

CHAPTER 2

LITERATURE REVIEW

Bacillus radicicola, the root-nodule bacteria of legumes, were first isolated, described, and named by Beijerinck, the father of microbial ecology. These organisms now constitute the genus Rhizobium--the name proposed by Frank in 1889 (Fred et al., 1932). From Beijerinck’s report in 1888 to the present, the Rhizobiaceae have been the object of intense investigation and are probably among the most widely studied of the soil microorganisms.

Interest in these bacteria stems from the unique nitrogen-fixing symbiotic association they have with their legume hosts. Legumes are among the world’s most important crop plants, second only to grains (Advisory Committee on Technology and Innovation 1979). Thus it seems only natural that the symbiont be intensely studied. While the actual mechanisms of host specificity remain elusive, questions concerning the life of these bacteria in the soil and the soil properties which influence their growth, persistance, and success or failure in nodulation can be answered. These answers can be readily applied to increasing legume yields through enhanced symbioses.

I. Use of Serological Techniques in Studies of Rhizobium

Serological techniques have been in use for many years to investigate the Rhizobiaceae. The discovery by Klimmer and Kruger (in Fred et al., 1932) that bacteria isolated from different species of legumes could be distinguished serologically, made serological methods extremely attractive for strain identification. Stevens (1923) and later Wright (1925) found that different strains isolated from the same species of plant, and therefore belonging to the same inoculation group, were serologically unrelated. In fact, Hughes and Vincent (1942) found strains isolated from different nodules on the same plant which were serologically unique. The results of these early investigations pointed to the great serological diversity now known to exist in the Rhizobiaceae.

A. Agglutination

Agglutination was one of the first methods to be applied to serological investigations of rhizobia. It is among the simplest of serological techniques to use and it has been widely exploited in many taxonomic and ecologic investigations. Bushnell and Sarles (1939) used the technique to define three types of antigens on rhizobia. They reported on the antigenic specificity between and within rhizobia from soybean, cowpea, and lupin cross-inoculation groups. They found no correlation between the ability of rhizobia from the three legumes to cross-inoculate and cross-agglutinate. This important observation was recently restated by Vincent (1977): strains which are related or apparently related serologically can be entirely unrelated in other characteristics. Bushnell and Sarles (1939) also confirmed the results of Stevens (1923) who found that due to the serological diversity within a species of Rhizobium all strains cannot be identified by the agglutination test.

Kleczkowski and Thornton (1944) used agglutination to study the serological relatedness between and within pea and clover strains of rhizobia. They tested six antisera (four clover, two pea) against 161 strains of R. trifolii, 29 R. leguminosarum, 5 each of R. meliloti and R. lupini, and 13 non-Rhizobium soil isolates. Partial cross-reactions occurred in the clover and pea groups which were removed after adsorption of antisera with the cross-reacting antigens. No cross-reactions were detected outside of the clover and pea groups; and none of the 13 soil isolates agglutinated. No “group” antigen common to all the strains was found and attempts to link effectiveness or ineffectiveness to any serological property failed.

Koontz and Faber (1961) used agglutination-adsorption (Edwards and Ewing, 1955) to characterize the somatic antigens of 25 strains of Rhizobium resulting in six distinct serogroups. Antigenic similarities and physiological characteristics could not be related.

Graham (1963), in a similar study to that of Kleczkowski and Thornton (1944), prepared antisera against somatic antigens and whole cell/flagellar antigens of 58 strains of root-nodule bacteria and 16 strains of agrobacteria. He tested the antisera by tube agglutination against 113 strains of Rhizobium, 20 strains of Agrobacterium and 20 strains of other, possibly related bacteria. On the basis of the agglutination reactions he categorized the rhizobia into three serologically distinct groups. Cross-reactions were more common when whole cell antisera were used than when agglutinations were run with somatic antisera.

Tube agglutination techniques utilize antigens obtained from pure cultures. Means et al. (1964) adapted the methods to type bacteria directly from root-nodules. They observed that the agglutination reaction of pure cultures and of root-nodule homogenates were identical for 15 of 17 strains tested, and recommended that this technique be used for a quick classification of nodules. This method was further modified into a micro-agglutination test (Damirgi et al., 1967). In micro-agglutination a drop of dilute nodule homogenate is mixed with a drop of dilute antiserum in a depression plate and allowed to react. In this way the number of serologic tests of even small nodules can be increased greatly.

B. Immunodiffusion

The technique of Ouchterlony double-diffusion has been widely used to study the antigens of root-nodule bacteria for both taxonomic and ecologic purposes (Dudman, 1964, 1971; Dudman and Brockwell, 1968; Gibbins, 1967; Humphrey and Vincent, 1965, 1969, 1975; Vincent and Humphrey, 1968, 1970, 1973). Strains which cross-react in agglutination tests because of minor similarities in their surface, particulate antigens (such as flagella) do not necessarily share other antigens as shown by immunodiffusion--a method which uses soluble antigens diffusing through gels (Eisen, 1974; Dudman, 1977).

The gel diffusion method permits the enumeration and comparison of antigens with minimal effort; but the confidence with which strains can be identified will increase in proportion to the number of antigens detected (Dudman, 1964, 1977; Eisen, 1974). Relationships between various antigens are established by observing the nature of the interaction at the junction of precipitin bands from the various wells, the number of precipitin bands being equal to the minimal number of separately diffusable soluble antigens present in the antigen well.

The somatic antigens of many Rhizobium strains diffuse slowly in the agar gels; they yield either no precipitin bands or only weak bands close to the antigen well since the location of bands is dependent upon the relative concentrations of diffusable antigens and antibodies (Eisen, 1974). Heating for various periods of time dissociates the poorly diffusable somatic antigen molecules and makes them more soluble; thus it is one of the easiest methods of antigen preparation for gel diffusion (Skrdleta, 1969; Dudman, 1971; Humphrey and Vincent, 1975). Gibbins (1967) found ultrasonic disruption prevented precipitin band formation; however, band formation was restored by heating the sonicated antigen preparation. Sonication is a useful method to liberate internal antigens which generally are not strain specific (Humphrey and Vincent, 1965; Vincent and Humphrey, 1970).

Dudman (1964) was the first investigator to adapt gel diffusion to studies of Rhizobium. His investigation of the extracellular soluble antigens of two strains of R. meliloti indicated that the two strains shared all extracellular antigens except for those strain-specific fast-diffusing polysaccharides, which were useful for identification purposes. Since the two strains did not cross-agglutinate he proposed that the strain-specific polysaccharides dominated their surfaces.

Humphrey and Vincent (1965) used gel-diffusion to show that whole-cells of R. trifolii strains grown on calcium-deficient medium yielded identical immunodiffusion patterns with mechanically disintegrated calcium-adequate bacteria. Earlier work by Vincent (1962) had shown that these strains required calcium for normal growth. The identical immunodiffusion patterns indicated that the walls of the untreated calcium-deficient bacteria were more fragile and the cells underwent autolysis with the release of their internal antigens.

In a later publication Humphrey and Vincent (1969) indicated that the somatic antigens of two strains of R. trifolii were strain specific. However, the internal antigens obtained by mechanically disrupting the cells were identical and could not be used to differentiate between strains.

Skrdleta (1969) utilizing gel-diffusion to study the serological relatedness of strains of R. japonicum divided the 11 strains into two somatic serogroups. The somatic antigens were more specific to differentiate between individual strains than those of flagella. Dazzo and Hubbel (1975) in contrast to the results obtained by Bushnell and Sarles (1939), Kleczkowski and Thornton (1944), and Koontz and Faber (1961) reported a correlation between serological properties and infectivity. They used immunodiffusion to analyze the antigenic relatedness of three infective and three non-infective strains: additional antigens were found in infective strains which were not found in non-infective strains.

C. Immunofluorescence (IF)

One of the most sensitive of the serological techniques available to study rhizobia is the fluorescent antibody (FA) technique. It allows for the visualization and investigation of the antigens of individual cells with the fluorescent microscope and requires only small quantities of both antigen and antibody (Schmidt, 1973). In contrast both agglutination and immunodiffusion require large amounts of antigen and antisera to give a visible reaction.

The FA technique originally developed by Coons et al. (1942) to visualize pneumococcal antigens in mouse tissue, was successfully adapted to studies of Rhizobium by Schmidt et al. (1968). For the first time individual cells could be identified directly in culture, in nodules, and in soil be differentiated from numerous other organisms. Using this technique, the classical approach of autecology: the study of an individual organism in its natural environment, could be applied to the study of rhizobia, or to any other soil microorganism desired.

Although immunofluorescence (IF) can be used to rapidly type the contents of root-nodules its most valuable feature is its potential ability to identify specifically bacteria directly from soil (Bohlool and Schmidt, 1979). None of the other serological techniques can be used to perform this function.

Fluorescent antibody (FA) has been used to identify strains of rhizobia (Schmidt et al., 1968; Bohlool and Schmidt, 1970, 1973b; Jones and Russel, 1972, May, 1979), to identify the nodule-bacteria (Schmidt et al., 1968; Trinick, 1969; Bohlool and Schmidt, 1973b; Jones and Russel, 1972; Lindemann et al., 1974, May 1979), to detect doubly infected nodules (Lindemann et al., 1974, May 1979), to study Rhizobium in soil (Schmidt et al., 1968; Bohlool and Schmidt, 1970, 1973b; Vidor and Miller, 1979b and c), to study population dynamics of R. japonicum in the rhizosphere (Reyes and Schmidt, 1979), and to make quantitative studies of Rhizobium in soil (Bohlool and Schmidt, 1973a; Schmidt, 1974; Reyes and Schmidt, 1979; Vidor and Miller, 1979b and c).

D. Enzyme-Linked Immunosorbant Assay (ELISA)

ELISA is the latest serological technique to be adapted for use in the identification of rhizobia (Kishinevsky and Bar-Joseph, 1978; Berger et al., 1979). The ELISA technique provides a colorimetric method for the identification of bacteria. A strain specific antiserum is conjugated to an enzyme such as alkaline phosphatase or peroxidase; and bacteria are coated with the enzyme-labelled antibody. After a period of incubation and subsequent washing, a chromogenic substrate is applied. The formation of an antigen-antibody complex is detected visually or spectrophotometrically. The ELISA is endowed with the specificity of antigen-antibody reactions and the sensitivity of enzyme-catalyzed reactions (Berger et al., 1979). It requires very small amounts of antiserum and no microscopic equipment is necessary. ELISA does not possess the flexibility of immunofluorescence; like agglutination and immunodiffusion, ELISA requires a purified antigen preparation, either from culture or from a root-nodule.

E. Antigens of Rhizobia

In general the antigens of cultures and nodules remain stable and antigenic stability is the major premise underlying the widely used serological practices described previously for serotyping root-nodules.

However, some differences between antigens of the nodule forms of Rhizobium and their parent cultures have been reported in the literature. Means et al. (1964) used antisera against cultured cells to examine the antigens of culture and nodule forms of 17 strains of R. japonicum. They found no detectable difference between the two forms among 15 strains. However, with one strain nodule-bacteria cross reacted with a wider range of antisera than the parent culture. Both heated and unheated preparations reacted the same.

Dudman (1971) used immunodiffusion to examine the antigens of cultures and nodule-bacteria of three strains of R. japonicum. Nodule-bacteria antigens from one strain lacked the full array of antigens of the cultured cells, and repeatedly formed spur reactions of partial identity with precipitin bands from the parent culture. Using this technique, the classical approach of autecology: the study of an individual organism in its natural environment, could be applied to the study of rhizobia, or to any other soil microorganism desired.

D. Enzyme-Linked Immunosorbant Assay (ELISA)

Pankhurst (1979) could detect no differences in the antigens expressed by cultures and nodules of a number of strains of Lotus rhizobia analyzed by immunodiffusion. However, he noted that there may have been a structural difference between the two forms. Whereas cultures had to be heat treated before their antigens would diffuse through the gels, the nodule forms of several strains were readily diffusable without heating. Heat treatment of the nodule suspensions generally intensified the slow diffusing somatic precipitin bands. He proposed that the readily diffusable nodule-bacteria antigens might have been due to an alteration in structure, form, or amount (though not antigenically different) of LPS as proposed by van Brussel et al. (1977) for nodule-bacteria of R. leguminosarum.

No differences have been reported between nodule-bacteria and cultured cell antigens when stained with FA’s prepared from the somatic antigens of cultured cells (Schmidt et al., 1968; Bohlool and Schmidt, 1973b; Lindemann et al., 1974, May 1979). However, no investigators have used immunofluorescence-adsorption to examine differences between nodule and culture forms of Rhizobium stained with fluorescent antibodies.

Serological markers also remain stable in soil. Brockwell et al. (1977) found serological markers to be unchanged during a three year investigation of rhizobia. Diatloff (1977) studied the stability of four rhizobial characters: colony color, effectiveness, sensitivity to four antibiotics, and antigenic stability. The antigenic and colony characteristics of the strains were not changed during a residence of five to twelve years in the soil. On the other hand, effectiveness and antibiotic sensitivity underwent slight modifications.

In summary the extensive use of serology to analyze rhizobia has revealed that heat-stable somatic antigens are more strain specific than those of whole cells (Koontz and Faber, 1961; Graham, 1963; Date and Decker, 1965; Means and Johnson, 1968; Schmidt et al., 1968; Humphrey and Vincent, 1969; Skrdleta, 1969; Dudman, 1971, 1977). However, exceptions do exist, gel diffusion of the somatic antigens from eight strains of R. meliloti (Humphrey and Vincent, 1975) revealed seven of the eight strains had identical heat-stable somatic, as well as heat-labile antigens. In this instance serology could not differentiate between strains.

II. Intrinsic Antibiotic Resistance

Josey et al. (1979) used the variation in intrinsic resistance to low levels of eight antibiotics as a characteristic (an “antibiotic fingerprint”) to identify 26 strains of R. leguminosarum. The major advantage of this method is that no alterations of the strain, which might interfere with its field performance are required. The fingerprint technique is a useful supplement to serological methods since, as with nutritional and biochemical tests, further delineation of strains that serologically cross-react may be possible. However, it is necessary to choose concentrations of antibiotics that will yield maximum strain differentiation. Unlike serological techniques this method requires isolation and culture steps which are time consuming.

III. Quantitative Techniques in Rhizobium Ecology

Only by studying the rhizobia directly in their natural habitat, rather than by indirect plant-dilution infection techniques (Date and Vincent, 1962) can the questions of survival, population densities, growth responses, interactions with other organisms, nutritional substrates, host specificity, and competition between strains for host sites be truly evaluated (Schmidt, 1978, 1979; Bohlool and Schmidt, 1979).

Although the need for a selective medium to enumerate Rhizobium has long been recognized (early literature reviewed by Fred et al., 1932), the physiological diversity of both the rhizobia and the resident soil microorganisms makes the prospects for the development of a truly selective medium specific for all rhizobia, unlikely (Schmidt, 1978). The work of Graham (1969b) and Pattison and Skinner (1973) demonstrate the difficulties inherent in making selective media for Rhizobium.

The “selective medium” most often used to detect the presence of rhizobia in soil has been the plant itself. Enumeration by the plant-dilution infection assay (or Most Probable Number, MPN) (Date and Vincent, 1962) has been the basis for virtually all ecological studies dealing with the persistence of Rhizobium in soil and their response to rhizosphere conditions (Schmidt, 1978). Unfortunately there exists a high degree of statistical uncertainty in plant-dilution infection/MPN methodology (Alexander, 1965). In addition the existence of host Rhizobium incompatibilities resulting in nodulation failures are well documented (Caldwell and Vest, 1968; Masterson and Sherwood, 1974; Sherwood and Masterson, 1974). Nodulation by only certain strains of Rhizobium might skew results when using plant-infection assays. This cannot happen with direct microscopic enumeration of FA stained cells.

Before 1973 all serological techniques, used in studies of Rhizobium, were qualitative. No means existed for the direct enumeration of rhizobia in soils at natural population levels. Breed slide counts require high numbers of rhizobia (10⁶ cells/gram) to be practicable (Bohlool, 1971; Schmidt, 1978). To work with natural populations it is necessary to remove the bacteria from interfering soil particles and to concentrate them for enumeration. Bohlool and Schmidt (1973a) described a soil release procedure and a quantitative membrane-filter FA technique (SRP), in which cells, recovered from soil on non-fluorescent membrane-filters and stained with the appropriate FA, were enumerated microscopically. The development of the SRP technique was a breakthrough in methodology for quantitative studies of microbial ecology in soil.

A. Quantitative Membrane-Filter Technique

Briefly, the quantitative membrane-filter (SRP) technique consists of dispersing by blending a soil sample in water. The soil suspension is then transferred to a narrow container, flocculant is added and the soil colloids allowed to settle. After settling, an aliquot of the supernatant is passed through a membrane-filter, stained with FA and the FA reactive bacteria enumerated (Bohlool and Schmidt, 1973a; Schmidt, 1974).

Since its development, the use of the quantitative technique in microbial ecology has not often been reported in the literature. Bohlool and Schmidt (1973a) followed the growth of a strain of R. japonicum (USDA 110) in autoclaved soil (Clarion) comparing filter counts to plate counts. The filter counts tended to underestimate at low cell numbers (approximately 30% of plate counts) and overestimate the plate counts at high numbers (approximately 150%), probably due to the accumulation of dead cells. The technique was used in a field study to examine the population level of the same strain in the rhizosphere of both inoculated and uninoculated (to examine the indigenous population) soybean plants. The rhizosphere levels of the uninoculated plants remained fairly constant (approximately 5 x 10³/gram of soil) while the rhizosphere populations of inoculated plants were both higher and more variable (attributed to uneven inoculation).

Schmidt (1974) followed the growth of Nitrobacter winogradski in a partially sterilized soil. He compared growth with nitrate formation; the two parameters correlated well during the exponential phase of growth, however, nitrate formation continued to increase when the growth of the population apparently leveled-off.

Several reports exist in the literature describing problems in implementing the membrane-filter quantitative technique. Reed and Dugan (1978) used the quantitative method, with indirect immunofluoresence, to determine the distribution of methane oxidizing bacteria in sediments from Cleveland harbor. They recovered only 10% of the methane oxidizers in the sediments. With several minor modifications Reyes and Schmidt (1979) obtained a 44% recovery, as compared to plate counts, of a strain of R. japonicum (USDA 123) from a sterilized Minnesota soil (Waukegan). The quantitative technique was originally developed using a different soil:strain combination (Clarion:USDA 110). Reyes and Schmidt (1979) did not compare recoveries of USDA 110 in sterilized Waukegan or USDA 123 in sterilized Clarion soil to determine if their problem with recovery was methodological, or related to the soil, or the strain. To aid recovery Vidor and Miller (1979a, b and c) substituted 1% CaCl₂ as a flocculant.

Wollum and Miller (1980) modified the density gradient centrifugation technique, used for studies of clay particles by Francis et al. (1972), to use for quantitative studies of Rhizobium. They examined the recoveries of two strains each, of the slow grower R. japonicum, and the fast grower R. phaseoli. High recoveries were obtained when the soils contained 10⁸ - 10⁹ cells/gram. Recoveries were better with temperate soils than with two South American Oxisols. The authors had difficulty in clearing the Oxisols; they suggested additional studies were needed.

B. Sorptive Interaction Between Microorganisms, Clay

Particles and Soils

The predominance of the solid phase is one of the main characteristics that distinguishes soil from other microbial habitats (Marshall, 1976; Stotzky and Rem, 1966). In habitats which have wide liquid:solid ratios, such as the oceans, much of the microbial activity is associated with solid, particulate materials (ZoBell, 1943: Jannasch, 1967, 1970; Pearl, 1975). In fact, attachment may be advantageous to microorganisms living in dilute environments. Nutrients may concentrate at the interface because of differences in charge between the solid surface and the surrounding solution, as well as to differences in the hydrophobic or hydrophyllic nature of the surface (ZoBell, 1943; Stotzky, 1966a and b; Fletcher, 1979).

Of the solid phase components in soil, the smallest mineral particles are the clays. It is due to their large surface area that clays exert the greatest influence on soil microorganisms (Stotzky and Rem, 1966; Stotzky, 1966a and b). Clay minerals are usually associated as aggregates or occur as coatings on larger particles (Brady, 1974). Clays differ from other particles normally present in natural microbial habitats in that they have an overall net negative charge, but also have positive charges at the broken edges. These charges are neutralized by ions from the surrounding solution, and by interactions with adjacent minerals (Brady, 1974). In many tropical soils the minerals and clay size particles are coated with an additional layer of oxides whose charges are pH dependent (Sanchez, 1976). The charges on these coatings vary with local soil conditions. Factors such as the type of clay, saturating ions, and the organization of the clay minerals within the soil matrix may be more important than the total amount of clay present in the soil habitat (Marshall, 1971; Stotzky, 19743).

The degree of sorption between microorganisms and soil particles is broadly related to the surface area and surface charge properties of the particles (Daniels, 1972). The positive and negative charges on microbial cells observed at different pH’s (Daniels, 1972; Lamanna and Mallette, 1965; Marshall, 1967) are due to the degree of ionization of surface components. Therefore the overall charge on the cell is determined by the isoelectric points or pH’s of these constituents. Most organic materials of biological origin have either no charge, or they are amphoteric and have a charge dependent on pH (Lehninger, 1970; Metzler, 1977).

Fast growing strains of Rhizobium (R. trifolii, R. meliloti, R. leguminosarum) exhibit a sharp increase in negative charge at high pH values (pH 11) and a slightly positive charge at low pH’s (pH 2) when assayed by electrophoretic mobility techniques (Marshall, 1967); this is generally true of most bacteria (Lamanna and Mallette- 1965; Daniels, 1972). The slow growing strains of Rhizobium (R. japonicum, R. lupini) possess either no charge at low pH (pH 2) or a negative charge at all other pH’s. The rhizobia can therefore be separated into two groups on the basis of their surface ionogenic character (Marshall, 1967, 1968), as well as by differences in growth rate, G+C, flagellation (Jordan and Allen, 1974), carbohydrate utilization (Fred et al., 1932, Graham and Parker, 1964; Vincent, 1977), glucose-6-phosphate dehydrogenase (Martinez-Drets and Arias, 1972; Martinez-Drets et al., 1977), and internal antigens (Humphrey and Vincent, 1965; Vincent and Humphrey, 1970; Vincent et al., 1973).

Marshall et al. (1971) proposed two major mechanisms for the sorption of marine bacteria to glass surfaces: (1) a random attachment concerning both motile and non-motile bacteria which involved polymeric bridging between the cells and the solid substrate; (2) an instantaneous reversible mechanism involving only motile bacteria which they believed was due to electrostatic attractions and van der Waals forces (physico-chemical). Fletcher (1977) found that the attachment of a marine pseudomonad to polystyrene petri dishes as dependent on the number of cells present, the time allowed for attachment, the growth phase of the culture (cell age and morphology), and temperature. Log phase cultures had the greatest facility for attachment, followed by stationary and death phase cultures, respectively. She found that the results could be described by a model based on physical/chemical adsorption (Langmuir adsorption isotherms). The model indicated that non-biological processes were playing a major role in initial events of bacterial adhesion. Fletcher’s model is similar to those describing molecular adsorption from solutions onto surfaces, in which the process is controlled by the concentration of the solution (or bacterial culture), time, and temperature. The relationship between solution concentration and extent of adsorption is termed the adsorption isotherm. A Langmuir Isotherm assumes: (1) adsorption is limited to a monomolecular layer; (2) adsorption is localized so that adsorbed components are confined to specific sites; and (3) the heat of adsorption is independent of surface coverage (Fletcher, 1977).

Scheraga et al. (1979) found that bacteria added to autoclaved marine sediments were adsorbed almost instantaneously. They plotted their data according to Fletcher (1977) and obtained Langmuir-type adsorption isotherms. This would indicate that the initial events in bacterial adsorption to sediments are similar to those observed with polystyrene. Thus, it seems likely that both biological and nonbiological forces are involved in sorptive interactions between microorganisms and their environment.

Few reports exist describing actual sorptive forces in soil. The problem is not necessarily one of methodology but one of deciding which fractions in soil are most influential, for example clay, organic matter, clay:organic matter complexes, oxide coatings, all fractions interacting simultaneously, or particles of larger size coated with these substances. The factors which are most influential in sorptive processes in one soil may have no bearing on those operative in another soil.

Niepold et al. (1979) advanced a model system to describe various forces which may influence the recovery of bacteria from soil. Bacteria may attach to soil particles by capillary (Hattori, 1973; Hattori and Hattori, 1976), electrostatic and adhesive forces (Marshall, 1971). Niepold et al. (1979) reasoned that detachment of bacteria from soil particles might be influenced by the chemical properties of the extraction fluid. They adsorbed the hydrogen bacterium Alcaligenes eutrophus to three types of model materials with differing capillary properties (“Mosy”, a porous material used for arrangements of cut flowers, pore sizes 0.1-0.5 mm in diameter), electrostatic properties (DEAE-cellulose), and adhesive properties, i.e. physico-chemical (125-200 mm diameter glass beads). In these studies A. eutrophus was more strongly held by capillary forces than by adhesive forces. Based on viable counts they found recovery to be lowest when extraction fluids contained detergents such as Tween 80, Triton X-100, Sodium Dodecyl Sulfate (SDS), and sodium desoxycholate. Recovery was also low when extraction fluids contained organic solvents such as DMSO, or dioxane. The highest recoveries were obtained with Tris buffer (pH 7.5). The low recoveries with detergents and organic solvents were attributed to the toxic effects these compounds had on the cells. Litchfield et al. (1975) also found SDS to be toxic when used to quantitate bacteria in marine sediments by viable counts.

Once extracted from soil, bacteria must be separated and recovered for enumeration. Niepold et al. (1979) investigated various methods for the separation of bacteria from model materials and soils. Filtration of soil suspensions through filter paper, settling for eight hours, and slow speed centrifugation (325 X g, 5 minutes) resulted in the lowest recoveries. In contrast, letting the soil suspension settle for 15 minutes prior to dilution for plate counts, resulted in greater recoveries. A flocculant to remove the soil colloids from solution was never tried. They adsorbed eight strains of hydrogen bacteria of differing size, motility, and slime formation to each of three soils. The efficiency of extraction of the adsorbed bacteria was strongly dependent on the bacterial strain used but was not significantly dependent upon soil type.

Rubertschik et al. (1936) used plate counts to demonstrate the ability of sediments to remove bacteria from suspension. The degree of sorption (e.g. removal) varied with the sediment and the species of bacteria. Shaking the mixtures for one minute did not increase desorption, i.e. the viable counts did not increase.

Hornby and Ullstrop (1965) found that for dilution plating from soil suspensions, agitation by blending or rapid stirring gave better sampling precision than shaking with a reciprocating shaker. They also found that a viscous solution, such as 1% carboxy-methyl cellulose or 0.2% agar gave better reproducibility than soil suspended in water. Sodium hexa-meta-phosphate (Na-HMP) is a powerful soil deflocculant (Michaels, 1958; Lahav, 1962). The many negative charges of phosphate ions combine with the broken edges of clay particles (positively charged) and neutralize their charges, thus soil particles are dispersed by mutual repulsion. Gamble et al. (1952) briefly blended (45 seconds) 10 grams of soil in 100 mls of 5% NaHMP, added 400 mls of water to produce a total dilution of 1:50, and then blended for another two minutes before plating. They felt this procedure would release the bacteria for plate counts. However, they never compared their method with any others.

Both Balkwill et al. (1975) and Faegri et al. (1977) used a combination of sequential homogenizing procedures with blendors, and sedimentations by centrifugation to extract and concentrate bacteria from soils. Faegri et al. (1977) enumerated bacteria extracted from Norwegian soils by direct counts of acridine orange (AO) stained cells (AO is a non-specific fluorescent dye) recovered on black membrane-filters. Depending upon the soil type almost equal numbers of cells were obtained in the first two extractions. Fewer cells were recovered following a third extraction.

From the foregoing discussion, it appears that there are many different opinions as to what method is best to recover and enumerate bacteria from soil. Depending upon the type of counting procedure to be employed, the type(s) of microorganism(s) to be enumerated and the type of soil which the bacteria inhabit, it is problematical whether only one or more of the described procedures could work. Except for Wollum and Miller (1980) all the investigations cited above have dealt mainly with temperate soils. There is a need, therefore, to determine which methods will function in tropical soils for the quantitative recovery of Rhizobium using the FA-membrane filter procedure.

dry completely on each filter prior to FA staining (May, 1979). Gelatin coated filters could be stored dry, either in a desiccator, or left in a drying-oven (60⁰C) for long periods of time prior to staining. This greatly simplified the processing of large numbers of samples. All FA enumerations were made with a Zeiss universal microscope. Incident illumination was from an HBO-200 (OSRAM) light source, and a Zeiss Fluorescein Isothiocyanate (FITC) filter.

Recovery of TAL-620 from Eight Tropical Soils

To determine the sorptive nature of various tropical soils for a strain of chickpea Rhizobium, eight soils, representative of three soil orders common to the tropics, were chosen: Wahiawa (Oxisol); Molokai (Oxisol); Lualualei (Vertisol); PLP, Burabod, LPHS, Makiki, and Waimea (all Inceptisols). An exponentially-growing culture of TAL-620, enumerated with a Petroff-Hauser chamber, was adjusted to 2 x 10⁷ cells/ml with saline (= Inoculum). The actual inoculum size was verified by FA-membrane-filter counts of the inoculum. Each tube of ten grams of non-sterile soil received two ml of inoculum

(= 4 x 10⁶ cells/gram soil). Two tubes containing ten grams of silica sand served as inoculated non-soil controls. After incubation for two hours at room temperature (24⁰C) the recovery of TAL-620 was assayed using the procedure of Bohlool and Schmidt (1973a) with the modifications previously described (see Appendix Table 21).

Soil Titrations

This series of experiments was designed to illustrate the sorptive capacities of soils, both tropical (5 soils) and temperate (2 soils), for fast growing rhizobia. In these experiments the number of bacteria added to each treatment was identical; the variable was the increasing quantity of

soil. The six treatments used are listed below: Treatment 1 served as a non-soil control; each treatment was run in duplicate.

The following soils were tested: Burabod, Clarion, Hubbard, Lualualei, Molokai, Wahiawa, Waimea. Both the TAL-620 and Hawaii-5-0 strains were used. The cultures were adjusted to approximately 2 x 10⁷ cells/ ml with a Petroff-Hauser counting chamber; one ml was added to the non-sterile soils and incubated for two hours at room temperature (24⁰C). The number of cells contained in the inoculum was verified by IF-membrane-filter counts. The influence of the various treatments, i.e. increasing soil, on the recovery of strains was assayed by SRP (see Appendix Table 21).

Use of Different Diluents/Extractants and Flocculants to Increase

Recovery of TAL-620 from Wahiawa Soil

The Wahiawa soil was chosen as the model problem soil. It consistently gave one of the poorest recoveries. In these experiments different extracting solutions were substituted for the H₂O-Tween 80 extractant normally used in the SRP procedure. Mid-exponential phase cultures of TAL-620 were enumerated by Petroff-Hauser counts and adjusted to give 3 x 10⁶ to 1 x 10⁷ cells/gram of non-sterile soil. The final inoculum size was confirmed by FA-filter counts. The inoculated soils incubated two hours at ambient temperature (24⁰C) prior to assay for recovery by SRP.

The nonionic detergent Nonidet P40, (0.5% solution) was substituted for Tween 80. Solutions of salts at different concentrations were tried (1M KCl, 3M NaCl), low pH (0.4M HCl), organic solvents (100% methanol, 10% ethanol), two different anion extractants CuSO₄: Ag₂SO₄ (used for NO₃^- and NO₂^- extraction from soils, Jackson, 1962), and NaHCO₃ (used for the extraction of soil phosphorus, Jackson, 1962), solutions of the chelator EDTA (0.01 and 0.1M), two solutions of peptone (1%, 2%), and two solutions containing partially hydrolyzed gelatin (PHG) were also substituted.

To prepare PHG a 1% solution of Bacto-Gelatin was adjusted to pH 10.3 with 1N NaOH and autoclaved for ten minutes at 121⁰C to partially hydrolyze the gelatin. When required the 1% solution was diluted to 0.1% either with water, or with 0.1M phosphate buffered saline (PBS) (see Appendix Table 22); thimerosal was added (final concentration 1:10,000) to prevent bacterial growth in all gelatin solutions.

Results and Discussion

Reports in the literature indicate that the soil release procedure (SRP) (see Appendix Table 21) does not fully release and extract Rhizobium and other bacteria from temperate soils (Reyes and Schmidt, 1979; Vidor and Miller, 1979a; Schmidt, 1974). In addition, seven soils representative of those commonly found in the tropics were highly sorptive for strains of fast-growing rhizobia when assayed by this procedure. The recovery of TAL-620, a strain of chickpea Rhizobium, from eight tropical soils is summarized in Table 6. All soils except one (Waimea) were highly sorptive for the added bacteria; recoveries were extremely low, in several cases less than 1% of the added level.

The Wahiawa Oxisol was the most highly sorptive of the eight soils and gave the poorest recovery. Poor recovery of Rhizobium was not related to soil order as both Oxisols (highly weathered, “old” soils) and Inceptisols (little weathered, “young” soils) retained the bacteria. (The soil order is the broadest level in soil classification and is equivalent to the phylum in biological classification.) Recovery of bacteria from soil may be related to the finer classification of soils such as at the sub and great group level, where both mineralogy and soil forming conditions are considered: from a typic eutrandept (Waimea) 100% of the inoculated bacteria were recovered, while recovery from hydric dystrandepts (PLP, LPHS, Burabod) and an andic ustic humitropept (Makiki) were much poorer.

The capacity of 5 tropical and 2 temperate soils to sorb bacteria and their recovery and enumeration by SRP was shown by the soil titration experiments. Figures 6, 7, and 8 show the inverse relationship between increasing soil content and recovery; as the quantity of soil in each treatment increased, recovery of added Rhizobium decreased. The strain of lentil Rhizobium Hawaii-5-0 was strongly sorbed by the Wahiawa soil as shown in Figure 6. This indicates that the poor recovery of TAL-620 was not a strain specific phenomenon. In fact Hawaii 5-0 was more strongly adsorbed by the Wahiawa soil than TAL-620. The Waimea Inceptisol (see Figure 7) and the two midwestern soils (Figure 8) had less sorptive capacity relative to the other soils; the decrease in recovery of added bacteria was not as great.

Seventeen different extractants were substituted for H₂O in the SRP recovery procedure, these included 2 nonionic detergent, salts, low pH, alcohols, solutions of peptone, EDTA, and two solutions of partially hydrolyzed gelatin. Of these 17 extractants (see Tables 7 and 8) four gave recoveries greater than 30% (see Table 8): 0.5M NaHCO₃ (33% recovery), 1% peptone (33% recovery), and 0.1% PHG in phosphate buffered saline (60% recovery). It was important to select the proper concentration for the

CHAPTER 5

MODIFIED MEMBRANE FILTER - IMMUNOFLUORESCENCE FOR ENUMERATION

OF RHIZOBIUM FROM TROPICAL SOILS

Introduction

Immunofluorescence (IF) provides a direct method for in situ autecological studies of microorganisms; it allows for the simultaneous detection and identification of the desired organism in its natural habitat. The technique can be made quantitative by separating the bacteria from soil particles and concentrating them on non-fluorescent membrane-filters for IF enumeration (Soil Release Procedure, SRP, see Appendix Table 21). In applying SRP to study Rhizobium in tropical soils I encountered great difficulty in releasing bacteria from soil particles and recovering them for IF enumeration (see Chapter 2).

Most tropical soils were highly sorptive for Rhizobium when assayed by SRP. However, as discussed by Niepold et al. (1979) the chemical composition of the extraction fluid greatly influenced the degree of sorption. Extraction solutions containing proteins (Partially Hydrolyzed Gelatin, PHG) were most successful in increasing recovery of rhizobia from a Hawaiian Oxisol, chosen as the model problem soil. Therefore, the failure to recover rhizobia sorbed to soil is directly related to the techniques used to extract and enumerate them. The bacteria are not irreversibly bound. All that is required is the proper methodology to recover the cells. In addition, results obtained with partially hydrolyzed gelatin solutions indicated that ion exchange may be important in the recovery of bacteria from tropical soils.

The present work describes efforts to optimize recovery of Rhizobium from tropical soils. Experiments to select the best diluting solution to use with PHG were undertaken. In the course of these studies I realized that blending the soils (SRP, Appendix Table 21) especially the well aggregated Oxisols led to a breakdown of the stable aggregates and to a very large increase in surface area. This increased surface, by creating more area for sorptive interactions, probably led to lower recoveries. I reasoned that the chemical extractants might work better when used with a less disruptive dispersion method. The final outcome of these investigations was the development of a modified soil release procedure (MSRP). The soils were dispersed by shaking on a wrist-shaker in flasks containing glass beads and a chemical extractant using PHG.

Materials and Methods

Source and Maintenance of Cultures

Two strains of fast growing rhizobia, TAL-620 (Rhizobium for chickpea, see Chapter 1, Table 1), Hawaii-5-0 (Rhizobium leguminosarum, May, 1979), and two strains of slow-growing rhizobia, USDA 31 and USDA 110 (both R. japonicum) (Dr. D. F. Weber, USDA Beltsville, Md.) were used in these experiments. All strains were maintained on a modified YEMS medium (see Appendix Table 19a), and when required were grown in broth of the same composition. Exponential phase cultures were enumerated by direct counts with a Petroff-Hauser chamber. The cultures were diluted in saline to the desired inoculum size. For experiments, one or two ml of the inoculum was added to moistened soil. Strict aseptic techniques were followed at all steps when autoclaved soils were to be inoculated.

Chemical Reagents

The chemical reagents have already been described (see Chapter 2, Materials and Methods). Granulated gelatin was obtained from three sources: Difco Bacto-Gelatin (2 lots, control #459821, and #464194), Difco, Detroit, Michigan; Fisher Bacterilogical Gelatin (Lot number illegible), Fisher Scientific, Fairlawn, N. J.; U.S.P. Granular Gelatin (No lot number), Pioneer Chemical Co., Inc., Long Island City, N. Y.

Soil Samples, Soil Sterilization

The soils used in these experiments, and their method or preparation and distribution were described previously (see Chapter 2, Materials and Methods). Soil sterilization, when needed was done by autoclaving for 1.5 hours at 121⁰C (Bohlool, 1971).

Preparation of Fluorescent Antibodies, FA Staining, Epifluorescence

Enumeration

Fluorescent antibodies were prepared by the method of Schmidt

et al. (1968). The soil release enumeration procedure (SRP) (Bohlool and Schmidt, 1973a; Schmidt, 1974) is described in Appendix Table 21). Modifications to the published procedures were described previously (see chapter 2, Materials and Methods).

Preparation of Partially Hydrolyzed Gelatin (PHG)

A 1% solution of gelatin (10 g/L H₂0) was adjusted to pH 10.3 with 1N NaOH, and autoclaved 10 minutes at 121⁰C to partially hydrolyze the gelatin. The hydrolyzed solution was stored at 4⁰C until used; thimerosal was added, 1:10,000 final concentration, as a preservative. All experiments unless indicated otherwise used Difco Bacto-Gelatin Lot No. 464194 (will be referred to as gel #1).

SRP - Effect of Different Strength Gelatin Solutions

To determine what concentration of gelatin would give maximum recovery of added bacteria, a 1% solution of PHG was used either undiluted (1%) or was diluted with water to give solutions containing 0.01%, 0.05%, 0.08%, 0.1%, 0.15% PHG. All solutions were adjusted to pH 7, and thimerosal was added, 1:10,000, to prevent bacterial growth. A mid exponential phase culture of TAL-620 was enumerated by direct counts with a Petroff-Hauser chamber, and adjusted to

1 x 10⁷ cells/ml; one ml portions were inoculated into 25 x 200 mm screw-cap tubes containing 10 g of non-sterile Wahiawa soil. Duplicate tubes were inoculated for each gelatin concentration tested (12 tubes). The soils, after incubating for two hours at 24⁰ C, were assayed with SRP for recovery of inoculated Rhizobium. The procedures were the same as described in Appendix Table 21 except that the various PHG solutions were substituted for H₂O-Tween 80.

SRP - Recovery of TAL-620 from Wahiawa Soil, 0.1 PHG (in H₂O) at

Different pH’s

Solutions of 0.1% PHG (1% diluted to 0.1% with H₂O) were adjusted to pH 4, 6, 7, 8, and pH 9 with 1N HCl and 1N NaOH. Thimerosal was added, 1:10,000, to prevent bacterial growth. A mid exponential phase culture of TAL-620 was enumerated by direct counts with a Petroff-Hauser chamber, and adjusted to 1 x 10⁷ cells/ ml. One ml was inoculated into 10 g of non-sterile Wahiawa soil to give approximately 1 x 10⁶ cells/g soil; duplicate tubes were used for each PHG solution. The inoculated soils were incubated for two hours at 24^oC prior to assay for recovery. The assay procedure was the same as that in Appendix Table 21 except that PHG solutions were substituted for the H₂O-Tween 80 extractant.

SRP - Recovery of TAL-620 from Wahiawa Soil, 0.1% PHG, Use of

Different Diluents

A 1% solution of PHG was prepared as described previously. After autoclaving, the solution was adjusted to pH 7 and thimerosal was added 1:10,000. The following solutions were used to test for percentage of recovery in conjunction with PHG (final strength of PHG - 0.1%): H₂0; 0.1M Na-EDTA; 0.5M NaHCO₃; 0.001M NaHMP; 0.1M PBS. A mid exponential phase culture of TAL-620 enumerated, as described above, was adjusted to 1 x 10⁷ cells/ml; one ml was inoculated into 10 g of non-sterile Wahiawa soil to yield approximately 1 x 10⁶ cells/g of soil; duplicate tubes were used per each PHG solution tested. After a two hour incubation at 24^oC the soils were assayed for recovery with SRP. Procedures were the same as those described in Appendix Table 21 except that PHG solutions were substituted for H₂O-Tween 80.

Development of a Modified Soil Release Procedure (MSRP)

Rather than blending the soil, the modified procedure required shaking the soil and extracting solution together on a wrist-action shaker, in a screw-cap flask with glass beads. Several PHG-diluent combinations were tested: 0.1% PHG - H₂0; 0.1% PHG - O.1M Na₂HPO₄ (pH 9); 0.1% PHG - 0.1M (NH₄)₂HPO₄ (pH 8.3); 0.1% PHG - 0.1M PBS (pH 7.1). Thimerosal was added to all gelatin solutions, final strength 1:10,000. A mid exponential phase culture of TAL-620 was adjusted to 1 x 10⁷ cells/ml as described previously; one ml was inoculated into 10 g of non-sterile Wahiawa soil to give approximately 1 x 10⁶ cells/g of soil. Duplicate tubes were used for each PHG solution tested. The inoculated soils were incubated for two hours at 24^oC prior to assay for recovery.

The assay procedure was the same as that described in Appendix Table 21 with the following modifications: the soils and extracting solution were added to 250 ml screw cap flasks containing 25 - 30 g of 3 mm glass beads. The flasks were shaken for five minutes on a Burrel wrist-action shaker to disperse the soil, all other procedures (flocculation, filtration, enumeration, etc.) were the same as those described in Appendix Table 21.

MSRP - Effect of the Decreasing Hydrated Radius of Four Monovalent

Cations Upon Recovery of TAL-620 from Wahiawa Soil

Solutions of 0.2M strength prepared from LiCl, NaCl, KCl and NH₄Cl were mixed with 1% PHG (prepared as described previously) to yield the following extractant solutions: 0.1% PHG - 0.2M LiCl; 0.1% PHG - 0.2M NaCl; 0.1% PHG - 0.2M KCl; 0.1% PHG - 0.2M NH₄Cl. A mid exponential phase culture of TAL-620 was adjusted to 3 x 10⁷ cells/ml as described previously; one ml was added to 10 g of non-sterile Wahiawa soil, in duplicate tubes. After a two hour incubation at 24^oC the soils assayed for recovery by MSRP.

MSRP - Effect of Gelatin Type (Manufacturer) and Shaking Time on

Recovery of TAL-620 from Wahiawa Soil

Four 1% solutions were prepared from each of the granulated gelatin preparations listed above under Chemical Reagents; thimerosal was added as a preservative and the solutions were stored at 4⁰C until used. All gelatin solutions were adjusted to 0.1% PHG with O.1M (NH₄)₂HPO₄ (pH 8.3). A mid exponential phase culture of TAL-620 was adjusted to 2 x 10⁶ cells/ml, one ml was inoculated into duplicate tubes containing 10 g of non-sterile Wahiawa soil. A two hour incubation at 24⁰C preceeded the recovery assay. The soils and extractants were shaken for five minutes.

The effect of shaking time was evaluated using Difco gel #1. A 1% PHG solution was prepared as described previously; this solution was adjusted to 0.1% with O.1M (NH₄)₂HPO₄ (final pH of extractant = 8.3). Four dispersion times were tried. The soils and extractant were shaken for 5, 15, 30, and 60 minutes.

MSRP - Soil Titrations

The procedures used to construct the titrations were described in the Materials and Methods section of Chapter 2. The SRP assay discussed in Chapter 2 and this assay (MSRP) were conducted simultaneously. For these experiments the MSRP extractant consisted of 0.1% PHG - O.1M (NH₄)₂HPO₄ (final pH = 8.3); the soil and extracting solution were shaken for five minutes.

Growth of Fast and Slow Growing Rhizobium in Sterile Wahiawa Soil

The growth of TAL-620 was followed for 20 days in sterile Wahiawa soil. The bacteria were enumerated directly from the soil by both viable counts and MSRP (MSRP = 0.1% PHG - O.1M (NH₄)₂HPO₄,shaking for 5 minutes). One set of data was analyzed by viable counts, SRP and MSRP. For viable counts soils were mixed with 95 ml of sterile H₂O in sterile screw cap flasks containing 25 - 30 g of 3 mm glass beads, and shaken for five minutes. After dispersion, the appropriate dilutions were plated onto YEMS medium (see Appendix Table 19a) using the Miles and Misra drop plate technique (Vincent, 1970). The procedure was modified to use a Pipetman P-20 adjustable micropipeter (Rainin Instrument Co., Inc., Woburn, Mass.) and disposable tips (sterilized by autoclaving). The pipetor was adjusted to dispense 20 pl of solution; one disposable tip was used per dilution.

A mid exponential phase culture of TAL-620 was adjusted to 1 x 10⁶ cells/ml; and one ml was inoculated into 10 g of sterile Wahiawa soil to yield approximately 1 x 10⁵ cells/g. When necessary the soils were moistened with several drops of sterile distilled water. The tubes were sampled randomly, four tubes were assayed per data point.

The growth of two strains of R. japonicum was followed for seven days (USDA 31) and fourteen days (USDA 110) in sterile Wahiawa soil. The bacteria were enumerated by viable count (as described), SRP, and MSRP. Mid exponential phase cultures of USDA 31 and USDA 110 were enumerated in a Petroff-Hauser chamber, adjusted to approximately 10⁵ cells/ml, and one ml was aseptically inoculated into the soils to give approximately 10⁴ cells/g. Population levels were determined after five days incubation at 28⁰C (USDA 31, USDA 110), seven days (USDA 31), and fourteen days (USDA 110). Duplicate tubes were used for each measurement.

Growth of USDA 110 in Sterile Clarion Soil

The growth of USDA 110 was followed in sterile Clarion soil for 4 days using plate counts, SRP and MSRP. A mid exponential phase culture of USDA 110 was enumerated with a Petroff-Hauser chamber and adjusted to 2 x 10⁶ cells/ml; one ml was inoculated aseptically into tubes containing 10 g of autoclaved Clarion soil to give approximately 2 x 10⁵ cells/g. The cells were counted three and five days after inoculation. The tubes were incubated at 28⁰C; duplicate tubes were used for each measurement.

Statistical Methods

A one way analysis of variance (ANOVA) (Sokal and Rohlf, 1969) was used to determine if the monovalent cation treatments had a significant effect on extraction of Rhizobium from soils and to determine if the type of gelatin was important, as well as the shaking/extraction period.

Results and Discussion

The results discussed in Chapter 2 indicated the difficulty in quantitatively recovering bacteria from tropical soils when using SRP (see Appendix Table 21); seven of eight tropical soils were highly sorptive for added rhizobia. Several other investigators have also experienced difficulty in recovering bacteria quantitatively from soil and sediments (Vidor and Miller, 1979a; Reyes and Schmidt, 1979; Reed and Dugan, 1978). However, the substitution of solutions of partially hydrolyzed gelatin (PHG) led to increased recovery of added bacteria.

Solutions of gelatin, due to their protein origins are amphoteric, i.e. they have pH dependent charges. The charges on the gelatin molecules can interact with both soil colloids and microbial cells. Partial hydrolysis of the gelatin increases the number of small peptides which can interact with the soils exchange complex and therefore satisfy charges within the matrix. By satisfying charges on both bacteria and soil particles the two should not bind due to any charge interactions. Preliminary evidence indicated that ion exchange phenomena might be involved in the extraction of bacteria from the soil matrix. PHG solutions mixed with phosphate (sodium) buffered saline gave greater recovery than PHG mixed with H₂O (see Table 8). Therefore, experiments were designed to establish the optimum concentration of PHG; to establish the importance of the extracting solution, and to determine the proper combination of ions for increasing recovery, as well as to determine if ion exchange processes were involved.

A solution of 0.1% PHG gave the best quantitative recovery of TAL-620 from the Wahiawa soil (see Table 9). Concentrations of gelatin greater than 0.1% prevented flocculation of the soil colloids. I did not determine if the lack of flocculation was due to the increased viscosity of the more concentrated PHG solutions, relative to H₂O or to an increase dispersive effect. The addition of more flocculant did not precipitate the soil colloids.

In addition to selecting the proper PHG concentration, it was necessary to select the optimum pH for extraction (see Table 10). A low pH gelatin solution (pH 4) gave poorer quantitative recovery than solutions at higher pH. Solutions at pH 8 and pH 9 gave quantitative recovery of added Rhizobium. However, good recovery was obtained at pH 6 and pH 7. Preliminary results had demonstrated that high pH would disperse the Wahiawa Oxisol. However, high pH alone did not lead to quantitative recovery of Rhizobium (Table 8, NaHCO₃ solutions, pH 8.2 - 8.3).

Previous results (see Chapter 2, Table 8) demonstrated the importance of selecting the proper diluent to mix with the gelatin solution, e.g. H₂O vs. PBS. I decided to mix several of the solutions which had given slightly increased recoveries when substituted for H₂O-Tween 80 in SRP with solutions of PHG. Both sodium-EDTA and sodium bicarbonate solutions were not satisfactory when mixed with PHG (see Table 11) as recoveries were below 50% of the inoculum. A combination of PHG and the soil deflocculant sodium hexa-meta-phosphate did not increase recovery of added Rhizobium. In addition, extractant solutions of PHG and water gave quite variable results from 20% recovery up to 80%.

During the course of these experiments, trends in the data indicated that

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