DEVICE FOR ASSESSING NEMATODE VITALITY AND METHOD FOR USING SAME

Abstract: Method and device for the in vitro testing of potential nematicidal and nematistatic agents, comprising treatment of nematodes with test substances, followed by assessment of the ability of the treated nematodes to move through a chemattractant gradient set up on a coated solid support, said coating comprising a gellable polymeric substrate incorporating a stabilizing agent.

Description: TITLE OF THE INVENTION; DEVICE FOR ASSESSING HBtATODB VITALITY AND METHOD FOR DSISG SAME BACKGROUND OF THE INVENTION Field of the Invention This invention is directed to a device for assess¬ ing the vitality of nematodes as a measure of the ef¬ ficacy of potential nematicidal and nematistatic agents. The assay device comprises a solid support coated with a gellable polymer intimately mixed with parti¬ cular chemicals that serve to stabilize the assay. Included in the device are disks impregnated with a particular chemical that is an attractant for nema¬ todes. Included in the invention are methods for us¬ ing the aforementioned device in chemotaxis assays, such assays being useful for assessing the efficacy of particular compounds as nematicidal or nematistatic agents. Brief Description of the Background Art The invasion of plant roots by parasitic nematodes leads to destruction of the plant. This is a serious problem in the agricultural industry world wide. For -2- example, the genus Meloidoqyne can infect more than 2000 different species of plants. R.S. Hussey, Advanced Treatise on Meloidogyne, vol. 1, North Caro¬ lina State Univ./Graphics, Raleigh, N.C. , 1985. It is well known that the invasion of host cells is preceded by the accumulation of plant-parasitic nematodes around plant roots. H.B. Jansson et al., J. Gen. Microbiol. 112:89 (1979). It has long been recognized that one approach to preventing invasion of plant roots by nematodes is to interrupt the mechan¬ isms of such accumulation. It is recognized that plant-parasitic nematodes utilize specific recognition mechanisms for finding their hosts and prey in the soil. The general consen¬ sus is that, in nematodes, the primary food-finding mechanisms are governed by chemotactic factors emanat¬ ing from the host. This process, which is called chemotaxis, mediates movement toward or away from chemical gradients. In no cases known to the inven¬ tors have the chemattractants emanating from plants or microorganisms been isolated and identified. Other stimuli, such as thermal, vibratory or tactile stimuli are believed to play minor roles, if any, in food- finding behavior. N.A. Croll, et al., in The Behavior of Nematodes: Their Activity, Census, and Responses, Edward Arnold, Publisher, London, 1970; B.M. Zucker- man, et al., Ann. Rev. Phytopathol. 2* 95- 113 (1984). There is no direct evidence on the manner in which reception of chemotactic signals leads to an oriented response, but a recently proposed hypothesis on ne a- tode che oreception speculates that carbohydrates exuded by chemosensory sensillae bind to the cuticle glycocalyx and thereafter interact with the chemotac¬ tic factors, and thereby play a role in chemorecep- tion. B.M. Zuckerman, 1984 supra. Another hypothesis speculates that, following binding of plant chemat¬ tractants to receptors in the sensory process, there results an influx of both sodium and calcium ' ions via ion pumps, thereby causing the generation of action potentials. K.A. Wright, J. Nematol. 15:151- 158 (1983). At present parasitic nematodes are attacked by chemical agents with nematicidal or nematistatic pro¬ perties, in order to prevent their invasion of plant roots. The commonly used compounds are toxic, not only to plant parasitic nematodes, but to animals (in¬ cluding man) as well. For example, one of the o-(methylcarbamoyl) oxi es, aldicarb (Temik 10G Union Carbide), is regis¬ tered for use on several crops for the control of in¬ sect and nematode pests. The nematicidal properties of aldicarb against the root-knot nematode M. incogni¬ ta were first described by Spurr, et al. , (Plant Pis. Rep. 5f:424-5 (1966) ). The nematicides carbofuran (C.P.DiSanzo, J. Nema¬ tol 5_:22-7 (1973) and carbaryl (D.W. Fenwick, Tropic Agric. Trinidad 2_5_:125-6 (1968)) appear to interfere with the orientation that is necessary for survival of plant parasitic nematodes. The possibility of intervening in the detection by nematodes of chemotactic signals produced by their hosts offers attractive options for novel and non- environmentally hazardous control of plant parasitic nematodes. The development of such methods of con- trol, as well as the discovery of new nematicidal and nematistatic agents that are more selectively toxic for nematodes, requires rapid screening procedures to allow evaluation of large numbers of potential nemati¬ cidal or nematistatic compounds. Field testing pro¬ cedures are incompatible with such mass screening pro¬ grams since they are cumbersome, expensive and poten¬ tially hazardous. Thus, there is a need for a rapid, reproducilbe, inexpensive and safe in vitro assay method for screening the effects of a large variety of substances on nematode vitality. Many researchers have utilized nematode movement as a criterion for in vitro screening of nematicidal and nematistatic compounds. Nematodes exposed to a toxicant are classified as either motile or non-mot¬ ile. Lack of motility often is equated with inability to infect or with death, and the potency of a candi¬ date compound is thereupon evaluated. A. Hough et al., J. Nematol. 7:221-229 (1975). One complication of such assays is that under laboratory conditions, nematodes exposed to low con¬ centrations of a toxicant may recover and become active when the toxicant is removed. For example, the in- vitro evaluation of various nonfumigant nematicides with cholinesterase-inhibiting properties can be mis¬ leading when their action is assessed by nematode motility tests which involve removal of the toxicant prior to assay. Furthermore, cholinesterase-inhibit¬ ing toxicants exhibit reversible reaction with the substrate, and treated nematodes often recover fully after the toxicant is removed. C.C. Yu et al., J. Agric. Food Chem. 2.5:537-540 (1972). An additional complication to in vitro assays is that many organophosphates and carbamates will kill at very high doses. It is believed that the mode of ac¬ tion of sublethal doses of these compounds is nematis¬ tatic, and it is known that intoxication with carba- mate nematicides is reversible at sublethal dosages. A. Hough, 1975, supra. Thus, there is need for a reproducible in vitro assay method that is sufficiently rapid so as to obvi¬ ate the complications of reversibility of the toxic effects of some nematicidal and nematistatic agents. Assays employed in studies of chemotactic agents are potentially suitable for the rapid in vitro as¬ sessment of the potential of candidate nematicides and nematistatic agents. Nematode chemotaxis assay methods and devices are disclosed by S.W. Ward (Proc. Nat. Acad. Sci.70:817-21 (1973)), J. Balan et al., (Nematologica 22:306-11 (1976)), J.-C. Prot (Rev. Ne atol. 1:21-6, 135-142 (1978); (Rev. Nematol. 2_ :11_ 21 (1979)), H.-B. Jansson, et al.,(J. Gen. Microbiol. 112:89-93 (1979)) and J. Schmidt, et al., (Environ. Entomol. 2:605-7 (1978)), and are reviewed by D.B. Dusenbery (J. Nematol 1J3:168- 173 (1983)). All de¬ vices, except that of Prot supra, comprise Petri dishes coated with a layer of agar, agarose or Sepha- dex, the material of the coating being suspended ini¬ tially in either sterile water or water-based HEPES buffer containing a detergent. Generally, the chemat¬ tractant in a suitable solvent is placed upon the sur¬ face of the coating at a prescribed location. The point of application of the chemattractant is usually covered with a disc of paper or agar. During a diffu- sion period of from 24 to 48 hours, gradients of the chemattractant are formed in the coating. A water or buffer control is applied to another site on the coat¬ ing. Thereafter, a suspension of nematodes is placed at an appropriate site on the plate, and movement of nematodes toward the attractant is followed. The device of Prot supra comprises a U-tube con¬ taining in both arms a continuous column of agar. The method comprises the addition of a chemattractant to one arm of the tube, and the formation of a gradient during a 48-hour diffusion period; water is added to the other arm as a control. Thereafter, nematodes are introduced into the bottom center of the agar column, and the number of nematodes moving to the chemattrac¬ tant is determined and compared to the number moving to the control. In spite of these methods and devices, it would still be useful to provide improved devices, and methods for using same, that aid in rapidly assessing the efficacy of large numbers of potential nematicidal and nematistatic agents in coated-plate motility assay systems that are based upon the principle of chemo- taxis. Furthermore, the identification and use of specific nematode chemattractants in such assay sys¬ tems may permit the discovery or development of new compounds which offer greater selectivity for plant parasitic nematodes and at the same time less toxicity for beneficial insects, as well as for animals and man. SPMMARY OF THE IHVENTION The present invention is directed to a coated solid support for the assay of vitality and motility of nematodes. The present invention is also directed to in vitro methods for using the aforementioned de¬ vice to assess the potential of chemicals as nematici¬ dal or nematistatic agents. The in vitro assay is rapid, as compared to field studies with plants, is highly reproduceable, is simple to set up, and is amenable to scale-up. Inclusion of assay stabilizing (i.e., anti-reversibility) agents into the material with which the solid support is coated provides great reliabilty to the assay. The device of the invention comprises a solid sup¬ port, including but not limited to, glass or plastic Petri dishes, coated with a gellable polymer. Gell¬ able polymers include, but are not limited to, agar, agarose, and dextrans. Gellable polymers are inti¬ mately mixed with chemicals constituting particular assay stabilizing agents. At appropriate sites on the coated solid supports are placed blocks or discs of agar intimately mixed with particular chemattractants. The device is then incubated for a time sufficient to form a gradient of chemattractants surrounding the agar block or disc. Methods for using the aforementioned device to assess the efficacy of potential nematicidal and nema¬ tistatic agents comprise contacting a suspension of nematodes with a solution of the aforementioned agent under appropriate conditions of time, temperature and shaking, then applying a small aliquot of the suspen- sion of treated nematodes to the afore-described coat¬ ed support at an appropriate distance from the chem¬ attractant gradients. After allowing the liquid to dry, the movement of nematodes toward the chemattrac¬ tant gradients is followed qualitatively and quantita¬ tively. These aspects of the invention provide multi-step protocols that are applicable to all potential nemati¬ cidal or nematistatic agents, regardless of their chemical nature, thus having the advantage of offering uniformity of materials for any desired assay. The procedures are applicable generally with only minor modifications or adjustments being required for any specific anti-nematode agent or any particular genus of nematode. The relative potencies of. different agents on various genera of nematodes can thus be re¬ liably compared. The assay stabilizing agents employed have the advantage of producing great reliability and reproduc- ibility to the assay for motility and viability of treated nematodes. These assay stabilizing agents also have the advantage of being suitable for use with a variety of chemattractant salts such as are found in soils surrounding plant roots. BRIEF DESCRIPTION OF TEE DRAWINGS Figure 1 is a top view f a device for an agar plate chemotaxis assay of the motility and viability of nematodes. Figure 2 is a photograph of the movement of nema¬ todes toward a chemattractant (CaCl 2 ) i an agar plate chemotaxis assay. Figure 3 is a graph depicting the number of nema¬ todes accumulating under a CaCl, chemattractant block as a function of time. Figure 4 is a graph depicting the concentration of calcium ions at various distances from the site of application of CaCl, on an agar chemotaxis assay plate. DESCRIFTIOH OF THE PREFERRED EMBODIMEHTS The approach of the invention, which provides an effective device and protocol for the in vitro testing of potential nematicidal and nematistatic agents, com¬ prises treatment of nematodes with such agents, and assessing the effects of these agents on the vitality of the organisms using a chemotaxis assay device. A. Preparation of nematodes Parasitic nematodes are isolated from appropriate plant roots, e.g., M. incognita from tomato plants, and M. javanica and M. hapla from eggplants, as fol¬ lows. Nematode eggs are released from egg masses on in¬ fected roots using the technique of McClure et al. , Journal of Ne atoloqy 5:230 (1973), which is incorpor¬ ated herein by reference. Active second stage juve¬ niles are harvested from the eggs, and stored at 10°- 25°C, preferably 14°-16°C, in a solution of an anti¬ biotic, typically streptomycin sulfate. Prior to use, nematodes are warmed to room temper¬ ature, isolated, and washed with sterile water by cen- trifugation. Thereafter, suspensions of appropriate density are prepared, typically, 15,000-60,000 organ¬ isms per ml. B. Treatment of Nematodes Aliquots of the washed nematodes are added to suitable containers containing the particular nemati¬ cidal or nematistatic agents in appropriate concentra¬ tions. It is expected that those with ordinary skill in the art will, by experimentation, select ratios of numbers of nematodes to concentrations of test sub¬ stances appropriate to elicit the maximum effects of such agents on the nematodes. Typically, about 25,000-30,000 nematodes in 0.5 ml. of solution are contacted with from 0 to 1 mg of suspected nematicidal or nematistatic agents. Nematicidal or nematistatic agents are added to the treatment container in any suitable formulation: as a solution of the chemical, or in the form of car¬ rier particles impregnated with a solution of the chemical. Thereafter, nematodes are contacted with the chem¬ ical agents for a suitable period, typically 12-16 hours, at an appropriate temperature, typically room temperature, with shaking. C. Chemotaxis Assay Device 1. Preparation of Chemattractant Source ' Chemical chemattractants are intimately mixed with 0.5-3%, preferably 1.5%, tempered molten agar prepared with doubly-distilled water, and the molten mass pour¬ ed- into Petri dishes, typically 10 x 100 mm but other sizes may be appropriate. The concentration of such chemical chemattractants in the agar is selected ac¬ cording to the nature of the nematodes; typically, 5-50 mM concentrations of chemattractant salts can be employed. Once the agar is solidified, circular blocks (typically 10 mm diameter, but other sizes may be appropriate) are excised and stored. The preferred chemattractants are salts of the alkaline earth metals. Most preferred are calcium chloride and calcium sulfate, although the chloride and sulfate salts of magnesium, manganese, and cobalt cations are also suitable. Alternatively, thick filter paper (Whatman No. 3 MM) is saturated with chemical chemattractants in the appropriate concentration. Thereafter, the paper is air dried, and disks of the appropriate diameter ex¬ cised and stored for use. 2. Preparation of Coated Solid Supports Solid supports are coated with a thin layer of a gellable polymeric substrate. In a preferred embodi¬ ment, Petri dishes are thinly (typically, l-5mm, pre¬ ferably 3mm thick) coated with 2% Bacto agar. Other polymeric substrates include, but are not limited to any galactan, cross-linked dextran, allyl dextran, hydroxypropylated dextran, or silica gel. It is an important aspect of this invention that the aforementioned coating has incorporated within it a chemical agent that stabilizes the assay. The term ■ stabilizes" is intended to mean a prevention of the reversibility of nematode movement, following chem- attraction, that may be a problem in chemotaxis assays in which particular chemattractant salts are employed. Stabilization agents comprise chloride and sulfate salts of monovalent alkali metals. The preferred monovalent alkali metals include, but are not limited to, sodium, potassium, and lithium cations, although cesium and rubidium ions are also suitable. The se¬ lection of the anion of the stabilizing salt is based upon the nature of the chemattractant salt employed. The anion in both cases should be the same. The concentration of the stabilizing salt is based upon the concentration of the chemattractant salt gra¬ dient in the chemotaxis assay plate. For example, where the concentration of CaCl 2 in the aforementioned chemattractant block is 25 mM prior to gradient forma¬ tion (see infra), the maximum concentration of calcium ions in the agar immediately adjacent to the block after gradient formation will be about 2.5 mM and that of chloride ion about 5.0 mM (see Figure 4). Under these conditions, the concentration of NaCl as a sta¬ bilizing agent in the coating should be at least equal to the anion (i.e., chloride) concentration provided by the gradient, i.e., 5mM, but most preferably twice that concentration is recommended. It is hypothe¬ sized, but the inventors do not intend to be bound by the hypothesis, that these concentrations of NaCl de¬ liver chloride ion concentrations that effectively negate the chloride anion gradient created by the cal¬ cium chloride in the attractant block. It should be recalled that chloride ion gradients themselves are a repellent for M. javanica (Prot, supra). This provid¬ es an explanation for why only 5-10 mM soidu chloride need be incorporated into the aforementioned coating where the maximum chloride ion concentration adjacent to the chematractant block is 5mM. 3. Establishing a Chemattractant Gradient A circular block or disk containing the chemat¬ tractant is placed at an appropriate site on the sur¬ face of the aforementioned coated solid support. "Ap¬ propriate site" as used herein is intended to mean a point sufficiently near to the nematode application site that the chemattractant gradient (see infra) will overlap with the nematode application site. Typical¬ ly, a 1.0 cm disk or block will be placed about 1.5 cm from the center of a 10 cm coated plate (see Fig. 1). Although more than one chemattractant block can be placed on a coated plate, it is preferred to use a single chemattractant block so as to avoid attraction for nematodes by competing gradients, which may pro¬ duce ambiguous results. The chemattractant block is contacted with the coated plate under conditions appropriate to estab¬ lishing a chemattractant gradient. It is preferred that the contact be from 18 to 24 hours at room tem¬ perature, preferably 20 hours. As seen in Figure 4 infra, starting with an initial CaCl ? concentration in the chemattractant block of from 1 to 25 mM, linear gradients were formed within 20 hours. ■ 4. Chemotaxis Assay A small aliquot of a suspension of treated or un¬ treated nematodes, typically 20ul, delivering 10-10,000, preferably 500-1,000, nematodes, is added to the aforementioned (3. supra) nematode application site on the assay plate. After the liquid evaporates, the plates are incu¬ bated at room temperature, preferably in the dark so as to avoid the possible influences of lumattraction. Nematode movement is examined at intervals over a per¬ iod of from 2 to 8 hours, preferably 4 to 6 hours. Viability of treated nematodes is preferably as¬ sessed simply by (1) examining the general shift of population relative to the site of application of ne¬ matodes, and (2) counting the number of nematodes un¬ der the attractant block. A variety of means are available to assess nematode movement, and the inven¬ tors do not intend to be limited to any one method. Examples of suitable methods include, but are not li¬ mited to observation by the naked eye or by optical means. Having now generally described the invention, the same will be more clearly understood by reference to the following examples, none of which are intended to be limiting unless so specified. Example I relates to that aspect of the invention^ that comprises the chemotaxis assay itself. Example II relates to that aspect of the invention that comprises treatment of nematodes with suspected nematicidal or nematistatic agents, followed by as¬ sessment of viability by the chemotaxis assay. Example III relates to that aspect of the inven¬ tion that comprises an assay for the formation of the CaCl- chemattractant gradient in coated plates follow¬ ing application of the disk or block containing the chemattractant. All literature reference cited in these examples are incorporated into this application by reference. EXAMPLE I The Chemotaxis Assay M. incognita was isolated from tomato plants, and M. javanica and M. hapla from eggplants. Eggs were released from egg masses on infected roots using the technique of McClure (supra). Second stage juveniles were obtained by placing the eggs on a wire mesh IS screen containing six layers of Kimwipes and incubat¬ ing in a Petri dish containing a solution of 1% (w/v) streptomycin sulfate. Juveniles were harvested every two days and refiltered through Kimwipes as above. The nematodes were stored at 15°C in a solution of 1% streptomycin sulfate. Prior to use for chemotaxis, the nematodes were warmed to room temperature and iso¬ lated by centrifugation (2,000 x g for 5 minutes). After washing the organisms once with sterile distil¬ led water, they were adjusted to a concentration of about 25,000 nematodes per ml. The chemotaxis assay was set up as follows. CaCl 2 , 25 mM, was mixed with 25 ml of molten, tempered (44°C) 1.5% Noble agar prepared with distilled water, and the mix poured into a 15 x 100mm Petri plate. Once the agar solidified, 1.0cm disks or blocks were excised and placed on a 15 x 100mm Petri plate con¬ taining 20ml of Bacto agar (Difco), made up to contain 5-10 mM NaCl, as shown in Figure 1. Each Bacto agar plate contained one control water agar block and one chemattractant block, each placed 1.5cm from the mid- line of the plate. The plate was incubated at room temperature for 20 hours to allow a chemical gradient to be formed (cf.. Example 2 and Figure 4 infra). The chemotaxis assay was initiated by placing ap¬ proximately 500 nematodes in 20ul along the midline of the assay plate (Figure 1). The assays were performed in triplicate. After the liquid had evaporated, plates were in¬ cubated in the dark at room temperature, and nematode movement examined over six hours. Chemattraction, as well as viability, was assessed by (1) examining the general shift of the population relative to the mid¬ line and to the chemattractant agar block and the con¬ trol water agar block (Figure 2), and (2) by counting the number of nematodes under the attractant and con¬ trol agar blocks (Figure 3) . EXAMPLE II Effect of Pretreatment of Nematodes With Nematicidal Agents on the Chemotaxis Assay Aldicarb (450 ug of the active component of TEMIK 15G , Union Carbide, in a gypsum carrier (15% aldicarb plus 85% inert carrier)) was dispensed into 1.5ml Ep- pendorf centrifuge tubes. A control tube contained gypsum granules without aldicarb. Oxamyl (from 0 to 5 ul of a 23 ug/ml water solu- tion of Vydate L , DuPont) was dispensed into 1.5 ml Eppendorf tubes. To each tube was added 25,000-30,000 nematodes suspended in 0.5 ml of water. The tubes were then incubated at room temperature for 16 hours on a rota¬ tor. Bacto agar (2%) plates made up in 10 mM NaCl, with 25 mM CaCl, attractant blocks were prepared as in Example I supra. Gradients were formed over 20 hours. Aliquots (20 ul) of the treated nematode suspen¬ sion were added to triplicate chemotaxis assay plates, as in Example I supra. Plates were incubated in the dark at room temperature, and nematode movement was assessed at 2, 4, and 6 hours. The results are shown in Table I. It is clear from the results shown in Table I that, in the water and gypsum controls, there was a rapid movement of large numbers of nematodes through CaCl, gradients, with the peak occurring at 4 hours. In contrast, nematodes treated with aldicarb or oxamyl essentially failed to exhibit significant motility, even as late as six hours. TABLE I Chemotaxis Assay of Nematodes afte Treatment with Aldicarb and Oxamyl Treatment 2hrs 4hrs 6hrs Water control 173(7)** 400(37) 193(29) Gypsum control 149(106) 233(68) 154(37) Aldicarb 0 0 0 Oxamyl (2.3ppm) 3 33(1) 30(0) Oxamyl (4.6ppm) 0 4(1) 11(2) * The values represent the approximate numbers of organisms under the chematractant blocks ** The values in parentheses represent water agar block controls. EXAMPLE III Calcium Diffusion Assay Agar blocks (1.0 cm diameter) containing various concentrations of CaCl 2 and supplemented with 45CaCl 2 (New England Nuclear Corporation) at a known specific activity were placed in the standard chemotaxis agar plate containing 10 mM NaCl, as shown in Example I and Figure 1. The plates were incubated for 20 hours un¬ der standard conditions to establish the CaCl 2 gra¬ dient. Agar slices were removed from various positions on the agar plate, and placed in scintillation fluid (Aquasol) to determine the amount of radioactivity. The calcium concentration at each position on the plate was determined by dividing the counts in the agar slices at a particular position on the plate by the counts in an agar slice of the same volume taken from the CaCl 2 block that had been placed on the plate. This ratio was divided by the specific activi¬ ty of the radioactive CaCl 2 to obtain the molar con¬ centration of CaCl 2 at each position on the agar plate. The molar concentrations of chloride ions are, of course, twice those of the CaCl 2 . • The results, depicted in Figure 4, demonstrate the linearity of the CaCl 2 gradient emanating from the chemattractant block and encompassing the center line of the plate, i.e., the site of application of the nematodes. Thus, the nematodes encountered the chloride ion component of the chemattractant at all times, begin¬ ning with the moment of their application to the chemotaxis assay plate. The minimum effective concentration of CaCl, to attract Meloidogyne nematodes was 5 mM in the chemat¬ tractant block initially. Following 20 hours of gra- dient formation, the Ca 2+ concentration in the center of the plate where the nematodes were applied was about 15 uM (of.. Figure 4). Thus, the threshold level for attraction of Meloidogyne to calcium appears to be approximately 15 uM.

Claims: WHAT IS NEW AND DESIRED TO BE COVERED BY LETTER PATENTS ARE: 1. A device for the detection of nematode vital¬ ity comprising: (a) a solid surface coated throughout with an intimate mixture of a gellable poly¬ meric substrate and an assay stabilizing salt; (b) said coating also containing, at parti¬ cular loci, concentration gradients of a chemattractant inorganic salt. 2. The device of claim 1 wherein said solid sur¬ face comprises a Petri dish. 3. The device of claim 2 wherein said Petri dish is composed of glass. 4. The device of claim 2 wherein said Petri dish is composed of plastic. 5. The device of claim 1 wherein said solid sur¬ face comprises a plate. 6. The device of claim 5 wherein said plate is composed of glass. 7. The device of claim 5 wherein said plate is composed of a plastic. 8. The device of claim 1 wherein said gellable polymeric substrate is selected from the group con¬ sisting of agar, galactan, cross-linked dextran, hy- droxypropylated dextran and silica gel. 9. The device of claim 1 wherein said assay sta¬ bilizing salt comprises a monovalent cation linked to an anion identical to the anion of said chemattractant inorganic salt. 10. The device of claim 1 wherein said stabiliz¬ ing salt is selected from among the chloride salts of sodium, potassium, lithium, rubidium and cesium. 11. The device of claim 10 wherein said stabiliz¬ ing salt comprises sodium chloride. 12. The device of claim 1 wherein said stabiliz¬ ing salt is selected from among sulfate salts of so¬ dium, potassium, lithium, rubidium, and cesium. 13. The device of claim 12 wherein said stabiliz¬ ing salt comprises sodium sulfate. • 14. The device of claim 1 wherein said chemat¬ tractant inorganic salt comprises an alkaline earth metal salt. 15. The device of claim 14 wherein said chemat¬ tractant salt comprises a divalent cation linked to an anion identical to the anion of said assay stabilizing salt. 16. The device of claim 14 wherein said chemat¬ tractant salt comprises chloride salts of divalent cations selected from among beryllium, magnesium, cal¬ cium, strontium, barium, cobalt, and manganese. 17. The device of claim 14 wherein the chemat¬ tractant salt is calcium chloride. 18. The device of claim 14 wherein said chemat¬ tractant salt comprises sulfate salts of divalent cat¬ ions selected from among beryllium, magnesium, cal¬ cium, strontium, barium, cobalt, and manganese. 19. The device of claim 18 wherein said chemat¬ tractant salt comprises calcium sulfate. 20. A method for assessing the potency of nemati¬ cidal and nematistatic agents comprising: (a) contacting a suspension of nematodes with said agents under appropriate con¬ ditions of suspension medium, concentra¬ tion, time, temperature, and physical orientation; (b) transferring said treated nematodes to appropriate sites on the surface of the device of claim 1; (c) detecting the numbers of nematodes mi¬ grating to the chemattractant source. 21. The method of claim 20 wherein said agents are contacted with said nematodes in solid form. 22. The method of claim 20 wherein said agents are contacted with said nematodes in soluble form. 23. The method of claim 20 wherein the concen¬ tration of said agents in. contact with said nematodes range from zero to saturation. 24. The method of claim 20 wherein said treat¬ ed nematodes are transferred to sites on the surface of said device within the outer limits of said chemat¬ tractant gradient. 25. The method of claim 20 wherein said detection is by counting of said nematodes. 26. The method of claim 25 wherein said detection is by optical means. 27. The method of claim 25 wherein the migration of said nematodes is detected by observation of mass movement of nematodes.

Inventors: CHANG CHANGTUNG P, HORWITZ ARNOLD, CHANG, CHANGTUNG, P., HORWITZ, ARNOLD
Applicants: INT GENETIC ENG, INTERNATIONAL GENETIC ENGINEERING, INC.
Application year: 1987
Main classification: C12M1
IPC codes: C12M1, C12Q1, G01N33