EP1169471A2

EP1169471A2 - Method for screening compounds using nematode worms

Info

Publication number: EP1169471A2
Application number: EP00919099A
Authority: EP
Inventors: Richard Feichtinger; Veerle Rottiers; Thierry Bogaert; Isabelle Maillet
Original assignee: Devgen NV
Current assignee: Devgen NV
Priority date: 1999-04-15
Filing date: 2000-04-14
Publication date: 2002-01-09
Also published as: WO2000063424A3; GB2350896B; GB2350896A; GB0009364D0; WO2000063424A2; HK1030450A1; AU3984400A; JP2002542464A; GB9908676D0; CA2369734A1

Abstract

The invention provides improved methods of screening compounds for potential pharmacological activity using nematode worms, principally but not exclusively.C. elegans. Specifically, the invention relates to methods in which the compound under test is added directly to a nematode food source organism and therefore taken up by the nematodes during feeding.

Description

METHOD FOR SCREENING COMPOUNDS USING NEMATODE WORMS

The invention relates to the screening of compounds in the drug discovery process. More specifically, it relates to an improved method of compound screening using the nematode worm C. elegans .

C. elegans is a nematode worm which occurs naturally in the soil but can easily be grown in the laboratory on nutrient agar seeded with a food source such as E. coli bacteria. Each worm grows from an embryo to an adult worm of about 1mm long in three days or so. As it is fully transparent at all stages of the life cycle, cell divisions, migrations and differentiation can be easily seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue types including muscle, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of a wild-type worm are relatively easily observed, either directly by microscopy or by using selective staining procedures .

These characteristics of C. elegans make it an extremely useful tool in the drug discovery process. In particular, C . elegans may be used in the development of high-throughput compound screens, useful in the identification of potential candidate drugs, in which worms are exposed to the compound under test and any resultant phenotypic and/cr behavioural changes are recorded.

Such screening methods are critically dependent upon the ability to expose C. elegans to the compound under test. Usually this is achieved by culturing C. elegans in the presence of the compound so that the worms take up the compound. The standard method for culturing C. elegans in the presence of test compounds is described by Rand and Johnson in Methods in Cell Biology 48: 147-204. In this standard method, compounds are mixed with heated, liquid agar. After cooling to room temperature the plates are seeded with E . coli bacteria, the food source for the nematodes. The compound enters the nematode as it feeds on the bacteria and is thus able to perform its action. This standard method has the disadvantage that because the compound is mixed with the agar a comparatively large amount of compound is needed in order to perform the screen. Furthermore, compounds unstable at the temperature of liquid agar will loose their activity before the nematodes encounter them.

The present inventors have developed a new screening method which requires much smaller amounts of compound and is more suitable for use in screening large compound libraries.

Accordingly, the invention provides a method of compound screening in nematode worms, which method comprises the steps of:

(a) providing a nematode food source on solid growth medium, wherein the nematode food source additionally contains an amount of the compound to be screened, (b) allowing the nematode worms to feed on the said food source containing the compound whilst preventing the nematode worms from feeding on any food source which does not contain the compound, and

(c) measuring or observing any changes in the phenotype and/or behaviour of the nematode worms. Caenorhabdi tis elegans is the preferred nematode worm for use in the method of the invention. However, it will be appreciated that the method may also be carried out with other nematodes and in particular with_-other microscopic nematodes, preferably microscopic nematodes belonging to the genus

CaenorήaJdi is. As used herein the term "microscopic" nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening. The nematode may be a wild-type strain or a non wild-type strain such as, for example, a mutated strain, a transgenic strain, or a mutant transgenic strain. Transgenic strains may carry transgenes encoding proteins of another species origin, e.g. transgenes encoding human proteins. Optionally, the nematode may express a reporter gene such as, for example, lacZ, green fluorescent protein (including any of the variant GFPs and GFP equivalents known in the art) , luciferase, acetohydroxyacid synthase, alkaline phosphatase, β-glucuronidase, chloamphenicol acetyltransferase, horseradish peroxidase, nopaline synthase or octapine synthase in some or all cell types .

In order to perform the compound screen nematode worms are grown on a solid growth medium which has been seeded with a nematode food source to which has been added a sample of a compound to be screened. The worms take up the compound as they feed on the food source.

The method preferably uses E . coli as a food source for the nematodes but other food sources known for use with nematode worms could also be used. These include other bacterial species, yeasts and slime moulds (e.g. Dictyostelium discoideum) . The food source can be living organisms, dead organisms, synthetic food sources or extracts from living organisms. The food source can even be beads of any particular material on which nematodes such as C. elegans can feed.

Suitable solid growth media include NGM agar which is routinely used for the culture of C. elegans in the laboratory. However, since it is no longer necessary to add the compound directly to the solid medium, as with the standard protocol described above, other polymer materials known to support growth of nematodes can be used. These polymer materials can be chosen in such a way that they give more or less adherence to the walls of microtiter plates.

The method of the invention can be performed in various formats, including single small agar plates (e.g. 3.5 cm diameter) as well as microtiter plates (e.g. 12, 24, 96 and 384 well plates). To apply this method in the smaller well plate (e.g. 96 and 384 well plates) , additional problems have to be overcome, such as the formation of a meniscus by the agar. The problem of the formation of an optically distracting meniscus can be overcome with the use of microtiter plates manufactured or coated with a specific wall material. These plates, described as WIM plates for Without Interfering Meniscus, are provided by Swiss Aircraft and Systems Company. Other plates designed to prevent disturbing meniscus formation can also be used with equivalent effect. Alternatively, or in combination, the polymer solution may be modified to achieve adhesive properties resulting in a decrease or absence of the optically distracting meniscus. Most of the other problems arising from using microtiter plates can be solved by applying microtiter plates in specific materials or coated with specific materials. It is an essential feature of the method of the invention that an amount of the compound to be tested is added to the nematode food source rather than to the solid growth medium. There are several different ways in which this can be achieved. In a first method a layer of food source is seeded onto the solid growth medium and an appropriate amount of the compound under test is then spread on top of the food source layer. For example, when E . coli is used as the food source 3.5cm diameter plates are seeded with a small drop (6μl) of E . coli culture that grows to a small round lawn of about 4 mm diameter. Compounds dissolved in a suitable solvent e.g. DMSO are then put on top of the bacterial lawn to cover to lawn completely. In an alternative method, the compound can be mixed directly with the food source prior to seeding onto the solid growth medium.

The final concentration of compound added to the food source can be optimised by performing test screens with serial dilutions of the compound, as described the examples given herein. A final concentration of the compound should be chosen which is sufficient to have an effect on the worms but which does not cause any non-specific effects e.g. which is not lethal to the food source organisms to avoid worm starvation. Where the compounds are to be dissolved in an organic solvent such as DMSO care must also be taken to ensure that the final concentration of the solvent is kept low, typically below ~0.5% (equivalent to lOμl total volume of solvent per 2ml agar) . Higher concentrations of DMSO, especially more than 1%, are not tolerated well by C. elegans .

Any changes in the phenotype and/or behaviour of the worms following exposure to the compound can be readily observed over time under dissecting scopes, or any other method of visual detection. Other suitable detection methods which may be used to observe or measure changes in the phenotype and/or behaviour of the worms include light microscopy, differential interference contrast optics, fluorescence microscopy, immunological detection, radiation detection, calorimetric detection, fluorescence detection or luminescence detection. In a typical compound screen the worms are maintained in culture in the presence of the compound under test throughout several generations and observations of phenotype and behaviour of the worms are made at regular intervals of time over the successive generations. In the examples given herein the brood of one single L4 hermaphrodite worm is grov/n up to adulthood under the permanent influence of a test compound. Other compound screens may be performed by plating several worms and observing the effects of test compounds on the parental generation only, on a shorter timescale.

The compounds to be tested using the screening method of the invention may be any chemical substance, including natural substances, extracts isolated from natural substances, compounds isolated from natural substances, compounds from combinatorial libraries or selected compounds or drugs from the pharmacopoeia. The compounds can be proteins, peptides or nucleic acids, for example double-stranded DNA, single- stranded DNA, single-stranded or double-stranded RNA. The DNA, RNA, protein or peptide can be added to the bacteria layer or, in an alternative embodiment, can be produced by the food source organisms themselves. According to the method of the invention the compound to be screened is added to the food source rather than to the solid growth medium. It is therefore important to ensure that the nematodes are prevented from feeding on any food source which does not contain the compound to be tested, since this would decrease the overall value of the screening method.

When a layer of food source containing the compound to be tested is placed on the surface of a solid growth medium (as described above) worms feeding on the food source repeatedly move in to and out of the food source layer. By doing this, the worms are able to spread some of the food source organisms outside of the area originally contacted with the food source/compound (hereinafter referred to as the 'contact area') by leaving them with their tracks. Diffusion of the compound outside of the original contact area is often found to be limited, in which case any food source organisms able to grow outside the contact area will contain the compound at a much reduced concentration. If this is allowed to occur the worms will preferentially feed on the food source organisms growing outside the original contact area and hence avoid uptake of the compound under test. Furthermore, the applied compound frequently forms a kind of film on the food source. The food source is able to grow beneath, where it has food supplies from the nutrient rich agar. The film with the compound is then lifted and the nematodes can feed on the fresh food source, without or with less feeding on the food source containing the compound. The possibility that the worms are able to feed on food source which does not contain any of the compound under test or contains the compound at a very low concentration will obviously tend to decrease the value and general application of the compound screening method. Accordingly, it is an essential part of the method of the invention that the nematodes are prevented from feeding on any food source organisms which do not contain the compound under test.

The present inventors have developed several ways of overcoming this problem. In one embodiment the problem is overcome by limiting the growth of the food source organisms under the conditions of the compound screening assay. This can be accomplished by adding growth inhibitor substances to the food source and/or the solid growth medium in order to prevent further growth of the food source organisms. Suitable growth inhibitors are antibiotics such as, for example, carbenicillin, tetracycline or kanamycin.

Alternatively, the food source can be inhibited in replication by irradiation either prior to applying it to the solid growth medium, or after it has been applied to the solid growth medium. In the preferred embodiment of the invention growth of the food source organisms can be controlled during the course of the screening assay by using cold-sensitive organisms as the food source. Cold- sensitive bacteria are preferred for use in this embodiment but cold-sensitive variants of other types of food source organisms could also be used.

One may select for cold-sensitive E . coli strains that show reduced growth under the conditions used for the culture of nematode worms (typically a few days at 20°C, but other conditions of growth may be selected) . Preferentially, bacterial strains are selected that grow only a very limited amount at this temperature. In this circumstance, the growth of the cold-sensitive bacteria outside the original contact area is extremely limited and is not enough to support feeding of the nematode. This ensures that the worms feed only on those bacteria that have been soaked with compound, in the meanwhile taking up compounds while feeding on these bacteria. Suitable cold-sensitive bacterial strains for use in this embodiment of the invention include E . coli strains JS10, N282, PS1583, PS1500, PS1518, PS1537 and PS1518, all of which can be obtained from the E . coli Genetic Stock Center, Yale University. All of these strains show clearly reduced growth at low temperatures . As an alternative to the cold-sensitive E . coli strains listed above, cold-sensitive variants of E. coli strain OP50, which is commonly used as a food source for the culture of C. elegans , can be constructed using standard mutagenesis and selection techniques known in the art ( E. coli strain OP50 can be obtained from the C. elegans Genetics Center, University of Minnesota, St Paul, Minnesota, USA) . First, E . coli strain OP50 are mutagenized using a standard mutagenesis technique such as EMS (ethyl methane sulfonate mutagenesis, described in "Methods in Cell Biology, Vol 48 page 31-35") . A large number of colonies of mutagenized bacteria (approximately 3000) are then patched onto duplicate agar plates. One plate is grown at 20°C, while the other plate is grown at 37°C. Colonies grown at 37°C of which the sister colony at 20°C showed no or very little growth are then re-tested at both temperatures. Following this procedure the inventors were able to isolate 5 colonies that showed strongly reduced growth at 20°C. Liquid cultures were made from each of these and tested on NGM agar for suitability as a food source for nematode worms. Two strains, referred to as OP50csl and OP50cs2, were found to be usable as worm food for the purposes of the invention. These strains were deposited on 25th March 1999 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in the Belgian Coordinated Collections of Microorganisms (BCCM) /Laboratorium voor Microbiologie-Bacterienverzameling (LMG) bacteria collection, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium under accession numbers LMG P-18933 (OP50csl) and LMG P-18934 (OP50cs2) . The approach described above could be used to construct cold-sensitive variants of other E . coli strains which are suitable for use as a food source for nematodes.

The cold sensitive strains derived from E . coli strain OP50 grow a little slower than OP50 at 37°C.

They show significant growth reduction at 25°C which is even more pronounced at 20°C. At 4°C they do not show any growth at all. They should preferentially be stored at room temperature rather than the usual 4°C because they do not survive prolonged exposure to this temperature.

The invention will now be further understood with reference to the following non-limiting experimental examples :

Example 1-screening compounds from a combinatorial library . 248 individual compounds from a combinatorial library were tested on wild-type C. elegans .

Screening Protocol The screen was carried out using 3.5cm plates containing 4ml NGM agar. NGM agar (nematode growth medium) for 1 litre is: 15g agar (Agar SERVA high gel strength, Nr. 11396), lg peptone (Difco Bacto Peptone, Nr. 0118), 3g NaCl_, 1 ml cholesterol solution (5 mg/ml in EtOH)with sterile addition after autoclaving of: 9.5 ml 0.1 M CaCl₂ 9.5 ml 0.1 M MgS0₄, 25ml 1M

KH₂P0₄/K₂HP0₄ buffer pH 6 and 5 ml nystatin solution (dissolved 10 mg/ml in 1:1 EtOH abs: CH₃COONH₄ 7.5M). The high quality agar with loss on drying of less than 15% is preferred to avoid cracking on the margins of the wall of the plates, especially when the method is performed in a microtiter plate format.

The plates were seeded with 6μl of E . coli OP50cs2 overnight culture. Overnight cultures of OP50cs2 typically grow to OD 0.47. If necessary, depending on the food demand of the worms and/or the size of the plates used for the assay, the overnight culture can be concentrated by centrifugation to OD -0.95. The bacteria are left to grow for one week at room temperature to form a thin lawn. lOμl of compound in DMSO is then pipetted on top of the bacterial lawn to cover the lawn completely. The solution is allowed to soak in for a few hours or overnight. The DMSO dilutes completely in the agar, resulting in a final concentration of 0.5% if lOul were pipetted on 2ml agar. It is preferably not to exceed this concentration, as higher concentrations of DMSO, especially more than 1%, are not well tolerated by C. elegans .

One L4 hermaphrodite worm per plate is placed onto the bacterial lawn, at the same time taking care not to transfer any non-cold-sensitive bacteria onto the screening plate. The plates are then incubated at 21°C to allow the brood of the single L4 hermaphrodite worm to grow up to adulthood and are checked at regular intervals of time to identify any changes in phenotype and/or behaviour. By doing this a whole generation of worms grown under the permanent influence of the compound can be scored for changes in phenotype and/or behaviour. As an alternative, several hermaphrodite worms could be plated onto the same__-bacterial lawn in order to observe effects of the compound on the parental generation only. In this case the timescale of the assay can be shortened accordingly.

For a first round of screening all 248 compounds were used at a concentration of O.lmg/lOμl DMSO. For MW 500 this calculates to 20mM stock, with a dilution of the compound in 4ml agar of 50μM. With many compounds this high dose is observed to create nonspecific problems, for example death of the food source bacteria leading to worm starvation. Compounds exhibiting these effects as well as compounds which are lethal to the worms at this dose can be re- screened at a lower concentration, typically dilutions of 1/3, 1/10 and 1/30 of the stock in DMSO.

Example 2-screening compounds from the pharmacopoeia

47 different drugs from the pharmacopoeia were tested using the method of the invention, following the protocol described in Example 1. Briefly, plates containing 4ml NGM agar were seeded with E . coli OP50cs2 and allowed to grow for one week at room temperature. lOμl of the appropriate dilution of compound in DMSO was the pipetted onto the bacterial lawn .

For comparison purposes a subset of 23 of these compounds were tested using the standard protocol known from the literature (described by Rand and Johnson in Methods in Cell Biology, vol 48, 147-204) in which the compound is added to the growth medium. For each compound a concentration series in agar plates was made up as follows:

1. Prepare lOOmM and 5mM stock solutions of each compound in DMSO.

2. Pipette the appropriate amounts of the stock solutions (given in Table 1) into 12 well plates and add 2ml molten NGM agar.

3. Shake for 1 minute to mix the compound and agar.

Table 1

The plates are then seeded with a lawn of E. coli OP50 according to the standard protocol, taking care to add sufficient food source bacteria to allow the brood of the single hermaphrodite worm to grow up to adulthood. One L4 hermaphrodite worm per plate is put onto the bacterial lawn and the plates incubated at 21°C. The plates are checked at regular intervals of time for any changes in phenotype and/or behaviour. Each dilution of compound was tested in triplicate.

Results A comparison was made between the results obtained using the method of the invention, using cold- sensitive bacteria in this instance, and the standard method known from the literature. For the majority of compounds the method of the invention was found to be more efficient than the standard method. Table 2 lists the calculated concentration ratio needed to get the same effect with the compound using the standard method (in 2ml agar) as compared to the method of the invention (on 4ml agar) for all the compounds that showed a comparable effect in both screening methods. For one of the compounds tested, pancuronium, no effect was observed using 1.66mg of compound in the highest concentration of the standard assay whilst there was an observable effect on the phenotype of the worms using 0.033mg (and O.lmg) in the method of the invention. It is therefore concluded that the method of the invention enables the identification of effects of compounds that could not have been identified using the standard screening method known in the art. Using the new method it is possible to screen a compound library using -O.lmg of compound, whereas using the standard method ~2mg would be needed per concentration series (assuming a molecular weight of 500) . This represents a clear advantage in using the new method since very often such large amounts of compound are not available, particularly when the compound is a newly synthesised compound or a compound from a combinatorial library.

Table 2

Example 3

1) Preparation of 12 well plates

12 well plates are filled with 2 ml NGM agar, and tested before use in the following way:

1. The plates are examined random for cracks at delivery and during storage.

2. One plate of the batch is seeded with E.coli OP50 _rcs2 grown for one week. 10 μL DMSO is added to six of the wells.

3. Six wells are filled with 20 N2 worms (3 on DMSO) and the six remaining with 3 N2 worms (3 on DMSO) . The worms are profiled on day 0, day 1 and day 4 for progeny. For verification purposes 10-minute video recordings are made of one well with and one well without solvent.

2) Seeding of plates 12 well and 24 well setup

Plates are seeded one week before use minimum three weeks before use maximum.

The bacterial strain used is streaked from an -80°C glycerol stock (provided with a batch number) on a TY-plate. This plate is grown overnight at 37°C and stored at 20°C for four weeks maximum. 1. Seed 50 ml of LB with a colony of OP50 cs2 and let grow overnight at 37 °C in a shaker. 2. Measure optical density at 600 nm of a lOx diluted culture, using LB as blank. Calculate which dilution you have to make to have an O.D. of 1.4-1.5. Make this dilution and use it to seed the plates 3. Put 10 μL drops of the diluted OP50 cs2 culture in the middle of the well if using 12 well plates and 5 μl if using 24 well plates. A Robot such as a Tecan robot can be used for seeding 12 well plates.

4. Let the bacteria grow at 20°C for one week.

5. Plates with bacteria can be stored at 10°C for three weeks.

3) Preparation of worms

The preparation of worms starts one week in advance. Day 0 is the day of dispensing and profiling:

Day -8: Grow at 15°C nematodes obtained from stock plates containing many dauer larvae by transferring pieces of agar to fresh 9cm plates containing a lawn of E . coli OP50 as food.

Day -4: Bleach the nematodes collected from the 10 plates and dispense the eggs on 20 fresh plates seeded with E . coli OP50cs2. The nematodes need to be grown on the same bacterial strain that is loaded with compound in the microwell plate.

1. Each 9cm plate should give enough young adult worms to fill 2 12-well plates with 20 worms per well so this would be enough for 40 12-well plates . 2. Half the plates can be used to bleach again to stage worms for a second day of profiling in the week, but bleach only two times in row maximum because of possible mutations due to the bleach procedure.

3. Leave the washed off plate overnight at 20°C; if the bleached eggs do not hatch after one day you can wash off LI from these plates and put them on fresh plates to have staged worms on time.

4. You should always set up several plates from a stock plate and put them at 15°C to have a backup

_-if the bleached worms do not grow properly.

Day -1: Check the stage of the worms from the bleach to be able to put them on a higher temperature to reach the young adult stage in time.

Day 0: Check the stage of the worm from the bleach

Wash off the plates with M9 and concentrate the worms in approximately 2 ml by centrifugation (3 min, 1300 rpm) .

Dispense the worms using a worm dispenser apparatus' (e.g. the device commercially available from Union Biometrica, Inc, Somerville, MA, USA which has properties analogous to flow cytometers, such as fluorescence activated cell scanning and sorting devices (FACS)). Use appropriate profile (12 or 24 well) , for the fastest dispensing obtain a flow-rate of 15-20 events per second. First dispense some worms onto the lid of a plate and examine the worms on presence of L4, adjust the TOF and EXT boundaries accordingly.

For profiling on day 0 and 1 use 20 worms per well; for profiling of progeny on day 4 use 1 worm per well 4) Preparation and addition of the compounds; 12 well profiling

Compounds are prepared and put on the plates one day in advance. Prepare stock-solutions and two dilutions of the compounds tested; the default concentration of the stock solution is lOOmM in 90μL of the appropriate solvent. The two dilutions are by default 10 mM and 1 mM in ..the appropriate solvent. If a compound does not dissolve, recover the powder by vacuum extraction and use another solvent like acetone or DMSO. Test the tendency of a compound to form a crust or crystals by pipetting 5 μl of the solution onto a test plate with OP50 . If a crust is observed further diluting of the compound is recommended.

Put lOμL of compound solution on the bacteria grown in each well of the 12-well plates. Let the compound diffuse though the agar overnight at 10°C. Final concentration of the compounds will be then 500μM, 50μM and 5μM respectively.

Put the 12-wells plates with compounds at 20^CC before use. 5) Putting on the compounds : 24 well quick screen 400mM compounds.

Compounds are prepared and put on the plates one day in advance .

Compounds are dissolved in DMSO. Use only 400 mM stock solutions.

A 4 times dilution (lOOmM) of the compounds is made in

DMSO and 5 μl of both solutions is added into separate wells of a 24 well plate containing NGM medium and a spot of OP50 cs2 bacteria. The 24 well plate will then have the following layout:

Put the 24 well plate with compound overnight at 10°C before use.

Claims

Claims :

1. A method of compound screening in nematode worms, which method comprises the steps of: (a) providing a nematode food source on solid growth medium, wherein the nematode food source additionally contains an amount of a compound to be screened;

(b) allowing the nematode worms to feed on the said food source containing the compound whilst preventing the nematode worms from feeding on any food source which does not contain the compound; and

(c) measuring or observing any changes in the phenotype and/or behaviour of the nematode worms and/or the offspring of the nematode worms.

2. A method as claimed in claim 1 wherein said measurements or observations of changes in phenotype and/or behaviour are made at several time intervals.

3. A method as claimed in claim 1 or claim 2 wherein the nematode is a microscopic nematode.

4. A method as claimed in claim 3 wherein the microscopic nematode is of the genus Ca enorhabdi ti s .

5. A method as claimed in claim 4 wherein the microscopic nematode is C. elegans .

6. A method as claimed in any one of the preceding claims wherein the nematode is a wild-type strain, a mutated strain or a transgenic strain.

7. A method as claimed in any one of the preceding claims wherein the food source is a microorganism.

8. A method as claimed in claim 7 wherein said microorganism is E . coli , Dictyostelium discoideum or a yeast.

9. A method as claimed in 7 wherein the microorganism is a cold-sensitive bacterial strain.

10. A method as claimed in claim 9 wherein the cold-sensitive bacterial strain is derived from E. coli strain OP50.

11. A method as claimed in claim 10 wherein the cold-sensitive bacterial strain is E . coli OP50csl or OP50cs2.

12. A method as claimed in any one of claims 7 to 11 wherein the microorganism shows limited growth or no growth on the solid growth medium.

13. A method as claimed in claim 12 wherein the solid growth medium and/or the nematode food source additionally contains a growth inhibitor substance.

14. A method as claimed in any one of claims 7 to 11 wherein the microorganism is dead or inhibited in replication prior to contact with the nematode.

15. A method as claimed in any one of claims 7 to 11 wherein the microorganism has been treated with radiation.

16. A method as claimed in claim 15 wherein the microorganism was treated with radiation before being placed onto the solid media.

17. A method as claimed in claim 15 wherein the microorganism was treated with radiation after being placed on the solid media.

18. E . coli strain OP50csl.

19. E . coli strain OP50cs2.