Rudolph Glaser was the first to successfully devise a culture method for an entomopathogenic nematode (Steinernema) on artificial medium (Glaser, 1931). However, Glaser was unaware of the need for the symbiotic bacterium that kills the insect host by septicaemia, supplies nutrients for the nematode, and, produces antibiotics to suppress secondary invaders. The absence of the bacterium resulted in the nematode's failure to become a biological control agent of the Japanese beetle (Gaugler et al., 1992). Subsequent rearing efforts with other EPN species were made primarily with insect hosts (Dutky et al., 1964). This method is still widely (employed in research laboratories and by some in cottage industry. However, in vivo production costs are far too high for large- scale commercial production. A major breakthrough in mass production was made by Bedding (1984) who developed a monoxenic semi-solid culture technique that achieved much higher and more consistent yields. A semi-automated harvesting process further reduced production costs. This technique has been the choice for a number of decentralized societies, such as China, and small commercial operations (Georgis, 1990). In scaled-up production by the Bedding technique, costs fall with increasing scale up to an output level of approximately 10x10 12 infective juveniles/month. Above this point, labour costs become significant. Production by monoxenic liquid fermentation costs less than other methods, and these costs decrease more rapidly up to a capacity of approximately 50x1012infective juveniles/month (Friedman, 1990).

Steinernematids and heterorhabditids can be cultured monoxenically on a variety of artificial media. The cultures are raised either in flasks or autoclavable bags. The prepared medium is coated in to 1 ml porous polyether polyurethane foam chips, which provide a large surface area for the bacteria to multiply and hence the IJs. It is ensured that pores are still visible holding sufficient medium inside and the medium added to foam come out of the pores if squeezed. Flasks are plugged with non-absorbent cotton and autoclaved. Foam chips with sterilized medium are inoculated with primary phase of bacteria (Xenorhabdus for Steinernema and Photorhabdus for Heterorhabditis). The bacterial cultures are maintained in 5ml of nutrient broth in test tubes for 24 h on a shaker and inoculated on a sterilized medium (one tube per flask) aseptically.

The bacteria and media are mixed well and incubated at 25oC for 3 days to allow the bacterial multiplication. Infective juveniles are surface sterilized and added to flasks at 10⁶ and 10⁷ per flask for steinernematids and heterorhabditids, respectively) to start new cultures. Cultures are established in 2-3 weeks. Incubation of cultures at temp (25°C) is important ,besides proper moisture and maintenance of bacterial purity (streak on to NBTA or MacConkey agar and identify contaminants) concentration, 100,000 IJs/ml, conical flasks with aeration facility are more suitable. Harvest of nematodes is done by taking out foam pieces on to a 20-mesh sieve and placing in a pan of water to submerge the pieces. Infective juveniles migrate to the water, which is collected in a beaker, let it settled down, decanted and more sterile water is added. The process is repeated 2-3 times and stored in tissue culture flasks at a concentration of 10-20 thousands nematodes per ml.

In vitro: Plant and animal protein media were evaluated in vitro for production of indigenous isolates of S. carpocapsae, S. bicornutum and S. tami and one of H. indica. Wout's medium, modified egg yolk, Soyflour + cholesterol and modified dog biscuit yielded highest number of IJs of S. carpocapsae, S. tami and H. indica PDBC EN 6.71. Maximum yield of S. carpocapsae PDBC EN 6.11 and PDBCEN 6.61 IJ was obtained on modified dog biscuit (Hussaini et al., 2000a,2002e). Costs of 10-60% more has been envisaged with use of nematode-based products than with chemical insecticides (Georgis & Hague, 1991) Technological improvements in production, formulation, packaging and shelf life of the products may change the scenario. Since nematode products are safe to apply and do not contaminate the environment, some clients opt for a biological control method even at a higher cost. Also, at least in some situations, the nematodes become established; recycle and their offspring continue to control the target insect. Thus, the higher short-term cost may be lower in the long run when continued control by the recycling nematode is obtained. Thus, by the judicious use of nematodes and chemicals, it may be possible to reduce the cost of control and protect the environment at the same time.

In vitro Solid culture: Mass production on artificial media was realized 30 yrs before Dutky et al 1964 established in vivo methods. Solid culture was pioneered by Rudolf Glaser who was the first to artificially culture a parasitic nematodes .Glaser and his co workers in one of the most ambitious and least known experiments in biological control, produced and released billions of S. glaseri throughout New Jersey against Japanese beetles from 1939 to 1942 ( Fleming 1948). Regretably early workers were unaware of the nematode's bacterial partner. Nematode production was carried out in shallow trays of veal pulp medium with salicylic acid and formalin to repress contaminating microbes (Mc Coy and Girth 1938) including apparently the natural symbiont Xenorhabdus poinarii (Gaugler et al 1992 ). Today need for monoxenicity is universally recognized as one of the cornerstones of nematode in vitro culture (Poinar and Thomas 1966). Laboratory: others would extend Glaser's accomplishments by developing alternative media to costly animal tissue homogenates such as dog food medium (House et al 1965) . Regardless of the growth medium cultures were produced on the substrate surface because of the need for gas exchange viz., cultures were two dimensional perfectly suited for lab cultures but a limitation that precluded commercial production Bedding (1981&84) development of practical solid culture technology was a seminal step in nematode production because it leapt from two to three dimensional substrates. Beddings flask cultures involved thinly coating crumbed polyurethane foam sponge with poultry offal homogenate .The porous foam afforded an outstanding area to volume ratio for growth while providing adequate gas exchange .Next advance was to adopt large autoclavable bags to replace flasks.

The nematodes can be mass-produced by in vivo or in vitro methods. G. mellonella is the most commonly used insect for in vivo mass production of EPN because it produces high nematode yields, is widely available commercially, and is very susceptible to infection.

Facilities and manpower available and the requirements for each option vary greatly, number of nematodes are required. In vivo culture is most appropriate for maintenance of colonies and production of lJs for lab and small-scale field tests. For large-scale production in vitro methods are practical. Which nematodes are needed, and length of storage before use. Steinernematids could be stored better than heterorhabditids. In vivo production of large numbers of heterorhabditids within a short enough time interval to avoid long storage periods may be difficult.

Nutritional composition: The nutritional composition of the medium is an important component in nematode production and could determine the final yield (Friedman 1990). Sub optimal composition presumably produces inferior bacteria that in turn result in poor quality nematodes reduced pathogenicity and low storage ability. The content and class of lipids in EPN can vary in amount and composition depending on culture conditions. Many studies of EPN behaviour and ecology over the past 10 years were originally undertaken to discover why these lethal insect parasites provided unpredictable field efficacy. Lab tests found a single species of EPN S. carpocapsae able to infect hundreds of species of insects. However field tests against many of these were disappointing, poorer than expected field results have been attributed to several aspects of behaviour and ecology which were understood at the time of application. Most research has focused on the IJs as this stage is applied by users to kill insects. IJs do not feed mate or go through development outside the host. Several EPN are currently under evaluation for mass production and field efficacy for biological control of insect pests. However quality and quantity of in vitro produced nematodes vary considerably depending on medium, temperature and production method. In addition nematode production should be cost effective. We investigated nematode yield, production time total lipid content and fatty acid composition of H. bacteriophora produced in artificial media supplemented with different lipid sources. Lipid source significantly affected lipid quantity and quality in H. bacteriophora. Media supplemented with expectable insect lipids produced yields 1.9 times higher than did beef fat or lard-supplemented media. Moreover the developmental rate in media supplemented host lipids was 1.7 times faster than that in media supplemented with beef fat or lard . Nematodes grown in media supplemented insect lipids accumulated significant lipid proportion per dry biomass than those grown in media supplemented with other lipid sources. H. bacteriophora produced in media supplemented with insect lipids, olive oil, or canola oil had similar fatty acid patterns with oleic acid (18:1) as the major lipid fatty acid. We recommend addition of fatty acid mixtures that resemble natural host lipids for mass producing EPN. This would provide nematode quality similar to in vivo produced nematodes and could improve yield.

EPNs can be mass-produced by in vivo or in vitro methods. In the in vivo process, an insect serves as a bioreactor; in the in vitro process, artificial media are used. The in vivo process is very simple and requires only minimal initial investment. The equipment used is also simple: trays and shelves. The wax moth larvae are most commonly used to rear nematodes because of their commercial availability. The methods of nematode infection, inoculation, and harvesting have been previously described. Using the in vivo process, yields between 0.5 x 10⁵ and 4 x 10⁵ IJs/larva, depending on the nematode species, have been obtained. During the past few years, a distinct cottage industry has emerged that utilizes the in vivo process for nematode mass production for sale, especially in the home lawn and garden markets. However, the in vivo process lacks any economy of scale; the labor, equipment, and material (insect) costs increase as a linear function of production capacity. Perhaps even more important is the lack of improved quality while increasing scale. The in vivo nematode production is increasingly sensitive to biological variations and catastrophes as scale increases.