Chapter 5: Conclusion
Culture System: The use of smaller culture tubes and lack of modified aeration may be reducing the nematode growth potential in the culture systems. Experimental population of controls appear to correspond with populations reported by Lu and Goetsch (1993) of 80,000 nematodes per milliliter. Populations of controls in experiments 4, 5 and 6 are comparable with reported populations. The major difference between this assay and other reported assays were the lack of indirect aeration, the use of complete chemically-defined media and the specific testing of proteins or peptides for their ability to inhibit reproduction.
Identified Inhibition: The presence of fractions KRS-1297-5 and KRS-1297-6 in cultures significantly reduced the average population below control levels. Further testing should be done to characterize and identify the active portions of these extracts. SDS-gels have revealed several bands of protein which may be a related to the active portion of the fraction. There is no indication exactly which band is responsible for the observed activity. Size fractionation studies, however, suggest the active component has a molecular weight greater than 50,000.
URP-Extracts: Only one of the URP extracts, URPPCM #28, reduced populations of C. elegans. URPPCM #28 was a crude extract. The inhibitory activity seen in this crude extract supports the need for further study. Further purification of the extract needs to be done prior to any further testing. The gelling of the extract was the most probable reason for the apparent inhibitory activity. Further use of the non-nutrient M-9 buffer as an assay medium for reproductive reduction should be discontinued. The population trends show only starvation. The M-9 buffer does not allow reproduction. Only controlled-nutrient media promoting reproduction like CbMM, should be used to look for reproductive inhibitors.
Financial Costs: The cost of the assay would appear to be reasonable except for the labor involved in recording populations every three to four days. It may be necessary to use a less accurate method of estimating populations for initial tests of supplements. A spectrophotometric method relating reflectance to population would be recommended following either Patel and McFadden (1976) or Watson et al. (1974). Cost details are provided in Appendix A.
Solutions for Problems Encountered: The stock nematode cultures must be maintained on a standardized HLE supplement, no older than 3 to 6 months old. Use of older stock HLE promotes laggardism and inhibits proper growth in CbMM defined media. The initial health of the culture is the most important factor controlling population productivity in this assay. Stocks must be kept at a standard age to help promote the reproducibility of data. Sterility must be maintained at all times.
Assay System: The population averages corresponding to KRS experiment # 5 and KRS experiment # 6 suggest proper use and construction of the defined media. More experiments are needed to provide baseline data for this assay system. Baseline data from this assay format should be compared to previously used methods. The testing of pure samples is more likely to provide better results. The previous experiments, however, have shown its use toward isolation of activity of partially purified extracts.
The advantage of the described system is based on the defined media, the reduced aeration and the ability to use small culture volumes. These assays require small amounts of test substances and still allow variability of culture size to satisfy many needs. Because this assay controls many of factors affecting growth and reproduction, a wide range of semi-purified and purified substances may be used in this assay to relate their direct affect on nematode reproduction.
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