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[
1994]
Nematodes have been cultured continuously in the laboratory since 1944 when Margaret Briggs Gochnauer isolated and cultured the free-living hermaphroditic species Caenorhabditis briggsae. Work with C. briggsae and other rhabditid nematodes, C. elegans, Rhabditis anomala, and R. pellio, demonstrated the relative ease with which they could be cultured. The culturing techniques described here were developed for C. elegans, but are generally suitable (to varying degrees) for other free-living nematodes. Whereas much of the early work involved axenic culturing, most of these techniques are no longer in common use and are not included here. In the 1970s C. elegans became the predominant research model due to work by Brenner and co-workers on the genetics and development of this species. An adult C. elegans is about 1.5 mm long, and under optimal laboratory conditions has a life cycle of approximately 3 days. There are two sexes, males and self-fertile hermaphrodites, that are readily distinguishable as adults. The animals are transparent throughout the life cycle, permitting observation of cell divisions in living animals using differential interference microscopy. The complete cell lineage and neural circuitry have been determined and a large collection of behavioral and anatomical mutants have been isolated. C. elegans has six developmental stages: egg, four larval stages (L1-L4), and adult. Under starvation conditions or specific manipulations of the culture conditions a developmentally arrested dispersal stage, the dauer larva, can be formed as an alternative third larval stage. Many of the protocols included here and other experimental protocols have been summarized in "The Nematode Caenorhabditis elegans". We also include a previously unpublished method for long-term chemostat cultures of C. elegans. General laboratory culture conditions for nematode parasites of animals have been described, but none of these nematodes can be cultured in the laboratory through more than one life cycle. Marine nematodes and some plant parasites have been cultured xenically or with fungi. Laboratory cultivation of several plant parasites on Arabidopsis thaliana seedlings in agar petri plates has also been reported.
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[
1977]
The workshop on nematodes presented current research from four laboratories on the development and physiology of C. elegans.
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Caenorhabditis elegans is a free-living soil nematode, about 1 mm in length, that is found around the world. It is currently a common laboratory model for many aspects of cellular, developmental, and molecular biology. Its popularity comes from its transparency (allowing all nuclei to be followed in living animals at all stages of development), its anatomical simplicity (1000 cells), its small genome (100 Mbp), an invariant somatic cell lineage, ease of laboratory culture, rapid generation time, and a mode of reproduction which facilitates classical genetic analysis. An interested beginner needs only a petri plate, some Escherichia coli, and a stereo dissecting microscope to begin study of this fascinating creature.
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[
Methods Cell Biol,
1995]
The nematode Caenorhabditis elegans is a small, rapidly growing organism that can easily be raised in the laboratory on the bacterium Escherichia coli. Because C. elegans is a self-fertilizing hermaphrodite, it is possible to readily grow large quantities of the organism in swirling liquid cultures and also possible to propagate severely incapacitated mutants. The rapidity of growth and the ability to self-fertilize necessitate special measures to establish a synchronous culture.
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[
1998]
In this study initial data were generated to develop laboratory control charts for aquatic toxicity testing using the nematode Caenorhabditis elegans. Tests were performed using two reference toxicants: CdCl2 and CuCl2. All tests were performed for 24 h without a food source and for 48 h with a food source in a commonly used nematode aquatic medium. Each test was replicated 6 times with each replicate having 6 wells per concentration with 10 +/- 1 worms per well. Probit analysis was used to estimate LC50 values for each test. The data were used to construct a mean laboratory control chart for each reference toxicant. The coefficient of variation (CV) for three of the four reference toxicant tests was less than 20%, which demonstrates an excellent degree of reproducibility. These CV values are well within suggested standards for determination of organism sensitivity and overall test system credibility. A standardized procedure for performing 24 h and 48 h aquatic toxicity studies with C. elegans is
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[
WormBook,
2005]
C. elegans presents a low level of molecular diversity, which may be explained by its selfing mode of reproduction. Recent work on the genetic structure of natural populations of C. elegans indeed suggests a low level of outcrossing, and little geographic differentiation because of migration. The level and pattern of molecular diversity among wild isolates of C. elegans are compared with those found after accumulation of spontaneous mutations in the laboratory. The last part of the chapter reviews phenotypic differences among wild isolates of C. elegans.
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[
1960]
For the purpose of the present chapter the noun 'cultivation' is to be taken as the maintenance, in the laboratory, of a population of organisms belonging to a desired species through successive generations and subcultures over a prolonged period of time (weeks, months, or years). This is a deliberate restriction of the term. The noun 'culture' is most aptly used for a population within a circumscribed vessel or container (test-tube, Petri dish, U.S. Bureau of Plant Industry watch glass, etc.); it is also used in a looser, more general way (as "in culture") to cover conditions of substantial growth whether or not leading to cultivation in the strict sense
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[
1974]
The free-living nematode, Caenorhaditis briggsae, is being used in our laboratory to study the complex events associated with biological aging. Our approach to this problem involved first the defining of parameters characterizing senescence in this animal, and then evaluating the effects on these aging signs of a drug reported to have a modifying effect on some aspects of the aging processes. Reference in this report to this preparation, Gerovital H3 (2% procaine hydrochloride, 0.16% benzoic acid, 0.14% potassium metabisulfite, buffered to pH 3.3 from Rom-Amer Pharmaceuticals, Ltd., Beverly Hills, California) is by its active ingredient, "Procaine".
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[
Methods Cell Biol,
1995]
Although Caenorhabditis elegans was originally chosen as a model organism for cell biology with serial section electron microscopy (EM) methods in mind, these methods have remained a daunting challenge. There is an apocryphal story that Nichol Thomson originally advised Sydney Brenner that C. elegans was unsuitable for electron microscopy and that Brenner should choose another species. Other experienced microscopists have probably shared similar dark thoughts from time to time. Nonetheless, the worm's very small size, simple organization, and cablelike nervous system have permitted Brenner's colleagues to characterize every cell and cell contact in the wild-type animal, potentiating the genetic characterization of cellular development in remarkable detail. We attempt to provide an adequate background for anyone to initiate EM studies of C. elegans. Two decades ago, as the first of Brenner's postdoctoral fellows left his laboratory to establish new worm laboratories, it was standard practice to include an EM component in their studies. Their combined efforts to characterize the adult animal's cell types and the essential steps in its development helped to erect a lovely scaffold of key manuscripts, capped by the description of the "Mind of the Worm" in some 600 micrographs and 175 drawings. Many of these works required technical heroics or suffered long delays before publication. Most people later chose to leave electron microscopy behind in pursuit of molecular quarry. The fruits of their molecular and genetic studies should soon stimulate a renewed flowering of electron microscopy. We hope to smooth your entry or reentry into these techniques. We also summarize our methods for three-dimensional (3D) image reconstruction, based largely on film techniques introduced by John White and Randle Ware. Digital imaging techniques seem poised to make 3D reconstruction more accessible, and may simplify the exchange of morphological data between laboratories. We discuss several computer systems that the C. elegans community could adopt for high-resolution studies of structure and function. In addition, we briefly cover several specialized specimen preparation techniques for electron microscopy, including freeze fracture and electron microscopic immunocytochemistry.