Worm Breeder's Gazette 16(5): 26 (February 1, 2001)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Hall of Sciences (RISE), Drew University, Madison NJ 07940
In the routine propagation of C. elegans, steam sterilization of the culture medium is standard practice, but in some circumstances it is inconvenient, impractical, or prone to error. In preliminary experiments in this lab, thiabendazole (TBZ) was added to C. elegans cultures in the expectation that the life cycle would be interrupted because of the well-known efficacy of the compound in preventing the development and hatching of nematode eggs. Surprisingly, the propagation of C. elegans appeared to be unaffected. Subsequent investigation showed that TBZ is without ovicidal efficacy against C elegans at concentrations that are fully effective against the eggs of the parasitic nematode Haemonchus contortus (Fasiuddin and Campbell, 2000). The qualitative observations here reported suggest the possibility of exploiting this finding to achieve a useful degree of microbial control in laboratory cultures.
TBZ was added to modified Nematode Growth Medium (Avery and Horowitz, 1990) at 20 µg/ml -- a concentration known to be ovicidal for nematodes (Egerton, 1969) and to have a broad spectrum of antifungal activity (Robinson et al., 1969). Methylene Blue, at 0.0002%, was added as a marker to prevent inadvertent mix-up in routine transfer operations (qualitative observations in this lab had indicated that such a concentration did not suppress the growth of the Escherichia coli seeded onto agar plates as food for C. elegans). The medium was boiled briefly to dissolve and clarify the agar, but it was not autoclaved. TBZ and Methylene Blue were added while the medium was still hot. TBZ was prepared as a solution of 10 mg/ml in 100% dimethyl sulfoxide and added to the medium so as to give a 500 fold dilution. An aqueous solution of Methylene Blue, 0.1%, was similarly added to give a 500 fold dilution. After dispensing the medium into 90-mm petri dishes, a suspension of E. coli (OP 50) was spread on the solidified medium and allowed to form a bacterial lawn in the usual way. Three days after the initial (October 8, 1999) preparation of the plates, 2 plates were inoculated with C. elegans; and single plates were inoculated with worms on day 28, 46, 55, 63 and 88. The plates were held in a humidity chamber at room temperature, without further addition of bacteria. Periodic microscopic examination of the dishes revealed prolific propagation of C. elegans, followed by the persistence of low numbers of motile worms (presumably dauer larvae) at intervals up to day 367 after inoculation of the first 2 plates. At that time, medium from each dish was transferred, without sterile technique, to conventional agar plates, resulting in abundant propagation of worms in all cases.
Another batch of 17 culture plates was prepared in a similar non-aseptic way, but without the addition of Methylene Blue. This batch has remained in a refrigerator for 9 months, during which time small compact colonies (presumably bacterial) have appeared, but there has been no trace of the mycelial fungal contaminants so commonly observed when sterilization and handling procedures have been imperfect.
The preparation of these two batches of non-sterilized culture plates was not accompanied by the simultaneous preparation of autoclaved plates, or plates with dimethyl sulfoxide or Methylene Blue as the only additive; but throughout the test period conventional plates, without additives, were being routinely made and used. Nor was any attempt made to expose the plates to known air-borne contaminants or to inoculate them with bacteria or fungi. Nevertheless, the absence of visible mycelial growth was in marked contrast to the degree of contamination customarily seen when sterility measures have been less than rigorous, and the year-long persistence of C. elegans was also striking.
If these findings can be confirmed in quantitative trials, supplementation of a medium with TBZ in a dimethyl sulfoxide vehicle (with an antibacterial adjunct) may provide a convenient "limited contamination" medium for the routine maintenance of C. elegans. Other antimycotic agents are available for use in culture systems. In the present context, the most useful characteristics of TBZ are its very long shelf life at room temperature, its stability in media when boiled or even autoclaved, and the degree to which this otherwise antinematodal agent is tolerated by the nematode C. elegans. Methylene Blue has some degree of antibacterial efficacy and, at the concentration used here, was tolerated by both the worm and the bacterium on which it feeds. Other compounds with selective antibacterial activity may be more effectively combined with TBZ to suppress unwanted bacteria in C. elegans cultures.
References.
Avery, L and H. R. Horvitz. 1990. Effects of starvation and neuroactive drugs of feeding in Caenorhabditis elegans. J. Experimental Zoology 253: 263- 270.
Egerton, J. R. 1969. The ovicidal and larvicidal effect of thibendazole on various helminth species. Texas Reports on Biology and Medicine (Suppl. 2) 27: 561 - 580.
Fasiuddin, A. and W. C. Campbell. 2000. Haemonchus contortus (Nematoda: Trichostrongylidae) is much more sensitive than Caenorhabditis elegans to the ovicidal action of thiabendazole. Journal of Parasitology 86: 628-630.
Robinson, H. J., R. H. Silber and O. E. Graessle. 1967. Thiabendazole: toxicological, pharmacological and antifungal properties. Texas Reports on Biology and Medicine (Suppl. 2) 27: 537-560.