A Rapid Colorimetric Assay for the Quantitation of the Viability of Free-living Larvae of Nematodes in Vitro

Overview

Journal Parasitol Res

Specialty Parasitology

Date 2007 May 12

PMID 17492469

Citations 19

Authors

Catherine E James

Mary W Davey

Affiliations

Soon will be listed here.

Abstract

With increasing drug resistance in gastrointestinal parasites, identification of new anthelmintics is essential. The non-parasitic nematode Caenorhabditis elegans is used extensively as a model to identify drug targets and potential novel anthelmintics because it can be readily cultured in vitro. Traditionally, the assessment of worm viability has relied on labour-intensive developmental and behavioral assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) colorimetric assay uses metabolic activity as a marker of viability in mammalian cell culture systems and has been applied for use with filarial nematodes. In the present study, this assay has been optimized and validated to rapidly assess the viability of C. elegans after drug treatment. Living, but not dead, C. elegans take up MTT and reduce it to the blue formazan, providing visual, qualitative, and quantitative assessment of viability. MTT at a concentration of 5 mg/ml with 3 h incubation was optimal for detecting changes in viability with drug treatment. We have applied this assay to quantitate the effects of ivermectin and short-chain alcohols on the viability of C. elegans. This assay is also applicable to first-stage larvae of the parasitic nematode Haemonchus contortus. The advantage of this assay is the rapid quantitation in screening drugs to identify potential anthelmintics.

Citing Articles

Anthelmintic activity of crude and separated extract of (Xanthorrhoeaceae) against bovine adults parasites of and infected larvae of drug resistant strains of the free-living nematode .

Kalmobe J, Vildina J, Boursou D, Menga H, Kouam S, Ndjonka D J Parasit Dis. 2024; 48(4):891-903.

PMID: 39493485 PMC: 11527861. DOI: 10.1007/s12639-024-01701-2.

Evaluation of a natural compound extracted from Dolichandrone atrovirens as a novel antioxidant agent using Caenorhabditis elegans.

Yellurkar M, Singh V, Sai Prasanna V, Das P, Nanjappan S, Velayutham R PLoS One. 2021; 16(9):e0257702.

PMID: 34551009 PMC: 8457486. DOI: 10.1371/journal.pone.0257702.

High-content approaches to anthelmintic drug screening.

Zamanian M, Chan J Trends Parasitol. 2021; 37(9):780-789.

PMID: 34092518 PMC: 8364491. DOI: 10.1016/j.pt.2021.05.004.

Nematocidal Effects of a Coriander Essential Oil and Five Pure Principles on the Infective Larvae of Major Ovine Gastrointestinal Nematodes In Vitro.

Helal M, Abdel-Gawad A, Kandil O, Khalifa M, Cave G, Morrison A Pathogens. 2020; 9(9).

PMID: 32916863 PMC: 7558654. DOI: 10.3390/pathogens9090740.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Jesudoss Chelladurai J, Martin K, Chinchilla-Vargas K, Jimenez Castro P, Kaplan R, Brewer M PLoS One. 2020; 15(8):e0237150.

PMID: 32760111 PMC: 7410292. DOI: 10.1371/journal.pone.0237150.

References

Geldhof P, Murray L, Couthier A, Gilleard J, McLauchlan G, Knox D . Testing the efficacy of RNA interference in Haemonchus contortus. Int J Parasitol. 2006; 36(7):801-10. DOI: 10.1016/j.ijpara.2005.12.004. View

Burglin T, Lobos E, Blaxter M . Caenorhabditis elegans as a model for parasitic nematodes. Int J Parasitol. 1998; 28(3):395-411. DOI: 10.1016/s0020-7519(97)00208-7. View

Marks D, Belov L, Davey M, Davey R, Kidman A . The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells. Leuk Res. 1992; 16(12):1165-73. DOI: 10.1016/0145-2126(92)90114-m. View

Gill M, Olsen A, Sampayo J, Lithgow G . An automated high-throughput assay for survival of the nematode Caenorhabditis elegans. Free Radic Biol Med. 2003; 35(6):558-65. DOI: 10.1016/s0891-5849(03)00328-9. View

Dias N, Nicolau A, Carvalho G, Mota M, Lima N . Miniaturization and application of the MTT assay to evaluate metabolic activity of protozoa in the presence of toxicants. J Basic Microbiol. 1999; 39(2):103-8. View

Brenner S . The genetics of Caenorhabditis elegans. Genetics. 1974; 77(1):71-94. PMC: 1213120. DOI: 10.1093/genetics/77.1.71. View

Dengg M, van Meel J . Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds. J Pharmacol Toxicol Methods. 2004; 50(3):209-14. DOI: 10.1016/j.vascn.2004.04.002. View

Rand J, Johnson C . Genetic pharmacology: interactions between drugs and gene products in Caenorhabditis elegans. Methods Cell Biol. 1995; 48:187-204. DOI: 10.1016/s0091-679x(08)61388-6. View

Boyd W, Cole R, Anderson G, Williams P . The effects of metals and food availability on the behavior of Caenorhabditis elegans. Environ Toxicol Chem. 2004; 22(12):3049-55. DOI: 10.1897/02-565. View

10.

Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63. DOI: 10.1016/0022-1759(83)90303-4. View

11.

Ved R, Saha S, Westlund B, Perier C, Burnam L, Sluder A . Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans. J Biol Chem. 2005; 280(52):42655-42668. PMC: 3910375. DOI: 10.1074/jbc.M505910200. View

12.

Nikolaou S, Hartman D, Presidente P, Newton S, Gasser R . HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus. Int J Parasitol. 2002; 32(6):749-58. DOI: 10.1016/s0020-7519(02)00008-5. View

13.

Anderson G, BOYD W, Williams P . Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans. Environ Toxicol Chem. 2001; 20(4):833-8. View

14.

Anderson G, Cole R, Williams P . Assessing behavioral toxicity with Caenorhabditis elegans. Environ Toxicol Chem. 2004; 23(5):1235-40. DOI: 10.1897/03-264. View

15.

Fujii M, Tanaka N, Miki K, Hossain M, Endoh M, Ayusawa D . Uncoupling of longevity and paraquat resistance in mutants of the nematode Caenorhabditis elegans. Biosci Biotechnol Biochem. 2005; 69(10):2015-8. DOI: 10.1271/bbb.69.2015. View

16.

Comley J, Townson S, Rees M, Dobinson A . The further application of MTT-formazan colorimetry to studies on filarial worm viability. Trop Med Parasitol. 1989; 40(3):311-6. View

17.

Avery L, Horvitz H . Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans. J Exp Zool. 1990; 253(3):263-70. DOI: 10.1002/jez.1402530305. View

18.

Goldstein B, Takeshita H, Mizumoto K, Sawa H . Wnt signals can function as positional cues in establishing cell polarity. Dev Cell. 2006; 10(3):391-6. PMC: 2221774. DOI: 10.1016/j.devcel.2005.12.016. View

19.

Thompson G, de Pomerai D . Toxicity of short-chain alcohols to the nematode Caenorhabditis elegans: a comparison of endpoints. J Biochem Mol Toxicol. 2005; 19(2):87-95. DOI: 10.1002/jbt.20060. View

20.

Comley J, Rees M, Turner C, JENKINS D . Colorimetric quantitation of filarial viability. Int J Parasitol. 1989; 19(1):77-83. DOI: 10.1016/0020-7519(89)90024-6. View