Efficacy of common laboratory disinfectants and heat on killing trypanosomatid parasites
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 12 August 2008
Accepted: 22 September 2008
Published: 22 September 2008
The disinfectants TriGene, bleach, ethanol and liquid hand soap, and water and temperature were tested for their ability to kill bloodstream forms of Trypanosoma brucei, epimastigotes of Trypanosoma rangeli and promastigotes of Leishmania major. A 5-min exposure to 0.2% TriGene, 0.1% liquid hand soap and 0.05% bleach (0.05% NaOCl) killed all three trypanosomatids. Ethanol and water destroyed the parasites within 5 min at concentrations of 15–17.5% and 80–90%, respectively. All three organisms were also killed when treated for 5 min at 50°C. The results indicate that the disinfectants, water and temperature treatment (i.e. autoclaving) are suitable laboratory hygiene measures against trypanosomatid parasites.
Demonstration of the efficacy of disinfectants against animal and human pathogens has become a requisite part of the documentation associated with licensed handling. In order to obtain a licence for working with trypanosomatid parasites, authorities request verification that the disinfectants and autoclaving conditions indicated in many standard operating procedures to inactivate the pathogens are indeed capable of efficient killing of the organisms. As such data has not been readily available either from manufacturers or as publication, in recent years each laboratory has been required to carry out inactivation experiments independently before further work can be undertaken. The purpose of this report is to confirm that disinfectants commonly used in laboratories and heat treatment result in killing of trypanosomatid parasites.
We tested the commercial disinfectant TriGene (MediChem International Ltd., U.K.), and bleach (sodium hypochlorite (NaOCl) solution; Fisher Scientific, U.K.) and ethanol as general laboratory disinfectants for their ability to kill bloodstream forms of Trypanosoma brucei (clone 427-221a ), epimastigotes of Trypanosoma rangeli (Choachi strain ) and promastigotes of Leishmania major (Friedlin strain ). In addition, we also investigated the effect of dilution in water, liquid hand soap (RBS HDS 10; Medline Scientific LTD., U.K.) and heat treatment on the parasites. The parasites were incubated at a cell density of 1 × 106/ml with various concentrations of the reagents in appropriate medium (T. brucei, Baltz medium plus 20% heat-inactivated foetal calf serum (iFCS) ; T. rangeli, Liver Infusion Tryptose medium plus 10% iFCS ; L. major, medium 199 plus 10% iFCS ) in a final volume of 1 ml at room temperature. The controls contained the corresponding amount of water (except for experiments testing the effect of dilution in water where the controls contained only medium). After 5 min incubation, live cells were counted using a Neubauer haemocytometer. The 50% lethal concentration (LC50), i.e. the reagent concentration necessary to kill 50% of the cells compared to the control, was determined by linear interpolation . The 100% lethal concentration (LC100), i.e. the lowest concentration of a reagent at which all cells were killed, was determined microscopically. For heat treatment, parasites at a cell density of 1 × 106/ml in 1 ml appropriate medium were incubated at different temperature using a digital heater block (Grant Instruments, U.K.). Samples incubated at room temperature served as controls. After 5 min incubation, live cells were counted using a Neubauer haemocytometer. The 50% lethal temperature (LT50), i.e. the temperature necessary to kill 50% of the cells compared to the control, was determined by linear interpolation . The 100% lethal temperature (LT100), i.e. the lowest temperature at which all cells were killed, was determined microscopically.
LC50 and LC100 values of disinfectants and water for bloodstream forms of T. brucei, epimastigotes of T. rangeli and promastigotes of Leishmania major.
Effect of temperature on bloodstream forms of T. brucei, epimastigotes of T. rangeli and promastigotes of L. major.
In this study we have shown that bloodstream forms of T. brucei, epimastigotes of T. rangeli and promastigotes of L. major are quite fragile organisms which can be easily killed with disinfectants commonly used in laboratories and by heat treatment. All three parasite species exhibited very similar sensitivities for the reagents tested and temperature. As these three parasites are representatives of the Salivaria group (T. brucei brucei, T. brucei rhodesiense, T. brucei gambiense, T. congolense and T. vivax), the Stercoraria group (sibling species T. cruzi and T. rangeli) and the Leishmania genus (L. major, L. donovani, L. infantum, L. mexicana, L. braziliensis, L amazonensis etc.), our findings likely indicate that all other pathogenic trypanosomatids display similar susceptibilities for these disinfectant and temperature treatments. In conclusion, common laboratory disinfectant (at the indicated concentrations) and temperature treatment can be used for effective inactivation of waste liquid and general laboratory ware that has been contaminated with trypanosomatid parasites.
We thank Professor Edmundo Grisard for providing epimastigotes of T. rangeli Choachi strain.
- Hirumi H, Hirumi K, Doyle JJ, Cross GAM: In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei. Parasitology. 1980, 80: 371-382.View ArticlePubMedGoogle Scholar
- Grisard EC, Campbell DA, Romanha AJ: Mini-exon gene sequence polymorphism among Trypanosoma rangeli strains isolated from distinct geographical regions. Parasitology. 1999, 118: 375-382. 10.1017/S0031182098003928.View ArticlePubMedGoogle Scholar
- Ivens AC, Blackwell JM: Unravelling the Leishmania genome. Curr Opin Genet Dev. 1996, 6: 794-710. 10.1016/S0959-437X(96)80024-4.View ArticleGoogle Scholar
- Baltz T, Baltz D, Giroud C, Crockett J: Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985, 4: 1273-1277.PubMed CentralPubMedGoogle Scholar
- Hendricks LD, Wood DE, Hajduk ME: Haemoflagellates: commercially available liquid media for rapid cultivation. Parasitology. 1978, 76: 309-316.View ArticlePubMedGoogle Scholar
- Huber W, Koella JC: A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop. 1993, 55: 257-261. 10.1016/0001-706X(93)90083-N.View ArticlePubMedGoogle Scholar
- Wolfreys K, Field MC: Verification of the efficiency of killing Trypanosoma brucei by TriGene reagent. Web publication. 2005, [http://homepage.mac.com/mfield/lab/PDFs/TriGENE+verification.pdf]Google Scholar
- Beitz E: Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy. Biol Cell. 2005, 97: 373-383. 10.1042/BC20040095.View ArticlePubMedGoogle Scholar
- Allen CL, Field MC: Verification of the efficiency of killing Trypanosoma brucei by autoclaving. Web publication. 2004, [http://homepage.mac.com/mfield/lab/PDFs/Autoclave+verification.pdf]Google Scholar
- Ritenour MA, Sargent SA, Bartz JA: Chlorine use in citrus packinghouses. Packinghouse Newsletter. 2000, 192: 4-7. [http://postharvest.ifas.ufl.edu/Packing+House+NewsLetters/PHNL+192.pdf]Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.