Nov 13, 2023
Assessment of the in vitro trypanocidal activity
- 1Ahmadu Bello University
External link: https://www.mdpi.com/2036-7481/8/1/6963
Protocol Citation: Muhammad Muhsin Fathuddin 2023. Assessment of the in vitro trypanocidal activity. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxqd1lpk/v1
Manuscript citation:
Fathuddin, M. M., & Inabo, H. I. (2017). In vitro antitrypanosomal potential of chloroform leaf extract of Punica granatum L. on Trypanosoma brucei brucei and Trypanosoma evansi. Microbiology Research, 8(1). MDPI AG. Retrieved from http://dx.doi.org/10.4081/mr.2017.6963
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 05, 2023
Last Modified: November 13, 2023
Protocol Integer ID: 90433
Keywords: Antitrypanosomal activity, Trypanosoma brucei brucei, Rapid Matching Method
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Abstract
The aim of this experiment is to assess the potential of a plant extract to fight off Trypanosoma species, including Trypanosoma brucei brucei, Trypanosoma evansi, etc., by testing it at concentrations ranging from 006.25 to 400.00 mg/mL. The results obtained indicate that the plant extract may be a useful tool in the management of trypanosomiasis, but further studies are required to confirm its effectiveness.
Guidelines
The experimental animals need to be handled in accordance with Good Laboratory Practices and Guidelines for Laboratory Animal Facilities and the recommendations of the Committee on Animal Use and Care
Safety warnings
Handling the experimental animals may require training to reduce the risk of injury to the animal and the researcher
Ethics statement
This experimental work needs the Committee on Animal Use and Care approval before commencement
Before start
The experimental animals should be screened for any ailment at the commencement of the experiment using the laboratory’s recommended procedure and cleared of any ailment.
Extract Preparation
Extract Preparation
To produce a stock Sample solution of100 mg/mL for the extract, weigh1 g (1000 mg ) of the extract, which was solubilized in 1 mL of dimethylsulfoxide (DMSO) solution and made up to 9 mL in dextrose saline/normal saline.
Serial dilutions were made for the remaining concentration of50 mg/mL , 25 mg/mL , 12.5 mg/mL and 6.25 mg/mL of the extracts were made in Dextrose saline (freshly prepared).
Antitrypansomal Activity
Antitrypansomal Activity
5m
The infected rat to undergo euthanasia must have attained a blood parasitemia of log 8.4 or higher
CITATION
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Euthanatized animal’s blood was dissolved in heparin (1 mL of heparin per 10 mL of blood) and mixed with glucose (0.1 g of glucose per 10 mL of blood).
Then, aseptically, a clean micropipette transfers the blood (50 µL ) to a clean, sterile microtiter plate into multiple wells.
To the two wells containing blood, the same volume (i.e., 50 µL ) of the highest drug concentration was added, respectively.
Step 4 was repeated for the other different concentrations available.
The negative control blood was mixed with the dilutant used to prepare the various concentrations (i.e., dextrose-saline or water).
The positive tests were also performed with the standard recommended concentrations
of a commercially available trypanocide drug.
At 00:05:00 5-minute interval, a drop of blood is taken from each well and smeared on a clean glass slide. then observe under a microscope at 400x for signs of motility unique to Trypanosoma spp.
CITATION
CITATION
The above step is repeated with each well. Till motility stopped in all wells
Protocol references
Herbert, W. J., & Lumsden, W. H. (1976). Trypanosoma brucei: a rapid "matching" method for estimating the host's parasitemia. Experimental parasitology, 40(3), 427–431. https://doi.org/10.1016/0014-4894(76)90110-7
Chappuis, F., Loutan, L., Simarro, P., Lejon, V., & Büscher, P. (2005). Options for field diagnosis of human African trypanosomiasis. Clinical microbiology reviews, 18(1), 133–146. https://doi.org/10.1128/CMR.18.1.133-146.2005
Fathuddin, M. M., & Inabo, H. I. (2017). In vitro antitrypanosomal potential of chloroform leaf extract of Punica granatum L. on Trypanosoma brucei brucei and Trypanosoma evansi Microbiology Research, 8(1) MDPI AG. Retrieved from http://dx.doi.org/10.4081/mr.2017.6963
Citations
Step 10
Herbert W.J., Lumsden W.H.R.. Trypanosoma brucei: A rapid “matching” method for estimating the host's parasitemia
https://doi.org/10.1016/0014-4894(76)90110-7Step 10
Chappuis F, Loutan L, Simarro P, Lejon V, Büscher P. Options for field diagnosis of human african trypanosomiasis.
https://doi.org/10.1128/cmr.18.1.133-146.2005Step 3
Herbert W.J., Lumsden W.H.R.. Trypanosoma brucei: a rapid "matching" method for estimating the host's parasitemia
https://doi.org/10.1016/0014-4894(76)90110-7