Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening
The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening. Some in vitro testings has been developed and are currently available for antimycobacterial screening. The aim...
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| Format: | Article |
| Language: | English |
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Elsevier
2019-08-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844019359237 |
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| author | Maya Dian Rakhmawatie Tri Wibawa Puspita Lisdiyanti Woro Rukmi Pratiwi Mustofa |
| author_facet | Maya Dian Rakhmawatie Tri Wibawa Puspita Lisdiyanti Woro Rukmi Pratiwi Mustofa |
| author_sort | Maya Dian Rakhmawatie |
| collection | DOAJ |
| description | The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening. Some in vitro testings has been developed and are currently available for antimycobacterial screening. The aim of the study was to compare Resazurin Microplate Assay (REMA) and Crystal Violet Decolorization Assay (CVDA) for testing mycobacteria susceptibility to isoniazid and rifampicin as well as for antimycobacterial screening of natural products (NP). Mycobacterium tuberculosis strain H37Rv and Mycobacterium smegmatis strain mc2 155 were used as tested mycobacteria. Serial two-fold dilutions from 0.0625 to 1.0 μg/mL for the isoniazid and rifampicin and from 6.25 to 100.0 μg/mL for the NP A and B were prepared. Tested mycobacteria were then incubated with tested drugs or NPs in each growth medium at 37 °C for 7 days for M. tuberculosis and 3 days for M. smegmatis. MIC values against M. tuberculosis were interpreted 24–48 h after adding resazurin or at least 72 h after adding crystal violet, whereas MIC values against M. smegmatis were interpreted 1 h after adding resazurin or 24 h after adding crystal violet. The MIC values against M. tuberculosis interpreted by REMA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively. Moreover, the MIC values against M. smegmatis interpreted by REMA were 0.0625, >1, 6.25, and 100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.125, >1, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, NP B respectively. In conclusion, REMA is faster and easier than CVDA to interpret MIC values, however CVDA produces higher MIC values than REMA for rifampicin and NP B in M. smegmatis susceptibility testing. Therefore, REMA and CVDA can be used for antimycobacterial screening. |
| first_indexed | 2024-04-12T01:02:35Z |
| format | Article |
| id | doaj.art-abe4b21122e2482ea2ecc35f9cdc52a4 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-12T01:02:35Z |
| publishDate | 2019-08-01 |
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| spelling | doaj.art-abe4b21122e2482ea2ecc35f9cdc52a42022-12-22T03:54:23ZengElsevierHeliyon2405-84402019-08-0158e02263Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screeningMaya Dian Rakhmawatie0Tri Wibawa1Puspita Lisdiyanti2Woro Rukmi Pratiwi3 Mustofa4Doctoral Program in Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia; Department of Biomedical Sciences, Faculty of Medicine, Universitas Muhammadiyah Semarang, Semarang, IndonesiaDepartment of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, IndonesiaResearch Center for Biotechnology, Indonesian Institute of Sciences, Bogor, IndonesiaDepartment of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, IndonesiaDepartment of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia; Corresponding author.The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening. Some in vitro testings has been developed and are currently available for antimycobacterial screening. The aim of the study was to compare Resazurin Microplate Assay (REMA) and Crystal Violet Decolorization Assay (CVDA) for testing mycobacteria susceptibility to isoniazid and rifampicin as well as for antimycobacterial screening of natural products (NP). Mycobacterium tuberculosis strain H37Rv and Mycobacterium smegmatis strain mc2 155 were used as tested mycobacteria. Serial two-fold dilutions from 0.0625 to 1.0 μg/mL for the isoniazid and rifampicin and from 6.25 to 100.0 μg/mL for the NP A and B were prepared. Tested mycobacteria were then incubated with tested drugs or NPs in each growth medium at 37 °C for 7 days for M. tuberculosis and 3 days for M. smegmatis. MIC values against M. tuberculosis were interpreted 24–48 h after adding resazurin or at least 72 h after adding crystal violet, whereas MIC values against M. smegmatis were interpreted 1 h after adding resazurin or 24 h after adding crystal violet. The MIC values against M. tuberculosis interpreted by REMA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively. Moreover, the MIC values against M. smegmatis interpreted by REMA were 0.0625, >1, 6.25, and 100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.125, >1, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, NP B respectively. In conclusion, REMA is faster and easier than CVDA to interpret MIC values, however CVDA produces higher MIC values than REMA for rifampicin and NP B in M. smegmatis susceptibility testing. Therefore, REMA and CVDA can be used for antimycobacterial screening.http://www.sciencedirect.com/science/article/pii/S2405844019359237Resazurin microplate assayMycobacteriumNatural product screeningCrystal violet decolorization assayAntimicrobial susceptibility testing |
| spellingShingle | Maya Dian Rakhmawatie Tri Wibawa Puspita Lisdiyanti Woro Rukmi Pratiwi Mustofa Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening Heliyon Resazurin microplate assay Mycobacterium Natural product screening Crystal violet decolorization assay Antimicrobial susceptibility testing |
| title | Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| title_full | Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| title_fullStr | Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| title_full_unstemmed | Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| title_short | Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| title_sort | evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening |
| topic | Resazurin microplate assay Mycobacterium Natural product screening Crystal violet decolorization assay Antimicrobial susceptibility testing |
| url | http://www.sciencedirect.com/science/article/pii/S2405844019359237 |
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