Currently i am try to development suspension cell culture. This my first experience in plant cell. i wanna check viability of cell. For that i make 0.25% evans blue solution in 0.1 mM CaCl2 solution ( Ph 5.6). I wanna comapre staining with unstained cell , stain cell and dead cell so i treat some of my cell with 70% ethanol overnight. then i stain cell both ethanol treated and cell without ethanol treatment for 10 min with concentration of 200 microlitre per ml. And wash with distil water twice. when i see under microscope. I doesn't observe any different between three cell type i.e unstained cell , ethanol treated cell and cell without ethanol treatment. Can any one suggest me better idea.
Hi Manoj, I have no experience with plant cells (I am a medic, pathologist, my experience is in immunohistochemistry) - so I am out of my depth - but maybe I could start a discussion on this topic and some plant experts decide to respond.
I thought that your problem is interesting as you have no difference in staining in three cell suspensions which theoretically should be different. Evan's blue is used in human pathology - it is non-toxic, non-permeable, should not stain viable, non-damaged cells, but only those with compromised membranes.
I looked at some early literature on peri-vital staining with non-toxic dyes in plants. There is a paper using betanin and Evan's blue (describes two concentrations for EB: lower with no washing and higher followed with H2O wash for better observation under microscope). It seems that the short length of staining with a wash may give you a more discerning method.
" The Use of Non-permeating Pigments for Testing the Survival of Cells" D. F. GAFF AND 0. OKONG'O-OGOLA1 Journal of Experimental Botany, Vol. 22, No. 72, pp. 756-8, August 1971 (You can get pdf via Google search)
https://watermark.silverchair.com/22-3-756.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAnEwggJtBgkqhkiG9w0BBwagggJeMIICWgIBADCCAlMGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQM-bH7aeg0MGqpdleMAgEQgIICJFlBZRDxrdlmYR18N1haB46rnl7OEvSJR_-je05gHCE6lLSD3YTiZl9-vu7yL6sUA5k6Xu8TUOBVg1QaTKPcTu64DocOjWiZlKSrv9L1-pld4l5jegWSKz_0PzOmzGoPZc8YikIJVnDo5U6Kknwr-Jw0RegvSnC0maedE1jgmIdd_QyTL41ksyHMv6_qE1oy4JRXHVkzE0kiuQgIOoUx1gZHjaf3I_BDbWZLJ8bwCZ4GY_hg2OF6gSge7pkpOYjEUzLrYqGAKcrde6RmzzT9CUzMQ-l-aYY5eGBcGzTZhVuthjQKwlxi80tQTchIwhAQ2NArz8UlJsiExA1OgJf-pMSWdZH0U-i0afgyJmcrhweAyV16b3w5-EC24ORSXzKYKFImy_oIdbMH5liwYXQCXnypPMHe3zAZyMuNqsUaiEgb6LN_e9d1zTC7BdosD4XrJPdmCbLI6xYRle1jpSRBJScFyEQBlckAtLIvWp6976uiws473IKjZV4YDihFltZsFRAzg9p5ZU2IxO3tvhCbQcAqvtzKNrAoZNaDCurtBdSn8tEVAMJO4jr0iofgBTQiSskqhcci7JjIske0zGQbY6668zDAUfH4JQw9HwcQLXRbosSOXZTiEV7j0BpEVvHveZoLvEPZmUY1opOWRLx-F_CEB9rQv9a8h_mp8fGD4y8ORS_g0qul3uaV3Ouloczi98ecRTawiHT153sBqPHQl_2S_1FT
Long treatment with ethanol (overnight) fixes any cell, plant or animal, and changes properties of cell membranes. The authors used heat treatment: " Sections from roots which had been killed in boiling water showed a uniform red coloration in all the cells" for betanin staining, maybe you could add heat treatment to your experiments.
Good luck with your project - hope some plant expert and your supervisor helps.
Thank you Maria Jeziorska for your interest,i will try to kill my plant by heat treatment
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