Plant Tissue Trypan Blue Staining During Phytopathogen Infection   

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Original research article

A brief version of this protocol appeared in:
The Plant Journal
Jan 2002


In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than ever. The protocol is applied specifically to detect cell death on Arabidopsis leaves during the course of infection with necrotrophic fungus Botrytis cinerea.

Keywords: Trypan Blue staining, Plant cell death, Botrytis cinerea, Arabidopsis thaliana, Chloral hydrate


One of the most common methods to detect dead plant tissue is trypan blue staining (Keogh et al., 1980). This diazo dye is also used in histology and medicine to measure tissue viability through allowing the visualization of cell death1 (Keogh et al., 1980; Cooksey, 2014). Most microscopic procedures involving trypan blue staining require a long subsequent clearing step using chloral hydrate (CHL), a small organic compound currently used such as a carcinogen, an anesthetic and an analgesic in laboratory animals (Keogh et al., 1980; Lu and Greco, 2006; Salmon et al., 1995). CHL is not approved by the FDA in the USA or the EMA in the European Union for any medical indication ( Only 250 mg or 50 mg of choral hydrate are sufficient to produce adult or pediatric sedation respectively, and its toxicity has also been measured in neonatals (, Salazar et al., 2009). The LD50 (median lethal dose) for an adult is estimated to be a 4-h exposure to 0,440 mg/L vapour concentration, which is also the duration currently recommended for de-staining of plant leaves at a concentration of 250 g/100 ml. Long-term use of chloral hydrate also results in a rapid development of tolerance to its effects and possible addiction, as well as adverse effects including rashes, gastric discomfort and severe kidney, heart, and liver failure (Gelder et al., 2005).

Through avoiding the use of CHL, this protocol allows researchers to stain for plant cell death with trypan blue more rapidly and safely, substantially reducing the risk to researchers. Here we demonstrate the utility of this method by monitoring the course of infection of Col-0 leaves with Botrytis cinerea (B.c), the second phytopathogen fungus on scientific/economic importance with a broad host range, and a high capacity to produce hydrogen peroxide in plants (Rolke et al., 2004; Dean et al., 2012; Lehmann et al., 2015). This protocol has been also applied successfully to other Arabidopsis accessions.
1Note: Trypan blue is a synthetic compound derived from toluidine, invented by Paul Ehrlich, winner of the Nobel prize in Physiology and Medicine, 1904 (

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Fernández-Bautista, N., Domínguez-Núñez, J. A., Moreno, M. C. and Berrocal-Lobo, M. (2016). Plant Tissue Trypan Blue Staining During Phytopathogen Infection. Bio-protocol 6(24): e2078. DOI: 10.21769/BioProtoc.2078.

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Jinping Zhao
Texas A&M University
Hi, I am doing trypan blue staining in Nicotiana benthamiana according to the protocol. In the Recipe of staining buffer it says that the final concentration of trypan blue is 10 mg/ml (1%), however 40 mg of trypan blue resolved in the liquid mixture makes a concentration of 1 mg/ml (0.1%). Which concentration should be used?
6/19/2017 10:54:43 AM Reply
Marta Berrocal-Lobo
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Spain

Dear user,
You can try with 0,04g in 40ml of mix, this is the minimum for Arabidopsis leave staining, however with Nicotiana you might need more, the leaves are thicker.
Let me know your results. I will review the text to fix some error.
We observed with Nicotiana sometimes some whitering on leaves during ethanol step.
Good luck

8/23/2017 4:13:31 AM