Received: 16 September 2021 | Revised: 29 November 2021 | Accepted: 31 December 2021 DOI: 10.1002/jobm.202100462 REVIEW Surface sterilization for isolation of endophytes: Ensuring what (not) to grow Pramod K. Sahu 1 | Jyotsana Tilgam 1 | Sushma Mishra 2 | Saima Hamid 3 | Amrita Gupta 4 | Jayalakshmi K. 1 | Satish K. Verma 5 | Ravindra N. Kharwar 5 1 ICAR‐National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan, Uttar Pradesh, India 2 Plant Biotechnology Laboratory, Dayalbagh Educational Institute (Deemed‐to‐be‐University), Agra, Uttar Pradesh, India 3 Department of Plant Biotechnology and Microbial Ecology, University of Kashmir, Hazratbal, Srinagar, Jammu & Kashmir, India 4 Department of Biotechnology, Amity Institute of Biotechnology, Amity University, Lucknow, Uttar Pradesh, India 5 Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Correspondence Pramod K. Sahu, ICAR‐National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan, Uttar Pradesh 275103, India. Email: pramod15589@gmail.com and pramod.sahu@icar.gov.in Abstract Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug‐producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mi- micking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides “what to grow” (the endophytes) and “what not to grow” (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%–10%), ethanol (70%–90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X‐100, Tween 80, and Tween 20. This review com- prehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length. KEYWORDS endophyte isolation, ethanol, sodium hypochlorite, surface contamination, surface sterilization, surfactants 1 | NEED FOR ISOLATION OF ENDOPHYTIC MICROBIOTA Plants are reservoirs of diverse endophytic microbes which are known to colonize within the plant tissues, causing no apparent harmful effects to the plant health [1–3]. In the past few years, the plant‐endophyte asso- ciation has gained immense popularity owing to its widespread applications such as enhancing plant growth and development, alleviation of biotic and abiotic stres- ses, and production of economically important plant metabolites [4–7]. Different mechanisms of the J Basic Microbiol. 2022;1–22. www.jbm-journal.com © 2022 Wiley‐VCH GmbH | 1 Abbreviations: PBS, phosphate‐buffered saline; SDW, sterile distilled water.