Comprehensive Tools for Culturing Blastocystis: A Standardized Resource for Research and Diagnostics

IF 2.2

Current protocols Pub Date : 2025-08-14 DOI:10.1002/cpz1.70175

Daisy Shaw, Constance Denoyelle, Kevin S. W. Tan, C. Graham Clark, Hisao Yoshikawa, Eric Viscogliosi, Eleni Gentekaki, Kateřina Jirků, Anastasios D. Tsaousis

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Abstract

Blastocystis spp. is a widely prevalent anaerobic protozoan of uncertain pathogenicity found in the gastrointestinal tracts of over 1 billion people worldwide. Despite its potential significance in health and disease, Blastocystis spp. remains challenging to culture axenically due to its anaerobic nature and the diversity of its genetic subtypes. This manuscript presents a standardized toolkit for culturing Blastocystis spp. in xenic and axenic conditions, detailing protocols for the preparation of appropriate liquid and solid media, cryopreservation, and inoculation. By providing a comprehensive set of tools and methodologies, this work aims to streamline research on Blastocystis spp., enabling reproducibility, subtype characterization, and advancements in understanding its role in the gut microbiome and host health. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Establishment of xenic Blastocystis culture from stool samples in liquid medium

Basic Protocol 2: Subculturing from existing Blastocystis liquid culture

Support Protocol 1: Preparation of modified Jones’ medium for Blastocystis culturing

Support Protocol 2: Preparation of trypticase-yeast extract-serum-gastric mucin-9 (TSGYM-9) medium for Blastocystis culturing

Support Protocol 3: Preparation of liver extract-yeast extract-serum-gastric mucin (LYSGM) medium for Blastocystis culturing

Basic Protocol 3: Subculturing xenic Blastocystis cultures in diphasic medium

Support Protocol 4: Preparation of Robinson's medium for Blastocystis culturing

Support Protocol 5: Preparation of Ringer's agar slants for Blastocystis culturing

Basic Protocol 4: Axenization of Blastocystis cultures

Basic Protocol 5: Subculturing axenic Blastocystis cultures in diphasic medium

Support Protocol 6: Preparation of Boeck and Drbohlav's Locke-egg serum (LES) medium for Blastocystis culturing

Support Protocol 7: Preparation of Boeck and Drbohlav's diphasic modified medium (BDMM) for Blastocystis culturing

Basic Protocol 6: Establishment of axenic Blastocystis cultures in Iscove's modified Dulbecco's medium (IMDM)

Basic Protocol 7: Establishment of axenic Blastocystis cultures in soft IMDM agar

Basic Protocol 8: Establishment of axenic Blastocystis cultures on solid IMDM agar

Basic Protocol 9: Optimized method for establishing axenic Blastocystis cultures on solid IMDM agar

Basic Protocol 10: Establishment of axenic Blastocystis cultures in semi-solid Locke's agar

Basic Protocol 11: Cryopreservation of xenic Blastocystis cultures

Basic Protocol 12: Cryopreservation of axenic Blastocystis cultures

Basic Protocol 13: Inoculation of liquid medium with xenic Blastocystis cultures from frozen stocks

Basic Protocol 14: Inoculation of liquid medium with axenic Blastocystis cultures from frozen stocks

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囊胚培养综合工具：研究和诊断的标准化资源

囊虫是一种广泛存在的致病性不确定的厌氧原生动物，在全球超过10亿人的胃肠道中发现。尽管囊虫在健康和疾病中具有潜在的意义，但由于其厌氧特性和遗传亚型的多样性，囊虫的无性培养仍然具有挑战性。这篇手稿提出了一个标准化的工具包，培养囊胚spp.在xenic和axenic条件下，详细说明了适当的液体和固体培养基，低温保存和接种的制备方案。通过提供一套全面的工具和方法，本工作旨在简化囊胚的研究，实现可重复性，亚型表征，并在了解其在肠道微生物群和宿主健康中的作用方面取得进展。©2025作者。Wiley期刊有限公司发表的当前方案基本方案1：从粪便样品中建立液体培养基中的异种囊胚培养基本方案2：从现有囊胚液体培养基中继代培养支持方案1：制备用于囊胚培养的改良Jones培养基支持方案2：制备用于囊胚培养的胰蛋白酶-酵母提取物-血清-胃粘蛋白-9 （tsgum -9）培养基支持方案3：制备肝提取物-酵母提取物-血清-胃黏液（LYSGM）培养基用于胚泡培养基本方案3：在双相培养基中传代培养异种胚泡培养支持方案4：制备罗宾逊培养基用于胚泡培养支持方案5：制备林格氏琼脂斜面用于胚泡培养基本方案4：胚泡培养的绝育基本方案5：在双相培养基中传代培养绝育胚泡培养支持方案6：制备Boeck和Drbohlav的lock- egg血清（LES）培养基用于囊胚培养支持方案7：制备Boeck和Drbohlav的双相修饰培养基（BDMM）用于囊胚培养基本方案6：在Iscove的改良Dulbecco培养基（IMDM）中建立无菌囊胚培养基本方案7：在软IMDM琼脂中建立无菌囊胚培养基本方案8：在固体IMDM琼脂上建立无菌囊胚培养基本方案9：在固体IMDM琼脂上建立无菌囊胚培养的优化方法基本方案10：在半固体Locke琼脂上建立无菌囊胚培养基本方案11：异种囊胚培养物的低温保存基本方案12：无菌囊胚培养物的低温保存基本方案13：用冷冻储存的异种囊胚培养物接种液体培养基基本方案14：用冷冻砧木无菌囊胚培养液接种液体培养基

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来源期刊

Current protocols

CiteScore

4.00

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0.00%

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