State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, China.
PLoS One. 2019 Nov 6;14(11):e0224635. doi: 10.1371/journal.pone.0224635. eCollection 2019.
Contamination control and removal are very important technical aspects of microbiological research. Bacterial contamination is very common in fungal cultures. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment; however, there are drawbacks to using antibiotics, including incomplete removal, limited antibacterial spectra, tendency toward recontamination, effects to fungal strains, and potential risks to the environment. Therefore, in the present work, we developed a new method for bacterial removal from fungi cultured on solid medium, the Cabin-Sequestering (CS) method, based on the different culture characteristics between fungi and bacteria. First, 3-5 mm round or square holes (the "cabin") are excavated on a solid medium plate. The fungal strain containing possible bacterial contamination is inoculated into the cabin. The cabin is then covered with a sterilized coverslip, followed by incubation at the appropriate temperature. After 7-10 days of culturing, fungal hyphae grow out along the edge of the coverslip; however, the contaminating bacteria cannot pass through the space formed between the medium and the coverslip and, thus, remain in the cabin. The newly grown fungal hyphae around the coverslip are re-inoculated into fresh culture plates, where they form bacteria-free fungal colonies. The CS method is easy handling, with a short experimental cycle and rare recontamination. When necessary, it can also be used in combination with antibiotics in bacterial removal operations.
污染控制和去除是微生物研究非常重要的技术方面。细菌污染在真菌培养中非常常见。目前，抑制细菌的常用方法是抗生素处理；然而，使用抗生素存在一些缺点，包括不完全去除、抗菌谱有限、容易再次污染、对真菌菌株的影响以及对环境的潜在风险。因此，在本工作中，我们根据真菌和细菌的不同培养特性，开发了一种从固体培养基上培养的真菌中去除细菌的新方法，即 Cabin-Sequestering（CS）方法。首先，在固体培养基板上挖 3-5 毫米的圆形或方形孔（“舱室”）。将可能含有细菌污染的真菌菌株接种到舱室中。然后，用消毒盖玻片覆盖舱室，在适当的温度下孵育。培养 7-10 天后，真菌菌丝沿着盖玻片的边缘生长；然而，污染的细菌无法通过培养基和盖玻片之间形成的空间，因此仍留在舱室内。盖玻片周围新生长的真菌菌丝被重新接种到新鲜的培养平板中，在那里它们形成无细菌的真菌菌落。CS 方法易于操作，实验周期短，很少发生再次污染。必要时，它也可以与抗生素联合用于细菌去除操作。
