Prakash Om, Waghmare Ujjwala, Chauhan Ashvini, Patil Yogesh
Symbiosis Centre for Climate Change and Sustainability, Symbiosis International (Deemed University), Lavale, India.
Environmental Biotechnology School of the Environment, Florida A&M University, Tallahassee, Florida, USA.
Appl Environ Microbiol. 2025 Jun 18;91(6):e0172824. doi: 10.1128/aem.01728-24. Epub 2025 May 14.
Using a buffered medium is considered essential for enriching and cultivating novel microbial taxa, studying their pH range and optima, and conducting different physiological experiments. Experimental evidence showed that different buffer compounds impact microbial physiology and cell growth differently, and some of them exert toxic and inhibitory effects on organisms under study. Laboratory growth media supplemented with incompatible buffers could also suppress the organism's growth. Therefore, the selection of buffers without the knowledge of their implications on cell growth and physiology in such experiments yields an inaccurate estimate of their physiological abilities and pH range and optima. In this paper, the authors argue against the use of buffered medium to enrich and isolate novel taxa and suggest determining the pH range and optima using unbuffered medium for taxonomic description and physiological characterization. Based on previous literature and our observations, we recommend the use of rich universal laboratory growth medium with their pH adjusted using 1 N NaOH and/or 1 N HCl for such studies, except in cases where the organism cannot grow in such media. However, the pH of the growth medium must be continuously monitored, and in cases where the medium's buffering capacity is compromised, a suitable pH buffer with only a neutral effect on cell growth must be used for more accurate physiological experiments with that organism in the future. Based on the inhibitory effects of buffers on different cells (prokaryotes and eukaryotes) and physiological activities, in this manuscript, we also recommend that the compatibility of the buffers should be first screened before starting any physiological experiments, and any buffer compound should be avoided in the culture medium during the designing of the culturomics protocols for the cultivation of novel taxa from natural samples.
使用缓冲介质被认为对于富集和培养新的微生物分类群、研究它们的pH范围和最适pH值以及进行不同的生理实验至关重要。实验证据表明，不同的缓冲化合物对微生物生理和细胞生长的影响不同，其中一些对所研究的生物体具有毒性和抑制作用。添加了不相容缓冲剂的实验室生长培养基也可能抑制生物体的生长。因此，在不了解缓冲剂在此类实验中对细胞生长和生理影响的情况下选择缓冲剂，会对其生理能力、pH范围和最适pH值产生不准确的估计。在本文中，作者反对使用缓冲介质来富集和分离新的分类群，并建议使用未缓冲的介质来确定pH范围和最适pH值，以进行分类描述和生理特征分析。基于先前的文献和我们的观察，我们建议在进行此类研究时使用丰富的通用实验室生长培养基，并用1 N NaOH和/或1 N HCl调节其pH值，除非生物体无法在这种培养基中生长。然而，必须持续监测生长培养基的pH值，并且在培养基缓冲能力受损的情况下，未来对该生物体进行更准确的生理实验时，必须使用对细胞生长仅具有中性作用的合适pH缓冲剂。基于缓冲剂对不同细胞（原核生物和真核生物）和生理活动的抑制作用，在本手稿中，我们还建议在开始任何生理实验之前首先筛选缓冲剂的兼容性，并且在设计从自然样品中培养新分类群的 culturomics 方案时，应避免在培养基中使用任何缓冲化合物。
