Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor‐intensive, skill‐dependent, low‐throughput and inevitably causing dilution‐to‐extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high‐throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony‐forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose‐degrading colonies were screened out among 4766 colonies on an enlarged 150‐mm‐diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony‐forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP‐reared communities. In addition to avoidance of dilution‐to‐extinction, operation‐friendly USI efficiently inoculated microbial samples on the agar plate in a high‐throughput and single‐cell form, which eliminated masking or out‐competition from other species in associated groups, thereby improving rare species cultivability.

中文翻译：

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高通量超声波喷雾接种法可促进稀有微生物菌落的培养

当前获得微生物菌落的方法仍然依赖于传统的稀释和扩散板（DSP）程序，该程序耗费大量人力，依赖技术，通量低并且不可避免地导致稀有微生物被稀释到灭绝。本文中，我们提出了一种新颖的超声喷雾接种（USI）方法，该方法将微生物悬浮液分散到数百万个包含单个细胞的气溶胶中，这些气溶胶最近可以自由沉积在凝胶板上，以实现高通量的菌落培养。与DSP相比，USI显着提高了琼脂平板上菌落的分布均匀性和通量，改善了稀有大肠杆菌的最小菌落形成丰度混合在1％至0.01％的湖泊样品中。将这种新型USI应用于湖泊样品，在150毫米直径的LB平板上，从4766个菌落中筛选出16个降解纤维素的菌落。同时，只有在使用常用的DSP程序时才能偶尔观察到它们。16S rRNA测序进一步表明，USI将集落形成物种从11个（通过DSP）增加到23个，其中包括在DSP培育社区中完全检测不到的7种微生物。除避免稀释至灭绝外，操作友好的USI还以高通量和单细胞形式有效地将琼脂平板上的微生物样品接种到琼脂板上，从而消除了相关组中其他物种的掩盖或竞争，从而改善了稀有物种的可培养性。