Label-free 1D microfluidic dipstick counting of microbial colonies and bacteriophage plaques.
Label-free 1D microfluidic dipstick counting of microbial colonies and bacteriophage plaques.
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对微生物菌落和噬菌斑进行无标记一维微流体试纸计数。
DOI：
10.1039/d2lc00280a
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发表时间：
2022
期刊：
影响因子：
6.1
通讯作者：
Dönmez SI
中科院分区：
文献类型：
--
作者：
Dönmez SI
计数活的细菌细胞和功能性噬菌体是微生物学的基础研究，监测，生物制药和诊断。菌落形成单位（CFU）和噬菌斑形成单位（PFU）计数仍然需要在培养皿或平板中的琼脂上进行缓慢且费力的固体培养。在这里，我们表明，试纸微流控条可以在没有生长指示剂染料的情况下用于快速和简单的CFU ml−1和PFU ml−1测量。我们首次证明，含氟聚合物微毛细管与数字成像相结合，使噬菌体噬菌斑被迅速计数的浸渍和测试格式。微流体长度尺度提供了2D固体琼脂培养基表面的线性一维替代方案，其中菌落或噬菌斑清晰可见为“虚线”或“间隙”。使用Raspberry Pi成像的廉价开源暗场生物传感器系统允许在400 μm内径微毛细管内的线性液体基质中在4-8小时内对菌落或噬菌斑进行无标记检测和计数。我们获得了完整的定量一致性之间的一维微流控菌落计数与传统的二维固体琼脂培养皿板的S。aureus和E.大肠杆菌，T2噬菌体和噬菌体K，但高达6倍的速度。延时暗场成像允许详细的动力学分析的微毛细管中的菌落生长，提供了新的见解微流体微生物学和菌落生长，不可能与培养皿。令人惊讶的是，E。大肠杆菌菌落出现较早，随后菌落沿毛细血管沿着扩展较快。金黄色。这可以通过微毛细管内为1D菌落生长提供的微环境来解释，该微环境与营养物（葡萄糖）扩散和细菌生长动力学之间的质量平衡有关。计数单个菌落在液体培养基中是不可能的运动菌株，迅速蔓延沿着毛细管，但列入软琼脂抑制蔓延，使这种新的简单的浸渍和测试计数方法适用于运动和非运动细菌。无标记试纸菌落和噬菌斑计数具有许多分析微生物任务的潜力，并且1D菌落计数的创新与其他微流体微生物学相关。
Counting viable bacterial cells and functional bacteriophage is fundamental to microbiology underpinning research, surveillance, biopharmaceuticals and diagnostics. Colony forming unit (CFU) and plaque forming unit (PFU) counting still requires slow and laborious solid culture on agar in Petri dishes or plates. Here, we show that dip-stick microfluidic strips can be used without growth indicator dye for rapid and simple CFU ml−1 and PFU ml−1 measurement. We demonstrate for the first time that fluoropolymer microcapillaries combined with digital imaging allow bacteriophage plaques to be counted rapidly in a dip-and-test format. The microfluidic length scales offer a linear 1-dimensional alternative to a 2D solid agar medium surface, with colonies or plaques clearly visible as “dashes” or “gaps”. An inexpensive open source darkfield biosensor system using Raspberry Pi imaging permits label-free detection and counting of colonies or plaques within 4–8 hours in a linear, liquid matrix within ∼200 μm inner diameter microcapillaries. We obtained full quantitative agreement between 1D microfluidic colony counting in dipsticks versus conventional 2D solid agar Petri dish plates for S. aureus and E. coli, and for T2 phage and phage K, but up to 6 times faster. Time-lapse darkfield imaging permitted detailed kinetic analysis of colony growth in the microcapillaries, providing new insight into microfluidic microbiology and colony growth, not possible with Petri dishes. Surprisingly, whilst E. coli colonies appeared earlier, subsequent colony expansion was faster along the microcapillaries for S. aureus. This may be explained by the microenvironment offered for 1D colony growth within microcapillaries, linked to a mass balance between nutrient (glucose) diffusion and bacterial growth kinetics. Counting individual colonies in liquid medium was not possible for motile strains that spread rapidly along the capillary, however inclusion of soft agar inhibited spreading, making this new simple dip-and-test counting method applicable to both motile and non-motile bacteria. Label-free dipstick colony and plaque counting has potential for many analytical microbial tasks, and the innovation of 1D colony counting has relevance to other microfluidic microbiology.
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