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Abstract

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.

Original languageEnglish
Pages (from-to)2820-2831
Number of pages12
JournalLab on a Chip
Volume22
Issue number15
Early online date6 Jul 2022
DOIs
Publication statusPublished - 26 Jul 2022

Bibliographical note

EPSRC EP/R022410/1 and EP/2010807/1 and Republic of Turkiye Postgraduate Study Award PEP/R022410/1EP/S010807/1

Funding Information:
We thank Danish Malik from Loughborough University for Phage K and host. We thank the Turkish Ministry of National Education, Republic of Turkey for Postgraduate Study Abroad Program to support Sultan İlayda Dönmez. This project was partially funded by Engineering and Physical Science Research Council (EPSRC) grants EP/R022410/1 and EP/S010807/1. Images from Servier Medical Art ( https://smart.servier.com ) were adapted and used for Fig. 1 and S1, under a Creative Commons Attribution license.

Funding Information:
We thank Danish Malik from Loughborough University for Phage K and host. We thank the Turkish Ministry of National Education, Republic of Turkey for Postgraduate Study Abroad Program to support Sultan İlayda Dönmez. This project was partially funded by Engineering and Physical Science Research Council (EPSRC) grants EP/R022410/1 and EP/S010807/1. Images from Servier Medical Art (https://smart.servier.com) were adapted and used for Fig. 1 and S1, under a Creative Commons Attribution license.

Publisher Copyright:
© 2022 The Royal Society of Chemistry.

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • General Chemistry
  • Biomedical Engineering

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