Chemotactic behaviour is an important part of the lifestyle of motile bacteria and enables cells to respond to various environmental stimuli. The Hard Agar Plug (HAP) method is used to study the chemotactic behaviour of bacteria, including the fastidious microaerophile Campylobacter jejuni, an intestinal pathogen of humans. However, the traditional HAP assay is not quantitative, is unsuitable for chemotaxis observation over short time periods and for the investigation of repellent taxis, and is prone to false-positive and -negative results. Here we report an accurate, rapid, and quantitative HAP-based chemotaxis assay, tHAP, for the investigation of bacterial chemotactic responses. The critical component of the new assay is the addition of triphenyltetrazolium chloride (TTC). Enzymatic reduction of TTC to TFP-Red (1, 3, 5-Triphenylformazan) enables colourimetric detection of actively metabolising bacterial cells. Quantitative assessment of chemotaxis is achieved by colourimetric measurement or viability count over a period of 10 min to 3 h. Using the tHAP assay, we observed the dose-responsive chemotactic motility of C. jejuni cells along different concentrations of attractants aspartate and serine. Importantly, we have also designed a competitive tHAP assay to differentiate between repellents and attractants and to identify chemoeffectors that do not activate metabolism.
Importance: The modified tHAP assay described here enables the exploration of the chemoresponse of Campylobacter jejuni towards chemorepellents, and catabolizable and non-catabolizable chemoattractants.
Keywords: Campylobacter; Chemoreceptor; Chemotaxis; Hard-plug agar (HAP) assay; Motility; Triphenyltetrazolium chloride (TTC).
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