Abstract
BackgroundMany investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed.Methodology/Principal FindingsAccording to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L−1 (13 and 21 nmol L−1), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium.Conclusions/SignificanceAgar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.
Highlights
Agar is an indigestible gelling material that is derived from seaweeds such as Gelidium crinale and Gracilaria vermiculophylla
Peak 1 from both fractions Fr-A and Fr-B was identical to 5hydroxymethylfuran-2-carboxylic acid (5-HMFA) using FD-MS, EI-HR-MS and 1H NMR [21,22]
We examined whether media solidified with the washed agar with or without supplemental 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and FA at their original concentrations in agar affect on bacterial cell growth, behaviour and physiological responses using the swarming strain Escherichia coli K12 strain W3110 [25]
Summary
Agar is an indigestible gelling material that is derived from seaweeds such as Gelidium crinale and Gracilaria vermiculophylla. Some studies have found that replacing agar with gellan gum in solid media accelerates the growth of slow-growing bacteria [11,12,13,14,15], those from soil or rhizosphere that utilize only extracellular polysaccharides, xanthan and gellan as their carbon source [14,16]. We have found that some slow-growing soil bacteria in the soil bed from a boreal larch forest generated minute colonies on 1.0% agar plates containing modified Winogradsky’s (MW) medium, they proliferated well and formed swarmed colonies on 1.0% gellan plates in MW [17]. MW plates containing 1.0 or 1.5% gellan gum that had been previously washed with MeOH (washed gellan) slightly repressed the swarming of P. collierea V5G’5 [17]. The reason why agar is unsuitable for the growth of some bacteria has not been addressed
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