Polyacrylamide hydrogels as substrates for studying bacteriawz

Since its introduction by Robert Koch in 1882, agar has been the most commonly used substrate for the growth and study of bacteria. Agar consists of alternating blocks of D-galactose and 3,6-anhydro-L-galactose and is a polysaccharide with a variety of characteristics that are useful for culturing bacteria: (1) it is biocompatible; (2) it is inert to metabolism and degradation by bacteria; (3) it remains gelled at the range of temperatures commonly used for bacterial culture; and (4) it forms a hydrogel with a large volume fraction of bound water that hydrates cells in contact with the polymer. Other classes of hydrogels, including gellan, alginate, xanthan gum, guar gum, and most recently Eladiumt have been used as substrates for bacterial culture; however, they have not supplanted agar. These polymers share at least one shortcoming in common with agar for bacterial studies: chemical variability. The heterogeneity in the structure and the length of the polysaccharide chains of agar is influenced by the conditions for its isolation from marine algae. The variability of agar makes it difficult to define and reproduce the chemical and physical properties of this hydrogel for bacterial studies. Another disadvantage of agar for microbiological studies is the limited variability of surface chemistry that can be presented to cells. This characteristic is particularly important, as the chemistry of surfaces in contact with the outer cell wall influences bacterial physiology, behaviour, and growth. The chemical modification of agar is possible, but is not a widely used route for controlling the surface chemistry of this hydrogel. The introduction of classes of biocompatible synthetic polymers with defined chemical and physical properties for microbiological studies may transcend the limitations of agar and find applications in bacterial culture and cell biology. Polyacrylamides (PAs) are a class of biocompatible hydrogels that have been instrumental in studying the influence of substrate stiffness on mammalian cell morphology. Importantly, the physical properties of PA—including stiffness, porosity, and shear modulus—can be controlled during its synthesis. The most common approach for the synthesis of PA hydrogels is via the free-radical polymerization of acrylamide (1) in the presence of the cross-linkerN,N0-methylenebisacrylamide (2) (Fig. 1). Different PA building blocks are commercially available, inexpensive, and enable control over the chemical and physical properties of PA (Fig. 1). Furthermore, several efficient approaches have been described for the synthesis of N-substituted acrylamide analogues that can be incorporated into PA hydrogels to introduce new surface chemistry. Another strategy for the synthesis of chemically diverse PA substrates is the copolymerization of 1 and 2 with acrylic acid or an acrylamide analogue containing a succinimidyl ester and the subsequent chemical modification of these moieties. Despite the ease of preparing PA substrates with defined chemical and physical properties, and a growing body of literature describing studies in mammalian cell biology, this hydrogel has been relatively unexplored for the study and culture of bacterial cells. In this manuscript we introduce and characterise PA hydrogels as a platform for the culture, study, and isolation of bacteria. We demonstrate the effects of surface chemistry and stiffness on bacterial cell growth and characterise growth rates. We demonstrate that PA chemistry affects community growth and spreading and discuss an approach for removing cells from substrates using reversible PA gels. We observed that the free-radical polymerization of 1 and 2 produced polymers containing monomers and oligomers that were not incorporated into the polymer network and were toxic to bacteria. To remove these compounds, we incubated the gels in water prior to infusing them with liquid nutrient