Extraction and quantification of solutes in solidified agar culture media

A method is described for determining the concentration of certain solutes in solidified culture media. The method is based upon the finding that under specified conditions the concentration of solute in an agar gel (C(g)) is related to the concentration of solute in a centrifugally extracted gel supernatant (C(s)) by the ratio, C(g)/C(s), which is characteristic for each solute. The method avoids direct assays of the gels and instead involves assaying the supernatants from inoculated and uninoculated (control) gels with conventional liquid assay techniques and then calculating solute concentrations in the inoculated gels by use of the C(g)/C(s) ratios determined from the controls. Uninoculated agar gels containing known concentrations of various solutes and similar gels inoculated with Neurospora crassa or Escherichia coli were centrifuged at various times, and the supernatants were assayed for solute concentrations. The solute concentrations in the supernatants from the inoculated gels multiplied by the C(g)/C(s) ratios for those solutes determined at the same times for the uninoculated controls gave calculated solute concentrations in the inoculated gels. The differences between these calculated solute concentrations and those initially present in the inoculated gels indicated the amounts of solutes utilized from the gels by the microorganisms at various incubation times.