Abstract: The present disclosure is directed towards using lyophilized reagent beads for a fully closed multi-step reaction to complete antibiotic susceptibility tests and amplification assays. The lyophilized reagent beads include a lyophilized amplification reagent bead and an lyophilized antibiotic bead. The lyophilized reagent bead may be embedded in solid wax layer inside the test tube to enable cell growth in antibiotic prior to release of amplification reagents. A microfluidic manifold device may be used to insert the sample into the amplification tube and enable a fully closed system.