Rapid antibiotic susceptibility testing system based on bacterial immobilization using gelling agent, antibiotic diffusion and tracking of single bacterial cells

A method for observing the responses of one or more microbial cells to a bioactive agent, comprising:

• contacting a gelling agent-containing liquid medium with the one or more microbial cells;
• solidifying the gelling agent-containing liquid medium to form a gelled matrix in which the one or more microbial cells are immobilized by the gelled matrix;
• providing a bioactive agent into the gelled matrix and allowing the bioactive agent to diffuse into the gelled matrix so that the bioactive agent is delivered to the immobilized one or more microbial cells;
• incubating the immobilized one or more microbial cells for a certain time;
• and
• tracking at least one individual microbial cell directly among the one or more microbial cells by a microscopy and imaging an increase in an area occupied by growth of the at least one individual microbial cell.

A method for an antibiotic susceptibility testing (AST), comprising:

• providing one or more microbial cells;
• providing a gelling agent capable of immobilizing the one or more microbial cells, wherein the gelling agent is selected from the group consisting of agar, agarose, gelatin, alginate, collagen, fibrin, and mixtures thereof;
• providing a bioactive agent capable of inhibiting growth of the one or more microbial cells;
• forming a solid thin film comprising the one or more microbial cells, the gelling agent, and the bioactive agent, wherein the one or more microbial cells are immobilized by the gelling agent;
• incubating the one or more microbial cells;
• tracking at least one individual microbial cell directly among the one or more microbial cells by a microscopy and imaging an increase in an area occupied by growth of the at least one individual microbial cell according to an incubation time and a concentration of the bioactive agent, wherein an image from the imaging step is taken as a microscopic picture from the microscopy;
• based on the image, determining a minimum inhibitory concentration (MIC) by analyzing the occupied area or a morphology based on the growth of the at least one individual microbial cell;
• and
• obtaining the result of the AST against the bioactive agent based on the step of determining the MIC.

A method for analyzing one or more microbial cells interacting with a bioactive agent, comprising:

• providing one or more microbial cells;
• providing a gelling agent capable of immobilizing the one or more microbial cells, wherein the gelling agent is selected from the group consisting of agar, agarose, gelatin, alginate, collagen, fibrin, and mixtures thereof;
• providing a bioactive agent capable of inhibiting growth of the one or more microbial cells;
• forming a solid thin film comprising the one or more microbial cells, the gelling agent, and the bioactive agent, wherein the one or more microbial cells are immobilized by the gelling agent;
• incubating the one or more microbial cells;
• and
• tracking at least one individual microbial cell directly among the one or more microbial cells by a microscopy and imaging an increase in an area occupied by growth of the at least one individual microbial cell.