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Agar Dilution Methods

In agar dilution methods, solutions containing a specific number of microbes are placed on a nutrient-rich agar plate and inoculated with varied concentrations of an antimicrobial agent. The plate is incubated for around 24 hours and the turbidity, growth sediment or amount of microbial colonies present is assessed. Microbes are classified as susceptible, intermediate or resistant to antimicrobial activity.

  1. Minimum Inhibitory Concentration

    • The aim of agar dilution is to determine the smallest concentration of antimicrobial agents, such as antibiotics, disinfectants or preservatives, required to destroy or prevent microbe growth, which is called the minimum inhibitory concentration (MIC). MIC is measured in milligrams per liter, and values obtained are used to measure the effectiveness of antimicrobial agents -- for example, the monitoring of antibiotic resistance.

    Colony Suspension Method

    • This method involves transferring a small sample of selected microbe colonies to a glass tube containing saline solution. The tube is shaken vigorously and left to stand. Turbidity is measured as a sign of microbe growth by comparing the tube contents with a laboratory standard such as McFarland standard. The comparison is best made against a white background in a room with adequate lighting. The colony suspension method takes approximately five minutes per sample.

    Growth Method

    • In the growth method, selected microbe colonies are placed in a tube containing a nutrient-rich medium. The tube is then incubated between 95 and 99 degrees F while being shaken at the same time. This process takes roughly two to six hours, depending on the growth rate of the microbe. Upon completion, turbidity is assessed using the same standard as the colony suspension method.

    Growth Method Using Overnight Cultures

    • Microbe colonies in nutrient-rich media are incubated overnight. Subsequent to incubation, the optical density of each sample is measured at 600 nm. The sample is then diluted with nutrient media at a ratio of 1 in 100 and separated into five further dilutions at 1 in 10. Four of the new dilutions are spread onto an agar plate and incubated overnight. The following day the amount of colonies formed is counted and colony forming units per milliliter is calculated.

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