Principle
Antiseptics, disinfectants and antibiotics are used in different ways to combat microbial growth. Antiseptics are used on living tissue to remove pathogens. Disinfectants are similar in use but are used on inanimate objects. Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria. Many antibiotics are chemically altered to reduce toxicity, increase solubility, or give them some other desirable characteristic that they lack in their natural form. Other substances have been developed from plants or dyes and are used like antibiotics. A better term for these substances is antimicrobials, but the term antibiotic is widely used to mean all types of antimicrobial chemotherapy.
Many conditions can affect a disc diffusion susceptibility test. When performing these tests certain things are held constant so only the size of the zone of inhibition is variable. Conditions that must be constant from test to test include the agar used, the amount of organism used, the concentration of chemical used, and incubation conditions (time, temperature, and atmosphere). The amount of organism used is standardized using a turbidity standard. This may be a visual approximation using a McFarland standard 0.5 or turbidity may be determined by using a spectrophotometer (optical density of 1.0 at 600 nm). For antibiotic susceptibility testing the antibiotic concentrations are predetermined and commercially available. Each test method has a prescribed media to be used and incubation is to be at 35-37o C in ambient air for 18-24 hours.
The disc diffusion method for antibiotic susceptibility testing is the Kirby- Bauer method. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. There is also a zone of intermediate resistance indicating that some inhibition occurs using this antimicrobial but it may not be sufficient inhibition to eradicate the organism from the body.
The standardized methods for antiseptic and disinfectant testing are more rigorous and more difficult to reproduce in a student laboratory. Two common tests are the Phenol Coefficient Test (a comparison of the effect of the chemical and phenol on several organisms) and the Use Dilution Test (testing the chemical under actual conditions of use). A disc diffusion test can be used to approximate the Use Dilution Test. The chemical under consideration is used to saturate a filter paper disc. This disc is then used to introduce the chemical to the agar for testing. The actual zone sizes have not been standardized as in the Kirby-Bauer method, but a comparison of zone sizes for the same chemical among organisms will provide a n approximate e