Antibiotic Sensitivity Virtual Lab | Disc Diffusion Method

Theoretical Background/Context

• The introduction of various antimicrobials for treating a variety of infections showed the necessity of performing antimicrobial susceptibility testing as a routine procedure in all microbiology laboratories.
• In laboratories it can be made available by using antibiotic disk which will diffuse slowly into the medium where the suspected organism is grown.
• The basic principle of antibiotic susceptibility testing has been used in microbiology laboratories for over 80 years.
• Various chemical agents such as antiseptics, disinfectants, and antibiotics are employed to combat with the microbial growth.
• Among these, antibiotics are generally defined as the substances produced by the microorganism such as Penicillium, which has the ability to kill or inhibit the growth of other microorganisms, mainly bacteria.
• Antimicrobial susceptibility tests (ASTs) basically measure the ability of an antibiotic or other antimicrobial agent to inhibit the in vitro microbial growth.
• There are many different procedures that microbiologists use to study the effects of various antimicrobial agents in treating an infection caused by different microorganisms.
• Mueller Hinton Agar is considered as best for routine susceptibility testing since it has batch-to-batch reproducibility, low concentration of inhibitors of sulphonamide, trimethoprim, and tetracyclines and produces satisfactory results for most of the non-fastidious pathogens.
• Fastidious organisms which require specific growth supplements need different media to grow for studying the susceptibility patterns.
• The Kirby Bauer test is a qualitative assay whereby disks of filter paper are impregnated with a single concentration of different antibiotics or any chemicals that will diffuse from the disk into the agar. The selected antibiotic disks are placed on the surface of an agar plate that has already been inoculated with test bacteria.
• During the incubation period, the antibiotics/chemicals diffuse outward from the disks into the agar.
• This will create a concentration gradient in the agar which depends on the solubility of the chemical and its molecular size.
• The absence of growth of the organism around the antibiotic disks indicates that the respected organism is susceptible to that antibiotic and the presence of growth around the antibiotic disk indicates the organism is resistant to that particular antibiotic.
• This area of no growth around the disk is known as a zone of inhibition, which is uniformly circular with a confluent lawn of growth in the media.
• The diameters of the zone of inhibition are measured (including disk) using a metric scale or a sliding caliper.
• The measured zone diameter can be compared with a standard chart for obtaining the susceptible and resistant values.
• There are zones of intermediate resistance which means that the antibiotic may not be sufficient enough to eradicate the organism from the body.
• Factors affecting Antibiotic Susceptibility Testing:
• Many conditions can affect the accuracy of the AST results, which is described in detail below:

pH

• The pH of the medium is an important factor which influences the accuracy of antibiotic susceptibility testing.
• If the pH of the medium is too low than the desired pH, certain drugs such as aminoglycosides, quinolones, and macrolides lose their potency, on the other hand, antibiotic classes such as tetracyclines appear to have excess activity a lower Ph and vice versa happens in the case of the higher pH.

Moisture

• The presence of moisture content on the medium can counteract with accuracy of the susceptibility testing.
• It is important to remove the excess moisture present in the agar surface, by keeping it in the laminar flow hood for a few minutes.
• Effects of Medium Components
• If the media selected for the antibiotic susceptibility contains excessive amounts of thymine or thymidine compounds, they will reversibly inhibit the action of certain antimicrobial agents such as trimethoprim groups.
• This reversible inhibition yields smaller or less distinct or even no zones and will be misinterpreted as resistant antibiotics.
• MHA is low in thymine and thymidine content and it can be used successfully to study the susceptibility of antibiotics.
• Also, the medium containing excessive cation reduces the zone size, while low cation content results in unacceptably large inhibition zones.

Amount of organism

• The amount of the organism used for the susceptibility testing is standardized using a turbidity standard.
• This is obtained by a visual approximation using the McFarland standard of 0.5 or else it can be determined by using a spectrophotometer with an Optical density of 1 at 600 nm wavelength.
• In addition to this, the antibiotic concentration for the susceptibility testing is pre-determined and is commercially available.

Principle of Work

• The disc susceptibility test determines the susceptibility of a microbial species against different antibiotic agents using diffusion simulation techniques, which can be studied through diffusion simulator platforms in an antibiotic sensitivity virtual lab environment.