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Antibiotic Sensitivity Virtual Lab | Disc Diffusion Method

General Aim of Antibiotic Sensitivity Virtual Lab

The antibiotic sensitivity virtual lab test determines the susceptibility of a microbial species against different antibiotic agents.

 

Method of Antibiotic Sensitivity Virtual Lab

Part 1: Sterilize the bacteriological loop 1- Sterilize the bacteriological loop by holding it above the flame till it becomes red and hot. 2- Let the sterile loop cool down by holding it still. N.B. Do not wave it around to cool it or blow on it. Part 2: Antibiotic sensitivity test (Disc diffusion method) Using a sterile wire loop, pick 3–5 well-isolated colonies of similar appearance to the test organism. Then, emulsify these colonies in 4 ml of sterile physiological saline (or nutrient broth). In a good light match the turbidity of the suspension to the turbidity standard (0.5 McFarland test standard). N.B. When comparing turbidities, it is easier to view against a printed card or sheet of paper. Using a sterile swab, insert it in the test suspension. Remove excess fluid by pressing and rotating the swab against the side of the tube above the level of the suspension. Streak the swab evenly over the surface of the plate of Mueller Hinton agar in three directions, rotating the plate approximately 60o to ensure even distribution. Cover the petri dish. Allow 3–5 minutes (no longer than 15 minutes) for the surface of the agar to dry. Using sterile forceps, or a multidisc dispenser, place the appropriate, evenly distributed on the inoculated plate. You may use a template as shown in the figure will help to ensure the discs are correctly placed. N.B. The discs should be about 15 mm from the edge of the plate and no closer than about 25 mm from disc to disc. No more than 6 discs should be applied (90 mm diameter petri dish). N.B. Each disc should be lightly pressed down to ensure its contact with the agar. It should not be moved once in place. 10- Within 30 minutes of applying the discs, invert the plate and incubate it aerobically at 35o C for 16–18 h in this antibiotic sensitivity virtual lab procedure. 11- After overnight incubation, using a ruler on the underside of the plate measure the diameter of each zone of inhibition in mm. N.B. The endpoint of inhibition is where growth starts. Part 3: Interpretation of zone sizes Using the Interpretative Chart, interpret the zones sizes of each antimicrobial, reporting the organism as ‘Resistant’, ‘Intermediate/Moderately susceptible’, ‘Susceptible’.

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.