Question

The disk diffusion assay is good for determining the effectiveness of many antibiotics of fixed concentrations against a particular pathogen. Based on the introduction material, your experience in this lab, and group discussion, how would you design an experiment to determine what concentration of antibiotic was most effective at inhibiting the growth of, or killing certain pathogens?

Answer 1

Testing the effectiveness of antimicrobial drugs against specific organisms is important in identifying their spectrum of activity and the therapeutic dosage. This type of test, generally described as antimicrobial susceptibility testing (AST), is commonly performed in a clinical laboratory

DISK DIFFUSION METHOD PRINCIPLE: This method is based on the principle that antibioticimpregnated disk, placed on agar previously inoculated with test bacterium, pick up moisture and the antibiotic diffuses radially outward through agar medium producing an antibiotic concentration gradient, the concentration of the antibiotic at the edge of the disk is high and gradually diminishes as the distance from the disk increases to a point where it is no longer inhibitory for the organism, which then grows freely. A clear one is formed around the antibiotic disk after inoculation if the agent inhibits bacterial growth. MEDIA: The diffusion method is performed by Mueller-Hinton Agar The inoculums for disk diffusion method are prepared by a suitable broth such as tyrptic soy broth Media is prepared using distilled water and deionized water Heat with frequent agitation and boil to dissolve the medium completely. Sterilize by autoclaving at 120 degree for 15 minutes. Ph should be between 7.2 to 7.4. Cool medium to 40-50 degree. Allow to solidify. INOCULUM From a pure bacterial culture take 4 or 5 colonies. Transfer to 5ml tyrpticase soy broth. Incubate the broth at 30degree until it achieves the turbidity of 0.5 Macfarland standards. Compare the turbidity of the test bacteria suspension with that of 0.5 Macfraland against a black background. INNOCULATION OF PLATE: Dip a sterile cotton swab into bacterial suspension. Inoculate the agar by streaking with the swab containing the inoculums. Rotate the plate by 60degree and repeat the rubbing procedure. Allow the surface medium to dry.

ANTIMICROBIAL DISK: The disk diameter is approximately 6mm. using sterile forceps place antibiotic disk on the surface of the inoculated plate. Immediately press downlightly to ensure complete contact between the disk and agar.

Incubate plates in an inverted position at 30 degree celcius. Observe for the zone of inhibition after 16-18 hours. READING AND MEASUREMENTS: The zone of inhibition is the point at which no growth is visible. Record the presence of individual colonies with zones ofinhibition. Read and record the diameter of the zones of inhibition using a ruler graduated at 0.5mm INTERPRETATION OF RESULTS: Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standards for pathogens. Report result as RESISTANT(R), INTERMEDIATE (0), SUSCEPTIBLE(S)

other methods include: dilution method, Kriby- Bauer disk diffusion method