15 Examination of Bacterial Susceptibility Tests - Disk Method 1. Measure the diameter in mm of the zone of inhibition of growth around each disk as illustrated in the diagram below. Record your observations in the table on page 17. 2. Interpret the measurements according to the criteria in the zone diameter interpretive chart on page16. Each organism is evaluated as "resistant," "intermediate," or "susceptible" to the Antimicrobic tested. Note any evidence of resistant mutants growing within the general zone of inhibition. If such colonies are noted, the organism should be graded as resistant to the Antimicrobic tested. Bacterial growth in a contiguous lawn Inhibition of bacterial growth appears as a clear zone in the bacterial lawn Inhibition of bacterial growth around an antibiotic disk. disk Diameter of zone of inhibition16 Zone Diameter Interpretive Chart Zone Diameter Interpretive Standards (mm) Antimicrobial Code Disc Agent Potency Resistant Intermediate Susceptible Penicillin P-10 10 U for staphylococci for L. monocytogenes for enterococci for nonenterococcal streptococci for N. gonorrhoeae * ≤28 ≤19 ≤14 ≤19 ≤26 -- -- -- -- -- ≥29 ≥20 -- ≥28 ≥47 Streptomycin S-10 10µg ≤11 12-14 ≥ 15 Chloramphenicol C-30 30µg for Haemophilus species for other organisms ≤25 ≤12 26-28 13-17 ≥29 ≥18 Ampicillin AM-10 10µg for gram-negative enteric organisms for staphylococci for enterococci for nonenterococcal streptococci for Listeria monocytogenes for Haemophilus species ≤13 ≤28 ≤16 ≤21 ≤19 ≤21 -- -- -- -- -- 22-24 ≥17 ≥29 -- ≥30 ≥20 ≥25 Tetracycline Te-30 30µg for Haemophilus species for N. gonorrhoeae for other organisms ≤25 ≤30 ≤14 26-28 -- 15-18 ≥29 ≥38 ≥19 Cephalothin CF-30 30µg ≤14 -- ≥18 Colistin CL-10 10µg ≤8 9-10 ≥11 Methicillin DP-5 5µg for staphylococci ≤9 10-13 ≥14 *Penicillin is not the antibiotic of choice for gram-negative enteric organisms17 Antimicrobic Agent Staphylococcus aureus I II Escherichia coli I II Penicillin (P) 10 units Chloramphenicol (C) 30 µg Ampicillin (AM) 10 µg Tetracycline (Te) 30 µg18 Demonstration of Minimal Inhibitory Concentration (MIC) 1. Materials: a. Broth cultures of two strains of E. coli (I and II) and two strains of S. aureus (I and II) adjusted to a turbidity of McFarland's 0.5 standard = (1.5 x 108 !"#$%&'"!" ) b. TSA plate c. Serial dilutions of Chloramphenicol and Ampicillin in broth (1 ml/tube). 2. Procedure: a. Resuspend organisms in broth b. Transfer 1.0 ml of suspension to each tube beginning with the control tube and from lowest to highest concentration of antimicrobic to each series of antimicrobics c. Discard pipette in jar d. Streak out one loopful from antimicrobic-free tube (tube 6) on TSA plate e. Incubate at 37°C overnight Examination of MIC Examine tubes for growth and record in the table on page 19. Determine the MIC of each antimicrobic for the organism tested. The MIC is the lowest concentration of the antimicrobic that causes complete inhibition of bacterial growth. The inhibition may be either bacteriostatic or bactericidal. Demonstration of Minimal Lethal Concentration (MLC) 1. Materials: a. Trypticase soy agar plate 2. Procedure: a. Inoculate a segment of the Trypticase soy agar plate with one loopful of broth from each tube showing no growth in the MIC test. The first tube showing growth should also be inoculated onto the Trypticase soy agar plate. b. Incubate at 37°C overnight Examination of MLC Agar Plate Examine the plate to determine which tube provided viable inoculums. The MLC is the lowest concentration of antimicrobic which prevents growth when the organism is subcultured in the absence of the antimicrobic. Record the MLC in the Table on page 19.19 Antimicrobic µg/ml Staphylococcus aureus I II Escherichia coli I II Chloramphenicol Tube 1 16 2 8 3 4 4 2 5 1 6 none MIC MLC Ampicillin Tube 1 6.4 2 3.2 3 1.6 4 0.8 5 0.4 6 none MIC