Standards for Antimicrobial Disk Susceptibility Tests

1. Standards for Antimicrobial Disk Susceptibility Tests D Dr MaryamSotoudeh Tehran heart center

2. OBJECTIVE This document describes • Indications for Performing Susceptibility Tests • preparation of Mueller-Hinton Agar Medium • Turbidity Standard for Inoculum Preparation • Inoculum Preparation • Inoculation of Test Plates • Storage of Antimicrobial Disks • Reading Plates and Interpreting Results

3. Indications for Performing Susceptibility Tests

4. The responsibility of the microbiology laboratory includes : 1.Microbial detection and isolation 2.Determination of microbial susceptibility to antimicrobial agents.

5. Which organisms? Many bacteria, have unpredictablesusceptibilities to antimicrobial agents.so their susceptibilities can be measured in vitroto help guide the selection of the most appropriate antimicrobial agent.(Oxacillin for staph. aureous ) Susceptibility tests are not performed on bacteria that are predictably susceptible to antimicrobial agent commonly used to treated infection . (penicillin for Group A β-hemolytic streptococcus)

6. Which drugs? Organism identification or group(no vancomycin for gr- bacilli) Antimicrobial Susceptibility tests methods (cefotaxim resistance in P.aeroginosa cannot be detected by disk diffusion) Site of infection (Nitrofurantoin only achieve effective level in the urinary tract) Availability of Antimicrobial agent Cost of individual antibiotics

7. Disk diffusion method(Kirby-bauer test)

8. Disk diffusion method(Kirby-bauer test): provides the greatest flexibility and cost-effectiveness. Widely used since 1966 when the first standard method was originally described by Bauer et al. It is appropriate for rapidly growing organisms and certain fastidious bacterial pathogens.

9. Disk diffusion testing (Kirby-bauer test): It is depends on the formation of a gradient of antimicrobial concentration as the antimicrobial agent diffuses radially into the agar. The drug concentration decrease at increasing stances from the disk .

10. Disk diffusion method components: McFarland 0.5 standard suspension of bacteria (for Inoculum preparation) Mueller-Hinton agar plate Filter paper disk containing a specific amount (not concentration ) of antibiotic

11. Standard preparation

12. The number of bacteria tested must bestandardized regardless of susceptibility method used. Too few bacteria false-susceptible Too many bacteria false-resistant The most widely used method of inoculums standardization is McFarland turbidity standards.

13. McFarland standards preparation: Add specific volume of 1% sulfuric acid and 1.175% barium chloridewith constant stirring to maintain a suspension.

14. McFarland standards preparation: McFarland 0.5 standards preparation: 99.5 ml of 1% sulfuric acid and 0.5 ml of 1.175% barium chloride . aliquots suspension in 4- to 6-mL into screw-cap tubes of the same size as used in growing or diluting the bacterial inoculum.

15. tubes should be tightly sealed and stored in the dark at room temperature. • The suspensionshould be vigorously agitated on a mechanical vortex mixer before each use and inspected for a uniformly turbid appearance.

16. If large particles appear, the standard should be replaced • The barium sulfate standards should be replaced or their densities verified monthly

17. McFarland standards should have an optical density (O.D.) of 0.08-0.1 at 625 nm.

20. Inoculum Preparation

21. Inoculum Preparation

22. Growth Method

23. Important : let sterilized loop cool before pick up your sample

24. The turbidity of broth culture is adjusted with sterile saline or broth • To perform this step properly, either a photometric device can be used or, if done visually, adequate light is needed to visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines. (Wickerham Card )

25. Direct Colony Suspension Method As a convenient alternative method, the inoculum can be prepared by making a direct broth or saline suspension of isolated colonies selected from an 18- to 24-hour agar plate (a non selective medium, such as blood agar, should be used).

26. Direct Colony Suspension Method The suspension is adjusted to match the 0.5 McFarland turbidity standard as Growth Method This approach is the recommended method for testing the fastidious organisms Haemophilus spp. Neisseriagonorrhoeae, and streptococci and for testing staphylococcifor potential methicillin or oxacillin resistance

27. Inoculation of Test Plates

28. Mix McFarland Standard well Standardize inoculumsuspension Janet FickHindler, MCLS MT(ASCP) UCLA Medical Center Los Angeles, CA

29. Procedures Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard Sample 0.5 McFarland Standard

32. Procedures streak a lawn of bacteria on Mueller-Hinton agar

34. NOTE: • Extremes in inoculum density must be avoided. • Never use undiluted overnight broth cultures or other unstandardizedinocula for streaking plates.

35. MEDIUM

36. Mueller-Hinton medium The most frequent basal culture medium for testing bacteria that grow aerobically.

37. Preparation of Mueller-Hinton Agar

38. Preparation of agar medium Prepare MHA ,according to the manufacturer's instructions, using distilled water or deionized water. Heat with frequent agitation and boil to dissolve the medium completely. Sterilize by autoclaving at 121°C for 15 min. Immediately after autoclaving, allow it to cool in a 45 to 50 °C water bath

39. The pH of each batch of Mueller-Hinton agar should be checked when the medium is prepared. • It should be 7.2 - 7.4 at room temperature after gelling.

40. The pH can be checked by one of the following means: • Macerate a sufficient amount of agar to submerge the tip of a pH electrode. • Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup. • Use a properly calibrated surface electrode

41. Pouring the Culture Plates Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed petri dishes on a Level, horizontal surface to give a uniform depth of approximately 4 mm.

42. Volume of agar medium

43. The agar medium should be allowed to cool to room temperature and, unless the plate is used the same day, stored in a refrigerator (2 to 8 °C).

44. Plates should be used within seven days after preparation unless adequate precautions, such as wrapping in plastic, have been taken to minimize drying of the agar. • A representative sample of each batch of plates should be examined for sterility by incubating at 30 to 35 °C for 24 hours or longer

45. Plates may be stored in the refrigerator inside airtight plastic bags at 2-8°C for up to 4 weeks.

46. Moisture • If, just before use, excess surface moisture is present, the plates should be placed in an incubator (35 °C) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually 10 to 30 minutes). • The surface should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the petri dish covers when the plates are inoculated.

47. Application of Disks to Inoculated Agar Plates

49. Because some of the drug diffuses almost immediately, a disk should not be relocated once it has come into contact with the agar surface. Instead, place a new disk in another location on the agar

50. Application of Disks to Inoculated Agar Plates The plates are inverted and placed in an incubator set to 35 °C within 15 minutes after the disks are applied.