1. Standards for Antimicrobial Disk Susceptibility Tests
Dr Maryam Sotoudeh
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 unpredictable susceptibilities to antimicrobial agents.so their susceptibilities can be measured in vitro to 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. With S. pyogenes from penicillin-allergic patients, erythromycin or another macrolide may be tested to detect strains resistant to those agents.
Susceptibility tests are also important in studies of the epidemiology of resistance and in studies of new antimicrobial agents.
When the nature of the infection is not clear and the specimen contains mixed growth or normal flora (in with little relationship to the infectious process), susceptibility tests are often unnecessary and the results may be misleading

7. 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

8. Disk diffusion method (Kirby-bauer test)

9. 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.

10. 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 .

11. 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

12. Standard preparation

13. 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.

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

15. McFarland 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.

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

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

18. McFarland standards should have an optical density (O. D. ) of 0. 08-0
McFarland standards should have an optical density (O.D.) of at 625 nm.

19. McFarland Standard No.
0.5 1 2 3 4
Barium chloride (ml)
0.05
0.1
0.2
0.3
0.4
Sulfuric acid (ml)
9.95
9.9
9.8
9.7
9.6
Approx. cell density (1X10^8 CFU/mL)
1.5 6 9 12
Transmittance at wavelength of 600 nm
74.3
55.6
35.6
26.4
21.5
Absorbance at wavelength of 600 nm
0.132
0.257
0.451
0.582
0.669

21. Inoculum Preparation

22. Direct Colony Suspension Method
Inoculum Preparation
Growth Method
Direct Colony Suspension Method

23. Growth Method
At least 3 to 5 well-isolated colonies of the same morphological type are selected from an agar plate culture
The top of each colony is touched with a loop, and transferred into a tube containing 4 to 5 mL of a suitable broth medium, such as tryptic soy broth.
The broth culture is incubated at 35 °C until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually 2 to 6 hours).

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

25. 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 )

26. 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).

27. 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. Neisseria gonorrhoeae, and streptococci and for testing staphylococci for potential methicillin or oxacillin resistance

28. Inoculation of Test Plates

29. Mix McFarland Standard well Standardize inoculum suspension
Janet Fick Hindler, MCLS MT(ASCP) UCLA Medical Center Los Angeles, CA

30. Sample 0.5 McFarland Standard Procedures
Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard
Use a Wickerham Card as background when adjusting the turbidity
Sample
0.5 McFarland Standard

31. within 15 minutes after adjusting the turbidity, a sterile cotton swab is dipped into the suspension.
The swab should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab.

32. The Mueller-Hinton agar plate is inoculated by streaking the swab
This procedure is repeated by streaking two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum.

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

34. As a final step, the rim of the agar is swabbed
The lid may be left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any excess surface moisture to be absorbed before applying the drug-impregnated disks.

35. NOTE: Extremes in inoculum density must be avoided.
Never use undiluted overnight broth cultures or other unstandardized inocula for streaking plates.

36. MEDIUM

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

38. Preparation of Mueller-Hinton Agar

39. 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
Check the pH of each preparation after it is sterilized, which should be between 7.2 and 7.4 at room temperature. Decrease in PH lead decrease in activity of AG,Erythro,Clinda and increase in activity of tetra.
Cool the agar medium to 40-50°C. Pour the agar into sterile glass or plastic petri dish 150 mm in diameter on a flat surface to a uniform depth of 4 mm.
Allow to solidify

40. 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

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

42. 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

43. 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.

44. Volume of agar medium
Pour 60 to 70 mL of medium for plates with diameters of 150 mm
Pour 25 to 30 mL of medium for plates with diameters of 100 mm

45. 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).

46. 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

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

48. 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.

49. Application of Disks to Inoculated Agar Plates

50. Disks are no closer than 24 mm from center to center.
The predetermined battery of antimicrobial disks is dispensed onto the surface of the agar plate.
Each disk must be pressed down to ensure complete contact with the agar surface.
Disks are no closer than 24 mm from center to center.
no more than 12 disks should be placed on one 150-mm plate
nor more than 5 disks on a 100-mm plate.

51. 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

52. 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.

53. Application of Disks to Inoculated Agar Plates
With the exception of
Haemophilus spp.
N. Gonorrhoeae
streptococci
the plates should not be incubated in an increased CO2 atmosphere, because the interpretive standards were developed by using ambient air incubation
and CO2 will significantly alter the size of the inhibitory zones of some agents.

54. Storage of Antimicrobial Disks
Cartridges containing commercially prepared paper disks are generally packaged to ensure appropriate anhydrous conditions.

55. Storage of Antimicrobial Disks
Refrigerate the containers at 8 °C or below
or freeze at -14 °C or below, in a freezer until needed of use.

56. Storage of Antimicrobial Disks
Sealed packages of disks that contain drugs from the β-lactam class should be stored frozen, except for a small working supply, which may be refrigerated for at most one week.
Some labile agents (e.g., imipenem, cefaclor, and clavulanic acid combinations) may retain greater stability if stored frozen until the day of use.

57. so they may equilibrate to room temperature before opening.
Antimicrobial Disks
The unopened disk containers should be removed from the refrigerator or freezer one to two hours before use
so they may equilibrate to room temperature before opening.
This procedure minimizes the amount of condensation that occurs when warm air contacts cold disks.

58. a disk-dispensing apparatus, should be fitted with a tight cover and supplied with an adequate desiccant.
The dispenser should be allowed to warm to RT before opening.
When not in use, the dispensing apparatus containing the disks should always be refrigerated

59. Only those disks that have not reached the manufacturer’s expiration date stated on the label may be used. Disks should be discarded on the expiration date

60. Incubate overnight Add disks
Janet Fick Hindler, MCLS MT(ASCP) UCLA Medical Center Los Angeles, CA

61. Reading Plates and Interpreting Results

62. READING AND MEASUREMENT OF ZONES OF INHIBITION
The zone of inhibition is uniformly circular point at which no growth is visible to the unaided eye

63. Reading Plates
Zones are measured to the nearest whole millimeter, using sliding calipers or a ruler, which is held on the back of the inverted petri plate.
The petri plate is held a few inches above a black, non-reflecting background .

64. If oxacillin is being tested against Staphylococcus spp
If oxacillin is being tested against Staphylococcus spp. or vancomycin against Enterococcus spp., 24 hours of incubation are required before reporting as susceptible
other agents can be read and reported at 16 to 18 hours. Transmitted light (plate held up

65. Modify methods for fastidious bacteria
If blood was added to the agar base (as with streptococci), the zones are measured from the upper surface of the agar illuminated with reflected light, with the cover removed.

66. Record the presence of individual colonies (arrow) within zones of inhibition.
Purity of the isolate must be confirmed
If the isolate was pure, the individual colonies are resistant mutants of the same SPP. and report as resistant

67. Ignore occurrence of fuzzy/hazy zones (arrow)in 2 instances:
1.Swarming of proteous SPP
2.In Sulfonamid and trimethoprim disks ,antagonists in the medium may allow some slight growth; therefore, ignore slight growth (20% or less of the lawn of growth) and measure the more obvious margin to determine the zone diameter
In other instances haze of growth should not be ignored.

68. Common interpretation problems
If individual colonies are apparent, the inoculum was too light and the test must be repeated
Do not read plates on which growth of test bacteria have isolated colonies or less than semi-confluent growth

69. Do not read zones of inhibition of two adjacent disks that overlap to the extent that measurement of the zone diameter cannot be made.

70. Do not read zones showing distortion from circular .

71. Common interpretation problems
An agar gel that is too thick leads to smaller zones
Solution:Use McFarland 0.5/ photometer
Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/
Source:

72. Interpretation of Disk Diffusion Test Results

73. A classification based on an in vitro response of an organism to an antimicrobial agent at levels of that agent corresponding to blood or tissue levels attainable with usually prescribed doses of that agent
Interpretive categories have been established by CLSI(clinical and laboratory standard institute ),formerly as NCCLS (national committee for clinical laboratory standards), including:

74. In vitro estimates of antimicrobial activity
Susceptible : implies that an infection due to a specific isolate can be treated with recommended dosage of antibiotic.
Resistant : implies that the isolate will not respond to achievable concentration of the antibiotic using normal doses.
Intermediate : implies that an infection due to a specific isolate can be treated with an antibiotic if treated with high doses or if the infections in an anatomic site where the antibiotic is concentrated . (β-lactam AB in urine).