Standards for Antimicrobial Disk Susceptibility Tests

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Dr Maryam SotoudehTehran heart center

OBJECTIVEThis 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

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Indications for Performing Susceptibility Tests

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The responsibility of the microbiology laboratory includes : 1.Microbial detection and isolation

2.Determination of microbial susceptibility to antimicrobial agents.

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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)

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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 antibiotics7

Disk diffusion method (Kirby-bauer test)

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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.

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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 .

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Disk diffusion method components:

1. McFarland 0.5 standard suspension of bacteria (for Inoculum preparation)

2. Mueller-Hinton agar plate3. Filter paper disk containing a specific

amount (not concentration ) of antibiotic

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Standard preparation

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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.

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McFarland standards preparation:

• Add specific volume of 1% sulfuric acid and 1.175% barium chloride with constant stirring to maintain a suspension .

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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.

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• 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.

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• If large particles appear, the standard should be replaced

• The barium sulfate standards should be replaced or their densities verified monthly

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

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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 3 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

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Inoculum Preparation

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Inoculum Preparation

Growth Method

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Growth Method

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).

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

• 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).

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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

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Inoculation of Test Plates

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Mix McFarland Standard wellStandardize inoculum suspension

Janet Fick Hindler, MCLS MT(ASCP) UCLA Medical Center Los Angeles, CA

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Procedures

Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard

0.5 McFarland StandardSample 30

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streak a lawn of bacteria on Mueller-Hinton agar

Procedures

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NOTE: Extremes in inoculum density must be avoided.

Never use undiluted overnight broth cultures or other unstandardized inocula for streaking plates.

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MEDIUM

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Mueller-Hinton medium

• The most frequent basal culture medium for testing bacteria that grow aerobically.

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Preparation of Mueller-Hinton Agar

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Preparation of agar medium

1) Prepare MHA ,according to the manufacturer's instructions, using distilled water or deionized water.

2) Heat with frequent agitation and boil to dissolve the medium completely. Sterilize by autoclaving at 121°C for 15 min.

3) Immediately after autoclaving, allow it to cool in a 45 to 50 °C water bath

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• 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.

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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

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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. 43

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

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• 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).

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• 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

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

 

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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.

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Application of Disks to Inoculated Agar Plates

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nor more than 5 disks on a 100-mm plate.

no more than 12 disks should be placed on one 150-mm plate

Disks are no closer than 24 mm from center to center.

Each disk must be pressed down to ensure complete contact with the agar surface.

The predetermined battery of antimicrobial disks is dispensed onto the surface of the agar plate.

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• 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

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Application of Disks to Inoculated Agar Plates

The plates are inverted andplaced in an incubator set to 35 °C within 15 minutes after the disks are applied.

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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.

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Storage of Antimicrobial Disks

• Cartridges containing commercially prepared paper disks are generally packaged to ensure appropriate anhydrous conditions.

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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.

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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.

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Antimicrobial DisksThe 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. 57

• 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

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• 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

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Add disks

Incubate overnight

Janet Fick Hindler, MCLS MT(ASCP) UCLA Medical Center Los Angeles, CA

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Reading Plates and Interpreting Results

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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

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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 .

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

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. 65

• 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

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• 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.

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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

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

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Do not read zones showing distortion from circular .

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An agar gel that is too thick leads to smaller zonesSolution:Use McFarland 0.5/ photometer

Common interpretation problems

Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/71

Interpretation of Disk Diffusion Test Results

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• 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:

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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).

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