Antibiotic Susceptibility test and Minimum Inhibitory Concentration tests

A. DISC DIFFUSION METHOD FOR THE ANTIMICROBIAL SUSCEPTIBILITY TESTING

principle:

A standardized inoculum of bacteria is swabbed onto the surface of a Mueller Hinton agar (MHA) plate. Filter paper disc impregnated with antimicrobial agents are placed on the agar. After overnight incubation, the diameter of the zone of inhibition is measured around each disc. By referring to the tables in the CLSI disc diffusion standard, a qualitative report of susceptible, intermediate or resistant is obtained.

Quality control:

      A. QC strains

     1.  Escherichia coli ATCC 25922     2. Staphylococcus aureus ATCC 25923

     3. Enterococcus faecalis ATCC 29212           

B. Monitoring accuracy

      1. Test QC strains by following routine procedure, and record results. Record lot number and expiration date of discs and agar.

      2. Compare to expected results (CLSI QC tables). Note any out of control result and document; proceed with corrective action, if necessary.

      3. Perform daily and weekly QC testing.

PROCEDURE:

1. Brings agar plates and canisters of discs to room temperature before use. Agar plates may be removed from refrigerator and placed in a 35 C ambient air incubator with lids slightly ajar to evaporate excess moisture. Do not leave in incubator for longer than 30 min.
2. Inoculum preparation

Using a loop or swab, transfer colonies as follows

1. Direct colony suspension method: - pick several colonies from a fresh (18 – 24 hr) nonselective agar plate to broth or 0.9% NaCl.
2. log phase method

a.       Pick four or five isolated colonies to 3.0 to 5.0 ml of broth.

b.      Incubate at 35 C for 2 to 8 hr until growth reaches the turbidity at or above that of a 0.5 McFarland standards.

3. For either the log phase or direct colony suspension method, vortex well and adjust turbidity visually with sterile broth or 0.9% NaCl to match a 0.5% McFarland standard.
2. Inoculation of agar plates
1. Within 15 minutes of adjusting turbidity, dip a sterile cotton swab into the inoculum and rotate against the wall of the tube above the liquid to remove excess inoculum.
2. Swab entire surface of agar plate three times, rotating plates approximately 60˚ between streaking to ensure even distribution. Avoid hitting the slides of the plate to avoid aerosols. Finally, run swab around the edge of the agar to remove any excess moisture.
3. Allow inoculated plate to stand for 3 to 15 min before applying discs.
3. Application of discs
1. Apply disc to agar surface with dispenser or manually with a sterile forceps.
2. Apply gentle pressure with sterile forceps or needle to ensure complete contact of disc with agar.
3. Do not place discs closer than 24mm from center to center (no more than 12 discs on 150 mm plates and 5 discs on 100 mm plates.
4. Do not relocate a disc once it has made contact with agar surface. Instead, place a new disc in another location on the agar.
4. Incubation
1. Invert plates and incubate within 15 min of disc application.           
2. Incubate for 16 to 18 at 35˚C in an ambient air incubator.
5. Reading plates
1. Read plates only if lawn of growth is confluent or nearly confluent.
2. Hold inverted plate a few inches above a black nonreflecting surface.
3. Illuminate plate with reflected light.
4. Use a sliding caliper or ruler held on the back of the plate to measure the diameter of inhibition zone to nearest whole millimeter.
5. When measuring zones for sulfonamides, trimethoprim, or trimethoprim- sulfamethoxazole, disregard light growth (20% less of lawn of growth) and measure edge of the more obvious margin of the zone.
6. Discrete colonies growing within the inhibition zone may represent a mixed culture or resistant variants; subculture single colonies from the primary culture plate, re-identify, and retest for susceptibility. If the discrete colonies are still apparent, measure the colony – free inner zone.

G. Interpretation and Reporting

Use criteria specified by the CLSI to interpret the zone of inhibition for each         antimicrobial agents and report categorical result as either susceptible(S), intermediate (I), or resistant (R).

Figure 1: Antibiotic Susceptibility test of a bacterial isolate.

precautions

The following common sources of error should be investigated to verify that:

• Zone diameters were measured and transcribed correctly;
• The turbidity standard has not expired, is stored properly, meets performance requirements, and was adequately mixed prior to use;
• All materials used were within their expiration dates and stored at the proper temperature;
• The incubator is at proper temperature and atmosphere;
• Other equipment used (e.g., pipettors) are functioning properly;
• Discs are stored desiccated and at proper temperature;
• The control strain has not changed and is not contaminated;
• Inoculum suspensions were prepared and adjusted correctly; and inoculum for the test was prepared from a plate incubated for the correct length of time and in no case more than 24 hours old.

B. DETERMINATION OF MINIMUM INHIBITORY CONCENTRATIONS OF ANTIBIOTICS (FLUOQUINOLONES)

PRINCIPLE

Minimum inhibitory concentration are considered the gold standard for determining the susceptibility of organisms to antimicrobials and therefore used to judge the performance of all other methods of susceptibility testing. MICs are widely used to give a definitive answer when a borderline result is obtained by other methods of testing or when disc diffusion methods are not appropriate and are important in the evaluation of antibiotics breakpoints.

Quality control

       A. QC strains

     1.  Escherichia coli ATCC 25922

  B. Monitoring accuracy

      1. Test QC strains by following routine procedure, and record results. Record lot number and expiration date of antibiotic powder.

      2. Compare to expected results (CLSI QC tables). Note any out of control result and document; proceed with corrective action, if necessary.

            3. Perform daily and weekly QC testing

PROCEDURE

1.      Antibiotic powder, solvents and diluents

1.1.   Obtain standard powder from the pharmaceutical company or a reputable supplier such as Sigma (Poole, Dorset, UK).

1.2.    Obtain information from the supplier regarding expiry date, potency, solubility, stability as a powder and in solution, storage conditions and any relevant information.

1.3.   Always prepare stock solutions following the manufacturer’s recommendations.

1.4.   Freeze and thaw stock solutions only once and then discard them.

1.5.   Appendix X shows solvent, diluents and storage conditions for antibiotics.

2.      Preparation of antibiotic stock solutions and dilution range

2.1.   Choose a suitable range of antibiotic concentrations for the organisms to be tested (see suggested ranges Appendix X).

2.2.   Prepare stock solutions using the formula 1000/P x V x C = W. Where P = potency given by the manufacturer (µg/mg), V = volume required (mL), C = final concentration of solution (multiples of 1000) (mg/L), and W = weight of antibiotic in mg to be dissolved in volume V (mL).

2.3.   For preparation of further stock solutions and dilution range, from the solution, prepare as described in table 3.

Dilution range for each antibiotic is prepared similarly. Solvents, diluents, dilution range and storage condition for antibiotic solution is described in appendix.

4.  Preparation of agar dilution plates

4.1        Prepare Mueller-Hinton agar following the manufacturer's instructions.

4.2        Add 1 ml of working antibiotic solution to each container containing 19 ml of cooled molten agar (ensure that the medium is cooled to 45°C before adding to the antibiotic), including the antibiotic-free control. Mix well before pouring into 90 mm Petri dishes.

4.3    Allow agar to set and then dry surface of the plates for 10 min in a fan assisted drying cabinet (without ultraviolet light) or in an incubator (time needed depend on the efficiency of the incubator).

4.4    Store plates at 4-8°C protected from light until inoculated. Ideally, plates should be used on the day of preparation. If plates are to be stored at 4 -80˚C before use, the stability of the drug must be determined by individual laboratories as part of the routine quality control programme.

5. Preparation of Inoculum

The inoculum should be adjusted so that 10⁴ cfu/spot are applied to the plates. The following procedure describes a method for preparing the desired inoculum by comparison with a 0.5 McFarland standard.

5.1. Preparation of the McFarland standard

Add 0.5 ml of 0.048 M BaCl₂ (1.17% w/v BaCl₂.2H₂O) to 99.5 ml of 0.18 M H₂SO4 (1% v/v) with constant stirring. Distribute the standard into screw cap tubes of the same size and with the same volume as those used in growing the broth cultures. Seal the tubes tightly to prevent loss by evaporation. Store protected from light at room temperature. Vigorously agitate the turbidity standard on a vortex mixer before use. Standards may be stored for up to six months after which time they should be discarded.

5.2. Preparation of inoculum

Touch at least four morphologically similar colonies with a sterile loop. Transfer growth into Mueller-Hinton broth or equivalent that has been shown not to affect the performance of the test and incubate broth with shaking at 35-37°C until the visible turbidity is equal to or greater than the 0.5 McFarland standard. Alternatively an overnight broth culture can be used. Direct colony suspension method can also be used.

5.3. Adjustment of the organism suspension to the density of the 0.5 McFarland standards.

Adjust the density of the organism suspension prepared to equal that of the 0.5 McFarland standards by adding sterile distilled water. To aid comparison, compare the test and standard against a white background with a contrasting black line. Suspensions should contain between 10⁷ and 10⁸ cfu/ml depending on genera. For the agar dilution method further dilution of suspension in sterile distilled water (1:10 for Enterobacteriaceae) is carried out before inoculation.

6. Quality Control

Appropriate controls, depending on genera, must be included with every batch of MIC determinations.

7. Inoculation

Use a multipoint inoculator to deliver 1-2 µl of suspension on to the surface of the agar. Allow the inoculum to be absorbed into the agar before incubation.

8. Incubation conditions

Incubation 35-37°C in air for 18-20 h

9. Reading and interpretation

After incubation ensure that all of the organisms have grown on the antibiotic-free control plate. The MIC is defined as the lowest concentration of antibiotic at which there is no visible growth of the organism. The growth of one or two colonies or a fine film of growth should be disregarded. The MIC for the control strain should be within plus or minus one two-fold dilution of the expected MIC.

Figure 2: Minimum Inhibitory Concentration test

PRECAUTIONS

• The turbidity standard has not expired, is stored properly, meets performance requirements, and was adequately mixed prior to use.
• All materials used were within their expiration dates and stored at the proper temperature;
• The incubator is at proper temperature and atmosphere.
• The control strain has not changed and is not contaminated.
• Inoculum suspensions were prepared and adjusted correctly.
• Inoculum for the test was prepared from a plate incubated for the correct length of time and in no case more than 24 hours old.

Source: Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement ed. CLSI document M100-S20. Wayne, PA: CLSI; 2010.