Antimicrobial Susceptibility Testing

1. Introduction

Appropriate antimicrobial drug use has unquestionable benefit, but physicians and the public frequently use these agents inappropriately. Inappropriate use results from physicians providing antimicrobial drugs to treat viral infections, using inadequate criteria for diagnosis of infections that potentially have a bacterial aetiology, unnecessarily prescribing expensive, broad-spectrum agents, and not following established recommendations for using chemo prophylaxis. The availability of antibiotics over the counter, despite regulations to the contrary, also fuel inappropriate usage of antimicrobial drugs in India. The easy availability of antimicrobial drugs leads to their incorporation into herbal or "folk" remedies, which also increases inappropriate use of these agents.

Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the development of antibiotic resistance. The association between increased rates of antimicrobial use and resistance has been documented for nosocomial infections as well as for resistant community acquired infections. As resistance develops to "first-line" antibiotics, therapy with new, broader spectrum, more expensive antibiotics increases, but is followed by development of resistance to the new class of drugs.

Resistance factors, particularly those carried on mobile elements, can spread rapidly within human and animal populations. Multidrug-resistant pathogens travel not only locally but also globally, with newly introduced pathogens spreading rapidly in susceptible hosts. Antibiotic resistance patterns may vary locally and regionally, so surveillance data needs to be collected from selected sentinel sources. Patterns can change rapidly and they need to be monitored closely because of their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by physicians in that area.

The results of in-vitro antibiotic susceptibility testing, guide clinicians in the appropriate selection of initial empiric regimens and, drugs used for individual patients in specific situations. The selection of an antibiotic panel for susceptibility testing is based on the commonly observed susceptibility patterns, and is revised periodically.

2. Principle

The principles of determining the effectivity of a noxious agent to a bacterium were well enumerated by Rideal ,Walker and others at the turn of the century, the discovery of antibiotics made these tests(or their modification)too cumbersome for the large numbers of tests necessary to be put up as a routine. The ditch plate method of agar diffusion used by Alexander Fleming was the forerunner of a variety of agar diffusion methods devised by workers in this field .The Oxford group used these methods initially to assay the antibiotic contained in blood by allowing the antibiotics to diffuse out of reservoirs in the medium in containers placed on the surface.

With the introduction of a variety of antimicrobials it became necessary to perform the antimicrobial susceptibility test as a routine.  For this, the antimicrobial contained in a reservoir was allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms.  Even now a variety of antimicrobial containing reservoirs are used but the antimicrobial impregnated absorbent paper disc is by far the commonest type used.  The disc diffusion method of AST is the most practical method and is still the method of choice for the average laboratory.  Automation may force the method out of the diagnostic laboratory but in this country as well as in the smaller laboratories of even advanced countries, it will certainly be the most commonly carried out microbiological test for many years to come. It is, therefore, imperative that microbiologists understand the principles of the test well and keep updating the information as and when necessary. All techniques involve either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth.

Even automated techniques are variations of the above methods.

3.

Factors Influencing Antimicrobial Susceptibility Testing

pH

The pH of each batch of Müeller-Hinton agar should be checked when the medium is prepared.  The exact method used will depend largely on the type of equipment available in the laboratory. The agar medium should have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is too low, certain drugs will appear to lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other agents may appear to have excessive activity (e.g., tetracyclines).  If the pH is too high, the opposite effects can be expected.  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.

 

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.

Effects of Thymidine or Thymine

Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in false-resistance reports.  Müeller-Hinton agar that is as low in thymidine content as possible should be used.  To evaluate a new lot of Müeller-Hinton agar, Enterococcus faecalis ATCC 29212, or alternatively, E. faecalis ATCC 33186, should be tested with trimethoprim/sulfamethoxazole disks.  Satisfactory media will provide essentially clear, distinct zones of inhibition 20 mm or greater in diameter.  Unsatisfactory media will produce no zone of inhibition, growth within the zone, or a zone of less than 20 mm.

Effects of Variation in Divalent Cations

Variation in divalent cations, principally magnesium and calcium, will affect results of aminoglycoside and tetracycline tests with P. aeruginosa strains.  Excessive cation content will reduce zone sizes, whereas low cation content may result in unacceptably large zones of inhibition. Excess zinc ions may reduce zone sizes of carbapenems.  Performance tests with each lot of Müeller-Hinton agar must conform to the control limits.

Testing strains that fail to grow satisfactorily

Only aerobic or facultative bacteria that grow well on unsupplemented Müeller-Hinton agar should be tested on that medium.  Certain fastidious bacteria such as Haemophilus spp.,

 N. gonorrhoeae, S. pneumoniae, and viridans and ß-haemolytic streptococci do not grow sufficiently on unsupplemented Müeller-Hinton agar.  These organisms require supplements or different media to grow, and they should be tested on the media described in separate sections.

4. Methods of Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods are divided into types based on the principle applied in each system. They include:

Diffusion                                Dilution                                              Diffusion&Dilution

Stokes method                             Minimum Inhibitory Concentration                       E-Test method

Kirby-Bauer method       i) Broth dilution

                                        ii)Agar Dilution
4.1 Disk Diffusion

Reagents for the Disk Diffusion Test

1. Müeller-Hinton Agar Medium

Of the many media available, Müeller-Hinton agar is considered to be the best for routine susceptibility testing of nonfastidious bacteria for the following reasons:

*          It shows acceptable batch-to-batch reproducibility for susceptibility testing.

*          It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.

*          It gives satisfactory growth of most nonfastidious pathogens.

*          A large body of data and experience has been collected concerning susceptibility tests performed with this medium.

Although Müeller-Hinton agar is reliable generally for susceptibility testing, results obtained with some batches may, on occasion, vary significantly.  If a batch of medium does not support adequate growth of a test organism, zones obtained in a disk diffusion test will usually be larger than expected and may exceed the acceptable quality control limits.  Only Müeller-Hinton medium formulations that have been tested according to, and that meet the acceptance limits described in, NCCLS document M62-A7- Protocols for Evaluating Dehydrated Müeller-Hinton Agar should be used.

Preparation of Müeller-Hinton Agar

Müeller-Hinton agar preparation includes the following steps.

1.         Müeller-Hinton agar should be prepared from a commercially available dehydrated base according to the manufacturer's instructions.

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

3.         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.  This corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of 100 mm.

4.         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).

5.         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.

6.         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.

2. Preparation of antibiotic stock solutions

Antibitiotics may be received as powders or tablets. It is recommended to obtain pure antibiotics from commercial sources, and not use injectable solutions.  Powders must be accurately weighed and dissolved in the appropriate diluents (Annexure III) to yield the required concentration, using sterile glassware. Standard strains of stock cultures should be used to evaluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted in 5 ml volumes and frozen at -20ºC or -60ºC.

Stock solutions are prepared using the formula (1000/P) X V X C=W, where P+potency of the anitbiotic base, V=volume in ml required, C=final concentration of solution and W=weight of the antimicrobial to be dissolved in V.

Preparation of dried filter paper discs

Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are placed in a Petri dish and sterilized in a hot air oven.

The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter of 2 mm. This delivers 0.005 ml of antibiotics to each disc.

Storage of commercial antimicrobial discs

Cartridges containing commercially prepared paper disks specifically for susceptibility testing are generally packaged to ensure appropriate anhydrous conditions.  Discs should be stored as follows:

*          Refrigerate the containers at 8°C or below, or freeze at -14°C or below, in a nonfrost-free freezer until needed.  Sealed packages of disks that contain drugs from the ß-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.

*          The unopened disc 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.

*          Once a cartridge of discs has been removed from its sealed package, it should be placed in a tightly sealed, desiccated container.  When using a disc-dispensing apparatus, it should be fitted with a tight cover and supplied with an adequate desiccant.  The dispenser should be allowed to warm to room temperature before opening.  Excessive moisture should be avoided by replacing the desiccant when the indicator changes color.

*          When not in use, the dispensing apparatus containing the discs should always be refrigerated.

*          Only those discs that have not reached the manufacturer's expiration date stated on the label may be used.  Discs should be discarded on the expiration date.

Turbidity standard for inoculum preparation

To standardize the inoculum density for a susceptibility test, a BaSO₄ turbidity standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle suspension), should be used.  A BaSO₄ 0.5 McFarland standard may be prepared as follows:

1.      A 0.5-ml aliquot of 0.048 mol/L BaCl₂ (1.175% w/v BaCl₂ . 2H₂O) is added to 99.5 ml of 0.18 mol/L H₂SO₄ (1% v/v) with constant stirring to maintain a suspension.

2.      The correct density of the turbidity standard should be verified by using a spectrophotometer with a 1-cm light path and matched cuvette to determine the absorbance.  The absorbance at 625 nm should be 0.008 to 0.10 for the 0.5 McFarland standard.

3.      The Barium Sulfate suspension should be transferred in 4 to 6 ml aliquots into screw-cap tubes of the same size as those used in growing or diluting the bacterial inoculum.

4.      These tubes should be tightly sealed and stored in the dark at room temperature.

5.      The barium sulfate turbidity standard should be vigorously agitated on a mechanical vortex mixer before each use and inspected for a uniformly turbid appearance.  If large particles appear, the standard should be replaced.  Latex particle suspensions should be mixed by inverting gently, not on a vortex mixer

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

Disc diffusion methods

The Kirby-Bauer and Stokes' methods are usually used for antimicrobial susceptibility testing, with the Kirby-Bauer method being recommended by the NCCLS. The accuracy and reproducibility of this test are dependent on maintaining a standard set of procedures as described here.

NCCLS is an international, interdisciplinary, non-profit, non-governmental organization composed of medical professionals, government, industry, healthcare providers, educators etc. It promotes accurate antimicrobial susceptibility testing (AST) and appropriate reporting by developing standard reference methods, interpretative criteria for the results of standard AST methods, establishing quality control parameters for standard test methods, provides testing and reporting strategies that are clinically relevant and cost-effective

Interpretative criteria of NCCLS are developed based on international collaborative studies and well correlated with MIC’s and the results have corroborated with clinical data. Based on study results NCCLS interpretative criteria are revised frequently. NCCLS is approved by FDA-USA and recommended by WHO.

Procedure for Performing the Disc Diffusion Test

 Inoculum Preparation

 Growth Method

The growth method is performed as follows

1.         At least three to five 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 the growth is transferred into a tube containing 4 to 5 ml of a suitable broth medium, such as tryptic soy broth.

2.         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)

3.         The turbidity of the actively growing broth culture is adjusted with sterile saline or broth to obtain a turbidity optically comparable to that of the 0.5 McFarland standard.  This results in a suspension containing approximately 1 to 2 x 10⁸ CFU/ml for E.coli ATCC 25922.  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.

Direct Colony Suspension Method

1.         As a convenient alternative to the growth method, the inoculum can be prepared by making a direct broth or saline suspension of isolated colonies selected from a 18- to 24-hour agar plate (a nonselective medium, such as blood agar, should be used).  The suspension is adjusted to match the 0.5 McFarland turbidity standard, using saline and a vortex mixer.

2.         This approach is the recommended method for testing the fastidious organisms, Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for potential methicillin or oxacillin resistance.

 Inoculation of Test Plates

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

2.         The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the swab over the entire sterile agar surface.  This procedure is repeated by streaking two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum.  As a final step, the rim of the agar is swabbed.

3.         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.

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

 

Application of Discs to Inoculated Agar Plates

1.         The predetermined battery of antimicrobial discs is dispensed onto the surface of the inoculated agar plate.  Each disc must be pressed down to ensure complete contact with the agar surface.  Whether the discs are placed individually or with a dispensing apparatus, they must be distributed evenly so that they are no closer than 24 mm from center to center. Ordinarily, no more than 12 discs should be placed on one 150 mm plate or more than 5 discs on a 100 mm plate.  Because some of the drug diffuses almost instantaneously, a disc should not be relocated once it has come into contact with the agar surface.  Instead, place a new disc in another location on the agar.

2.         The plates are inverted and placed in an incubator set to 35°C within 15 minutes after the discs are applied. With the exception of Haemophilus spp., streptococci and

            N. gonorrhoeae, 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.

 

Reading Plates and Interpreting Results

1.         After 16 to 18 hours of incubation, each plate is examined.  If the plate was satisfactorily streaked, and the inoculum was correct, the resulting zones of inhibition will be uniformly circular and there will be a confluent lawn of growth.  If individual colonies are apparent, the inoculum was too light and the test must be repeated.  The diameters of the zones of complete inhibition (as judged by the unaided eye) are measured, including the diameter of the disc. 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, nonreflecting background and illuminated with reflected light.  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.  If the test organism is a Staphylococcus or Enterococcus spp., 24 hours of incubation are required for vancomycin and oxacillin, but other agents can be read at 16 to 18 hours.  Transmitted light (plate held up to light) is used to examine the oxacillin and vancomycin zones for light growth of methicillin- or vancomycin- resistant colonies, respectively, within apparent zones of inhibition.  Any discernable growth within zone of inhibition is indicative of methicillin or vancomycin resistance.

2.         The zone margin should be taken as the area showing no obvious, visible growth that can be detected with the unaided eye.  Faint growth of tiny colonies, which can be detected only with a magnifying lens at the edge of the zone of inhibited growth, is ignored.  However, discrete colonies growing within a clear zone of inhibition should be subcultured, re-identified, and retested.  Strains of Proteus spp. may swarm into areas of inhibited growth around certain antimicrobial agents.  With Proteus spp., the thin veil of swarming growth in an otherwise obvious zone of inhibition should be ignored.  When using blood-supplemented medium for testing streptococci, the zone of growth inhibition should be measured, not the zone of inhibition of hemolysis.  With trimethoprim and the sulfonamides, antagonists in the medium may allow some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth), and measure the more obvious margin to determine the zone diameter.

3.      The sizes of the zones of inhibition are interpreted by referring to Tables 2A through 2I (Zone Diameter Interpretative Standards and equivalent Minimum Inhibitory Concentration Breakpoints) of the NCCLS M100-S12: Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement, and the organisms are reported as either susceptible, intermediate, or resistant to the agents that have been tested. Some agents may only be reported as susceptible, since only susceptible breakpoints are given.

4.2 Dilution Methods

Dilution susceptibility testing methods are used to determine the minimal concentration of antimicrobial to inhibit or kill the microorganism. This can be achieved by dilution of antimicrobial in either agar or broth media. Antimicrobials are tested in log₂ serial dilutions (two fold).  

Minimum Inhibitory Concentration (MIC)

Diffusion tests widely used to determine the susceptibility of organisms isolated from clinical specimens have their limitations; when equivocal results are obtained or in prolonged serious infection e.g. bacterial endocarditis, the quantitation of antibiotic action vis-a-vis the pathogen needs to be more precise.  Also the terms ‘Susceptible’ and ‘Resistant’ can have a realistic interpretation.  Thus when in doubt, the way to a precise assessment is to determine the MIC of the antibiotic to the organisms concerned.

There are two methods of testing for MIC:

(a) Broth dilution method

(b) Agar dilution method.

Broth Dilution Method

The Broth Dilution method is a simple procedure for testing a small number of isolates, even single isolate.  It has  the added advantage that the same tubes can be taken for MBC tests also:

Materials

Sterile graduated pipettes of 10ml, 5ml, 2ml and 1ml Sterile capped 7.5 x 1.3 cm tubes / small screw-capped bottles, Pasteur pipettes, overnight broth culture of test and control organisms ( same as for disc diffusion tests), required antibiotic in powder form (either from the manufacturer or standard laboratory accompanied by a statement of its activity in mg/unit or  per ml. Clinical preparations should not be used for reference technique.),            required  solvent for the antibiotic, sterile Distilled Water  -  500ml and suitable nutrient broth medium.

Trimethoprim and sulphonamide testing requires thymidine free media or addition of 4% lysed horse blood to the media

A suitable rack to hold 22 tubes in two rows i-e 11 tubes in each row.

Stock solution

Stock solution can be prepared using the formula

                         1000

                        -------  x V x C= W

                           P

Where P=Potency given by the manufacturer in relation to the base

V= Volume in ml required

C=Final concentration of solution (multiples of 1000)

W= Weight of the antimicrobial to be dissolved in the volume V

Example: For making 10 ml solution of the strength 10,000mg/l from powder base whose potency is 980 mg per gram,the quantities of the antimicrobials required is

                        W =       1000

                                     -------  x 10 x 10=102.04mg

                                       980

Note:the stock solutions are made in higher concentrations to maintain their keeping qualities and stored in suitable aliquots at -20^oC .Once taken out,they should not be refrozen or reused.

Suggested dilution ranges of some antimicrobials are shown in Annexure II.

Method

Prepare stock dilutions of the antibiotic of concentrations 1000 and 100 µg/L as required from original stock solution (10,000mg/L). Arrange two rows of 12 sterile 7.5 x1.3 cm capped tubes in the rack. In a sterile 30ml (universal) screw capped bottle, prepare 8ml of  broth containing the concentration of antibiotic required for the first tube in each row from the appropriate stock solution already made. Mix the contents of the universal bottle using a pipette and transfer 2ml to the first tube in each row. Using a fresh pipette ,add 4 ml of broth to the remaining 4 ml in the universal bottle mix and transfer 2ml to the second tube in each row. Continue preparing dilutions in this way but where as many as 10 or more are required the series should be started again half the way down. Place 2ml of antibiotic free broth to the last tube in each row. Inoculate one row with one drop of an overnight broth culture of the test organism diluted approximately to 1 in 1000 in a suitable broth and the second row with the control organism of known sensitivity similarly diluted. The result of the test is  significantly affected by the size of the inoculum.The test mixture should contain 10⁶ organism/ml.If the broth culture used has grown poorly,it may be necessary to use this undiluted. Incubate tubes for 18 hours at 37^oC. Inoculate a tube containing 2ml broth with the organism and keep at +4^oC in a refrigerator overnight to be used as standard for the determination of complete inhibition.

Calculations for the preparation of the original dilution.

This often presents problems to those unaccustomed to performing these tests. The following method advocated by Pamela M Waterworth is presented. Calculate the total volume required for the first dilution. Two sets of dilution are being prepared (one for the test and one for the control), each in 2ml volumes i-e a total of 4 ml for each concentration as 4ml is required to make the second dilution, the total requirement is 8ml. Now calculate the total amount of the antibiotic required for 8ml. For 64 g/l concentration, 8x64mg/l =512µg in 8 ml. Place a decimal point after the first figure (5.12) and take this volume in ml (i.e 5.12 ml) of the dilution below 512mg/l and make upto 8ml with broth. In this example given above, the series has to be started again mid way at 2 mg/l which would be obtained in the same way:

            8ml of 2mg/l=16µg in 8ml.

            1.6 ml of 10 mg/ l + 6.4 ml of broth.

Reading of result

MIC is expressed as the lowest dilution, which inhibited growth judged by lack of turbidity in the tube.

Because very faint turbidity may be given by the inoculum itself, the inoculated tube kept in the refrigerator overnight may be used as the standard for the determination of complete inhibition.

Standard strain of known MIC value run with the test is used as the control to check the reagents and conditions.

 

Minimum Bactericidal Concentrations(MBC)

The main advantage of the ‘Broth dilution’ method for the MIC determination lies in the fact that it can readily be converted to determine the MBC as well.

Method

Dilutions and inoculations are prepared in the same manner as described for the determination of MIC. The control tube containing no antibiotic is immediately subcultured (Before incubation) by spreading a loopful evenly over a quarter of the plate on a medium suitable for the growth of the test organism and incubated at 37^oC overnight. The tubes are also incubated overnight at 37^oC. Read the MIC of the control organism to check that the drug concentrations are correct. Note the lowest concentration inhibiting growth of the organisms and record this as the MIC. Subculture all tubes not showing visible growth in the same manner as the control tube described above and incubate at 37^oC overnight. Compare the amount of growth from the control tube before incubation,which represents the original inoculum. The test must include a second set of the same dilutions inoculated with an organism of known sensitivity .These tubes are not subcultured; the purpose of the control is to confirm by its MIC that the drug level is correct,whether or not this organism is killed is immaterial.

Reading of result

These subcultures may show

• Similar number of colonies- indicating bacteriostasis only.

·         A reduced number  of colonies-indicating a partial or slow bactericidal activity.

·         No growth- if the whole inoculum has been killed

·         The highest dilution showing at least 99% inhibition is taken as MBC

 

Micro-broth dilution test

This test uses double-strength Müeller-Hinton broth, 4X strength antibiotic solutions prepared as serial two-fold dilutions and the test organism at a concentration of 2x10⁶/ml. In a 96 well plate, 100 ml of double-strength MHB, 50 ml each of the antibiotic dilutions and the organism suspension are mixed and incubated at 35°C for 18-24 hours. The lowest concentration showing inhibition of growth will be considered the MIC of the organism.

 

Reading of result

MIC is expressed as the highest dilution which inhibited growth judged by lack of turbidity in the tube. Because very faint turbidity may be given by the inoculum itself,the inoculated tube kept in the refrigerator overnight may be used as the standard for the determination of complete inhibition. Standard strain of known MIC, run with the test is used as the control to check the reagents and conditions.

 

The Agar dilution Method

Agar dilutions are most often prepared in petri dishes and have advantage that it is possible  to test  several organisms on each plate .If only one organism is to be tested e.g M.tuberculosis,the dilutions can be prepared in agar slopes but it will then be necessary to prepare a second identical set to be inoculated with the control organism.The dilutions are made in a small volume of water and added to agar which has been melted and cooled to not more than 60^oC.Blood may be added and if ‘chocolate agar’ is required,the medium must be heated before the antibiotic is added.

It would be convenient to use 90 mm diameter petri dishes and add

one ml of desired drug dilutions to 19 ml of broth.The factor of agar dilution must be allowed for in the first calculation as follows.

final volume of medium in plate = 20 ml

Top antibiotic concentrations   = 64mg/l

Total amount of drug    = 1280µg to be added to1 ml of water

2ml of 1280 µg /ml will be required to start the dilution = 2560µg in 2 ml

               = 1.28ml of 2000µg /ml ± 0.72 ml of    water.

                        1 ml of this will be added to 19 ml agar.

(Note stock dilution of 2000µg /ml is required for this range of MIC)

The quickest way to prepare a range of dilutions in agar is as follows:

Label a sterile petri dish on the base for each concentration required. Prepare the dilutions in water placing 1 ml of each in the appropriate dish. One ml water is added to a control plate. Pipette 19 ml melted agar, cooled to 55^oC to each plate and mix thoroughly. Adequate mixing is essential and if sufficient technical expertise is not available for the skilled manipulation, it is strongly recommended that the agar is first measured into stoppered tubes or universal containers and the drug dilution added to these and mixed by inversion before pouring into petri dishes. After the plates have set they should be well dried at 37^oC with their lids tipped for 20 to 30 minutes in an incubator. They are then inoculated either with a multiple inoculator as spots or with a wire loop or a platinum loop calibrated to deliver 0.001ml spread over a small area. In either case the culture should be diluted to contain 10⁵ to 10⁶ organisms per ml. With ordinary fast growing organisms, this can be obtained approximately by adding 5 µl of an overnight broth culture to 5ml broth or peptone water.

It is possible to test spreading organism such as P.mirabilis by this method either by cutting ditches in the agar between the inocula, or by confining each with small glass or porcelain cylinders pressed into the agar. Although swarming of P.mirabilis can be prevented by the use of higher concentration of agar in the medium, this is not recommended for determination of MIC because of the difficulty of ensuring adequate mixing of the drug with this very viscous medium. Selective media should not be used and electrolyte deficient media will give false results because of the effect of variation in the salt content on the action of many antibiotics.

Reading of results

The antibiotic concentration of the first plate showing ³ 99% inhibition is taken as the MIC for the organism.

4.3 Dilution and Diffusion

E test also known as the epsilometer test is an ‘exponential gradient’ testing methodology where ‘E’ in E test refers to the  Greek symbol epsilon (e).The E test(AB Biodisk) which is a quantitative method for antimicrobial susceptibility testing applies both the dilution of antibiotic and diffusion of antibiotic into the medium.. A predefined stable antimicrobial gradient is present on a thin inert carrier strip. When this E test strip is applied onto an inoculated agar plate, there is an immediate release of the drug. Following incubation , a symmetrical inhibition ellipse is produced. The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC value over a wide concentration range (>10 dilutions) with inherent precision and accuracy .

 

E  test  can  be  used  to  determine  MIC  for  fastidious  organisms  like  S. pneumoniae,

 ß-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also be used for Nonfermenting Gram Negative bacilli (NFGNB) for eg-Pseudomonas sp. and Burkholderia pseudomallei.

Resistance of major consequence may be detected for e.g., the test is very useful in detecting glycopeptide resistant Enterococci (GRE) and glycopeptide intermediate S.aureus (GISA) and slow growing pathogens such as Mycobacterium tuberculosis. Further it can be used for detection of extended spectrum beta lactamases (ESBL). In conclusion E test is a simple, accurate and reliable method to determine the MIC for a wide spectrum of infectious agents.