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    What's New in Antimicrobial Susceptibility Testing?: Dilution MIC Test Broth Dilution

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    Viswanathan Ananthanarayanan
    1. The document discusses various methods for antimicrobial susceptibility testing (AST), including broth and agar dilution tests, disk diffusion tests, and E-test. 2. The broth microdilution and agar dilution tests directly measure the minimum inhibitory concentration (MIC) of antibiotics, but are time-consuming. Disk diffusion tests provide indirect qualitative results by measuring inhibition zone diameters. 3. E-test combines aspects of dilution and diffusion methods by using plastic strips containing continuous gradients of antibiotics, allowing direct reading of MIC results without interpretive charts. Studies have shown E-test to highly correlate with standard dilution methods and to be applicable to fastidious bacteria.

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    What's New in Antimicrobial Susceptibility Testing?: Dilution MIC Test Broth Dilution

    Uploaded by

    Viswanathan Ananthanarayanan
    42 views3 pages
    1. The document discusses various methods for antimicrobial susceptibility testing (AST), including broth and agar dilution tests, disk diffusion tests, and E-test. 2. The broth microdilution and agar dilution tests directly measure the minimum inhibitory concentration (MIC) of antibiotics, but are time-consuming. Disk diffusion tests provide indirect qualitative results by measuring inhibition zone diameters. 3. E-test combines aspects of dilution and diffusion methods by using plastic strips containing continuous gradients of antibiotics, allowing direct reading of MIC results without interpretive charts. Studies have shown E-test to highly correlate with standard dilution methods and to be applicable to fastidious bacteria.

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    1. The document discusses various methods for antimicrobial susceptibility testing (AST), including broth and agar dilution tests, disk diffusion tests, and E-test. 2. The broth microdilution and agar dilution tests directly measure the minimum inhibitory concentration (MIC) of antibiotics, but are time-consuming. Disk diffusion tests provide indirect qualitative results by measuring inhibition zone diameters. 3. E-test combines aspects of dilution and diffusion methods by using plastic strips containing continuous gradients of antibiotics, allowing direct reading of MIC results without interpretive charts. Studies have shown E-test to highly correlate with standard dilution methods and to be applicable to fastidious bacteria.

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    42 views3 pages

    What's New in Antimicrobial Susceptibility Testing?: Dilution MIC Test Broth Dilution

    Uploaded by

    Viswanathan Ananthanarayanan
    1. The document discusses various methods for antimicrobial susceptibility testing (AST), including broth and agar dilution tests, disk diffusion tests, and E-test. 2. The broth microdilution and agar dilution tests directly measure the minimum inhibitory concentration (MIC) of antibiotics, but are time-consuming. Disk diffusion tests provide indirect qualitative results by measuring inhibition zone diameters. 3. E-test combines aspects of dilution and diffusion methods by using plastic strips containing continuous gradients of antibiotics, allowing direct reading of MIC results without interpretive charts. Studies have shown E-test to highly correlate with standard dilution methods and to be applicable to fastidious bacteria.

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    of 3

    What’s New in Antimicrobial Susceptibility Testing?

    Myrna T. Mendoza, M.D.*

    (*Head, Section of Infectious Diseases, Department of Medicine, University of the Philippines-Philippine General
    Hospital and National Kidney and Transplant Institute; Clinical Microbiology Consultant, VRP Medical Center,
    Cardinal Santos Medical Center and National Kidney and Transplant Institute)

    (Phil J Microbiol Infect Dis 1998; 27(3):113-115)

    Specific antimicrobial treatment for infection is usually guided by the offending pathogen
    and its predicted antimicrobial susceptibility pattern. The antimicrobial susceptibility test (AST)
    performed on etiologic organism provides valuable information for patient care. The AST results
    in most cases can predict the clinical outcome and or response to therapy.
    The widely used susceptibility testing methods are the disk diffusion and broth agar
    dilution tests. The minimum inhibitory concentration (MIC) of a particular drug to a particular
    organism can be quantitatively measured in-vitro through the broth agar or micro/or macro-
    dilution test. These testing methods have been standardized and the National Committee of
    Clinica1 Laboratory Standards (NCCLS) provides susceptibility test guidelines. Most laboratories
    in training tertiary hospitals in the Philippines follow the NCCLS technical guidelines.

    ANTIMICROBIAL SUSCEPTIBILITY TESTING METHODS

    Microbial susceptibility testing guidelines are based on MIC determinations. These are
    correlated with the pharmacological properties of the antimicrobial drug and the clinical results
    when the drug is used. In MIC determinations, a bacteriological breakpoint is usually drawn
    between susceptible and resistant organisms. This breakpoint has been defined as the minimum
    concentration at which the drug is predicted to be clinically effective against the etiologic agents.

    Dilution MIC Test

    Broth Dilution

    In the broth dilution MIC method, various concentrations of an antimicrobial drug are
    inoculated with a standard suspension of test bacteria. Following an overnight incubation at 35°C,
    the MIC is determined by observing the lowest concentration of the drug that will inhibit visible
    growth of the test bacteria. For full range MIC testing, 5- 8 concentrations representing a
    therapeutically achievable range for an antimicrobial are usually tested. Recent modifications,
    however, provide only 1-3 concentrations of each drug and results are reported.
    More tests can be done at the same time by limiting dilution steps. This method can be
    done by using the test tube dilutions (macrodilution) or the plastic microdilution tray
    (microdilution). The broth microdilution MIC test method has been well standardized for most
    fastidious rapidly growing bacteria.

    Agar Dilution

    Different concentrations of antimicrobial agents are incorporated onto Mueller-Hinton


    (MH) agar plates. These are inoculated with an inoculum of test organisms adjusted to match 0.5
    McFarland standards. Inoculation is done either by a calibrated loop or an inoculum-replicating
    device (replicator). Plates are incubated at 35°C overnight and read by observing the lowest drug
    concentration that inhibits visible bacterial growth. This concentration is reported as the MIC.
    Simultaneous testing of several isolates is possible with this method. However, this method is
    labor intensive and time consuming.
    The dilution tests are generally not recommended for routine susceptibility test in the
    clinical laboratory

    Disk Diffusion Susceptibility Test

    This is the standard AST used in most bacteriology laboratories in the country. In the disk
    diffusion susceptibility test; a standardized inoculum of the organism is swabbed onto the surface
    of a Muller-Hinton agar plate, then filter paper disks impregnated with antimicrobial agents are
    placed on the agar. After overnight incubation at 35°C, the diameter of the zone of inhibition (ZI)
    of bacterial growth around each disk is measured. The size of the ZI is inversely proportional to
    the MIC of the organism. This method is an indirect measure of the susceptibility based on MIC-
    zone size correlation. Based on the zones of inhibition a qualitative report of "susceptible,"
    “intermediate" or "resistant" can be determined for rapidly growing non-fastidious aerobic
    bacteria.
    Bauer et al first described this simple method in 1966. The test however, must be
    performed under controlled and standardized conditions to provide accurate results.
    Recommendations on the standardized technique specifically the appropriate culture media to
    use, inoculum size and concentration, disk potency and its proper storage are provided by
    NCCLS. Different interpretive charts of ZI are also regularly updated and revised for a better
    clinical correlation. A disadvantage is that interpretive charts vary with the organism being tested.
    Current revisions include guidelines for the use of the disk diffusion test for some fastidious
    aerobic bacteria.

    E-test (PDM Epsilometer AB Biodisk)

    AB Biodisk has recently introduced a new quantitative antimicrobial susceptibility test


    method. The test provides an Epsilometer test (E-test) device for direct quantification of
    antimicrobial susceptibility of microorganisms. This method combines the ease of disk diffusion
    and accuracy of the MIC broth dilution techniques. The E-test utilizes a rectangular plastic strip
    device that contains predefined, continuous exponential gradient of antibiotic concentrations that
    correspond to MIC dilutions. This plastic test device has a reading and interpretive scale
    corresponding to 2-fold MIC dilutions indicated on the surface of the strip. In this method, the
    strip is applied onto the surface of an inoculated MH agar plate. The drug on the strip is
    immediately released and diffused in the agar. After 24 hours of incubation, an elliptical zone of
    inhibition of bacterial growth is seen around the test strip. The zone edge intersects the plastic
    strip at a specific level corresponding to the inhibitory concentration of the drug that inhibits the
    microorganism (See Figure). This concentration is a direct measure of the susceptibility of the
    microorganism to the particular test drug. Basically a diffusion technique, the test agar and
    inoculum suspension concentration are similar to the disk diffusion technique. The antibiotic
    gradient strip is stable, giving reproducible inhibitory concentrations. The high correlation of the
    E-test results with the broth or agar MIC dilution tests has been shown by several studies since
    1988.
    There are also supportive evidences that E-test is applicable to fastidious microorganisms
    such as S. pneumoniae and H. influenzae. It can also be used for epidemiologic surveillance of
    clinically important resistant organisms such as MRSA and multiply-resistant nosocomial
    organisms.
    This new method of AST, which is based on dilution and diffusion tests, will give direct
    quantification of the MIC without the need of interpretive charts. E-test is now acknowledged and
    accepted worldwide because it can be easily adapted in the routine bacteriology laboratory and
    can provide more accurate and reliable AST results. This is going to be particularly helpful in
    patients with life-threatening infections such as infective endocarditis, meningitis and sepsis.
    Since it is not inexpensive, it is recommended locally to confirm questionable or unusual AST
    results based on disk diffusion tests.

    E-test plate showing susceptibility test result of an isolate of Pseudomonas aeruginosa:


    Piperacillin-tazobactam 2 ug (S), Gentamicin 2 ug (S), Aztreonam 1.5 ug (S),
    Amikacin 4 ug (S) and Ciprofloxacin 0.225 ug (S).

    REFERENCES

    1. Wood GL, Washington JA. Antibacterial Susceptibility Tests: Dilution and Disk Diffusion Methods. In Murray P.R (ed.):
    Manual of Clinical Microbiology. Washington DC: ASM Press 1995; 1327-1341.
    2. National Committee for Clinical Laboratory Standards, 1996. Performance Standards for Antimicrobial Susceptibility
    Testing. M100-S5. Villanova, Pa.
    3. Macias E, Mason E, Ocera H, La Rocco M. Comparison of E. test with standard broth microdilution for determining
    antibiotic susceptibilities of penicillin-resistant strains of S. pneumoniae. J Clin Microbiol 1994; 32:430-432.
    4. Kiska D, Kerr A, Jones M, et al. Comparison of antimicrobial susceptibility. Methods for detection of penicillin resistant S.
    pneumoniae. J Clin Microbiol 1995; 33:229-232.
    5. Tenover F, Baker CN, Smenson J. Evaluation of commercial methods for determining antimicrobial susceptibility of S.
    pneumoniae. J Clin Microbiol 1994; 34:10-14.
    6. Jorgensen JH, Homell AW, Maher L. Quantitative antimicrobial susceptibility testing of Hemophilus influenzae and S.
    pneumoniae by using the E-test 1991; 29:109-114.

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