Broth dilution testing involves challenging the organism of interest with antimicrobial agents in a liquid environment. Each antimicrobial agent is tested using a range of concentrations, commonly expressed as micrograms (µg) of active drug per milliliter (mL) of broth (i.e., µg/ mL). The concentration range examined for a particular drug depends on specific criteria, including the safest therapeutic concentration possible in a patient’s serum. Therefore, the concentration range examined often varies from one drug to the next, depending on the pharmacologic properties of the antimicrobial agent. Additionally, the concentration range may be based on the level of drug required to reliably detect a particular resistance mechanism. In this case, the test concentration for a drug may vary depending on the organism and its associated resistances. For example, to detect clinically significant resistance to cefotaxime in S. pneumoniae, the dilution scheme uses a maximum concentration of 2 µg/ mL; however, to detect cefotaxime resistance in Escherichia coli, the required maximum concentration is 16 µg/ mL or higher.

Typically, the range of concentrations examined for each antibiotic is a series of doubling dilutions (e.g., 16, 8, 4, 2, 1, 0.5, 0.25 µg/mL); the lowest antimicrobial concentration that completely inhibits visible bacterial growth, as detected visually or with an automated or semiautomated method, is recorded as the minimal inhibitory concentration (MIC).

Procedures. The key features of broth dilution testing procedures are shown in Table1. Because changes are made in these procedural recommendations, the CLSI M07 series, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,” should be consulted annually.

Table1. Summary of Broth Dilution Susceptibility Testing Conditions

Medium and Antimicrobial Agents. With in vitro susceptibility testing methods, certain conditions must be altered when examining fastidious organisms to optimize growth and facilitate expression of bacterial resistance. For example, the Mueller-Hinton preparation is the standard medium used for most broth dilution testing, and conditions in the medium (e.g., pH, cation concentration, thymidine content) are well controlled by commercial manufacturers. However, media supplements or different media are required to obtain good growth and reliable susceptibility profiles for bacteria such as S. pneumoniae and H. influenzae. Although staphylococci are not considered fastidious organisms, media supplemented with sodium chloride (NaCl) enhance the expression and detection of methicillin-resistant isolates (see Table 1).

Broth dilution testing is divided into two general categories: microdilution and macrodilution. The principle of each test is the same; the only difference is the volume of broth in which the test is performed. For microdilution testing, the total broth volume is 0.05 to 0.1 mL; for macrodilution testing, the broth volumes are usually 1 mL or greater. Because most susceptibility test batteries require testing of several antibiotics at several different concentrations, the smaller volume used in microdilution allows this to be conveniently accomplished in a single microtiter tray (Figure 1).

Fig1. Microtiter tray used for broth microdilution testing. Doubling dilutions of each antimicrobial agent in test broth occupies one  vertical row of wells. 

The need for multiple large test tubes in the macro dilution method makes that technique substantially cumbersome and labor intensive when several bacterial isolates are tested simultaneously. For this reason, macrodilution is rarely used in most clinical laboratories, and subsequent comments about broth dilution focuses on the microdilution approach.

A key component of broth testing is proper preparation and dilution of the antimicrobial agents incorporated into the broth medium. Most laboratories that perform broth microdilution use commercially supplied microdilution panels in which the broth is already supplemented with appropriate antimicrobial concentrations. Therefore, antimicrobial preparation and dilution are not commonly carried out in most clinical laboratories (the details of this procedure are outlined in the CLSI M07-A6 document). In most instances, each antimicrobial agent is included in the microtiter trays as a series of doubling twofold dilutions. To ensure against loss of antibiotic potency, the antibiotic microdilution panels are stored at −20°C or lower, if possible, and are thawed immediately before use. Once thawed the panels should never be refrozen, which may result in substantial loss of antimicrobial action and potency. Alternatively, the antimicrobial agents may be lyophilized or freeze dried with the medium or drug in each well; upon inoculation with the bacterial suspension, the medium and drug are simultaneously reconstituted to the appropriate concentration.

Inoculation and Incubation. Standardized bacterial suspensions that match the turbidity of the 0.5 McFar land standard (i.e., 1.5 × 10⁸ CFU/mL) usually serve as the starting point for dilutions ultimately achieving the required final standard bacterial concentration of 5 × 10⁵ CFU/mL in each microtiter well. It is essential to prepare the standard inoculum from a fresh, overnight, pure culture of the test organism. Inoculation of the microdilution panel is accomplished using manual or automated multiprong inoculators calibrated to deliver the precise volume of inoculum to each well in the panel simultaneously (see Figure 1).

Inoculated trays are incubated under optimal environ mental conditions to optimize bacterial growth without interfering with the antimicrobial activity (i.e., avoiding environmentally mediated results). For the most commonly tested bacteria (e.g., Enterobacteriaceae, P. aeruginosa, staphylococci, and enterococci), the environmental condition consists of room air at 35°C (see Table 1). Fastidious bacteria, such as H. influenzae, require incubation in 5% to 10% carbon dioxide (CO2). Similarly, incubation durations for some organisms may need to be extended beyond the usual 16 to 20 hours (see Table 1). However, prolonged incubation times beyond recommended limits should be avoided, because antimicrobial deterioration may result in false or elevated resistance patterns. This is a primary factor that limits the ability to perform accurate testing with some slow-growing bacteria.

Reading and Interpretation of Results. After incubation, the microdilution trays are examined for bacterial growth. Each tray should include a growth control that does not contain antimicrobial agent and a sterility control that was not inoculated. Once growth in the growth control and no growth in the sterility control wells have been confirmed, the growth profiles for each antimicrobial dilution can be established and the MIC determined. The detection of growth in microdilution wells is often augmented through the use of light boxes and reflecting mirrors. When a panel is placed in these devices, bacterial growth, manifested as light to heavy turbidity or a button of growth on the well bottom, is more reliably visualized (Figure 2).

Fig2. Bacterial growth profiles in a broth microdilution tray. The wells containing the lowest concentration of an antibiotic that completely inhibits visible growth (arrow) are recorded in micrograms per milliliter (µg/mL) as the minimal inhibitory concentration (MIC). 

When the dilution series for each antibiotic is inspected, the microdilution well containing the lowest drug concentration that completely inhibits visible bacterial growth is recorded as the MIC. Once the MICs for the antimicrobials in the test battery for an organism have been recorded, they are usually translated into one of the interpretive categories, specifically susceptible, intermediate, or resistant (Box 1). The interpretive criteria for these categories are based on extensive studies that correlate the MIC with serum-achievable levels for each antimicrobial agent, particular resistance mechanisms, and successful therapeutic outcomes. The interpretive criteria for an array of antimicrobial agents are published in the CLSI M07 series document, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (M100 supplements).” For example, using these standards, an isolate of P. aeruginosa with an imipenem MIC of less than or equal to 4 µg/mL would be classified as susceptible; one with an MIC of 8 µg/mL would be classified as intermediate; and one with an MIC of 16 µg/mL or greater would be classified as resistant to imipenem.

Box1. Definitions of Susceptibility Testing  Interpretive Categories *

After the MICs are determined and their respective and appropriate interpretive categories assigned, the laboratory may report the MIC, the category, or both. Because the MIC alone will not provide most physicians with a meaningful interpretation of data, either the category result with or without the MIC is usually reported.

In some settings, the full range of antimicrobial dilutions is not used; only the concentrations that separate the categories of susceptible, intermediate, and resistant are used. The specific concentrations that separate or define the different categories are known as breakpoints, and panels that only contain these antimicrobial concentrations are referred to as breakpoint panels. In this case, only category results are produced; precise MICs are not available, because the full range of dilutions is not tested.

Advantages and Disadvantages. Broth dilution methods provide data for both quantitative results (i.e., MIC) and qualitative results (i.e., category interpretation). Whether this is an advantage is the subject of debate. On one hand, the MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to treat a patient with a specific antimicrobial agent. For example, the penicillin MIC for S. pneumoniae may determine whether penicillin or alternative agents will be used to treat a patient with meningitis. On the other hand, for most antimicrobial susceptibility testing methods, a category report is sufficient and the actual MIC data are superfluous. This is one reason other methods (e.g., disk diffusion) that focus primarily on producing interpretive categories have been maintained among clinical microbiologists.

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