Tests to Guide Antimicrobial Use: Videos & Practice Problems

Antimicrobial susceptibility testing, including the Kirby-Bauer disk diffusion and E test, helps determine bacterial resistance and minimal inhibitory concentration (MIC), the lowest antibiotic concentration that inhibits growth. The broth dilution test further identifies MIC and minimal bactericidal concentration (MBC), the lowest concentration that kills bacteria. These tests guide effective antibiotic use by distinguishing bacteriostatic from bactericidal effects. Understanding zones of inhibition, antibiotic diffusion, and serial dilutions in nutrient broth is essential for interpreting susceptibility, resistance, and optimizing treatment strategies against infections.

Disk Diffusion Methods Including Kirby-Bauer Test

When determining the effectiveness of antimicrobial drugs against infections, it is essential to understand how to identify which drugs will work and the appropriate dosage. Two key concepts in this process are the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). The MIC is defined as the lowest concentration of an antibiotic that inhibits bacterial growth, meaning it stops bacteria from multiplying but does not necessarily kill them. In contrast, the MBC is the lowest concentration of an antibiotic that kills the bacteria outright.

One common method to assess antibiotic effectiveness is the disk diffusion method, often referred to as the Kirby-Bauer test. This test involves spreading bacteria on an agar plate and placing small paper discs impregnated with different antibiotics on the surface. After incubation, bacterial growth appears as a cloudy area, while clear zones around some discs indicate where bacteria could not grow. These clear areas are called the zone of inhibition, representing regions where the antibiotic concentration was sufficient to prevent bacterial growth.

The size of the zone of inhibition reflects the bacteria's susceptibility to the antibiotic: a larger zone indicates greater susceptibility. For example, if the zone around disc C is larger than that around disc B, the bacteria are more susceptible to the antibiotic on disc C. If there is no zone of inhibition, as with disc A, the bacteria are resistant to that antibiotic. However, while the Kirby-Bauer test shows whether an antibiotic inhibits growth, it does not provide the exact MIC because the antibiotic concentration at the edge of the zone is unknown. Additionally, it cannot determine the MBC, as it is unclear whether bacteria within the zone are dead or merely not growing.

To estimate the MIC more precisely, the E-test (or epsilon test) is used. This method employs a plastic strip with a gradient of antibiotic concentrations placed on an agar plate inoculated with bacteria. After incubation, the zone of inhibition forms an ellipse around the strip. The point where bacterial growth intersects the strip corresponds to the MIC, which can be read directly from the scale on the strip, typically expressed in micrograms per milliliter (\(\mu g/mL\)). This allows for a quantitative measurement of the lowest antibiotic concentration that inhibits bacterial growth.

Despite providing an estimate of the MIC, the E-test still does not reveal whether the antibiotic is bactericidal or merely bacteriostatic. Determining the MBC requires additional testing beyond these diffusion methods. Understanding these tests and their limitations is crucial for selecting effective antimicrobial therapies and appropriate dosages to treat infections successfully.

Broth Dilution Test

The broth dilution test is a valuable method for determining both the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of antibiotics against bacterial infections. Unlike simpler tests such as the disc diffusion or E-test, the broth dilution test provides detailed insight into whether an antibiotic merely inhibits bacterial growth (bacteriostatic) or actually kills the bacteria (bactericidal). Some antibiotics can act as bacteriostatic at lower concentrations and bactericidal at higher concentrations, and this test helps distinguish those effects.

The procedure begins by preparing a series of test tubes containing liquid nutrient broth with serial dilutions of the antibiotic. These dilutions range from a high concentration to a low concentration, for example, from 8.0 to 0.25 micrograms per milliliter. Bacteria are then added to each tube and incubated. After incubation, the tubes are observed for turbidity, which indicates bacterial growth. Clear tubes show inhibition of growth, while cloudy tubes indicate bacterial proliferation.

The minimal inhibitory concentration (MIC) is identified as the lowest antibiotic concentration at which no visible bacterial growth occurs. For instance, if tube C with 2.0 µg/mL is the lowest concentration without turbidity, then 2.0 µg/mL is the MIC. However, the MIC does not confirm whether bacteria are killed or just inhibited.

To determine the minimal bactericidal concentration (MBC), samples from the clear tubes (those with no growth) are transferred onto antibiotic-free agar plates. After incubation, the presence or absence of bacterial colonies reveals whether the bacteria were killed. If colonies grow, the antibiotic concentration was only inhibitory; if no colonies grow, the bacteria were killed at that concentration. For example, if no growth occurs from the sample taken from tube B (4.0 µg/mL), then 4.0 µg/mL is the MBC.

This test is more complex than diffusion methods but provides comprehensive data on antibiotic efficacy, distinguishing between bacteriostatic and bactericidal effects. The broth dilution test is essential for precise antibiotic susceptibility testing, especially when determining the appropriate therapeutic dosage to effectively treat infections.

Tests to Guide Antimicrobial Use Example 1

The broth dilution test using a microtiter plate is a common method to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of an antibiotic against bacteria. In this test, equal amounts of broth are added to wells one through eight, with increasing concentrations of antibiotic added to wells three through eight. The antibiotic concentrations are measured in micrograms per milliliter (µg/mL). A bacterial sample is added to wells two through eight, while well one serves as a negative control with no bacteria added.

After incubation, wells that appear cloudy indicate bacterial growth, while clear wells indicate inhibition of growth. The MIC is identified as the lowest antibiotic concentration in the broth that prevents visible bacterial growth. In this case, the MIC corresponds to the concentration in well five, which is 25 µg/mL, since it is the lowest concentration where no cloudiness (growth) is observed. Well one, which has no bacteria, is excluded from this determination.

To determine the MBC, samples from wells without visible growth are plated onto agar without antibiotics and incubated again. This step tests whether bacteria were merely inhibited or actually killed. Growth on these plates indicates surviving bacteria, while no growth indicates bactericidal activity. Here, colonies grew from samples taken from wells five and six, meaning those concentrations were inhibitory but not bactericidal. Wells seven and eight showed no growth on the agar plates, indicating bactericidal effects. The MBC is the lowest concentration that kills bacteria, which is 100 µg/mL in well seven.

Wells one and two serve as controls to validate the experiment. Well one, containing no bacteria and no antibiotic, confirms that the broth is sterile and free from contamination. Well two, containing bacteria but no antibiotic, confirms that the bacteria can grow in the broth under normal conditions. These controls ensure the reliability of the MIC and MBC results.

Understanding MIC and MBC is crucial in microbiology and clinical settings to determine the effective antibiotic dosage needed to inhibit and kill bacterial pathogens. The MIC is the minimal concentration that stops bacterial growth, while the MBC is the minimal concentration that kills the bacteria, both essential for guiding antibiotic therapy.

Based on the image, which drug do you think would be most effective at inhibiting the bacteria on the agar?

• A. 1
• B. 2
• C. 3
• D. 4

Answer: (C)

In the image, drugs 1, 3, & 4 are streptomycin, polymyxin B, & imipenem, respectively. Drugs 2, 5, & 6 are penicillin G, bacitracin, & vancomycin, respectively. Knowing that, what can you reasonably conclude from this test?

• A. A broad-spectrum drug would be the most effective at treating this infection.
• B. Drugs that target the cell wall are the most effective against this bacterium.
• C. Drugs 3 and 4 work synergistically.
• D. The bacterium on the plate is likely a Gram-negative bacterium.

Answer: (D)

What can an E-test tell you that a standard Kirby-Bauer test cannot?

• A. An E-test can tell you whether a particular antibiotic is effective against a particular bacterium.
• B. An E-test can tell you the MIC.
• C. An E-test can tell you the MBC.
• D. An E-test can tell you both the MIC & the MBC.

Answer: (B)

Here’s what students ask on this topic:

What is the Kirby-Bauer disk diffusion test and how does it determine antibiotic effectiveness?

The Kirby-Bauer disk diffusion test is a method used to evaluate the effectiveness of antibiotics against bacteria. In this test, bacteria are spread on an agar plate, and small paper discs containing different antibiotics are placed on the surface. After incubation, bacteria grow except where the antibiotic inhibits them, creating a clear zone called the zone of inhibition. The size of this zone indicates how susceptible the bacteria are to the antibiotic—the larger the zone, the more effective the drug. However, this test does not provide the exact minimal inhibitory concentration (MIC) or indicate whether the antibiotic kills the bacteria (bactericidal) or just inhibits growth (bacteriostatic).

How does the E test differ from the Kirby-Bauer test in determining antibiotic susceptibility?

The E test is a modified version of the Kirby-Bauer test that uses a plastic strip with a gradient of antibiotic concentrations. When placed on an agar plate inoculated with bacteria, it creates an elliptical zone of inhibition. The point where bacterial growth intersects the strip indicates the minimal inhibitory concentration (MIC), which is the lowest antibiotic concentration that inhibits bacterial growth. Unlike the Kirby-Bauer test, the E test provides a quantitative MIC value, helping to guide precise antibiotic dosing. However, like the Kirby-Bauer test, it does not determine if the antibiotic is bactericidal or bacteriostatic.

What is the broth dilution test and how does it determine both MIC and MBC?

The broth dilution test involves adding serial dilutions of an antibiotic to liquid growth media containing bacteria. After incubation, tubes are checked for bacterial growth indicated by cloudiness. The minimal inhibitory concentration (MIC) is the lowest antibiotic concentration that prevents visible growth. To determine the minimal bactericidal concentration (MBC), samples from tubes without growth are plated on antibiotic-free media. If bacteria grow, they were inhibited but not killed; if no growth occurs, the bacteria were killed. The MBC is the lowest concentration that kills the bacteria. This test provides detailed information on whether an antibiotic is bacteriostatic or bactericidal but is more complex and time-consuming than diffusion tests.

Why is it important to distinguish between bacteriostatic and bactericidal antibiotics in treatment?

Distinguishing between bacteriostatic and bactericidal antibiotics is crucial for effective treatment. Bacteriostatic antibiotics inhibit bacterial growth, allowing the immune system to eliminate the infection, while bactericidal antibiotics kill bacteria directly. In severe infections or immunocompromised patients, bactericidal drugs may be preferred to ensure rapid bacterial clearance. Understanding whether an antibiotic is inhibitory or lethal helps clinicians choose the right drug and dosage, optimizing therapy, reducing resistance development, and improving patient outcomes.

What are the advantages and limitations of the Kirby-Bauer and broth dilution tests?

The Kirby-Bauer test is simple, cost-effective, and provides quick results on bacterial susceptibility by measuring zones of inhibition. However, it only offers qualitative data and does not provide exact MIC values or distinguish between bacteriostatic and bactericidal effects. The broth dilution test, while more complex and time-consuming, provides quantitative MIC and MBC values, allowing precise determination of antibiotic effectiveness and whether bacteria are killed or just inhibited. This detailed information guides optimal antibiotic selection but requires more resources and expertise, limiting its routine use in some clinical settings.