Susceptibility Testing
Susceptibility testing is a crucial laboratory procedure used to evaluate the effectiveness of antimicrobial agents, such as antibiotics, against specific microorganisms, typically bacteria. The process begins by selecting the microorganism of interest, often isolated from a clinical sample like blood or urine. A standardized suspension of the microorganism is prepared, usually adjusted to match a concentration like the 0.5 McFarland standard, to ensure consistency in testing. This suspension is then spread across the surface of an agar plate, where antimicrobial agents are applied in various forms, such as impregnated disks (as in the Kirby-Bauer method), E-test strips, or microdilution wells. The inoculated plates are incubated under controlled conditions to allow the microorganisms to grow and interact with the antimicrobial agents. After incubation, the zones of inhibition—areas where bacterial growth is prevented—are measured to assess the effectiveness of the antimicrobial agents. These measurements are compared against standardized interpretation charts, which classify the microorganism's response as susceptible, resistant, or intermediate. The results of susceptibility testing guide healthcare providers in selecting the most effective antibiotics for treating infections, helping to ensure successful patient outcomes and reduce the spread of antibiotic resistance.
Figure 31 Kirby-Bauer Test showing Zones of Inhibition
Kirby-Bauer Method
The Kirby-Bauer Disk Diffusion Method is a widely used technique for assessing the effectiveness of antibiotics against specific bacterial strains. This method involves placing antibiotic-impregnated paper disks onto an agar plate that has been uniformly inoculated with the bacteria in question. The bacteria are typically prepared in a suspension to match a standardized concentration which ensures that the bacterial density is consistent across tests.
Once the agar plate is inoculated, the antibiotic disks are carefully placed on the surface. The plate is then incubated. During incubation, the antibiotics diffuse outward from the disks, creating a gradient of concentrations in the agar. Bacteria that are susceptible to a particular antibiotic will be unable to grow near the disk, resulting in a clear area known as the zone of inhibition. After incubation, the zones of inhibition around each disk are measured, typically in millimeters. The size of these zones is then compared to standardized charts that categorize the bacteria as susceptible, intermediate, or resistant to the antibiotic. This classification helps determine which antibiotics are likely to be effective in treating infections caused by the bacteria. The Kirby-Bauer method is simple, cost-effective, and provides valuable information for guiding clinical decisions regarding antibiotic therapy.
Antimicrobial Agents
Antimicrobial agents are substances used to kill or inhibit the growth of microorganisms, including bacteria, viruses, fungi, and parasites. These agents play a critical role in treating infections and preventing the spread of diseases. Antimicrobial agents can be either bactericidal/fungicidal (killing the microorganism) or bacteriostatic/fungistatic (inhibiting the growth of the microorganism, allowing the immune system to eliminate the infection). The effectiveness of these agents depends on various factors, including the microorganism's susceptibility, the concentration of the agent, and the site of the infection.
Figure 32 Common Antibiotics and their target bacteria
Antibiotics
These are used to combat bacterial infections. They can work in various ways, such as by disrupting bacterial cell walls (e.g., penicillin), inhibiting protein synthesis (e.g., tetracycline), or interfering with DNA replication (e.g., ciprofloxacin). Antibiotics are specific to bacteria and do not work against viruses or fungi.
Antivirals
These agents are designed to treat viral infections by inhibiting the replication of viruses within the host's cells. Examples include acyclovir for herpes viruses and oseltamivir for influenza.
Antifungals
These drugs target fungal infections by disrupting the cell membrane or interfering with fungal cell processes. Common antifungals include fluconazole, which treats yeast infections, and terbinafine, used for fungal infections of the skin and nails.
Lab Experiment
Objective
In this lab you will evaluate the antibiotic susceptibility of three bacterial species (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) using the Kirby-Bauer disk diffusion method. The experiment aims to determine the effectiveness of various antibiotics, including Penicillin, Tetracycline, Erythromycin, Ciprofloxacin, Streptomycin, Kanamycin, Chloramphenicol, and Novobiocin, by measuring the zones of inhibition (ZOI) around antibiotic disks placed on Mueller-Hinton Agar (MHA) plates. In addition to standard antibiotics, the experiment also seeks to compare the antibacterial efficacy of common household products by soaking sterile disks in these substances and placing them on inoculated MHA plates. The results will provide insight into the resistance patterns of the tested bacteria and the comparative effectiveness of household products against these strains, contributing to a broader understanding of antimicrobial susceptibility.
Materials
· Escherichia coli (Gram-negative)
· Staphylococcus aureus (Gram-positive)
· Pseudomonas aeruginosa (Gram-negative)
· Mueller-Hinton Agar (MHA) plates (3)
· Sterile saline
· Micropipette
· Lawn creation tool
· 8 chambered disk dispenser
· Antibiotic disks (Penicillin, Tetracycline, Erythromycin, Ciprofloxacin, Streptomycin, Kanamycin, Chloramphenicol, Novobiocin)
· Spectrophotometer (Spec20) set to 625 nm
· 0.5 McFarland standard
· Sterile forceps
· Ethanol
· Bunsen burner
· Incubator set to 35°C ± 2°C
· Metric ruler
· Hydrogen peroxide
· Test chemicals for comparison (with known active ingredients)
Kirby-Bauer Protocol
1. Preparation of Bacterial Suspensions
a. Suspend bacterial colonies in sterile saline to match a 0.5 McFarland standard.
i. Using a sterile loop or pipette, collect a small amount of the bacterial culture from an overnight agar plate or broth culture.
ii. Transfer the bacteria into a tube containing sterile saline. The initial volume of saline should be around 4-5 mL.
iii. Mix the suspension thoroughly using a vortex mixer or by gently shaking the tube until the bacterial cells are evenly distributed.
b. Measure the absorbance of each suspension using a Spectrophotometer at 625 nm, the absorbance of each suspension should be between 0.08 and 0.1.
i. Place a cuvette filled with sterile saline (blank) into the Spec20 spectrophotometer to calibrate it to zero absorbance at 625 nm.
ii. Place your bacterial suspension into the Spec20 and measure the absorbance at 625 nm. The goal is to achieve an absorbance reading between 0.08 and 0.1, which corresponds to a 0.5 McFarland standard.
1. If the absorbance is too high (above 0.1), dilute the suspension by adding more sterile saline, mix well, and re-measure the absorbance.
2. If the absorbance is too low (below 0.08), add more bacteria from the culture, mix well, and re-measure the absorbance.
2. Inoculation of MHA Plates
a. Label three MHA plates with the names of the test bacteria.
b. Using a micropipette transfer 100μl of the bacterial suspension to the appropriate MHA plate.
c. Use a sterile lawn creation tool to spread the bacteria across the entire surface of the MHA plate to create a uniform lawn of bacteria.
d. Repeat steps 2.b and 2.c for each bacterial strain.
e. Use the disk dispenser to uniformly stamp 8 different anti-bacterial disks on to each inoculated plate.
f. Place the plates in the incubator at 37 °C for 18-24 hours.
3. Measuring the Zone of Inhibition
a. Identify each antibiotic disk on the agar plate.
b. Start measuring from the center of the disk to the point where bacterial growth resumes, which is marked by a distinct line of dense bacterial growth or a visible change in turbidity compared to the surrounding area. Calculate the diameter by multiplying the measured radius by two.
i. If necessary, measure the ZOI in multiple directions (e.g., vertically and horizontally) to ensure accuracy, especially if the zone is not perfectly circular.
Write down the measured diameter of the ZOI for each antibiotic disk on a piece of paper or directly onto a data sheet.
c. Compare the measured diameter of each ZOI against a standard interpretation chart or table provided by recognized guidelines to determine if the bacteria are susceptible, resistant, or have intermediate susceptibility to the antibiotic.
Compare results with standards
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Common Product susceptibility
1. Preparation of MHA Plates
a. Label three MHA plates with the names of the test bacteria.
b. Quadrant each plate by drawing crossing lines across the bottom of the plate.
i. Label each quadrant with the name of the household product being used, one of the quadrants will be Hydrogen Peroxide (H2O2) to serve as a positive control.
c. Using the same bacterial suspensions in the Kirby-Bauer test, use a micropipette to transfer 100μl of the bacterial suspension to the appropriate MHA plate.
d. Use a sterile lawn creation tool to spread the bacteria across the entire surface of the MHA plate to create a uniform lawn of bacteria.
e. Repeat steps 1.b - 1.d for each bacterial strain on respective plates.
2. Placement of Household Product Disks:
a. Choose three common household products.
b. Identify the active ingredients of each product.
c. Using sterile forceps, submerge one disk into one of the chosen products
d. Place each household product disk onto the appropriate quadrant of the inoculated plates. Ensure disks are spaced sufficiently to avoid overlapping zones of inhibition.
e. Lightly press the disks to ensure contact with the agar surface.
f. Repeat steps, 2.c - 2.e with each product used
g. Place a disk soaked in hydrogen peroxide as the positive control on each plate.
h. Place the plates in the incubator at 37 °C for 18-24 hours.
3. Measuring the Zone of Inhibition
a. Identify each product disk on the agar plate.
b. Start measuring from the center of the disk to the point where bacterial growth resumes, which is marked by a distinct line of dense bacterial growth or a visible change in turbidity compared to the surrounding area. Calculate the diameter by multiplying the measured radius by two.
i. If necessary, measure the ZOI in multiple directions (e.g., vertically and horizontally) to ensure accuracy, especially if the zone is not perfectly circular.
Write down the measured diameter of the ZOI for each antibiotic disk on a piece of paper or directly onto a data sheet.
c. Evaluate the effectiveness of each household product against the bacterial strains.
i. Lookup and compare the measured ZOI with standard values to determine bacterial susceptibility (resistant, intermediate, or susceptible).
1. Search for the product or active ingredient along with the bacterial type for susceptibility.
Compare results against standards
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Review Questions
1. What is the purpose of the Kirby-Bauer disk diffusion assay?
2. Explain why Mueller-Hinton agar is used in the Kirby-Bauer assay.
3. Compare the expected zones of inhibition for Gram-positive and Gram-negative bacteria when exposed to broad-spectrum antibiotics.
4. Why might Pseudomonas aeruginosa exhibit broader resistance to antibiotics compared to other bacteria?
5. Design an experiment using the Kirby-Bauer method to test the effectiveness of a new antibacterial agent. Include controls and variables.
6. Discuss the effectiveness of common household products compared to hydrogen peroxide (H2O2) as a control.
7. Analyze differences in susceptibility among bacterial strains and products.
8. Consider practical implications for antimicrobial use in household settings.
9. If you observed no zone of inhibition around an antibiotic disk for a bacterial strain, what could be some possible explanations?
10. Based on the results from the Kirby-Bauer assay, how would you determine the best antibiotic to prescribe for a urinary tract infection caused by E. coli?