Untitled

• The aim is to introduce students to methods for evaluating antibiotic activity.
• Objectives include: understanding antibiotic activity evaluation methods, understanding disk diffusion for measuring inhibition zones, classifying bacteria as sensitive, intermediate, or resistant, determining minimum inhibitory concentration (MIC), and determining minimum bactericidal concentration (MBC).

Working Beside a Flame

• Open flames should be lit by a lab expert.
• Flames should never be left unattended.
• Lab work should be conducted close to the flame in an aseptic area.
• Use a beaker to close the flame, with assistance available if needed.
• For any issues, a lab specialist can provide help.

Antimicrobial Susceptibility Testing

• Antimicrobial susceptibility can be assessed using disc diffusion testing.
• Cultures of bacterial strains provided include: Salmonella spp. (B17), Staphylococcus aureus (B3), Escherichia coli (B285), E. coli (G651), Pseudomonas aeruginosa (B287), Klebsiella spp. (B14), and Clostridium perfringens (SDB 2).
• Agar cultures are used to prepare a 0.5 McFarland suspension of each bacterium in sterile saline.
• Suspensions are diluted according to BSAC guidance: for Gram-negative bacteria, the dilution is 1:100; for Gram-positive bacteria, it's 1:10.
• To conduct the procedure for disc diffusion testing, inoculate 100 µl of diluted bacterial suspension onto ISO plates using a sterile swab and allow to dry for 2 minutes.
• Dispense antibiotic-impregnated plastic discs (4 different discs) onto the inoculated ISO agar using a disc dispenser (Oxoid, Cambridge, UK).
• Incubate agar plates overnight at 37°C in air or anaerobically for C. perfringens.
• The inoculum on agar plates should support semi-confluent colony growth after overnight incubation.
• A denser inoculum results in reduced zones of inhibition, while a lighter inoculum has the opposite effect.
• After incubation, measure the zone of inhibition (mm).
• Compare your data with the control zones of inhibition diameters (mm) of Gram-negative bacterial isolates for BSAC disc diffusion susceptibility testing
• The bacterial isolates are either sensitive (S), intermediate (I), or resistant (R) to each of the antimicrobial agents, based on zone of inhibition.
• Measure the zone diameters for each antibiotic (mm) using a ruler and record them in the provided tables.
• It's important to determine whether each bacterial isolate is susceptible, intermediate, or resistant to each antimicrobial agent.
• The results must be recorded in the table alongside the corresponding zone diameter.

Minimum Inhibitory Concentration (MIC) Testing

• MIC is the minimum concentration of an antimicrobial that inhibits the growth of a bacterial pathogen.
• E-testing involves a susceptibility testing methodology with an antibiotic-impregnated test strip.
• When placed on an agar plate inoculated with the test organism, the antimicrobial agent diffuses into the agar, creating an elliptical inhibition pattern.
• The MIC can be read where the growth of the organism intersects the strip, indicating the lowest concentration of the antimicrobial agent.
• Broth dilution MIC testing procedure: prepare a series of test tubes which contain ISO broth, S. aureus cells, and a range of ciprofloxacin concentrations and then also prepare two control tubes:
  • A culture inoculated into drug-free ISO broth ('control', C+).
  • A drug free ISO broth only (C-).
• Incubate overnight at 37°C in air.
• Determine the ciprofloxacin MIC against each strain of S. aureus.
• The MIC is the lowest concentration of ciprofloxacin resulting in no visible growth.
• The presence of visible growth in broth dilution assays should be recorded using '+' for visible growth and '-' for no visible growth and recorded in a logbook.

Minimum Bactericidal Concentration (MBC) Testing

• MBC is the lowest drug concentration needed to reduce bacterial viability by ≥99.9%.
• Determine MBC by sub-culturing MIC tubes lacking visible growth, then calculating viability by inoculating these cultures onto solid agar media.
• Aseptic technique inoculates 0.5 mL of ISO broth from tubes lacking growth after the MIC test into 4.5 mL of fresh ISO broth (10⁻¹ dilution factor).
• Step 1 is repeated to produce a 10⁻² dilution.
• 50 µl of the undiluted culture and each of the dilutions are transferred onto separate ISO agar plates via the spread plate technique.
• The inoculated agar plates are incubated overnight at 37°C.
• The number of colony forming units on each of the three plates is counted to determine the total viable count (TVC)
• TVC equals the average number of cfu multiplied by 1/volume inoculated onto plates, multiplied by 1/sample dilution.
• The percentage kill for each concentration of ciprofloxacin can be calculated using the initial viable count for each S. aureus from the MIC experiment ('control', C+): % Kill = 100 - [(TVC from antibiotic containing tube / TVC from control) * 100].
• The MBC for ciprofloxacin of each S. aureus strain is then determined.

Coursework Assessment

• Students must write a scientific abstract of 300-350 words on the antimicrobial susceptibility tests conducted in the lab.
• The abstract should include the following sections: Background, Objectives, Methods, Results, and Conclusion.
• A scientific abstract of 300-350 WORDS that details the Background/Objectives, Methods, Results, and Conclusions should be written using data generated in the practical lab and datasets.
• Quantitative data from the experiments should be included in the abstract, written in a passive and scientific style.
• Include five keywords.
• Publications in the Journal of Antimicrobial Chemotherapy provides guidance of the style and structure of the abstract.
• The abstract does not need to cover every aspect of the experiment but should not include citations or references.
• Use credible scientific resources, such as review articles, journal articles, or book chapters, format in Times New Roman font (size 12), single space, justified text, and normal margins.
• Use a descriptive title that reflects the study, shows the selected range of data, and describes the conclusions
• Include background to the study, its context, and purpose.
• The introduction from the practical manual must NOT be copied.
• Include the primary objectives of the study, use credible resources, and exclude citations.
• Abstract must have a clear description of the disc diffusion testing method, the MIC, and the MBC.
• Important results/outcomes based on experiments, depth, and breadth should be evident.
• Avoid explaining the purpose of the method.
• Add percentages, values, and units.
• The results section should the longest.
• Conclusions should reflect synthesis and analysis.
• Answer objectives set in the introduction.
• Describe wider perspective beyond the experiments.
• Include five keywords to reflect the critical aspects of the study.
• Assessment criteria include presentation, structure and style (20%), content/knowledge (40%) and synthesis, evaluation and/or reflection (40%).

Data example

• Sample: pleural fluid collected.
• From 79-year-old man, with medication of insulin.
• Condition: diabetes, aminoglycosides hypersensitivity results
• Fucidic acid (18 mm), Ampicillin (32 mm), Amoxicillin (37 mm), Azithromycin (12 mm) are tested against the fluid samples.
• Table 2 has data from a minimum inhibitory concentration experiment (mg/L) on Streptococcus pneumonia and Streptococcus agalactiae.
• Table 3 presents data regarding a minimum bactericidal concentration (MBC) experiment.
• TVC (CFU/ml) values are listed of the total viable count
• A formula can be used to determine the MBC of norfloxacin antibiotic for the provided bacterial strains.
• The formula is % Kill = 100 - [(TVC from antibiotic containing tube/TVC from control) * 100].