University of Hertfordshire School of Pharmacy Pharmaceutical Microbiology and Manufacture 2011-12

Pharmaceutical Microbiology and Manufacture Practical (Level 5) Micro Lab 3: Antibiotic activity

INTRODUCTION
The advantages of effective antimicrobial chemotherapeutics are self evident. However, the selection of the most appropriate antibiotic is a significant problem in the management of acute and chronic infection. Evaluation of prescribing trends has determined that more than 50% of the antibiotic prescribing is inappropriate. Bacterial resistance to antibiotics is well established and may be classified as either intrinsic or acquired. Intrinsic (or innate) resistance refers to the inherent properties of the bacterium being responsible for preventing antibiotic action, i.e. Gram-positive and Gram-negative bacteria. Acquired resistance is when previously susceptible bacteria become resistant after exposure to the antibiotic in question by mutation e.g. MRSA. Hence, tests to determine antibiotic susceptibility are important to determine which antibiotic can be used for the treatment of disease. Both qualitative and quantitative tests can be used to determine the type and dose of antibiotic to which bacteria are susceptible.

Aim
The aim of this experiment is to introduce the students to the ways in which the activity of antibiotics can be evaluated.

Learning outcomes

1) Understand different methods for evaluation of antibiotic activity 2) Determine the minimum inhibitory concentration of an antibiotic 3) Determine the minimum bactericidal concentration of an antibiotic 4) Understand the disk diffusion method for measuring zones of inhibition 1|Page

University of Hertfordshire School of Pharmacy Pharmaceutical Microbiology and Manufacture 2011-12

Experimental Methods
Safety

Please refer to the safety instructions and to the microbiology code of practice listed in the PMM module handbook. If you are in any doubt about the safety of any of the chemicals or experimental techniques you are required to use, please contact a member of staff before undertaking any such work. Every microbe encountered must be considered pathogenic, hence all cultures must be considered to be contaminated with a pathogenic microbe. Bags and coats are not permitted in the lab an appropriate attire including a lab coat completely covering your trunk up to your neck must be worn at all times. It is not permitted to touch your face or put anything in your mouth while in the lab. You must keep your space clean, clear and uncluttered, any accidents including spillages, cuts and abrasions must be reported to a member of staff immediately. Before leaving the lab you must swab down your workspace with disinfectant fluid and wash your hands with a suitable germicidal soap. All breaches of safety and professionalism as per the RPSGB student code of conduct are taken very seriously and will result in your ejection from the class and may result in further disciplinary action.

Materials and equipment Sterile test tubes, sterile universal bottles, sterile nutrient broth, penicillin G solution, nutrient agar plates, S. aureus culture NCTC 6571 (reference strain), S. Aureus culture (unknown strain), Antimicrobial susceptibility test system disc dispenser (Oxoid), various antibiotic sensitivity discs, Various other cultures of bacteria.

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University of Hertfordshire School of Pharmacy Pharmaceutical Microbiology and Manufacture 2011-12

PART 1 Minimum Inhibitory Concentration (MIC) The MIC is the lowest concentration of drug required to prevent bacterial multiplication (Bacteriostasis). Measuring the MIC is an extremely common method for determining the antimicrobial activity of a chemotherapeutic agent. Various methods can be employed to measure MIC, but in this practical the MIC will be determined using broth suspensions.

1) Collect the nutrient agar spread plates for the two S. aureus strains (10-5 and 10-6 dilutions) and perform the viable count 2) A series of test tubes containing nutrient agar broth, inoculating bacteria and range of concentrations of the antibiotic penicillin G were previously prepared and incubated over night at 37C. Determine the MIC, which is the lowest concentration that resulted in no visible growth. 3) Two control test tubes were also run to validate the experiment: i) culture inoculated into drug free nutrient broth, ii) Non-inoculated drug free nutrient broth

PART 2 Minimum Bactericidal Concentration (MBC) The MBC is defined as the lowest drug concentration that is capable of reducing bacterial viability by 99.9%. The MBC is typically evaluated by sub-culturing the MIC tubes that do not show any visible growth and then calculating the viability.

1. Using aseptic technique, pipette 0.5 mL of nutrient broth from the tubes lacking growth after the MIC test and inoculate into 4.5 mL of fresh nutrient broth (10-1 dilution factor) 2. Repeat step 1 to produce a 10-2 dilution. 3. Using the spread plate technique, transfer 0.1 mL of the original nutrient broth (neat) and both of the dilutions onto separate plates. 4. Incubate the plates overnight at 37C.

Count the number of colony forming units on each of the three plates (viable count) and determine the MBC for penicillin G. Using the initial viable count from the MIC experiment, calculate the percentage kill for penicillin G. 3|Page

University of Hertfordshire School of Pharmacy Pharmaceutical Microbiology and Manufacture 2011-12

PART 3 Antibiotic sensitivity The antibiotic sensitivity of a range of bacteria will be assessed using the Stokes disc sensitivity method.

1) The Stokes Disc Sensitivity Method involves seeding a bacillus (B30) culture onto nutrient agar plates. 2) An antibiotic is added via a plastic disc or strip using the antimicrobial susceptibility test system disc dispenser (Oxoid, Cambridge, UK). 3) Antibiotics to include Salmonella (B17), S. Aureus (B3), E. coli (B285), P. aeruginosa (B14), proteus vulgaris (B265). 4) Incubate plates over night at 37C 5) After incubation you can measure the zone of inhibition and compare with the standard international tables.

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