GeNei TM. Antibiotic Sensitivity. Teaching Kit Manual KT Revision No.: Bangalore Genei, 2007 Bangalore Genei, 2007

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1 GeNei Bacterial Antibiotic Sensitivity Teaching Kit Manual Cat No. New Cat No. KT Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 4 Materials Provided 5 Procedure 6 Result 9 Appendix 10 Ordering Information 11 1

3 Objective: To determine sensitivity of bacteria to various antibiotics. Principle: The principle of antibiotic sensitivity test is based on the Bauer-Kirby disc diffusion method. It is a standard qualitative test wherein the bacterial culture is spread onto the surface of Mueller-Hinton agar, followed by addition of antibiotic impregnated discs to the agar surface. The antibiotic diffuses through the agar to form a concentration gradient. This concentration gradient influences the growth of the bacterial strain. If an organism is susceptible to an antibiotic, a clear zone appears around the disc where the growth has been inhibited, called the zone of inhibition. If resistant, no clear zone of inhibition appears. The diameter of the zone of inhibition surrounding the antibiotic disc is measured to determine whether the micro-organism is sensitive (S), intermediately sensitive (I) or resistant (R) to a particular antibiotic. The size of zone of inhibition depends on: The rate of diffusion of the antibiotic through agar and The concentration of the antibiotic present in the disc. Hence, by determining the susceptibility of a pathogen, clinicians can select the most appropriate agent for treating the disease. The test also helps in studying microbial strains. 2 3

4 Kit Description: Using this kit, students will perform antibiotic sensitivity tests for three different strains - Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Five different antibiotic discs- Chloramphenicol, Tetracycline, Kanamycin, Gentamycin and Vancomycin are provided against which the sensitivity of each of the strains will be tested. By measuring the diameter of zone of inhibition, students will determine the sensitivity of each of the strains against the five antibiotics using the zone size interpretative chart. Zone Size Interpretative Chart: Antimicrobial Conc. Resistant Intermediate Sensitive Agent (mg) (mm) (mm) (mm) Chloramphenicol Tetracycline Kanamycin Gentamycin Vancomycin KT68 : The kit is designed to carry out 5 antibiotic sensitivity tests. Three different strains will be tested against five antibiotics each. Duration of experiment: Experiment is carried out over a span of 3 days, approximate time taken on each day is indicated below: Day 1: 2 hours (Preparation of media and Revival of strains) Day 2: 8 hours (Bacterial antibiotic sensitivity test) Day 3: 1 hour (Result) Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Quantity Materials KT68 Store (5 expts.) Antibiotic discs Chloramphenicol 20 discs Tetracycline 20 discs Kanamycin 20 discs 4 C Gentamycin 20 discs Vancomycin 20 discs Lyophilized Strains Escherichia coli 1 vial Staphylococcus aureus 1 vial 4 C Pseudomonas aeruginosa 1 vial Cotton Swab (to be sterilized) 20 Nos. RT Mueller- Hinton media 15 g RT Agar 10 g RT Materials Required: Equipment : Incubator (37 C), Shaker. Glassware : Conical flask, Measuring cylinder, Petri plates, Test tubes. Reagent : Distilled water. Other Requirements: Forceps, Micropipette, Tips. 4 5

5 Note: Read the entire procedure before starting the experiment. All microbiological operations should be done strictly under aseptic conditions. Revive the strains as soon as the lyophilized vials are opened. Take care not to cross contaminate the strains while reviving them. Carry out 5 experiments within 2 weeks of reviving the strain. Sterilize the cotton swabs prior to starting the experiment. Flame the forceps before placing each of the antibiotic discs, on the MH agar plate. For preparation of media, refer appendix. Procedure: Day 1: Revival of strains 1. Prepare six Mueller-Hinton (MH) agar plates. 2. Label the MH agar plates as E.coli, S. aureus and P. aeruginosa (in duplicates). 3. Break open the lyophilized vials. Rehydrate the E.coli, S. aureus and P. aeruginosa strains with 0.1 ml of Mueller-Hinton broth. Note: Revived strains can be stored at 4 C for about 2 weeks. Handle these master plates with care as they have to be used for the subsequent four experiments. 4. Streak the three strains onto the respective labeled MH agar plates. 5. Incubate the plates overnight at 37 C. Day 2: Antibiotic sensitivity test 6. Take three sterile test tubes. Add 5ml of MH broth into each and label them as E. coli, S. aureus and P. aeruginosa. 7. Pick single colony of each strain from the MH agar plates and inoculate into the respective labeled test tubes. 8. Incubate the test tubes at 37 C for 6-8 hours (in a shaker). 9. Label another set of three MH plates as E. coli, S. aureus and P. aeruginosa. 10. Take a sterile cotton swab and dip it into the E. coli culture test tube. Gently press the cotton swab against the side of test tube to drain out excess inoculum. 11. Spread the E. coli culture with the cotton swab onto the E. coli labeled MH agar plate. Ensure even distribution of the inoculum over the agar surface. 12. Repeat steps 10 & 11 for S. aureus and P. aeruginosa strains. 13. Allow the agar surfaces to dry for 5 minutes. 14. Pick the Chloramphenicol antibiotic disc supplied, with a sterile forceps and place it on the agar surface of the E. coli plate. Press the disc gently with the forceps to ensure firm contact with the agar surface. 15. Similarly place the Tetracycline, Kanamycin, Gentamycin and Vancomycin antibiotic discs onto the E. coli plate. (Refer fig. 1) 6 7

6 16. Repeat steps 14 & 15, to place the antibiotic discs onto S. aureus and P. aeruginosa plates. Note: Use a different pair of forceps for each strain. 17. Invert and incubate all plates overnight at 37 C. 18. Observe the plates for zone of inhibition surrounding the disc. 19. Measure the zone of inhibition using a ruler on the underside of the petri plate and tabulate your results as follows: Antibiotics Chloramphenicol Tetracycline Kanamycin Gentamycin Vancomycin Zone of Inhibition (in mm) E. coli S. aureus P. aeruginosa Result: Compare the results obtained with that of standard (zone size interpretative chart) to determine the sensitivity of each of the organism to each of the five antibiotics and record the results as follows: Antibiotics Chloramphenicol Tetracycline Kanamycin Gentamycin Vancomycin Denote as follows: S : Sensitive I : Intermediately sensitive R : Resistant Organism E. coli S. aureus P. aeruginosa Bacterial lawn Zone of inhibition Antibiotic disc Fig 1: Antibiotic sensitivity testing plate 8 9

7 Appendix: Preparation of Muller-Hinton broth: Suspend 21 g of MH powder in one litre of distilled water and warm gently to dissolve. Sterilize at 121ºC for 15 minutes using an autoclave. Preparation of MH Agar: Add 15 g of agar to 1000 ml of MH broth, autoclave to sterilize it. Following aliquots of media are required for one experiment (Excludes preparation of media for master plates): MH Broth 3 x 5 ml MH Agar 60 ml Ordering Information Product Size Cat # Bacterial Antibiotic 1 Pack KT68 Sensitivity Teaching Kit (Consumables for 5 experiments) Sales: Customer Support: 10 11

8 Notes: 12 13

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