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Antibiotics are chemicals that a

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									Exercise 8. Antibiotics and the Kirby Bauer Disk Diffusion Method Laboratory Techniques in Biological Sciences Biology Unit-Philippine Science High School-Main Campus
Antibiotics are chemicals that are produced by living organisms which, even in minute amounts, inhibit the growth of or kill another organism. A simple method using paper discs saturated with the chemicals to be tested can be used to determine the resistance or sensitivity of an organism to different antibiotics. Following inoculation of an agar medium, discs are placed onto a surface of the medium so that the antibiotic will diffuse into the medium. Inhibition of the organism is evident following incubation as a clear region around the disc, called a ‘zone of inhibition’, in which no growth has occurred. Materials: Dyed nutrient agar in petri dish (3 plates/ group) Escherichia coli broth 3 kinds of antibiotics (e.g. Streptomycin, Ampicillin, Gentamicin, Erythromycin, Chloramphenicol, Tetracycline etc.) Spectrophotometer 5 sscrew cap test tubes of sterilized distilled water Distilled water blanks Paper discs Forceps Sterile cotton swabs Aseptic Technique Materials Beaker with 70% alcohol Procedure:

Note- Sterilize the following before proceeding with the experiment (Dyed nutrient agar, petri plates, distilled water in test tubes, paper discs in beakers, wrapped forceps, and wrapped cotton swabs) 1. Prepare the nutrient agar in each petri plate. The group needs three petri plates with dyed nutrient agar. Label and seal the plates and set aside for use later. 2. Measure the OD value of Escherichia coli. The class will use the bacteria in its midlog phase. Please do not forget to calibrate the spectrophotometer using distilled water blanks. Also, subtract the OD value of a clear broth to the OD values of E. coli. 3. Prepare the antibiotics (3 kinds) by mixing 500 mg of any antibiotic to a 10 ml of sterile distilled water in a small beaker. Mix the solution well until the antibiotic is fully dissolved. 4. Paper discs are prepared by cutting filter papers with a puncher to produce circular discs. Prepare at least 20 paper discs. Mark with an abbreviation of the antibiotic (i.e. use the standard abbreviation below) the center of the paper discs (e.g. AM for ampicillin). Six paper discs should be marked with antibiotic 1 abbreviation, another six with antibiotic 2 abbreviation, and the last six with antibiotic 3 abbreviation. Put the paper discs in a small beaker ready for sterilization. 5. As soon as the E. coli cultures are ready to use, dip one swab into the broth of E. coli using aseptic technique. Swab the entire surface of the three plates. Go over the plate at least twice in each direction. Discard the swab in the beaker of disinfectant provided. 6. Immerse six antibiotic 1 paper discs to antibiotic solution 1. Do this to the rest of the paper discs with their corresponding antibiotic solution.

Exercise 8. Antibiotics and the Kirby Bauer Disk Diffusion Method Laboratory Techniques in Biological Sciences Biology Unit-Philippine Science High School-Main Campus
7. Add the antibiotic discs to each plate using forceps. Remove the cover of the petri dish and set the disc on top of the inoculated agar. Gently tap each antibiotic disc onto the surface of the agar using the sterile forceps to assure good contact. The three petri dishes should contain the three types of antibiotics discs with 1 replicate for each type. 8. Incubate the plates at 37°C for 48 hours. 9. Examine each plate for the presence of zones of inhibition. Measure the diameter of each zone in millimeters using the ruler. Tabulate the values properly. Compare the diameter of each zone to the chart to determine if the organism is sensitive or resistant to the antibiotic.

Results of Antibiotic Sensitivities in Plate 1: Organism:
Antibiotic Abbreviation on the disc S10 Zone Diameter Resistant Diameter 11mm 11mm AM Gentamicin GM Erythromycin E Chloramphenicol C Tetracycline Te 14mm 15-18mm 19mm 12mm 13-17mm 18mm 13mm 14-22mm 23mm 12mm 13-14mm 15mm Intermed Diameter 12-14mm 12-14mm Sensitive Diameter 15mm 15mm Sensitive Intermed Resistant

Streptomycin Ampicillin

Exercise 8. Antibiotics and the Kirby Bauer Disk Diffusion Method Laboratory Techniques in Biological Sciences Biology Unit-Philippine Science High School-Main Campus Results of Antibiotic Sensitivities in Plate 2: Organism:
Antibiotic Abbreviation on the disc S10 Zone Diameter Resistant Diameter 11mm 11mm AM Gentamicin GM Erythromycin E Chloramphenicol C Tetracycline Te 14mm 15-18mm 19mm 12mm 13-17mm 18mm 13mm 14-22mm 23mm 12mm 13-14mm 15mm Intermed Diameter 12-14mm 12-14mm Sensitive Diameter 15mm 15mm Sensitive Intermed Resistant

Streptomycin Ampicillin

Results of Antibiotic Sensitivities in Plate 3: Organism:
Antibiotic Abbreviation on the disc S10 Zone Diameter Resistant Diameter 11mm 11mm AM Gentamicin GM Erythromycin E Chloramphenicol C Tetracycline Te Reference: A Laboratory Manual for Microbiology, Third ed., John M Larkin, with some additions by Ruth A. Gyure for WISTR workshop July 2006, Western CT State University. 14mm 15-18mm 19mm 12mm 13-17mm 18mm 13mm 14-22mm 23mm 12mm 13-14mm 15mm Intermed Diameter 12-14mm 12-14mm Sensitive Diameter 15mm 15mm Sensitive Intermed Resistant

Streptomycin Ampicillin


								
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