Disc Diffusion Susceptibility Methods

Transcription

1 Disc Diffusion Susceptibility Methods Introduction When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a zone of inhibition. Principle Antiseptics, disinfectants and antibiotics are used in different ways to combat microbial growth. Antiseptics are used on living tissue to remove pathogens. Disinfectants are similar in use but are used on inanimate objects. Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria. Many antibiotics are chemically altered to reduce toxicity, increase solubility, or give them some other desirable characteristic that they lack in their natural form. Other substances have been developed from plants or dyes and are used like antibiotics. A better term for these substances is antimicrobials, but the term antibiotic is widely used to mean all types of antimicrobial chemotherapy. Many conditions can affect a disc diffusion susceptibility test. When performing these tests certain things are held constant so only the size of the zone of inhibition is variable. Conditions that must be constant from test to test include the agar used, the amount of organism used, the concentration of chemical used, and incubation conditions (time, temperature, and atmosphere). The amount of organism used is standardized using a turbidity standard. This may be a visual approximation using a McFarland standard 0.5 or turbidity may be determined by using a spectrophotometer (optical density of 1.0 at 600 nm). For antibiotic susceptibility testing the antibiotic concentrations are predetermined and commercially available. Each test method has a prescribed media to be used and incubation is to be at o C in ambient air for hours. The disc diffusion method for antibiotic susceptibility testing is the Kirby- Bauer method. The agar used is Meuller-Hinton agar that is rigorously tested for composition and ph. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. There is also a zone of intermediate resistance indicating that some inhibition occurs using this antimicrobial but it may not be sufficient inhibition to eradicate the organism from the body.

2 The standardized methods for antiseptic and disinfectant testing are more rigorous and more difficult to reproduce in a student laboratory. Two common tests are the Phenol Coefficient Test (a comparison of the effect of the chemical and phenol on several organisms) and the Use Dilution Test (testing the chemical under actual conditions of use). A disc diffusion test can be used to approximate the Use Dilution Test. The chemical under consideration is used to saturate a filter paper disc. This disc is then used to introduce the chemical to the agar for testing. The actual zone sizes have not been standardized as in the Kirby-Bauer method, but a comparison of zone sizes for the same chemical among organisms will provide an approximate effectiveness of the chemical. Procedure Kirby-Bauer Antimicrobial Susceptibility Test Organisms to be tested: Staphylococcus aureus Procedure 1. Students will work independently in the laboratory exercise. 2. Obtain a plate culture of one of the organisms to be tested. 3. Using a sterile loop, emulsify a colony from the plate in the sterile saline solution. Mix thoroughly making sure that no solid material from the colony is visible. 4. Repeat this procedure until the turbidity of the saline solution matches that of the standard available for your class. 5. Dip the swab into the broth culture of the organism. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab to streak a Mueller-Hinton agar plate or a nutrient agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. End by using the swab to streak the outside diameter of the agar. 6. Allow the plates to dry for about 5 minutes.

3 7. Antibiotic disks can be placed on the surface of the agar using a dispenser that dispenses multiple disks at the correct distance apart, or by obtaining individual disks and placing them on the surface of the agar using flame sterilized forceps. a. Dispenser method: 1. Obtain the dispenser containing the correct antibiotic disks for the organism you are using. 2. Place the dispenser over the surface of the plate and using the lever/plunger dispense the disks. 3. Using sterile forceps or a loop, gently press the disks onto the surface of the agar, taking care not to press them into the agar. b. Dispensing individual disks: 1. Obtain 6 of the appropriate individual disk dispensers. 2. Using the levers, dispense the disks at equal distances apart on the surface of the agar. 3. Using flame sterilize forceps or a sterile loop gently press the disks onto the surface of the agar disks may also be individually placed onto the surface of the agar using sterile forceps. 8. Invert the plates and incubate for 24 hours at 37 C. 9. Using a metric ruler measure the diameter of the zone of inhibition (if present) for each antibiotic used. 10. Compare the measurement obtained from the individual antibiotics to the table of standards to determine if the bacterial species tested is resistant or sensitive to the antibiotic. 11. Use the data you collected and that of the rest of the class to fill in the table below. Discard the plates in the biohazard container. Antibiotic Staph. aureus

4 Zone Diameter (mm) Interpretation Chart Antibiotic Resistant Intermediate Susceptible Tetracycline = = 19 Ciprofloxacin = = 21 Enoxacin = = 18 Erythromycin = = 23 Penicillin Staphylococci = 28 = 29 Oxacillin Staphylococci = = 13 Tobramycin = = 15 Ceftriaxone = = 21 Kanamycin = = 18 Clindamycin = = 21 Piperacillin Gram negatives = = 21 Ampicillin Gram negative enterics Staphylococci = 13 = = 17 = 29

5 Antiseptic/Disinfectant Susceptibility Test Organisms used Staphylococcus aureus Bacillus cereus Pseudomonas aeruginosa 1. Students work individually on this laboratory exercise. 2. Obtain one of the organisms to be tested, 5 nutrient agar plates, and a sterile swab. 3. Dip the swab into the broth culture of the organism. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab to streak a nutrient agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. End by using the swab to streak the outside diameter of the agar. Repeat this procedure for the remaining plates. 4. Place a disc soaked in an antiseptic or disinfectant in the center of each plate. Be sure to label the plates with the organism and chemical used. 5. Incubate the plates in the standard upside down position until the next lab period. 6. Measure the diameter of the zone of inhibition for each chemical. The class will share data so you can fill in the table provided. 7. Discard the plates in the biohazard container. Chemical Staph. aureus Bacillus cereus Ps. aeruginosa

6 Review Questions What conditions must be held constant when doing disc diffusion procedures? Define Antiseptic Disinfectant Antibiotic Zone of inhibition According to your results, which chemical is the most effective? On what do you base this conclusion? What are the standard tests used for determining the effectiveness of antiseptics and disinfectants? What is the standard method used for antimicrobial susceptibility testing?