13. Containment of Microorganisms: Antibiotic Sensitivity Testing Introduction Antibiotics are chemical compounds produced by microorganisms that inhibit growth or kill other microorganisms. Antibiotics are more specific and limited in the types of organisms that they kill than are antiseptics & disinfectants. For example, a given antibiotic will generally be effective in killing only some bacterial species, whereas antiseptics/disinfectants are lethal to a wide range of microbes. KIRBY-BAUER DISK DIFFUSION METHOD CULTURES To compare how effective one antibiotic is to another, or to measure the degree of antibiotic resistance in a bacterium, a procedure called the Kirby-Bauer test can be done. To do this, a pure culture of bacteria is isolated from an infected person. This pure culture is then spread over the surface of a special medium, called Mueller-Hinton agar, to create a lawn of bacteria. Mueller Hinton is a very rich medium that allows the growth of a large number of bacteria and was adopted as a standard medium for hospitals and research laboratory for comparison purposes. Small filter paper disks, impregnated with standardized amounts of antibiotic, are gently pressed on to the surface of the agar (your group will use a multi-disk dispenser). The plates are incubated overnight while the antibiotic diffuses from the disk into the agar. After incubation, the plates are examined for the presence of zones of inhibition (clear rings around the antibiotic disk). If the chemical agent being tested inhibits the test microbe there will be a clear zone of inhibition (ZI) surrounding the disk where no microbial growth has occurred due to the presence of the agent. If there is no inhibition, growth extends up to the rim of the disks on all sides, and the organism is reported as resistant (R). In general the larger the diameter of the ZI, the more effective the test chemical is. This procedure has been standardized for antibiotics.
Staphylococcus aureus culture tested with several antibiotics http://phil.cdc.gov/phil/home.asp
13-1
The lab guide was adapted from the protocol published by the South East Asia Regional Office of WHO: http://www.searo.who.int/en/Section10/Section17/Section53/Section482.htm Lab report due on ________________ Relationship to Class Instruction: Chapter 21 Purpose of Laboratory Evaluate the effect of several antibiotics on Gram (+) and Gram (-) bacteria using the Kirby Bauer test. Throughout the procedure use aseptic techniques and wash your hands immediately after you are done. Report spills at once. Materials and Methods Materials Staphylococcus epidermidis overnight culture in TSB Escherichia coli overnight culture in TSB Sterile cotton swabs Sterile forceps 2 Mueller Hinton plates per group/2-3 students Disks of antibiotics in dispensers Biohazard bags 1/table Antibiotics Group I (closest to door)
Group II (closest to fume hood)
Ampicillin Chloramphenicol Erythromycin
Cefaclor Penicillin G Streptomycin
Ciprofloxacin
Sulfisoxazole
Tetracycline
Trimethoprim
Sulfisoxasole and trimethoprim act synergistically (interact positively). Place them in adjacent locations. Procedure Special Safety Precautions Discard all biohazard material appropriately: Petri dishes in the biohazard bag Swabs in the biohazard bag Part I
the back to divide each plate into 5 sections.
1. Obtain 2 plates and the cultures of E. coli and S. epidermidis. Draw pie lines on 13-2
2. Fill table 1 with name and concentration of antibiotic. (The concentration is written on the tube and on the disk.)
11. Repeat procedure for S. epidermidis with a new plate. 12. Allow the plates to dry for 2-5 min. Part II
3. Open the sterile envelope and remove the dropper. Be careful not to touch anything with the tip of the dropper.
13. The antibiotic disks are placed on the inoculated plates using a pair of sterile forceps. Remove the forceps from the alcohol beaker and pass through flame of a Bunsen burner. When the alcohol has burned off, use sterile forceps to remove aseptically the disk from the dispenser and place it on each plate. Touch each disk to assure it will not fall when the plate is inverted for incubation. The Instructor will demonstrate proper use of the antibiotic Disc Dispenser.
4. Flame the tube open while holding the dropper. 5. Withdraw about 0.25ml culture (3-4 drops) from tube. Markings are indicated on the stem of the dropper. 6. Close the tube. 7. Pipet 0.25ml culture on the plate. 8. Remove a single swab from autoclaved envelope. Keep the rest of the swab sterile by not opening the envelope more than necessary and refolding the top. Spread culture with swab using a back and forth motion and turning the plate 90째 twice.
14. A maximum of five disks can be placed on a 9-10 cm diameter plate. Five disks may be spaced evenly, approximately 15 mm from the edge of the plate. Each disk should be pressed down gently to ensure even contact with the medium.
9. The goal is to cover the plate with bacterial culture. Cover the dish and let dry 5 to10 minutes before proceeding with part II.
15. Bring your Kirby-Bauer test plate to the front desk after checking it has been labeled properly. The plates will be incubated at 37째C overnight.
10. Discard the swab in the designated beaker.
Results After overnight incubation, the diameter of each zone (including the diameter of the disk) should be measured and recorded in mm. Enter the results in table 1. The results should then be interpreted according to the critical diameters by comparing with standard tables (Table 2), when possible. The measurements can be made with a ruler on the under surface of the plate without opening the lid. The end-point of inhibition is judged by the naked eye at the edge where the growth starts. Zone diameters, to the nearest whole mm for various antimicrobial agents with disk content specified for each one for interpretation as susceptible, intermediate and resistant are given in Table 2. The result of the susceptibility test, as reported to the clinician, is the classification of the microorganism in one of two or more categories of susceptibility. The simplest system comprises only two categories, susceptible and resistant. This classification, although offering many advantages for statistical and epidemiological purposes, is too inflexible for the clinician to use. Therefore, a three-category classification is often adopted. The Kirby-Bauer 13-3
method and its modifications recognize three categories of susceptibility and it is important that both the clinicians and the laboratory workers understand the exact definitions and the clinical significance of these categories. Susceptible: An organism is called "susceptible" to a drug when the infection caused by it is likely to respond to treatment with this drug, at the recommended dosage. Intermediate susceptibility covers two situations. It is applicable to strains that are "moderately susceptible" to an antibiotic that can be used for treatment at a higher dosage because of its low toxicity or because the antibiotic is concentrated at the focus of infection (e.g. urine). The term also applies to those strains that are susceptible to a more toxic antibiotic that cannot be used at a higher dosage. In this situation the intermediate category serves as a buffer zone between susceptible and resistant. Resistant: This term implies that the organism is expected not to respond to a given drug, irrespective of the dosage and the location of the infection. Lab Report Introduction: Describe the use of the Kirby Bauer test in a clinical setting. Mention advantages and drawbacks of the method. Materials and Methods: Describe the bacterial cultures and the antibiotics that were used. Results: Enter diameters of the zone of inhibition in the table provided in the lab results worksheets. Discussion: Compare the diameters that were measured in class with the standard table. Record for each antibiotic and culture whether the bacteria were susceptible, intermediate or resistant. Compare Gram +ve and Gram –ve bacteria susceptibility to the antibiotics tested. Explain your results. Conclusions
13-4