ANTIMICROBIAL SUSCEPTIBILITY TESTING

ANTIBIOTIC FILTER-PAPER DISC AGAR DIFFUSION TECHNIQUE : Kirby-Baeur Method MINIMAL INHIBITORY CONCENTRATION (MIC):Tube Broth Dilution Method

For some microorganisms, susceptibility to chemotherapeutic agents is predictable. However, for many microorganisms (Pseudomonas, Staphylococcus aureus, and gram-negative enteric bacilli such as Escherichia coli, Serratia, Proteus, etc.) there is no reliable way of predicting which antimicrobial agent will be effective in a given case. This is especially true with the emergence of many antibiotic-resistant strains of bacteria. Because of this, antibiotic susceptibility testing is often essential in order to determine which antimicrobial agent to use against a specific strain of bacterium. Experiment 1

Disc Diffusion Technique (Kirby-Baeur Method) A standardized procedure commonly used in clinical labs to determine antimicrobial susceptibility is the Kirby-Bauer disc agar diffusion method. In this test, the in vitro response of bacteria to a standardized antibiotic impregnated filter-paper disc has been correlated with the clinical response of patients given that drug. Materials: Culture plate Sterile tryptone water Mueller Hinton agar Antibiotic discs

1. You are provided with the organisms cultured on nutrient agar and sterile tryptone water. 2. Emulsify a few colonies of the test organism into tryptone water. O.D of 0.1 at 600nm. 3. Incubate at 37 C for 15 minutes. 4. After incubation, by using sterile L-shaped spreader, spread the test organism evenly onto the whole surface of the Muller Hinton agar. 5. Allow the inoculum to dry at room temperature for a few minutes with the petri dish lid in place. 6. Using sterile forceps or needle, place the four antibiotic discs provided onto the surface of Muller Hinton Agar at equal distance (See figure). Gently press each disc down to ensure good contact with agar surface. 7. Incubate at 37 C overnight. 17 | P a g e

8. The next day, measure the diameter of the inhibition zone surrounding each antibiotic to the nearest mm and compare with the standard table of the inhibition zone size given. 9. The result should be reported as sensitive, intermediate or resistant.

Abbreviation of antibiotics used : AMP = Ampicilin (10g) TE CN CIP = = = Tetracycline (30g) Gentamicin (10g) Ciprofloxacin (5g)

Antibiotic disc

Muller-Hinton Agar

Figure: 4 antibiotic discs arrangement

Zone diameter (in mm)

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Antibiotics Ampicilin Tetracycline Gentamicin (AMP 10g) (TE 30g) (CN 10g)

Resistant 13 14 12 15

Intermediate 14 - 16 15 - 18 13 - 14 16 - 20

Sensitive 17 19 15 21

Ciprofloxacin (CIP 5g)

Standard table of the inhibition zone

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Experiment 2 Minimal Inhibitory Concentration (MIC) - Tube Broth Dilution Method

In this test, a series of culture tubes are prepared, each containing a liquid medium and a different concentration of antibiotic. The tubes are then inoculated with the test organism and incubated for overnight at 37 C. After incubation, the tubes are examined for turbidity (growth). The lowest concentration of antibiotic capable of preventing growth of the test organism is the minimum inhibitory concentration (MIC). Materials: Gentamicin solution (40 g/ml) Sterile test tubes Micropipettes Overnight broth culture (Staphylococcus aureus) Method: 1. 2. Set up 2 rows of 8 sterile test tubes each. Do serial doubling dilution of the stock gentamicin solution in the sterile test tubes to give concentrations of 40, 20,10, 5 . down to 0.3 g/ml. 3. The procedure is as follow: Fill up all the first row of the test tubes except tube no. 1 with 1.0 ml of sterile distilled water. Then add 1.0 ml of the stock gentamicin solution (40 g/ml) to the first and second test tube. Mix well, and then transfer 1.0 ml of the mixture from the second to the third tube. Follow the same step, until the last tube and discard the last 1.0 ml. 4. 5. For the second row of the test tubes, add 0.9 ml nutrient broth to all of the test tubes. Dispense 0.1 ml of the gentamicin 40 g/ml into the first tube (4ug/ml) and 0.1 ml of each gentamicin dilution to the corresponding nutrient broth tubes. 6. 7. 8. Inoculate 50ul of an overnight broth culture into each tube. Mix well. Incubate all the tubes at 37 C overnight. After overnight incubation, the tube containing the lowest concentration (highest dilution) of the antibiotic which does not show any turbidity represents the MIC. 9. ** Subculture all the tubes that show no visible growth (no turbidity) onto blood agar plate. Incubate all the plates at 37 C overnight to determine the Minimum Bactericidal Concentration (MBC). ( ** This step is not performed in this practical.)

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