ANTIBIOTIC SENSITIVITY TEST : DETERMINATION OF MIC TOOLS AND MATERIALS • Tools: mortar and stampler, large and small test tubes, tube rack, 1 mL and 10 mL volume pipettes, sterile petri dishes, 100 mL measuring flasks, Oose, spirit and incubator. • Materials: Test preparations, various gram-positive and gram-negative bacterial suspensions, double strength nutrient broth (NB), nutrient broth (NB), test preparation solvent, distilled water, nutrient agar (NA). EXPERIMENT PROCEDURE DILUTION METHOD 1. Put the test preparation into a measuring flask, dissolve it with a little solvent.Then add sterile distilled water up to the mark. If the test preparation is solid, grind it first in a mortar, before placing it in a measuring flask. 2. Plan the dilution and calculate the concentration of the mixture for each each large tube and small tubes. 3. Make graded dilutions of the test solution with distilled water in tubes big reaction. 4. Fill the first small test tube with 1 mL NB Double Strength, meanwhile subsequent test tubes with 1 mL of regular NB. 5. Pipette 1 mL of the final dilution into tube 1 containing NB double strength, shake until homogeneous. 6. Pipette 1 mL of the mixture from tube 1 to tube 2, shake until homogeneous. 7. Repeat these steps until the last tube. Discard 1 mL of mixture from the last tube. 8. Add 1 dose of bacteria into each small tube, shake until homogeneous. 9. Repeat steps 3 to 7 for the other bacteria. 10. Create positive controls and negative controls. The positive control consists of 1 mL of NB and 1 dose of bacteria (one of them only). The negative control contained only 1mL of NB 11. Incubate all small tubes at 37oC for 18-24 hours. Observe the turbidity that occurs, compare it with the positive and negative controls. 12. Determine where the KHM is. MIC is located in the last clear tube or before the first turbid tube. DIFFUSION METHOD 1. Put the test preparation into a measuring flask, dissolve it with a little solvent. Then add sterile distilled water up to the mark. If the test preparation is solid, grind it first in a mortar, before placing it in a measuring flask. 2. Plan the dilution and calculate the concentration of the mixture in each large tube and petri dish. 3. Make graded dilutions of the test preparation solution with deep distilled water large test tubes. 4. Divide the bottom surface of the cup into areas of equal size. Give it a name tag bacteria to be used in each area. 5. Pipette 1 mL of each dilution into petri dishes, add 19 mL of liquid NA at a temperature of 40-50oC, shake for a few moments, then let sit until it solidifies. 6. Scratch each bacteria in a separate area using a brush. Create a positive control that occurs from 20 mL of NA in a petri dish, streaked by the bacteria used in a separate area. 7. Incubate all petri dishes at 37oC for 18-24 hours. Observe the growth of bacteria from visible colonies. Compare the morphology of the colonies with the positive control. 8. Determine where the KHM is. MIC was located in the last petri dish where no bacterial colonies were visible.