DETERMINATION OF COMPOUND ACTIVITY ANTIMICROBIAL TOOLS AND MATERIALS • Petri dishes, test tubes, Oose, 25mL and 10mL measuring flasks • Incubator, caliper, vortex, stopwatch, metal sparer, spectrophotometer, pH meter • Spirtus, cotton swab, sterile distilled water, standard phenol, paper discs, Mc Farland III • Test bacterial suspension: Staphylococcus aureus, Escherichia coli, Bacillus subtillis, Pseudomonas aeruginosa • Nutrient Ager (NA), Nutrient Broth (NB), physiological NaCl 0.9%, buffer solution pH 8.0 (diluent solution), standard chloramphenicol. • Test antibiotics, Test disinfectants, Test samples EXPERIMENT PROCEDURE PAPER DISK DIFFUSION 1. Pour 20 ml of liquid NA at a temperature of 40-50oC into each petri dish, then let it sit until it solidifies. 2. Using a sterile cotton swab, smear the test bacterial suspension over the entire surface of the petri dishes until evenly distributed. Leave it for approximately 1 hour. 3. Place the antibiotic discs on the agar surface at such a distance that it is hoped that there will be no accumulation of inhibition zones. 4. Incubate all petri dishes at 37oC for 18-24 hours. Measure the zone of inhibition that occurs using a caliper. PHENOL COEFFICIENT Media Creation Nutrient Broth was put into 12 test tubes measuring 20 x 150 mm, the volume of each was made 5 ml. The composition per liter consists of 10 g peptone, 5 g meat extract, and 5 g NaCl; Final pH 6.8. Making Inoculum Pseudomonas aeruginosa or Staphylococcus aureus bacteria had previously been grown on slanted nutrient agar (Nutrient Agar) and incubated at 37°C for 24-48 hours. The test bacteria dilution stage is as follows: 1. Prepare a test tube containing 2 ml of 0.9% physiological NaCl 2. Transfer the P. aeruginosa or S. aureus culture (choose one) into the NaCl solution with a cycle, and equalize the turbidity with Mc Farland III solution (109 microbes/mL) 3. The bacterial suspension is now estimated to contain 109 microbes/mL 4. Prepare 3 test tubes each containing 4.5 ml of 0.9% physiological NaCl 5. Pipette 0.5 mL of the previous microbial suspension (109 microbes/mL), transfer to one of the test tubes containing 4.5 ml of NaCl. The microbial suspension now concentrates 108 microbes/mL 6. Perform a second dilution by taking 0.5 ml of microbial suspension 108 and transferring it to a second tube containing 4.5 NaCl.The microbial suspension now concentrates 107 microbes/mL 7. The final dilution is carried out by transferring 0.5 mL of the microbial suspension 107 into the final tube of NaCl. The microbial suspension was equivalent to 106 microbes/ml. This concentration of bacterial suspension will be used to carry out this practical test. Preparation of Phenol Raw A 5% phenol standard stock solution was prepared by weighing 2.5 g of phenol in 50 ml of sterile distilled water. Then the concentration was diluted to 1:80 by pipetting 12.5 ml of 5% phenol solution added to 37.5 ml of sterile distilled water in a sterile tube measuring 25 x 150 mm. Making Disinfectant Solution Dilution of the disinfectant solution is carried out in sterile tubes measuring 25 x 150 mm. The stages are as follows: • Prepare 4 sterile tubes containing distilled water with different volumes, namely 9 ml, 7 ml, 4.5 ml and 7 ml, in sequence. • Perform the first dilution by pipetting 1 ml of disinfectant solution ontoin 9 ml of distilled water so that the concentration becomes 1:10 • The next dilution is to transfer 1 ml of 1:10 disinfectant into a tube containing 7 ml of distilled water. The disinfectant concentration in this tube is 1:80 • Transfer 0.5 ml of disinfectant 1:80 into 4.5 ml of distilled water so that the concentration now 1:100 • Pipette 0.5 ml of 1:100 disinfectant into a tube containing 7 ml of distilled water so that the concentration in this tube is 1:150 • The disinfectants that will be used next are those with concentrations of 1:80, 1:100, and 1:150. Therefore, equalize the volume of each to 5 ml.