MICROBIAL CONTAMINATION TESTS AND TESTS STERILITY TOOLS AND MATERIALS • Nutrient Agar Media (NA), Fluid Thioglycolate Media (FTM), Tryptic Media Soybean Broth (TSB), Sabouraud Dextrose Agar (SDA) Media + chloramphenicol 100 mg/1 liter of media • 0.9% NaCl solution • Sterile petri dishes, sterile test tubes, test tube rack, sterile 10 mL graduated pipette,micropipette and 1mL tips, tissue, alcohol, Bunsen • Samples to be examined. • Suspension of Bacillus subtilis and Candida albicans EXPERIMENT PROCEDURE TEST FOR MICROBIAL CONTAMINATION 1. Melt the medium in a water bath. Once liquid, leave it in a water bath at 45500Cfor approximately 30 minutes. 2. Make a decimal dilution of the sample using 0.9% NaCl sterile, until a 10% dilution is obtained. 3. Mark the petri dish with the name of the medium and the dilution level. 4. Pipette 1 mL of each dilution and place into the appropriate petri dish. 5. Pour in the liquid agar with a temperature of approximately 500C and homogenize by shaking the cup with a clockwise movement 5x and a counterclockwise movement 5x. Make sure it doesn't spill. Do a duplicate for each dilution and also do media control for each medium. Information : NA media for determining the number of aerobic bacteria SDA media for determining the number of molds and yeasts 6. Let it solidify, then incubate at the appropriate temperature. NA media at 370C, observe 1 – 5 days SDA media at a temperature of 200C, observe 1 – 7 days 7. Count the number of bacterial, mold and yeast colonies that grow on the agar plates and express them in cfu/mL units. STERILITY TEST The sterility test includes several testing stages as follows: 1. Media Sterility Test The media sterility test is used to see whether the media to be used is sterile or not. The results obtained must be sterile (the media remains clear/no microbial growth). Method: Put 10 mL of FTM and TSB media into a test tube. Incubate at 370C for FTM and 200C for TSB, observe for 1 -14 days. Do it in duplicate. 2. Fertility Test This fertility test is to see whether the media to be used meets the requirements to grow the test microbes or not. Method: Inoculate 1 mL of the test microbial suspension into 9 mL of media in a test tube. Bacillus subtilis bacteria are inoculated on FTM media and incubated at 35-370C, observed for 1 - 14 days. Candida albicans was inoculated on TSB media and incubated at 20-250C, observed for 1 - 14 days. This work was carried out in duplicate. 3. Bacteriostatic and Fungistatic Tests Before carrying out a sterility test using direct inoculation on a preparation, first determine the level of bacteriostatic and fungistatic activity that may be present in the preparation to be examined. Method: Inoculate 1 mL of microbial suspension and 1 mL of preparation into 9 mL of media in a test tube. FTM media is inoculated with Bacillus subtilis and the preparation, incubate at a temperature of 35-370C, observe for 1 – 14 days. TSB media is inoculated with Candida albicans and the preparation, incubate at a temperature of 20-250C, observe for 1 – 14 days. This work was carried out in duplicate.If the test preparation shows bacteriostatic/fungistatic activity (the test preparation inhibits the growth of the test microbes, which is indicated by the absence of growth of the test microbes/the medium remains clear), then a sterility test is carried out by filtration. By filtering, the bacteriostatic/fungistatic substances contained in the test preparation will pass through the membrane. Meanwhile, if there are microbes in the test preparation, the microbes will be retained on the membrane. This membrane will be inoculated into the media. 4. Sterility Test for Direct Inoculation Method Method: inoculate 1 mL of test preparation into 9 mL of media in a test tube.FTM and TSB media, each inoculated with 1 mL of test preparation.Incubate at 370C for FTM and 200C for TSB, observe for 1 – 14 days. This work was carried out in duplicate.