Preservative Cativy Test - Lecture Note

PRESERVATIVE ACTIVITY TEST TOOLS AND MATERIALS Biohazard cabinet equipment , Autoclave, Oven, Incubator temperature (35 ± 2)oC and (25 ± 2)oC, Vortex mixer, Glass beads, scale measuring pipette, Petri dish, Hemocytometer and microscope available), Spectrophotometer/colorimeter (if available), Colony counter, Top-loading scale, pH meter(IfMedia Dry media with equivalent function, which has been tested for sterility and ability to grow appropriate standard microbes, consists of: • Nutrient Agar or equivalent (Tryptic Soy Agar/TSA) • Letheen Agar or Tryptic Soy Agar + 1% Tween 80 (TSAt) • Mycophil Agar pH 4.7 or Potato Dextrose Agar + antibiotics(PDAa)/SDAa or Sabouraud Dextrose Agar + 1% Tween 80 (SDAt) Letheen Broth or Peptone Saline + 1% Tween 80 • Chloride Buffer Solution or equivalent • Sterile Diluent 1 Solution (0.1% Peptone in 0.9% NaCl) • Sterile Diluent 2 Solution (0.05% Tween 80 in 0.9% NaCl) • Mc.Farland (BaSO4) no.2 (if available) Test Microbes Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans,Enterobacter aerogenes, Aspergillus niger EXPERIMENT PROCEDURE MAINTENANCE OF MICROBIAL TESTS Test microbes are maintained on appropriate media: • Bacteria with Nutrient Agar (TSA or equivalent) • Molds and yeasts with Mycophil Agar pH 4.7 (PDAa/SDAa or othersequivalent). MICROBIAL TEST SETUP 1. Live microbes used in the test must be no more than the fifth derivative of the original ATCC culture. 2. Stretch stock cultures of Pseudomonas aeruginosa, Staphylococcus aureus, and Enterobacter aerogenes onto TSA agar slants and incubate at 35±2 oC for 18-24 hours. 3. Streak the Candida albicans culture stock onto an SDAa agar slant and incubateat 25±2 oC for 48 hours. 4. Stretch the Aspergillus niger culture stock onto SDAa agar slants and incubate at 25 ± 2 oC for 7-14 days or until sporulation is complete. HARVESTING TEST MICROBIAL CULTURES 1. Wash each bacterial and yeast culture with 2 x 2.5 mL of diluent 1; and culture the mold with 2 x 2.5 mL of diluent solution 2, remove the culture from the surface of the agar with the help of sterile glass beads.Transfer the suspension into a sterile test tube and mix using a vortex mixer so that it is evenly distributed. 2. Adjust each wash with the same diluent to produce 108 cfu/mL of bacterial suspension and 107 cfu/mL of yeast and mold suspension. Compare the suspension with the standard Mc solution. Farland (BaSO4) no.2, absorbance measurement or other methods related to Total Plate Number (ALT) as will be explained in points C and D. DETERMINATION OF THE NUMBER OF TEST BACTERIA USING THE ALTSPREAD METHOD 1. Surface Pour approximately 15 - 20 mL of melted TSA into a sterile Petri dish and allow to solidify. 2. Fill each of 10 sterile tubes with 9 mL of diluent 1. 3. Inoculate 1 mL of each bacterial suspension (Pseudomonas aeruginosa, Staphylococcus aureus, and Enterobacter aerogenes) into the first test tube containing 9 mL of diluent 1 (mark as 10-1 ). Repeat the dilution process 10-times up to 10-10. Mix using a vortex mixer to guarantee a homogeneous distribution. 4. Pipette 0.5 mL of suspension from a 10-4 to 10-10 dilution and distribute overTSA surface, carried out in duplicate. 5. After the suspension is absorbed by the medium, invert the plate and incubate at 35 ± 2 oC for 24-48 hours. 6. Note the growth of between 30-300 colonies. Total colonies are the sum of the colonies from both Petri dishes, multiplied by the dilution factor.Use a suspension containing 108 cfu/mL of test bacteria. 7. Use the bacteria immediately or store in the refrigerator at 2-8°C for no more than 72 hours. DETERMINATION OF THE NUMBER OF MOLDS AND YEASTS BY TEST METHOD ALT-WAY POUR 1. Melt SDAa and keep the temperature at 45-50 oC in a water bath. 2. Fill each of 10 sterile test tubes with 9 mL of diluent (diluent 1 for Candida albicans and diluent 2 for Aspergillus niger). 3. Inoculate 1 mL of Candida albicans into the first test tube containing diluent 1 and Aspergillus niger into the first test tube containing diluent 4. Pipette 1 mL of suspension from dilution 10-4 to 10-10 into a sterile petri dish, do it in duplicate. Pour about 15-20 mL of melted SDAa into each cup, cover, shake gently and let it solidify. 5. Incubate at 25 ± 2 oC for 48 hours (yeast) and 72 hours (mould) in positionthe cup is turned upside down. 6. Note the growth of between 30-300 colonies. Total colonies are the average of colonies from both Petri dishes, multiplied by the dilution factor. 7. Use a suspension containing 107 cfu/mL of yeast and mold for the test. 8. Use microbes immediately or store in the refrigerator at 2-8°C for no more than 72 hours. SAMPLING TEST SETUP Prepare 5 x 100 g samples in 5 suitable glass containers. Mark appropriately (3 containers for bacterial test microbes and 2 containers for mold and yeast). i. Liquid cosmetics. Use the product directly for testing. ii. Cream with water and lotion base. Dissolve using chloride buffer solution with the same ratio (1:1). Heat at 40-45°C and homogenize using a vortex mixer with the help of glass beads. iii. Solid and powder. Mix the product with equal parts chloride buffer solution. Heat at 40-45°C and homogenize using a vortex mixer with the help of glass beads. iv. Oil/fat based products. Mix 100 g of sample with 20 g of mineral oil to form a paste. Add 80 mL of chloride buffer solution. Heat at 40-45°C and homogenize using a vortex mixer with the help of glass beads. v. Aerosols. Store the entire container in the refrigerator (2-8C) for 30 minutes. Transfer 50 g of aerosol into a container containing 50 mL of chloride buffer solution. Homogenize using a vortex mixer with the help of glass beads. SAMPLING TEST PROCEDURE 1. Samples must be verified for the absence of bacterial, yeast and mold growth, using the test method "Determination of Yeast Mold Numbers and Total Plate Number Test in Cosmetics". This must be done before proceeding to further processes. 2. Inoculate each sample container with 1 mL of suspension of each test microbe containing 1.0 to 9.9 x 106 cfu/mL (for bacteria) or 1.0 to 9.9 x 105 cfu/mL (for yeast and mold) [suspension volume should not exceed 1% of the number of samples tested]. Homogenize using a vortex mixer. 3. Transfer 20 g each of the inoculated samples (as stated in NO 2) into 4 different containers to be tested within a period of 2, 7, 14 and 28 days. For validation purposes, testcontrols must be carried out simultaneously with sample tests: days 0, 2, 7, 14, and 28 • Inoculate 100 g of sample without preservative or 100 g of sterile physiological NaCl solution containing 1% Tween 80 with 1 mL of suspension of one of the test microbes to produce 1.0 to 9.9 x 106cfu/mL (for bacteria) or 1.0 up to 9.9 x 105cfu/mL (for yeast and mold) as a control. • Transfer 1 mL of sample within 30 minutes from each container to be tested on days 0, 2, 7, 14 and 28 days. Carry out the test by carrying out a dilution series in multiples of 10 using a peptone saline solution containing 1% polysorbate 80. • Note: untested samples are stored at room temperature (20-25°C) during testing. • Determine the number of live microbes using the ALT method using a surface spread method on TSAt for bacteria and the ALT method using a pour method using SDAt for yeast and mold. Do it in duplicate. • Incubate bacteria at 35 ± 2oC for 24 - 48 hours, yeast and mold at 25 ± 2oC for 3 - 5 days. Calculate the number of surviving microbes per g or mL of sample. • Identification of microbes by Gram staining