LUBRIZOL TEST PROCEDURE

Test Procedure #: 6TV0001 Edition: August 25, 2009 Previous Edition: August 9, 2006

Total Viable Count with Enrichment Microbiological Procedure

SAFETY PRECAUTIONS Knowledge of microbiological and aseptic techniques is required for this procedure. General aseptic and safety procedures should be followed in accordance with the American Society for Microbiologys reference book Methods for General and Molecular Bacteriology. Wear a laboratory coat, safety glasses and protective gloves while conducting procedures. Disinfect work area and benchtop surfaces according to Laboratory SOPs before and after executing procedures to maintain a clean work environment. After removing gloves, thoroughly wash hands with soap and water. SCOPE This test method is designed to ascertain whether a given product, process intermediate, or raw material meets microbiological quality specifications and to indicate the presence or absence of any objectionable organisms. The method is designed for routine microbiological control purposes and is not intended as a test for sterility or preservative activity. This method is not intended for process water, or samples not requiring enrichment. PRINCIPLE This test method is designed to detect the presence of microorganisms in a cosmetic raw material. The Total Viable Count (TVC) pour plate procedure is a quantitative estimate of the level of microorganisms present in the test sample. The enrichment procedure is designed to detect low levels of microbial bioburden in a test sample.
The information contained herein is believed to be reliable, but no representations, guarantees or warranties of any kind are made as to its accuracy, suitability for particular applications or the results to be obtained. The information often is based on laboratory work with small-scale equipment and does not necessarily indicate end product performance or reproducibility. Because of the variations in methods, conditions and equipment

MATERIALS: Equipment and Supplies 1. Autoclave 2. Calibrated Balance (0.00g) 3. Calibrated Inoculating Loops-10l 4. Quebec Colony Counter 5. Disposable Latex or Nitrile Gloves 6. Disinfectant (70% Isopropyl Alcohol or Approved Laboratory Disinfectant) 7. Incubator at 32 2C 8. Incubator at 26 2C 9. Sharpie or Permanent Markers 10. Sterile Disposable Petri Dishes, 100x15mm 11. Sterile Syringes (or Serological Pipets), 10cc (10ml) 12. Sterile Syringes (or Serological Pipets), 1cc (1ml) 13. Automatic Pipettor to Dispense 1-10g or ml 14. Automatic Pipettor to Dispense 100-1000l 15. Sterile Racked Pipet Tips 100-1000l 16. Scissors 17. Ethanol 18. Bunsen burner 19. Vortex Mixer 20. Water Bath or Incubator at 47 2C for Holding Liquefied Media 21. Light Microscope 22. Glass Slide 23. Immersion Oil MATERIALS: Media and Microorganisms Media can be prepared in-house following manufacturers directions or purchased from: General Laboratory Products 365 Remington Boulevard Bolingbrook, IL 60440 Phone: 630.759.8500, Fax: 630.759.5342

Lubrizol Advanced Materials, Inc. / 9911 Brecksville Road, Cleveland, Ohio 44141-3247 / TEL: 800.379.5389 or 216.447.5000
used commercially in processing these materials, no warranties or guarantees are made as to the suitability of the products for the applications disclosed. Full-scale testing and end product performance are the responsibility of the user. Lubrizol Advanced Materials, Inc. shall not be liable for and the customer assumes all risk and liability for any use or handling of any material beyond Lubrizol Advanced Materials, Inc.s direct control. The SELLER MAKES NO WARRANTIES, EXPRESS OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. Nothing contained herein is to be considered as permission, recommendation, nor as an inducement to practice any patented invention without permission of the patent owner.

For further information, please visit www.personalcare.noveon.com

Lubrizol Advanced Materials, Inc. is a wholly owned subsidiary of The Lubrizol Corporation * Trademark owned by The Lubrizol Corporation Copyright 2009 / The Lubrizol Corporation

2 Media Notes: 1. Refer to Table 1: Sample Preparation for product specific media requirements. 2. All dehydrated media should be sourced from Difco/BBL or equivalent. Pre-poured plates and enrichment broths must be tempered to room temperature (RT) before use. 3. Liquefied agar should be tempered to 47 2C in a water bath or incubator before use. 4. Media can only be re-melted once and must be discarded if not used for testing. 1. Neutralizing Diluents A. Tryptone-Azolectin-Tween Broth (TAT), a neutralizing enrichment broth. B. Letheen Broth (LB), a neutralizing enrichment broth. C. Dey Engley Neutralizing Broth (D/E), a neutralizing enrichment broth. D. Span 80 or Tween 80 additive for water-immiscible samples. E. Tryptic Soy Broth (TSB), used for Growth Promotion Testing. F. Dilution Blanks (TSB, 0.9% Sterile Saline, 0.1% Peptone Water or Equivalent), used for determining bacterial titers for Growth Promotion Testing and Verification of Preservative Neutralization. 2. Molten Agar for Plating and Pre-Poured Plates for Enrichment Streaking All are non-selective to recover a broad spectrum of microorganisms A. Letheen Agar (LA), contains neutralizers to inactivate preservatives. B. Microbial Content Test Agar (MCTA), contains neutralizers to inactivate preservatives. C. Trypticase Soy Agar with Lecithin and Polysorbate 80 (TSALT), contains neutralizers to inactivate preservatives. D. Sabouraud Dextrose Agar (SDA), no neutralizers, for the isolation of mold and yeast. 3. Selective Media for Isolate Identification (pre-poured plates) A. Pseudomonas Isolation Agar (PIA), for isolation and differentiation of Pseudomonads. B. MacConkeys Agar (MAC), for isolation and differentiation of Gram negative organisms. C. Sabouraud Dextrose Agar (SDA), for the isolation of mold and yeast. D. Mannitol Salt Agar (MSA), for the isolation and differentiation of staphylococci. E. Trypticase Soy Agar (TSA), no neutralizers, for microorganism maintenance. 4. Reagents A. Gram Stain Reagents (Difco, BBL or Equivalent), for staining isolated microorganisms by the differential Gram method. B. Oxidase Reagent (BBL, Difco or Oxoid), for the differentiation of Gram negative, oxidase positive organisms. C. Rabbit Coagulase Plasma (BBL or Equivalent), BBL Staphyloslide Latex Test, Sure-Vue Color Staph ID or approved alternative, for detecting coagulase activity by Staphylococci. D. Rabbit Coagulase Plasma (BBL or Equivalent), for detecting the presence of germ tubes by Candida albicans. E. 3% W/V Potassium Hydroxide (KOH), for KOH String Test, for preliminary confirmation of Gram negatives. 5. Microorganisms- For Growth Promotion Testing and Verification of Preservative Neutralization A. Pseudomonas aeruginosa ATCC 9027 (Culti-Loops or Stock Cultures) B. Staphylococcus aureus ATCC 6538 (Culti-Loops or Stock Cultures) C. Escherichia coli ATCC 10536 (Culti-Loops or Stock Cultures) D. Candida albicans ATCC 10231 (Culti-Loops or Stock Cultures) GENERAL NOTES 1. Proper aseptic techniques must be followed while performing these procedures. 2. Plate in duplicate from a 1:10 or 1:100 sample dilution. Refer to Table 1: Sample Preparation. 3. When possible, corrugated boxes should not be brought into the Microbiology Laboratory. 4. The exterior of product containers should be disinfected with an ethanol soaked paper towel prior to testing. 5. Sample manipulation and plating should be done in a laminar flow hood, in a separate plating room or area protected from traffic flow or air borne contamination.

TABLE 1

SAMPLE PREPARATION
Product Category Water Miscible Dilution 1:10 Minimum Sample Weight 10.0g Neutralizing Diluent Neutralizing Broth: LB or TAT Broth LB or TAT Broth supplemented with Span or Tween 80 Plating Media LA Plates, SDA for Fungal Count if specified LA Plates, SDA for Fungal Count if specified

Water Immiscible1

1:10

1.0g

Water Immiscible Samples require dispersion in Span or Tween 80 prior to the addition of a neutralizing diluent.

4 PROCEDURE Sample Preparation 1. Refer to Table 1: Sample Preparation for details on dilution scheme, sample weight and media. 2. Label the appropriate number of neutralizing diluent containers and the bottom of petri dishes with a sample number, dilution factor and date. 3. Shake sample well to achieve homogeneous mixture prior to removing aliquot and adding to neutralizing broth. 4. For water-miscible 10 gram samples - transfer by means of a sterile spatula, sterile pipet or other sterile sampling device, 10 grams of the sample to a dilution bottle containing 90 ml of diluent. Mix sample by shaking or vortexing to achieve adequate dispersion of sample in neutralizing diluent. 5. For water-immiscible 10 gram samples - transfer by means of a sterile spatula, sterile pipet or other sterile sampling device 10 grams of the sample to a dilution bottle containing 10 g of sterile Span or Tween 80. Mix sample and Span/Tween with a sterile stir pipet, rod or spatula to achieve adequate dispersion. Add 80 ml of neutralizing diluent to achieve a final volume of 100 ml. 6. For water-immiscible 1.0 g samples - transfer by means of a sterile spatula, sterile pipet or other sterile sampling device, 1ml or 1g of the raw material to a dilution bottle containing one ml of sterile Span or Tween 80. Mix sample with a sterile stir rod or spatula to achieve adequate dispersion in the emulsifying agent. Add the appropriate amount of neutralizing diluent to achieve a final volume of 100 ml. 7. After diluting the sample, wait 10-15 minutes before plating to achieve adequate preservative neutralization. PROCEDURE Total Viable Count 1. From the sample-diluent mixture, aseptically transfer one ml into each of two sterile petri dishes. If higher counts are expected, then plate 0.1 ml of the dilution into each of two additional petri dishes to achieve the next level serial dilution. 2. Aseptically pour 18-20 ml molten tempered non-selective plating media into duplicate petri dishes to determine the total viable count. 3. If Yeast and Mold is designated in the specification or a Y&M count is desired, aseptically pour 18-20 ml molten tempered SDA into an additional set of petri dishes to determine the fungal count. 4. Ensure complete mixing of the dilution and agar by rotating plates to sufficiently disperse sample. 5. Allow the agar to solidify at room temperature. 6. Bacterial Count: Invert the non-selective plates and incubate at 32 2C for a minimum of 72 hours. After the incubation period, examine the plates for growth and count all visible colonies with the aid of a Quebec Colony Counter. 7. Fungal Count: Incubate the SDA plates at 26 2C for a minimum of 72 hours. After the incubation period, examine the plates for growth and count all visible colonies with the aid of a Quebec Colony Counter. 8. Agar and Diluent Sterility Control: Each batch of agar or diluent used for plating needs to be evaluated to determine that the media is sterile. Prepare two plates by pouring 18-20ml of molten, tempered agar into sterile petri dishes. Allow the agar to solidify. Invert the plates and incubate them with the samples being tested. After the 72 hour incubation period, if microbial growth is indicated on the control plates, the media is contaminated and the test results may be invalid. Any positive results from the test samples evaluated with that lot of agar should be repeated. PROCEDURE Enrichment for the Detection of Low Levels of Microorganisms, Including Gram Negatives and/or Objectionable Organisms 1. After the 1:10 or 1:100 sample dilution has been plated, incubate the remaining broth for enrichment. 2. Loosen the cap on the jar slightly to provide adequate aeration and incubate the product dilution at 32 2C for a minimum of 48 hours. 3. After the 48 hour incubation period, remove the enrichment broth from the incubator. 4. Tighten the cap on the jar and mix by shaking or vortexing. 5. Remove the cap on the jar and using a calibrated 10L inoculating loop, streak a portion of the broth onto the surface of an agar plate. A four quadrant streak plate can be prepared. Streak one plate of LA, MCTA or TSALT and MAC. 6. Incubate the plates inverted at 32 2C for 24 hours. 7. Retain jars at room temperature or refrigeration until the enrichment plates are interpreted, in case further investigation is needed. 8. After the incubation period, remove the plates from the incubator and examine for growth.

5 9. Broth Sterility Control: Each lot of broth used for the enrichment procedure needs to be evaluated to determine that the media is sterile. Loosen the cap of one jar without added test sample, and incubate at 32 2C with the test enrichment broths. After the 48 hour incubation period, absence of growth should be confirmed by streaking a loopful (with a 10l loop) onto the surface of a non-selective agar plate. Incubate the inverted plates for 24 hours at 32 2C. Microbial growth on the plates streaked with the control broth indicates that the broth is contaminated, and the test results may be invalid. Any positive results should be repeated. INTEPRETATION OF RESULTS 1. At a minimum of 72 hours, count the number of colony forming units (cfu) on each TVC pour plate with the aid of a Quebec colony counter. Average the count from the duplicate dilution plates, then multiply by the dilution factor (inverse of dilution) to obtain the final cfu/g or ml in the test sample. An example of a TVC calculation is given in the table below.
cfu/ Plate 1 87 8 cfu/ Plate 2 94 9 Average cfu/Plate 90.5 8.5 Dilution 1:10 1:100 TVC cfu/ g or ml 9.05 x 10 2 8.5 x 10
2

3. If yeasts are recovered, a Germ tube test can be used for the preliminary identification of Candida albicans. If the Germ tube test is positive, identification to Genus and species is required. 4. If molds are recovered, a tentative identification can be completed using a mycology text. For example, Identifying Filamentous Fungi, G. St-Germain and R. Summerbell. Star Publishing Co. 1996. Interpretation of Colonial Morphology on Selective Media 1. MAC: MAC agar is a selective media for the detection, isolation and differentiation of lactose fermenting from lactose non-fermenting Gram negative bacilli. Isolated colonies of coliform bacteria are brick red in color and may be surrounded by a zone of precipitated bile. Pseudomonas, Burkholderia and other Gram negative Genera can grow on MAC agar. 2. PIA: PIA agar is a selective media for the isolation of Pseudomonas. PIA can also be used for the differentiation of Pseudomonas aeruginosa from other pseudomonads based on pigment formation. On PIA, P. aeruginosa will typically appear green to blue-green. Pseudomonas is usually the only genus that will grow on PIA, and are generally oxidase positive. 3. MSA: MSA agar is a selective media for the isolation and differentiation of staphylococci. Typical pathogenic staphylococci (coagulase positive) will produce yellow colonies with yellow zones around the colonies. Coagulase negative staphylococci will produce small red colonies with no color change to the media surrounding them. 4. SDA: SDA agar is a selective media for the isolation of yeast and mold species. The high dextrose level and acidic pH make the agar

2. If no colonies are present on the TVC pour plates, the results can be reported as < 10 or <100 cfu/g or ml for the 1:10 and 1:100 dilutions respectively. 3. The enrichment plates are reported as growth (+) or no growth (-). Colonies present on an enrichment plate do not need to be quantified. Any growth obtained on an enrichment plate must be investigated. 4. Recovered organisms are characterized by colonial morphology, Gram staining and may be identified by other appropriate biochemical techniques. PRELIMINARY IDENTIFICATION METHODS 1. If Gram negative rods are recovered, an oxidase test and identification to Genus and species is required. A KOH string test may be used as an additional biochemical test to confirm the isolation of a Gram negative organism. 2. If Gram positive cocci are recovered, a coagulase test is required. Coagulase positive organisms require identification to Genus and species.

selective for fungi.

Note: All of the above media are selective but other types of organisms can be recovered on them occasionally.

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6. GROWTH PROMOTION TESTING Growth promotion testing must be completed on all batches of media prepared in-house. ATCC organisms can be used for growth promotion testing. For purchased prepared media, a supplier certification for growth promotion is acceptable. Media should not be used until growth promotion testing is completed with acceptable results.

Prepared Plates 1. For each batch of media, inoculate duplicate plates to deliver approximately 100 cfu/plate. For MCTA, TSALT or LA S.aureus (ATCC 6538) and E.coli (ATCC 10536) or P.aeruginosa (ATCC 9027) are recommended. For MAC E.coli (ATCC 10536) and S.aureus (ATCC 6538) are recommended. For PIA P.aeruginosa (ATCC 9027) is recommended. For SDA C.albicans (ATCC 10231) and S.aureus (ATCC 6538) are recommended. For MSA S.aureus (ATCC 6538), S.epidermidis (ATCC 12228) and E.coli (ATCC 25922) are recommended. 2. Invert and incubate the plates at 32 2C for 24 hours. 3. After the incubation period, remove the plates from the incubator and examine for bacterial growth. 4. Count and record the number of colonies on each plate. 5. The growth should not be inhibited on the test media in comparison to the TSA control. Neutralizing Enrichment Broths 1. For each batch of media, inoculate duplicate neutralizing enrichment broths to deliver approximately 100 cfu per sample. In addition, inoculate control broths from a recent batch previously evaluated for growth promotion. S.aureus (ATCC 6538) and E.coli (ATCC 10536) or P.aeruginosa (ATCC 9027) are recommended. 2. Incubate the enrichment broths at 32 2C for 48 hours. 3. After the incubation period, remove the samples from the incubator. 4. Tighten the cap on the jar or test tube and mix by shaking or vortexing. 5. Remove the cap on the jar and using a calibrated inoculating loop, streak a portion of the broth onto the surface of a TSA plate.

6. Invert and incubate the plates at 32 2C for 24 hours. 7. After the incubation period, remove the plates from the incubator and examine for bacterial growth. 8. Results should be reported as growth (+) or no growth (-). 9. The growth should not be inhibited on the test media in comparison to the control. DISPOSAL 1. All plates, equipment and broth bottles should be autoclaved at 121C/15 lbs pressure before disposal. 2. Dispose according to local waste disposal requirements. REFERENCES McConville, John F. 1974. Methods for Performing Aerobic Plate Counts of Anhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispensing Agents. Appl. Microbiology 27:5-7 Current US Pharmacopoeia (USP), Microbial Limits Tests Philipp Gerhardt, R.G.E. Murray, Willis A. Wood, Noel R. Krieg, Methods for General Molecular Bacteriology, ASM Press, Washington D.C.