Microbiological Analysis of Finished Products and Raw Materials 1.0 PURPOSE 1.

1 To assay test samples for the presence of microorganisms and to obtain a quantitative value for any growth found. 1.2 The colony forming unit (cfu) gives rise to one colony, quantification o f cfu/ml by this method will determine the number of bacteria in a material. 2.0 SCOPE

2.1 Applies to all samples subject to microbiological scrutiny, and under ev aluation for microbial contamination, the colonies that develop are counted and multiplied by the degree of dilution or dilution factor 2.2 Applies to all water samples tested. 3.0 EQUIPMENT & PERSONNEL

3.1 An adequately equipped microbiological testing laboratory. Vertical Laminar flow hood Balance Salmonella spp. (ATCC 14028) Water bath (45-47C) C. albicans (ATCC 10231) if needed A. brasiliensis (ATCC 16404) if needed Colony counter Tryptic soy broth with 0.5% lecithin and 4% Polysorbate 80 S. aureus (ATCC 6538) Disinfectant Incubators (20-25C, 30-35C & 35-37C) P. aeruginosa (ATCC 9027 ) Sterile pipettes Molten TSA with 0.5% lecithin and 4% Polysorbate 80 E. coli (ATCC 8739) Inoculation loops Molten Sabouraud dextrose agar (SAB-80) with 0.5% lecith in and 4% Polysorbate 80 Sterile Petri plates (15x100mm) TAT broth Microfuge tubes for frozen storage of ATP Positive Control Lactose broth Refrigerator (2-8 C) Freezer BIS plates EMB plates PSA plates MAC plates MSA plates TSA plates SAB plates 4.0 PROCEDURE Testing: Perform all procedures using aseptic techniques: 1. Refer to monographs for each drug product being tested, or SOP for each co smetic being tested, to ensure that proper testing is completed. First of Batch and End of Run testing will be performed on all products that receive micro test ing. Raw materials require only one sample to be tested. 2. Record all product information and results Attachment 1. Testing Procedure: 1. Label TSB, LB and plates with the sample number. 2. Prepare Positive controls each day testing is performed by placing one l oop of each organism from the stock culture into the appropriate broth: TSB----------S. aureus, P. aeruginosa and C. albicans LB------------E. coli and Salmonella spp. 3. Use an un-inoculated bottle of 90ml TSB and 90 ml LB as negative control s. 4. If more than one lot of media is used in one day, positive and negative controls must be performed for each lot used in testing. 5. Accurately weigh out or pipette (liquids) 10g/ml of product into 90 ml TSB and /or 90 ml LB. If a product (raw material) causes the broth to clump or

solidify, use 1g instead of 10g. 6. Vigorously shake the container(s) for about 10 seconds. 7. Counts: a. If a total plate count is needed, pipette 1 ml of the TSB broth dilutio n into each of two (2) pre-labeled petri plates. For the negative control, pipet 1 ml from the un-inoculated TSB broth. For positive controls pipet 1 ml from t he broths containing P aeruginosa and S. aureus. Pour 25-30 ml of TSA-80 and al low plates to solidify. Incubate plates at 30-35C for 48 hrs. b. If a total yeast and mold count is needed, pipet 1 ml of TSB broth dilu tion into each of two (2) prelabeled petri plates. For the negative control pip et 1 ml from the uninoculted TSB broth. For the positive control, pipette 1 ml from the broth containing C. albicans. Pour 25-30 ml of SAB-80 and allow plates to solidify. Incubate plates at 20-25C for 5-7 days. The control plates can be read after 48 hours. c. If screening for indicator organisms, incubate broth dilution(s) at 30 -35C fro 24-48 hours. 8. After incubation, observe the plates (if necessary) and broth (if nec essary) for growth: a. If there is growth on the plates, count the number of colony forming un its (cfus) on both plates, average the cfus from both plates and then multiply the average by the dilution factor. This gives you the total number of cfus/g/ml. If the result is out of specification, an OOS investigation will be launched . If there is no growth on the plates record the total number of cfus/g/ml as <10, or <100 if 1 g was used. The number of cfus on the positive control plates shoul d be >250 and should be reported as such. Report the negative control as <1 cfu /ml. b. The presence of turbidity in the broth(s) indicates growth has occurred. If you are unsure as to whether or not turbidity is present due to the nature o f the product report the result as indeterminable, otherwise report the results as positive or negative. 9. If the results of the broth(s) show growth (positive or indeterminable) then streak one loop from the broth dilution which is in question to the approp riate media. Incubate MSA at 35-37C and all the other medias at 30-35C for 24-48 hours. MSA, PSA,, SAB, and MAC TSB LB MAC, EMB, and BIS 10. After incubation, observe the plates for growth characteristics specifie d organisms below: PSA P. aeruginosa NA S. aureus E. coli NA Salmonella spp. MSA EMB MAC BIS Yellow-green fluorescent colonies NA NA NA NA NA NA

Dark yellow colonies NA NA NA Metallic green colonies Pink to red colonies 1 NA NA NA Clear colonies 2 Black colonies 3

Note 1: Lactose positive Note 2: Lactose negative Note 3: Colonies will have a metallic sheen Note 4: C. albicans appear as white colonies on SAB, which may or may not exhib it star-like features Note 5: A. brasiliensis appear as white mycelia with black spores on SAB 11. If there is no characteristic growth of the organism(s) being sc reened for, record the results as presumptive negative for their presence. If t here is characteristic growth of the specified organisms, an OOS investigation w ill be launched. 12. Report positive controls as presumptive positive for each organi sm.

Attachment 1

(attachment 1-MBL 1100-USP61-62)