US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD SOP for Growing a Pseudomonas aeruginosa Biofilm using the CDC Biofilm Reactor SOP Number: MB-19-01 Date Revised: 04-13-11 ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 1 of 17 EPA/OPP MICROBIOLOGY LABORATORY ESC, Ft. Meade, MD Standard Operating Procedure for Growing a Pseudomonas aeruginosa Biofilm using the CDC Biofilm Reactor SOP Number: MB-19-01 Date Revised: 04-13-11 Initiated By: Date: / / Print Name: Technical Review: Date: / / Print Name: Technical Staff QA Review: Date: / / Print Name: QA Officer Approved By: Date: / / Print Name: Branch Chief Effective Date: / / Controlled Copy No.: Withdrawn By: Date: / / ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 2 of 17 TABLE OF CONTENTS Contents Page Number I.0 SCOPE AND APPLICATION 3 2.0 DEFINITIONS 3 3.0 HEALTH AND SAFETY 4 4.0 CAUTIONS 4 5.0 INTERFERENCES 4 6.0 PERSONNEL QUALIFICATIONS 5 7.0 SPECIAL APPARATUS AND MATERIALS 5 8.0 INSTRUMENT OR METHOD CALIBRATION 8 9.0 SAMPLE HANDLING AND STORAGE 8 10.0 PROCEDURE AND ANALYSIS 8 II.0 DATA ANALYSIS/CALCULATIONS 14 12.0 DAT A MANAGEMENT/RECORD MANAGEMENT 14 13.0 QUALITY CONTROL 14 14.0 NONCONFORMANCE AND CORRECTIVE ACTION 14 15.0 REFERENCES 14 16.0 FORMS AND DATA SHEETS 15 ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 3 of 17 1.0 SCOPE AND APPLICATION: 1.1 This SOP describes the methodology used for growing a repeatable Pseudomonas aeruginosa biofilm in a CDC biofilm reactor. This SOP is based on ASTM method Designation: E2562-07, Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor, with the exception of Section 10.3 - Sampling the Biofilm. The resulting Pseudomonas aeruginosa biofilm can be used in antimicrobial product efficacy testing. 2.0 DEFINITIONS: 2.1 ASTM = ASTM International, formerly American Society for Testing and Materials 2.2 ATCC = American Type Culture Collection 2.3 CDC = Centers for Disease Control and Prevention 2.4 OD = outer diameter 2.5 ID = inner diameter 2.6 Reagent grade water - all references to reagent grade water or water as a diluent or reagent shall mean distilled water or water of equal quality. Reagent grade water should be free of substances that interfere with analytical methods. Any method of preparation of reagent grade water is acceptable provided that the requisite quality can be met. Reverse osmosis, distillation, and deionization in various combinations all can produce reagent grade water when used in the proper arrangement. See Standard Methods for the Examination of Water and Wastewater for details on reagent grade water. 2.7 Biofilm - a structured community of microorganisms encapsulated within a self- developed polymeric matrix and adherent to a living or inert surface. Often, it is characterized by structural heterogeneity, genetic diversity, complex community interactions, and microbial and environmental by-products that are determined by the environment in which it lives. 2.8 Coupon - sampling-size support on which microorganisms grow (e.g., polycarbonate, borosilicate, stainless steel, etc.) ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 4 of 17 2.9 Residence Time - the time that it takes for the entire volume of the reactor to exchange once (during continuous flow operation) and is equal to the inverse of the dilution rate. For Example: a volume of 100 mL with a flow rate of 10 mL/min has a residence time of 10 minutes. Residence time is proportional to the volume and inversely proportional to the flow rate. 2.10 CSTR - continuously stirred tank reactor 2.11 Continuous Flow Operation - CSTR Mode, commonly referred to as a chemostat where growth is broadly controlled by the dilution rate. 2.12 Standard Method Dilution Water [SMDW] - Standard Methods Buffered dilution water - Method 9050 C.lar (0.0425 g/1 KH2P04 and 0.405 g/1 MgCl2 6H20) autoclaved for 15 minutes at 120°C. Prepared according to Method 9050 C.la, Standard Methods for the Examination of Water and Waste Water, 19th Edition. 3.0 HEALTH AND SAFETY: 3.1 All manipulations of the test organism are required to be performed in accordance with biosafety practices stipulated in SOP MB-01, Biosafety in the Laboratory. 4.0 CAUTIONS: 4.1 Strict adherence to the protocol is necessary for the validity of results. 4.2 Use appropriate aseptic techniques for all test procedures involving the manipulation of test organisms and associated test components. 4.3 It may be necessary to change silicon tubing and filters on the reactor and carboys after five to six autoclaving processes. 5.0 INTERFERENCES: 5.1 It may be necessary to use only the biofilm on the coupon side that faces the rotating baffle, or vice visa. Therefore, it is critical to maintain the orientation of the carriers once they have been removed from the polypropylene rods. An erroneous count or processing can occur if the appropriate side is not evaluated. A rim of the coupon could be marked with non-toxic alcohol/waterproof permanent marker such as VWR Lab Marker (VWR Scientific, Cat. No. 52877) to aid orientation identification. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 5 of 17 6.0 PERSONNEL QUALIFICATIONS: 6.1 Personnel are required to be knowledgeable of the procedures in this SOP. Documentation of training and familiarization with this SOP can be found in the training file for each employee. 6.2 Basic microbiology training is required to perform this test method. 7.0 SPECIAL APPARATUS AND MATERIALS: 7.1 Test Organism: Pseudomonas aeruginosa (e.g., ATCC #15442) 7.2 Media and reagents: 7.2.1 Standard Method Dilution Water [SMDW], 7.2.2 Bacterial Plating Medium, R2A Agar for sterility and performance test when necessary. 7.2.3 Bacterial Liquid Growth Broth, soybean-casein digest medium, or an equivalent general bacterial growth medium. Tryptic Soy Broth (TSB) is recommended. NOTE: Tryptic Soy Broth (TSB) concentrations in this protocol differ from the manufacturer's recommendation. Two different concentrations are used in the protocol, 300 mg/L for the inoculum and batch reactor operation and 100 mg/L for the continuous flow reactor operation. In addition, a concentrated TSB solution is used to prepare the 20L volume of TSB (100 mg/L) for the continuous flow reactor operation. 7.3 Inoculating loop. 7.4 Petri dish - 100 x 15 mm, plastic, sterile and empty to put beneath polypropylene rods while sampling. 7.5 Culture tubes and culture tube closures - any with a volume capacity of 10 mL and diameter no less than 6 cm. 7.6 Pipetters - continuously adjustable with volumes of 100 |iL to 1000 |iL. 7.7 Vortex - any vortex that will ensure proper agitation and mixing of culture tubes. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 6 of 17 7.8 Sonicator or Ultrasonic cleaner/water bath for cleaning the CDC Reactor parts and coupons. 7.9 Bunsen burner - used to flame inoculating loop and other instruments. 7.10 Environmental shaker - capable of maintaining temperature of 3 6°C + 1 °C. 7.11 Analytical balance - sensitive to 0.01 g. 7.12 Sterilizers - any steam sterilizer capable of producing the conditions of sterilization is acceptable. However, the ability to accommodate a 20 liter carboy is a significant limitation to be considered. 7.13 Colony counter - any one of several types may be used, such as the Quebec, Buck, and Wolfhuegel. A hand tally for the recording of the bacterial count is recommended if manual counting is done. 7.14 Pump - Masterflex L/S Computerized Drive Model 755-50 with Easy-Load II pump head, model 77201-60. The pump is driven by Linkable Instrument Network v2.0. The pump head is capable of holding tubing with ID 3.1 mm and OD 3.2 mm. Use Masterflex Norprene tubing (Cole Palmer 06404-16). 7.15 Magnetic stir plate-top plate 10.16x10.16 cm, capable of rotating at 125 + 60 rpm. 7.16 Silicon Tubing - two sizes of tubing: one with ID 3.1 mm and OD 3.2 mm and the other with ID 7.9 mm and OD 9.5 mm. Both sizes must withstand sterilization. 7.17 Glass flow break - any that will connect with tubing of ID 3.1 mm and withstand sterilization. 7.18 Clamp - used to hold flow break, extension clamp with 0.5 cm minimum grip size. 7.19 Clamp stand - height no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tubing above reactor. 7.20 Reactor Components (for schematic illustrations of CDC reactor components, see reference 15.3). ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 7 of 17 7.20.1 Berzelius pyrex beaker - 1000 mL without pour spout, 9.5 + 0.5 cm diameter. 7.20.2 Berzelius pyrex beaker - 1000 mL without pour spout, 9.5 + 0.5 cm diameter. Pyrex barbed outlet spout added at 400 +20 mL mark. Angle the spout 30-45 degrees to ensure drainage. Spout should accommodate silicon tubing with an ID of 8 - 11 mm. NOTE: The rods and baffle described in 7.20.4 and 7.20.7, respectively, will displace approximately 50 mL of liquid when the system is completely assembled. Therefore, an outlet spout at the 350 mL mark will result in approximately a 300 ml operating volume. The user is encouraged to confirm the actual liquid volume in the reactor, when the rods and baffle are in place, before use. The measured volume is used to calculate an exact pump flow rate. 7.20.3 Reactor top - UHMW (ultra-high molecular weight) polyethylene top (10.1 cm diameter tapering to 8.33 cm) equipped with 3 holes accommodating 6-8 cm long pieces of stainless steel or other rigid autoclavable tubing with OD of 5-8 mm for media inlet, air exchange and inoculation port. Center hole, 1.27 cm diameter, to accommodate the glass rod used to support the baffle assembly. Eight rod holes, 1.905 cm diameter, notched to accommodate stainless steel rod alignment spike (0.236 cm OD). 7.20.4 Polypropylene rods - Eight polypropylene rods, 21.08 cm long, machined to hold three coupons at the immersed end. 316 stainless steel set screws imbedded in side to hold coupons in place. Rods fit into holes in reactor top and lock into preformed notches. 7.20.5 Twenty-four cylindrical coupons (i.e., borosilicate, polycarbonate, stainless steel) - with a diameter of 1.27 +/- 0.013 cm, thickness of approximately 3.0 mm. 7.20.6 Small Allen wrench - for loosening set screws. 7.20.7 Stir blade assembly (baffled stir bar) - Teflon blade (5.61 cm) fitted into cylindrical Teflon holder (8.13 cm) and held in place with a magnetic stir bar (2.54 cm). Teflon holder fits onto a glass rod (15.8 cm), fitted into the reactor top. The glass rod is held in place with a Swagelock fitting and acts as a support for the moving blade assembly. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 8 of 17 7.21 Carboys - two 20 L autoclavable carboys, to be used for waste and nutrients. 7.22 Carboy lids - 2 Carboy Lids: one carboy lid with at least 3 barbed fittings to accommodate tubing ID 3.1 mm (one for nutrient line, one for bacterial air vent (filter), and one for injecting TSB medium concentrate). One carboy lid with at least two 1 cm holes bored in the same fashion (one for effluent waste and one for bacterial air vent). 7.23 Large magnetic stir bar (e.g., 108x27 mm heavy duty large magnetic stir bar, e.g., VWRNo. 58948-972) 7.24 Bacterial air vent (filter - Life Sciences PN 4210) - autoclavable 0.2 micrometer pore size, to be spliced into tubing on waste carboy, nutrient carboy and reactor top, recommended diameter 37 mm. 7.25 Micro-90 Concentrated Cleaning Solution for Critical Cleaning; International Products Corporation. 8.0 INSTRUMENT OR METHOD CALIBRATION: 8.1 Refer to the laboratory equipment calibration and maintenance SOPs (SOP EQ series) for details on method and frequency of calibration. 8.2 The MasterFlex pump is driven through the Linkable Instrument Network software. The calibration may be by flow, volume or weight. To calibrate by flow rate, after making all the necessary connections as the pump will be used, pump the liquid to be used into, say a 2 liter volumetric flask. Obtain the time it took to fill the flask to the marker. Then, open the software tool, go to Setup/Calibrate Tubing/Tubing Calibration, and enter the volume, time taken to fill the flask, and the density of the fluid at the appropriate data field. Upon pushing the New Data button, the necessary calculation will be made and the flow rate appearing. Push Save the New Calibration button to save the flow rate. 9.0 SAMPLE HANDLING AND STORAGE: N/A 10.0 PROCEDURE: SUMMARY - This method is used for growing a repeatable Pseudomonas aeruginosa biofilm in a CDC Biofilm Reactor. The biofilm is established in TSB (300 mg/L) growth media by operating the reactor in batch mode (i.e., no flow) for 24 hours. A steady state population is reached while the reactor operates for an additional 24 hours with continuous flow of TSB (100 mg/L) growth media. The residence time of the TSB (100 ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 9 of 17 mg/L) growth media in the reactor is set to select for biofilm growth, and is species and reactor parameter specific. During the entire 48 hours, the biofilm experiences continuous fluid shear stress from the rotation of a baffled stir bar. Controlling the rate at which the baffle turns determines the shear stress at the surface of the coupons. 10.1 Test Microbe - culture initiation, maintenance, and quality control. 10.1.1 Culture Initiation - for initiating Pseudomonas aeruginosa ATCC #15442, follow steps outlined in SOP MB-05, Test Microbes, section 10.1 10.1.1.1 Record all manipulations on the Biofilm - Organism Culture Tracking Form (see 14.1). 10.1.2 Organism Tracking: Supply Control Number. 10.1.2.1 All cultures (dehydrated ampules as received from ATCC) are given a supply control number upon receipt (see SOP QC-09, Control Numbers). 10.1.2.2 The supply control number will consist of the date received (R) and the date the ampule expires (E). For example: For a P. aeruginosa dehydrated ampule received on 06-21-11 and expiring on 10-07-14, the supply control number would be R062111E100714. 10.1.3 Organism Tracking: Microbe Received and Microbe Expiration Number (MRME). 10.1.3.1 The MRME number will consist of the date received (MR) and the date the reconstituted microbe expires (ME) - i.e., one year later. 10.1.3.2 A culture received on 06-21-11 and reconstituted on 07-28-11 would be notated MR062111ME072812. 10.1.3.3 Once reconstituted, the organism can only be transferred for a period of one year. 10.1.3.4 Once expired, the stock cultures must be autoclaved and discarded and a new culture initiated from a new lyophilized ampule from ATCC. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 10 of 17 10.1.4 Additional Culture Notation. The MRME culture notation will have a suffix "PB." The "P" designates Pseudomonas aeruginosa and the "B" designates a Biofilm culture. Thus, the final culture notation after reconstitution would be MRXXXXXXMEXXXXXX-PB. 10.1.5 Culture Transfer Notation of Test Microbes. Additional suffix notations following the "-PB" designation are added to every daily entry on the Biofilm - Organism Tracking Form to uniquely identify and track any test culture (a full MRME tracking number uniquely identifies a test organism within the one year tracking period for that culture - see 10.1.3.3). 10.2 T est Organi sm Culturi ng. 10.2.1 Use the tracking notation -PB-XX-A where XX indicates the transfer month within the allowed 12 month culture cycle (e.g., "01" would indicate the month the organism was initiated not necessarily the month of the calendar year, and would only be January if the organism was initiated in January), and "A" would indicate [1st Transfer) the initial transfer from a CTA stab to a glass test tube containing 10 mL TSB (300 mg/L). (See 10.2.4). 10.2.2 Use the tracking notation -PB-XX-B where XX indicates the transfer month within the allowed 12 month culture cycle (see above), and "B" indicates [2nd Transfer] the inoculation of 100 mL of sterile TSB (300 mg/L) with 1 mL suspension culture from the 1st Transfer test tube. (See 10.2.6). 10.2.3 Use the tracking notation -PB-XX-C where XX indicates the transfer month within the allowed 12 month culture cycle (see above), and "C" indicates [3rd Transfer] the inoculation of the Batch Phase of the CDC Biofilm Reactor. (See 10.2.8). 10.2.4 Begin test culture by using a plastic loop to aseptically transfer a representative sample from a CTA stock culture, just by a touch, into a 20 x 150 mm glass test tube, in duplicate, containing 10 mL TSB (300 mg/L). Lightly vortex the tube as the loop is held in the TSB. 10.2.5 Incubate bacterial suspension in both tubes in an incubator at 36±1°C for 20-24 hours. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 11 of 17 10.2.6 Conduct OD @ 650nm for both test tubes and a blank using 300 mg/L TSB. Use 1 mL of the content of the tube with 0.060 OD or higher to spike the 100 mL of TSB (300 mg/L) in duplicate 250 mL flasks. 10.2.7 Incubate bacterial suspension in both flasks in an environmental shaker at 36±1°C and 150 revolutions per minute (RPM) for 20-24 hours. 10.2.8 Conduct OD @ 650nm for both flasks and a blank using 300 mg/L TSB. Use 1 mL of the content of the flask with higher OD to inoculate the Batch Phase (see 10.4.1) of the CDC Biofilm Reactor (culture notation "C" from above) containing about 325 mL of TSB (300 mg/L). 10.2.9 Viable bacterial density should equal approximately 108 CFU/mL, and may be checked by serial dilution and plating. 10.2.10 Record all manipulation on the Biofilm - Organism Culture Tracking Form (see 14.1). 10.3 Reactor Preparation. 10.3.1 Preparation of Coupons (polycarbonate, borosilicate, porcelain, etc,). NOTE: Coupons can be used once and discarded or used repeatedly with proper cleaning and sterilization in-between each use. Check each coupon for scratches, chips, other damage or accumulated debris before each use. Discard those with visible damage to surface topography. 10.3.1.1 For glass coupon, sonicate coupons individually in 50 mL centrifuge plastic tubes for 60±5 seconds in 3 - 5 mL of a 1:100 dilution of laboratory soap (e.g., Micro-90 Concentrated Cleaning Solution) and tap water. The soapy water must completely cover the coupons. Drain and completely rinse off soapy water. 10.3.1.2 Whilethe coupons are still in their separate tubes, add2-3 mL of 1 N HC1 and let stand at room temperature for about 30 minutes. 10.3.1.3 Adequately rinse the coupon with reagent grade water. Once the coupons are clean, care must be given to prevent oils and other residue from contaminating the surface. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 12 of 17 10.3.1.4 Place a coupon into each hole in the reactor rods, leaving the top of the coupon flush with the inside rod surface. Tighten the set screw. 10.3.1.5 Place rods into reactor top loosely (not yet fitted into notches). 10.3.2 Preparation of Reactor Top. 10.3.2.1 Place baffle onto glass rod suspended from the reactor top. 10.3.2.2 Place assembled top into the reactor beaker. 10.3.2.3 Connect the bacterial air vent by fitting the vent to a small section of appropriately sized tubing, which is then attached to one of the rigid tubes on the reactor top. 10.3.2.4 The glass flow break is spliced into the nutrient tubing line near the reactor top. 10.3.3 Sterilization of the Reactor System. 10.3.3.1 Cover the end of the inj ection ports, the nutrient tubing that connects to the nutrient carboy and the overflow (waste) tubing with aluminum foil. Cover any extra openings on the reactor top with aluminum foil. 10.3.3.2 Sterilize the empty reactor system for 20 minutes on gravity cycle. 10.3.3.3 If effluent sample needs to be taken from the reactor for any reasons, insert a plastic 3-way stopcock (e.g., Kendall Argyle EZ-FLO [173518]) in the closed position into the overflow (waste) line, add 350 mL sterile batch culture medium (300 mg/L TSB) to the cooled reactor. 10.4 Growth of Biofilm in CDC Reactor. 10.4.1 The Batch Phase. 10.4.1.1 Place prepared cooled reactor (from 10.3.3.3 above) onto a stir plate. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 13 of 17 10.4.1.2 Inoculate the reactor with 1 mL of bacteria from the culture prepared previously (see 10.2.5). Aseptically inject the inoculum into the beaker through one of the available reactor top rigid stainless steel tubes using a 1 mL sterile pipette. 10.4.1.3 Turn on the magnetic stir plate. The rotation speed should equal 125 RPM. The reactor system incubates in batch mode at room temperature (21±2°C) for 24±1 hours. NOTE: The speed at which the baffled stir bar rotates directly determines the strength of the shear stress that the biofilm experiences. Biofilm accumulation on the coupons is sensitive to changes in the baffle's rotational speed. The baffle rotational speed is a critical factor that must be controlled. 10.4.2 Continuous Flow Operation (CSTR mode) 10.4.2.1 Prepare continuous flow nutrient broth by dissolving bacterial liquid growth medium (100 mg/L TSB) in 20 liters sterile reagent grade water. Dissolve and sterilize the broth in a smaller volume to prevent caramelization. Aseptically pour the concentrated broth into the carboy of sterile water (prepared with a 4" magnetic stir bar placed inside the carboy) to make a total of 20 liters. For example, prepare a concentrated TSB solution (40 g/L) and add 50 mL of the concentrated TSB to the 20L sterile water. 10.4.2.2 After adding the concentrated TSB to the 20L of sterile water, place the carboy on a large stir plate and mix vigorously for > 5±1 minutes. 10.4.2.3 Aseptically connect the nutrient tubing line to the carboy containing the continuous flow nutrient broth. 10.4.2.4 Pump a continuous flow of nutrients into the reactor at a flow rate equal to 11,7±0.2 mL/min. Place the end of the reactor drain into the waste carboy and open the e-way stopcock. The drain spout at the 325 mL mark on the beaker allows overflow to occur, maintaining a constant bacterial liquid growth broth concentration of 100 mg/L in the reactor during CSTR (continuously stirred tank reactor) mode. ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 14 of 17 NOTE: Flow rate is calculated by dividing the reactor volume by the residence time. The residence time is 30 minutes. The reactor volume is approximately 350 mL (see NOTE, section 7.24.2). The operator should set an exact flow rate based upon the measured fluid volume in the reactor when the rods are in place to achieve a 30 minute residence time. 10.4.2.5 Operate the reactor in CSTR mode for 24±2 hours. 10.5 For conducting disinfectant efficacy evaluations, proceed to Standard Operating Procedure for the Evaluation of Disinfectants against Pseudomonas aeruginosa Biofilm Grown in a High Shear Continuous Flow Reactor Model. 11.0 DATA ANALYSIS/CALCULATIONS: N/A 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT: 12.1 Data will be recorded promptly, legibly, and in indelible ink on the forms indicated. Completed forms are archived in notebooks kept in secured file cabinets in D217. Only authorized personnel have access to the secured files. Archived data is subject to OPP's official retention schedule contained in SOP ADM-03, Records and Archives. 13.0 QUALITY CONTROL: 13.1 The OPP Microbiology Laboratory conforms to 40CFR Part 160, Good Laboratory Practices and Standards. Appropriate quality control measures are integrated into each SOP 13.2 For quality control purposes, the required information is documented on the appropriate form(s). 14.0 NONCONFORMANCE AND CORRECTIVE ACTION: 14.1 Any deviation from the standard protocol and the reason for the deviation will be recorded on the appropriate record sheet; corrective action will be expeditious. 15.0 REFERENCES: 15.1 Official Methods of Analysis (2006) 21st ED., AOAC INTERNATIONAL, Method 966.04, Gaithersburg, MD, Chapter 6 ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 15 of 17 15.2 Standard Methods for the Examination of Water and Wastewater. 21st Edition. Eaton, A.D., Clesceri L.S., RiceE.W., Greenberg A.E. (Eds.) 2005. American Public Health Association, 1015 15th Street, NW, Washington, DC 15.3 ASTM International, 2007. E-2562-07: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow Using CDC Biofilm Reactor. 15.4 Hamilton, M., Heersink, J., Buckingham-Meyer, K., Goeres, D. (Eds.) 2003. 1st Ed. The Biofilm Laboratory, Step-by-Step Protocols for Experimental Design, Analysis, and Data Interpretation. Center for Biofilm Engineering, Montana State University, Bozeman, MT. 16.0 FORMS AND DATA SHEETS: 16.1 Biofilm - Organism Culture Tracking Form 16.2 Biofilm - Test Microbe Confirmation Sheet ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 16 of 17 BIOFILM - ORGANISM CULTURE TRACKING FORM OPP Microbiology Laboratory Organism: Pseudomonas aeruginosa Supply Control Number: Source and Strain no.: ATCC #: Lot N umber: Date Time Init. Subculture Source T ransler* Media Inoculated (and # inoc.) Media Prep No. Incubation Conditions Comments Monthly Daily TSB = trypticase soy broth, NA = nutrient agar, NB = nutrient broth MB-19-01 Fl.doc ------- SOP No. MB-19-01 Date Revised 04-13-11 Page 17 of 17 BIOFILM - TEST MICROBE CONFIRMATION SHEET OPP Microbiology Laboratory Organism: Pseudomonas aeruginosa MRME Number: Source and Strain no.: ATCC #: Notes: Source: Tube/Plate ID Date/ Initials Staining Results* Media Information Results Name Prep. No. Inc. Time/ Temp. Date/Initials Colony Characteristics Vitek Log# * Record Gram stain results, GNR = Gram Negative Rods MB-19-1 F2.doc ------- |