US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Growing a Pseudomonas aeruginosa Biofilm using the CDC Biofilm Reactor SOP Number: MB-19-02 Date Revised: 08-06-13 ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 1 of 14 SOP Number MB-19-02 Title Growing a Pseudomonas aeruginosa Biofilm using the CDC Biofilm Reactor Scope Describes the methodology used for growing a repeatable Pseudomonas aeruginosa biofilm in a CDC biofilm reactor. Application The resulting Pseudomonas aeruginosa biofilm can be used in antimicrobial product efficacy testing. This SOP may be used for additional organisms like S. aureus; however, the growth parameters (e.g., media concentrations, baffle speed, residence time) may need to be adjusted. Approval Date SOP Developer: Print Name: SOP Reviewer Print Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Date SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 2 of 14 TABLE OF CONTENTS Contents Page Number 1. DEFINITIONS 3 2. HEALTH AND SAFETY 3 3. PERSONNEL QUALIFICATIONS AND TRAINING 3 4. INSTRUMENT CALIBRATION 3 5. SAMPLE HANDLING AND STORAGE 4 6. QUALITY CONTROL 4 7. INTERFERENCES 4 8. NON-CONFORMING DATA 4 9. DATA MANAGEMENT 5 10. CAUTIONS 5 11. SPECIAL APPARATUS AND MATERIALS 5 12. PROCEDURE AND ANALYSIS 7 13. DATA ANALYSIS/CALCULATIONS 10 14. FORMS AND DATA SHEETS 10 15. REFERENCES 10 ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 3 of 14 1. Definitions Additional abbreviations/definitions are provided in the text. 1. CDC = Centers for Disease Control and Prevention 2. Biofilm = e.g., microorganisms living in a self-organized community attached to surfaces, interfaces, or each other, embedded in a matrix of extracellular polymeric substances of microbial origin, while exhibiting altered phenotypes with respect to growth rate and gene transcription. 3. Coupon = materials used to support the growth of biofilm (e.g., polycarbonate, borosilicate, stainless steel, etc.) 4. 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: an operating volume of 325 mL with a flow rate of 10.8 mL/min has a residence time of 30 min. Residence time is proportional to the volume and inversely proportional to the flow rate. In addition, refer to section 12. 5. Batch phase = establishment of the biofilm by operating the reactor without the flow of nutrients 6. LIN = Linkable Instrument Network 7. Continuous Flow Operation = continuously stirred tank reactor (CSTR) mode, where growth is broadly controlled by the dilution rate. 2. Health and Safety Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study Director and/or lead analyst should consult the Safety Data Sheet for specific hazards associated with products. 3. Personnel Qualifications and Training Refer to SOP ADM-04, OPP Microbiology Laboratory Training. 4. Instrument Calibration 1. Refer to SOPsEQ-Ol, EQ-02, EQ-03, EQ-04, EQ-05, andEQ-10 for details on method and frequency of calibration. 2. Confirm the operating volume of reactor. a. Fully assemble the reactor (including rods with coupons and baffle apparatus) and place on a stir plate set to the appropriate speed (e.g., 125 ± 5 rpm). Clamp the effluent tubing on the reactor beaker. b. Remove one of the rods and fill the reactor with water, higher than the level of the glass spout. c. Remove the clamp on the effluent tubing and allow the excess fluid to drain out of the reactor. d. Carefully pour the remaining water into a graduated cylinder; this ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 4 of 14 remaining water is the operating volume of the reactor. e. Use the operating volume of the reactor to determine the appropriate flow rate that will result in a residence time of 30 min (see section 1.4). 3. The MasterFlex pump can be driven through the LIN software. a. To calibrate by flow rate: prepare the pump for its intended use and pump the liquid to be used into an appropriate sized flask or graduated cylinder. Determine the time required to fill the container to the appropriate volume. i. Using LIN: open the software, go to Setup/Calibrate Tubing/Tubing Calibration and enter the volume, time required to fill the vessel, and the density of the fluid in the appropriate data fields. Select the New Data button; the necessary calculation will be made and the flow rate will appear. Push Save the New Calibration button to save the flow rate. 5. Sample Handling and Storage Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01, Chain of Custody Procedures. 6. Quality Control For quality control purposes, the required information is documented on the appropriate form(s) (see section 14). 7. Interferences 1. 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. If baffle speed is not maintained correctly, it may impact the quality of the biofilm. 2. Due to the deterioration of the materials, it may be necessary to change the tubing and filters on the reactor and carboys after 5 -6 autoclaving processes. 3. Inspect all parts of the reactor system frequently because reuse of worn parts may cause variability in the data. 4. Do not place any plastic or rubber pieces of the reactor system under UV light due to potential degradation of the material. 5. Overuse of carriers or carriers not prescreened adequately may cause variability in the results. 8. Non- conforming Data 1. Management of non-conforming data will be specified in the study protocol; procedures will be consistent with SOP ADM-07, Non- Conformance Reports. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 5 of 14 9. Data Management 1. Data will be archived consistent with SOP ADM-03, Records and Archives. 10. Cautions 1. Use appropriately sized secondary containment for contaminated waste to prevent a spill of biohazardous material. 11. Special Apparatus and Materials 1. Test organism. Pseudomonas aeruginosa (ATCC No. 15442)1 obtained directly from ATCC. 2. Growth medium. Tryptic soy broth (TSB). Three concentrations are used: 300 mg/L for inoculum and batch phase operations, 40 g/L to prepare the 20 L volume of TSB, which is 100 mg/L, and used for CSTR operation. 3. Micropipettes. For performing culture transfers. 4. Ultrasonic water bath. Any bath capable of maintaining a homogeneous sound distribution of 45 kHz with a variable power setting and a volume large enough to accommodate 50 mL conical tubes in a wet environment. For cleaning the CDC Reactor parts and coupons. 5. Environmental shaker. Capable of maintaining temperature of 3 6 ± 1°C, for growing the second transfer of biofilm culture. 6. Peristaltic 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 inner diameter (ID) 3.1 mm and outer diameter (OD) 3.2 mm. Use MasterflexNorprene tubing (Cole Palmer 06404- 16). 7. Magnetic stir plate. Top plate 10.16 x 10.16 cm, capable of rotating at 125 ± 60 rpm. 8. 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. 9. Glass flow break. Any that will connect with tubing of ID 3.1 mm and withstand sterilization. 10. Clamp. Used to hold flow break, extension clamp with 0.5 cm minimum grip size. 11. Clamp stand. Height no less than 76.2 cm, used with clamp to suspend glass flow break vertically and stabilize tubing above reactor. 12. Reactor Components. For schematic illustrations of CDC reactor 1 MLB revision, not in ASTM E2562-12. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 6 of 14 components, see reference 15.1. a. Berzeliuspyrex 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 11.12d and 11.12f, 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 a reactor operating volume of approximately 300 mL. The user is encouraged to confirm the actual liquid volume in the reactor when the rods and baffle are in place (operating volume of the reactor) before use. Calculate an exact pump flow rate using the measured operating volume of the reactor. b. 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). c. 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. d. Coupons. 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. e. Small Allen wrench. For loosening set screws. f. Stir blade assembly (baffledstir 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. 13. Carboys. Two 20 L autoclavable carboys, used for waste and nutrients. a. Carboy lids. Two carboy lids. One carboy lid with at least 3 ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 7 of 14 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). 14. Bacterial air vent (filter - Life Sciences PN 4210). Autoclavable 0.2 |im pore size, to be spliced into tubing on waste carboy, nutrient carboy and reactor top; recommended diameter 37 mm. 15. Detergent. Micro-90 Concentrated Cleaning Solution for Critical Cleaning; International Products Corporation. For cleaning coupons and reactor parts. 12. Procedure and Analysis This method is used for growing a Pseudomonas aeruginosa biofilm in a CDC Biofilm Reactor. The biofilm is established in TSB (300 mg/L) by operating the reactor in batch phase (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). The residence time of the TSB (100 mg/L) growth medium in the reactor selects 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. 12.1 Culture preparation Refer to SOP MB-02 for the test microbe culture transfer notation. Refer to Attachments 1-3 for stock culture generation and QC. a. Defrost a single cryovial at room temperature and briefly vortex to mix. Add 10 |iL of the thawed frozen stock (single use) to a tube containing 10 mL of TSB (300 mg/L), vortex, and incubate at 36 ± 1 °C for 24 ± 2 h2. i. Culture tracking: add "-BF-A" after the number of the frozen stock culture vial in the organism transfer number to denote the first transfer (A) of a biofilm culture (BF) (see section 14 for culture tracking form). b. Inoculate a flask containing 100 mL of TSB (300 mg/L) with 1 mL of culture from the first transfer (-BF-A) tube.3 Incubate the flask in an environmental shaker at 36 ± 1°C and 150 revolutions per minute (RPM) for 24 ± 2 h.4 i. Culture tracking: add "-BF-B" after the number of the frozen stock culture vial in the organism transfer number to denote the second transfer (B) of a biofilm culture (BF) 2 MLB revision, not in ASTM E2562-12. 3 MLB revision, not in ASTM E2562-12. 4 MLB revision, not in ASTM E2562-12. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 8 of 14 (see section 14 for culture tracking form). 12.2 Coupon preparation5 Coupons may be used repeatedly with proper cleaning and sterilization between each use. Check each coupon under 20X magnification for scratches, chips, other damage or accumulated debris before each use. Discard those with visible damage to surface topography. a. For glass coupons, sonicate coupons individually in plastic 50 mL conical tubes for 30 s in 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. b. Rinse coupons with reagent grade water and sonicate for 30 s in reagent grade water. Repeat rinsing and sonication with reagent grade water until no soap is left on the coupons. c. Once the coupons are clean, prevent oils and other residue from contaminating the surface. d. 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. e. Place rods into reactor top loosely (not yet fitted into notches). 12.3 Preparation of reactor top a. Invert the reactor top and place baffled stir bar onto glass rod positioned in the center of the reactor top. b. Place assembled top into the reactor beaker. c. Connect the bacterial air vent by fitting the vent to a small section of appropriately sized tubing and attach to one of the rigid tubes on the reactor top. d. Splice the glass flow break into the nutrient tubing line near the reactor top. 12.4 Sterilization of reactor system Cover the end of the injection 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 or plastic caps. Steam-sterilize the empty reactor system for 20 min. 12.5 Growth of biofilm in CDC reactor - batch phase If an effluent sample needs to be taken from the reactor, insert a sterile plastic 3-way stopcock (e.g., Kendall Argyle EZ-FLO [173518]) in the closed position into the overflow (waste) line. Making sure that the overflow (waste) line is clamped, aseptically add 500 mL of the batch culture medium (TSB, 300 mg/L) to the 5 Note: this procedure is listed in ASTM E2562-12 for polycarbonate coupons. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 9 of 14 cooled reactor (e.g., carefully remove one rod, pour the medium into the reactor through the rod opening, and re-insert the rod). c. Secure the rod alignment pins into the reactor top notches. d. Place prepared reactor on a stir plate. e. Clamp the flow break in an upright position. f. Inoculate the reactor with 1 mL of culture from the second transfer (-BF-B) (see 12.1b) through one of the available rigid stainless steel tubes in the reactor top. g. Turn on the magnetic stir plate. The rotation speed should equal 125 ± 5 RPM. Incubate the reactor system in batch phase at room temperature (21 ± 2°C) for 24 ± 2 h. i. Culture tracking: add "-BF-C" after the number of the frozen stock culture vial in the organism transfer number to denote the third transfer (C) of a biofilm culture (BF) (see section 14 for culture tracking form). 12.6 Growth of biofilm in CDC reactor - CSTR mode a. Prepare the growth media for the CSTR mode (100 mg/L TSB). i. For example, add 50 mL of concentrated TSB (40 g/L) to 20 L sterile water. ii. After adding the concentrated TSB to the 20 L of sterile water, shake the carboy to thoroughly mix the contents. b. Aseptically connect the nutrient tubing line to the carboy containing the CSTR mode growth medium. c. Pump a continuous flow of nutrients into the reactor at a flow rate of 11.7 ± 0.2 mL/min or appropriate flow rate per the reactor's operating volume. Connect the end of the reactor drain to the waste carboy and remove the clamp (open the 3-way stopcock if present). The drain spout on the beaker allows overflow to occur, maintaining a constant bacterial liquid growth medium concentration of 100 mg/L in the reactor during CSTR mode. NOTE: Flow rate is calculated by dividing the reactor volume by the residence time (30 minutes). The reactor volume (with the 8 rods and baffled stir bar operating at 125 rpm) is approximately 325 mL (see NOTE, section 11.12a). Set an exact flow rate based on the operating volume of the reactor to achieve a 30 min residence time. d. Operate the reactor in CSTR mode for 24 ± 2 h. 6 MLB revision, not in ASTM E2562-12. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 10 of 14 i. Culture tracking: add "-BF-D" after the number of the frozen stock culture vial in the organism transfer number to denote the fourth step (D) in production of a biofilm culture (BF) (see section 14 for culture tracking form). e. For conducting disinfectant efficacy evaluations, proceed to SOP MB-20, Evaluation of Disinfectants against Pseudomonas aeruginosa Biofilm Grown in a High Shear Continuous Flow Reactor Model. 13. Data Analysis/ Calculations N/A 14. Forms and Data Sheets 1. Attachment 1: Culture Initiation Flow Chart for P. aeruginosa 2. Test Sheets. Test sheets are stored separately from the SOP under the following file names: Biofilm Organism Culture Tracking Form MB-19-02 F1 .docx Test Microbe Confirmation Sheet (Quality MB 02 F3 docx Control) - 15. References 1. ASTM International, 2012. E-2562-12: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow Using CDC Biofilm Reactor. 2. Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD. P. aeruginosa p. 164. 3. Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and Company. Part no. 882020191JAA. Revision 07/2011. 4. Package Insert - Oxidase Reagent Droppers. Becton, Dickinson and Company. Part no. LOO 1133. Revision 06/2010. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 11 of 14 Attachment 1 Culture Initiation and Stock Culture Generation Flow Chart for P. aeruginosa (D Rehydrate ampule. Incubate © Transfer entire rehydrated pellet to TUBE A. Ampule TSB TUBEA TUBEA (pre-incubation) (post-incubation) ©StockCulture Generation Inoculate TSA plates with 100 jliL culture from TUBE A; incubate. Harvest inoculum from plates. ¦ HE Prepare frozen stock cultures © Culture ID & Quality Control BAP Selective media A Gram Additional Stain confirmation steps (see Attachment 3) Al. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the organism control number. a. Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa (ATCC 15442) from ATCC within 18 months. b. Open ampule of freeze dried organism as indicated by ATCC. Using a tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into the original tube of broth designated as "TUBE A." Mix well. c. Incubate broth culture (TUBE A) at 36 ± 1°C for 24 ± 2 hours. Record all ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 12 of 14 manipulations on an organism culture tracking form (see section 14). d. Using a sterile spreader, inoculate a sufficient number of TSA plates (e.g., 5 to 10 plates per organism) with 100 |iL each of the culture. Incubate plates at 36 ± 1°C for 24 ± 2 h. e. Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol) to the surface of each agar plate. Re-suspend the cells in this solution using a sterile spreader or a sterile swab and aspirate the cell suspension from the surface of the agar. Transfer the suspension into a sterile vessel. Repeat by adding another 5 mL of cryoprotectant to the agar plates, re-suspend the cells, aspirate the suspension and pool with the initial cell suspension. f. Mix the pooled contents of the vessel thoroughly. Immediately after mixing, dispense approximately 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials). Perform QC of stock cultures concurrently with freezing (see section A2: QC of Stock Cultures). g. Place and store the cryovials at -70°C or below; these are the frozen stock cultures. Stock cultures may be used up to 18 months; reinitiate using a new lyophilized culture. These cultures are single-use only. A2. QC of Stock Cultures. a. Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on a plate of BAP. In addition, streak a loopful onto selective media (MSA and Cetrimide). Incubate all plates at 36 ± 1°C for 24 ± 2 hours. b. Following the incubation period, record the colony morphology as observed on the BAPs and selective media plates (including the absence of growth) and Gram stain. See Attachment 2 for details on cell and colony morphology, colony characteristics on selective media, and stain reactions. c. For each organism, perform a Gram stain (refer to 15.3) from growth taken from the BAPs according to the manufacturer's instructions. Observe the Gram reaction by using brightfield microscopy at 1000X magnification (oil immersion). d. For additional confirmation steps refer to the Confirmation Flow Chart for P. aeruginosa (see Attachment 3). Refer to 15.4 for additional instructions. e. Record all confirmation results on the Test Microbe Confirmation Sheet (Quality Control) (see section 14). ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 13 of 14 Attachment 2 Typical Growth Characteristics of strains of P. aeruginosa (see ref. 15.2). P. aeruginosa* Gram slain reaction (-) Typical Growth Characteristics on Solid Media Mannitol Salt No Growth Cclrimide circular, small, initially opaque, turning fluorescent green over time; agar fluorescent yellowish green Blood agar (BAP) flat, opaque to off-white, round spreading (1). metallic sheen, slightly beta hemolytic Typical Microscopic Characteristics Cell dimensions 0.5-1.0 nm in diameter by 1.5-5.0 |im in length* Cell appearance straight or slightly curved rods, single polar flagella, rods formed in chains *After 24±2 hours (1) Test organism may display three colony types: a) circular, undulate edge, convex, rough and opaque; b) circular, entire edge, convex, smooth and translucent; c) irregular, undulate edge, convex, rough, spreading, and translucent. Pyocyanin is not produced. ------- SOP No. MB-19-02 Date Revised 08-06-13 Page 14 of 14 Attachment 3 Confirmation Flow Chart for P. aeruginosa P. aeruginosa Identification Not P. aeruginosa P. aeruginosa ------- |