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

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Withdrawn By: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

Contents	Page Number

















SOP No. MB-19-01
Date Revised 04-13-11
Page 3 of 17


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.


2.1	ASTM = ASTM International, formerly American Society for Testing and

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 120C. Prepared according to Method 9050,
Standard Methods for the Examination of Water and Waste Water, 19th Edition.


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.


4.1	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.


5.1 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


6.1	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.


7.1	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

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 6C + 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

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

7.17	Glass flow break - any that will connect with tubing of ID 3.1 mm and withstand

7.18	Clamp - used to hold flow break, extension clamp with 0.5 cm minimum grip

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

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.


8.1	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.



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 Record all manipulations on the Biofilm - Organism Culture
Tracking Form (see 14.1).

10.1.2	Organism Tracking: Supply Control Number.	All cultures (dehydrated ampules as received from ATCC) are
given a supply control number upon receipt (see SOP QC-09,
Control Numbers).	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).	The MRME number will consist of the date received (MR) and
the date the reconstituted microbe expires (ME) - i.e., one year
later.	A culture received on 06-21-11 and reconstituted on 07-28-11
would be notated MR062111ME072812.	Once reconstituted, the organism can only be transferred for a
period of one year. 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

10.2 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

10.2.2	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

10.2.3	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 361C 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
361C 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.	For glass coupon, sonicate coupons individually in 50 mL
centrifuge plastic tubes for 605 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.	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.	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	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.	Place rods into reactor top loosely (not yet fitted into notches).

10.3.2	Preparation of Reactor Top.	Place baffle onto glass rod suspended from the reactor top.	Place assembled top into the reactor beaker.	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.	The glass flow break is spliced into the nutrient tubing line
near the reactor top.

10.3.3	Sterilization of the Reactor System.	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.	Sterilize the empty reactor system for 20 minutes on gravity
cycle.	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. Place prepared cooled reactor (from above) onto a stir

SOP No. MB-19-01
Date Revised 04-13-11
Page 13 of 17	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.	Turn on the magnetic stir plate. The rotation speed should
equal 125 RPM. The reactor system incubates in batch mode
at room temperature (212C) for 241 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) 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.	After adding the concentrated TSB to the 20L of sterile water,
place the carboy on a large stir plate and mix vigorously for >
51 minutes.	Aseptically connect the nutrient tubing line to the carboy
containing the continuous flow nutrient broth.	Pump a continuous flow of nutrients into the reactor at a flow
rate equal to 11,70.2 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. Operate the reactor in CSTR mode for 242 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.



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.


13.1	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).


14.1 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.


15.1 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.


16.1 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

OPP Microbiology Laboratory	


Pseudomonas aeruginosa

Supply Control Number:

Source and Strain no.:


Lot N umber:




Subculture Source

T ransler*

Media Inoculated
(and # inoc.)

Media Prep No.





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

OPP Microbiology Laboratory	


Pseudomonas aeruginosa

MRME Number:

Source and Strain no.:






Media Information



Prep. No.

Inc. Time/




* Record Gram stain results, GNR = Gram Negative Rods
MB-19-1 F2.doc