Building and Using a Biofilm Continuous Flow Stirred Reactor

---

Subject Area(s): microbiology
Intended Audience: high school biology, independent study/science fair, introductory undergraduate microbiology, advanced college level microbiology
Type: laboratory exercise
Revision Date: June 24, 2004

  1. CONTENT

This exercise describes the construction of a continuous flow, stirred biofilm reactor.  The materials used are both economical and readily available. The resulting reactor will be suitable for growing bacterial biofilms that can be subsequently analyzed, as described in other exercises.

A continuous flow through biofilm reactor gives a more realistic growth of biofilms with fewer planktonic cells than does growth in a batch reactor.  However, either method of growing biofilms is adequate to perform the companion exercises.

2. PREREQUISITES

Students should be able to define a biofilm, describe the differences between biofilm (surface-attached) and planktonic (free-floating) bacteria, and describe why bacteria tend to grow on surfaces.

3. INSTRUCTIONAL OBJECTIVE

Given readily accessible materials, detailed instructions and figures, and a finished reactor system, a student will be able to construct a simple reactor system that is suitable for growing biofilms on standard glass microscope slides.

4. INSTRUCTIONAL PROCEDURES

a. A finished and working continuous flow, stirred reactor system will be shown to the students.
b. Students will be provided with a complete kit of materials required for assembling the reactor. This kit will include all reactor parts, and any necessary tools or other construction supplies, detailed written assembly instructions, and detailed illustrations.
c. The individual parts of an unassembled reactor will be shown and explained relative to their context as part of the finished reactor.
d. Students will review their parts kit, written assembly instructions and associated illustrations, followed by a brief period for feedback between students and instructor in order to clarify construction details.
e. Students will begin constructing the reactor by following the assembly instructions (Attachment A) and soliciting help and feedback from the instructor as needed.
f. Students will test their reactor by filling it with water and operating it on the stir plate for several minutes, observing the relationship between stir bar speed and reactor mixing hydrodynamics.

5. MATERIALS AND EQUIPMENT  

1 - wide mouth polycarbonate sample jar with cap (Cole-Parmer U-06101)
3 - #1 (1-holed) rubber stoppers
4 - #6 rubber stoppers
4 - 1x3 inch microscope slides (1mm thick)
1 - fine tooth modeling saw  
1 - magnetic stir bar (a 1 inch teflon bar works well)
360 ml (approx) 1/10 Nutrient Broth, liquid Luria-Bertani (LB) broth, or any other desired medium
1 - Millipore sterile air vent (50 mm) (Millex-FG 50)
1 - influent port (#1 1-holed rubber stopper with glass tubing inserted for in flow)
1 - effluent port (#1 1-holed rubber stopper with glass tubing inserted for out flow)
*The ports in the reactor lid and side can be cut with a laser engraver, drill, or drill press with a 1 1/16 inch paddle-type bit and a 1/2 inch paddle-type bit.

Cole-Parmer  http://www.coleparmer.com
Millipore  http://www.millipore.com/

a. Four of the access ports in the lid of the polyethylene jar may be cut with a 1 1/16 inch paddle bit mounted in a drill press. Four 1 1/16 inch holes are placed in a crosswise pattern and centered midway between the center and the edge of the lid (see diagram).

b.  Two other access ports in the lid of the polyethylene jar may be cut with a 1/2 inch paddle bit mounted in a drill press.  One of these 1/2 inch holes is placed in the center of the lid (see diagram) and will be used as an air vent.  The other 1/2 inch hole is placed in the outer diameter of the lid, and is used for inflow (see diagram).

c.  A third hole of 1/2 inch (use same type 1/2 inch paddle bit) is bored in the reactor side for the effluent (out flow) port. The position of this port determines the volume of the reactor. 

d. The microscope slides are supported in #6 rubber stoppers mounted in the holes in the lid of the reactor. The slides are slipped into 1cm deep slots cut into the narrow end of the rubber stopper. A useful tool for cutting these slots is a fine toothed modeling saw (available at craft and hobby shops).

Note:  Erie Scientific Company manufactures printed microscope slides available in a large variety of formats.  The ER-243 is printed to expose 10, 7mm diameter wells on the slide surface.  When inserted in a biofilm producing environment,  these provide 10 distinct regions for biofilm formation of known diameter. The wells are easily scraped to recover the adherent cells.  If the depth of the biofilm is measured in one of these wells, the approximate biofilm volume (p r² h) can also be easily determined. Erie Scientific Company, 20 Post Road, Portsmouth Industrial Park, Portsmouth, New Hampshire 03801-5691.

Two useful methods of feeding the reactor are gravity feed and by peristaltic pump.  Planktonic bacteria are kept to a minimum by having the reactor residence time be less than the organism doubling time.   Residence time is controlled by the media flow rate.   Most bacteria will be on the microscope slide coupons and on the walls of the reactor.

Waste should be collected and disposed of as with any other  microbiological waste.

6. ASSESSMENT / EVALUATION

Assessment will be made by the instructor through visual evaluation of each student's reactor system and by testing it in operation.

7. FOLLOW-UP ACTIVITIES

This exercise results in the construction of a flow through biofilm growth reactor system that can be used for biofilm growth experiments described in other exercises.

Companion exercises: 

1. Gram staining.
2. Harvesting biofilm associated cells from the glass slides by scraping and sonication and finding their enumeration by the drop plate method.

8. ATTACHMENTS

a.  Detailed instructions and illustrations for assembling the reactor system.

9.  REFERENCES 

Effects of culture conditions and biofilm formation on the iodine susceptibility of Legionella pneumophila
Cargill KL, Pyle BH
Can J Microbiol 1992; 38:423-429

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection
Yu FP, Pyle BH, McFeters GA
J Microbiol Meth 1993; 17:167-180

---

Educational Program Curricula and Teaching Resources
Supported in part by the Waksman Foundation for Microbiology
Developed in collaboration with Dr. John Lennox, Penn State University-Altoona
© 1999-2006 Center for Biofilm Engineering, http://www.erc.montana.edu

NOTE: Links marked by the 3-arrow sign take you off the BiofilmsOnline.com web site. The Center for Biofilm Engineering is not responsible for content of sites marked with the 3-arrow sign.

Return to top

Return to Laboratory Exercises

Return to Education homepage