Model System for Growing a Standard Biofilm: Static Glass Coupon Reactor

Instructor Version (go to Student Version)

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	July 19, 2004

CONTENT

This exercise describes a method by which standardized and reproducible biofilms can be grown with readily available and inexpensive materials. These biofilms can be used in a variety of exercises contained within this collection or may be used as a starting point for student created independent investigations.

PREREQUISITES

Students should be able to define a biofilm, describe the differences between biofilm (surface-attached) and planktonic (suspended bacterial cells) bacteria, and be able to describe why bacteria usually grow on surfaces. Students should also be familiar with standard methods handling and transferring microbial cells with aseptic technique.

INSTRUCTIONAL OBJECTIVE

Given readily available materials, detailed instructions and diagrams, the student will be able to grow biofilms on 1 X 3 inch glass slides (coupons) which are uniform and reproducible. These model biofilms may be used in a variety of laboratory exercises including harvesting and enumerating cells, microscopic observation and demonstrating resistance of biofilms to antimicrobics.

INSTRUCTIONAL PROCEDURES

1. Photographic or diagrammatic images of biofilms will be shown to the students. These should illustrate the complex community in which biofilm cells typically grow. See biofilm images at the ASM MicrobeLibrary
2. Students will be provided with materials required for growing a biofilm along with detailed instructions and diagrams for carrying out this procedure.
3. The students will be given an opportunity to review the materials and instructions and to ask questions concerning the procedure.
4. The resulting biofilm can be used for several other exercises. (See companion exercises).

MATERIALS AND EQUIPMENT

Quantity	Description
As Necessary	square petri plates (10 x 10 cm) of Trypticase Soy Agar (BD BBL 211043), poured to a depth of at least 5 mm (approximately 40 ml/plate)
As Necessary	tubes containing 9 ml of 1/10 strength sterile Trypticase soy broth (BD BBL 211768) at 1/10 normal strength
As Necessary	Whatman #2 filter paper cut to fit into the square petri dishes. These filter paper inserts can be wrapped in aluminum foil (clusters of 10) and autoclaved
As Necessary	sterile pipettes, 1 ml size
As Necessary	an overnight culture of Pseudomonas putida or other organism in a liquid medium such as Tryptic soy broth (1/10 normal strength is preferable)
As Necessary	glass slides (1x3 inch) Note: Erie Scientific manufacturers preprinted slides with wells of standard size.  http://www.cel-line.com/brand/standard.html .  If these slides are used, the student can later on sample a surface area of known dimensions for example, in the slides with an 8mm diameter, each well has a surface area of 50.26 mm1.
1	forceps
1	beaker of alcohol for flame sterilizing forceps and glass slides
As Necessary	gloves

ASSESSMENT / EVALUATION

Assessment may be made by the instructor through a naked eye or microscopic examination of a representative biofilm. An estimation of the cell density of the biofilm can be made by having students scrape a known region of the slide and enumerate the cell number present by the techniques described in the exercises called Harvesting and Dispersing of Cells from Biofilms and Drop Plate Method for Counting Biofilm Cells.

FOLLOW-UP ACTIVITIES

The production of these model biofilms can lead to a large number of other exercises, including plate counting, drop plate counting, and any other exercise in which a population of cells of biofilm origin is required (antimicrobic resistance testing for example)

Companion exercises:

This procedure may be linked to several other exercises, including Harvesting and Dispersing of Cells, Dilution and Enumeration, Drop Plate Method for Counting Biofilm Cells and Gram Staining by the Flow Through Gram Stain Method.

REFERENCES

Charaf UK, Bakich SL, and Falbo DM, 1999, A Model Biofilm for Efficacy Assessment of Antimicrobials Versus Biofilm Bacteria, IN Biofilms: The Good, The Bad and the Ugly. J. Wimpenny, P Gilbert, J. Walker, M. Brading and R. Bayston, Eds. Published by BioLine for the Biofilm Club, Cardiff University, UK, ISBN 0-9520432-6-2.

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Educational Program Curricula and Teaching Resources

Supported in part by the Waksman Foundation for Microbiology
Developed in collaboration with Dr. John Lennox, Education Editor, Penn State Altoona
© 1999-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu<