Chapter 11 Lab Exercises
Section 14 Model System for Growing a Standard Biofilm: Static Glass Coupon Reactor
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Model System for Growing a Standard Biofilm: Static Glass
Coupon Reactor
Introduction:
The ability to create biofilms with typical characteristics
that are consistently reproducible is of great importance to
researchers in medicine, industry and academia. The technique
described here was devised in an industrial laboratory to
create uniform, reproducible biofilms for testing the
effectiveness of cleansing and disinfecting products. Prior to
the creation of this technique, industries wishing to test
their products typically would spray planktonic cells onto
sterile glass slides, dry them and then spray them with the
selected product. Often the “kill” rate observed in
these laboratory tests and the rate observed on naturally
occurring microbial populations on surfaces were markedly
different. This observation is explained by the unique
properties of biofilms that include many phenotypic differences
from planktonic cells including resistance to antimicrobic
substances.
Supplies Needed:
Quantity |
Description |
1 |
square petri plate (10 x 10 cm) of Trypticase Soy
Agar |
2 |
tubes containing 9 ml of 1/10 strength sterile
Trypticase soy broth |
1 |
piece of sterile Whatman #2 filter paper wrapped in
aluminum foil |
2 |
sterile pipettes, 1 ml size |
4 |
glass slides (1x3 inch) |
1 |
pair forceps |
1 |
beaker of alcohol for flame sterilizing forceps and
glass slides |
1 |
gloves |
1 |
Overnight culture of Pseudomonas
putida |
Instructions:
Constructing a Model Biofilm - Period 1
- Select one of the square petri plates containing
Trypticase Soy Agar (TSA).
- Onto the surface of the agar, aseptically place one
sheet of sterile filter paper. Do this by opening the
aluminum foil package being careful not to touch the
sterile filter paper sheets inside. Sterilize a pair of
forceps by dipping them in a container of alcohol and
flaming them. When the flames go out the forceps are
sterile. Pick up one filter paper sheet and transfer it to
the surface of the TS agar plate. Place the sheet so that
no air bubbles are trapped under the filter paper. Trapped
bubbles can be removed by pushing them with the sterile
forceps to the edge of the filter paper.
- Dilute the overnight culture by pipetting 1 ml of the
culture into 9 ml of 1/10 normal strength TSB. Thoroughly
mix the culture by vortexing or by rolling the tube
containing the culture between the palms of your
hands.
- Uniformly moisten the filter paper with 1 ml of the
diluted bacterial culture.
- Select a clean 1 X 3 inch microscope slide and using
the forceps, immerse the slide in a container of alcohol
and sterilize it by flaming. When the flame goes out the
slide is sterile. Allow the slide to cool for a few seconds
and then carefully lay the slide on top of the filter
paper. Press the slide down with the forceps, being careful
that no air bubbles are trapped beneath the slide.
- Repeat step 5 three more times. If you are careful, you
should be able to place four 1” X 3” slides
side by side on the plate
(See Figure 1).
- Between Periods 1 and 2 If possible, remoisten the plate
after 24 hours with 1 ml of 1/10 strength TSB. Note: If
scheduling problems make this impossible this step may be
omitted, but more robust biofilms are produced if this is
done.
Period 1
- After two days the biofilms may be harvested by
carefully lifting the slides from the filter paper surface
with a pair of sterile forceps (flamed). The biofilm should
appear as a slimy layer coating the under surface of the
slide. Lifting too rapidly may disturb the biofilm causing
large sections to slough off the slide.
- Be sure to dispose of all materials according to your
teacher’s instructions.
Illustration:
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 1. Cross sectional diagram of the
construction of a Static Glass Coupon Reactor.
Reference
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.
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
©1999-2008 Center for Biofilm Engineering,
http://www.biofilm.montana.edu