Chapter 11 Lab Exercises
Section 7 Colorimetric Measurement of Biofilm Density
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Colorimetric Measurement of Biofilm Density
Supplies Needed:
Materials
Quantity |
Description |
As Necessary |
sterile, 24-well polysterene plates |
As Necessary |
Tryptic Soy broth or LB broth, 1/10 strength |
As Necessary |
95% ethanol |
As Necessary |
1 % aqueous crystal violet dye |
Equipment
Quantity |
Description |
1 |
laboratory spectrophotometer or colorimeter |
As Necessary |
cuvettes for the above |
1 |
vacuum suction device (see diagram in Student
Instructions) |
As Necessary |
pipettes - 2 ml and 5 ml volumes (need as many
pipettes as you have wells) |
As Necessary |
i. PiPumps - 2 ml and 10 ml volumes or other
pipetting devices (Note: Under no circumstances should
mouth pipetting be permitted.) |
As Necessary |
rubber/latex gloves |
Instructions:
Growing the biofilm
- With a sterile pipette, and a 2 ml PiPump, transfer 1.8
ml of 1/10 strength Tryptic Soy Broth or LB broth into 2
wells of a 24 well polysterene plate.
- Inoculate one of the two wells with 0.2 ml of a
microbial pure culture, a soil suspension1, or some other
material as indicated by your instructor.
- Place the plate on a platform shaker or a tilt table.
Agitation has been observed to produce denser and more
robust biofilms. Incubate for 24 hours or longer.
- Soil suspensions may be made by transfering 1 gram of
soil into 100 ml of sterile distilled water. Shake the
suspension and allow it to settle for 1-2 minutes. With a
sterile pipette withdraw a 0.2 ml sample from the
liquid.
Estimating biofilm mass
- From this step on, use latex or rubber gloves.
- Using the vacuum device suction liquid from the culture
in the two wells, but do not let the wells dry out. Be
careful not to damage the biofilm adhering to the side and
floor of the well.
- Wash the wells by filling the wells with tap water and
dumping the wash water into the sink or into a designated
waste container, depending on the virulence of the organism
used.
- Repeat step f twice more.
- With a 5 ml pipette add 2 ml of 1% crystal violet (CV)
dye solution to each well and stain for 5 minutes.
- Using the same vacuum device (step e), remove the CV
dye from each well.
- Wash the wells 3 times, as in steps f and g.
- Drain all of the wells.
- Observe the distribution of CV dye on the walls of the
microtiter wells.
- Add 2 ml of 95% ethanol to each well to re-elute the CV
dye. If a platform shaker is available, shake gently for
two minutes. If a platform shaker is not available,
manually shake the polysterene wells until all the crystal
violet dye is re-eluted.
- Transfer the ethanol/CV solution to an appropriate
cuvette with a pipette and add 2 ml of fresh ethanol to
bring the volume to 4 ml.
- Measure the absorbance of the ethanol/CV solution at A
570-600. Remember that you have diluted the original
culture volume 1:2, so it is necessary to double the
absorbance reading.
- The absorbance of the uninoculated well represents the
negative control. This value should be subtracted from the
value for the inoculated well.
Illustrations:
Permissions
J. Lennox, Penn State Altoona
Figure 1. A 24 well dilution series.
Three students did an independent study project using the
technique described here to determine the optimum nutrient
concentration for the production of biofilm on 24 well
polysterene plates. The following graph is a result of their
effort.
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 2. Illustration of vacuum suction device
Permissions
J. Lennox, Penn State Altoona
Figure 3. An example of the effect of nutrient
concentration on biofilm formation.
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.biofilm.montana.edu