Colorimetric Measurement of Biofilm Density

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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:  December 13, 2004

1. CONTENT

This exercise is a research technique modified for teaching laboratories, by which the amount of biofilm growing in the wells of polysterene plates can be estimated by colorimetric analyses.

2. PREREQUISITES

Students should be able to define biofilm, be able to describe the differences between biofilm (surface attached) and planktonic (free-floating) cells, and be able to describe why bacteria tend to grow on surfaces. Students should also be familiar with the use of a simple laboratory spectrophotometer or colorimeter. Students should understand standard safety procedures and methods for the safe handling and disposal of microorganisms. For this reason, gloves should be worn during this exercise and a nonpathogenic microorganism should be used.

3. INSTRUCTIONAL OBJECTIVE

Given materials readily available in many school laboratories and detailed instructions, the student should be able to estimate the relative density of biofilm growing on the wall of a 24 well polysterene plate. This technique has many applications in determining the effect of nutrient concentration, strain differences in biofilm production, the effect of temperature, antimicrobics and other environmental variables.

4. INSTRUCTIONAL PROCEDURES

a. Students will be provided with all materials required for the growth and analysis of biofilms in polysterene plates, with detailed instructions and diagrams for carrying out this procedure.
b. If this has not been done previously, students will be introduced to the theory and operation of a laboratory spectrophotometer or colorimeter.
c. Students will be given an opportunity to review the materials and instructions and ask questions concerning the procedure.

Preparation of the 24 well polysterene plate cultures
d. Students will be provided with a sterile 24 well plate, an appropriate sterile broth medium (1/10 strength Tryptic Soy Broth or LB Broth), and a culture of an appropriate microorganism.
e. The students will aseptically transfer 2 ml aliquots of the medium to designated wells of the plate using sterile pipettes.
f. These wells will be aseptically inoculated with an appropriate pure culture and the plates will be incubated.
g. At the end of the incubation period the wells are drained using a vacuum device. Do not let the wells dry out.
h. The wells are then washed with distilled or tap water at least three times. Depending on the pathogenicity of the organisms used, the wash water may be disposed of down the drain or into a container that will be subsequently disinfected (by autoclaving or other means).
i. Crystal violet dye (1%) is then added to each of the wells. The volume of dye should be enough to cover the depth of the culture (approx 3 ml). Crystal violet stains microbial cells intensely. After staining the wells for 5 minutes, the crystal violet is suctioned out using the same device as in step g above.
j. The wells are again washed (at least three times) with proper disposal of the wash water.
k. A 2 ml volume of 95% ethanol is added to each well. Note: Since crystal violet is very soluble in ethanol all of the biofilm-associated dye is re-eluted.
l. The 2 ml of ethanol dye mixture is transferred to a cuvette. An additional 2 ml of ethanol is added to the cuvette, bringing the final volume to 4 ml. Since the original culture volume is 2 ml and the ethanol dye mixture volume is 4 ml, the actual absorbance is twice the measured value.

5. MATERIALS AND EQUIPMENT

a. sterile, 24-well polysterene plates
b. Tryptic Soy broth or LB broth, 1/10 strength
c. 95% ethanol
d. 1 % aqueous crystal violet dye

Equipment
e. laboratory spectrophotometer or colorimeter
f. cuvettes for the above
g. vacuum suction device (see diagram in Student Instructions)
h. pipettes - 2 ml and 5 ml volumes (need as many pipettes as you have wells)
i. PiPumps - 2 ml and 10 ml volumes or other pipetting devices (Note: Under no circumstances should mouth pipetting be permitted.)

6. ASSESSMENT / EVALUATION

This exercise may be assessed either by the student or the instructor. One can evaluate the consistency of measurements on identical samples by calculating standard deviation, the smaller the SD the greater the precision of the measurement.

7. FOLLOW-UP ACTIVITIES

This protocol can be used in any number of exercises. Students could be asked to design exercises to determine:

a. The effect of nutrient concentration on biofilm formation
b. The effect of shaking on biofilm formation.
c. The effect of surfactants (e.g. sodium dodecyl sulfate, SDS) on biofilm formation.
d. The effect of temperature, pH, osmotic pressure and other environmental factors on biofilm accumulation.
e. The effect of anti-microbics such as disinfectants, antibiotics and heavy metals on biofilm formation.
f. The effect of grazing by protozoa, nematodes or rotifers on biofilm mass.

8. ATTACHMENTS

a.  Detailed student instructions and illustrations forthe colorimetric measurement of biofilm mass.
b. Illustration for making the vacuum suction devise used in this exercise.

9. REFERENCES

George A. O’Toole, Leslie A. Pratt, Paula I. Watnick, Dianne K. Newman, Valerie B. Weaver and Roberto Kolter, 1999, Genetic Approaches to Study Biofilms, IN, Methods in Enzymology: Volume 310, pp. 91-109, Ron J. Doyle Ed. Academic Press, San Diego.
A reprint of this paper can be obtained by emailing the Center for Biofilm Engineering, publications@erc.montana.edu.

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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
©2002-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu

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