||high school biology, independent study/science fair, introductory undergraduate microbiology, advanced college level microbiology|
|Revision Date||November 20, 2003|
The basic methods of conducting serial dilutions and of plate counting should be reviewed prior to carrying out this exercise.
|4||sterile dilution tubes with caps (18 x 150 mm), each containing 9 ml of PBS. Note: number of dilution tubes needed may vary according to the biofilm being sample|
|4-5||R2A agar (Difco) plates per sample|
|1 ml||automatic pipetting device (Rainen digital EDP 2, Emeryville, CA), or any accurate microliter pipetting device|
|1 ml||sterile 1 ml pipette or pipette tips|
|As Necessary||phosphate buffered saline (PBS)|
To 900 ml of distilled water add 8.0 g sodium chloride, NaCl; 0.2 g potassium chloride, KCl; 0.2 g potassium phosphate, monobasic, KH2PO4; 0.1 g magnesium chloride, hexahydrate, MgCl2.6H2O; and 1.15 g sodium phosphate, dibasic, Na2HPO4.
Dissolve completely and add 0.10 g calcium chloride, CaCl2, dissolved in a little water. Adjust to pH 7.4 with either HCl or NaOH as appropriate. Adjust total volume to 1 liter by adding distilled water. Sterilize by autoclaving2.
|As Necessary||Sharpie marking pens|
|1||biohazard bag for disposal of plates, pipette tips, etc.|
Substitute 4 sterile microcentrifuge tubes containing 900 µl of PBS for the 4 sterile dilution tubes with caps
This exercise results in the ability to enumerate the bacteria from a biofilm and can be used to analyze biofilm growth experiments described in other exercises.
How to optimize the drop plate method for enumerating bacteria
Herigstad B, Hamilton M, Heersink J
J Microbiol Meth, 2001; 44(2):121-129
Measuring antimicrobial effects on biofilm bacteria: From laboratory to field
Zelver N, Hamilton M, Pitts B, Goeres D, Walker D, Sturman P, Heersink J
in R.J. Doyle, et al. (eds), Biofilms: Methods in Enzymology, Academic Press, San Diego, CA, 1999, pp.608-628.
1Example exercises of how to grow biofilms on objects can be found at http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/. See Buried Slide Technique, Microbial Fishing. This referred site is not maintained by the Biofilm Institute and the Biofilm Institute is not responsible for the site contents.
2Standard methods for the examination of water and wastewater : including bottom sediments and sludges 18th ed. ©1992 American Public Health Association, New York. A reprint of this paper can be obtained by emailing the Center for Biofilm Engineering, firstname.lastname@example.org. Request paper 01-021
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