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Drop Plate Method for Counting Biofilm Cells
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: November 20, 2003
1. CONTENT
This exercise describes a technique (drop plating) for enumerating biofilm
cells that can be used instead of standard spread or pour plating
techniques. The materials for the technique are economical and readily available.
An entire dilution series
with replicates can be obtained on just two plates of medium. Instead of
the typical 0.1 ml of sample being plated, 5 x 10 µl aliquots are placed on
the surface of the plate in drops. The plate is duplicated and the number
of colonies appearing in the 10 x 10 µl drops are averaged to determine
the plate count.
2. PREREQUISITES
Students should be able to define a biofilm, describe the differences between biofilm (surface-attached) and planktonic (free-floating) bacteria, and describe why bacteria tend to grow on surfaces.
The basic methods of conducting serial dilutions and of plate counting
should be reviewed prior to carrying out this exercise.
3. INSTRUCTIONAL OBJECTIVE
Given readily accessible materials, detailed instructions and figures, and a finished
drop plate, a student will be able to follow the technique and protocol
to enumerate and analyze the biofilms they have grown.
4. INSTRUCTIONAL PROCEDURES
a. A final result from the drop plate method will be shown to the students.
b. Students will be provided with a complete kit of materials required
to complete the drop plate method. This kit will include all lab
materials, instructions, and an illustration.
c. The individual parts of a drop plate will be shown and explained relative to their context as part of
a completed drop plate method.
d. Students will review their parts kit, written instructions and associated illustrations, followed by a brief period for feedback between students and instructor in order to clarify
method protocols.
e. Students will begin performing the method by following the instructions (Attachment A) and soliciting help and feedback from the instructor as needed.
f. Students will test their method by repeating it and
comparing results.
5. MATERIALS AND EQUIPMENT (for Standard Method)
biofilm1
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 sampled
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
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.
Vortex mixer
Sharpie marking pens
small ruler
biohazard bag for disposal of plates, pipette tips, etc.
6. ASSESSMENT / EVALUATION
Assessment will be made by the instructor through visual evaluation of each student's
drop plate results.
7. FOLLOW-UP ACTIVITIES
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.
8. ATTACHMENTS
a. Detailed student instructions and illustrations for
completing a drop plate method .
9. REFERENCES
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.
____
1 Example 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.
2 Standard 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, publications@biofilm.montana.edu. Request paper 01-021
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
NOTE: Links marked by the 3-arrow sign take you off the BiofilmsOnline.com web site. The Center for Biofilm Engineering is not responsible for content of sites marked with the 3-arrow sign.
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Industrial Associates
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Biofilm Engineering
at Montana State
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