Drop Plate Method for Counting  Biofilm Cells

Instructor Version (go to Student Version)

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

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.

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.

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.

INSTRUCTIONAL PROCEDURES

1. A final result from the drop plate method will be shown to the students.
2. 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.
3. The individual parts of a drop plate will be shown and explained relative to their context as part of a completed drop plate method.
4. 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.
5. Students will begin performing the method by following the instructions (Attachment) and soliciting help and feedback from the instructor as needed.
6. Students will test their method by repeating it and comparing results.

MATERIALS AND EQUIPMENT (for Standard Method)

Materials

Quantity	Description
As Necessary	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 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.

Equipment

Quantity	Description
1	Vortex mixer
As Necessary	Sharpie marking pens
1	small ruler
1	biohazard bag for disposal of plates, pipette tips, etc.

MATERIALS AND EQUIPMENT (for Alternate Method)

Substitute 4 sterile microcentrifuge tubes containing 900 µl of PBS for the 4 sterile dilution tubes with caps

ASSESSMENT / EVALUATION

Assessment will be made by the instructor through visual evaluation of each student's drop plate results.

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.

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.

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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, publications@biofilm.montana.edu.  Request paper 01-021

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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, Penn State University-Altoona
© 1999-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu