Chapter 11 Lab Exercises
Section 8 Drop Plate Method for Counting Biofilm Cells
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Drop Plate Method for Counting Biofilm Cells
Supplies Needed for Standard Method:
Quantity |
Description |
|
biofilm |
4 |
sterile dilution tubes with caps (18 x 150 mm) |
1 |
1 ml automatic pipetting device
|
4-5 |
sterile 1 ml pipettes or pipette tips |
As Necessary |
Sterile buffered water |
4-5 |
R2A agar plates per sample |
1 |
100 µl automatic pipetting device |
4-5 |
100 µl pipette tips |
1 |
Sharpie marking pens |
1 |
small ruler |
1 |
Vortex mixer |
Instructions:
Serial Dilutions
- Pipette 1 ml of bacterial suspension (from the
Harvesting exercise) into a dilution tube containing 9 ml
of sterile buffered water.
- Recap tube and vortex the tube for approximately 8
seconds.
- Using a new tip, pipette 1 ml of vortexed dilution tube
into a second dilution tube containing 9 ml of sterile
buffered water.
- Repeat process until there are four serial dilutions of
the original 9 ml sample. See Figure 1.
Supplies Needed for Alternate Method:
Quantity |
Description |
As Necessary |
biofilm |
4 |
sterile dilution tubes with caps (18 x 150 mm) |
1 |
1 ml automatic pipetting device
|
4-5 |
sterile 1 ml pipettes or pipette tips |
As Necessary |
Sterile buffered water |
4-5 |
R2A agar plates per sample |
1 |
100 µl automatic pipetting device |
4-5 |
100 µl pipette tips |
1 |
Sharpie marking pens |
1 |
small ruler |
1 |
Vortex mixer |
Instructions:
Serial Dilutions
- Pipette 1 ml of bacterial suspension (from the
Harvesting exercise) into a dilution tube containing 9 ml
of sterile buffered water.
- Recap tube and vortex the tube for approximately 8
seconds.
- Using a new tip, pipette 1 ml of vortexed dilution tube
into a second dilution tube containing 9 ml of sterile
buffered water.
- epeat process until there are four serial dilutions of
the original 9 ml sample. See Figure 2.
For both methods continue with these instructions
Drop Plating
- Label the bottom of the R2A agar plates by dividing
them into fourths with a ruler and marking pen (see Figure
3). Each serial dilution of the sample will occupy one
quadrant of each plate. Prepare plates in
duplicate.
- Set the automatic pipetteman to pull up 100 µl of
sample and expel 10 µl with each push of the button
(slow setting).
- Vortex sample for approximately 8 seconds.
- Pick up sample with pippetteman. Expel sample in
five evenly spaced 10 µl drops onto the quadrant of
one of the petri plates that have been labeled for that
particular dilution of the sample.
- Repeat this procedure with the duplicate plate.
Note: If only a dedicated 10 ml pipette is available,
5 drops of 10 ml each should be placed on the plate.
- Vortex sample again for approximately 8 seconds.
- Pick up 100 µl of the next dilution and repeat
steps 4-6.
- Repeat steps 4-6 for each tube in the dilution
series.
- Let the drops soak into the media before turning plates
over for incubation.
- Incubate overnight (17-20 hours) at 35-37˚C.
- Remove plates when colonies have developed and count
the dilution which contains 3-30 colonies per 10 µl
drop. Viable cell counts are expressed as colony
forming units (CFU)/surface area.
Example Calculation
Calculate LOG10 CFU/cm2 according to the following
formula:
LOG (CFU/cm2) = LOG [(average CFU/drop volume)(dilution
counted)(volume scraped into/surface area*)] *scraped
slide
= LOG [(average of plate counts/ 0.01 ml)(10dilution)(10
ml/1.267cm2)]
For example, if the average units per drop was 16.6 and they
were counted in the third dilution, then the LOG (CFU/cm2) =
LOG [(16.6 / 0.01)(103)(10/1.267)] = 7.12
Illustrations:
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 1. Dilution series followed by drop-plating
technique for the Standard Method.
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 2. Dilution series followed by drop-plating
technique for the Alternate Method.
Permissions
J. Lennox, Penn State Altoona
Figure 3. Example of a colonized agar plate,
divided into four quadrants and containing five evenly
spaced drops of diluent
References:
How to optimize the drop plate method for enumerating
bacteria
Herigstad B, Hamilton M, Heersink J
J Microbiol Meth, 44(2): 121-129 (2001)
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.
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