Chapter 11 Lab Exercises
Section 11 Harvesting and Dispersing of Cells from Biofilms (Alternative Method)
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Harvesting and Dispersing of Cells from Biofilms
(Alternative Method)
Supplies Needed
Quantity |
Description |
1 |
wooden applicator stick |
4 |
microcentrifuge tubes 1.5 ml |
1 |
flask containing sterile distilled water or
phosphate buffered saline, for making dilution
tubes
|
1 |
mechanical pipetting device and sterile pipette or
an automatic pipetter, pipette and pipette tip |
1 |
biohazard bag for disposal of used tips |
1 |
Vortex mixer |
1 |
Branson or equivalent sonic cleaning water
bath |
1 |
forceps/hemostat |
1 |
microscope slide, coupon or other object with
biofilm attached |
Instructions:
Harvesting Cells
- Using a sterile pipette and mechanical pipetting
device, dispense 0.9 μl (900μl ) of distilled water or
phosphate buffered saline (PBS) into sterile
microcentrifuge tubes (1.5ml). These will be used as
dilution blanks.
- Select a slide or coupon bearing an established
biofilm. Hold the slide with a flame sterilized
hemostat/forceps.
- Remove the cap from the tube containing the sterile
wooden applicator sticks and gently shake the sticks so
that one end extends a short distance from the tube. Being
careful to touch only one stick, remove it from the
tube.
- Using the applicator stick as a scraper, rub the
surface of the coupon for approximately 15 seconds. Be sure
to hold the stick perpendicular to the coupon surface so
that the flat end of the stick is doing the scraping.
Occasionally transfer the material removed to one of the
0.9 ml microcentrifuge tubes prepared previously. Swirl the
applicator stick vigorously to remove the biofilm.
Note: you may not be able to see any
visible material on the stick or in the diluent as the
stick is rinsed. Repeat this process at least 3-4 times (as
indicated by your instructor) to ensure full coverage of
the coupon surface or defined area being sampled.
- Using a sterile pipette and automatic pipetting device,
rinse the scraped area once with 10 μl aliquots of
sterile distilled water of PBS. This rinse water
should be added to the 0.9 ml dilution blank. The total
volume of rinse water should be 1 ml, (for example rinse 4
times with 10μl aliquots, then add 60 ml to
bring the tube up to 1 μl).
- Dispose of the contaminated applicator stick, pipette,
scraped slide in the biohazard bag provided.
Dispersing Cells: Sonicator Method
This technique uses a Fisher Scientific, a Branson
Ultrasonic Corporation or similar sonic water bath cleaner
delivering approximately 50-60 hz, to disrupt biofilms and
disperse cells.
- Insert the microcentrifuge tubes (containing the
biofilm harvested in the previous section) into a floating
device, such as a thin piece of StyrofoamTM with holes for
the tubes to be inserted. The floating device will
hold the tubes upright while in the sonic water
bath.
- Sonicate the tubes for 2 minutes. Turn off
sonicator before removing tubes.
Note: The time required for disruption of
the biofilm varies with the material and the appropriate
time for any particular specimen should be determined by
the instructor.
- The cells should now be dispersed and ready for the
next procedure.
- The sonicated microcentrifuge tubes should be vortex
mixed before proceeding to dilution and plating to ensure
that the dispersed cells are uniformly
distributed.
Illustrations:
Permissions
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 1. Scraping a coupon.
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.erc.montana.edu