Chapter 11 Lab Exercises
Section 10 Harvesting and Dispersing of Cells from Biofilms (Standard 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 (Standard
Method)
Supplies Needed:
Quantity |
Description |
1 |
wooden applicator stick |
4 |
sterile culture tubes |
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: Standard Method
- Using a sterile pipette and mechanical pipetting
device. Dispense 9ml of distilled water or
phosphate buffered saline (PBS) into sterile serological
tubes. 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 9
ml dilution tube 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 mechanical pipetting
device, rinse the scraped area several times with aliquots
of sterile distilled water or PBS. This rinse water should
be added to the 9 ml dilution blank. The total volume of
rinse water should be 1 ml, making the final volume of the
tube 10 ml (for example rinse 4 times with 0.25 ml
aliquots, and add these to the dilution tube).
- 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.
- Place a 150 ml beaker in the sonic water bath and fill
the beaker with water so that the volume of the beaker is
brought up to the level of water in the cleaner.
- Place your tube (containing the biofilm harvested in
the previous section) in the beaker and sonicate 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 dilution tube should be vortex mixed before
proceeding to dilution and plating to ensure that the
dispersed cells are uniformly distributed.
Illustration:
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