Building and Using a Biofilm Continuous Flow Stirred Reactor.
This exercise describes the construction of a Continuous Flow Stirred Reactor from readily available and inexpensive materials. While similar to the Batch reactor described in Ex. 1 this device is more like a chemostat than a culture tube. Nutrients are constantly fed so that the culture growth phase can be controlled. Once established, conditions such as nutrient concentration and waste product levels remain more or less constant. If the flow rate is greater than the growth rate, only cells growing on the 1 x 3 microscope slide and the walls of the CFSR remain in the reactor.
A Flow Cell is a type of biofilm reactor, which permits the continuous microscopic observation of a developing biofilm from irreversible attachment to maturity and dispersal. It is a type of Plug Flow reactor in which conditions at any point in the cell remain relatively constant although conditions in other part of the cell may be quite different. The flow cell described can be built of easily obtained materials and provides one of the most dramatic real time illustrations of biofilm structure and growth obtainable.
Commercial Drip Flow reactors are quite expensive, this one can be made from materials in the lab or purchased at the local grocery or hardware store. A drip flow reactor is a type of Plug Flow reactor, which mimics the common habitat in nature in which water flows over a surface carrying nutrients which feed the biofilm growing on the surface. Rock walls, shower curtains, sinks and toilet bowls above the water line, are all examples of this sort of natural biofilm producing habitat.
Model System for Growing a Standard Biofilm: Static Glass Coupon Reactor.
This protocol was developed by scientists at S.C. Johnson to provide reproducible biofilms for testing disinfectant and cleansing products. A culture is established as a sort of sandwich growing between a filter paper and a microscope slide. When the slide is lifted from the filter paper, the biofilm, which adheres to the slide can be used in a variety of ways.
This exercise employs the fact that the extracellular polysaccharide matrix of a biofilm stains densely with dyes like crystal violet (CV) and safranin. Biofilms are grown in a 24 well plate (or 96 well if you have a plate reader). The wells are washed and then stained with CV. The wells are then washed to remove unbound CV and then the CV bound to the biofilm matrix is eluted and measured in a spectrophotometer. With a little practice it is dramatic and reproducible.
The Drop Plate method was developed to make it possible to do serial dilutions on a minimal number of plates. If precision pipeters are available a student can do a 10-4 dilution in replicate on two plates and a 10-8 dilution on 4.
Typical staining procedures require the slide to be heat fixed prior to staining. Since the biofilm matrix is > 95% water, heat fixing destroys precisely what one wishes to observe. In this Gram stain modification, the slide is never dried or heated. The biofilms are already "fixed" to the slide by their matrix.
Harvesting and Dispersing of Cells from Biofilms (Standard Method).
The two methods differ only in the method of dispersing the cells once they have been harvested. The standard method uses a Branson Ultrasonic Corporation or similar sonic water bath cleaner delivering approximately 50-60 hz, to disrupt biofilms and disperse cells, the alternative method uses a TurboMixTM High Performance Attachment ™ for the Vortex Genie mixer which can vortex 12 samples in microcentrifuge tubes simultaneously.
Harvesting and Dispersing of Cells from Biofilms (Alternative Method).
The two methods differ only in the method of dispersing the cells once they have been harvested. The standard method uses a Branson Ultrasonic Corporation or similar sonic water bath cleaner delivering approximately 50-60 hz, to disrupt biofilms and disperse cells, the alternative method uses a TurboMixTM High Performance Attachment ™ for the Vortex Genie mixer which can vortex 12 samples in microcentrifuge tubes simultaneously.
The point of this exercise is to collect mixed species biofilms from either natural water sources (streams, ponds, salt marshes etc) or laboratory or domestic sources (aquariums, rain barrels, traps in sinks (gross!) etc.).
This exercise uses a modified light microscope to measure the thickness of a biofilm prepared by any growth or collection method. The microscope is equipped with a pointer and a piece of polar coordinate graph paper so that by focusing on the surface and the base of the biofilm and recording the difference in degrees one can determine depth. Instructions for calibrating the microscope are included.
This classroom exercises introduces students to the reality of Pseudomonas infections in Cystic fibrosis patients. The students are given some information from which they must draw inferences and conclusions. Then additional information is given permitting them to see more of the picture. The exercise, written by a CF medical specialists takes small portions of a couple of classes.
In this exercise students construct a column containing sand or small glass beads. The column is charged with a culture of a nonpathogen that produces a great deal of EPS. The flow rate of liquid thorough the column is measured at intervals as the biobarrier forms.
Buccal Epithelial Cells with Adherent Bacterial Cells Revealed by Negative Staining.
Students harvest squamous epithelial cells from their cheek with a tongue depressor and stain the cells with nigrosine dye. This dye reveals the biofilm cells growing on the surface of the epithelial cells.
Building a Biofilm Model from a Confocal Microscope Stack.
Images taken by a Confocal Scanning Laser Microscope are of limited depth of field and are stored individually in the memory of a computer like so many individual slices of bread. The computer can reassemble these "slices" to produce a three dimensional "whole loaf". Paul Stoodly has provided a "confocal stack" from which individual students or classes can "cut out" and reassemble their own model of a biofilm. Once assembled the students can answer questions about the original such as direction of flow, overall dimensions, architecture and the presence of water channels.
In this exercise, students construct a miniature pond similar to those first built by Sergei Winogradsky. Given an appropriate container, some mud or soil, and samples of a carbon, nitrogen and sulfur source students can make columns which in time may become truly spectacular as highly pigmented photosynthetic microorganisms grow as a biofilm at the mud glass interface.
Dental Biofilms: Detection and Quantification of Plaque.
n this exercise, students use a dental disclosing agent to show the extent of plaque attached to their teeth. A guide is given which aids students in quantifying the amount of plaque present.