Introduction

During the 2006 Carsky Symposium, a roomful of microbiologists was asked how many had had a class or a unit of a class focused on biofilms during their training. Not one hand was raised. Then they were asked how many had had as much as one lecture on biofilms and the response was less that one fifth of the audience. In general we tend to teach what we have been taught, and biofilms are not part of the curriculum.

Well, if that is the case, perhaps ignoring biofilms is justified on the basis that they just aren't that important. Why do we argue for this "paradigm shift" approach to biofilm education? The answer to these questions is the objective of Biofilms: The Hypertextbook project, but perhaps the sketch presented here will give the sense of the importance of biofilms.

Since its inception in 1990 the faculty and staff of the Center for Biofilm Engineering have exhibited a vigorous commitment to biofilm education at all levels. Each year many undergraduate, and graduate students study with the scientists, and engineers of the Center on honors projects, and Masters and PhD thesis. Visiting scientists and Post Doctoral students from around the world come to learn new skills and make contributions to our knowledge of biofilms. Dozens of students have been trained in the basics of biofilm microbiology through the Biofilm Systems Training Laboratory classes and twice annually the center hosts the noted Montana Biofilm Science & Technology Meeting (MBM), formerly known as TAC (Technical Advisory Conference), meetings for the Center's Industrial Associates. At MBM meetings scientists, technicians and management personnel from industry come to learn about the latest discoveries in slime management. On three occasions the Center has sponsored International Symposia to which the top research scientists in the world have contributed.

Despite these efforts, and those of other biofilm research centers throughout the world, there is a feeling within the community that information concerning biofilms is not entering the undergraduate curriculum at a rate commensurate with the importance of the topic. Even a cursory examination of undergraduate microbiology textbooks will reveal a paucity of information on the subject. Although in recent years the treatment has increased the typical textbook still allots only 1-3% of its pages to biofilms and the material is typically scattered throughout the text so that few students will be likely to acquire a coherent notion of the range of topics affected by biofilms and of their importance.

Why Biofilms?

Those of us committed to biofilm education see the world of microbiology in a fundamentally different way. The term paradigm shift is overused today, often for discoveries that are trivial but for those in the biofilm community the term aptly describes the world as we see it.

There was a time, not so long ago, when what we today call biofilms were thought of as unusual, sometimes amusing (picture someone slipping on a slimy rock in a stream) but certainly peripheral to the mainstream of microbiological thought. There were indications of a new phenomenon in the academic literature. In 1933, for example, Arthur Henrici wrote "it is quite clear that, for the most part, water bacteria are not free floating organisms, but grow upon submerged surfaces", and in the 1940's Claude ZoBell made many observations indicating the ubiquity and importance of microbial growth on surfaces. Then during the 1980s and 90s the number of academic centers devoted to investigating this new concept began to increase and the professional literature began to grow at an exponential rate. Today the concept is well established and at microbiology meetings both research and educationally oriented, it is not unusual to hear faculty members saying something like, "I would really like to add more content on biofilms but I don't know what to drop out and I just don’t have the time".

So why hasn't the biofilm concept entered the mainstream of microbiology education? There are many reasons, the lack of materials to support the teachers' interest, the time involved in bringing oneself up to date on a rapidly expanding subject, the dilemma of deciding what to omit from the traditional curriculum and the lack of exposure to the concept during undergraduate and graduate training.

It is now generally accepted that most of the worlds biomass is composed of bacteria and that greater than 90 percent of prokaryotes live in biofilms. Taken together, one can then argue that biofilms are the dominant life forms on the planet and have been so since life began. The biofilms called stromatolites are among the earliest fossils known and dated at greater than 3 billion years old. Of course, these stromatolites are remnants of cyanobacterial communities so one can reasonably argue that biofilms were in large part responsible for our present atmosphere and particularly its oxygen and nitrogen content. This oxygen rich atmosphere was in turn a prerequisite for energy efficient metabolism and the evolution of multicellular life.

Much of the interest in biofilms centers on their role in health and disease. Estimates from the US Centers for Disease Control and Prevention, for example, are that 65 percent of hospital acquired infections are due to biofilms with an associated treatment cost in excess of $1 billion per year as a result of extended hospital stays and increased antibiotic resistance.

Biofilms growing in or on pipelines, heat exchangers, ship hulls, and other surfaces of industrial importance cost industry billions of dollars annually in repair and replacement and increased energy expenditure.

On the other hand, microbial filtering in water treatment and waste disposal plants, bioremediation of chemical spills, and the formation of biobarriers that retard the spread of ground water contamination are valuable economic processes contributing to human heath and welfare.

Taking these and myriad other facts into account, it is the contention of the people who have developed Biofilms: The Hypertextbook that biofilms represent a concept as fundamental as immunology, genetics, or molecular biology. Each of these subjects is typically thought to be worthy of a chapter or unit in most introductory microbiology texts. The failure of publishers to respond to this need is the rationale for this text and manual.

The intent of the manual is to aid teachers of microbiology in integrating biofilm topics into their classes. The Hypertextbook may be used in part to supplement an introductory text or in its entirety to serve as the basis of an undergraduate class and laboratory, perhaps at the upper undergraduate level.

This material, at least in the blue version is rather closely tied to the research literature and makes reference to the laboratories and researchers making many of these discoveries about biofilms. As such one might view this material as an intermediate step between the reference free nature of most introductory textbooks and the reference laden journal articles used to teach many graduate level classes. In part our intent is to give students the sense of research as a fascinating human endeavor, and also the concept that the people doing this research are not so different than the student reading the Hypertextbook. That in a few years, if so inclined, they too could be making contributions to this, or some other exciting field of research.

Here are some of the features that instructors may find useful.

1. Chapter Outlines and/or Summaries
2. Tips on integrating the Hypertextbook into an undergraduate class. The manual also attempts to answer the questions teachers may have about biofilms such as "If I add biofilm materials, what do I leave out". Or, is it better to deal with biofilms as a unit or to integrate this knowledge throughout the class?
3. Insights and comments from biofilm professionals that we hope will be of value to teachers new to the subject. These include historical perspectives contributed by the investigators involved in the discovery.
4. Pointers to other graphic and textual materials not in the hypertext book, including images that may be used to supplement lectures or handouts. These will include directions to video clips, images and graphics materials, which are in the public domain.
5. A growing collection of laboratory exercises are included and recommendations are given as to how these may be substituted for more traditional exercises found in introductory laboratory manuals.
6. The textual and laboratory materials are keyed to the ASMs Core Themes and Concepts for an Introductory Microbiology Courses at
7. Short case histories, which may be used by teachers to illustrate particular aspects of the biofilm story.
8. A Biofilm Timeline indicating the developmental history of the biofilm concept.
9. An annotated list of references for teachers beyond those listed in the Hypertextbook itself. These are provided for instructors or advanced students who are interested in a more detailed treatment of a particular topic or perhaps in technical aspects of the research involved.

We hope that teachers find that Biofilms: The Hypertextbook and this manual will fill a gap in their continuing effort to keep their classes interesting, and current. We hope that instructors will share their experiences with us and continue to make suggestions for the continuing modification and improvement of this educational resource. A sample concept map for biofilms. Concept maps are a useful method of showing the relationships among concepts. Each link in a concept map depicts two ideas, connected by some operational link. The most general concept is placed at the top (Biofilm) and subsidiary concepts are placed below (harmful and beneficial) and are connected by lines representing connecting relationships (produce, form, etc.). Teaching your students the skill of concept mapping may take a period of your class, but it can give them a skill that will last them a lifetime and improve their performance, not only in your class but also in every other class they ever take. The "bible" for learning about concept mapping is the wonderful little book called "Learning How to Learn" by Novak and ______, published by ______________________ © ______. Concept maps also make useful homework assignments. They permit the instructor to determine how a student is organizing the information and provide feedback, which can inform review sessions, future lectures or homework assignments.

Time Line

A. Van Leeuwenhoek

Notes: Chapter 11

Safety Notes

Alternative autoclaving methods - Pressure cookers are one alternative, however, they can be dangerous and may not be large enough to autoclave a class load of reactors efficiently.

It is possible to "sterilize" media in a microwave (two minutes is recommended). However, that will not get rid of spores. An alcohol rinse of the reactor would disinfect it. The combination of both of these techniques could prove adequate for a high school lab setting However, a note of caution would be that these are not completely sterile, so it would be possible to have pathogenic microorganisms present. Therefore, precautions must be taken in the handling of the samples taken from the reactor.

Gloves

Gloves should be selected consistent with the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxic substances, hazardous chemicals and other physically hazardous agents. Temperature resistant gloves must be worn when handling hot material or dry ice.

Safety glasses? Splash shields or safety glasses with solid side shields should be required for anticipated splashes, sprays or splatters of infectious or other hazardous materials to the face.

Disposing of materials? Proper disposal of spent media, used slides, and reactor vessels. Items should be bleached and allowed to sit for a period of time and then washed or thrown away. Use Biohazard Bags for contaminated waste. It may be possible to arrange for the local hospital to autoclave the waste.

Exercise Notes

Harvesting and Dispersing Cells from Biofilms Harvesting Cells: Alternative Method
For small surface areas and using automatic (digital) pipettes.

1. The procedure is as "Harvesting Cells: Alternate Method" except that sterile 1.5 ml microcentrifuge tubes are used as dilution blanks. These are prepared by the aseptic addition of 900 μl of sterile distilled water or PBS.
2. Scraping is as shown in the standard technique. The biofilm removed from the coupon is transferred with the stick and swirling into the microcentrifuge tube.
3. The scraped area of the coupon is rinsed with 100 μl aliquots of sterile distilled water of PBS and the volume of the microcentrifuge tube is brought up to 1 ml.

Dispersing Cells: TURBO MIX™ Method
This technique employs a TurboMix™ High Performance Attachment for the Vortex-Genie 2® (pictured at right), and is used in place of the BransonTM or equivalent sonic cleaning water bath. However, this device requires that the dilutions be made in 1.5 ml microcentrifuge tubes.

Instructions:

1. Place your microcentrifuge tube containing the harvested biofilm material into the TurboMixTM attachment of the Vortex-Genie.
2. Lower the shield of the TurboMix and vortex for 2 minutes.
3. The cells should now be dispersed and ready for the next procedure.
4. The dilution tube should be vortex mixed before proceeding to dilution and plating to ensure that the dispersed cells are uniformly distributed.