A Biofilm Primer

Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces. These microorganisms are usually encased in an extracellular polysaccharide that they themselves synthesize. Biofilms may be found on essentially any environmental surface in which sufficient moisture is present. Their development is most rapid in flowing systems where adequate nutrients are available.
Bar = 10 micrometers
This biofilm formed from mixed culture of Pseudomonas aeruginosa, P. fluorescens and Klebsiella pneumoniae. The image was taken with a confocal laser microscope and was generated as 27 overlaid optical sections of 6 micrometer thickness.

  Microbial Mats are specialized microbial communities composed mainly of photosynthetic procaryotes. Thus the principle distinction between microbial mats and other biofilms is their dependence on photosynthetic primary productivity as their source of energy.

 This microbial mat in a roadside puddle formed quickly during the height of summer in Pennsylvania. It demonstrates that it is not necessary to travel to exotic locations in order to study complex and interesting biofilm communities.


Biofilms may form:
  1. on solid substrates in contact with moisture.
  2. on soft tissue surfaces in living organisms.
  3.  at liquid air interfaces.
Typical locations for biofilm production include rock and other substrate surfaces in marine or freshwater environments.

The pristine lake shown in this picture is in the northern Rocky Mountains of Montana. Biofilm communities such as that shown below, form here and are composed of a range of different types of organisms, both autotrophic and heterotrophic. Algae derive their energy from photosyhnthesis and their carbon from dissolved carbon dioxide. Bacteria, which are generally heterotrophic, obtain their energy from organic matter produced by the algae or from organic matter washing into the lake from the surrounding terrestrial habitat.

These organisms form the basis for the food webs that nourish larger organisms such as insect larvae, which are consumed by fish that are in turn consumed by birds like eagles. Hence, biofilms are an important link in the energy budget of many natural communities.  This is one kind of biofilm from a pristine aquatic alpine ecosystem as seen through a conventional microscope. The larger, roughly spherical cells that appear green to brown are algae while the smaller dark cells are associated bacteria. Both types of cells produce a polymeric extracellular slime layer which encloses the cells. This complex aggregate of cells and polysaccharide is the biofilm community.


Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce. The ASM biofilm image collection contains many such images in its medical slide set.

  One image from the collection, shown here, is a scanning electron micrograph (SEM) of the mucous layer of a mouse ileum illustratin the nature of the relationship of organisms within the layer and on the villi. Note the way the mucous layer covers the villi and the relative depth of the layer.


Here, human dental plaque has been exposed to 5 % sucrose for 5 minutes, after which Gram's iodine (0.33% Iodine in 0.66% KI) was applied.

  The sucrose solution was applied to the left central incisor (which appears on the right) while the right central incisor served as a control.

 Iodine selectively binds to alpha-1,4 glucans (iodophilic polysaccharide, i.e. glycogen or amylose) which results in brown to purple staining.

 The ability of oral bacteria to store iodophilic polysaccharides or glycogen-like molecules inside their cells is associated with dental caries since these storage compounds may extend the time during which lactic acid formation may occur. It is this prolonged exposure to lactic acid which results in decalcification of tooth enamel.

 Plant tissues also commonly have microbial populations associated with their external tissues.

  One such plant microbe association is called the rhizosphere and is a relationship between the plant roots and root hairs and a complex microbial community. The rhizosphere association is mutualistic. Plant roots secrete significant amounts of sugars, amino acids, vitamins and plant hormones which vastly stimulate microbial growth in the immediate vacinity of the root. This relationship may also be important to the plant in that the microbial population may facilitate the absorption of nutrients by the plant from the soil.


Beneficial and detrimental attributes of biofilms

Humans have made considerable use of microbial biofilms, primarily in the area of habitat remediation. Water treatment plants, waste water treatment plants and septic systems associated with private homes remove pathogens and reduce the amount of organic matter in the water or waste water through interaction with biofilms.

 This image is a scanning electron micrograph of the naturally occuring biofilm on sand grains in the clog mat of a septic system infiltration mound.

Scale Bar= 150 micrometers.

The biofilm is composed of mineral particals, a variety of microorgasims, and a network of slime, or glycocalyx (indicated by the arrows), that binds the microorgasims and particals together.

  On the other hand biofilms can be a serious threat to health especially in patients in whom artificial substrates have been introduced. It is a common,experience that patients with indwelling catheters for urine excretion, for continuous ambulatory peritoneal dialysis (CAPD) or for any other reason are subject to frequent and persistent bouts of infection. These recurrent infections are due to the accumulation of mixed biofilms on the artificial surfaces provided by the catheter or other implant. The glycocalyx in which the bacteria live protects them from the effects of antibiotics and accounts for the persistence of the infection even in the face of vigorous chemotherapy. In vitro experiments suggest that bacteria encased in biofilms may be 50 to 500 times more resistant to chemotherapy than planktonic bacteria of the same strain. Fragments of biofim that slough off at intervals can spread the infection to distant locations within the body.

 This micrograph shows a large number of Staphylococcus epidermidis cells covered with glycocalyx and adhereing to the surface of a catheter segment.

How do biofilms form?

Place a clean sterile glass slide in a stream of water containing at least minimal nutrients, and over the course of days or weeks a microbial ecosystem will form consisting of a variety of microorganisms arranged in a complex relationship to one another and embedded in a mass of extracellular polysaccharides of their own making.

 The formation of this biofilm is far from a random process. To the contrary, the formation of a biofilm follows a course the nature of which can be predicted and recorded.


Typically, within minutes, an organic monolayer adsorbs to the surface of the slide substrate. This changes the chemical and physical properties of the glass slide or other substrate. These organic compounds are found to be polysaccharides or glycoproteins. These adsorbed materials condition the surface of the slide and appear to increase the probability of the attachment of planktonic bacteria.

 Free floating or planktonic bacteria encounter the conditioned surface and form a reversible, sometimes transient attachment often within minutes.

This attachment called adsorption is influenced by electrical charges carried on the bacteria, by Van der Waals forces and by electrostatic attraction although the precise nature of the interaction is still a matter of intense debate. In some instances, as for example, in the association between a pathogen and the receptor sites of cells of its host there may be a stereospecificity which though still reversible is stronger than that achieved strictly by ionic or electrostatic forces.

 If the association between the bacterium and its substrate persists long enough, other types of chemical and physical structures may form which transform the reversible adsorption to a permanent and essentially irreversible attachment.

The final stage in the irreversible adhesion of a cell to an environmental surface is associated with the production of extracellular polymer substances or EPS. Most of the EPS of biofilms are polymers containing sugars such as glucose, galactose, mannose, fructose, rhamnose, N-acetylglucosamine and others.

 This layer of EPS and bacteria can now entrap particulate materials such as clay, organic materials, dead cells and precipitated minerals adding to the bulk and diversity of the biofilm habitat. This growing biofilm can now serve as the focus for the attachment and growth of other organisms increasing the biological diversity of the community.

  The microbial inhabitants with in biofilms and microbial mats in a significant sense behave as multicellular assemblages. Far from being the homogeneous populations usually assumed in planktonic pure cultures, biofilms as simple as colonies on agar surfaces and as complex as pathogen interactions with host cells and the bacterial populations inhabiting the surface of mud flats behave in many respects like the tissues of a multicellular organism.

 The accompanying illustration presented here courtesy of the Center for Biofilm Engineering gives some concept of the structure and complexity of a typical biofilm.

 At one time, most scientists thought that biofilms consisted of bacteria randomly distributed within a uniform slimy matrix. However, once scientists began using new imaging tools like the Confocal Scanning Laser Microscope, which allows viewing of hydrated living biofilms, they found that biofilm structures take a wide variety of forms depending on their age and growing conditions.

 This structural complexity allows bacteria deep within the biofilm to have access to nutrients carried by the convective flow of water. Some of the features typically seen in biofilms are labeled in this picture.