Chapter 2 Biofilm Formation and Growth
Section 3 Biofilm Development
Page 2 Conditioning Film

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Biofilm Development

The Conditioning Film

Within moments of placing a clean sterile slide into a water source, be it a pond, stream or an aquarium, a film begins to form that consists of proteins and polysaccharide molecules adsorbed to the glass surface. Similar films develop on a professionally cleaned tooth surfaces or on the surface of indwelling urinary catheters. It is to this so called 'conditioning film' that the first cells of the insipient biofilm attach. Such conditioning films begin to form so rapidly, perhaps in seconds, that there is a question as to whether bacteria can attach to a barren substrate lacking such a film. In any case, the accumulation of the conditioning film continues to develop for hours following first exposure.

Diagram of the deposition of the conditioning film.
Figure 1. Diagram of the deposition of the conditioning film.
Similar to the diagram page 59 of Allison, Gilbert, Lappin-Scott and Wilson Community structure and Co-operation in biofilms.

Conditioning films form on both hydrophilic (eg. glass) and hydrophobic (eg. plastic) surfaces. A great deal of effort time and money has been invested by industries attempting to find surfaces to which biofilms will not adhere. Such a material would be of enormous value in many industrial applications including water and oil pipeline manufacturing, heat exchangers, air conditioning systems and also in medical applications such as indwelling catheters, cardiac pacemaker probes, prosthetic implants (hip, knee joints) and hydraulic dental drills. To date, no surface has been discovered that will reliably inhibit the formation of biofilms, although mistakes, costly in lawyer's fees and human lives, have been made on the assumption that such materials had been found. Great wealth awaits the fortunate individual or group who making this discovery.

Bacterial Approach and Contact

Figure 2. Van der Waals forces and electrostatic charges.
Note: Drawing needed illustrating the two forces acting on a bacterium as it approaches a surface.

Two forces act on a bacterium suspended in the fluid stream close to the substrate wall. The first, the van der Waals force, is attractive and acts at a distance of a few hundred nm (a few tenths of a mm). This force tends to bring small objects close to the substrate wall. The second force is due to the fact that bacterial cells and many environmental surfaces possess a net negative electrostatic charge. These negative charges produce a repulsive force as the bacterium approaches the surface at a distance of about 10-20 nm. When the bacterial cell comes to within about 5 nm the probability of physical contact by cell surface receptors, such as pili and adhesions, increases greatly (DLVO Theory - Discussion Chapter 8).

It is also in this layer close to the substratum wall that flow velocity becomes much reduced. This region called the hydrodynamic boundary layer is caused by drag and frictional forces and provides a low shear, low turbulence area in which physical contact between the bacterium and the wall may occur. Physical contact with the conditioning film may bring about a transient and potentially reversible attachment to the substrate.

Any increase in kinetic energy may boost the probability of a bacterium overcoming the electrostatic force and this may be a major function of bacterial flagellae. In addition, projections such as pili that extend through the boundary layer increase the likelihood of bacterial attachment to the substrate wall.

Reversible attachment

Video photo micrography has been of enormous importance in deciphering the events surrounding the attachment of bacteria to surfaces.  The best evidence suggests that attachment occurs as a two-step process.  Reversible attachment, which is tenuous and often transient and irreversible attachment that is much more stable. 

In some cases, following their initial attachment to a surface the bacteria can be seen to be rapidly spinning, vibrating or actually moving across the substrate surface.   Spinning is an indication that the cells are attached to the substrate by their flagella, but since the flagellum is now “fixed”, the cell body rotates as the flagellar motor continues to rotate.  Sometimes, cells in contact with the surface seem to be vibrating, that is they areexhibiting Brownian motion, caused by the constant and random impact of water molecules striking the loosely attached bacterial cell.Videos of Pseudomonas aeruginosa reveal that after initial attachment, cells move over the surface with a jerky type of motion which has been called twitching motility.  This motion is now known to be due to the action of Type IV pili that extend, attach to the substrate, and then contract drawing the bacterial cell along the surface.  As the population of cells increases on the surface, video micrography shows that many of the cellsbegin to move toward one another forming aggregations or microcolonies. These microcolonies some times disaggregate once again, but more often they persist, forming the earliest stages of the tower and mushroom architecture of the mature biofilm.

Media 1
(Film from Kolter showing initial stages of attachment.)

Media 2
O'Toole video - Not sure if we are inserting this

O’toole and Kolterhave isolated a collection of sad mutants (surface attachment deficient) in Pseudomonas aeruginosa which are unable to form an initial attachment to surfaces and therefore are unable to make normal appearing biofilms.  The mutants fell into two clusters, one being deficient in the ability to produceflagellae and the other deficient in making the polar located Type IV pili.  The wild type strain forms a monolayer of cells on a polyvinylchloride plastic surface over an eight-hour period.  Cells then aggregate to form microcolonies distributed throughout he monolayer.  Cells bearing the sad mutation formed the monolayer, but do not aggregate to form microcolonies indicating a role for Type IV pili in the formation  ofbiofilms.  O’toole and Kolter also observed that in the other class of surface attachment deficient cells, carrying the flgK mutation, deficient in flagellae formation, very few cells attached to the surface indicating a role for flagellae in the initial attachment of P. aeruginosato surfaces.

Irreversible attachment

At some point following initial contact, the cells become encased in a polymeric matrix that the cells produce. The use of reporter genes such as the Green Fluorescent Protein (Chapter 8) indicate that attachment causes the  quorum sensing induction of genes  responsible for the production of compounds found in the matrix of developing biofilms such as the genes of the alginate pathway. Alginate is a major component of matrix EPS  (Extracellular Polymeric Substance) in this organism. In fact it has been shown that attachment causes a major change in phenotypic expression in biofilm forming organisms that may involve a change in expression of at least 30% of the genome