Electrostatic Charge and Bacterial Adhesion
   
   

 

Not all surfaces are obvious

Although this may sound a little strange it needs to be pointed out that surfaces are an important feature of very many habitats. If you think about it, some surfaces are very obvious like rocks in a river, the leaves and stems of plants, skin, hair etc etc. However, some are not so obvious eg the surface of water.

Two examples

By now you probably think you have a fairly good handle on what constitutes a surface and how it is exploited by microbes.

Obvious examples include the rock in a stream. Anyone who has ever picked up such a rock will have noticed that it is slimey. This is the microbial biofilm growing on it.

But habitats and their associated interfaces (surfaces) are not always that obvious. Take, for example, the surface of a pond. This is a habitat defined by the surface of the water, the things dissolved in the water and the nature of the air immediately above it. This habitat will be exploited by a community which includes many microbes. These microbes find it advantageous to stick to the water surface because of the right gaseous environment, it's near the air, and because organic and inorganic material is concentrated there.

But what are they attached to? The answer is partly to the interface and partly to each other. If you look closely at the surface of a pond you will see this biofilm even with the naked eye.

Most microbes do not roam free

It is a widely held view that the world is teeming with microbes. While this is generally true, they are not as evenly distributed throughout the world as you might think. Take the air, for example. If asked, most people would say that the "air" is full of bacteria but this, in fact, is very far from the truth. Few microbes have a truely free-roaming lifestyle, they much prefer to stick to a surface.

 
 
   
The benefits of attachment

Sticking to a surface has very real benefits for many microorganisms.

First, there are very few habitats that don't contain at least one surface and by sticking to it, a microbe can manage to physically stay in it's chosen environment for which, we must presume, it has become adapted. eg a rock on a river bed

Second, surfaces are where the food is. It doesn't matter if the nutrient is organic or inorganic, the laws of physics dictate that molecules tend to adsorb to interfaces and interfaces are surfaces. This means that in any environment the concentration of any particular molecule is very likely to be higher on, or in close proximity to, a surface.

Third, different species of microbes often act together to exploit a habitat. These consortia are usually present as a biofilm growing on a surface. In this case the surface provides the physical support which allows the community to stay together.

 
     
Microbes in the mouth

The mouth comprises a number of quite distinct habitats most of which are bathed in saliva. In order to survive in the mouth bacteria must attach to one of its surfaces or risk being swallowed.

Bacteria attaching to exposed smooth surfaces in the mouth must be quite firmly attached to resist the flow of saliva.

Any build-up of cells due to multiplication is more easily dislodged because the mass of bacteria experiences a greater shear force. Anyone who has ever waded into a river knows that as you get deeper into the water the more you feel the flow of the river.

This does not mean that the exposed, smooth, surfaces of teeth are devoid of attached bacteria because some species have evolved efficient adhesion mechanisms. It does mean, however, that any significant build-up is inhibited and that plaque accumulation is limted to sheltered sites such as interproximal areas, the gingival margin and fissures. Bacteria will also accumulate in defects as shown on the right.

 
How smooth is smooth?

This is a picture of a smooth surface of a tooth taken using a scanning electron microscope. Note that the surface is far from smooth at the microbial level. Enamel prisms orientated perpendicular to the tooth surface create major indentations approximately 5-10 microns in diameter which are sufficient to provide sheltered sites enabling the adhesion of microbes and the development of microcolonies. Similar sites can be provided by areas of enamel erosion.

 
Initial attachment

Before plaque can accumulate, the tooth has to be colonised by bacteria which then multiply and attract further colonisers. These "first colonisers" are known as pioneer species and, in the mouth comprise:

1. Streptococcus oralis

2. Streptococcus mitis

3. Streptococcus sanguis

The surfaces of these cells and, in fact the surfaces of nearly all cells, are negatively charged because of the presence of proteins and other wall and cell membrane components which contain phosphate, carboxyl and other acidic groups. Furthermore, nearly all non-biological surfaces are also negatively charged. Sometimes this is due to the accumulation of organic material which adsorbs to the surface from the environment and sometimes because the surface is inherently negatively charged because of its chemistry.

 
Why understanding adhesion is important

Quite how these pioneer species, and all the other bacteria in the mouth, actually adhere to the tooth has been the subject of a considerable amount of research over the last 20 years. Interest in bacterial adhesion is, of course, not confined to the mouth because it is a vitally important first step in many diseases and knowledge of how it works may lead to the means to prevent it and the subsequent disease.

The surface charge

Now, you would be forgiven for thinking that, as a bacterial cell approached a surface, its negative charge would interact with the negative charge of the surface, or other cell, and that this would cause it to be repelled. Unfortunately it is not as simple as this because the surface charges attract oppositely charged ions from the surrounding fluid.

These ions which are attracted to the surface form a mobile layer known as the "electric double layer". Think of it as a cloud of positively charged ions adjacent to the cell or surface.

So when two charged particles approach each other it is their electric double layers which start to overlap. This might sound a bit pedantic but the significance will become clear when we look at the effect of ionic strength.

 
The Electric Double Layer
   
Measuring the force of repulsion

Since we are considering two surfaces with the same charge then they will, of course, repel each other. If, however, they were of opposite charge, they would attract each other. The calculation is exactly the same.

As you might expect the calculation is not easy and there are a number of factors to take into account. However, the equation can be simplified to the one shown on the right.

In this equation lots of the factors are constants which can be combined and estimated as 332

Q1 and Q2 are the total charges which are being brought together

D is the dielectric constant of the surrounding fluid which in this case is water and is "1"

r is the distance separating the surfaces

 
 

So the force repelling the two charged surfaces is inversely proportional to the distance between them.

In other words the closer they get together the bigger the force, just as you might expect.

The force of repulsion is sensitive to the ionic strength of the surrounding fluid

The force due to the overlapping electric double layers as the bacterial cell approaches the surface is affected by the amount of salt in the surrounding fluid. This is because the depth of the electric double layer shrinks as the ionic strength increases.

   

The relevant equation is shown on the right. This fairly complicated formula includes various factors such as:

 

 

D the dielectric constant (again)

T the temperature

e the size of the ions

v the valence of the ions

n the concentration of the ions

x is the depth of the electric double layer

The temperature and the dielectric constant will always be fairly constant in the mouth. Also, for the moment let's suppose that the salt can vary in concentration but that the type of salts present remain the same.

 

 

This means that D, T, and e, are constant. Pi is also a constant. So if we combine all these constants into one big constant , include the number 8 and call it "k" as shown on the right then the equation simplifies to the one shown below.

This is much simpler and shows clearly that the electric double layer (x) shrinks if the salt concentration (n) increases.

   
The electric double layer shrinks as the ionic strength of the solution increases

What this simplified equation means is that if you increase the salt concentration then the cells can get closer to each other, or a surface, before the electric double layers overlap. The effect of this on bacterial adhesion and aggregation is dramatic.

 
The effect of Van der Waals forces

Before we can deduce any effect on adhesion we now need to look at the attraction force. The only significant attractive force known to be present is van der Waals force which is due to an interaction between oscillating dipoles on the surface molecules. This force is unaffected by ionic strength.

Van der Waals attractive force is a very powerful force but it only operates over a small distance. Significantly less than the repulsive force due to overlapping electric double layers. It is, however, very strong and if the cell can get close enough to the surface, the van der Waals force will hold it very tightly.

Note that when the cell gets very close to the surface the van der Waals force starts to get very big indeed. In fact, at very small distances the van der Waals attractive force is enormously bigger than the electrostatic repulsion.

 
How these two forces affect bacterial adhesion

Bacteria are almost the same size as colloidal particles.
Modern thinking on how bacteria stick to surfaces and to each other is greatly influenced by the work done by two groups of researchers who worked independently of each other on the chemistry of colloids. They were interested to know how colloidal particles managed to stay suspended in a solution and the factors which caused them to precipitate. This work has enormous implications in the modern treatment of waste water!

The workers in one of these groups were called Deryagin and Landau and in the other were called Verway and Overbeek. Between them they worked out the mathematics which described the forces acting on colloidal particles, some of which you have seen above. Their theory became known as the DLVO theory after the initials of their names.

Many years later, microbiologists interested in how bacteria attach to surfaces realised that bacteria were almost colloidal particles and that the DLVO theory might well be a useful model.

 
The DLVO Theory

The DLVO theory in its simplest form looks at the two main forces acting on charged colloidal particles in a solution. The two forces are:

1. electrostatic repulsion

2. van der Waals attractive force

By adding these two opposite forces together it is possible to describe the overall force acting on a colloidal particle as it approaches another particle or a charged surface.

It is very important to realise that the overall shape of this resultant force depends on the ionic strength of the fluid because this affects the depth of the electric double layer and, therefore, the electrostatic repulsion.

Now back to bacteria

This graph shows the two forces acting on a bacterial cell as it approaches another cell. The same forces apply if it was approaching a surface.

If these two forces are added together we get a picture of the overall force acting on the cells. This is shown by the green line.

The sum of the two forces

The green line shows the force acting on the cells as they approach each other.

1.

At a separation distance of about 10nm the cells feel a small attraction.

2.

As they get closer still the electrostatic repulsion increases but the van der Waals force, being a short-range force, does not.

3.

If the cells get closer still they start to feel an increasingly stronger repulsion

4.

At a distance of about 2 nm the van der Waals force kicks in and becomes dominant at about 1nm

5.

At less than 1nm the cell starts to feel a strong and increasing attraction to the other cell or surface

 
The effect of ionic strength

The graph above shows what is happening at physiological ionic strength which approximates to the situation in the mouth. At higher ionic strength however, the electric double layer shrinks. This affects the strength of the repulsive force but not the van der Waals force.

Increased ionic strength reduces the electrostatic repulsion which means that the cells can get closer. If the ionic strength is high enough the cells feel no repulsion at all and slam together at high speed. Move the mouse cursor over the graph to see the effect of increasing the ionic strength.

So, adding salt to a bacterial suspension causes the cells to aggregate and fall out of suspension. It would also cause them to stick tightly to a surface.

The bottom line

The cells, however, can not get past the large electrostatic repulsion at a separation distance of between 3-5nm.

Cells are held briefly at about 10nm apart by the weak attractive force. Thermal motion, shear force or the cells own motility will easily break this attraction

In order to adhere firmly the cells must exploit this brief time when they are held 10nm from the surface. They do this in a variety of ways which are beyond the scope of this tutorial

 
     
And finally, remember "v" for valence?

There is one more interesting consequence of the DLVO theory and that is that the stability of a colloid depends not only on the concentration of electrolyte but also on its valence. The point at which a colloid becomes unstable and flocculates is known as the "critical coaggregation concentration" and its calculation is heavy going but after a number of reasonable assumptions can be simplified to the value of 0.8 divided by the sixth power of the valence of the electrolyte (opposite).

For a negatively charged colloid such as bacterial suspension the "ccc" in the presence of a monovalent salt such as sodium, is 0.8M which is above normal physiological values for this ion. For calcium, however, the "ccc" is 0.0125M or 12.5mM reflecting its divalent nature. For aluminium, valency of 3, the "ccc" is approximately 1mM. This explains why aluminium is used as a flocculant in water treatment plants.

The reason why this is important in the mouth is that saliva, and especially dental plaque fluid, contains quite large amounts of calcium. Normally, the concentration of calcium in saliva does not vary a great deal but it can fluctuate a lot in plaque fluid. Raised calcium concentrations in plaque fluid will encourage bacteria to aggregate and adhere more firmly to the tooth.

The DLVO theory predicts that this would happen, however it has not been tested experimentally and the clinical significance is not known...yet

Interestingly this also explains some observations made back in the 1970s by oral microbiologists investigating bacterial adhesion to teeth. Some researchers had noticed that the addition of small amounts of calcium enhanced bacterial adhesion to surfaces in a number of different assays. The amount often quited was 10mM which is very close to the "ccc" for calcium (12.5mM). The hypothesis widely held at the time was that calcium ions, being divalent, were able to bridge the gap between two negatively charged surfaces. This clearly was at odds with DLVO theory which predicts that the two surfaces would not approach close enough for something on the atomic scale to have such an effect. Nevertheless, this view still prevails in some quarters.

What is clearly happening is that the addition of small amounts of calcium are destabilising the bacteria being held in suspension. A similar effect would have been achieved with 0.8M sodium chloride but no one thought to try it until much later.

 

 

 

Not to scale obviously!

   

SUMMARY

1.

Most cells and biological surfaces are negatively charged

2.

In a fluid environment the surface negative charge attracts a layer of mobile counterions (cations) which is known as the "Electrical Double Layer"

3.

As bacteria approach a surface or another cell the electric double layers begin to overlap causing an electrostatic repulsive force

4.

As bacteria approach a surface they also experience an attractive force known as van der Waals force

5.

The combination of these two forces dictates how a bacterial cell adheres and is described by the DLVO theory

6.

The DLVO theory predicts that in a solution of physiological ionic strength bacterial cells will be held about 10nm away from a surface and will be unable to approach closer.

7.

The electrical double layer shrinks if the ionic strength of the surrounding fluid is increased which allows cells to get nearer to surfaces. This is the reason why adding salt to a bacteria suspension causes the cells to flocculate

8.

The stability of bacteria in suspension is very sensitive to the valence of the counterion, therefore, calcium has a greater effect on bacterial adhesion than sodium.

 

 

 

 

 

 

 

 

 

 

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