Stephan Curves: The Basics
   
The basic Stephan Curve

The Stephan Curve describes the change in dental plaque pH in response to a challenge. The type of challenge does not matter but it is usually some element of the diet. On the other hand the challenge could be an inert substance placed in the mouth with the aim of determining its effect on plaque pH. For example, mechanical stimulation of the salivary glands caused by masticating chewing-gum base. This would be of interest in an investigation into the effect of saliva flow on the pH-changes happening in plaque after a challenge by a cariogenic food.

The characteristics of the Stephan Curve are shown in the diagram. In this example, dental plaque was challenged with a fermentable carbohydrate by asking a volunteer to rinse with 10mls of 10% sucrose solution for 10 seconds. Average plaque samples were removed at intervals and the pH recorded.

Characteristically the Stephan Curve reveals a rapid drop in plaque pH, followed by a slower rise until the resting pH is attained. The time course varies between individuals and the nature of the challenge.

The initial drop is usually rapid with the lowest pH being attained within a very few minutes. However, pH recovery can take anything between 15 and 40 minutes depending to a large extent on the acid-neutralising properties of the individual's saliva.

 

 
The Stephan Curve

 

 

The initial drop in pH

The initial rapid drop in pH is due to the speed with which plaque microbes are able to metabolise sucrose. Larger carbohydrates, such as starch, would diffuse into plaque more slowly and would need to be broken down before assimilation by the microbes. In the case of starch, salivary amylase would produce a mixture of glucose and maltose together with incompletely digested material comprising the branch points of the starch molecule (limit dextrins). The glucose and maltose would then be taken up by plaque bacteria and metabolised. The rate of starch breakdown slows up glycolysis and, therefore, acid production producing a less steep drop in pH.

Plaque Metabolism

 

     
The lowest pH

The lowest pH achieved depends greatly on:

1.

the microbial composition of the dental plaque

2.

the nature of the fermentable carbohydrate source

3.

the rate of diffusion of substrates and metabolites into and out of the plaque.

 

Microbial composition

The presence of significant numbers of aciduric, acidogenic bacteria in plaque developing in a sheltered site with a low diffusion rate coupled with a readily fermentable carbohydrate such a sucrose or glucose would produce the lowest pH. Under these conditions a pH in the region of 4.5, or even lower, might be attained.

Carbohydrate nature

Conversely, a challenge with a carbohydrate which is metabolised more slowly to a plaque community with fewer aciduric, acidogenic microbes would result in less acid production and a higher terminal pH.

Rate of diffusion

The rate of diffusion of material into and out of plaque is governed by the density of the plaque and access by saliva. Thus less dense plaque fully exposed to saliva flow will more rapidly exchange metabolites with the surroundings. This will enable substrates to diffuse into the plaque rapidly and at the same time allow microbial by-products to diffuse out. The terminal pH following a challenge to the plaque will reflect the relative rates of diffusion of both substrate and metabolites.

 
Effect of microbial composition
 
 
Rate of Diffusion

The lowest pH attained is also determined by the rates of diffusion of substrates and metabolites. Molar fissures are the most caries-prone sites because they are very sheltered from saliva flow. The deepest parts are often inaccessible to toothbrushes which means that fissures frequently contain impacted food for extended periods of time. If the food contains carbohydrate the plaque is likely to have a lower resting pH than that found at other sites in the mouth. This lower resting pH will encourage the growth of aciduric microbes such as S. mutans. In fact, research has shown that fissure plaque has a greater proportion of aciduric microbes including S.mutans and lactobaccilli.

The rise in pH

The pH starts to rise after a few minutes due to:

1.

acid by-products diffusing out of the plaque

2.

salivary bicarbonate diffusing into the plaque and neutralising the acid by-products.

In addition, the low pH produced will inhibit microbial metabolism and thereby slow the rate of acid production. This will allow the processes of diffusion and neutralisation to exert a greater effect on plaque pH. Also, as before, plaque which is less dense and fully exposed to saliva flow will show a faster rate of pH recovery.

It normally takes at least 20 minutes for the plaque pH to reach its resting value but it can take considerably longer depending on the factors described above.

 

 
Effect of Saliva on Diffusion
Saliva flow rate

One of the most important factors governing the overall shape of a Stephan Curve, but particularly the pH recovery, is saliva flow rate. The effect of saliva on the Stephan Curve is easily demonstrated by isolating some dental plaque using cotton wool pads. This is shown in the diagram on the right.

Saliva exerts two effects. First, it dilutes and carries away metabolites diffusing out of the plaque. Second it supplies bicrabonate ions which diffuse into plaque and neutralise the by-products of fermentation (organic acids) in situ. The bicarbonate-mediated acid neutralisation effect is enhanced by the increase in salivary bicarbonate associated with increased saliva flow which co-incides with eating.

Acid neutralisation by bicarbonate is accelerated by salivary carbonic anhydrase. This is secreted by acinar cells of the parotid and submandibular glands and is the only example of a secreted carbonic anhydrase in mammals. Find out more from this review article.

 

 
Stephan Curve: Effect of Saliva
Methods of obtaining Stephan Curves
In Vitro   In Vivo

In the in vitro method plaque is removed from the mouth at intervals and the pH measured using a micro-pH electrode. Graphs similar to those shown above are obtained by this means. Success with the in vitro method depends to a great extent on the skill of the operator in obtaining 6-7 representative plaque samples and the compliance of the volunteer who has to allow sufficient plaque to accumulate. Obtaining multiple, similar plaque samples requires careful choice of sampling sites and avoiding harvesting too much plaque from any one site. The operator visits about 6 different sites in the mouth harvesting a small amount of plaque from each. The picture shows the amount of plaque required on the end of a microspatula. This plaque is homogenised in a small amount of distilled water and the pH recorded. This is repeated up to 5-6 times for each experiment. Great care must be taken to standardise the time taken to collect plaque and measure the pH to ensure that samples are equivalent.

 

In the in vivo method plaque pH is measured using electrodes placed within the mouth. The normal method used involves constructing a special dental bridge incorporating the pH electrode. This is placed in the mouth and the volunteer allows plaque to accumulate over the surface of the electrode. The device is connected to recording equipment which can either be carried around with the volunteer or the experiment is carried out within the confines of a laboratory.

 

Pros and cons

The main advantages of the in vivo method are that large amounts of data can be collected in real time for a long period and that the plaque is not disturbed meaning that the response of the same plaque to different challenges can be compared directly.

The main disadvantages are that volunteers are required to have a specific dentition which permits a bridge to be fitted and that the overall expense is high with the result that only a relatively few individuals can be examined.

Pros and cons

The main advantage of the in vitro method is that it is cheap and doesn't require much specialised equipment. This means that it can be performed on quite large numbers of volunteers. The main disadvantage is that it requires considerable skill to obtain reliable results.

   

 

 

 

 

 

 

SUMMARY

1.

Stephan Curves describe the changes in pH ocurring within dental plaque when it is subjected to a challenge, typically with a foodstuff

2.

When challenged with a fermentable carbohydrate the pH within plaque drops rapidly and then rises back to the resting pH more slowly

3.

Factors affecting the shape of the Stephan Curve include the microbial composition of the plaque; the nature of the fermentable substance; the rate of diffusion of bacterial metabolites, salivary components such as bicarbonate and the fermentable substance; salivary access to the plaque; saliva flow rate

4.

The relationship of the shape of the Stephan Curve to the Critical pH can be used to assess the relative cariogenicity of foods

5.

Stephan Curves can be constructed from data obtained by in vitro and in vivo methods.

Read more about Stephan Curves and their Clinical Relevance

   
 

[top]