Dilution theory and techniques

Dilution allows the number of living bacteria to be determined in suspensions that contain even very large numbers of bacteria.

The number of bacteria obtained by dilution of a culture can involve growth of the living bacteria on a solid growth source, the so-called dilution plating technique. The objective of dilution plating is to have growth of the bacteria on the surface of the medium in a form known as a colony . Theoretically each colony arises from a single bacterium. So, a value called the colony-forming unit can be obtained. The acceptable range of colonies that needs to be present is between 30 and 300. If there is less than 30 colonies, the sample has been diluted too much and there is too a great variation in the number of colonies in each milliliter (ml) of the dilution examined. Confidence cannot be placed in the result. Conversely, if there are more than 300 colonies, the over-crowded colonies cannot be distinguished from one another.

To use an example, if a sample contained 100 living bacteria per ml, and if a single milliliter was added to the growth medium, then upon incubation to allow the bacteria to grow into colonies, there should be 100 colonies present. If, however, the sample contained 1,000 living bacteria per ml, then plating a single ml onto the growth medium would produce far too many colonies to count. What is needed in the second case is an intervening step. Here, a volume is withdrawn from the sample and added to a known volume of fluid. Typically either one ml or 10 ml is withdrawn. These would then be added to nine or 90 ml of fluid, respectively. The fluid used is usually something known as a buffer , which is fluid that does not provide nutrients to the bacteria but does provide the ions needed to maintain the bacteria in a healthy state. The original culture would thus have been diluted by 10 times. Now, if a milliliter of the diluted suspension was added to the growth medium, the number of colonies should be one-tenth of 1,000 (= 100). The number of colonies observed is then multiplied by the dilution factor to yield the number of living bacteria in the original culture. In this example, 100 colonies multiplied by the dilution factor of 10 yields 1,000 bacteria per ml of the original culture.

In practice, more than a single ten-fold dilution is required to obtain a countable number of bacterial colonies. Cultures routinely contain millions of living bacteria per milliliter. So, a culture may need to be diluted millions of times. This can be achieved in two ways. The first way is known as serial dilution. An initial 10-times dilution would be prepared as above. After making sure the bacteria are evenly dispersed throughout, for example, 10 ml of buffer, one milliliter of the dilution would be withdrawn and added to nine milliliters of buffer. This would produce a 10-times dilution of the first dilution, or a 100-times dilution of the original culture. A milliliter of the second dilution could be withdrawn and added to another nine milliliters of buffer (1,000 dilution of the original culture) and so on. Then, one milliliter of each dilution can be added to separate plates of growth medium and the number of colonies determined after incubation. Those plates that contain between 30 and 300 colonies could be used to determine the number of living bacteria in the original culture.

The other means of dilution involves diluting the sample by 100 times each time, instead of 10 times. Taking one milliliter of culture or dilution and adding it to 99 ml of buffer accomplish this. The advantage of this dilution scheme is that dilution is obtained using fewer materials. However, the dilution steps can be so great that the countable range of 30-300 is missed, necessitating a repeat of the entire procedure.

Another dilution method is termed the "most probable number" method. Here, 10-fold dilutions of the sample are made. Then, each of these dilutions is used to inoculate tubes of growth medium. Each dilution is used to inoculate either a set of three or five tubes. After incubation the number of tubes that show growth are determined. Then, a chart is consulted and the number of positive tubes in each set of each sample dilution is used to determine the most probable number (MPN) of bacteria per milliliter of the original culture.

See also Agar and agarose; Laboratory techniques in microbiology; Qualitative and quantitative techniques in microbiology