Serial dilutions are made with the sample to be tested. These dilutions can be made with sterile liquids such as isotonic saline (0.7% w/v) or peptone (1%w/v). It is possible to use sterile water, but this is not the best way to test viability due to osmotic stress that water can have on organisms (Fugelsang, 1997). Each dilution is made in a sterile test tube which has a sterile closure. In order to make ten fold dilutions, it is necessary to put nine parts of the dilution media into the test tube and add one part of the sample that needs to be tested. This creates a 10^-1 dilution. This is repeated, usually to make the desired number of different dilution samples so that there is a concentration bracket around the concentration that will actually be analyzed. Once the dilutions have been made, the sample will be smeared onto plates and grown at the optimum temperature for the specific organism. When transferring the sample from the test tubes onto the plate, the sampler should start with the lowest concentration and move up to the highest concentration. This allows for the sampler to use the same sampling tool to inoculate the plate because the lower concentration will not change the results on the higher concentration plate since each sample is a ten-fold increase. After the plates have grown and established colonies, the sampler should take the plate that contains 30 to 300 CFU/plate and count it. It has been found that if the plate has less than 30 colonies, the test is not significantly reliable and a plate with more than 300 colonies is too difficult to count due to crowding of colonies. After getting the number of colony forming units and taking into account the amount of sample put onto the plate, a concentration of CFU/mL can be calculated. These tests can be repeated and concentration changes can be monitored and tracked. Pictorial representations of this procedure are shown in Figures 1 and 2.
Application in Wine Microbiology:
The use of dilution plating and counting colony forming units allow for wineries to track concentrations of microorganisms in the wine. This makes it possible to see when there is a change in the population of a certain microbe from a range of activities in a winery such as tracking yeast growth in a fermentation or the concentration of a prepared inoculum to be used for malolactic fermentations.
- Boulton, R. Singleton, V. Bisson, L. Kunkee, R. Principles and Practices. Chapman & Hall. 1997.
- Fugelsang, K. Wine Microbiology. The Chapman & Hall Enology Library, NY 1997.
- Steane, R. http://www.biotopics.co.uk/microbes/tech3.html