Tip plating requires one to make 10-fold dilutions from 10-1 to 10-5 of a sample. Using a P1000 pipetman, 900uL of sterile water is added into six labeled 1.5 mL centrifuge tubes. 100uL of the culture is added to the 10-1 tube, this is then mixed well and 100uL from the 10-1 tube is transferred to the 10-2 tube and so on until all dilutions are properly made. It is important to mix each dilution in the centrifuge tubes well before transferring to the next dilution. This is a more rapid version of serial dilution plating by using a single plate for all of the dilutions.
On a square Petri plate, mark the bottom side with all dilutions. Be sure to write right to left so that it is legible when flipped back over. Use a P20 pipetman, set for 10uL (this volume can change depending on the dryness of the plate). Start with dilution 10-5 and transfer 10uL on to the appropriately labeled spot on the plate. Once you have done this transfer 10uL of each dilution in order to the appropriate spot. When done, quickly tip the square plate to a ~45% angle and watch the liquid drain downward, in straight lines down the plate. Before the liquid reaches the other side of the dish, lay it down flat and let it dry for 10 minutes. Place in incubator when finished.
In theory, each colony emerges from a single cell. Ideally, one would count lanes that contain 20 to 150 colonies on tip plates. Less than 20 results in statistically insignificant numbers and greater than 150 become too many to accurately count per lane.
Application in Wine Microbiology:
This is an indirect method that represents how many cells were present in a given culture or sample. In wine microbiology tip plating can be used as a tool to determine how many cells are present in a spoiled wine or must. It can be used to monitor fermentations with multiple inoculums added by allowing you to compare tip plates containing different limiting agents to determine which inoculum is dominating the fermentation. Tip plating can also be used in wine microbiology to compare cleaning and sanitizing agents and their effectiveness on killing microbes. This indirect method of counting cells can be used for many different applications in wine laboratories and is not limited to just those applications stated above. Tip plating is a useful and necessary technique in wine microbiology.
- Bisson, L. F. (2010). VEN128L Lab Manual. University of California-Davis.
- Boulton, R. B., Singleton, V.L., Bisson, L., Kunkee, R. (1996). Principles and Practices of Winemaking. New York, Chapman & Hall.
- Pan, Y., Breidt Jr., F. (2006). Resistance of Listeria monocytogenes Biofilms to Sanitizing Agents. Applied and Environmental Microbiology, Dec. 2006, p. 7711-7717 Vol. 72, No. 12