Scientists use spot plates both to calculate the number of cells in cultures and to obtain information about the growth properties of strains on different media. The figure below
shows an example of a typical spot plate. Each row represents a dilution series from a different yeast culture. The same volume of diluted culture is used for each spot. The dilution series is planned so that the most dilute spots contains a small number of individual colonies that can be distinguished from one another, typically less than ten.
Each row on the plate represents a series of 1:10 dilutions of a liquid culture of S. cerevisiae. Five μL of each dilution was spotted on the plate. The plate was incubated for two days at 30 ̊C. Individual colonies are apparent at the highest dilution of each extract.
Most commonly, investigators make a series of 1:10 dilutions in sterile water and then spot a few microliters of each dilution in a row. In this experiment, 5 μL aliquots were spotted from the serial dilutions. Note that it is possible to count individual colonies in the most dilute samples. This in turn enables you to calculate the number of viable cells in the original culture. In the top row, you can distinguish 4 colonies in the sample that has been diluted 100,000-fold. The original culture would have contained 400,000 cells in 5 μL, which corresponds to 80 million cells per mL (8 x 107 cells/mL).
In this experiment, you will use spot plates to estimate the cell densities of log phase and stationary phase cultures of S. cerevisiae and S. pombe. Each group will receive four cultures:
CL - S. cerevisiae log phase culture
PL - S. pombe log phase culture
CS - S. cerevisiae stationary phase culturePS - S. pombe stationary phase culture
Spot the dilution series from each culture on a separate row of the plate. Be sure to LABEL the rows!
Preparing the spot plate
1. Alignment grids are useful for preparing good-looking spot plates! Obtain an alignment grid (right) and mark the target positions for culture dilutions. Place an orientation mark at one point along the circumference.
2. Label the plate with your initials and date with small letters around the BOTTOM rim of the dish. Put a hash mark on the edge of the plate to serve as an alignment marker.
3. Prepare a series of five 1:10 dilutions from each culture using sterile water. (Diagrams in your lab notebook are often helpful in designing dilution series.) To prepare a serial dilution, first pipette 90 μL sterile water into five microcentrifuge tubes. Next, use a P20 to transfer 10 μL from the original culture into the first tube. Eject the tip into the appropriate waste container.
4. Vortex the newly diluted culture to insure that the cells are uniformly distributed. With a fresh micropipette tip, transfer 10 μL from this tube to the second tube in the series. Repeat this step for tubes 3-5.
- Prepare the spot plate. Beginning with the last dilution in the series, spot 5 μL spots in a row. Vortex each dilution before spotting it, because cells may have settled. You will be able to use a single pipette tip for each dilution series, since you started with the most dilute cell suspension.
- Repeat step 3 for each culture that you are analyzing. Be careful to note in your lab notebook which culture has been spotted into each row on the plate!
- Leave the plate right side up for ~30 minutes, to allow time for the yeast to settle and attach to the medium.
8. When the cells have settled, invert the plates and incubate them at 30°C. Plates are inverted to prevent water droplets that form on the inner surface of the lid from falling on the colonies. Plates can also be kept at room temperature, but cells will grow more slowly.
9. When the colonies are large enough to count, the plates will be removed from the incubator by the staff and placed in the refrigerator or cold room for your analysis later.
10. At the next class: Record your data with the scanner. To do this, remove the top from each plate and invert both the plate and its lid. Place the bottom half of the dish on the scanner and leave the inverted lid on the bench. (The lid is inverted to avoid contamination from spores and microorganisms that may be present in the air.) Place a black piece of cardboard or a folder over the plates before lowering the lid of the scanner.
11. Use spots where you can count individual colonies to calculate the density of cells in the original cell culture, correcting for the dilutions and the volume of the spot (see above).