Introductory Microbiology (1+1)

Measurement of yeast growth

• An important component of fermentation processes is to continually monitor yeast growth and viability. The most common method for doing this is using the hemocytometer count method. In this method, samples are taken from the fermentation vessel, stained with methylene blue and then counted manually under a microscope using a hemocytometer.
• While this method is well known and documented, it is at best an estimate based upon a very small sample count. The hemocytometer, when viewed under a microscope, presents a grid of measurement areas.
• Because of the time involved for an operator to do manual counting, only a small number of actual grid cells are counted, with the results then being interpolated as an average number. Not only is the sample size very small, which yields low statistical significance, but it is known that up to 25% error can be introduced merely by “operator interpretation”.
• It was desired to develop a method for making the yeast counts more precise, increase the statistical significant by looking at a larger sample and it eliminate the time and potential operator error for the procedure.

Method
The flow camera(CAM) is ideally suited to automate this process. It can image count and measures thousands of individual yeast cells in the time it takes for an operator to count only tens of cells using the hemocytometer method. The visual spreadsheet software automatically produces a count of line, dead and budding yeast cells without any operator being involved. This normalizes out human error, and provides extremely precise and repeatable results. Further, the numers have a much higher statistical significance due to the larger data populations obtained by the flow CAM.
The yeast sample are taken from the fermentation vessel and prepared just as they are for the hemocytometer method. The sample is then run through the flow CAM is auto image mode at reverse frame per second as it flows through the flow cell. Every yeast cell is imaged, stored and measured during acquisition.
The flow CAM automatically captures each yeast cell as a single stored image from the fluid flow. During image capture, up to 26 different spatial and gray-scale measurements are recorded and indexed to the individual cell images. When the yeast cells are stained with the methylene blue, dead cells will uptake the stain, causing them to appears blue to the camera.
For the Flow CAM, differentiating between the line and dead cells is quite straight forward, and is based primarily on the “average blue” value recorded for the cell image (along with several shape measurements). The budding cells present a bit more difficult challenge, however, due to the fact that the resolution needed to accurately differentiate a single “line” cell from a “budding” cell is much higher than can be obtained from the flow CAM.