Hello Everyone,
I’ve created these videos primarily as instructional aides for new students, interns, and trainees in my research group. While many of the methods are generally applicable, I am of course producing these demonstrations with the materials, reagents, and equipment that are available in our lab. Also, I’ll occasionally reference the locations of items in our lab.
In this video, I go over how to measure the optical density of a bacterial cell culture using an Eppendorf Biophotometer. Determining the optical density at 600 nanometers (OD600) of a sample provides a simple means of monitoring the cell density of that culture. The machine is just measuring (Intensity of light in) - (Intensity of light out), but most biomolecules absorb relatively little light at 600nm so this is largely a measure of light scattering by suspended particulate matter. Your cells will comprise the vast majority of the suspended particulate matter, thus OD600 is an effective measurement of cell density.
Please note that the relationship between OD600 and absolute cell density (cells per unit volume) is heavily dependent upon (1) the size and shape of the cells and (2) the actual density and optical properties of the cells themselves. As a result, the same OD600 reading would mean different cell densities for different organisms.
Other considerations:
-- For the tapered “semi-micro” cuvettes like the ones I’m using, a sample volume of 0.8mL is sufficient to get a good measurement. Of course, this volume will depend on the make of the cuvettes that you are using.
-- When handling a cell cultures, try to work under a flame with good aseptic technique.
-- In order to make sure that the sample you aspirate is representative of the whole cell culture, make sure that the culture is well-mixed immediately before taking a sample and that you’re sampling from the middle (not the bottom or the top) of the culture.
-- A cuvette that has had a cell sample sitting in it for a while may have experienced some settling. Either re-suspend your sample by pipetting or take a new sample.
-- Bubbles throughout your sample volume may wildly alter your measurement. If there are bubbles stuck to the walls of the cuvette, try to get these up to the top by gently tapping the cuvette on the bench-top.
-- Unless you are trying to take a very precise measurement, it’s usually fine to blank with water (unless you’re using TB or a similarly dark growth medium). You can also blank with water and then subtract a “fudge factor” to account for your growth medium. The absorbance of the different growth media at 600nm should be fairly consistent.
About me: I am currently (at the time of uploading this video) a doctoral student in the Chemical and Biomolecular Engineering department at The Ohio State University. My advisor is Dr. David Wood, and our research group specializes in protein engineering and downstream processing for the biotechnology industry. I’m also very new at producing instructional videos so I apologize in advance if these are not of professional quality. For instance, in my first few videos I did not know how to get rid of the timestamp (which is wrong), however I posted them anyways.
Please keep comments civil and apolitical. You are of course welcome to ask questions, however it is unlikely that I will answer them if you are not a member of my research group.
Thanks for watching,
Steven