MEASURING THE OPTICAL DENSITY OF BACTERIA
Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD600 is measured. If you did not do an experiment in your Microbiology laboratory class that demonstrates how to determine the OD that corresponds to a log phase of growth, please read one of the laboratory protocols that I will have available in class. If you would like to do this experiment I can arrange for you to do it in the laboratory on one of the non-class days.
The protocol below is specific for the Beckman DU 640B.
1) Turn the machine on using the switch in the back of the machine (lower right-hand side as you face the machine). Turn on the monitor screen and printer. Wait for the machine to go through its start up routine.
2) Open the lid on the top of the machine. Choose the large cuvette holder and place it in the holder in front of the light source. The word FRONT should be toward the front of the machine.
3) Click on the “visible light” key to turn the light source on. Quit the diagnostic screen by clicking on the word QUIT in the upper right hand corner. The Main Menu will appear. Choose Single Wavelength mode from the menu that appears. When the next menu appears, make sure that the wavelength used to measure OD is 600. If the menu reads differently, highlight the number and click on it. A keypad will appear, enter 600. Click OK.
4) Place a 500 µl sample of your blank (broth that your bacteria are growing in) in a cuvette. Place the cuvette in the holder.
5) Shut the lid of the machine.
6) Click READ BLANK in the bottom left corner of the screen.
7) After the machine has read the blank, remove the cuvette, replace it with a cuvette containing 500µl of the bacterial culture.
8) Close the lid and click on READ SAMPLES in the upper left hand of the screen.
9) After the machine has read your sample, the data will appear on the screen. Click on Print in the upper right-hand corner of the menu bar to print the data. If you are measuring more than one sample, you do not need to print each time you measure a sample. The machine will collect data for you. You can print when you are finished all of the data collection. You also do not need to blank each time unless your samples are suspended in different solutions.
10) When you are finished reading samples, print your results by clicking on PRINT at the top right of the screen. QUIT the data screen. This will take you back to the main menu. Turn off the light source. You can turn off the machine while the Main Menu screen is active. Turn off the machine, the monitor and the printer.