Bacteria are grown in petri dishes upon a solid medium known as bacterial agar, where raised, circular colonies form. Their growth can be measured by simple observation of how dense their colonies are and how many are present, however more quantitative methods include the use of a counting chamber, or more frequently, viable plate counts. The latter is used most frequently as it also provides qualitative information such as the effect of varying growth conditions.
Things You'll Need
- Dilution media
- Pipettes and tips
- Bacterial culture media and agar plates
- Bunsen burner
- Glass spreaders
- 70 percent ethanol
Set up a series of tubes containing serial 1:10 dilutions of media. For example, set up a row of 10 tubes and add 10 microliters of the starting bacterial culture to 90 microliters of dilution medium. Shut the lid of the tube tightly and vortex gently to obtain a homogeneous mix. Transfer 10 microliters of this to the next tube containing 90 microliters of dilution medium, and so on. Ensure each tube is labelled with the correct dilution, i.e 10^1, 10^2, etc.
Dispense 10 microliters of the last dilution (to make a plating factor of 10) onto the agar plate. Using the spreading edge, distribute the bacterial solution across the entire surface of the agar plate. Repeat this for as many replicates as needed, however duplicates are usually sufficient. Replace the lid and let the agar plates dry for several minutes either on a laboratory bench beneath a flame, or in an incubator. Invert the plates onto their lids, and label the plates. Place the plates in the incubator which should be set to the appropriate temperature for the strain of bacteria. Leave to grow for 12 to 16 hours.
Colonies should be visible after 16 hours, however, some genetic modifications may require longer (e.g. color development). When colonies are observed, take the plates out and find ones that have between 30 and 300 colonies. Using a permanent marker, place a dot on the bottom of the petri dish (the side with the agar, not the lid) where a colony is visible through the agar, and count it. Repeat until all colonies are marked and counted.
To derive the quantity of bacteria in the starting culture for this experiment, use the formula:
Number of colonies counted X dilution factor (e.g. 10^7, or 10^9 etc) X dilution factor of plating (i.e. 10) = Number of colony forming units (CFU) per milliliter of starting culture.
- "At the Bench: A Laboratory Navigator;" Kathy Barker; 1998
- Western Washington University: Bacterial Enumeration
- Photo Credit Picturenet/Blend Images/Getty Images
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