In this article we will discuss about the principle, requirements and procedure for measuring the growth of bacteria by spectrophotometric method.
This method relates cell number to turbidity of a broth culture. Turbidity or optical density (OD) is directly proportional to cell concentration.
1. 10-12 hour E. coli broth culture maintained at lag phase by immersing in an ice bath.
2. Brain-Heart infusion broth (pH 7.4):
3. Water bath shaker incubator set at 37°C.
4. Bausch and Lomb Spectronic 20.
6. 13×100 mm cuvettes.
7. Sterile 5 ml pipettes.
8. Bunsen flame.
9. Inoculating loop.
10. Glass marking pencil.
1. Transfer aseptically 5 ml of broth culture to a flask containing 100 ml brain- heart infusion.
2. Find the initial OD (i.e. at To) at 600 nm wavelength which should be 0.08 to 0.01.
3. Keep the inoculated flask in the water bath shaker (120 rpm) at 37°C for 6 hours.
4. Transfer aseptically 5 ml of culture to cuvette after 30 minutes.
5. Determine the OD of sample at 600 nm.
6. Repeat the above steps after every 30 minutes interval for a period of 6 hours.
OD = 2-log of per cent transmittance, e.g. if the per cent transmittance of one of your samples is 53.5, the problem is to be solved as follows.
OD = 2 – log of 53.5
= 2- 1.7284
(0.7284 is the value found in log table) = 0.272.
Plot a graph with OD versus incubation time and calculate generation time for E. coli culture.