Bacterial Enumeration Worksheet

Below is absorbance data (Figure 1) collected using an Escherichia coli culture isolated from the well water. Using this data and the [cell] of the well water calculated previously, construct a standard curve. Consider all of the components of a graph when constructing it!

Table 1. Spectrophotometric data.

Dilution	ABS 595 nm
1/1	0.620
1/3	0.380
1/6	0.260
1/12	0.180
1/24	0.130

To determine the generation time of this E. coli isolate, a TSB tube is inoculated with 0.1 mL of an overnight TSB culture. After 2 hours of incubation at 37^oC in a shaking platform incubator, the absorbance (595 nm) of the culture is 0.160. The culture is then incubated for an additional 4.5 hr., after which the measured absorbance is 0.460. What is the generation time of this E. coli isolate? Remember that you first must use the standard curve to determine the density of E. coli cells at each of the measured absorbance values (be sure to show this on your graph), then use the appropriate formulas to (1) calculate the number of generations produced, and (2) calculate the generate time. You must show your calculations work below.