The Growth Rate (k) and Generation Time (g) of The E. Coli Cultures in Terms of Different Temperatures: a Lab Study

Microbial Growth

In this lab we measured the absorbance, at 15 minute intervals, of Escherichia coli in tryptic soy broth using a spectrophotometer. Using measurements of optical density (OD) provided by the spectrophotometer we are then able to calculate the values for growth rate (k) and generation time (g) of the E. coli cultures at different temperatures. OD readings were taken for cultures of E. coli incubated at 37?, 27?, 45?, and 55? at 15 minute intervals for 75 minutes. OD is a measure of the turbidity of the culture, as such it cannot differentiate between viable and dead cells. In part II of the lab we performed a serial dilution and spread plating of E. coli, after incubation we performed a viable count of the plates that had between 30-300 colonies in order to determine the number of colony forming units per ml.

For the growth at different temperatures experiment we expect that our data will indicate that the optimal growing temperature for E. coli is 37?. Our hypothesis is based on growing conditions for wild E. coli, E. coli bacteria are typically found in the intestines of humans and other animals, where the internal temperature is around 37?. At temperatures above (45?) and below (27?) 37? we expect some growth, though it will likely be slowed as the bacteria will have to adjust to these temperatures. At 55? we expect to see no growth as this temperature is far too high for the bacteria to survive.

For the viable counts experiment we expect that our spread plates from our dilution will show a decrease in colony count for the more dilute culture. For the serial dilution spread plates the dilutions from 1X10-1 to 1X10-4 the number of colonies was too numerous to count, the 1X10-5 dilution plate had 59 colonies present, and the 1X10-6 to 1X10-8 dilution plate colonies were too few to count. For the 10-5 dilution we calculated the number of colony forming units per ml as 5.9x107 CFU/ml. This was calculated by using a volume of 0.1 ml, a dilution factor of 105, and number of colonies equal to 59.

The OD values for each 15 minute interval, for the growth experiment, is shown below in Figure 1. Using this data we can calculate the k and g values for each temperature tested. With a starting time of 15 minutes (N0) and an end time of 75 minutes (N) the value for n may be calculated, which is required to determine k. The formula for k is k=n/t, where n=3.3*(log N - log N0) and t is the interval of time in hours. The k values (generations/hour) for 27?, 37?, 45?, and 55?, respectively, are 0.85 generations/hour, 1.63 generations/hour, 1.69 generations/hour, and 0.14 generations/hour respectively. Using k we calculated g (generation time) as g=1/k. This gives us a generation time of 1.17 hrs, 0.61 hrs, 0.59 hrs, and 6.97 hrs for each temperature respectively. Converting those values to hours and minutes give a generation time of 1 hour 10 minutes for 27?, 37 minutes for 37?, 35 minutes for 45?, and 6 hours 58 minutes for 55?. We also plotted absorbance over time for each temperature as seen below in Figure 2.

Our results for the growth experiment did not match the expected results. The results for 27? and 55? were consistent with our hypothesis, however the results for the bacteria kept at 45? and 37? did not agree with our expectations. Initially, the bacteria at these temperatures had performed as expected, from the interval 15-30 minutes our data shows that the 37? bacteria had an increase in OD readings higher than that of the 45? bacteria. After the 30 minute reading the 45? bacteria increased its OD by .036 versus .009 for the 37? bacteria. The reason for this unexpected result id not entirely clear. It may be due to human error in taking OD readings, especially if the machine is not properly blanked. Another possibility is fluctuations in temperature that could be caused by exposing the culture to room temperature for too long when taking samples. If the E. coli in the 37? water bath was exposed to room temperature air for too long its temperature may have dropped leading to a temporary stall in growth. Our data does indicate a stall in growth like this from 15-60 minutes overall OD increased by 0.051 then during the last interval we observed an increase in OD by 0.056, which was greater that the previous hour of growth.

For the serial dilution spread plates, the results were as expected.