5-6 Generating a growth curve
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In this experiment, the classic bacterial growth curve will be demonstrated. A culture of Escherichia coli will be sampled at hourly or half-hourly intervals from the time of inoculation of the culture (0-time) through a 7 to 9-hour incubation period. The periodic samplings will be plated to determine viable counts (as colony-forming units per ml of culture) over the incubation period such that a growth curve may be plotted. From the graph, we may note the stages in the growth of the culture as it grows into the stationary phase. Additionally we will be able to determine the growth rate and generation time of E. coli under our experimental conditions from two points in the exponential phase of the graph.
Figure 5-16 Bacterial Growth
Graphing of bacterial growth on a linear scale
By definition, bacterial growth is cell replication - i.e., growth of the culture. Most species of bacteria replicate by binary fission, where one cell divides into 2 cells, the 2 cells into 4, the 4 into 8, etc. If this cell division occurs at a steady rate - such as when the cells have adequate nutrients and compatible growing conditions - we can plot numbers of cells vs. time such as on the graph at right. Before too long, we will need to extend the paper vertically as the population continues to double. For a culture where cells divide every 20 minutes, one cell can result in 16,777,216 (i.e., 224) cells after just 8 hours - barring nutrient depletion or other growth-altering conditions.
Figure 5-17 Bacterial Growth
Graphing of bacterial growth with cell number on a log scale.
If we were to convert our vertical axis to a logarithmic scale - as on the graph at right - we will not need as many sheets of graph paper, and we will find that a steady rate of growth is reflected as a straight line. (On the vertical axis, the same distance on the paper is covered with each doubling.) This type of graph paper is called semilogarithmic graph paper on which we will be plotting our class results. The numbers we plot will fall on the graph at the same place the logarithms of these numbers would fall when plotted on conventional graph paper.
The example below shows the type of graph we may obtain from our class data. We can plot both colony-forming units (CFUs) per ml and absorbance on the same graph, remembering that the absorbance units should also be on a logarithmic scale. Rather than "connecting the dots," we draw the best straight line among our CFU/ml plots to represent the phases of growth - lag, exponential, and the start of the maximum stationary phase.
Figure 5-18 Two measurements of growth
Example data showing a plot of cell number by VPC and by turbidity.
For the growth rate formula we are about to use, we need to choose two points on the straight line drawn through the exponential phase, also making note of the time interval between them. As we will be converting our numbers to logarithms for the formula, why not choose two points for which the logs are easy to obtain? (For example, the log of 1X1010 is simply 10.)
Using the first formula, we find the growth rate which is the number of generations (doublings) per hour:
Figure 5-19 Caluclating the growth rate
Use this formula to determine the growth rate k
With the second formula, we find the generation time which is the time it takes for the population to double:
Figure 5-20 Generation time
The generation time is the reciprocal of the growth rate.
With a clear graph, one should be able to determine the generation time without the use of formulas. Just look for a doubling of the population and the time it takes for that to happen. For example - in the above graph - the time it takes to go from 3 X 109 to 6 X 109 appears to be approximately 30 minutes, which is close to the generation time determined above.
In preparation for this exercise, be sure to read the relevant material in your textbook, and look over the procedure below.
Samples (5-6 ml) which were taken at hourly or half-hourly intervals from a culture of E. coli growing in Nutrient Broth+0.2% yeast extract, incubated at 37°C on a shaker. These samples have been kept on ice for use in this experiment, and each pair will use one sample.
The following are provided for each pair of students:
7-9 nine ml dilution blanks
8 tubes of melted Plate Count Agar (PCA) in test tubes (15-20 ml/tube) - in 50°C water bath
8 empty, sterile petri dishes
Pipettors (P1000) and sterile tips
Spectrophotometer tube and spectrophotometer
The dilutions to be plated are as follows:
For Your Assignment: