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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.
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.
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.)
With the second formula, we find the generation time which is the time it takes for the
population to double:
When we graph the CFUs/ml and absorbance on the same graph, we would hope to see an
upward trend for both. Sometimes the absorbance continues to rise after the CFUs/ml level off
into the maximum stationary phase. What would be the cause of that?
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.
5. Remember to use a new tip each time you begin to use a more dilute concentration of cells
Period 1
Materials
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.
8 tubes of melted Plate Count Agar (PCA) in test tubes (15-20 ml/tube) - in 50°C water bath
1. Each pair will pick up one culture from the ice-water bath on the front table. Record the
number on the tube. It represents the age of the culture at which time the sample was
taken.
2. With the P1000 (blue) pipettor set at 1.0 ml, transfer 1 ml of the culture to the first nine
ml dilution blank (for the first 1/10 dilution to work with in Step 5).
3. Aseptically dump the remainder of the culture into the small spectrophotometer tube (to
work with in step 4).
4. With the culture in the spectrophotometer tube, one person in the pair will obtain and
record the absorbance reading of the culture while the other begins the next step.
5. With additional dilution blanks, make dilutions as specified below. Inoculate 1 ml from
each of the four specified dilutions into each of two petri plates; plate inoculations can be
made concurrently with preparation of the dilutions.
For 0-hour through 2-hour sampling times: 10-4, 10-5, 10-6, 10-7
For 2.5-hour through 4.5-hour sampling times: 10-5, 10-6, 10-7, 10-8
For 5-hour through 9-hour sampling times: 10-6, 10-7, 10-8, 10-9
1. For each plate, obtain a tube of melted PCA from the water bath and pour the contents
into the plate. Mix the medium and inoculum by carefully swirling and allow the plates to
solidify.
2. Incubate the plates inverted at 30°C until the next period.
Period 2
1. Each pair will determine the total plate count (no. of colony-forming units/ml of culture).
Be sure you are counting colonies of all sizes. (E. coli typically produces small, lens-
shaped colonies when growing below the surface.) Turn your result in to the instructor
along with the absorbance reading. Be sure you have indicated the sampling time! Results
will be compiled and presented next period.
1. Plot the plate count data on semi-logarithmic graph paper. Rather than generating a
growth curve by connecting the dots, draw the best straight lines through the lag and
exponential phases. (Transitions between the growth phases can be rounded out.)
2. Determine the growth rate and generation time for the particular strain and cultural
conditions in our experiment. Remember that the points you need to calculate these
values are to be taken from the best straight line drawn through the exponential phase. Do
not use individual data points from the class data. Also, be sure to indicate the proper
units (gen/hr or hr/gen). Show your calculations!
3. Plot the absorbance readings on semi-logarithmic graph paper. Note any similarities in
the graph generated and that for the plate count data. Both the absorbance and plate count
plots can be made on the same graph. Do not use the absorbance readings for any
calculations of growth rate or generation time.