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Lecture 4

BIOB11H3 Lecture Notes - Lecture 4: Bacterial Growth, Bacteriuria, Microbiological Culture


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Aarti Ashok
Lecture
4

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39
LABORATORY 3 THE BACTERIAL GROWTH CURVE
Pre-lab questions (to be handed in in prior to the start of the lab). The answers must be typed and not
handwritten. [2 Marks]
1. How would you dilute a NaCl solution that has an initial concentration of 15% (w/v) to a 50ml
solution at 0.1%?
2. You have 5ml of an overnight culture (2x108 cells per ml). How would you prepare a series of 1 or
10ml dilutions (maximum dilution 1:100) to obtain a final 200 ml culture at 1x103 cell/ml? Show
your work for each step.
3. If 0.1 ml (from a 1 ml sample) of a 105 dilution of pond water was plated and yielded 52 colonies,
how many bacteria were present per ml in the original water sample? Show your work.
4. If 0.1 ml of a urine culture from a 107 dilution yielded 37 colonies, how many bacteria were
present per ml in the original sample? Show your work.
NOTE: Refer to the following site to refresh calculations of dilutions:
http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html
Growth may be defined as an increase in cellular constituents, in some organisms. It leads to a rise in cell
number when microorganisms reproduce by processes like budding or binary fission. In the latter process,
individual cells enlarge and divide to yield two progeny of approximately equal size. Growth also results
when cells simply become longer or larger. However, it is not usually convenient to investigate the growth
and reproduction of individual microorganisms because of their small size. Therefore, when studying
microbial growth, microbiologists normally follow changes in the total population number.
Population growth is studied by analyzing the growth curve of a population in a confined space broth
culture (for bacteria) or in culture flasks (for tissue culture) for instance. When microorganisms are
cultivated in liquid medium, they usually are grown in a batch culture or closed system. Since fresh medium
is not provided during incubation, nutrient concentrations decline and concentrations of waste products
increase. The resulting curve has four distinct phases (Fig. 3.1).
Objectives for Week 3 - After completing these exercises, you should:
1. Be able to follow the growth of a liquid bacterial turbidimetrically.
2. Be able to determine viable cell numbers by plate counts during growth.
3. Understand how to plot and analyze bacterial growth data.
Reading:
Madigan et al (2018): Ch 5: p. 138; 140-143 and 149-152

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Fig. 3.1 Bacterial growth curve in batch culture
Lag Phase: During this phase, cells in a new culture adjust to the medium. Initiation of gene expression and
subsequent increases in enzyme production and cell size occur.
Exponential or Logarithmic Phase: During this phase, metabolic activities proceed at a constant rate, and
cell mass as well as the number of cells double at a constant rate. The time required for the population to
double is called the generation time (g) or doubling time and is usually expressed in hours. Because the
population is doubling every generation, the increase in population is always 2n where n is the number of
generations. The resulting population increase is exponential or logarithmic. The rate of growth during the
exponential phase in a batch culture can be expressed in terms of the growth rate constant (k) or the
number of generations/unit time. k = ln2/g and is often expressed as generations/h.
Stationary Phase: During this phase, waste products that can be toxic or alter the environment (i.e. make
it more acidic) accumulate and/or the availability of nutrients decreases causing the cells to increase their
generation time. Eventually division stops and the population reaches a plateau.
Death Phase: Cells of the population enter this phase when toxic substances accumulate and/or cell
starvation occurs. The rate of decline becomes exponential with time.
Measurement of bacterial population growth can be determined by a number of methods. These include
microscopic counts, plate counts, turbidimetric measurements, nitrogen or dry weight determinations, and
biochemical activity measurements. In this laboratory, you will monitor bacterial growth using
turbidimetric and plate counting methods.
Before coming to the lab, consult the literature to determine the optimum temperature for:
Escherichia coli (DH5)

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You will work in pairs. Since it is impossible to follow an entire growth curve in a 3-hour lab period, most
of your measurements will involve only the exponential and (perhaps) lag phases. To use your laboratory
time efficiently and effectively, there are several things you must do ahead of time.
1. Read the procedures very carefully and be sure that you understand what has to be done.
2. Practice calculating dilutions.
3. Prepare you data tables (i.e. those below) so that you can fill in the data as you track the bacterial
growth.
Table 1: Turbidimetric growth results (Do Not allow the tables to split between pages when preparing
the Lab Report)
Tube
Growth
time (min)
Dilution
Dilution Factor (DF)
Undiluted OD
(calculation)
1
0
2
20
3
40
4
60
5
80
6
100
7
120
Table 2: Plate count results
Tube
Growth time
(min)
Final plated
Volume (ml)
Final plated
Dilution
Final plated
DF
Number of
Colonies
Calculated
cfu/ml
1
0
0.1
2
20
0.1
3
40
0.1
4
60
0.1
5
80
0.1
6
100
0.1
7
120
0.1
THE MATERIALS AND PROCEDURES FOR TURBIDITY AND PLATE COUNT MEASUREMENTS (EXERCISES 1
AND 2 RESPECTIVELY) WILL BE DESCRIBED SEPARATELY BUT WHILE YOU ARE PERFOMING THAT LAB, YOU
WILL COLLECT SAMPLES FOR BOTH TYPES OF MEASUREMENTS AT THE SAME TIME. THE PLATE COUNT
STEPS CAN BE CARRIED OUT IN INTERVALS AFTER THE TURBIDIMETRIC SAMPLING AND READINGS.
NOTE: Dilution Factor = Final volume / Solute volume
Example: You want to make a 1:5 dilution of a culture in a final volume of 5 ml Using this formula, you
would ultimately add 1 ml of your culture to 4 ml of diluent (e.g., LB broth or 0.95% saline solution).
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