The document describes various aspects of bacterial growth in batch culture. It discusses the different types of cell division used by bacteria, including binary fission, budding, and filamentous growth. It then focuses specifically on binary fission and describes the typical growth phases seen in a bacterial growth curve: lag phase, exponential growth phase, stationary phase, and death phase. It also discusses various methods for measuring and quantifying bacterial growth, including direct counts, viable counts, turbidity measurements, and generating a growth curve by plotting measurements over time. Finally, it covers several environmental factors that can influence bacterial growth rates, such as temperature, pH, water availability, and oxygen levels.

1. Growth of microbes in batch
culture

2. The Phases of Growth
Growth in the bacterial context is normally
described as an increase in cell number.
Microorganisms, depending upon the specific
species, increase their numbers by binary fission,
budding or by filamentous growth. Binary fission is
the separation of the initial cell, the mother cell,
into two daughter cells of approximately equal size.
This is a very common method of multiplication and
most of the organisms we will investigate divide in
this manner.

3. Budding division
Budding division involves the
asymmetric creation of a
growing bud, on the mother
cell. The bud increases in size
and eventually is severed from
the parental cell. After division
is complete, the mother cell
reinitiates the process by
growing another bud. Yeast and
some bacteria (Caulobacter is
one example) use this form of
division.

4. Filamentous growth
Filamentous growth is characterized by
the formation of long, branching, non-
divided filaments, containing multiple
chromosomes. Sometimes the
filaments will have cross walls
separating chromosomes. As growth
proceeds, the filaments increase in
length and number. Under nutrient
limiting conditions, some filamentous
microorganisms will go through
developmental changes, with a fraction
of the filaments differentiating to form
spores. The structures and mechanisms
used to form these spores can be
spectacular. Streptomyces species and
many molds grow in this manner.

5. Binary fission
The best characterized type of growth is binary
fission and this is what we will focus on in this
experiment. When grown in liquid medium,
bacterial cultures progress through several
distinguishable phases, which can be
characterized by plotting the log of the cell
number vs. time. A typical growth curve has
phases of growth, lag phase, exponential
growth phase (also termed balanced growth),
stationary phase and death phase.
Binary fission requires:
• cell mass to increase
• chromosome to replicate
• cell wall to be synthesized
• cell to divide into two cells

6. Exponential growth
Exponential growth is a function of binary fission since at each
division there are two new cells. The time between divisions is
called generation time or the doubling time since this is the
time for the population to double. These can range from
minutes to days depending on the species of bacteria.
Growth rate is the change in cell number or mass per unit
time.
What do we mean by exponential growth? A population
doubles each generation is exponential growth.
Graphically on arithmetic coordinates the graph takes the
shape of a J - a curve with ever increasing slope - growth rate.
Plotted on semilogarithmic paper, where the Y axis is
logarithmic (base 10) and the X axis is arithmetic (either
generation or time), you get a straight line.

7. Exponential growth
Generation times - N = No2n where No is the original
number of cells and n is the number of generations.
g, generation time, equals t/n, time divided by
generation.
How do you calculate n?
N = No2n
log N = log No + nlog2
log N - log No = n log2
n = [log N - log No] / log2
n = [log N - log No] / 0.301
We also know that the slope of the semi-log line
equals 0.301 divided by the generation time.

8. A bacterial growth curve
The four phases of growth (lag (1), exponential (2), stationary
(3) and death phase(4)) are labeled.
• When an organism is inoculated into a fresh medium, it
needs to adapt to the new nutrients available, synthesize
RNA, protein and finally replicate its DNA before starting
division. These processes take time and there is no net
increase in cell numbers, thus a lag phase is observed.
• Once the appropriate enzymes for growth in a particular
medium have been expressed cells begin to multiply.
This period of maximal division can last for several hours
or days, depending upon the organism, and is called the
log or exponential growth phase .
• Eventually the increase in cell number ceases, either
because cells stop dividing or the rate of division equals
the rate of cell death, resulting in a stationary phase.
This is usually caused by limitation of a nutrient or the
accumulation of a toxic waste product. Depending on
the bacterium, stationary phase can last for several
hours to many days.
• The final chapter of a growth curve is the death phase .
An exponential decrease in the number of organisms
due to cell death occurs during this phase. Some
microorganisms never experience a death phase or it is
greatly delayed due to their ability to survive for long
periods without nutrients.

9. Measurement of Bacterial Growth
How do we measure growth?
• Direct microscopic counts - use the microscope and a slide with a grid engraved on
it. A coverslip and placed over the grid which captures a known volume of liquid.
Problems :
dead cells are difficult to distinguish, small cells are difficult to see
method not suitable for dilute samples
• Viable counts - count only cells that are able to divide and form offspring. Referred
to as plate counts or colony counts. Assumption each viable cell gives rise to a
colony.
• Dilutions - to cover a cell density that ranges from 30 - 300 colony forming units
per plate. Problems:
not all species of bacteria will form colonies on any particular medium, small
colonies are not counted.
• Turbidity - cell suspensions look cloudy because each cell scatters light as it passes
through a suspension of cells. Take advantage of the light scattering properties of a
suspension using a spectrophotometer which measures unscattered light as it
passes through. The scatter is proportional to cell number (density of cells) up to
high density cultures because cells begin to cause rescatter the light back into the
path of unscattered light. Therefore the optical density is not linear at high density
suspensions.
Need to develop a standard curve between OD and cell numbers (viable counts).

10. Measurement of Bacterial Growth
• Growth of microorganisms can be measured by following the increase in cell
number or the increase in a cellular macromolecule such as DNA or protein.
In most cases, the increase in cell number is determined. Cell numbers can
be measured in a variety of ways. For this experiment we will use the viable
plate count, which you are familiar with, and turbidometric measurement.
• So which wavelength of light do we use to measure cell numbers? Typically
the shorter the wavelength the greater the sensitivity of the measurement.
Some wavelengths cannot be used, however, because cellular constituents
absorb light, not scatter them. For example proteins absorb light at 280 nm.
Likewise the color of the medium affects absorbance. For this experiment
we will be measuring the growth of E. coli in either a yellow or a clear
medium, 600 nm works well. Beware, however, that the choice of
wavelength must be considered for each experimental condition.
• The sample is placed in a sample chamber. The chamber will contain a
holder, an entrance for the selected wavelength, and an exit, leading to the
detector. A critical piece of equipment used in the sample chamber is the
cuvette. A cuvette is a special test tube that holds the sample. Cuvettes
must be clean and free of aberrations, both of which could scatter light,
resulting in inaccurate readings. Most good cuvettes are expensive and must
be treated with care!

11. Measurement of Bacterial Growth
The actual operation of a spectrophotometer is much simpler than understanding its parts. There are a
few general, rules of thumb, when using any spectrophotometer.
1. Before you begin, be sure the wavelength selector is set for the wavelength of light you are going
to use. This involves setting the correct wavelength on the selector, making sure the correct lamp
is on for the wavelength you have selected and that you are using the correct cuvette.
2. Make sure to zero the spectrophotometer. When this is performed you are adjusting the machine
to 100% transmittance. This is done by using a sample that contains all components of a mixture
except the component to be measured. For example, if you are measuring the growth of B.
cereus in nutrient broth, the machine would be blanked with sterile, uninoculated nutrient broth.
Realize that the machine is being blanked to read 100% T (or 0 absorbance) with a specific
solution in a specific cuvette, with the cuvette in a specific orientation. This last consideration,
nullifies any aberrations in the cuvette.
3. With proper technique, nothing should be spilled into the spectrophotometer. If it is, clean it up
and immediately notify an instructor. This type of spillage usually happens if cuvettes are filled
over a spectrophotometer, a very bad habit to form!
4. Clean the cuvette after each use by rinsing with distilled water and allowing to dry upside down
in a test tube rack. For more stubborn stains use a dilute solution containing a mild soap (like
Ivory). In rare cases, difficult blemishes can be removed with dilute acid (1-5%) or with ethanol.
Never let a sample dry in a cuvette! Protein and nucleic acids form a strong bond with glass and
can be impossible to remove.
5. When measuring the turbidity [60] of a cell suspension, absorbance values in the range of 0.1--
0.8 are acceptable. Readings below 0.1 push the limits of the spectrophotometer, since it is not
sensitive enough in that range. Absorbance values above 0.8 result in microorganisms casting
shadows onto one another and not being seen by the spectrophotometer. If a reading is above
this range, the culture must be diluted with sterile medium. If a reading is below this range,
concentrate your culture using a centrifuge.
6. When graphing growth versus time, you must plot absorbance on a log scale. Even though
absorbance is a logarithmic unit, absorbance readings are proportional to cell mass which is
increasing exponentially during growth.

12. Generating a growth curve
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.
Graphing of bacterial growth
on a linear scale

13. Generating a growth curve
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.
Graphing of bacterial growth
with cell number on a log scale.

14. Generating a growth curve
If we were to convert our vertical axis to a
logarithmic scale - as in the previous slide - 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.
In the next slide you will see a logarithmic paper.

16. Generating a growth curve
The example on the right 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.
• 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.)
• Higher CFU/ml = Xt = 1X1010 (at 5.75 hours)
• Lower CFU/ml = X0 = 1X108 (at 2.75 hours)
• Time interval (in hours) between the 2 points = t = 3
We find the growth rate which is the number of generations
(doublings) per hour:

17. Generating a growth curve
• 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 previous 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.

18. Environmental factors
Temperature - as temperature
increases, the growth rate
increases until a point at which
the growth rate declines.
Minimum temperature - below
growth does not occur may be
due to the stiffening of the
cytoplasmic membrane.
optimum temperature where
the growth rate is maximum
Maximum temperature- above
which growth does not occur
which reflects when proteins
may be denatured, nucleic acids
and other cellular components
are irreversibly damaged.

19. Environmental factors
Classification of bacteria based on temperature optimum
psychrophiles - low temperature optima <15 C - may even be killed by brief warming or
thawing.
mesophiles - midrange temperature optima 25 - 40 C
thermophiles - high temperature optima 40 - 80 C
hyperthermophiles - very high temperature optima >80 C
Psychrophiles
open ocean water is between
1 and 3 C.
Artic and Antartic regions are cold.
Adaptation
membranes rich in unsaturated
fatty acids
Thermophiles
hot springs all over the world
fermenting compost
Adaption
thermostable proteins with usually
a few changes in the amino acid sequence when compared to a mesophile's protein
saturated fatty acids in their membranes

20. Environmental factors
Acidity and alkalinity
Most environments are between 5
and 9 and optima are between these
values, around neutral pH of 7.
Acidophiles
live at low pH
Obligate acidophiles such as
Thiobacillus.
cytoplasmic membrane actually
dissolves and the cell lysis at more
neutral pH.
Alkaliphiles
live at high pH such as soda lakes and
carbonate soils.
Important to biotechnology since
they have hydolytic proteases that
function at alkaline pH and are used
in household cleaners.

21. Environmental factors
Water availability - bacteria need water as a solvent.
Water availability is expressed as water activity - how
much water is available. Solutes and surfaces affect water
activity - both decrease it. Water moves from high water
activity values to lower values in the process of osmosis.
Different bacteria have different tolerances towards low
water activities. In fact, preservation process takes
advantage of lower water activity which causes
plasmolysis or pulling away of the membrane from the
cell wall. This inhibits cell growth.
Halophiles require 1-6% for mild halophiles and 7-15%
salt for moderate halophiles. Extreme halophiles require
15-30% salt.

22. Environmental factors
Oxygen -
1. Aerobes require oxygen up to 21% as in air.
2. Microaerophilic bacteria require reduced levels of oxygen
3. strict or obligate anaerobes require the absence of oxygen.
4. Facultative anaerobes can grow aerobically if oxygen is present and switch to fermentation or
anaerobic respiration if oxygen is absent. E. coli is a facultative anaerobe that grows aerobically and
using anaerobic respiration when necessary.
5. Aerotolerant anaerobes don't use oxygen for growth but tolerate its presence. Can grow on the
surface of solid medium with out the special anaerobic conditions required for the strict anaerobes.
Anaerobic culture conditions - add reducing reagents such as thioglycolate, bubble nitrogen gas
through your solutions to remove oxygen after autoclaving, add a dye such as resazurin to indicate
when oxygen is penetrating, use an anaerobic jar with an atmosphere containing hydrogen gas and
carbon dioxide.
Why go to such great troubles for the strict anaerobes? Because they contain lots of flavins which react
with oxygen to produce toxic oxygen species that are very reactive.
Oxygen species - singlet oxygen which the valence electrons become highly reactive and oxidize organic
matter readily.
Superoxide anion, hydrogen peroxide, and hydroxyl radical which are inadvertant byproducts during
respiration. These can all damage cell macromolecules by oxidation processes.
Measures to counter these toxic oxygens - catalase degrade hydrogen peroxide to oxygen and water.
peroxidases - destroys hydrogen peroxides too but requires NADH. no oxygen evolved.
super oxide dismutase produces hydrogen peroxide from super oxides.
Aerobes and facultative aerobes generally contain catalase and super oxide dismutase.

23. Environmental factors

24. Big thanks to
http://inst.bact.wisc.edu/inst/index.php?modul
e=book&type=user&func=displaychapter&chap
_id=43&theme=Printer
AND
http://microbiology.okstate.edu/faculty/demed
2/Notes/Microbial%20growthdoc.html