ONLINE LESSON 5: GROWTH AND DEATH OF MICROORGANISMS

INTRODUCTION
Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number
when microorganisms reproduce by processes like budding or binary fission. Growth also results
when cells simply become longer or larger. If the microorganism is coenocytic, that is, a
multinucleate organism in which nuclear division are not accompanied by cell divisions – growth
results in an increase in cell size but not cell number.

THE GROWTH CURVE

Binary fission and other cell division processes bring about an increase in the number of cells in
a population. Population growth is studied by analyzing the growth curve of a microbial culture.
When microorganisms are cultivated in liquid medium i.e. they are incubated in a closed vessel
with a single batch of medium. Because no fresh medium is provided during incubation, nutrient
concentrations decline and concentrations of wastes increase. The growth of microorganisms
reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus
the incubation time. The resulting curve has four distinct phases

Fig. 5: Microbial growth curve in a closed system

Lag Phase: When microorganisms are introduced into fresh culture medium, usually no
immediate increase in cell number occurs, so this period is called the lag phase. Although cell
division does not take place right away and there is no net increase in mass, the cell is
synthesizing new components. A lag phase prior to the start of cell division can be necessary for
a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and
ribosomes; these must be synthesized before growth can begin. The medium may be different
from the one the microorganism was grown in previously. Here, new enzymes would be needed
to use different nutrients. Possibly, the microorganisms have been injured and require time to
recover. Whatever the causes, eventually the cells retool, replicate their DNA, begin to increase
in mass and finally divide.

The lag phase varies considerably in length with the condition of the microorganisms and the
nature of the medium. This phase may be quite long if the inoculum is from an old culture or one
that has been refrigerated. Inoculation of a culture into a chemically different medium also
results in a longer lag phase. On the other hand, when a young, vigorously growing exponential
phase culture is transferred to fresh medium of the same composition, the lag phase will be short
or absent.

Exponential or Logarithm Phase: After the lag phase, microorganisms enter the exponential or
log phase. This is the time when the cells are growing and dividing at the maximal rate possible
given their genetic potential (metabolic activity is high as DNA, RNA, cell wall components, and
other substances necessary for growth are generated for division), the nature of the medium and
the conditions under which they are growing. Their rate of growth is constant during the
exponential phase: that is, the microorganisms are dividing and doubling in number at regular
intervals. The population is most uniform in terms of chemical and physiological properties
during this phase; therefore, exponential phase cultures are usually used in biochemical and
physiological studies.

Stationary Phase: Because it is a closed system, eventually, the population growth experienced
in the log phase begins to decline as the available nutrients become depleted and waste products
start to accumulate. The growth curve becomes horizontal. Bacterial cell growth reaches a
plateau, or stationary phase, where the number of dividing cells equal the number of dying cells,
or the population may simply cease to divide but remain metabolically active.
Microbial population enter the stationary phase for several reasons. One obvious factor is
nutrient limitation. Aerobic organisms are often limited by oxygen availability. Population
growth also may cease due to accumulation of toxic waste product. Spore forming bacteria
produce endospores in this phase and pathogenic bacteria begin to generate substances (virulence
factors) that help them survive harsh conditions and consequently cause disease.

Death Phase: As nutrients become less available and waste products increase, the number of
dying cells continues to rise. In the death phase, the number of living cells decreases
exponentially and population growth experiences a sharp decline. As dying cells lyse or break
open, they spill their contents into the environment making these nutrients available to other
bacteria. This helps spore producing bacteria to survive long enough for spore production.
Spores are able to survive the harsh conditions of the death phase and become growing bacteria
when placed in an environment that supports life. Some species of gram-negative cocci die
rapidly, so that there may be very few viable cells left in a culture after 72 hours or less. Other
species die so slowly that viable cells may persist for months or even years.

MATHEMATICS OF GROWTH

During the exponential phase each microorganism is dividing at constant intervals. Thus, the
population will double in number during a specific length of time called the generation time or
doubling time.

The generation time g (the time required for the population to double) can be determined from
the number of generations n that occur in a particular time interval t.

Total population N at the end of a given time period would be expressed as

N = 1 x 2n _______________ 1

The number of bacteria No inoculated at time zero is not 1 but more likely several thousand, so
the formula now becomes

N = No x 2n _______________ 2

Solving equation 2 for n, we have

 log10 N = log10 No + n log10 2

n = log10 N - log10 N0 ___________________ 3

log10 2

If we now substitute the value of log 10 2, which is 0.301, in the above equation, we can simplify
the equation to
n = log10 N - log10 N0
0.301
n = 3.3 (log10 N - log10 N0) _________________ 4
g=t=t
n 3.3 (log10 N - log10 N0) _______________ 5

During exponential growth, the growth rate (i.e. the number of generations per hour), termed R,
is the reciprocal of the generation time g. It is also the slope of straight line obtained when the
log number of cells is plotted against time

R = 3.3 (log10 N - log10 N0) _____________ 6

t

Quiz 1: During log phase growth of a bacterial culture, a sample is taken at 8:00 a.m. and found
to contain 1,000 cells per ml. A second sample is taken at 5:54p.m. and is found to contain
1,000,000 cells per ml. What is the generation time in hours?

Solution
N0 = 1,000
N = 1,000,000
t = 9 hrs 54 mins x 60 mins = 594 mins
g =?
n =?

g=t
n

g = 594 = 60 minutes = I hour

9.9

g = 1 hour
Quiz 2:
Suppose that a bacterial population increases from 103 cells to 109 cells in 10 hours. Find the
generation time.
g=t=t
n 3.3 (log10 N - log10 N0)

g = 10
3.3 (log109 - log103)

g = 10
19.8

= 0.5 hr/gen or 30mins/gen

Assignment

Solve the following:

1. Suppose the generation time of a bacterium is 90 mins and the initial number of cells in a
culture is 103cells at the start of the log phase. How many bacteria will there be after 8 hours of
exponential growth?

2. An inoculum of 107 bacterial cells was introduced into a flask of culture medium and
growth monitored. No change was seen for 18 minutes (the lag phase), then growth occurred
rapidly. After a further 76 minutes, the population had increased to 4.32 x 10 8cells. What is the
generation time of the culture?
 FACTORS AFFECTING MICROBIAL GROWTH
Bacteria require certain conditions for growth, and these conditions are not the same for all
bacteria. Factors such as oxygen, pH, temperature, and light influence microbial growth.
Additional factors include osmotic pressure, atmospheric pressure, and moisture availability. A
bacterial population's generation time, or time it takes for a population to double, varies between
species and depends on how well growth requirements are met. Q

1. Temperature

Microorganisms as a group are able to grow over a wide range of temperatures, from around
freezing to above boiling points. For any organism, the minimum and maximum growth
temperature defines the range over which growth is possible. Growth rates increase with
temperature until the optimum temperature is reached, then the rate falls again. The optimum
temperature is generally closer to the maximum growth temperature than the minimum.

Psychrophiles can grow at 0ºC, with optimal growth temperature occurring at 15ºC or below.
Such organisms are not able to grow at temperatures above 25ºC e.g. Bacillus psychrophilus,
Chlamydomonas nivalis. Psychrotrophs, on the other hand, although they can also grow at 0ºC,
have much higher optimal temperature of 20 - 30ºC and a maximum around 35ºC e.g. Listeria
monocytogenes, Pseudomonas fluorescens. Members of this group are often economically
significant due to their ability to grow on refrigerated foodstuffs. Mesophiles grow best around
20 -45ºC. These include bacteria that are part of the human microbiome which experience
optimum growth at or near body temperature (37°C) e.g. Escherichia coli, Neisseria
gonorrhoeae, Trichomonas vaginalis. Thermophiles have become adapted to not only surviving
but thriving at much higher temperatures. Typically, they are capable of growth at 55°C or
higher with optimum often between 55 and 65°C e.g. Geobacillus stearothermophilus, Thermus
aquaticus, Cyanidium caldarium, Chaetomium thermophile. Hyperthermophiles can tolerate
temperature in excess of 100°C with an optimum between 80 and about 113°C e.g. Sulfolobus,
Pyrococcus.
 2. pH

Microorganisms are strongly influenced by the prevailing pH of their surroundings. As with

temperature, we can define minimum, optimum and maximum values for growth of a particular
type. The pH range (between minimum and maximum values) is greater in fungi than it is in
bacteria. Most microorganisms grow best around neutrality - neutrophiles. The optimum pH is
between pH 5.5 and 8.0 e.g. Escherichia, Euglena, Paramecium. Many bacteria prefer slightly
alkaline conditions but relatively few are tolerant of acid conditions, and fewer still are
acidophilic. The optimum growth pH for Acidophile is between pH 0 and 5.5 e.g, Sulfolobus,
Ferroplasma. While alkalophile requires growth optimum between pH 8.0 and 11.5 e.g.
Bacillus alcalophilus.

3. Oxygen Concentration

Oxygen is present as a major constituent (20%) of our atmosphere, and most life forms are
dependent upon it for survival and growth. Such organisms are termed obligate aerobes e.g.
Micrococcus luteus, Pseudomonas, Mycobacterium, most protists and fungi. Not all organisms
are aerobes however; anaerobes are able to survive in the absence of oxygen, and for some this
is actually a necessity.

Obligate anaerobes cannot tolerate oxygen at all e.g. Clostridium, Bacteroides,

Methanobacterium, Trepomonas agilis. They are cultured in special anaerobic chambers, and
oxygen excluded from all liquid and solid media. Facultative anaerobes are able to act like
aerobes in the presence of oxygen but have the added facility of being able to survive when
conditions become anaerobic e.g. Escherichia, Enterococcus, Saccharomyces cerevisiae.
Aerotolerant anaerobes are organisms that are basically anaerobic; although they are not
inhibited by atmospheric oxygen, they do not utilize it e.g. Streptococcus pyogenes.
Microaerophiles require oxygen but are only able to tolerate low concentration of it (2-10%),
finding higher concentrations harmful e.g. Campylobacter, Treponema pallidum.

4. Osmotic pressure

Osmosis is the diffusion of water across a semipermeable membrane from a less concentrated
solution to a more concentrated one, equalizing concentrations, the pressure required to make
this happen is called the osmotic pressure. If a cell is placed in a hypertonic solution (one whose
solute concentration is higher), osmosis would lead to loss of water from the cell (plasmolysis).
This is the basis of using high concentrations of salt or other solutes in preserving foods against
microbial attack. In the opposite situation, water would pass from a dilute (hypotonic) solution
into the cell, causing it to swell and burst. The rigid cell walls of bacteria prevent them from
bursting; this, together with their minutes size, makes them less sensitive to variations in osmotic
pressure than other types of cell. They are generally able to tolerate NaCl concentrations of
between 0.5 and 3.0 per cent. Haloduric (‘salt tolerant’) e.g. Staphylococcus aureus are able to
tolerate concentrations ten times as high, but prefer lower concentrations, whereas halophilic
(‘salt loving’) forms are adapted to grow best in conditions of high salinity e.g. Halobacterium.

5. Light

Some bacteria require light for growth. These microbes have light-capturing pigments that are
able to gather light energy at certain wavelengths and convert it to chemical energy.
Cyanobacteria are examples of photoautotrophs that require light for photosynthesis. These
microbes contain the pigment chlorophyll for light absorption and oxygen production through
photosynthesis. Cyanobacteria live in both land and aquatic environments and can also exist as
phytoplankton living in symbiotic relationships with fungi (lichen), protists, and plants.

Other bacteria, such as purple and green bacteria, do not produce oxygen and utilize sulfide or
sulfur for photosynthesis. These bacteria contain bacteriochlorophyll, a pigment capable of
absorbing shorter wavelengths of light than chlorophyll. Purple and green bacteria inhabit deep
aquatic zones.

Other References:

Anon. Eucaryotic cell. Available at

https://www.toppr.com/guides/biology/cell-the-unit-of-life/eukaryotic-cell/

Bailey, R. (2020). "Phases of the Bacterial Growth Curve." ThoughtCo, Feb. 11, 2020.
Available at thoughtco.com/bacterial-growth-curve-phases-4172692.

Bruslind, L. (2019). Bacteria – Surface Structures. Available at https://bio.libretexts.org

Kim, B.H. and Gadd, G.M. (2008). Bacterial Physiology and Metabolism. 1 st edition. Cambridge
University Press, USA