M6-Growth and Reproduction

Prepared by W.R.
Jacinto 1
Asso. Prof., Biological Sciences Department
De La Salle University - Dasmarinas 4/25/22

§ Increase in cellular constituents
that may result in:
§ increase in cell number
§ increase in cell size GROWTH
§ Growth refers to population
growth rather than growth of
individual cells
2
If Streptococcus
pyogenes at the back of
your throat, a sore
throat.
Growth of
microorganisms in
refrigerator shortens the
shelf life of the food
Produce beer, wine,

cheese, yogurt and
other products.
De La Salle University - Dasmarinas 4/25/22 3

§ Most bacterial § Types of bacterial division:
chromosomes are circular
§ Binary Fission
§ DNA replication proceeds § Budding
in both directions from the
origin § Certain actinomycetes by
coniodiospores
§ Origins move to opposite ends
of the cell § Fragmentation
§ Cell elongates
§ Septation – formation of
cross walls between
daughter cells and cells
separate th
Willey et al. ( ) 9 ed. Copyright © McGraw-Hill
Global Education Holdings, LLC. Permission
required for reproduction or display.

5
• New cell wall is
6.3 synthesized
PEPTIDOGLYCAN during bacterial
growth by
SYNTHESIS AND inserting new
glycan units into
CELL DIVISION, P. preexisting wall
139 material (Figure
6.3).

•A hydrophobic alcohol
called bactoprenol
facilitates transport of new
glycan units through the
cytoplasmic membrane to
become part of the growing
cell wall (Figure 6.4).
•Transpeptidation bonds
the precursors into the
peptidoglycan fabric
(Figure 6.5).

§ Generation (doubling) time
§ time required for the population to double in size
§ varies depending on species of microorganism and
environmental conditions
§ range is from 10 minutes for some bacteria to several days for
some eukaryotic microorganisms
§ This is calculated during log growth phase
10
Microbial populations show a characteristic type of growth pattern called
exponential growth, which is best seen by plotting the number of cells
over time on a semilogarithmic graph (Figure 6.6).

Generation Number of To key, then Calculate Log10 of
Number cells press log10 cells
0 20 1 0
5 25 32 1.51
10 2 10 1,024 3.01
15 2 15 32,768
16 2 16 65,536
17 2 17 131,072
18 2 18 262,144
19 2 19 524,288
20 2 20 1,048,576 6.02

§ Population is doubling every generation
14
§ Number of generation = Log no. of cells (end) - Log no. of cells (start)
0.301 (constant)
§ Generation time = time lapse in minutes = min/generation

number of generations
§ If 100 cells growing for 5 hours produced 1,720,320 cells:
§ What is the number of generations?

§ What is the generation time ?

6.6 THE • Microorganisms
show a
GROWTH characteristic
growth pattern
CYCLE, P. (Figure 6.8)

when inoculated
142
into a fresh
culture medium.

There is usually a lag phase, then exponential growth commences. As essential
nutrients are depleted or toxic products build up, growth ceases, and the
population enters the stationary phase. If incubation continues, cells may begin
to die (the death phase).

§ Cell synthesizing new components
§ e.g., to replenish spent materials
§ e.g., to adapt to new medium or
other conditions
§ Varies in length
§ in some cases can be very short or
even absent
A. LAG PHASE § depends on harshness of medium

§ is it selective or enrichment medium?
§ what is the temperature of medium?
§ The general rule of thumb is that

microbes adapt to a shift to
improved conditions much more
rapidly than they do to a shift to
poorer conditions.
4/25/22 De La Salle University - Dasmarinas 19

Also called log phase or log
growth phase
Rate of growth and division is

constant and maximal
Population is most uniform in

terms of chemical and physical
properties during this phase
Bacteria from this stage would

be used for studies

§ Closed system population growth
ceases due to
§ Nutrient limitation
§ accumulation of a waste product.
§ Limited oxygen availability
§ Critical population density reached
§ Bacteria die off and liberate some
nutrients
C. STATIONARY § no change in the number of viable

cells, active cells stop reproducing
PHASE or reproductive rate is balanced by
death rate
§ Can last for long period
§ microbes in nutrient-poor
environments (like soils and many
aqueous environments) probably
spend most of their time in stationary
phase

§ Cell numbers begin to decline due to
§ DNA or protein damage or
§ perhaps exhaustion of energy reserves
§ Accumulation of toxic waste
§ Bacteria are dying off opposite to log growth phase

§ do not die all at once
§ Two alternative hypotheses

§ cells are Viable But Not Culturable (VBNC)
§ cells alive, but dormant, capable of new growth when conditions are right
§ Programmed cell death

§ fraction of the population genetically programmed to die (commit suicide)

§ The culture is kept in exponential growth phase for as long as
desired by the continuous addition of new medium and the
simultaneous removal of the same volume of old medium and
cells.
§ Under these conditions, all cells should be growing
exponentially and samples at various times should be identical.

A chemostat A reservoir of
sterile medium is added to the
culture at a constant rate. Spent
medium leaves the culture at
the same rate. Fresh nutrients
are constantly entering the
culture and waste products are
continuously removed.

ENVIRONMENTAL EFFECTS ON
MICROBIAL GROWTH:
25

§ Temperature is a major
environmental factor
controlling microbial
growth.
§ The cardinal
temperatures are the
minimum, optimum, and
maximum temperatures at
which each organism
grows (Figure 6.16).
§ Why?
A common curve of temperature

vs. Growth rate
Psychrophiles - optimum is 15C
and max is below 20 C;
Psychrotolerant, opt. 20C to 40C
Psychrotrophs – 0 oC to 35 oC
Mesophiles - optima in the 20 oC

to 45 oC range
Thermophiles have optima from

45 oC to 80 oC
Extreme thermophiles - optima,

above 80 C

§ Mesophiles, which have midrange temperature optima, are found in warm-
blooded animals and in terrestrial and aquatic environments in temperate and
tropical latitudes. Extremophiles have evolved to grow optimally under very
hot or very cold conditions.
§ Organisms with cold temperature optima are called psychrophiles, and the
most extreme representatives inhabit permanently cold environments.
§ Psychrophiles have evolved biomolecules that function best at cold
temperatures but that can be unusually sensitive to warm temperatures.
Organisms that grow at 0ºC but have optima of 20ºC to 40ºC are called
psychrotolerant.
§ Organisms with growth temperature optima between 45ºC and 80ºC are called
thermophiles, and those with optima greater than 80°C are called
hyperthermophiles.
§ These organisms inhabit hot environments up to and including boiling hot
springs, as well as undersea hydrothermal vents that can have temperatures in
excess of 100ºC.
§ Thermophiles and hyperthermophiles produce heat-stable macromolecules,
such as Taq polymerase, which is used to automate the repetitive steps in the
polymerase chain reaction (PCR) technique.

TABLE 1. TEMPERATURE FOR GROWTH OF BACTERIA.
Bacterium Habitat Cardinal

Temperature oC
Listeria Animals, soil, rotting Minimum Optimum Maximum
monocytogenes vegetation. water
1 30-37 45
Vibrio marinus Open ocean 4 15 30

Stenotrophomonas Soil 4 35 41
maltophilia
Thiobacillus novellus Places where reduced 5 25-30 42
sulfur are present
(many habitats)
Staphylococcus Skin 10 30-37 45

aureus
Escherichia coli Intestines 10 37 45
Clostridium Soil, food 15 45 55
perfringens
Streptococcus Mucous membranes 20 37 40
pyogenes
ENVIRONMENTAL EFFECTS ON
MICROBIAL GROWTH:
33

§ In water, the hydrogen ion concentration will range from 1 x 10-
14 M (a pH of 14) to 1 M (a pH of 0).
§ Microbes are found at almost any conceivable pH, with most
common bacteria growing at or near neutral pH (7).
§ The acidity or alkalinity of an environment can greatly affect
microbial growth. Figure 6.22 shows the pH scale.

• Some organisms have evolved to grow best at low or high pH, but most
organisms grow best between pH 6 and 8. The internal pH of a cell must
stay relatively close to neutral even though the external pH is highly
acidic or basic.
•Organisms that grow best at low pH are called acidophiles; those that
grow best at high pH are called alkaliphiles.
36

§ Acidophiles -pH optima 1-5.4
§ Neutrophiles - 5.5-7.9
§ Alkalophiles - 8.0-11

TABLE 4. PH RANGES FOR REPRESENTATIVE
BACTERIAL SPECIES
Organism Habitat Minimum Optimum Maximum
pH pH pH
Thiobacillus Areas rich in sulfur, often acidic 0.5 2.0-2.8 4.0-6.0
thiooxidans
Bacillus Acidic hot springs 2.0 4.0 6.0

acidocaldarius
Lactobacillus Animals, plants, decaying matter 4.0-4.6 5.8-6.6 6.8

acidophilus
Staphylococcus Surfaces of animals, nasal cavity, 4.2 7.0-7.5 9.3

aureus skin
Escherichia coli Intestines of animals 4.4 6.0-7.0 9.0
Clostridium Soils and sediments that are 5.0-5.8 6.0-7.6 8.5-9.0

sporogenes anaerobic
Pseudomonas Ubiquitous 5.6 6.6-7.0 8.0

aeruginosa
Streptococcus Pathogen of animals 6.5 7.8 8.3

pneumoniae
40
• Table 6.2 shows the water activity (aw) of several substances.

§ Water availability (water activity) is a measurement of how
much free water is available.
§ Pure water has a water activity of 100% and the activity
decreases as solutes are added to the solution.
§ Changes in osmotic concentrations in the environment may
affect microbial cells
§ hypotonic solution (lower osmotic concentration)
§ water enters the cell
§ cell swells may burst (plasmoptysis)
§ hypertonic (higher osmotic concentration)
§ water leaves the cell
§ membrane shrinks from the cell wall (plasmolysis) may occur

§ Water can be made unavailable by:
§ Evaporation
§ Freezing
§ Bound to solutes
§ Cause dehydration
§ Water unavailable to enzymes

§ Halophiles
§ Extreme – 15-30% NaCl
§ Moderate – 6-15% NaCl
§ Mild - 1-6% NaCl
§ Halotolerant – endures NaCl; organisms can tolerate some
reduction in the water activity of their environment but
generally grow best in the absence of the added solute.
§ Osmophiles – lives in high sugar
§ Xerophiles – microbes in dry environments
§ Some microorganisms (halophiles) have evolved to grow best

at reduced water potential, and some (extreme halophiles)
even require high levels of salts for growth.

TABLE 2. COMPARISON OF THE HALOTOLERANCE
OF SEVERAL SPECIES OF BACTERIA
Organism Habitat Minimum water activity

for growth
Caulobacter Dilute fresh and sea water 1.00
Pseudomonas Ubiquitous low salt environments 0.91
Salmonella/E. Animals 0.91

coli
Lactobacillus Animals and plants 0.90
Bacillus Soil 0.90
Staphylococcus Animals 0.85
Halobacterium High salt lakes such as Great Salt 0.75

Lake or the Dead Sea
• Water activity becomes limiting to an organism when the dissolved
solute concentration in its environment increases.
•To counteract this situation, organisms produce or accumulate
intracellular compatible solutes (Figure 6.24; Table 6.3) that maintain
the cell in positive water balance.
47

52

§ Many microbes and most eukaryotes need
oxygen for growth as it is the terminal
electron acceptor.
§ Others microbes can live without it and some
cannot even tolerate its presence.
§ Obligate aerobes
§ facultative anaerobes
§ Microaerophiles
§ aerotolerant anaerobes
§ strict anaerobes

• Aerobes require oxygen to live, whereas anaerobes do not and may
even be killed by oxygen.
•Facultative organisms can live with or without oxygen.
• Microaerophiles are aerobes that can use oxygen only when it is

present at levels reduced from that in air.
•Aerotolerant anaerobes can tolerate oxygen and grow in its presence
even though they cannot use it.
54

§ Strict aerobes and facultative anaerobes are able to use
oxygen in metabolic processes and generate more energy per
mole of energy source consumed.
§ Aerotolerant anaerobes and strict anaerobes do not use
oxygen in their metabolism and typically have a lower energy
yield and slower growth rates.

• A reducing agent
such as
thioglycolate can be
added to a medium
to test an organism's
requirement for
oxygen (Figure
6.25).

• TA restrict oxygen to the top one-third of the tube. Aerobes will grow
only at the top of the tube (1) Facultative anaerobes will growth
throughout (2 and 3) and strict anaerobes will only grow at the
bottom of the tube (4).
Organism Habitat Oxygen Relationship
Sulfolobus Hot sulfur springs Strict aerobe
acidocaldarius
Acinetobacter Skin Strict aerobe
calcoaceticus
Bifidobacterium Human intestines Strict anaerobe
bifidum
Magnetospirillum Fresh and marine water Microaerophile
magnetotacticum
Campylobacter jejuni Mucosal surfaces of animals Microaerophile
and birds
Lactobacillus Animals plants, fermented Aerotolerant anaerobe
acidophilus foods.
Enterobacter Intestines of warm-blooded Facultative anaerobe
aerogenes animals, fresh water
Vibrio fischeri Marine water, light organ of Facultative anaerobe
4/25/22 several marine
De La Salle University species
- Dasmarinas 59
•Several toxic forms
of oxygen can be
formed in the cell,
but enzymes are
present that can
neutralize most of
them (Figure 6.28).
Superoxide in
particular seems to
be a common toxic
oxygen species.
60
• Special
techniques are
needed to grow
aerobic and
anaerobic
microorganism
s (Figure
6.26).

NUTRITION AND CULTURE OF
MICROORGANISMS
66

§ Macronutrients
§ Micronutrients
§ Trace elements
§ Temperature
§ pH
§ Osmotic Pressure (Aw)
§ Oxygen

Element % of Source Function
Dryweight*
Macronutrients
Carbon 50 organic compounds Main constituent of cellular material

or CO2
Oxygen 20 H2O, organic Constituent of cell material and cell

compounds, water; O2 is electron acceptor in
CO2, and O2 aerobic respiration
Nitrogen 14 NH3, NO3, organic Constituent of amino acids, nucleic
compounds, N2 acids nucleotides, and
coenzymes
Hydrogen 8 H2O, organic Constituent of organic compounds
compounds, H2 and cell water. Also important in
energy generation as protons.
Phosphorus 3 inorganic Constituent of nucleic acids,
phosphates nucleotides, phospholipids, LPS,
(PO4) teichoic acids
Micronutrients
Sulfur 1 SO4, H2S, So, organic Constituent of cysteine, methionine,

sulfur glutathione, several coenzymes
compounds
Potassium 1 Potassium salts Main cellular inorganic cation and

cofactor for certain enzymes
Magnesium 0.5 Magnesium salts Inorganic cellular cation, cofactor
for certain enzymatic reactions
Calcium 0.5 Calcium salts Inorganic cellular cation, cofactor
for certain enzymes and a
component of endospores
Iron 0.2 Iron salts Component of cytochromes and
other proteins and a cofactor for
some enzymatic reactions

Just read on
Nitrogen Sulfur Phosphorus
In amino acids, In amino acids, In DNA, RNA, ATP, and
proteins thiamine, biotin membranes
Most bacteria Most bacteria PO43- is a source of
decompose proteins decompose proteins phosphorus
Some bacteria use Some bacteria use
NH4+ or NO3- SO42- or H2S
A few bacteria use N2
in nitrogen fixation

Element Example of Function
Cobalt Part of vitamin B12, which is used to carry
methyl groups
Zinc Structural role in many enzymes including
DNA polymerase
Mo Certain reactions involving nitrogen
assimilation. Found in nitrate reductase and
nitrogenase.
Cu Catalytic role in some enzymes that react
with oxygen for example cytochrome
oxidase.
Mn Required by a number of enzymes in
catalytic sites. Certain photosynthetic
enzymes use manganese to split water into
oxygen and protons.
Ni Several different enzymes including some
involved in carbon monoxide metabolism,
urea metabolism and methanogenesis

§ Some examples of growth factors include
§ Vitamins which are non-protein components of many enzymes
§ Amino acids for protein synthesis
§ Nucleic acids for DNA and RNA synthesis

§ Trace Elements
§ Inorganic elements required in small amounts
§ Usually as enzyme cofactors

chemotrophs obtain
Phototrophic - utilize
energy by the oxidation
light as a source of of either inorganic or
energy
organic compounds.
Autotrophs - obtain Heterotrophs - rely on

their carbon from pre-made organic
carbon dioxide compounds for carbon.
Organotrophs - obtain lithotrophs - obtain

their electrons from electrons from inorganic
organic compounds compounds

Nutritional Type Energy Carbon Electron Examples
Source Source Source
Photoautotrophic Light CO2 Inorganic Cyanobacteri
lithotrophs (H2O or a, some
H2S) purple
and green
bacteria
Photoheterotrophic Light Organic Organic Some purple
organotrophs compou compounds and green
nds bacteria
Chemoautotrophic Chemicals CO2 Inorganic Bacteria and
lithotrophs (H2, NH3, compounds many
H2S) archaea
Chemoheterotrophic Organic Organic Organic Most

organotrophs compounds compou compounds bacteria,
nds some
archaea

§ Parameters used as a measure of growth of a population of
bacteria. They include:
§ Change in cell number.
§ Change in the turbidity or light scattering of the culture.
§ Change in the amount of a cell component.

§ Microscopic Counts:
§ counting chambers
§ electronic counters – flow cytometry
§ on membrane filters
§ Viable Counting Methods:

§ Spread and pour plate techniques
§ Membrane filter technique
§ Turbidity for Most Probable Number (MPN)
§ Measurement of Cell Mass

§ Dry Weight Analysis
§ Measurement of cell components
§ Turbidity
77
§ Easy, inexpensive, and
quick
§ Useful for counting both
eukaryotes and prokaryotes
§ Cannot distinguish living
from dead cells
78
§ Cells filtered through special membrane that provides dark
background for observing cells
§ Cells are stained with fluorescent dyes
§ Useful for counting bacteria

§ With certain dyes, can distinguish living from dead cells
79
§ Microbial suspension forced through small orifice with a
laser light beam
§ Movement of microbe through orifice impacts electric
current that flows through orifice
§ Instances of disruption of current are counted = result in
count of individual cells.
81
§ (1) Without special staining techniques dead cells cannot be
distinguished from live cells.
§ (2) Small cells are difficult to see under the microscope, and some
cells are inevitably missed.
§ (3) Precision is difficult to achieve.
§ (4) A phase-contrast microscope is required if the sample is not
stained.
§ (5) Cell suspensions of low density (less than about 106
cells/milliliter) have few if any bacteria in the microscope field
unless a sample is first concentrated and resuspended in a small
volume.
§ (6) Motile cells must be immobilized before counting.
§ (7) Debris in the sample may be mistaken for microbial cells.

§ Whether or not a cell is alive
or dead isn’t always clear cut
in microbiology
§ Cells can exist in a variety of
states between ‘fully viable’
and ‘actually dead’
§ VBNC (Viable but not
culturable)
83
§ Spread and pour plate techniques
§ diluted sample of bacteria is spread over solid agar surface or
mixed with agar and poured into Petri plate
§ after incubation the numbers of organisms are determined by
counting the number of colonies multiplied by the dilution factor
§ results expressed as colony forming units (CFU)
84
§ Membrane filter technique (used in our lab
during water testing)
§ bacteria from aquatic samples are
trapped on membranes
§ membrane placed on culture media
§ colonies grow on membrane
§ colony count determines # of bacteria in
sample
86
§ If microbe cannot be cultured on plate media
§ Dilutions are made and added to suitable media
§ Turbidity determined to yield the most probable number
(MPN)
87
• Plate counts can be highly unreliable when used to assess
total cell numbers of natural samples such as soil and water.
Direct microscopic counts of natural samples typically reveal
far more organisms than are recoverable on plates of any
given culture medium.
• This is referred to as "the great plate count anomaly," and it

occurs because direct microscopic methods count dead cells
whereas viable methods do not, and different organisms in
even a very small sample may have vastly different
requirements for resources and conditions in laboratory
culture.

§ Dry weight
§ time consuming and not very sensitive
§ Quantity of a particular cell constituent

§ e.g., protein, DNA, ATP, or chlorophyll
§ useful if amount of substance in each cell is constant
§ Turbidometric measures (light scattering)

§ quick, easy, and sensitive
90
• Turbidity measurements are an indirect but very rapid and useful
method of measuring microbial growth (Figure 6.12). However, to relate
a direct cell count to a turbidity value, a standard curve must first be
established.
91

92
§ End of presentation

§ Madigan MT, Clark DP, Stahl D, Martinko JM. 2013. Brock
biology of microorganisms. 13th edition. NY,USA: Benjamin
Cummings.
§ Tortora G, Funke B, Case C. 2014. Microbiology: An
Introduction, 11th ed. Singapore : Pearson Education South Asia.
§ Willey et al. ( ) 9th ed. Copyright © McGraw-Hill Global Education
Holdings, LLC. Permission required for reproduction or display.

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M6-Growth and Reproduction

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Prepared by W.R.

De La Salle University - Dasmarinas 4/25/22

Produce beer, wine,

De La Salle University - Dasmarinas 4/25/22 3

De La Salle University - Dasmarinas 4/25/22 4

De La Salle University - Dasmarinas 4/25/22 6

De La Salle University - Dasmarinas 4/25/22 8

De La Salle University - Dasmarinas 4/25/22 11

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§ Generation time = time lapse in minutes = min/generation

§ If 100 cells growing for 5 hours produced 1,720,320 cells:

§ What is the number of generations?

De La Salle University - Dasmarinas 4/25/22 15

CYCLE, P. (Figure 6.8)

De La Salle University - Dasmarinas 4/25/22 16

De La Salle University - Dasmarinas 4/25/22 17

A. LAG PHASE § depends on harshness of medium

§ The general rule of thumb is that

4/25/22 De La Salle University - Dasmarinas 19

Rate of growth and division is

Population is most uniform in

Bacteria from this stage would

De La Salle University - Dasmarinas 4/25/22 20

C. STATIONARY § no change in the number of viable

4/25/22 De La Salle University - Dasmarinas 21

§ Bacteria are dying off opposite to log growth phase

§ Two alternative hypotheses

§ Programmed cell death

De La Salle University - Dasmarinas 4/25/22 22

De La Salle University - Dasmarinas 4/25/22 23

De La Salle University - Dasmarinas 4/25/22 24

De La Salle University - Dasmarinas 4/25/22

A common curve of temperature

Mesophiles - optima in the 20 oC

Thermophiles have optima from

Extreme thermophiles - optima,

De La Salle University - Dasmarinas 4/25/22 27

De La Salle University - Dasmarinas 4/25/22 29

Bacterium Habitat Cardinal

Vibrio marinus Open ocean 4 15 30

Staphylococcus Skin 10 30-37 45

De La Salle University - Dasmarinas 4/25/22

De La Salle University - Dasmarinas 4/25/22 34

De La Salle University - Dasmarinas 4/25/22

De La Salle University - Dasmarinas 4/25/22 37

Bacillus Acidic hot springs 2.0 4.0 6.0

Lactobacillus Animals, plants, decaying matter 4.0-4.6 5.8-6.6 6.8

Staphylococcus Surfaces of animals, nasal cavity, 4.2 7.0-7.5 9.3

Escherichia coli Intestines of animals 4.4 6.0-7.0 9.0

Clostridium Soils and sediments that are 5.0-5.8 6.0-7.6 8.5-9.0

Pseudomonas Ubiquitous 5.6 6.6-7.0 8.0

Streptococcus Pathogen of animals 6.5 7.8 8.3

De La Salle University - Dasmarinas 4/25/22

De La Salle University - Dasmarinas 4/25/22 41

De La Salle University - Dasmarinas 4/25/22 43

§ Some microorganisms (halophiles) have evolved to grow best

De La Salle University - Dasmarinas 4/25/22 44

Organism Habitat Minimum water activity

Pseudomonas Ubiquitous low salt environments 0.91

Salmonella/E. Animals 0.91

Bacillus Soil 0.90

Staphylococcus Animals 0.85

Halobacterium High salt lakes such as Great Salt 0.75

De La Salle University - Dasmarinas 4/25/22