UNIT 5

Microbial Growth and

Reproduction
 OBJECTIVES
 1. Explain binary fission in bacteria

 2. Define microbial growth

 3. Explain the growth cycle of bacteria

 4. Explain methods of quantitative estimation of growth of bacteria

 5. Describe the effect of environmental factors on microbial growth

 6. Describe the effect of nutritional and physical factors on microbial

growth.
 Bacteria Divide by BINARY FISSION
 Prokaryotes reproduce asexually by cell division called binary fission, which
involves the duplication of genetic material (or DNA) and then the division
into two parts, with each new organism receiving one copy of DNA.
 First, the bacterial chromosome begins to replicate, starting at the origin of replication
 Replication continues, one origin moves to the opposite side of the cell, and the cell
elongates
 Upon the completion of replication, the plasma membrane grows inward, and a new cell
wall is laid down

 The production of two genetically identical daughter cells is called cloning

 In order for the cell to divide in two (or half) the peptidoglycan structure must
be different in the hemispherical cap than in the straight portion of the cell
wall.
 Binary fission is the evolutionary precursor to mitosis
 Figure 6.12a Binary fission in bacteria.
Cell wall
Cell elongates and
DNA is replicated. Plasma
membrane

Cell wall and plasma DNA

membrane begin to constrict. (nucleoid)

Cross-wall forms,
completely separating the
two DNA copies.

Cells separate.

Each daughter cell can continue to grow at the same rate as its parent.

A diagram of the sequence of cell division

 Cell Count From Binary Fission

Generation Cell
Number Count
0 1
1 2
2 4
3 8
4 16
5 32
10 1,024
20 1,048,576

Let’s watch a time

lapse movie of E. coli
population growth. From the Virtual Microbiology Classroom on ScienceProfOnline.com
 MICROBIAL GROWTH
Bacterial Variations
 If binary fission produces clonal offspring, why are bacteria so genetically
diverse???
 Variations - Any change in the genotype of a bacterium or its phenotype is known as
variation.
 Genotypic variation can occur as a result of changes in the genes by way of mutation.
 Two factors contribute to genetic diversity among and within bacterial species
 Mutation – a gene will spontaneously mutate in a hundred million cell
divisions. Most of the mutants will die, but some will adapt to the environment
and emerge as a new variant. (antibiotic resistance??)
 Recombination – DNA is transferred from one bacteria (donor) to another
(recipient). This process occurs in three main ways: ??
Transformation, Transduction and Conjugation.
 MICROBIAL GROWTH
Growth of Bacteria

 Growth of bacteria (or bacterial cultures) is defined as an increase

in the number of bacteria in a population rather than in the size of
the individual cells

 Under optimal conditions, a single prokaryote cell divides to

produce two daughter cells every ~ 1-3 hours
 Each round of division is a generation

 Bacterial population growth is therefore rapid and exponential

 1 cell  2 cells  4 cells  8 cells  16 cells etc.
 A colony is formed from a single cell in 12 hours
 MICROBIAL GROWTH
Growth of Bacterial Populations – some important definitions
 Bacterial Population - a group of bacteria of the same species or subtype defined
by time and space.
e.g. we can study the population of Salmonella Typhi inhabiting the
gall bladder of a typhoid carrier.
This example is specific to a certain species of bacteria (Salmonella
Typhi) in the gall bladder (space), while the patient is infected with
typhoid (time)
 Bacterial Community – speaks to the total bacterial loading present in a given
sample and considers all possible species that are specific to that sample.
 Bacterial Colonies – a visible mass of bacteria growing on solid media, all
originating from a single mother cell. Therefore, a colony constitutes a clone of
genetically alike bacteria.
 Bacterial Culture – the growing of bacteria on/in a special medium, such as agar.
 PREPARATIONS OF BACTERIAL CULTURES

 Closed system = Batch Culture

 Closed culture vessel (a)
 One batch of culture medium

 Continuous culture (b)

 Culture expected to grow indefinitely by:
-continuously supplying fresh medium
-accumulated cells and waste products are removed at
the same time
-vessels are called fermenters
Used widely in industry for production of microbial mass
Batch Culture Techniques
 In this technique at first nutrient solution is prepared and it is
inoculated with inoculum (culture organism) and then nothing is
added in the fermentation tank except aeration.

 In batch culture, neither fresh medium is added nor used up

media is removed from the cultivation vessel. Therefore volume
of culture remains same.

 Since fresh media is not added during the course of incubation,

concentration of nutrition decreases continuously. Furthermore
various toxic metabolites also accumulates in the culture vessel.
Therefore batch culture technique gives characteristics growth
curve with lag phase, log phase, stationary phase and decline
phase.
 GROWTH CYCLE OF BACTERIA
Bacterial Population Growth Curve

From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Bacterial growth phases, Michal Komorniczak
Lag Phase

 A period of apparent inactivity in which the cells are adapting to

a new environment and preparing for reproductive growth,
usually by synthesizing new cell components
 ATP
 Ribosomal proteins
 rRNA
 tRNA
 Co-factors
 Enzymes
Lag Phase cont’d

 Varies in length depending upon the condition of the microorganisms

and the nature of the medium
 Assessment of medium – Receptors
 DNA synthesized – initiation of cell division
Exponential phase (Log Phase)
 Optimal growth rate and cell division dependent on
medium, O2, temperature, pH, genetic composition
 Regular, constant cell division (logarithmically)
 Smooth curve – division not synchronous
 Most useful phase for biochemical, physiological and DNA
replication studies
 Biotechnology applications – competent cells – uptake of plasmid
DNA
 Late log = optimal plasmid concentration
 The population is most uniform in terms of chemical and
physical properties during this period
 Stationary Phase
 When the population reaches ~109/ml (106 for
protozoan and algal cultures), cell division = cell
death (stasis)
 Nutrients become scarce
 O is depleted
2
 Toxic waste accumulates
 The number of viable microorganisms remains
constant either because metabolically active cells
stop reproducing or because the reproductive
rate is balanced by the rate of cell death
 Death Phase
 Nutrient deprivation
 Waste material accumulation
 Irreversible loss of ability to reproduce
 Viable cell mass decreases
 Often logarithmic
 Cells not viable when inoculated into fresh medium
 Cells have reached the carrying capacity of their
environment
 Quantitative Estimation of Growth
 The mathematics of growth-
Microbial growth can be described by certain
mathematical terms:
 Mean generation time (g) or doubling time is the time
required for the population to double
 Mean growth rate constant is the number of
generations per unit time, often expressed as
generations per hour
Slope of straight line (log phase) gives the growth
rate of the cells
 Generation times vary markedly with the species of microorganism
and environmental conditions
 they can range from 10 minutes for a few bacteria to several days
with some eukaryotic microorganisms
 Population size = 2n where n = the number of generations

Example: The generation time for E. coli in the laboratory is 15-20

mins, but in the intestinal tract, the generation time is estimated to
be 12 – 24 hours.

Pathogenic bacteria (such as Treponema pallidum and

Mycobacterium tuberculosis) tend to have especially long
generation times, and this adds to their virulence.
 Methods for Measurement of Cell Numbers
Measuring techniques involve:
 direct counts – directly measuring the number of
colonies or cells
 Viable plate counts
 Membrane filtration
 Microscopic counts - Petroff-Hausser Counting Chamber
 Electronic counters - Coulter Counter

 indirect viable cell counts – indirectly measuring some

parameter based on the cells’ activity
 measurements of metabolic activity
 measurements of a population’s dry weight
 measurements of the turbidity of a broth, especially by a
device known as a spectrophotometer.
 DIRECT

Viable Plate Counts (only live cells)

-involves plating out (spreading) a sample of a culture on a

nutrient agar surface.
- The sample or cell suspension can be diluted in a nontoxic
diluent (e.g. water or saline) before plating.
- If plated on a suitable medium, each viable unit grows and
forms a colony.
- Each colony that can be counted is called a colony forming unit
(CFU) and the number of CFU's is related to the viable number of
bacteria in the sample.
 Membrane filtration assay

 Membrane traps bacteria on the surface

 Membrane transferred to an agar plate
 Colonies grow  counted
 Can use selective media (e.g. Endo agar for coliform
counts in contaminated water supplies)
  Microscopic counts
Petroff-Hausser Counting Chamber
 A sample is placed on a cell counter
 A cell counter is a glass slide with depressed etched grids
(having 25 squares), and viewed through a microscope
 The slide is covered with a coverslip
 The number of bacteria in several of the large squares is
counted
 The mean number of bacteria per square is then
determined by calculations
 25 squares (area) = 1mm2
 Depth = 0.02mm
 Volume = 2 x 10-5 mL in 25 squares
 Determination of cell numbers:
If there is an average of 20 cells per square,
then the total number of cells across the entire
25 squares is:
20 cells x 25 squares = 500 cells for the entire
counter
The number of cells per mL is therefore:
500 cells / 2 x 10-5 ml = 2.5 x 107 cells/mL
 Electronic Counter
Coulter counter
A device that counts cells as they interrupt an
electrical current flowing across a narrow tube held in
front of an electronic detector
 Bestused for counting eukaryotic cells – such as RBC
and WBC
 Less accurate with small cells, due to:
High interference
Clumping
 INDIRECT

Cell mass increases as cell number increases

 Dry weight measurements

 To calculate the dry weight of cells, they must first be
separated from the medium.
 Separation can be done by some physical means:
 Filtration
 Centrifugation

 The cells are then dried and weighed

 Dry weight of filter paper containing pellets of microbial
cells is measured
 Dry weight of microbial cells = (Dry weight of filter
paper containing pellets of microbial cells is measured)
– (Dry weight of only filter paper of similar size)
 Spectrophotometric determination
 As bacteria multiply in media, the media becomes turbid
 The cloudiness or turbidity of a culture is caused by the individual
cells scattering light. (therefore, the amount of light that is
scattered, is proportional to cell number)
 The spectrophotometer is used to determine the amount of light
absorbed or transmitted
 Often written as % transmittance (as absorbance increases,
transmittance decreases)
 Linear relationship between absorbance and cell density
 Requires cultures to be ~107/ml and upwards (slight turbidity)
 A major disadvantage is that dead and live bacteria cannot be
distinguished
Continuous Culture
Techniques
Used to maintain cells in the exponential
growth phase at a constant biomass
concentration for extended periods of
time
Conditions are met by continual
provision of nutrients and removal of
wastes = OPEN SYSTEM
Constant conditions are maintained
Chemostat
A continuous culture device that maintains a
constant growth rate by:
supplying a medium containing a limited amount of
an essential nutrient at a fixed rate
removing medium that contains microorganisms at
the same rate
As fresh media is added to the chamber,
bacteria are removed
Limiting nutrients control growth rates
Cell density depends on nutrient concentration
 Turbidostat
 A continuousculture device that regulates the flow rate of
media through the vessel in order to maintain a
predetermined turbidity or cell density
 There is no limiting nutrient
 Absorbance is measured by a photocell (optical sensing device)
 Thenumber of cells in culture controls the flow rate and the rate of
growth of culture adjusts to this flow rate
Balanced and Unbalanced
Growth
Balanced (exponential) growth occurs when
all cellular components are synthesized at
constant rates relative to one another
Unbalanced growth occurs when the rates of
synthesis of some components change
relative to the rates of synthesis of other
components.
This usually occurs when the environmental
conditions change
 Environmental Factors
Affecting Microbial Growth
Solutes and Water Activity

 Osmoticconcentrations affect microbes (e.g.

plasmolysis in hypertonic solutions)
 Wateractivity (Aw) = measurement of availability
of water in particular environments
 Aw = Psolution/Pwater (P = vapor pressure)
= inversely related to osmotic pressure
 Ifthe solution has a high osmotic pressure (high
extracellular solute concentration), then its A w = low
 Energy is required by microbes to tolerate low Aw because in order to keep
water, solute concentration inside of cells must be kept high
= Osmotolerance
S. aureus can tolerate up to 3M NaCl
Archaebacteria halophiles tolerate 2.8-6.2M NaCl (Great Salt Lake, Dead
Sea)
 Avoidance of plasmolysis. Plasmolysis - the cytoplasm
pulls away from the cell wall due to the loss of water through osmosis. This
occurs in a hypertonic solution. The reverse process, cytolysis, can occur if
the cell is in a hypotonic solution resulting in a lower external osmotic
pressure and a net flow of water into the cell.
pH (Log scale of 0 – 14; each pH unit
= 10x change)

pH is the negative logarithm of the

hydrogen ion concentration
Acidophiles grow best between pH 0
and 5.5
Neutrophiles grow best between pH 5.5
and 8.0
Alkalophiles grow best between pH 8.5
and 11.5
Extreme alkalophiles grow best at pH 10.0 or
higher
Despite wide variations in habitat pH, the
internal pH of most microorganisms is
maintained near neutrality either by proton/ion
exchange or by internal buffering
Sudden pH changes can inactivate enzymes and
damage PMs
Reason for buffering culture medium, usually with a
weak acid/conjugate base pair (e.g. KH 2PO4/K2HPO4
– monobasic potassium/dibasic potassium)
Temperature
Microorganisms are sensitive to temperature
changes
Usually unicellular and poikilothermic
Enzymes have temperature optima
If temperature is too high, proteins denature, including
enzymes, carriers and structural components
Temperature ranges are enormous (-20 to 100oC)

 Poikilothermic – an organism, such as fish or reptile, that has a body temperature that varies with the
temperature of its surroundings.
Effects of Temperature on Growth

40oF 77oF 95oF

Most of our plates are incubated at 37 oC (98.6oF).

Conversion C to F = 1.8 x C + 32

From the Virtual Microbiology Classroom on ScienceProfOnline.com

 Growth temperature
 The minimum growth temperature is the lowest
temperature at which a species will grow
 The optimum growth temperature is the temperature at
which it grows best
 The maximum growth temperature is the highest
temperature at which growth is possible.
 Temperature Requirements
 5 groupings of Bacteria by their optimal temperature growth
requirements

1. Psychrophiles : cold loving -8 to 18 °C

2. Psychrotrophs: 0 to 30 °C
3. Mesophiles: moderate temp 10 to 48°C
4. Thermophiles: heat loving 40 to 72 °C
5. Hyperthermophiles: super heat 66 to 110 °C
Typical Growth Rates and Temperature
 Psychrotrophs
 Grow between 0°C and 20–30°C
 Cause food spoilage
Food Preservation Temperatures
Temperature Considerations

 food preservation
 refrigeration

inhibits
fast growing
mesophiles
 psychrophiles can still grow in
refrigeration, but at a
diminished rate
 freezingdestroys
microorganisms that require
water to grow
Organisms exhibit distinct cardinal
temperatures (minimal, maximal, and optimal
growth temperatures)
Ifan organism has a limited growth
temperature range = stenothermal (e.g. N.
gonorrhoeae)
Ifan organism has a wide growth temperature
range = eurythermal (Enterococcus faecalis)
Psychrophiles can grow well at 0C, have
optimal growth at 15C or lower, and
usually will not grow above 20C
Arctic/Antarctic ocean
Protein synthesis, enzymatic activity and
transport systems have evolved to function at
low temperatures
Cell walls contain high levels of unsaturated
fatty acids (semi-fluid when cold)
 Psychrotrophs (facultative psychrophiles) can also grow
at 0C, but have growth optima between 20C and
30C, and growth maxima at about 35C
 Many are responsible for food spoilage in refrigerators

 Mesophiles have growth minima of 15 to 20C, optima

of 20 to 45C, and maxima of about 45C or lower
 Majority of human pathogens
Thermophiles have growth minima
around 45C, and optima of 55 to 65C
Hot springs, hot water pipes, compost heaps
Lipidsin plasma membrane more saturated
than mesophiles (higher melting points)
Hyperthermophiles have growth minima
around 55C and optima of 80 to 110C
Sea floor sulfur vents
Oxygen concentration
Obligateaerobes are completely dependent on
atmospheric O2 for growth
Oxygen is used as the terminal electron acceptor for
electron transport in aerobic respiration
Facultativeanaerobes do not require O2 for
growth, but do grow better in its presence
Aerotolerant anaerobes ignore O2 and grow
equally well whether it is present or not
Obligate (strict) anaerobes do not tolerate
O2 and die in its presence
Microaerophiles are damaged by the
normal atmospheric level of O2 (20%) but
require lower levels (2 to 10%) for growth
 Oxygen tolerance is determined by an organism’s ability to
destroy toxic oxidizing products of oxygen reduction
 Remember, because oxygen has two unpaired outer
orbital electrons, it accepts electrons readily
 Toxiccompounds
 Superoxide radical:
O + e-  O2•-
2
 Hydrogen peroxide:
O • -
+ e -
+ 2H +
 H2O2
2
 Hydroxyl radical:
H O + e -
+ H +
 H2O + OH•
2 2
 These compounds are used deliberately by
phagocytic WBC to break down intracellular
microbes (respiratory burst)
 Solutionused by obligate aerobes and facultative anaerobes:
 Produce enzymes that convert these toxic oxidizing products to
non-toxic compounds
Superoxide dismutase

2O2•- + 2H+  O2 + H2O2

Catalase

2H2O2  2H2O + O2

 Aerotolerant microbes have Superoxide dismutase (SOD);

Obligate anaerobes lack SOD and catalase or have low
concentrations
SOD - an enzyme that catalyzes the conversion of superoxide into hydrogen peroxide and oxygen
 Laboratory considerations
Aerobic cultures
Shakenor sterile air introduced to
medium
 Anaerobic cultures
Remove oxygen
Include reducing agents in medium (e.g.
thioglycolate or cysteine
Dissolved oxygen is destroyed
Growth beneath surface
Replace oxygen with nitrogen gas and
CO2 gas
Gas-Pak jar
H2 + palladium catalyst + O2 
H2O
Bagsor pouches  CaCO3  CO2 rich
atmosphere
Pressure
Barotolerant organisms are adversely
affected by increased pressure, but not as
severely as are nontolerant organisms
Barophilic organisms require, or grow
more rapidly in the presence of, increased
pressure
Radiation
Ultraviolet radiation damages cells by
causing the formation of thymine dimers in
DNA
Photoreactivation repairs thymine
dimers by direct splitting when the cells
are exposed to blue light
Dark reactivation repairs thymine
dimers by excision and replacement in
the absence of light
Ionizing radiation such as X rays or
gamma rays are even more harmful to
microorganisms than ultraviolet radiation
Low levels produce mutations and may
indirectly result in death
High levels are directly lethal by direct
damage to cellular macromolecules or
through the production of oxygen free
radicals
Nutritional Factors of
Microbial Growth
 Prokaryotic cells use nutrients to synthesize components
essential for growth
 The major elements required are the chemical elements that
actually make up the cell, such as:
Carbon
Oxygen
Hydrogen
Nitrogen
Potassium, Sulphur, Magnesium etc
Trace elements (required in small mounts) including:
cobalt, zinc, copper, manganese
 Functions of major elements:
Carbon, Oxygen and Hydrogen = important
components of amino acids, lipids, nucleic acids
and sugars
Nitrogen = main component of amino acids and
nucleic acids
Sulphur = component of some amino acids
Potassium, Magnesium and Calcium = required for
enzyme function
 Growth Factor – a compound that is necessary for certain
bacteria to grow

 When a bacterium is unable to synthesize certain

components such as amino acids or vitamins on their own,
the growth medium they are in must contain the necessary
material in order for them to grow
Example: Neisseria gonorrhoeae - is fastidious, as they
have complicated nutritional requirements and require
growth factors that they are unable to produce on their
own
Sources:
 Heterotrophs – use organic carbon (medically important bacteria) already
produced by other organisms for its nourishment.
 Autotrophs – take inorganic substances into their bodies and transform them
into organic nourishment by the use of sunlight.
Example: bacteria create their food using inorganic sulfur compounds
gushing out of the vents from the hot interior of the planet.
 Carbon Fixation – autotrophs convert inorganic carbon to organic carbon
 Nitrogen Fixation – bacteria use N2 as a nitrogen source during nitrogen
fixation
 Phototrophs – harvest energy directly from sunlight (photosynthetic bacteria
such as green bacteria) and obtain carbon from CO 2
 Chemotrophs – extract energy from chemical compounds, such as hydrogen
sulphide and hydrogen gas (this is an ability that distinguishes prokaryotes
from eukaryotes)
Nutritional Diversity
 Photoautotrophs - use energy from sunlight to convert carbon
dioxide and water into organic materials to be used in cellular
functions such as biosynthesis and respiration.
Example - cyanobacteria

 Photoheterotrophs - obtain their energy from sunlight and carbon

from organic material and not carbon dioxide. Produce ATP but use
environmentally obtained organic compounds to build structures
and other bio-molecules.
Example- purple non-sulfur bacteria
 Chemolithoautotrophs – use inorganic compounds for energy and obtain carbon
from carbon dioxide.
Use chemicals (chemo) from rocks (litho) as the source
of energy to make their own (auto) food (troph)
Habitat: minimum sunlight, sulfur hot springs,
hydrothermal vents.
Example- Elemental sulfur granules present in the tissues of sulfur-oxidizing
bacteria from a submerged cave in central Florida.

 Chemoorganoheterotrophs – uses organic compounds as a source of energy and

carbon.
Breaks down complex organic compounds produced by
autotrophs, into simpler ones.
Typically known as decomposers
Example – most bacteria, Escherichia coli, Bacillus spp.