MICROBIAL GROWTH

MICR213
Dr. S.T Gumbi

INSPIRING GREATNESS
 Reproductive Strategies
▪ The reproductive strategies of eukaryotic
microbes
• asexual and sexual, haploid or diploid

▪ Bacteria and Archaea

• haploid only, asexual - binary fission, budding,
filamentous
• all must replicate and segregate the genome prior
to division
• Bacterial growth – exponential.

• Daughter cells may separate or remain attached in

characteristic arrangements of chains, clusters or
pairs.

• Other forms of reproduction include: budding,

fragmentation, conidia, or sporulation.
 The Prokaryotic Cell Cycle

▪ Cell cycle - sequence of events from formation

of new cell through the next cell division
• most bacteria divide by binary fission

▪ Two pathways function during cycle

• DNA replication and partition

• cytokinesis (Z ring)
 Growth
▪ Increase in cellular constituents - may result in:
• increase in cell number

• e.g., reproduction by budding or binary fission

• Increase in cell size

• e.g., coenocytic microorganisms - nuclear

divisions not accompanied by cell divisions

▪ Microbiologists usually study population growth

rather than growth of individual cells
 The Cell Cycle in E. coli
• E. coli requires ~40 minutes to replicate its DNA
and 20 minutes after termination of replication to
prepare for division
 The Growth Curve
▪ Observed when microorganisms cultivated in batch
culture
• culture incubated in a closed vessel with a single
batch of medium
▪ Exponential - plotted as logarithm of cell number
versus time

• Single parent cell gives rise to two progeny

cells
▪ Usually four distinct phases
 Lag Phase
• No cell division – acclimatization
• Cell synthesizing new components
• essential enzymes, cofactors, ATP
• replenish spent materials
• adapt to new medium or other conditions
• Varies in length
• older cells and stressed cells - longer to recover
• can be very short or even absent
• dependent on bacteria and environmental
conditions
 Exponential Phase
▪ Log phase – maximal growth

▪ Rate of growth is constant - steady increase

▪ Population - most uniform in terms of chemical

and physical properties during this phase
 Exponential Phase:
Balanced growth

• During log phase, cells exhibit balanced growth

• cellular constituents manufactured at constant rates

relative to each other
 Exponential Phase:
Unbalanced growth
▪ Rates of synthesis of cell components vary relative to
each other

▪ Occurs under a variety of conditions

• change in nutrient levels

• shift-up (poor medium to rich medium)

• shift-down (rich medium to poor medium)

• change in environmental conditions

• Maximal rate of exponential growth via binary
fission
• metabolic activity peaks

• Generation time = rate of bacterial reproduction

• time taken by one individual bacterium to divide
• varies according to type of bacterium and
environmental conditions

• Maximum cell concentration dependent on

organism and environment
• up to 1011 bacterial cells per ml
• Each individual cell
divides at a slightly
different time

• Curve rises
smoothly rather
than as discrete
steps
 Effect of nutrient
concentration on growth
 Stationary Phase

▪ Growth/cell division ceases – plateau reached

▪ Total number of viable cells remains constant

▪ metabolically active cells stop reproducing

▪ reproductive rate is balanced by death rate

 Possible reasons for entry
into stationary phase
▪ Nutrient limitation

▪ Limited oxygen availability

▪ Toxic waste accumulation

▪ Critical population density reached

Stationary phase: Starvation
responses
• Morphological changes (e.g., endospore formation)
• Decrease in size, protoplast shrinkage, and nucleoid
condensation

• RpoS protein assists RNA polymerase in transcribing

genes for starvation proteins
• increase cross-linking in cell wall
• Dps protein protects DNA
• chaperone proteins prevent protein damage

• Persister cells - long-term survival; increased

virulence
 Death Phase
▪ Unfavourable environmental conditions, starvation,
stress

▪ Cells dying, usually at exponential rate

▪ Death
• irreversible loss of ability to reproduce

▪ In some cases, death rate slows due to accumulation

of resistant cells
 Two alternative hypotheses

▪ Viable but non-culturable bacteria – VBNC

• Temporarily unable to grow - dormant

• Can resume growth – environment favourable

• Programmed cell survival

▪ Programmed cell death

• Programmed cell suicide by fraction of population

• Dead cells provide nutrients

Loss of Viability
 The Mathematics of Growth
• Generation (doubling) time
• Specific length of time required for the population to
double in size
• e.g., 2 cells after 20 min; 4 cells after 40 min, etc

• Population is doubling every generation - increase

in population = 2n; n = no. of generations

• Mean growth rate constant

• number of generations per unit time

• usually expressed as generations per hour

• Each individual cell
divides at a slightly
different time

• Curve rises smoothly

rather than as discrete
steps

• Population is doubling
every generation
 CALCULATING THE MEAN
GROWTH RATE
• N0 = initial population number
• Nt = population at time t
• n = number of generations in time t
• 2n = generation time

Nt = N0 × 2n (for populations using binary fission)

• Which converts down to…

n = (log Nt- log N0)/0.301

• Yes…you really should learn this equation

• To calculate n (number of generations):

• Log Nt = log N0 + n . log 2

n = log Nt – log N0
log 2

= log Nt – log N0
0.301
• Mean growth rate constant (k) – rate of
growth in the exponential phase
• number of generations per unit time

• usually expressed as generations per hour

•µ = n/t
= log Nt – log N0
0.301t
• Mean generation time (g) – how long does it take a population
to double?
• If the population doubles (t = g), then

• Nt = 2N0

• µ= log (2N0) – log N0

0.301g

= log 2 + log N0 – log N0

0.301g
µ = 1/g
g = 1/µ – reciprocal of mean growth rate
constant
 Measurement of
Microbial Growth

▪ Can measure changes in number of cells in a

population

▪ Can measure changes in mass of population

 Measurement of Cell
Numbers

• Direct cell counts

• counting chambers

• electronic counters

• on membrane filters
 Counting chambers
• Easy, inexpensive, and quick
• Useful for counting both eukaryotes and
prokaryotes
• Cannot distinguish living from dead cells (Bacteria)
 Electronic counters - Flow
Cytometry
▪ Microbial suspension forced through small
orifice

▪ Movement of microbe through orifice impacts

electric current flowing through orifice

▪ Instances of disruption of current are counted

 Direct Counts on
Membrane Filters
▪ Cells filtered through special membrane that
provides dark background for observing cells

▪ Cells are stained with fluorescent dyes

▪ With certain dyes, can distinguish living from dead
cells

▪ Useful for counting bacteria

• Viable cell counts
• plating methods (spread, streak and pour
plates)
• membrane filtration methods
 Viable counting methods
• Measure number of plate dilutions of population
viable cells on suitable solid medium

• Viable – alive and count number of colonies
reproducing

• Population size is calculate number of cells in
expressed as colony population (cfu)
forming units (CFU)
= no. of colonies x dilution
• Spread plate and pour factor
plate methods
• Simple and sensitive

• Number calculated from cfu and sample dilution

• 1 ml of 10-3 dilution = 1.59 × 105

• Therefore, original sample had 1.59 × 105 cfu/ml

No. of colonies × dilution factor (this is inverse of dilution)

• Widely used for viable counts of microorganisms in food,

water, and soil

• Inaccurate results obtained if cells clump together

• 30 -300 colonies
• Membrane filtration technique

• bacteria from aquatic samples are trapped on

membranes of known pore size

• membrane soaked in culture media

• colonies grow on membrane

• colony count determines number of bacteria in

original sample
▪ 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)
• Use the 3 number
combination (code)
in the MPN Index
Table to estimate
the number of
microorganisms
present in a sample
 Measurement of Cell Mass
• Dry weight
• Time consuming and not very sensitive
• Filamentous fungi

• Quantity of a particular cell constituent

• e.g., protein, DNA, ATP, or chlorophyll
• Useful if amount of substance in each cell is
constant
Turbidometric
• light scattering directly proportional to biomass and
indirectly proportional to cell number

• spectrophotometry

• quick, easy, and sensitive

• Cloudiness or turbidity of broth

 Continuous Culture of
Microorganisms

• Growth in an open system

• continual provision of nutrients

• continual removal of wastes

• Maintains cells in log phase at a constant

biomass concentration for extended periods

• Continuous culture system

 The Chemostat
• Rate of incoming
medium = rate of
removal of medium
from vessel

• An essential
nutrient is in
limiting quantities
 Dilution rate and microbial
growth
• Dilution rate – rate at
which medium flows
through vessel
relative to vessel size
• Note: cell density
maintained at wide
range of dilution rates
and chemostat
operates best at low
dilution rate
• Population density and generation time linked to
dilution rate
• Population density unchanged over wide dilution
rate range
• Generation time decreases as dilution rate
increases
• Growth rate increases
• Too high dilution rate – washout
• > maximal growth rate
• Too low dilution rate
• Increased cell density and growth rate
• Limited nutrient supply available
 The Turbidostat
• Regulates the flow rate of media through vessel to
maintain a predetermined turbidity or cell
density
• Photocell
• Dilution rate varies – not constant
• No limiting nutrient – all nutrients in excess
• Turbidostat operates best at high dilution rates
 Importance of continuous
culture methods
• Constant supply of cells in exponential phase
growing at a known rate

• Study of microbial growth at very low nutrient

concentrations, close to those present in natural
environment

• Study of interactions of microbes under conditions

resembling those in aquatic environments

• Food and industrial microbiology

 Influence of
Environmental Factors
• Physical and chemical factors required for growth
• light, temperature, pH, and osmotic pressure

• Most organisms grow in fairly moderate

environmental conditions

• Extremophiles
• grow under harsh conditions that would kill most
other organisms
 Solutes and Water Activity
• Changes in osmotic concentrations in the
environment may affect microbial cells

• Water activity (aw)

• amount of water available to organisms
• reduced by interaction with solute molecules
(osmotic effect)
higher [solute]  lower aw
• reduced by adsorption to surfaces (matric effect)
▪ Aw of 0.9 – 1.0 required for microbial growth

▪ Fungi grow at lower Aw than bacteria

▪ implicated in spoilage of dry foods such as bread
 Osmotolerant organisms
▪ Grow over wide ranges of water activity

▪ Osmophiles – high osmotic pressure for growth

• approx. 0.98 - spoilage of sweet food

▪ Use compatible solutes to increase their internal

osmotic concentration (Mechanosensitive
channels)
▪ solutes - compatible with metabolism and growth

▪ Proteins and membranes that require high solute

concentrations for stability and activity
 Halophiles
• Adapted to saline environments
• Some Archaea require 20 – 30% NaCl
• Halobacterium spp. from Dead Sea – 6.2 M NaCl
(29%)
• Extremely high concentrations of potassium
• Cell wall, proteins, and plasma membrane
require high salt to maintain stability and
activity
Effects of NaCl on microbial
growth
Halophiles
• grow optimally at
>0.2 M NaCl or
other salts

Extreme halophiles
• require salt
concentrations >2
M and 6.2 M
 pH
▪ Measure of the
relative acidity
of a solution

▪ Negative
logarithm of the
hydrogen ion
concentration
Acidophiles
• growth optimum
between pH 0 - 5.5
• Most fungi = pH 4-6
– acidophiles

Neutrophiles
• growth optimum
between pH 5.5 – 7
• Most bacteria and
protozoa –
neutrophiles

Alkalophiles
• growth optimum
between pH 8.5 -
11.5
▪ Most acidophiles and alkalophiles maintain an
internal pH near neutrality
• the plasma membrane is impermeable to proton
• exchange potassium for protons

▪ Acidic tolerance response

• pump protons out of the cell
• some synthesize acid and heat shock proteins that
protect proteins

▪ Many microorganisms change pH of their habitat by

producing acidic or basic waste products
• most media - buffers to prevent growth inhibition
 Temperature
• Organisms exhibit distinct
cardinal growth temperatures

• minimal

• maximal

• optimal
 Temperature
• Microbes cannot regulate their internal
temperature

• Enzymes have optimal temperature at which

they function optimally

• High temperatures may inhibit enzyme

functioning and be lethal
 Temperature Optima
• Psychrophiles
• 0 – 20 °C – optimum 15 °C (unsaturated fatty
acids)

• Psychrotrophs or facultative psychrophiles

• Prefer 20 – 30 °C, grow at wide range of 0 – 35 °C
• spoil refrigerated foods

• Mesophiles
• 20 – 45 °C
• human pathogens
• Thermophiles
• Can grow at >45°C
• 55 – 65 °C optimal temperature
• compost, hot water springs, deep sea volcanoes,
rifts, and ridges

• Hyperthermophiles
• 80 -115 °C (optimal temperatures)

• Not grow well below 55°C

 Adaptations of
thermophiles
• Protein structure stabilized by:
• e.g., more H bonds
• e.g., more proline
• e.g., chaperones
• Histone-like proteins stabilize DNA

• Membrane stabilized by:

• e.g., more saturated, more branched and higher
molecular weight lipids
• e.g., ether linkages (archaeal membranes)
 Oxygen and Bacterial
Growth
• Aerobe
• grows in presence of atmospheric oxygen (O2) which is
20% O2

• Obligate aerobe
• requires O2

• Anaerobe
• grows in the absence of O2

• Obligate anaerobe
• usually killed in presence of O2
• Microaerophiles
• requires 2–10% O2

• Facultative anaerobes
• do not require O2 but grow better in its presence

• Aerotolerant anaerobes
• grow with or without O2
 Oxygen Concentration
Need Prefer Ignore Oxygen is < 2 – 10%
oxygen oxygen oxygen toxic oxygen
 Basis of different oxygen
sensitivities
• Oxygen easily reduced to toxic products
▪ superoxide radical
▪ hydrogen peroxide
▪ hydroxyl radical

• Aerobes produce protective enzymes

▪ superoxide dismutase (SOD)
▪ Catalase
▪ Peroxidase
• Anaerobes
• Unable to grow in presence of oxygen
• Obligate anaerobes – do not tolerate oxygen
• all strict anaerobic microorganisms lack or have
very low quantities of
• superoxide dismutase (SOD)
• catalase

• Grown in special anaerobic flasks or cabinets in

presence of CO2 and N2 gas mixtures

• Oxygen toxic to Bacteroides, Clostridium,

Fusobacterium, Methanococcus
 Pressure
• Microbes that live on land and water surface live at
1 atmosphere (atm)
• Some Bacteria and Archaea live in deep sea with
very high hydrostatic pressures (>600 atm)
• Barotolerant organisms
• adversely affected by increased pressure, but not as
severely as non-tolerant organisms
• Barophilic organisms
• require or grow more rapidly in the presence of
increased pressure (nutrient recycling in deep sea)
• change membrane fatty acids to adapt to high pressures
• Thermobarophile
Radiation
 Radiation damage
• Ionizing radiation (short wave and high energy)
• X-rays and gamma rays

• mutations → death

• disrupts chemical structure of many molecules,

including DNA
• damage repaired by DNA repair
mechanisms
• Deinococcus radiodurans
• extremely resistant to DNA damage
 Radiation damage…
• Ultraviolet (UV) radiation
• mutations → death

• causes formation of thymine dimers in DNA

• DNA damage can be repaired by two mechanisms

• photoreactivation – dimers split in
presence of light
• dark reactivation – dimers excised and
replaced in absence of light
 Radiation damage…
• Visible light
• Bacteriochlorophyll (cytochromes and flavins):
High intensities generates singlet oxygen (1O2)
• powerful oxidizing agent

• carotenoid pigments
• protect many light-exposed microorganisms from
photooxidation
 Microbial Growth in
Natural Environments
• Microbial environments are complex,
constantly changing

• Microorganism exposed to overlapping

gradients of nutrients and environmental
factors

• often contain low nutrient concentrations

(oligotrophic environment)
 Growth Limitation by
Environmental Factors
▪ Leibig’s law of the minimum
• total biomass of organism determined by nutrient
present at lowest concentration

▪ Shelford’s law of tolerance

• above or below certain environmental limits, a
microorganism will not grow, regardless of the nutrient
supply
 Responses to low nutrient
levels
• Oligotrophic environments

• Organisms become more competitive in

nutrient capture and use of available resources

• Morphological changes to increase surface

area and ability to absorb nutrients

• Mechanisms to sequester certain nutrients

(Bacteriocins)
 Counting Viable but
Nonculturable Vegetative
Prokaryotes
▪ Stressed microorganisms - temporarily lose ability
to grow using normal cultivation methods

▪ Microscopic, isotopic and genetic methods for

counting viable but nonculturable cells have been
developed
 Biofilms
• Most microbes grow attached to surfaces (sessile)
rather than free floating (planktonic)

• Ubiquitous in nature

• Complex, slime enclosed colonies attached to

surfaces

• Can be formed on any conditioned surface

• Microbes reversibly attach to conditioned surface

and release polysaccharides, proteins, and DNA
Life in a biofilm
• Additional polymers are produced as biofilm
matures
• Interactions occur among the attached organisms

• A mature biofilm is a complex, dynamic

community of microorganisms

• Heterogeneity is differences in metabolic activity

and locations of microbes

• Interactions occur among the attached

organisms
• exchanges take place metabolically, DNA uptake and
communication
 Biofilm microorganisms
• Extracellular matrix and change in attached
organisms’ physiology protects them from
harmful agents such as UV light and antibiotics

• When formed on medical devices, such as

implants, often lead to illness

• Sloughing off of organisms can result in

contamination of water phase above the biofilm
such as in a drinking water system
 Quorum Sensing and
Microbial Populations
• Quorum sensing
• microbial communication and cooperation

• involves secretion and detection of chemical signals

• Concentration present allows cells to

access population density

• Bacterial cells in biofilms communicate in a

density-dependent manner
 Quorum Sensing
• Acylhomoserine lactone (AHL) - autoinducer
molecule produced by many Gram-negative
organisms

• AHL or other signal molecule diffuses across

plasma membrane

• At high concentrations it enters cell

• Once inside the cell it induces expression of

target genes that regulate a variety of functions
Processes sensitive to quorum
sensing: gram-negative
bacteria
• Bioluminescence (Vibrio fischeri)
• Synthesis and release of virulence factors
(Pseudomonas aeruginosa)
• Conjugation (Agrobacterium tumefaciens)
• Antibiotic production (Erwinia carotovora,
Pseudomonas aureofaciens)
• Biofilm production (P. aeruginosa)
 Quorum sensing: gram-
positive bacteria
• Often mediated by oligopeptide pheromone

• Processes impacted by quorum sensing:

• mating (Enterococcus faecalis)
• transformation competence (Streptococcus
pneumoniae)
• sporulation (Bacillus subtilis)
• production of virulence factors (Staphylococcus
aureus)
• development of aerial mycelia (Streptomyces
griseus)
• antibiotic production (S. griseus)
 Self Study questions
• Describe the following phases of bacterial
growth in a batch culture system
• Lag (4)
• Log/exponential (4)
• Stationery (4)
• Death (4)

• What do you understand about the viable but

non culturable bacteria and programmed
cell death (4)
• A bacterial population increases from 105 cells to
1011 cells in 15 hours. Calculate the mean
growth rate constant (k) and the mean
generation time (g). (8)

• Identify N0
• Identify Nt
• Identify time
• Which equation will you use?
• Provide one word that describe organisms that:
• grow under harsh conditions that would kill most organisms
• require high osmotic pressure for growth
• are adapted to saline environments
• require low pH (0 - 5.5) for growth optimum
• grows at temperatures >45°C
• grows in the absence of O2
• grow more rapidly in high hydrostatic pressure
• requires 2-10% atmospheric O2 (8)

• In your own understanding, how do Biofilms benefit

microbial species inside this community? (4)

• Name four processes that occur or G-ve and G+ve when

they receive quorum sensing molecule. (8)
• Provide one word that describe organisms that:
• grow under harsh conditions that would kill most
organisms
• require high osmotic pressure for growth
• are adapted to saline environments
• require low pH (0 - 5.5) for growth optimum
• grows at temperatures >45°C
• grows in the absence of O2
• grow more rapidly in high hydrostatic pressure
• requires 2-10% atmospheric O2 (8)

• In your own understanding, how do Biofilms benefit

microbial species inside this community? (4)
• Name four processes that occur or G-ve and G+ve when
they receive quorum sensing molecule. (8)