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Microbial Growth

Why study growth?

Important to understanding biology of an organism growth is essential to any organism's
existence
Information on growth is required for control microoganisms

Definitions of Growth
Steady increase in all the chemical components of an organism that may result in an increase
cell size, cell number or both

Increase in biomass as measured by changes in

Dry weight increase
Increase in absorbance
Increase in cellular constituents
Protein
Nucleic acids
other constituents e.g., peptidoglycan and chitin

Growth results in increased cell size and frequently cell division

Particularly relevant to unicellular organisms:

o In unicellular organisms cell growth results in increase in numbers
o In multicellular organisms cell growth results in an increase in organism size

I. Factors that Affect Growth

A. Chemical factors
Nutrients are substances used in biosynthesis and energy release and are therefore required
for growth
One must define nutritional requirements in order to cultivate the microbe in the laboratory
Chemical factors are supplied by i) the culture medium (pl. - media) that contains substrates
required for growth and ii) culture conditions (i.e., aerobic vs anaerobic conditions).

1. Macroelements (major elements - C, O, H, N, S, P, K, Ca, Mg, and Fe)

required in large amounts by the cell >95% of cells are composed of macroelements
(sometimes call macronutrients)
C, O, H, N, S, P are components of macromolecules

Carbon
o Life on earth is carbon based
o Half of the dry weight of a typical cell is carbon

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Nitrogen
o Nitrogen makes up approximately 14% of the dry weight of a typical cell
o Major constituent of protein and nucleic acids, some carbohydrates and lipids
o NH3, NO3-, N2 (nitrogen fixation) and organic N compounds (e.g., amino acids) from the
environment. Some bacteria use atmospheric nitrogen (N2) as a nitrogen source

Phosphorus
o component of phospholipids and nucleic acids, nucleotides such as ATP, some proteins
o available as organic and inorganic forms in the environment

Sulfur
o structural role in methionine and cysteine as well as a number of vitamins (thiamine,
biotin), coenzyme A and some carbohydrates
o available usually from inorganic sources SO42- or H2S and organic sulfur compounds such
as cysteine

K, Ca, Mg, and Fe are cations in cells and required for a variety of roles
e.g., - cofactors (K+, Ca2+, Mg2+, and Fe2+ or Fe3+)
- stabilize membranes and ribosomes (Mg2+)
- contribute to heat resistance of endospores (Ca2+)
- components of biomolecules such as cytochromes (Fe2+ and Fe3+)

2. Trace elements or Micronutrients

required in lesser or trace amounts.
Critical to cell function
Many are metals structural role with many enzymes - cofactors
often trace elements present in medium components or water provide all that is required for
growth
Co, Cu, Mn, Mo, Ni, and Zn are needed by most cells.
Some cells require Cr, Se, W, and V

3. Oxygen

a) Aerobic organisms
growth at full atmospheric O2 tensions (21% O2 in the atmosphere)
facultative organisms (under appropriate nutrient and culture conditions) can grow under
either aerobic or anaerobic condition
obligate aerobes - require O2 for growth
O2 is poorly soluble - forced aeration is often used in culture systems to provide O2

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b) Anaerobic organisms
obligate (strict) anaerobes - grow only in the absence of O2; sensitive to O2 and brief exposure
will kill these organisms; perhaps because these organisms are unable to detoxify some of the
products of O2 metabolism
lack a respiratory system and cant use oxygen as a terminal electron acceptor
These organisms do use oxygen found in cellular materials

Obligate anaerobiosis - prokaryotes, and a few groups of fungi and protozoa

Toxic forms of oxygen

Oxygen itself is not toxic to anaerobic organisms rather it is certain derivatives that are toxic
reduction of O2 in respiration produces several toxic products
singlet oxygen (1O2-) produced photochemically and biochemically (peroxidase activity). Outer
shell electrons become highly reactive; carry out spontaneous and undesirable oxidations in the cell
hydrogen peroxide (H2O2) Produced during aerobic respiration; damage cell components but not
as toxic as O2.-, or OH
superoxide (O2.-) Formed in small amounts during aerobic respiration; highly reactive and can
oxidize any organic compound in the cell
hydroxyl radical (OH) - most reactive, instantly oxidize any organic substance in the cell.
All cells contain flavoproteins, quinines, thiols, and iron-sulfur proteins that can react with O2 and
produce superoxide
Ionizing radiation is the major source of hydroxyl radicals. Small amounts of hydroxyl radicals
can be produced from H2O2.
A number of enzymes have evolved to detoxify oxygen species

Catalase
o destroys H2O2
o H2O2 + H2O2 2 H2O + O2
o Catalase test - 30% H2O2 place on cells. Cells with catalase activity produces vigourous
bubbling as O2 is released

Peroxidase
o destroys H2O2 but does not produce O2. May require a reductant such as NADH
o H2O2 + 2H+ 2 H2O

Superoxide dismutase (SOD)

o Destroys superoxide
o Indispensable to aerobic cells
o O2.- + O2.- + 2H+ H2O2 + O2
o Generally works in tandem with catalase: 4O2- + 4H+ 2H2O + 3O2

Superoxide reductase
o Found in some obligately anaerobic prokaryotes
o O2.- + 2H+ + cytochrome creduced H2O2 + cytochrome coxidized

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o Avoids production of O2 as found with SOD

o H2O2 may then be removed by peroxidase activity
Aerobes and facultative anaerobes usually produce superoxide dismutase and catalase

c) Aerotolerant anaerobes
tolerate O2 and grow in its presence even though they cant use oxygen.
Aerotolerant organisms can tolerate oxygen because they produce SOD or equivalent system that
neutralizes toxic oxygen species. Usually lack catalase activity

d) Microaerophiles
grow only at reduced O2 concentrations (2 to 10%)
These organisms have limited capacity to respire or have some oxygen-labile molecules;
sensitivity to oxygen may also be due to the sensitivity superoxide radicals and peroxides

O2 usually excluded from culture systems by one or a combination of the following mechanisms
Fill container to the top and seal
Boil medium to drive out O2
Use reducing agents that react with O2; reduces it to H2O (e.g., thioglycolate, cysteine, H2S)
Seal containers under O2 free gas
Use redox indicators such as resazurin to indicate the presence of O2.
Use O2 consuming devices (catalyst)
Work under a stream of O2 free gas or in an anoxic glove box/anaerobic chamber

4. Other required elements

Somemicrobesmayhaveparticularrequirementsthatreflecttheirspecificenvironment
(HalophilesrequireNa+)andmorphology(DiatomsandSilicondioxidebasedcellwalls)

5. Growth Factors
Some microbes have the enzymes and biochemical pathways needed to synthesize all cellular
components using minerals and sources of energy, carbon, nitrogen, phosphorus and sulfur.
Other microbes lack one or more enzymes necessary to synthesize essential constituents they get
these constituents or precursors from the environment
Growth factors are organic compounds that are essential cellular components or precursors of these
components but cannot be synthesized by the organism
Major Classes of Growth factors
1. amino acids
2. purine and pyrimidines
3. vitamins (e.g., thiamine, biotin, cobalamin, pyridoxine)

Other growth factors include heme (nonprotein component of many cytochromes) or

cholesterol
Understanding growth factor requirements has practical implications

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o Bioassays using microbes to detect the specific growth factor that they need. Growth-
response assay uses this approach to detect the amount of a growth factor in solution.
These assays can be specific, sensitive, simple and quantitative
o Manufacture of growth factors by specific microorganisms (e.g., Vitamin D by
Saccharomyces) in industrial fermentations
B. Physical (or environmental) Factors

1. The Effect of Temperature on Growth

Cardinal temperatures (Fig 6.1)

Depend on environmental factors such as pH and available nutrients

a) Minimum temperature - below which cells are inactive

reduced membrane fluidity perhaps affects nutrient transport or proton gradient formation

b) Optimum temperature
highest rate of growth and reproduction, always nearer maximum temperature

c) Maximum temperature - above which growth is not possible

Growth stops because of inactivation of one or more key proteins, damages transport carriers
or other proteins, or thermal disruption of membrane

Cardinal temperatures vary for different organisms

Medium composition can have a slight affect
Temperature optima usually vary from 0C to 75C

Pyrolobus fumarii (archaeon) - maximum temperature = 113C

Growth temperature range for a particular organisms usually spans 30 to 40C

Distinguish five groups of microbes based on temperature optima

i) Psychrophiles
Grow well at 0C and have an optimum temperature 15C and a maximum temperature
around 20C
heat sensitive and unable to survive temperate climates

Adaptations to Psychrophily
o Enzymes, transport systems and protein synthetic apparatus work well at low
temperatures

enzymes with low temperature optima

o greater amounts of -helix and lesser amounts of sheet secondary structure

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o greater amounts of polar amino acids and lesser amounts of hydrophobic amino acids

membranes contain higher amounts of unsaturated fatty acids

o some psychrophiles have membranes higher in polyunsaturated fatty acids

ii) Psychrotolerant (psychrotrophs, facultative psychrophiles)

grow at 0C but have optima of 20 - 30C

iii) Mesophiles
Optimum temperature between 25 and 40C
Minimum temperature between 15 and 20C
Maximum temperature 45C
Most common type of microbe

e.g., E. coli
Optimum temperature < 39C
Maximum temperature < 48C
Minimum temperature 8C

iv) Thermophiles
Optimum temperature between 50 and 60C
Minimum temperature around 45C
Maximum temperature 45C

Only prokaryotes grow above 60C

The most thermophilic organisms are Archaea
Nonphototrophic organisms are able to grow at higher temperatures than phototrophic forms

v) Hyperthermophiles
Optimum temperature > 80C
Extreme thermophiles are usually Archaea
The highest growth temperatures for an archaeon is 113C (Pyrolobus fumarii)

Adaptations to Thermophily

i) Enzymes and other proteins are heat stable

Subtle amino acid substitutions
Increased number of salt bridges
Densely packed hydrophobic interiors

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The presence of certain solutes such as di-inositol phosphate and diglycerol phosphate

ii) Macromolecules function optimally at high temperatures

iii) Membrane is heat stable

Membrane lipids are more branched, rich in saturated fatty acids and of higher molecular
weight
In some cases they have lipid monolayers (diglycerol tetraethers)

iv) DNA is stabilized by special histone like proteins

Review cell membrane structure Chapter 4

Why dont eukaryotes grow above 60C?

Applications of Thermophily
High temperature enzymes
e.g., feed pelleting process
PCR Taq DNA polymerase from Thermus aquaticus

2. The Effect of pH on Growth

All organisms have a characteristic pH range within which growth is possible. The range is
usually 2 3 pH units.
In nature, environmental pH ranges from 5 to 9
Few organisms can growth at pH < 2 and > 10
pH is a great influence on growth rate
pH is important because of its effect on proteins (charge is important to protein
conformation) as well as the plasma membrane

a) neutrophiles - pH optimum between 5.5 and 8

Most bacteria grow well within the pH range of 6 - 9

b) alkaliphiles - prefer growth under alkaline conditions (pH 8.0 to 11.5)

many produce enzymes that work well at high pH useful for the detergent industry

c) acidophiles - restricted to growth at low pH values between 0 and 5.5

Fungi are generally more acid tolerant than bacteria many grow at pH 4 to 6
Some Bacteria and Archaea are obligate acidophiles
e.g., Bacteria - Thiobacillus
Archaea - Sulfolobus
pH has an important effect on stability of acidophile plasma membrane

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Intracellular pH
o Intracellular pH is usually between pH 6 to 8 but internal pH as low as 4.6 and as high as
9.5 have been measured
o Maintained by pumping H+ across the membrane, internal buffering and synthesizing new
proteins (e.g., acid shock proteins and heat shock proteins) that function by pumping
protons or acting as chaperones

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3. Osmotic Effects on Growth

Microbes require water to grow their cells are 80 90% water
Water availability depends not only on amount of water present in any environment but also
the concentration of solutes present (e.g., salts, sugars,).
Water activity (aw) - amount of water that is free to react = availability of water in a substance
aw = a ratio of the vapour pressure of the air in equilibrium with a substance or solution to the
vapour pressure of pure water (1/100 the relative humidity of a solution)
aw ranges between 0 and 1
Most bacteria require an aw of 0.9 for active metabolism
Most organisms are adversely affected by very low water activity (They suffer from
plasmolysis)
In nature osmotic effects are of interest mainly in habitats with high salt concentration

a) Halophilic bacteria
A organism requiring salt (NaCl) for growth
microbes found in the sea (which is 3% NaCl) usually have a growth requirement for salt

Mild halophile salt requirements between 1 and 6%

Moderate halophile - salt requirements between 7 and 15%
Extreme halophiles - salt requirements between 15 and 30% (e.g., Archaebacteria such as
Halobacterium species)

Halotolerant organisms can withstand some reduction in aw but generally grow best without added
solute
Osmotolerant grow over a wide range of water activity
Osmophiles - require high solute (e.g., sugar) concentration for growth
Xerophiles able to grow in very dry environments (i.e., made dry by lack of water)

How does an organism grow under low aw?

Increases internal solute concentration
Pumps inorganic ions (e.g., K+) into the cell
Synthesize or concentrate an organic solute (e.g., proline, glycine betaine, sucrose,
trehalose, mannitol)
These substances must not inhibit biological processes; they are usually highly water
soluble

How does an organism grow under high aw?

II. Microbial Growth in Natural Environments

Most natural ecosystems are complex and constantly changing

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Low concentrations of usable nutrients (Oligotrophic)

Competition
Growth in an environment depends on the nutrient supply and the microbes tolerance for the
environment.

Liebigs law - the total biomass of an organism will be determined by the nutrient present in the
lowest concentration relative to the organisms requirements

Shelfords law there are limits to environmental factors below and above which a
microorganism cannot survive and grow regardless of the nutrient supply

Most bacteria are likely to experience starvation. How do they deal with nutrient limitation?

Reduction in cell size

Change in morphology increase surface area and ability to absorb nutrients
Shutdown of metabolism except for housekeeping maintenance genes

Biofilms
Most microbes are typically found in biofilms in nature
Biofilms consist of cells embedded in EPS (Chapter 4)
Microbes in biofilms share nutrients, communicate (e.g., quorum sensing), exchange genetic
information and are sheltered from adverse environmental factors (i.e., desiccation,
antibiotics, host immune response)
Microbes in biofilms can be 1000X more resistant to antimicobial compounds
Microbes in biofilms can carry out complex chemical processes (i.e., breakdown of plant cell
walls such as occurs in the rumen)

III Culture Media

A culture medium (pl = media) is a nutrient solution used to grow microorganisms in the
laboratory. The growth medium is the most important factor when culturing microbes
There are vast differences in the biosynthetic capacities of microorganisms and thus a need
for a variety of culture media. Knowledge of the microoganisms normal habitat is useful in
selecting an appropriate medium
Specialized media are used for a variety of purposes, including isolation and identification of
microorganisms, testing antibiotic sensitivities, water and food analysis, industrial
microbiology
Factors like temperature, pH, Oxygen and pressure must also be considered when culturing
micoroganisms

Inoculum (pl. = inocula) = microbes introduced into a culture medium to initiate growth. These
cells multiply and are referred to as the culture.

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Fastidious microorganisms - have very rigorous or complex requirements (e.g., for vitamins,
amino acids...)

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A. Chemical and Physical Types of Culture Media

1. Chemically defined (synthetic) media

the exact chemical composition of the medium is known
measured amounts of highly purified inorganic and organic chemicals are added to distilled
water

BM+G (chemically defined medium)

Ingredient g/L in dH2O
Glucose 2.0
(NH4)2SO4 2.0
K2HPO4 0.5
Monosodium glutamate 5.0
MgSO4.7H2O 0.3
MnSO4.H2O 0.05
CaCl2 0.08
ZnSO4.7H2O 0.005
CuSO4.5H2O 0.005
FeSO4.7H2O 0.0005

2. Complex media
certain components are of unknown composition and these components may change from
batch to batch.
Use of this type of medium results in the loss of control of nutrient composition

Luria Burtani (LB; Chemically undefined or Complex medium)

Ingredient g/L in dH2O
Yeast Extract 5.0
Tryptone 10.0
NaCl 5.0

Tryptic Soy Broth (TSB Chemically undefined or Complex medium)

Ingredient g/L in dH2O
Tryptone 17.0
Peptone 3.0
Glucose 2.5
NaCl 5.0
Dipotassium phosphate 2.5

Refer to appendix 8 of lab manual for other examples of complex media

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3. Liquid or solidified media

Both liquid and solidified media are routinely used in microbiology
Solidified media is particularly important for the establishment of pure cultures as well as
determination of cell number. It is often desirable to have cells produce colonies (visible,
isolated masses of cells) - Colonies come in different shapes, sizes, textures and colors, and
colonial morphology may be useful in identifying a microorganism
Agar is the most commonly used solidifying agent. It is extracted from red algae and is a
sulfated heteropolymer of D-galactose, 3,6-anhydro-L-galactose and D-glucuronic acid. Agar
is added to a final concentration between 1 and 2% with 1.5% w/v being the most commonly
used concentration.
Agar is particularly well suited for this application because it melts at a relatively high
temperature (90C) but does not solidify until it reaches 45C. Moreover, very few
microorganisms can hydrolyze agar.
Agar is melted during sterilization and the molten medium is poured into Petri dishes and
allowed to solidify

B. Functional Types of Culture Media

Complex media such as tryptic soy broth are called general purpose media or supportive
media because they sustain the growth of many microorganisms
For some particularly fastidious organisms additional components such as whole blood or
serum must be added. These media are referred to as enriched media and designed to better
mimic natural conditions (i.e., host for pathogens)

Selective medium
A medium with a composition favoring growth of certain types of microorganisms while
inhibiting growth of any other microorganisms that may be present.

Examples

Differential medium
A medium that contains substance(s) that permits for the differentiation of particular
metabolic activities during growth. Useful in distinguishing particular groups of
microbes and may provide information useful in identification

Examples

Selective and differential characteristics may be combined in a single medium

Examples

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C. Enrichment technique
Developed by Beijerinck
The use of culture media or conditions that favour growth of one type or group of
physiologically related microorganisms over all other microorganisms present in the sample

D. Notes on culturing microbes

not all microbes can be cultured in the laboratory
General usage media generally permit the growth of a wide variety of microbes.
At times it is desirable to use environmental or nutritional factors to selectively cultivate a
certain group or kind of microorganism.

Aseptic Technique
Series of steps used to minimize contamination during the manipulations of cultures and
sterile culture media
Sterilize all media and implements for handling materials of interest
Clean working area
Limit exposure to potential sources of contamination

Preparation of Pure Cultures

Streak plate technique

Dilution Deposition of individual cells or clumps of cells (known as colony forming
units or CFU) on agar medium
Cell growth multiplication resulting in the production of colonies (visible mass
of cells)
each isolated colony on the streak plate is assumed to have originated from a
single CFU (It is unknown whether the cells in the colony came from a single cell or a
clump of cells)

Preserving Bacterial Cultures

1. Refrigeration at 4C
short term solution - several weeks to several months
duration depends on type of medium

2. Glycerol stocks
Sterile glycerol is added to liquid cultures to a final concentration of 15 25%
The stocks are placed in small plastic tubes with tight fitting lids (i.e., preferably screw cap
tubes with gaskets in the lids)
The glycerol stocks are stored at -20C (1 to 2 years) or -80C (up to 10 years or more)

3. Lyophilization

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Freeze drying
Culture is quick-frozen at temperatures ranging from -50 to -90C and then dried under
vacuum on a lyophilizer; freeze dried cultures are stored in sealed glass ampules for extended
periods of time.
Microbial Culture Collections
Sources of microbial cultures
Cultures are distributed for a fee or free depending on the culture collection

ATCC American Type Culture Collection

DSMZ Deutsche Sammlung von Mikrooganismen und Zellkulturen

NCTC National Collections of Type Cultures and Pathogenic Fungi

NCIMB National Collections of Industrial and Marine Bacteria
EGSC E. coli Genetic Stock Centre
BGSC Bacillus Genetic Stock Centre
FGSC Fungal Genetic Stock Centre

IV. Growth of Microbial Cultures

i) Eukaryotic Cell Cycle review Biol 1010 notes

ii) Prokaryotic cell cycle

most often is accomplished by Binary Fission but budding, fragmentation and other
processes may occur

Mother cell two daughter cells

Generation time (g)

Binary fission in E. coli takes 20 minutes under optimal conditions

Required as many as 2000 chemical reactions
Length of time depends on a number of factors, including nutrition, genetics and
environment

Rapidly Growing Cells

In E. coli, the cell cycle takes 60 min to complete: 40 minutes for DNA replication and
partitioning and 20 min for septum formation and Cytokinesis
But E. coli can complete this entire process in 20 min under optimal conditions
This is possible because E. coli starts a second round of DNA replication (and sometimes a
third and a fourth round) before the first round of replication is completed.

A. Population Growth

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Growth rate
change in cell number or cell mass per unit time

Generation
interval for the formation of two cells from one cell

Generation time (doubling time)

time it takes for one cell to become two cells
time it takes for the population to double
depends on growth medium and conditions

1. The Mathematics of Growth (Growth Equations)

Growth by binary fission results in exponential growth of the population (Figure 6.13 & 6.14)

Geometric progression of the number 2

21222324

(1) Nt = N02n

Nt = final number of cells at time t

N0 = initial number of cells
n = number of generations that have occurred during period of exponential growth

Solving for n (where all logarithms are to the base 10)

log Nt = log N0 + n log 2 and

(2) n = log Nt - log N0 = log Nt - log N0

log 2 0.301

Growth rate can also be expressed as the mean growth rate constant (k). The specific growth rate
is a measure of the number of generations that occur per unit time

(3) k = n/t = log Nt - log N0

0.301t

Can now calculate the mean generation time (g) or mean doubling time.
When the population doubles t = g and Nt = 2N0; substitute 2N0 into (3)

(4) k = log (2N0) - log N0 = log 2 + log N0 log N0 = 1/g

0.301g 0.301g

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Therefore

(5) g = 1/k

Generation time can also be calculated from the slope of a line obtained in a semi-log plot of
exponential growth

(6) slope = 0.301/g ; g = 0.301/slope

How can we use growth rate information?

2. Culture Systems
"Fermentation" - cultivation of microorganisms in a controlled, enclosed system

i. Batch Culture
A fixed volume of liquid medium is inoculated and incubated for an appropriate period of time
with no further addition of microorganisms or growth substrates

closed environment
most common method of microbial cultivation
nutrient concentration is a determinant of growth rate and cell yield

The batch culture has a continually changing environment

o nutrients are depleted
o products produced
o cells change

Ultimately the culture quits growing due to nutrient limitation or product accumulation

e.g., test tube to flask to 100,000 L fermenter

ii. Fed Batch

A nutrient stock (limiting nutrient) is added at intervals or continuously to a batch culture

iii. Continuous Culture

Spent culture is replaced by fresh medium allowing continual growth of the culture.

Open system
system can be manipulated to reach an equilibrium or steady state where the cell density and
nutrient status remain constant

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Can control culture growth rate as well as yield of cells by manipulating dilution rate and the
level of the limiting nutrient, respectively
More sophisticated apparatus required
Superior productivity possible because of reduced downtime.

e.g., Chemostat
uses dilution rate and nutrient concentration to control growth and population density
growth rate (adjust dilution rate) and yield (adjust limiting nutrient) can be controlled
independently of each other

Compared to batch culture the chemostat allows:

experimenter to vary growth rate and population density independently of each other
can maintain population in exponential phase at a known growth rate for long periods of time
Can study microbial growth at very low nutrient concentrations close to those present in
nature

3. Bacterial Growth Curve

Growth of a batch culture population of cells can be monitored and plotted as a growth curve
A typical batch culture growth curve can be divided into 4 phases (Fig 6.15)

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i) Lag Phase
Initial phase during which time cells are adjusting their metabolism to prepare for a new cycle of
growth.
There is no increase in cell number - increase in cell size
The cells are transporting nutrients, synthesizing RNA and subsequently enzymes needed for
growth; replicating DNA
The length of this phase depends on the history of the culture and growth conditions
Examples:

ii) Exponential Phase (Log phase)

Cell are growing and dividing at the maximum growth rate possible given their genetic
potential, the nature of the medium and incubation conditions.
One cell gives rise to two and so on: Cell number is increasing as an exponential function of
time Log transformation of data results in a linear curve
During this phase the resulting cell population is most uniform with respect to chemical and
physiological properties; cells in this phase are most often used in biochemical and
physiological studies
Exponential growth is said to be balanced growth because all cellular components are made
at constant rates relative to each other. If the nutrient levels or some other environmental
parameter changes then unbalance growth results: growth during which the rates of synthesis
of the various cellular constituents vary relative to one another until a new balanced state is
reached.
Shift-up (culture is transferred from a nutritionally poor medium to a richer medium)
and shift-down (culture is moved to from a nutritionally rich medium to a poor
medium) experiments produce unbalanced growth.
In the shift-up experiment there is a lag in while the cells first produce more
ribosomes to enhance protein synthesis. There is then an increase in protein and
DNA synthesis followed by the rise in productivity.
In the shift down experiment:

Determinants of growth rate

Different nutrients and nutrient concentration allow for different growth rates. Growth rate
increases with increasing nutrient concentration. At some point nutrient transport systems
are saturated and growth rate can increase no further
Temperature, pH, Oxygen and other physical parameters
Genetic determinants
Small cells generally grow faster than larger cells (surface area to volume ratio)
Nutrient concentration affects maximum cell yield

iii) Stationary Phase

Closed system - cells cant grow indefinitely
No further net increase in cell number

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Total number of viable cells remains unchanged because i) growth rate = death rate (i.e.,
some cells in the population grow while others die. This is known as cryptic growth) or ii)
the population may not be dividing but remain metabolically active
Stationary phase is entered because 1) nutrient limitation, 2) oxygen limitation, 3) build up of
toxic wastes (e.g., organic acids), 4) a critical population level is reached, or 5) several of
these factors acting together

Cellular composition and activity changes

Prokaryotes have evolved a number of strategies to deal with starvation. A few genera will
produce endospores but most will reduce cell size, which is often accompanied by protoplast
shrinkages and nucleoid condensation. Morphological changes can also occur

e.g., Arthrobacter - log cells - rods

- stationary cells - coccoid

The most important changes are in gene expression and physiology.

o Different genes are turned on (e.g., catalase, exonuclease and acid phosphatases; survival
genes (sur) have been identified for E. coli)
o Most starving cells produced starvation proteins that make the cell more resistant to
environmental stresses (e.g., elevated temperature, osmotic pressure and toxic chemicals
such as hydrogen peroxide and chlorine) and harder to kill. The cells increase
peptidoglycan crosslinking and cell wall strength, produce proteins to protect their DNA
(DNA binding protein from starved cells Dps) and to prevent protein denaturation and
renature damaged proteins (Chaperone proteins).

vi) Death Phase (Senescence phase)

Exponential decline in viable cell numbers. Typically the rate of exponential decline is much
slower than that of exponential growth
In many instances this phase can be reversed if modify the environmental parameters
In many cases the decline is cell number is associated with a loss of intact cells. In other
cases this is not the case
A decline in viable cell numbers may be explained by simple cell death associated with
starvation or build up of toxins. But two other hypotheses have been proposed

i) Not all cells are culturable = Viable but nonculturable (VBNC) cells.
Cells are viable as demonstrated by the presence of metabolic activities but can't be
cultivated in the lab - detected by discrepancies between indirect and direct counts. VBNC
cells are genetically programmed to become dormant (genetic response triggered in starving
stationary phase cells) and when appropriate conditions become available (e.g., change in
temperature, passage through animals), the cells begin growing again.

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ii) Programmed cell death. A fraction of the microbial population is genetically programmed
to commit suicide nonculturable cells are dead and the nutrients that they leak enable
eventual growth of those cells in the population that did not commit suicide.

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4. Measurement of Growth
Enumeration of microbial populations or measuring mass

i) Measurement of Cell Numbers

a) Direct Counting (counts all cells - viable and dead)

Direct microscopic counts with counting chambers (Fig 6.20)

Use a chamber (e.g., Petroff-Hausser counting chamber) of defined volumes. Count cells the
aid of a microscope
can also use samples dried onto slides

Advantages
rapid
counts all cells in a sample (can often count individual cells in clumps)
can acquire cell morphology information with these methods

Disadvantages
can't determine which cells are viable unless they are treated in a special manner
(e.g.,fluorescent live/dead cell stains).
small cells are difficult to see
affected by debris in samples
not suitable for cell suspensions of low density (< 106/mL); precision difficult to achieve
motile cells are difficult to count
phase contrast microscopy required if sample not stained
may require expensive pieces of equipment
unable to perform further studies on the observed microbes without further cultivation

Filtration
known volume of a suspension filtered onto a black polycarbonate filter membrane.
cells are stained with fluorescent dyes and counted under the microscope

Coulter Counter
automated method of counting cell.
as cell pass through a aperture they disturb an electric field
perturbations are transformed into number and size data.
Most useful for larger cells

Fluorescence Activated Cell Sorter (FACS)

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b) Viable Counts (counts viable cells that can be cultured)

Viable Plate Count

counts viable cultivable bacteria
Viable count methods assume that each viable cell can grow and divide to yield one colony
Serial dilutions of cultures are prepared and these suspensions of bacteria are plated onto agar
medium
use spread plate or pour plate technique
Following incubation - count number of colonies in order to determine the number of colony
forming units (CFUs) per unit volume.
limit counting to plates with between 30 and 300 colonies
plates containing less than 30 colonies are not acceptable for statistical reasons
plates containing greater than 300 (TNTC) - plates are crowded and it becomes hard to
distinguish and count colonies.
Problems with culturability of particular microbes on the medium - may be selective!!!!

Spread Plate (Fig 6.17)

suspension of microbes is spread over the surface of agar medium.
spreading separates cells that grow and give rise to isolated colonies
assumes each colony arises from a single cell or clump of cells (CFU).
suspension of cells must be dilute enough otherwise the plate will be overgrown - too
many cell get confluent growth or a lawn of cells with no discrete colonies.
Usually spreading 0.1 mL of less on the plate

Pour Plate (Fig 6.17)

suspensions of cells (0.1 to 1.0 mL) are added to molten agar (42 to 45C)
Note - agar begins solidifies at approx. 42C.
molten agar is poured into a petri dish, allowed to solidify and incubated; the hot agar
may kill or injure sensitive cells

Advantages of viable plate counts

Counts only viable cells widely used in food, dairy, medical industries and research
Very sensitive detect presence of very few cells
Use of selective and/or differential media can restrict counts to a particular cell type
the techniques require inexpensive materials
once counts are completed you have viable cultures to use in subsequent experiments

Disadvantages of plate counts

these methods are selective and count only viable cells or cells that can be grown with the
culture techniques used (i.e., they underestimate actual cell number)
they do not distinguish between an individual cell and a cluster of cells and therefore
underestimate cell numbers

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takes time for data acquisition (i.e., Cells must grow for >12 h to be counted with the
viable count methods
size of colonies vary and it is easy to miss small colonies
subject to large errors if not done carefully require adequate replication
Most Probable Number (MPN)
another technique for counting viable CFU
dilute to extinction - such that not all aliquots transferred to tubes of growth medium will
contain a cell
following incubation one checks for growth and compares results to a table of statistical
probability for obtaining the observed results.

Membrane filtration
Aquatic samples are filtered through a membrane trapping cells on the membrane
The membrane is placed on an agar medium and incubated until each cell forms a colony
Useful for analyzing water samples especially when the populations are low

c. Indirect estimation of Bacterial Numbers

Microbial Dry Weight

Cells growing in liquid medium are collected by centrifugation or filtration, washed, dried in
a vacuum oven and weighed
Time consuming, not very sensitive but good for filamentous fungi

Turbidity (Spectophotometry)
rapid and sensitive method for obtaining estimate of culture density
The more cells that are present the more light that is scattered by a suspension
can measure transmittance of light and determine the optical density (OD) of a suspension
using a spectrophotometer
growth results in increased turbidity and OD proportional to cell number for unicellular
organisms

Can generate a standard curve to relate OD to CFU's/unit volume or some other measure of
growth (e.g., dry weight)

Metabolic Activity
Measures a metabolic product and assumes there is a direct relationship between the amount
of the metabolic product and the cell number.
Measurement of CO2 evolution

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