Microbial Nutrition, Growth, and

Growth Control

Nutrients
•Nutrients - chemicals required for synthesis of
chemicals present in living cells.
•Macronutrients - elements required in fairly
large amounts (KP COHNS CaFe, Na, Mg)
•Micronutrients (trace elements) - metals
needed in very small amounts
•Growth factors (vitamins) - organic compounds
required in small amounts

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 Iron
• Iron - major role in cellular respiration as
key component of cytochromes and iron-
sulfur proteins involved in electron
transport.
•Iron insoluble as Fe+3 in oxic conditions
(soluble as Fe+2 in anoxic conditions).
• Cells use siderophores that bind iron
and transport it into the cell.

Common Siderophores
•Siderophores secreted by the cell,
interact with Fe+3, transport Fe+3 back into
cell, reduce it to Fe+2 and release it as
soluble Fe+2
•Hydroxamate – widely distributed
•Enterobactin - E. coli and Salmonella
typhimurium
•Aquachelin – marine environment

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 Culture Media
• Culture media supply the nutritional needs of
microorganisms.

• Defined media - chemically defined.

• Complex media - chemically undefined.

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 Classes of Media

•Selective – inhibitory compounds inhibiting

growth of some organisms

•Differential – indicator compounds to detect

metabolic products

•Enriched – complex medium with additional

undefined nutrients e.g. blood

Solid Media
• Solid culture media ‘immobilizes’ cells
formation of visible, isolated masses –
colonies

•Typically 1.5% agar as gelling agent

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Cell Growth and Binary Fission

Generation - the formation of

two cells by binary fission.

Growth - increase in numbers of

cells (generations).

The general process of binary fission in a rod-shaped prokaryote.

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 Fts Proteins and Cell
Division
• fts = filamentous temperature sensitive.
• Fts proteins universal in prokaryotes and also
found in mitochondria and chloroplasts.
• FtsZ has structural similarities to tubulin.
• Several distinct Fts proteins form the divisome
(the division apparatus of prokaryotes).

Fts Proteins and Cell

Division
• Divisome in rod shaped cells is formed by attachment
of FtsZ molecules to form a ring around mid-point.
• Other cell division proteins then assembled with FtsZ.
• ZipA as a membrane anchor for FtsZ ring.
• FtsA as an ATPase for divisome assembly.
• FtsI complexes with FtsA and is involved in
peptidoglycan synthesis (binds penicillin which blocks
its functioning).
• FtsK assists in separation of replicated
chromosomes.

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 Events in Binary Fission

•FtsZ ring formation begins at end of DNA

replication and is between the nucleoids
•Min proteins (minicells) locate midpoint of rod
•FtsZ ring depolymerizes causing constrictions
•Energy for assembly and disassembly of ring
provided by FtsZ GTPase activity

Division Events
• DNA replication prior to FtsZ ring.
• FtsZ finds midpoint via Min proteins - MinC
inhibits ring formation until cell center is located.
• MinE attaches to center and inhibits MinC.
• FtsZ molecules recruited and ring forms.

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 Cell shape
•MreB proteins are actin-like.
•Form spiral cytoskeletal network length of rods
•MreB mutants are spheres
•Cocci have no mreB gene
•Other bacterial morphologies likely due to
variations in MreB arrangements

Exponential Growth

•Generation time is doubling time

•Growth readily plotted on semilogarithmic

graph with cell numbers on the log scale and
time on the arithmetic scale

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 The rate of growth of a microbial culture
Data plotted on an arithmetic (left
ordinate) and a logarithmic (right
(a) Data for a population that doubles every 30 min. ordinate) scale.

Method of estimating the generation times (g) of exponentially growing

populations with generation times in different hours from data plotted on
semilogarithmic graphs
(b) 2 h.
(a) 6 h

The slope of each line is equal to 0.301/g and n equals the number of generations that have
occurred in the time, t. All numbers are expressed in scientific notation; that is, 10,000,000 is
1 × 107, 60,000,000 is 6 × 107, and so on.

Growth Cycle
•All phases refer to events affecting the
population - not individuals
•Lag phase
•Exponential phase (log phase)
•Stationary phase
•Death phase

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 Typical growth curve for a bacterial population.

Lag phase
•Period of adjustment for new inoculum
•Cells need to adjust to new environmental
conditions
•Adjustment typically requires re-synthesis of
depleted essential constituents
•Length (time) depends on inoculum history
•Long if from culture at different phase of growth
•Long if from rich medium to minimal medium
•Long if cells damaged (e.g. heat, radiation)

Exponential Phase
•Cells in healthiest condition
•Doubling time dependent upon both physical
(e.g. heat) and chemical (e.g. richness of
medium) conditions
•Small cells expected to generally have shorter
generation times (>SA/V ratios and more
efficient nutrient / waste exchange)

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 Stationary Phase
•No net change in cell numbers
•Not static
•Doubling = dying
•Typically depletion of nutrient(s) or
accumulation of waste(s)

Death Phase
•Cells continue to metabolize
•Death rate >doubling rate
•Death is exponential (log death), but slower
than exponential growth

Typical growth curve for a bacterial population.

A viable count measures the cells in the culture that are capable of reproducing.
Turbidity, or optical density, is a quantitative measure of light-scattering by a
liquid culture.

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 Measuring Growth
•Total Cell Count - direct measurement that
counts all cells

•Viable Count (Plate Counts) - direct

measurement that counts only the living,
reproducing cells in medium used

•Turbidity - Indirect count that measures un-

scattered incident light

Direct microscopic counting procedure using the Petroff-Hausser counting

chamber. A phase-contrast microscope is typically used to count the cells to
avoid the necessity for staining

To calculate number
per milliliter of sample:
12 cells x 25 large squares x 50 x 103
= 1.5 x 107

The viable count. Two methods of performing a viable count (plate count).
In either case the sample must usually be diluted before plating. Note how in the
pour plate method, colonies form within the agar as well as on the agar surface

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 Procedure for viable counting using serial dilutions of the sample and the pour
plate method

Great Plate Count Anomaly

•Plate counts highly unreliable when assessing
natural samples (e.g. soil and water).
•Direct counts of natural samples far higher than
recoverable on plates of any specific medium.
•Microscopic methods count dead cells, viable
methods do not.
•Different organisms may have vastly different
requirements for resources and conditions in
laboratory culture.

Turbidity measurements of microbial growth. Measurements of turbidity are made in a

spectrophotometer or photometer. The photocell measures incident light unscattered by
cells in suspension and gives readings in optical density or photometer units

Typical growth curve data obtained in Klett units or optical density (OD) for two organisms
growing at different growth rates.

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 Continuous Culture: The
Chemostat
•Continuous culture devices maintain cell
populations in exponential growth for long
periods
•Rate at which the culture is diluted governs
growth rate and yield
•Population size governed by concentration of
growth-limiting nutrient entering the vessel

Schematic for a continuous culture device (chemostat).

In a chemostat, the population density is controlled by the concentration of limiting nutrient in

the reservoir, and the growth rate is controlled by the flow rate.

The effect of nutrients on growth in a batch culture (closed system).

Relationship between nutrient concentration, growth rate (green curve), and growth yield (red
curve). At low nutrient concentrations both growth rate and growth yield are affected.

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 Steady-state relationships in the chemostat.

The dilution rate is determined from the flow rate and the volume of the culture vessel. Thus,
with a vessel of 1000 ml and a flow rate through the vessel of 500 ml/h, the dilution rate would be
0.5 h-1. Note that at high dilution rates, growth cannot balance dilution, and the population washes
out. Note also that although the population density remains constant during steady state, the
growth rate (doubling time) can vary over a wide range. Thus, the experimenter can obtain
populations with widely varying growth rates without affecting population density.

ENVIRONMENTAL EFFECTS
ON MICROBIAL GROWTH:
TEMPERATURE

Cardinal Temperatures
•<Minimum - membrane gelling, transport too
slow to maintain growth
•>Maximum - protein denaturation, thermal
collapse of membrane
•Optimum - enzymatic reactions at maximum
efficiency

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 Effect of temperature on the cell

Effect of temperature on growth rate and the molecular consequences for the
cell. The three cardinal temperatures vary by organism.

Temperature Classes of
Organisms
•Psychrophiles - low optimal temperatures
•Mesophiles - mid-range optimal temperatures
•Thermophiles - high optimal temperatures
•Hyperthermophiles - very high temperatures
•Optimal temperatures affected by media
•Extremophiles - grow under either very hot or
very cold conditions

Relation of temperature to growth rates of microbes

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• Psychrophiles have biomolecules that
function best at cold temperatures but that may
be unusually sensitive to warm temperatures.
•Organisms that grow at 0ºC but have optima of
20ºC to 40ºC are psychrotolerant.

Mesophiles
•Mesophiles have midrange temperature
optima
•Found in warm-blooded animals and in
terrestrial and aquatic environments in
temperate and tropical latitudes

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 High Temperature Growth

•Thermophiles - growth optima between 45ºC

and 80ºC
•Hyperthermophiles - growth optima > 80°C
•Hot environments up to and including boiling hot
springs
•Hydrothermal vents with temperatures in excess
of 100ºC.

• Thermophiles and hyperthermophiles

produce heat-stable macromolecules
•Taq (Thermus aquaticus) polymerase
used in PCR (polymerase chain reaction)

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 Microbial Growth at Low or
High pH

pH and microbial growth

Although some microorganisms can live at very low or very high pH, the cell's
internal pH remains near neutrality.

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 Osmotic Effects on Microbial
Growth

Water Activity and Osmosis

Osmotic effects on Microbial Growth

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 Halophiles
•Halophiles - optimal growth at reduced
water potential
•Extreme halophiles - require high levels
of salts
•Halotolerant - tolerate some reduction in
water activity but grow best in absence of
the added solute

Effect of NaCl concentration on growth of microorganisms of

different salt tolerances or requirements. The optimum NaCl
concentration for marine microorganisms such as V. fischeri is about
3%; for extreme halophiles, it is between 15 and 30%, depending on the
organism.

Compatible Solutes
• Water activity becomes limiting to an organism
when the dissolved solute concentration in its
environment increases
•Organisms produce or accumulate intracellular
compatible solutes that maintain the cell in
positive water balance
•Compatible solutes raise intracellular solute
concentration to increase rate of osmosis into
cell
•Compatible solutes are “compatible” with the
cell’s metabolism

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Structures of some common and highly soluble compatible solutes in
microorganisms.

• Xerophiles - capable of growth in

extremely dry environments.

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 Oxygen and
Microbial Growth

Oxygen Classes of Microbes

•Aerobes require oxygen to survive
•Anaerobes do not require oxygen and may be
killed by its presence
•Facultative organisms can live with or without
oxygen.
•Aerotolerant anaerobes can tolerate oxygen
and grow in its presence even though they cannot
use it.
•Microaerophiles are aerobes that can use
oxygen, but only when present at reduced levels

Oxygen and Microbial Growth

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 Detection of Oxygen Growth
Classes
Thioglycolate - strong reducing agent
converts O2 to H2O

Resazurin - redox indicator is pink when

O2 is present

Growth versus oxygen concentration.

a. Aerobic
b. Anaerobic
c. Facultative
d. Microaerophiles
e. Aerotolerant anaerobes

(b) Anoxic glove bag for manipulating and incubating

a) Anoxic jar. A chemical reaction in the envelope in the
jar generates H2 + CO2. The H2 reacts with O2 in the jar
on the surface of a palladium catalyst to yield H2O; the
final atmosphere contains N2, H2, and CO2.
cultures under anoxic conditions. The airlock on the
right, which can be evacuated and filled with O2-free
gas, serves as a port for adding and removing
materials to and from the glove bag.

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 Control of Microbial
Growth

General Terms
•Sterilization - killing of all organisms,
including viruses
•Inhibition - effectively limiting growth
•Decontamination - treatment making
surfaces safe to handle
•Disinfection - elimination of targeted
pathogens on surfaces

Temperature Control of
Growth
•Death from heat due to denaturation of
macromolecules
•Death from heating is exponential and occurs
more rapidly as temperatures rise
•Moist heat more effective - penetrates more
rapidly
•Decimal Reduction Time (D) - time required
for a 10-fold reduction in population density at
a given temperature

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 Temperature and the rate of killing as
The effect of temperature on the viability of
a mesophilic bacterium.
indicated by the decimal reduction time for
two different microorganisms.

Data were obtained for decimal reduction times, D, at

The decimal reduction time, D, was obtained for
several different temperatures, as in Figure 20.1. For
the same mesophilic organism at three different
organism (a), a typical mesophile, exposure to 110° C
temperatures. D is the time at which only 10% of
for less than 20 sec resulted in a decimal reduction, while
the original population of organisms remains
for organism (b), a thermophile, 10 min were required to
viable at a given temperature. For 70° C, D = 3
achieve a decimal reduction.
min; for 60° C, D = 12 min; for 50° C, D = 42 min.

Autoclaving
•Steam heat under pressure (15 lbs/in2) at
121oC (sufficient to kill all endospores) for
15 min
•Entire object must be exposed for 15 min
•Large volume or viscous materials
require longer times to reach require
temperature throughout
•Heat, not pressure, kills

Autoclave and sterilization

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 Pasteurization
• Pasteurization reduces microbial load -
does not sterilize
•Kills most pathogens and inhibits growth
of spoilage microbes (increases shelf-life)
•Originally for milk to kill agents of TB, Q-
fever, brucellosis, typhoid fever

Types of Pasteurization
•Flash Pasteurization - rapid heating to 71oC
for 15 sec then rapidly cooled
•Can be done on continuous flow basis
•Less change in flavor
•Bulk Pasteurization - large vats at 63-66o C,
30 min

Radiation Sterilization,
• Controlled doses effectively inhibit microbial
growth
•UV radiation to decontaminate surfaces and
materials that do not absorb light
•Ionizing radiation penetrates solid or light-
absorbing materials (widely used for sterilization
and decontamination in medical and food
industries

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 The D10, or decimal reduction dose.

Human=10 Gy

Filter Sterilization

• Depth filters - remove microorganisms from air

or liquids
•Include HEPA (high efficiency particulate air)
filters and often used as prefilters
•Membrane filters - primarily used for sterilizing
heat-sensitive liquids

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 Filter Sterilization

Left: a filter system designed for small volumes. Right: a filter system designed for
larger volumes.

Chemical Growth Control

•cidal agents - chemicals that kill e.g. bacterio-
cidal, fungicidal, viricidal
•static agents - chemicals that do not kill but
inhibit growth e.g. bacteriostatic, fungistatic,
viristatic
•MIC (minimum inhibitory concentration) -
smallest amount of agent needed to inhibit growth
of a test organism

Three types of action of antimicrobial agents.

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 Determination of the minimum inhibitory concentration (MIC).

Agar diffusion method for assaying antibiotic susceptibility.

Agents for External Use

•Sterilants - chemical sterilizer (kills all microbial

forms)
•Disinfectants - compounds used only on
inanimate objects
•Sanitizers - compounds used to decontaminate
nonliving material

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Antimicrobial Agents Used in
vivo

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 Synthetic Antimicrobial
Drugs
•Salvarsan - Paul Ehrlich in 1900’s
•Synthetic arsenic agent effective against
syphilis
•First “magic bullet”
•Sulfa drugs - Gerhard Domagk in 1930’s
•Growth factor analogs - sulfanilamide is
analog of PABA, a precursor of folic acid

Salvarsan.

This arsenic-containing compound was one of the first useful antimicrobial

chemotherapeutic drugs. Salvarsan was used in the early 1900s to treat syphilis.

Naturally Occurring
Antimicrobial Drugs -
Antibiotics
•From diverse range of bacteria and fungi
•<1% clinically useful
•Many modified as “semi-synthetic antibiotics”
•Broad-spectrum - effective on both Gram (+)
and Gram (-)
•Narrow spectrum - small range

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 Antibacterial chemotherapeutic agents

Mode of action of major antimicrobial chemotherapeutic agents

THF, Tetrahydrofolate; DHF, dihydrofolate; mRNA, messenger RNA; tRNA, transfer RNA.

Antimicrobial spectrum of action

Antimicrobial chemotherapeutic agents each affect a limited

group of microorganisms

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