Microbial

Growth
Lecture 5
Chapter 6

1. What environmental factor may influence the growth of microorganisms?

TODAYS OBJECTIVES

2. Characterize types of bacteria in regards to temperature, oxygen, and pressure requirements;

3. Why is pH important for bacterial growth and how can we control pH in culture media;
4. Understand the relationship between temperature and food preservation;
5. Describe the enzymes observed in microbes that protect them against toxic O2 products;
6. Explain why carbon, nitrogen, phosphorous and sulfur are important for bacterial growth;
7. Describe the formation of biofilms and summarize their importance in natural environments,
industrial settings, and medicine;
8. Define quorum sensing and provide examples of cellular processes regulated by quorum
sensing;
9. Differentiate between chemically defined and complex media;
10.Justify the use of the different media types and techniques;
11.Describe binary fission as observed in bacteria and archaea.
12.Compare the three reproductive strategies used by bacteria other than binary fission.
13.Summarize the two major events in a typical bacterial cell cycle.
14.Define generation time.
15.Describe the four phases of a microbial growth curve observed when microbes are grown in a
batch culture
16.Describe three hypotheses proposed to account for the decline in cell numbers during the
death phase of a growth curve.
17.Correlate changes in nutrient concentrations in natural environments with the four phases of a
microbial growth curve
18.Evaluate direct cell counts, viable counting methods, and cell mass measurements for
determining population size.
19.Explain why plate count results are expressed in terms of colony-forming units (CFUs).

The Influence of Environmental

Factors on Growth
Bacteria are
adapted to a specific
environment;
Physical and
chemical changes in
the environment can
influence growth;
Environmental limits
varies among
species

Temperature
pH
Osmolarity
Oxygen
Pressure

The Requirements for

Growth
Physical requirements
Temperature
pH
Osmotic pressure

Chemical requirements
Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factors

Physical Requirements
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
ranges for
microbial growth
Adaptations of
thermophiles
Protein structure stabilized by a variety of
means
more H bonds
more proline
chaperones
Thermus aquaticus
Histone-like proteins stabilize DNA
Membrane stabilized by variety of means

more saturated, more branched and higher

molecular weight lipids
ether linkages (archaeal membranes)

 Organisms exhibit distinct cardinal growth

temperatures
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature

Temperature
Psychrotrophs
Grow between 0C and 20 to 30C
Cause food spoilage

Thermophiles
Optimum growth temperature of 50 to
60C
Found in hot springs and organic
compost

Hyperthermophiles
Optimum growth temperature >80C

Microbiology & Food preservatio

pH
Most bacteria grow between pH 6.5
and 7.5
Molds and yeasts grow between pH 5
and 6
Acidophiles grow in acidic
environments

pH
Acidophiles
growth optimum between
pH 0 and pH 5.5

Neutrophiles
growth optimum between
pH 5.5 and pH 7

Alkaliphiles
(alkalophiles)
growth optimum between
pH 8.5 and pH 11.5

Sulfur Caldron,
Yellowstone
National Park
Acidic condition

Lake Magadi,
Kenya
Alkaline condition

pH homeostasis
Most microbes 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 the pH of their habitat

by producing acidic or basic waste products

Solutes and Water Activity

Changes in osmotic concentrations in the

environment may affect microbial cells
hypotonic solution (lower osmotic concentration)
water enters the cell/cell swells may burst

hypertonic (higher osmotic concentration)

water leaves the cell/membrane shrinks from the cell wall
(plasmolysis) may occur

Microbes Adapt to Changes in Osmotic

Concentrations
Reduce osmotic concentration of cytoplasm in
hypotonic solutions
mechanosensitive (MS) channels in plasma
membrane allow solutes to leave

Increase internal solute concentration with

compatible solutes to increase their internal
osmotic concentration in hypertonic solutions
solutes compatible with metabolism and growth.

Osmotic Pressure
Hypertonic environments (higher in
solutes than inside the cell) cause
plasmolysis due to high osmotic
pressure
Extreme or obligate halophiles
require high osmotic pressure (high
salt)
Facultative halophiles tolerate
high osmotic pressure

Extremely Adapted Microbes

Halophiles

grow optimally in the presence

of NaCl or other salts at a
concentration above or about
0.2M.

Extreme halophiles

require salt concentrations of

2M and 6.2M
extremely high concentrations
of potassium
cell wall, proteins, and plasma
membrane require high salt to
maintain stability and activity

Plasmolysis

Chemical Requirements
Carbon
Structural backbone of organic
molecules
Chemoheterotrophs use organic
molecules as energy
Autotrophs use CO2

Chemical Requirements
Nitrogen
Component of proteins, DNA, and ATP
Most bacteria decompose protein
material for the nitrogen source
Some bacteria use NH4+ or NO3 from
organic material
A few bacteria use N2 in nitrogen
fixation

Chemical Requirements
Sulfur
Used in amino acids, thiamine, and biotin
Most bacteria decompose protein for the
sulfur source
Some bacteria use SO42 or H2S

Phosphorus
Used in DNA, RNA, and ATP
Found in membranes
PO43 is a source of phosphorus

Trace Elements
Inorganic elements required in small
amounts
Usually as enzyme cofactors
Include iron, copper, molybdenum,
and zinc

Oxygen
Obligate aerobesrequire oxygen (20% O2)
Facultative anaerobesgrow via
fermentation or anaerobic respiration when
oxygen is not available
Obligate anaerobesunable to use oxygen
and are harmed by it
Aerotolerant anaerobestolerate but
cannot use oxygen
Microaerophilesrequire oxygen
concentration lower than air (2-10% O 2)

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Basis of Different Oxygen Sensitivities

Oxygen easily reduced to toxic reactive oxygen
species (ROS) that can damage DNA, RNA,
proteins and lipids:
Singlet oxygen: (1O2) boosted to a higherenergy state and is reactive
Superoxide radicals: O2
Peroxide anion: O22
Hydroxyl radical (OH)
Aerobes produce protective enzymes
superoxide dismutase (SOD)
Catalase
peroxidase

Enzymes help neutralize these toxic reactive oxygen

species and detect and repair macromolecules damaged by

Organic Growth Factors

Organic compounds obtained from
the environment
Vitamins, amino acids, purines, and
pyrimidines

Biofilms
Most microbes grow attached to surfaces

(sessile) rather than free floating (planktonic),

forming microbial communities.
These attached microbes are members of
complex, slime enclosed communities called a
biofilm.
Biofilms are ubiquitous in nature in water.
Can be formed on any conditioned surface.
Share nutrients
Shelter bacteria from harmful environmental
factors

Biofilm Formation
Microbes reversibly attach to conditioned surface
and release polysaccharides, proteins, and DNA to
form the extracellular polymeric substance (EPS)
Additional polymers are produced as microbes
reproduce and biofilm matures

A mature biofilm is a
complex 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

Biofilms
Protects microbes from harmful agents
UV light, antibiotics, antimicrobials
1000x resistant to microbicides

When formed on medical devices, such as

implants, often lead to illness
Involved in 70% of infections
Catheters, heart valves, contact lenses, dental caries

Found in digestive system and sewage treatment

systems; can clog pipes
Sloughing off of organisms can result in
contamination of water phase above the biofilm such
as in a drinking water system

Cell to Cell Communication Within

the Microbial Populations
Bacterial cells in biofilms communicate in a
density-dependent manner called quorum
sensing
Produce small proteins that increase in
concentration as microbes replicate and
convert a microbe to a competent state
DNA uptake occurs, bacteriocins are released.

Quorum Sensing
Acylhomoserine lactone (AHL) is an autoinducer
molecule produced by many gram-negative
organisms
diffuses across plasma membrane
once inside the cell it induces expression of target
genes that regulate a variety of functions.

Processes regulated by quorum sensing involve

host-microbe interactions
symbiosis Vibrio fischeri and bioluminescence in squid
pathogenicity and increased virulence factor production
DNA uptake for antibiotic resistance genes

LABORATORY CULTURE OF
CELLULAR MICROBES
CULTURE MEDIA
Need to grow, transport, and store
microorganisms in the laboratory
Culture media is solid or liquid preparation
Must contain all the nutrients required by
the organism for growth
Classification

chemical constituents from which they are made

physical nature
function

Culture Media
Culture medium: nutrients
prepared for microbial growth
Sterile: no living microbes
Inoculum: introduction of microbes
into a medium
Culture: microbes growing in or on a
culture medium

Culture Media
Agar
Complex polysaccharide
Used as a solidifying agent for culture
media in Petri plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 100C
Solidifies at ~40C

Culture Media
Chemically defined media: exact
chemical composition is known
Fastidious organisms are those that require
many growth factors provided in chemically
defined media

Complex media: extracts and digests of

yeasts, meat, or plants; chemical
composition varies batch to batch
Nutrient broth
Nutrient agar

Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as
Escherichia coli

Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic
Bacteria

Anaerobic Growth Media and

Methods
Reducing media
Used for the cultivation of anaerobic
bacteria
Contain chemicals (sodium
thioglycolate) that
combine O2 to deplete it
Heated to drive off O2

Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.

Clamp with
Lid with
O-ring gasket clamp screw

Envelope containing
inorganic carbonate,
activated carbon,
ascorbic acid,
and water

Anaerobic indicator
(methylene blue)

Petri plates

CO2
H2

Figure 6.7 An anaerobic chamber.

Air
lock

Arm
ports

Special Culture Techniques

Capnophiles
Microbes that require high CO2
conditions
CO2 packet
Candle jar

Special Culture Techniques

Biosafety levels
BSL-1: no special precautions; basic
teaching labs
BSL-2: lab coat, gloves, eye protection
BSL-3: biosafety cabinets to prevent
airborne transmission
BSL-4: sealed, negative pressure; "hot
zone"
Exhaust air is filtered twice through HEPA
filters

Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

Selective and Differential

Media
Selective media
Suppress unwanted microbes and encourage
desired microbes
Contain inhibitors to suppress growth

Differential media
Allow distinguishing of colonies of different
microbes on the same plate

Some media have both selective and

differential characteristics
Bacterial
colonies

Hemolysis

Enrichment Culture
Encourages the growth of a desired
microbe by increasing very small
numbers of a desired organism to
detectable levels
Usually a liquid

Table 6.5 Culture Media

Bacterial Division
Bacteria and Archaea:
Haploid
Reproduce asexually

Increase in number of cells, not cell

size
Binary fission
Budding
Fragmentation of filaments
Conidiospores (actinomycetes)

Figure 6.12a Binary fission in bacteria.

Cell wall

Plasma membrane

Cell elongates and

DNA is replicated.
DNA (nucleoid)
Cell wall and
plasma membrane
begin to constrict.

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

Cells
separate.

A diagram of the sequence of cell division

Figure 6.12b Binary fission in bacteria.

DNA (nucleoid)

Partially formed cross-wall

A thin section of a cell of Bacillus

licheniformis starting to divide

Cell wall

Bacterial Cell Cycle

Cell cycle is sequence of events from

formation of new cell through the next cell
division
Two pathways function during cycle
Single origin of replication
DNA replication and partition
cytokinesis

Terminus
Proteins
needed for
DNA
synthesis

Move in
both
directions

Generation Time
Time required for a cell to divide
20 minutes to 24 hours

Binary fission doubles the number of cells

each generation
Total number of cells = 2number of generations
Growth curves are represented
logarithmically

GROWTH CURVE: WHEN ONE BECOMES

TWO AND TWO BECOME FOUR
Increase in cellular constituents that may
result in:

increase in cell number

increase in cell size

Growth refers to population growth rather

than growth of individual cells
Observed when microorganisms are cultivated
in batch culture
Has four distinct phases

Phases of Growth
Lag phase
Log phase
Stationary
phase
Death phase

Lag Phase
Cell synthesizing new components
e.g., to replenish spent materials
e.g., to adapt to new medium or other
conditions

Varies in length
in some cases can be very short or
even absent

Exponential Phase
Also called log phase
Rate of growth and division is
constant and maximal
Population is most uniform in
terms of chemical and physical
properties during this phase

Stationary Phase
Closed system population growth eventually
ceases, total number of viable cells remains
constant
active cells stop reproducing or reproductive rate is
balanced by death rate

Possible Reasons for Stationary Phase

Nutrient limitation
Limited oxygen availability
Toxic waste accumulation
Critical population density reached

Senescence and Death Phase

Two alternative hypotheses
cells are Viable But Not Culturable (VBNC)
cells alive, but dormant, capable of new growth
when conditions are right

Programmed cell death

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

58

Prolonged Decline in Growth

Bacterial population continually evolves
Process marked by successive waves of
genetically distinct variants
Natural selection occurs

59

Direct Measurement of Microbial

Growth
Direct measurementscount
microbial cells
Plate count
Filtration
Most probable number (MPN)
method
Direct microscopic count

Plate Counts
Count colonies on plates that have 30 to
300 colonies (CFUs)
To ensure the right number of colonies,
the original inoculum must be diluted
via serial dilution
Counts are performed on bacteria mixed
into a dish with agar (pour plate
method) or spread on the surface of a
plate (spread plate method)

Figure 6.16 Serial dilutions and plate counts.

Figure 6.17 Methods of preparing plates for plate counts.

The
pour
plate
method
The
pour
plate
method

1.0 or 0.1 ml

The
plate method
method
The spread
spread plate

0.1 ml
Inoculate plate
containing
solid medium.

Inoculate
empty plate.

Bacterial
dilution

Spread inoculum
over surface
evenly.
Add melted
nutrient agar.

Swirl to mix.

Colonies
grow on and
in solidified
medium.

Colonies grow
only on surface
of medium.

Filtration
Solution passed through a filter that
collects bacteria
Filter is transferred to a Petri dish and
grows as colonies on the surface

The Most Probable Number (MPN)

Method
Multiple tube test
Count positive tubes
Compare with a statistical table

Direct Microscopic Count

Volume of a bacterial suspension
placed on a slide
Average number of bacteria per
viewing field is calculated
Uses a special Petroff-Hausser cell
counter
Number of bacteria/ml =

Number of cells counted

Volume of area counted

Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.

Grid with 25 large squares

Cover glass
Slide

Bacterial suspension is added here

and fills the shallow volume over the
squares by capillary action.
Bacterial
suspension

Microscopic count: All cells in

several large squares are
counted, and the numbers are
averaged. The large square
shown here has 14 bacterial cells.

Cover glass
Slide
Location of squares

The volume of fluid over the

large square is 1/1,250,000
Cross section of a cell counter.
The depth under the cover glass and the area of a milliliter. If it contains 14
of the squares are known, so the volume of the cells, as shown here, then
bacterial suspension over the squares can be there are 14 1,250,000 =
17,500,000 cells in a milliliter.
calculated (depth area).

Estimating Bacterial Numbers by

Indirect Methods
Turbiditymeasurement of
cloudiness with a spectrophotometer
Metabolic activityamount of
metabolic product is proportional to
the number of bacteria
Dry weightbacteria are filtered,
dried, and weighed; used for
filamentous organisms

Figure 6.21 Turbidity estimation of bacterial numbers.

Light source

Spectrophotometer
Light

Blank
Scattered light
that does not
reach detector

Light-sensitive
detector

Bacterial suspension

Viable counting:
Alive or dead?
Whether or not a
cell is alive or
dead isnt always
clear cut in
microbiology
Cells can exist in a
variety of states
between fully
viable and
actually dead