MICROBIAL GROWTH AND CONTROL

MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

● Microbes that grow at 4 C but optima of 20-40 C

are called psychrotolerant
TEMPERATURE AND MICROBIAL GROWTH

EFFECT OF TEMPERATURE ON GROWTH

EFFECT OF TEMPERATURE ON GROWTH

● Cardinal Temperatures
○ Minimal: lowest temperature at which a
microorganism grows and multiplies;
membrane loses its fluidity (as it is
semi-fluid in nature) MICROBIAL LIFE IN THE COLD
○ Optimal: temperature at which growth ● Molecular adaptations to psychrophily
rate is fastest ○ Production of enzymes that function
○ Maximal: highest temperature that can optimally in the cold; features that may
support microbial growth; temperature at provide more flexibility
which growth ceases ■ More α-helices than 𝑏-helices
● Temperature is a major environmental factor ■ More polar and less
controlling microbial growth hydrophobic amino acids
■ Fewer weak bonds
CLASSIFICATION OF MICROORGANISMS BY TEMP. REQUIREMENTS ■ Decreased interaction between
protein domains
○ Transport processes function optimally
at low temperatures
■ Modified cytoplasmic
membranes (high unsaturated
fatty acid content – in the Fatty
Acid Chain, there are one or
more double bonds, promoting
flexibility)
● Antarctic habitats and microbes
○ Diatoms/green algae
● Psychrophiles: 0-15 C ○ Polaromonas (4 C)
● Mesophiles: prefer 20-45 C ○ Snow algae
● Thermophiles: grow in extreme >55 C, often ■ Can be seen as green
grow at 45 C but often higher structures but due to melting of
● Hyperthermophiles: some grow up to 95 C or the eyes, the spores carry
higher pigment called astaxanthin

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MICROBIAL GROWTH AND CONTROL
MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

which contributes to red ● Due to ends that are

pigments.
■ Chlamydomonas nivalis
MICROBIAL LIFE AT HIGH TEMPERATURES
● Above 65 C, only prokaryotic life forms exist
● Thermophiles: organisms with growth
temperature optima between 45 C and 80 C
● Hyperthermophiles: organisms with optima
greater than 80 C
○ Inhabit hot environments including
boiling hot springs and seafloor
hydrothermal vents that can have
temperatures in excess of 100 C
● Hyperthermophiles in hot springs
○ Chemoorganotrophs
chemolithotrophic species are present
○ High prokaryotic diversity (both Archaea
and Bacteria represented)
● Studies of thermal habitats have revealed that:
○ Prokaryotes are able to grow at higher
temperatures than eukaryotes
○ Organisms with the highest temperature
optima are Archaea
○ Non-phototrophic organisms can grow
at higher temperatures than
phototrophic organisms
● Molecular adaptations to thermophily
○ Enzyme and proteins function optimally
at high temperatures; features that
provide thermal stability
■ Critical amino acid substitutions
in a few locations could provide
more heat-tolerant folds
■ An increased number of ionic
bonds between basic and acidic
amino acids resist unfolding in
aqueous cytoplasm
■ Production of solutes (e.g.
di-inositol phosphate, diglycerol
phosphate) help stabilize
proteins
○ Modifications in the cytoplasmic
membranes to ensure heat stability
■ Bacteria: lipids rich in saturated
fatty acids
■ Archaea: lipid monolayer rather
than bilayer
linked
● Tend to be more
resistant to higher
temperatures than lipid
bilayer
● Hyperthermophiles produce enzymes widely
used in industrial microbiology
○ Example: Taq polymerase used in
polymerase chain reaction (PCR) and is
a heat-stable enzyme
■ PCR: Technique used to amplify
a particular gene to create
several thousand copies.
■ Thermus aquaticus (organism
& sourcing Taq polymerase)
OTHER ENVIRONMENTAL FACTORS AFFECTING GROWTH
ACIDITY AND ALKALINITY (pH & MICROBIAL GROWTH)
● pH of environment greatly affects microbial
growth
○ Has to be maintained on a particular
level
● Some organisms have evolved best to grow at
low or high pH, but most organisms grow best at
pH 6-8 (neutrophiles)
● Recalling general chemistry, higher H+ ions,
lower pH/more acidic solution; higher OH+ ions,
more basic solution
● Increasing acidity: acidophiles
○ Some are obligate acidophiles;
membranes of these organisms are
destroyed at neutral pH, thus requiring
more H+ protons

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MICROBIAL GROWTH AND CONTROL
MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

○ Stability of cytoplasmic membrane is OSMOTIC EFFECTS ON MICROBIAL GROWTH

critical
● Increasing alkalinity: alkaliphiles
○ Some have sodium motive force rather
than proton motive force
HOW DO CELLS RESPOND?
WATER ACTIVITY
● Water activity (aw) is synonymous to water
availability and is expressed in physical terms
𝑃 𝑠𝑜𝑙𝑢𝑡𝑒
aw= 𝑃 𝑤𝑎𝑡𝑒𝑟
○ Defined as ratio of vapor pressure of air
in equilibrium with a substance or
solution to the vapor pressure of pure
water
● Osmotic pressure depends on the surrounding
solute concentration and water availability

If a normal ion is being pumped out, potassium ions are

being pumped in to neutralize the pH in the organism. ● Solute concentration
● The internal pH of a cell must always stay ○ Refers to salt, sugars, etc. in the
relatively close to neutral even though the external environment.
external pH is highly acidic or basic
● Neutrophiles can exchange K (potassium) for Solute concentration within the
protons using antiport transport system or use a microbial cell is almost the same
Isotonic Solution
proton translocating ATPase with the concentration outside the
cell
Lower concentration of solutes
BUFFERS within the cell and higher
Microbial culture media typically contain buffers to Hypertonic Solution concentration of solutes outside
maintain constant pH: the cell leading to shrinkage or cell
● CO2/bicarbonate-buffered medium death
○ Using sodium bicarbonate-buffered Higher concentration of solutes
media, the same physiological buffering within the cell and lower
Hypotonic Solution
system surrounding mammalian cells concentration of solutes outside
○ Use CO2 incubators 5-7% the cell
● Phosphate-buffered medium
○ Not required CO2 environment ENVIRONMENTAL FACTORS AND GROWTH
○ Maintain pH in air
○ Detrimental to embryo development in Osmophiles Thrive in high solute
vitro Osmotolerant Tolerate high solute
● HEPES-buffered medium Halophiles Thrive in high salt
○ Used as a buffered medium for human Halotolerant Tolerate high salt
oocyte collection and embryo handling. Barophiles Thrive in high pressure
Barotolerant Tolerate high pressure
pH AFFECTS GROWTH BY:
● Disrupting plasma membrane OTHER INFORMATION
● Altering activity of the enzymes ● Cytoplasm has a higher solute concentration
● Altering activity of membrane transport proteins than the surrounding movement, thus the
tendency is for the water to move into the cell
SOME MICROORGANISMS (positive water balance)
● When a cell is in an environment with higher
● Lactic acid bacteria: pH 4-7
external solute concentration, water will flow out
unless the cell has a mechanism to prevent this.

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MICROBIAL GROWTH AND CONTROL
MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

Non-halophiles Cannot tolerate high salt

Can tolerante some reduction in
Halotolerant water activity but grow best in the
absence of added solute
Grow best at reduced water
Halophiles potential and have a significant
requirement for NaCl (>0.4M salt)
Require high levels (15-30%) of
Extreme halophiles
NaCl for growth (> 2M)
Live in environments high in sugar
Osmophiles
as solute CLASSIFICATION OF ORGANISMS BASED ON O2 UTILIZATION
Able to grow in very dry ● Thioglycollate broth: contains a reducing
Xerophiles
environments agent (resazurin) and provides aerobic and
anaerobic conditions
OSMOTIC EFFECT: HOW DO CELLS RESPOND?
Mechanisms for combating low water activity in
surrounding environment involves increasing the internal
solute concentration by:
● Use of mechano-sensitive channels when cell
is placed in hypotonic solutions
○ To prevent re-entry of water molecules
into the cytoplasm of the cell
● Pumping inorganic ions from environment into
the cell
● Synthesis/concentration of organic solutes
○ Compatible solutes: compounds used
by cells to counteract low water activity
in the surrounding environment when
placed in hypertonic solutions (e.g. When oxygen is utilized by the cells, these can be
amino acids, choline, etc.) converted into toxic forms that can kill the cells. To
counterattack these toxic forms, they have these
enzymes to convert into a safer compound. For obligate
OXYGEN AND MICROORGANISMS
aerobes, they have superoxide dismutase and catalase,
and so forth.
CLASSIFICATION OF ORGANISMS BASED ON O2 UTILIZATION
AEROBES TOXIC FORMS OF OXYGEN
Obligate Aerobes Require oxygen to live
Can use oxygen only when it is
Microaerophiles present at levels reduced from that TOXIC FORMS OF OXYGEN
in air Several toxic forms of oxygen can be formed in the cell:
Facultative organisms Can live with or without oxygen ● Single oxygen
ANAEROBES ● Superoxide anion
Can tolerate oxygen and grow in its ● Hydrogen peroxide
Aerotolerant
anaerobes
presence even though they cannot ● Hydroxyl radical
tolerate it
Do not require oxygen to live
Obligate Anaerobes CLASSIFICATION OF ORGANISMS BASED ON O2 UTILIZATION
● Utilization of O2 during metabolism yields toxic
by-products including O2, singlet oxygen, and/or
H2O2
● Toxic O2 products can be converted to harmless
substances if the organism has catalase or
peroxidase and superoxide dismutase (SOD)
○ SOD converts O2- into H2O2 and O2
○ Catalase breaks down H2O2 into H2O
and O2

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MICROBIAL GROWTH AND CONTROL
MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

○ Any organism that can live in or require

O2 has SOD and catalase

● Oxygen is easily reduced to toxic products

○ Superoxide radical
○ Hydrogen peroxide (damages proteins
and DNA) PHYSICAL AGENTS - HEAT, PRESSURE, RADIATION
○ Hydroxyl radical
● Sterilization of liquids (autoclave)
○ Moist heat killing
DETOXIFYING ENZYMES ○ 121 C
Enzymes are present to neutralize most of these toxic ○ 1 ATM
oxygen species by: ○ 20-45 minutes
● Catalase ● Sterilization of dry materials
● Peroxidase ○ 170 C
● Superoxide dismutase ○ 4-6 hours
● Superoxide reductase
Anaerobes lack superoxide dismutase or catalase, PASTEURIZATION
hence:
● Removing unwanted pathogens
● Chemical: thioglycollate; pyrogallol + NaOH; H2
● UHP (Ultra-high temperature process)
generator + catalyst
○ Amount of time (3-5 sec) to kill
● Physical: removal/replacement (GASPAK)
pathogens
● Why do we pasteurize? To increase shelf life
CONTROL OF MICROORGANISMS
UV RADIATION & IONIZING RADIATION
● This can integrate DNA mutations, particularly
Destruction of vegetative the formation of thymine dimers in DNA
Disinfection pathogens from objects (not ● Cells have their own mechanisms to combat
spores)
radiation via:
Complete removal of ALL viable
Sterilization ○ Secreting pigments/metabolization
organisms
Chemicals applied to body surfaces
● Ionizing Radiation (X-Rays, Gamma Rays)
Antisepsis to destroy or inhibit vegetative ○ As radiation increases, survival fraction
organisms decreases (decimal reduction dose –
Chemicals to destroy or inhibit only 10% of cells will survive)›
Chemotherapy vegetative pathogens within
tissues MECHANICAL FACTORS - AIR FILTRATION
● Laminar Flow Hood
○ Has a filter called HEPA filter
○ HEPA filter: High-efficiency particulate
absorbing filter & high-efficiency
particulate arrestance filter
○ Prevents microbial contamination &
capture tiny organisms

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MICROBIAL GROWTH AND CONTROL
MCROBIO (Lecture)
BIOMED | PROF. ESPIRITU | SEM 1 2023

CHEMICAL CONTROL OF MICROBIAL GROWTH Inoculate the organism then aseptically put an antibiotic
● Antimicrobial agent: natural/synthetic chemical disc which contains an antibiotic that inhibits growth of
which kills/inhibits growth of microorganisms the organism.
○ -cidal agents
● -static agents do not kill but only inhibit growth FACTORS INFLUENCING EFFECTIVENESS OF ANTIMICROBIAL AGENTS
● -lytic agents: lead to the decrease of total cell ● Population size
and viable cell count due to lysis of cytoplasmic ● Population composition
membrane ● Concentration or intensity of an antimicrobial
agent
● Exposure time
● Temperature
● Environment

WAYS TO MEASURE INHIBITION

MINIMUM INHIBITORY CONCENTRATION (MIC)

Using different concentrations of inhibitory agents. You

identify the lowest concentration that can accommodate
the organism. Turbid means that organisms can grow
and concentration is not inhibitory to cells. Clear means
cells were not able to grow anymore meaning this
concentration is the minimum inhibitory concentration to
inhibit cell growth.

PAPER DISC DIFFUSION ASSAY