TOPIC 3.

Microbial Nutrition, Growth, and Growth

Control



I. Nutrition and Culture
Microbial Nutrition
Nutrition
o Macronutrients are nutrients required in large amounts
o Micronutrients are nutrients required in small amounts
Carbon and Nitrogen
o All cells require carbon and most prokaryotes require organic compounds as their
source of carbon
o Following carbon the next most abundant element in the cell is nitrogen
Other Macronutrients: P, S, K, Mg, Ca, Na
o Phosphorous is required by the cells for the synthesis of nucleic acids and
phospholipids
o Sulfur is required for the synthesis of amino acids and vitamins
o Many enzymes require potassium for activity
o Magnesium functions to stabilize the ribosome
o Calcium stabilizes the cell wall
o Sodium is required by some but not all organisms
Iron and Trace Metals
o Iron is a key component of the cytochromes and iron-sulfur proteins involved in
electron transport
o Many other metals are required or utilized by microorganisms. These are called trace
elements
Growth Factors
o Growth factors are organic compounds that are required in small amounts by only
certain organisms
o Vitamins are the most commonly required growth factors
Culture Media
Classes of Culture Media
o Culture media are the nutrient solutions used to grow microbes in the laboratory
o Two classes of culture media are used: defined media and complex media
o The exact compostion of a defined media is known
o Complex media employ highly nutritious yet impure substances
o A selective medium contains compounds that inhibit the growth of certain
microorganisms
o A differential medium is one that allows for the differentiation of particular chemical
reactions
Nutritional Requirements and Biosynthetic Capacity
The defined media for L. mesenteroides requires more nutrients than the defined media for
E. coli. Therefore E. coli has the greater biosynthetic capacity

Chapter 6 Guide
Microbial Growth

I. Bacterial Cell Division
Cell Growth and Binary Fission
Cell Growth
o In microbiology, growth is defined as an increase in microbial cells
o Bacterial cell growth depends upon a large number of chemical reactions of a wide
variety of types
Binary Fission
o In a growing rod-shaped cell, elongation continues until the cell divides into two
cells. This process is called binary fission
o When one cell divides into two, one generation has occurred and the time required is
called the generation time
o In the period of one generation all cells increase proportionally, thus cells are said to
be in balanced growth
o The time required for a generation in a given bacterial species is highly variable
o
Peptidoglycan Synthesis and Cell Division
Biosynthesis of Peptidoglycan
o Bactoprenol transports peptidoglycan precursors across the cytoplasmic membrane
Transpeptidation
o Transpeptidation forms the peptide cross-link between muramic acid residues in
adjacent glycan chains
Transpeptidation and Penicillin
o Transpeptidation is the reaction that is inhibited by penicillin
o Humans are Eukarya and therefore lack peptidoglycan. The penicillin can be
administered in high doses and is typically nontoxic
II. Growth of Bacterial Populations
Growth Terminology and the Concept of Exponential Growth
Exponential Growth
o The pattern of population increase where the number of cells doubles during a
constant time interval is called exponential growth
The Consequences of Exponential Growth
o During exponential growth, the increase in cell numbers is slow at first but increases
at an ever faster rate
Other Growth Expressions
o The generation time can be determined from the slope of the straight line generated in
the semi-logarithmic plot of exponential growth
o The reciprocal of the generation time is called the division rate
The Microbial Growth Cycle
Lag Phase
o When a microbial population is inoculated into fresh media, growth only begins after
a period of time called the lag phase
o A lag is also observed when a microbial population is transferred from a rich media to
a poor media
Exponential Phase
o During exponential growth each cell divides to form two cells
o Rates of exponential growth vary greatly
Stationary Phase
o In batch culture, such as in a tube or flask, exponential growth is limited
o When exponential growth ceases the population reaches stationary phase
o Cryptic growth is the phenomenon where some cells in a population grow and other
cells in a population die
Death Phase
o If incubation continues after cells reach stationary phase, the cells will eventually die.
This is called death phase
o The phases of growth are the result of a population of cells, not individual cells
Continuous Culture: The Chemostat
The Chemostat
o The most common type of continuous culture device is the chemostat
o In a chemostat both growth rate and population density can be controlled
o In a batch culture the nutrient concentration can affect both growth rate and growth
yield
Varying Chemostat Parameters
o There is a rather wide range over which dilution rate controls growth rate
o Cell density in a chemostat is controlled by limiting a nutrient
Experimental Uses of the Chemostat
o In a chemostat cell populations may be maintained in exponential growth for long
periods
o The chemostat is used in microbial physiology as well as microbial physiology
o Chemostats have also been used for enrichment and isolation of bacteria
Measuring Microbial Growth
Measurements of Total Cell Numbers: Microscopic Counts
Total Cell Count
o The most common total count method is the microscopic cell count
o The second method of enumerating cells in liquid is with a flow cytometer
o Microscopic counting is a quick and easy way of estimating microbial cell number
o In microbial ecology, total cell counts are often performed on natural samples using
stains to visualize the cells
Viable Cell Counting
Viable Cell Counting
o A viable cell is one that is capable of dividing to form offspring
o A viable count is also called a plate count
o There are at least two methods for performing a plate count the spread-plate method
and the pour-plate method
Diluting Cell Suspensions before Plating
o With both the spread-plate method and the pour-plate method, it is important that
there are not too many or too few colonies developing on a plate
o In order to obtain the appropriate colony number, the sample must always be diluted
Sources of Error in Plate Counting
o The number of colonies obtained in a viable count depends on the inoculum size, the
viability of the culture, the suitability of the culture medium, and the incubation
conditions
o Viable counts can be subject to large errors
o Use of highly selective culture media and growth conditions allow one to target only
particular species
The Great Plate Count Anomaly
o Direct microscopic counts of natural samples reveal far more organisms than those
recoverable on plates
o Microscopic methods count dead cells
o Total cell counts on a single media are actually underestimates of the total cell
number
Measurements of Microbial Mass: Turbidimetric Methods
Optical Density
o Turbidity is measured with a spectrophotometer
o Optical density (OD) is a unit of turbidity at a specified wavelength
Relating OD to Cell Numbers
o For unicellular organisms, OD is related to cell number
o Turbidity measurements are quick and easy to perform
Temperature and Microbial Growth
Effect of Temperature on Microbial Growth
Cardinal Temperatures
o As temperature rises, chemical and enzymatic reactions proceed at more rapid rates
o Above a certain temperature cell components will be damaged
o For every microorganism, there is a minimum temperature, an optimum temperature,
and a maximum temperature. These three temperatures are called the cardinal
temperatures
o The cardinal temperatures of microorganisms differ widely. Some have temperature
optima as low as 4
o
C and others higher than 100
o
C
Temperature Classes of Organisms
o Psychrophiles have a low temperature optima
o Mesophiles have a midrange temperature optima
o Thermophiles have a high temperature optima
o Hyperthermophiles have a very high temperature optima
Microbial Growth at Cold Temperatures
Cold Environments
o Much of the Earths surface is cold. Microorganisms will inhabit any low
temperature environment in which some liquid water remains
Psychrophilic Microorganisms
o A psychrophile is an organism with an optimum growth temperature of 15
o
C or lower
o Organisms that grow at 0
o
C but have a temperature optima of 20-40
o
C are called
psychrotolerant
o Psychrophiles are found in environments that are constantly cold
Psychrotolerant Microorganisms
o Psychrotolerant microorganisms are widely distributed in nature
Molecular Adaptations to Psychrophily
o Psychrophiles produce enzymes that function optimally in the cold and may be
denatured at moderate temperatures
o The cytoplasmic membrane of psychrophiles are modified so that they function at low
temperatures
Freezing
o As long as liquid water is available, microbial growth is possible below the freezing
point of water
o Freezing does not necessarily kill microbial cells
Microbial Growth at High Temperatures
Thermal Environments
o Organisms whose growth optimum exceeds 45
o
C are called thermophiles
o Organisms whose growth optimum exceeds 80
o
C are called hyperthermophiles
Hyperthermophiles in Hot Springs
o Hyperthermophiles are present in boiling hot springs. Both chemoorganotrophic and
chemolithotrophic species can be found
Thermophiles
o Many thermophiles are also present in hot springs and other thermal environments
Protein Stability at High Temperatures
o Enzymes and other proteins of thermophiles and hyperthermophiles are heat stable
Membrane Stability at High Temperatures
o The cytoplasmic membrane of thermophiles and hyperthermophiles are heat stable
Thermophily and Biotechnology
o Thermophiles and hyperthermophiles may be useful in industry and biotechnology.
Several processes can be run more efficiently or rapidly at high temperatures
Other Environmental Factors Affecting Growth
Microbial Growth at Low or High pH
Acidophiles
o Organisms that grow at low pH (typically below pH 6) are called acidophiles
o Low pH is required for the stability of the cytoplasmic membrane of an acidophile
Alkaliphiles
o Microorganisms that have a growth pH optima of 9 or higher are called alkaliphiles
Internal Cell pH
o The optimum pH for growth is a measure of the extracellular environment
o The intracellular environment remains relatively close to neutral pH
Buffers
o Buffers are added to microbial culture medias to keep the pH relatively constant
Osmotic Effects on Microbial Growth
Water Activity and Osmosis
o Water availability is expressed in physical terms as water activity
o Water diffuses from regions of high water concentrations to regions of low water
concentrations in the process of osmosis
Halophiles and Related Organisms
o Organisms that have a requirement for NaCl are called halophiles
o Halotolerant organisms can tolerate some reduction in the water activity but grow
best in the absence of added solute
o Organisms capable of growth in very salty environments are called extreme
halophiles
o Organisms capable of growth in environments high in sugar are called osmophiles
o Organisms capable of growth in very dry environments are called xerophiles
Compatible Solutes
o Solutes used inside the cell for the adjustment of water activity are called compatible
solutes
Oxygen and Microbial Growth
Oxygen Classes of Microorganisms
o Aerobes can grow at full oxygen tensions
o Microaerophiles are aerobes that can use oxygen but only when it is present at lower
than atmospheric levels
o Many aerobes are facultative, meaning that they can grow under oxic or anoxic
conditions
o Some organisms cannot respire oxygen. They are called anaerobes
o Aerotolerant anaerobes can tolerate oxygen and grow in its presence
o Obligate anaerobes are inhibited or killed by oxygen
Culture Techniques for Aerobes and Anaerobes
o Many aerobes require extensive aeration
o Obligate anaerobes require reduction of oxygen
o Thyoglycholate broth is a complex medium that contains a small amount of agar.
Thyoglycholate reacts with oxygen so oxygen can only penetrate the top of the tube

Chapter 27 Guide
Microbial Growth Control

Physical Antimicrobial Control
Heat Sterilization
Heat Sterilization
o The most used method for controlling microbial growth is the application of heat
Measuring Heat Sterilization
o All microorganisms have a maximum growth temperature, beyond which viability
decreases
o At very high temperatures virtually all macromolecules lose structure and functional
capacity, a process known as denaturation
o The decimal reduction time or D is the time required for a 10-fold reduction in the
population density at a given temperature
o Thermal death time is the time it takes to kill all cells at a given temperature
Endospores and Heat Sterilization
o Some bacteria produce highly resistant cells called endospores
o Bacterial endospores are able to survive heat that would rapidly kill vegetative cells
of the same species
o The water content of the endospore coupled with the concentration of small acid-
soluble spore proteins (SASP) determines the heat resistance of the endospores
o The nature of the medium in which heating takes place also influences the killing of
both vegetative cells and endospores
The Autoclave
o The autoclave is a sealed heating device that allows the entrance of steam under
pressure
o It is not the pressure inside the autoclave that kills the microorganisms but the high
temperature that can be achieved when steam is placed under pressure
Pasteurization
o Pasteurization uses precisely controlled heat to reduce the microbial populations
(microbial load) in milk and other heat-sensitive liquids
o Pasteurization of milk is usually achieved by passing the milk through a heat
exchanger
o Milk can also be heated in large vats to 6366C for 30 minutes
Radiation Sterilization
Ionizing Radiation
o Ionizing radiation is electromagnetic radiation of sufficient energy to produce ions
and other reactive molecules.
o Ionizing radiation generates electrons, hydroxyl radicals, and hydride radicals
Radiation Practices
o Common sources of ionizing radiation include cathode ray tubes that generate
electron beams, X-ray machines, and radioactive nuclides
63
Co and
137
Cs
o Radiation is currently used for sterilization and decontamination in the medical
supplies industry
o Certain foods and food products are also routinely irradiated to ensure sterilization,
pasteurization, or insect deinfestation
Filter Sterilization
Filter Sterilization
o Filter sterilization passes liquid or gas through a filter, a device with pores too small
for the passage of microorganisms but large enough to allow the passage of the liquid
or gas
Membrane Filters
o The most common type of filter for liquid sterilization in the microbiology laboratory
is the membrane filter
o Membrane filters are composed of polymers such as cellulose acetate, cellulose
nitrate, or polysulfone, manufactured to contain a large number of tiny holes, or pores
o Another type of membrane filter in common use is the nucleation track (Nucleopore)
filter
Chemical Antimicrobial Control
Chemical Growth Control
Effect of Antimicrobial Agents on Growth
o Bacteriostatic agents prevent the growth of bacteria
o Bacteriocidal agents kill the bacterial cell
o Bacteriolytic agents induce killing by cell lysis
Measuring Antimicrobial Activity
o Minimum inhibitory concentration (MIC) is the smallest amount of antimicrobic
needed to inhibit the growth of a test organism
o The MIC is not a constant for a given antimicrobial agent
o Another common assay for antimicrobial activity is the disk diffusion technique
Chemical Antimicrobial Agents for External Use
Sterilants
o Chemical sterilants, also called sterilizers or sporicides, destroy all forms of
microbial life, including endospores
Disinfectants, Sanitizers, and Antiseptics
o Disinfectants are chemicals that kill microorganisms, but not necessarily endospores,
and are also used on inanimate objects
o Sanitizers are agents that reduce microbial numbers to levels considered to be safe
o Antiseptics and germicides are chemical agents that kill or inhibit growth of
microorganisms and are sufficiently nontoxic to be applied to living tissues
Antimicrobial Efficacy
o Many disinfectants are neutralized by organic material
o Biofilms may slow or prevent penetration of antimicrobial agents
o Only sterilants are effective against bacterial endospores
o Some bacterial cells are resistant to the disinfectant
Antimicrobial Agents Used In Vivo
Synthetic Antimicrobial Drugs
Growth Factor Analogs
o A growth factor analog is a synthetic compound that is structurally similar to a
growth factor, but prevents the cell from functioning normally
Sulfa Drugs
o Sulfa drugs were the first widely used growth factor analogs shown to specifically
inhibit the growth of bacteria
o Sulfanilamide is an analog of p-aminobenzoic acid, which is part of the vitamin folic
acid
o Sulfanilamide is active only in Bacteria because Bacteria synthesize their own folic
acid, whereas most animals obtain folic acid from their diet
Isoniazid
o Isoniazid is effective only against Mycobacterium
o Isoniazid interferes with the synthesis of mycolic acid, a mycobacterial cell wall
component
Nucleic Acid Base Analogs
o Nucleic acid base analogs are used in the treatment of viral and fungal infections and
are also used as mutagens
Quinolones
o The quinolones are antibacterial compounds that interfere with bacterial DNA gyrase,
preventing the supercoiling of bacterial DNA, a required step for packaging DNA in
the bacterial cell
Naturally Occurring Antimicrobial Drugs: Antibiotics
Antibiotics and Selective Antimicrobial Toxicity
o Antibiotics are naturally occurring antimicrobial compounds
o Narrow-spectrum antibiotics are effective against a subgroup of Bacteria
o Broad-spectrum antibiotics are effective against both gram-positive and gram-
negative Bacteria
Antibiotics Affecting Protein Synthesis
o Many antibiotics inhibit protein synthesis by interacting with the ribosome and
disrupting translation
o Two antibiotics can inhibit the same step in protein synthesis, but have different
mechanisms of inhibition
o Many antibiotics inhibit ribosomes of organisms from only one phylogenetic domain
Antibiotics Affecting Transcription
o A number of antibiotics specifically inhibit transcription by inhibiting RNA synthesis
Lactam Antibiotics: Penicillins and Cephalosporins
Penicillins
o Alexander Fleming characterized an antibacterial product of the fungus Penicillium
chrysogenum as the antibiotic penicillin
o Penicillin G is active primarily against gram-positive Bacteria
o Many semisynthetic penicillins are quite effective against gram-negative Bacteria
Mechanism of Action
o The -lactam antibiotics are potent inhibitors of cell wall synthesis
o An important feature of cell wall synthesis is the transpeptidation reaction, which
results in the cross-linking of two glycan-linked peptide chains
o The transpeptidase enzymes bind to penicillin or other -lactam antibiotics. Thus,
these transpeptidases are known as penicillin binding proteins (PBPs)
o Because the cell wall and its synthesis are unique to Bacteria, the -lactam
antibiotics have very high selectivity and are not toxic to eukaryotic cells
Cephalosporins
o Cephalosporins, produced by the fungus Cephalosporium sp., differ structurally from
the penicillins
o The cephalosporins bind irreversibly to the PBPs and prevent the cross-linking of
peptidoglycan
o Cephalosporins have a broader spectrum of antibiotic activity than the penicillins
o Cephalosporins are generally more resistant to the -lactamases
Antibiotics from Prokaryotes
Aminoglycosides
o Aminoglycoside antibiotics contain amino sugars bonded by glycosidic linkages to
other amino sugars
o Aminoglycosides inhibit protein synthesis at the 30S subunit of the ribosome
o Aminoglycosides are clinically useful against gram-negative Bacteria
Macrolides
o Macrolide antibiotics, such as erythromycin, contain large lactone rings connected to
sugar molecules
o Erythromycin is a broad-spectrum inhibitor of protein synthesis
o Erythromycin targets the 50S subunit of the ribosome
Tetracyclines
o The tetracyclines were some of the first broad-spectrum antibiotics, inhibiting almost
all gram-positive and gram-negative Bacteria
o The basic structure of tetracyclines consists of four rings
o Tetracycline is a protein synthesis inhibitor that interferes with the 30S ribosomal
subunit function
Daptomycin
o Daptomycin is a cyclic lipopeptide with a unique mode of action
o Daptomycin binds to bacterial cell membranes and induces rapid depolarization of the
membrane
Platensimycin
o Platensimycin works by selectively inhibiting lipid biosynthesis
Control of Viruses
Antiviral Drugs
Antiviral Chemotherapeutic Agents
o The most successful and commonly used agents for antiviral chemotherapy are the
nucleoside analogs
o Almost all nucleoside analogs, or nucleoside reverse transcriptase inhibitors
(NRTI), work by inhibiting the elongation of the viral nucleic acid chain
o Several other antiviral agents target reverse transcriptase
o The protease inhibitors prevent viral replication by binding the active site of HIV
protease, inhibiting processing of viral polypeptides and virus maturation
Antimicrobial Drug Resistance and Drug Discovery
o Antimicrobial drug resistance is the acquired ability of a microorganism to resist
the effects of a chemotherapeutic agent to which it is normally susceptible
Resistance Mechanisms
o The organism may lack the structure an antibiotic inhibits
o The organism may be impermeable to the antibiotic
o The organism may be able to alter the antibiotic to an inactive form
o The organism may modify the target of the antibiotic
o The organism may develop a resistant biochemical pathway
o The organism may be able to pump out an antibiotic entering the cell (efflux pump)
o Antibiotic resistance can be genetically encoded by the microorganism at either the
bacterial chromosome or in a resistance (R) plasmid
Mechanism of Resistance Mediated by R Plasmids
o Most drug-resistant bacteria isolated from patients contain drug-resistance genes
located on R plasmids
o R plasmids can make enzymes that chemically modify the antibiotics either by
phosphorylation, acetylation, or adenylylation
o In the case of the penicillins, R plasmids encode a penicillinase enzyme
Origin of Resistance Plasmids
o Evidence indicates that the existence of R plasmids predated the antibiotic era
o R plasmids with antibiotic resistance genes have been detected in some
nonpathogenic gram-negative soil bacteria
Spread of Antimicrobial Drug Resistance
o The widespread use of antibiotics in medicine and agriculture provides selective
conditions for the spread of R plasmids
o Inappropriate use of antimicrobial drugs is believed to be a major cause of antibiotic
resistance in disease-causing microorganisms
o Indiscriminant, nonmedical use of antibiotics has also contributed to the emergence of
resistant strains
Antibiotic-Resistant Pathogens
o Failures to properly use antibiotics and monitor resistance has led to pathogenic
microorganisms with resistance to some chemotherapeutic agents
o A few pathogens have developed resistance to all known antimicrobial agents
o A few reports suggest that if the use of a particular antibiotic is stopped, the resistance
to that antibiotic will be reversed over the course of several years
The Search for New Antimicrobial Drugs
New Analogs of Existing Antimicrobial Compounds
o New analogs of existing antimicrobial compounds often work because the new
compounds have a proven mechanism of action
o The application of automated chemistry methods to drug discovery has dramatically
increased our ability to rapidly generate new antimicrobial compounds
o According to pharmaceutical industry estimates, about 7 million candidate
compounds must be screened to yield a single useful clinical drug
Computer Drug Design
o Novel antimicrobial compounds are much more difficult to identify than analogs of
existing drugs
o New antimicrobial compounds must work at unique sites in metabolism and
biosynthesis, or be structurally dissimilar to existing compounds