Medical Microbiology BIOL 2161

Chapter Outline
Ch. 9 – Microbial Growth
I. 9.1 How Microbes Grow. The bacterial cell cycle involves the formation of new cells through the replication of DNA
and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual,
although extensive genetic recombination in the form of horizontal gene transfer takes place. Most bacteria have a
single circular chromosome; however, some exceptions exist. For example, Borrelia burgdorferi, the causative agent
of Lyme disease, has a linear chromosome.
A. Binary Fission. There are 5 stages of binary fission
1. Replication of the circular chromosome begins at the origin and goes in both directions
2. The cell begins to elongate, FtsZ proteins migrate to center of cell
3. Duplicated chromosomes separate and move away from each other. FtsZ protein forms a ring between
the two separating cells
4. FstZ directs the formation of a septum that divides the cell and allows for plasma membrane to reform
5. After the septum is complete the cell pinches in two forming two daughter cells.
B. Generation Time or Doubling Time is the time between the same points of the life cycle in two successive
generations. For example, the typical generation time for the human population is 25 years. is defined as the
time it takes for the population to double through one round of binary fission. Bacterial doubling times vary
enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in
the laboratory, bacteria of the same species may need several days to double in especially harsh environments.
Mycobacterium tuberculosis, the causative agent of tuberculosis, has a generation time of between 15 and 20
hours and M. leprae, which causes Hansen’s disease (leprosy) takes up to 14 days.
C. Growth Curve Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are
added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve. An
example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment.
The culture density is defined as the number of cells per unit volume. In a closed environment, the culture
density is also a measure of the number of cells in the population.
1. The Lag Phase. The beginning of the growth curve represents a small number of cells, referred to as an
inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth. The initial
phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of
growth. The number of cells does not change during the lag phase; however, cells grow larger and are
metabolically active, synthesizing proteins needed to grow within the medium.
2. The Log Phase. In the logarithmic (log) growth phase, sometimes called exponential growth phase, the
cells are actively dividing by binary fission and their number increases exponentially. For any given
bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and
so forth) is genetically determined, and this generation time is called the intrinsic growth rate. During
the log phase, the relationship between time and number of cells is not linear but exponential; however,
the growth curve is often plotted on a semilogarithmic graph which gives the appearance of a linear
relationship. Cells in the log phase show constant growth rate and uniform metabolic activity. For this
reason, cells in the log phase are preferentially used for industrial applications and research work. The
log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and
common antibiotics that affect protein, DNA, and cell-wall synthesis.
3. Stationary Phase. Waste products accumulate, and nutrients are gradually used up. In addition, gradual
depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions
slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as
the stationary phase. In this phase, the number of new cells created by cell division is now equivalent to
the number of cells dying; thus, the total population of living cells is relatively stagnant. During the
stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the
synthesis of peptidoglycans, proteins, and nucleic acids; thus, stationary cultures are less susceptible to
antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells
undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are
synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also
associated with the expression of virulence factors, products that contribute to a microbe’s ability to
 survive, reproduce, and cause disease in a host organism. For example, quorum sensing in
Staphylococcus aureus initiates the production of enzymes that can break down human tissue and
cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.
4. The Death Phase. As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in
greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells,
leading to an exponential decrease in the number of cells. This is the aptly named death phase,
sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing
surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are
characterized by a slow metabolic rate. Persister cells are medically important because they are
associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic
treatment.
5. Sustaining Microbial Growth. In many cases, though, it is advantageous to maintain cells in the
logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat
is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled
amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as
nutrients flow in to maintain an optimal growth environment.
D. Measurement of Bacterial Growth. Estimating the number of bacterial cells in a sample, known as a bacterial
count, is a common task performed by microbiologists. Two major approaches are used to measure cell number.
The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell
presence or activity without actually counting individual cells. Both direct and indirect methods have advantages
and disadvantages for specific applications.
1. Direct Cell Count - counting the cells in a liquid culture or colonies on a plate. It is a direct way of
estimating how many organisms are present in a sample.
a. The simplest way to count bacteria is called the direct microscopic cell count, which involves
transferring a known volume of a culture to a calibrated slide and counting the cells under a
light microscope.
i. The calibrated slide is called a Petroff-Hausser chamber and is similar to a
hemocytometer used to count red blood cells. The central area of the counting chamber
is etched into squares of various sizes. A sample of the culture suspension is added to
the chamber under a coverslip that is placed at a specific height from the surface of the
grid.
ii. It is possible to estimate the concentration of cells in the original sample by counting
individual cells in a number of squares and determining the volume of the sample
observed. Cells in several small squares must be counted and the average taken to
obtain a reliable measurement.
iii. The advantages of the chamber are that the method is easy to use, relatively fast, and
inexpensive. On the downside, the counting chamber does not work well with dilute
cultures because there may not be enough cells to count.
iv. Newly developed fluorescence staining techniques make it possible to distinguish viable
and dead bacteria. These viability stains (or live stains) bind to nucleic acids, but the
primary and secondary stains differ in their ability to cross the cytoplasmic membrane.
The primary stain, which fluoresces green, can penetrate intact cytoplasmic
membranes, staining both live and dead cells. The secondary stain, which fluoresces red,
can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live
cells fluoresce green because they only absorb the green stain, whereas dead cells
appear red because the red stain displaces the green stain on their nucleic acids. Counts
of live cells are needed when assessing the extent of an infection, the effectiveness of
antimicrobial compounds and medication, or contamination of food and water.
b. Another technique uses an electronic cell counting device (Coulter counter) to detect and count
the changes in electrical resistance in a saline solution. A glass tube with a small opening is
immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second
electrode is located outside of the tube. As cells are drawn through the small aperture in the
glass tube, they briefly change the resistance measured between the two electrodes and the
 change is recorded by an electronic sensor; each resistance change represents a cell. The
method is rapid and accurate within a range of concentrations; however, if the culture is too
concentrated, more than one cell may pass through the aperture at any given time and skew the
results. This method also does not differentiate between live and dead cells.
c. Viable Plate Count aka simply plate count, is a count of viable or live cells. It is based on the
principle that viable cells replicate and give rise to visible colonies when incubated under
suitable conditions for the specimen. The results are usually expressed as colony-forming units
per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have
landed on the same spot to give rise to a single colony. Furthermore, samples of bacteria that
grow in clusters or chains are difficult to disperse and a single colony may represent several
cells. Some cells are described as viable but nonculturable and will not form colonies on solid
media. For all these reasons, the viable plate count is considered a low estimate of the actual
number of live cells. These limitations do not detract from the usefulness of the method, which
provides estimates of live bacterial numbers.
d. The serial dilution of a culture is an important first step before proceeding to either the pour
plate or spread plate method. The goal of the serial dilution process is to obtain plates with
CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10
to simplify calculation. The number of serial dilutions is chosen according to a preliminary
estimate of the culture density.
i. A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the
first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a
dilution factor of 10, or 1:10, compared with the original culture. From this first dilution,
the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution
solution. The dilution factor is now 1:100 compared with the original culture. This
process continues until a series of dilutions is produced that will bracket the desired cell
concentration for accurate counting.
ii. From each tube, a sample is plated on solid medium using either the pour plate method
or the spread plate method. The plates are incubated until colonies appear. Two to
three plates are usually prepared from each dilution and the numbers of colonies
counted on each plate are averaged. In all cases, thorough mixing of samples with the
dilution medium (to ensure the cell distribution in the tube is random) is paramount to
obtaining reliable results.
iii. A very dilute sample—drinking water, for example—may not contain enough organisms
to use either of the plate count methods described. In such cases, the original sample
must be concentrated rather than diluted before plating. This can be accomplished
using a modification of the plate count technique called the membrane filtration
technique. Known volumes are vacuum-filtered aseptically through a membrane with a
pore size small enough to trap microorganisms. The membrane is transferred to a Petri
plate containing an appropriate growth medium. Colonies are counted after incubation.
Calculation of the cell density is made by dividing the cell count by the volume of filtered
liquid.
e. The number of microorganisms in dilute samples is usually too low to be detected by the plate
count methods described thus far. For these specimens, microbiologists routinely use the most
probable number (MPN) method, a statistical procedure for estimating of the number of viable
microorganisms in a sample. Often used for water and food samples, the MPN method
evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.
2. Indirect Cell Count – Besides direct methods of counting cells, other methods, based on an indirect
detection of cell density, are commonly used to estimate and compare cell densities in a culture.
a. The foremost approach is to measure the turbidity (cloudiness) of a sample of bacteria in a
liquid suspension. The laboratory instrument used to measure turbidity is called a
spectrophotometer. In a spectrophotometer, a light beam is transmitted through a bacterial
suspension, the light passing through the suspension is measured by a detector, and the amount
of light passing through the sample and reaching the detector is converted to either percent
 transmission or a logarithmic value called absorbance (optical density). As the numbers of
bacteria in a suspension increase, the turbidity also increases and causes less light to reach the
detector. The decrease in light passing through the sample and reaching the detector is
associated with a decrease in percent transmission and increase in absorbance measured by the
spectrophotometer. Measuring turbidity is a fast method to estimate cell density as long as
there are enough cells in a sample to produce turbidity.
b. Measuring dry weight of a culture sample is another indirect method of evaluating culture
density without directly measuring cell counts. The cell suspension used for weighing must be
concentrated by filtration or centrifugation, washed, and then dried before the measurements
are taken. The degree of drying must be standardized to account for residual water content. This
method is especially useful for filamentous microorganisms, which are difficult to enumerate by
direct or viable plate count.
c. Recent methods measure cell activity by following the production of metabolic products or
disappearance of reactants. Adenosine triphosphate (ATP) formation, biosynthesis of proteins
and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of
cells.
E. Alternative Patterns of Cell Division. Binary fission is the most common pattern of cell division in prokaryotes,
but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production
of spores in aerial filaments.
1. In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament,
leading to the generation of many new cells at once. The new cells often split from the parent filament
and float away in a process called fragmentation. Fragmentation is commonly observed in the
Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil.
2. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited
by the giant bacterium Epulopiscium. Several daughter cells grow fully in the parent cell, which
eventually disintegrates, releasing the new cells to the environment.
3. Other species may form a long narrow extension at one pole in a process called budding. The tip of the
extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell.
Budding is most common in yeast, but it is also observed in prosthecate bacteria and some
cyanobacteria.
4. The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in
fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low
nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments, elongated
cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give
rise to new colonies.
F. Biofilms. In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a
variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds.
Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment
containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for
example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the
human mouth may contain hundreds of bacterial species.
1. Biofilm Structure
a. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams,
eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-
moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats
downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-
like shape. The structure of biofilms may also change with other environmental conditions such
as nutrient availability.
b. Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal
clusters of microorganisms embedded in a matrix interspersed with open water channels. The
extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the
organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm,
accounting for 50%–90% of the total dry mass.
 c. EPS is a hydrated gel composed primarily of polysaccharides and containing other
macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the
integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste,
and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also
shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans,
white blood cells in the human body).
2. Biofilm Formation. Free-floating microbial cells that live in an aquatic environment are called planktonic
cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate,
where they become sessile (attached to a surface).
a. Steps of Biofilm Formation
i. The first stage involves the attachment of planktonic cells to a surface coated with a
conditioning film of organic material. At this point, attachment to the substrate is
reversible.
ii. Irreversible attachment
iii. Growth and cell division
iv. As cells express new phenotypes that facilitate the formation of EPS, they transition
from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures,
including an extensive matrix and water channels. Appendages such as fimbriae, pili,
and flagella interact with the EPS, and microscopy and genetic analysis suggest that such
structures are required for the establishment of a mature biofilm.
v. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a
planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is
referred to as dispersal.
b. Metabolism within the biofilm. Within a biofilm, different species of microorganisms establish
metabolic collaborations in which the waste product of one organism becomes the nutrient for
another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that
promote the growth of anaerobes.
c. The mechanism by which cells in a biofilm coordinate their activities in response to
environmental stimuli is called quorum sensing. Quorum sensing—which can occur between
cells of different species within a biofilm—enables microorganisms to detect their cell density
through the release and binding of small, diffusible molecules called autoinducers. When the
cell population reaches a critical threshold (a quorum), these autoinducers initiate a cascade of
reactions that activate genes associated with cellular functions that are beneficial only when the
population reaches a critical density. For example, in some pathogens, synthesis of virulence
factors only begins when enough cells are present to overwhelm the immune defenses of the
host. Although mostly studied in bacterial populations, quorum sensing takes place between
bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida albicans, a
common member of the human microbiota that can cause infections in immunocompromised
individuals. The signaling molecules in quorum sensing belong to two major classes.
i. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones
ii. Gram-positive bacteria mostly use small peptides
3. Biofilm and Human Health.
a. The human body harbors many types of biofilms, some beneficial and some harmful. For
example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role
in warding off infections by pathogens. However, other biofilms in the body can have a
detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can
contribute to dental and periodontal disease. Biofilms can also form in wounds, sometimes
causing serious infections that can spread. The bacterium Pseudomonas aeruginosa often
colonizes biofilms in the airways of patients with cystic fibrosis, causing chronic and sometimes
fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body,
causing infections in patients with in-dwelling catheters, artificial joints, or contact lenses.
b. Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-
floating counterparts. Several hypotheses have been proposed to explain why. Cells in the deep
 layers of a biofilm are metabolically inactive and may be less susceptible to the action of
antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics
and antiseptics, preventing them from reaching cells in the deeper layers of the biofilm.
Phenotypic changes may also contribute to the increased resistance exhibited by bacterial cells
in biofilms. For example, the increased production of efflux pumps, membrane-embedded
proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an
important mechanism of antibiotic resistance among biofilm-associated bacteria. Finally,
biofilms provide an ideal environment for the exchange of extrachromosomal DNA, which often
includes genes that confer antibiotic resistance.

II. 9.2 Oxygen Requirements for Microbial Growth. As discussed previously, oxygen was not always present in Earth’s
atmosphere. The Great Oxygenation Event or the Oxygen Revolution, caused a massive extinction. Most organisms
could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable ions and
molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with
which they come in contact. Singlet oxygen (O2•), superoxide (O2−), peroxides (H2O2), hydroxyl radical (OH•), and
hypochlorite ion (OCl−), the active ingredient of household bleach, are all examples of ROS. The organisms that were
able to detoxify reactive oxygen species harnessed the high electronegativity of oxygen to produce free energy for
their metabolism and thrived in the new environment.
A. Oxygen Requirements of Microorganisms. We can easily observe different requirements for molecular oxygen
by growing bacteria in thioglycolate tube cultures. A test-tube culture starts with autoclaved thioglycolate
medium containing a low percentage of agar to allow motile bacteria to move throughout the medium.
1. Types of Metabolisms
a. Obligate (strict) aerobes require O2 for growth; they use O2 as a final electron acceptor in
aerobic respiration. Growth will be restricted to the very top of the broth and often form a film
or cap. Ex: B. subtillis, Mycobacterium tuberculosis, the causative agent of tuberculosis and
Micrococcus luteus, a gram-positive bacterium that colonizes the skin. Neisseria meningitidis,
the causative agent of severe bacterial meningitis, and N. gonorrhoeae, the causative agent of
sexually transmitted gonorrhea.
b. Facultative anaerobes are organisms that can switch between aerobic and anaerobic types of
metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic
respiration, but in the presence of O2 they switch to aerobic respiration. Ex: Staphylococcus
epidermis, and E. coli
c. Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of
metabolism but they are insensitive to the presence of O2. They live by fermentation alone
whether or not O2 is present in their environment. Ex: lactobacilli and streptococci
d. Obligate (strict) anaerobes do not need or use O2 as a nutrient. In fact, O2 is a toxic substance,
which either kills or inhibits their growth. The most common approach is culture in an anaerobic
jar. Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO 2).
An anaerobic chamber is an enclosed box from which all oxygen is removed. Gloves sealed to
openings in the box allow handling of the cultures without exposing the culture to air Ex:
Clostridium spp
e. Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However,
they are poisoned by high concentrations of oxygen found at normal atmospheric levels. They
gather in the upper part of the test tube but not the very top. Ex: Campylobacter jejuni
2. The optimum oxygen concentration, as the name implies, is the ideal concentration of oxygen for a
particular microorganism. The lowest concentration of oxygen that allows growth is called the minimum
permissive oxygen concentration. The highest tolerated concentration of oxygen is the maximum
permissive oxygen concentration. The organism will not grow outside the range of oxygen levels found
between the minimum and maximum permissive oxygen concentrations.

B. Detoxification of Reactive Oxygen Species. Aerobic respiration constantly generates reactive oxygen species
(ROS), byproducts that must be detoxified. Even organisms that do not use aerobic respiration need some way
 to break down some of the ROS that may form from atmospheric oxygen. Three main enzymes break down
those toxic byproducts:
1. superoxide dismutase
2. peroxidase
3. catalase

III. 9.3 The Effects of pH on Microbial Growth

A. The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that
an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH.
These values can cover a wide range, which is important for the preservation of food and to
microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5,
but the minimum growth pH is closer to 4.2.
B. Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the
neutral pH of 7. Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are
neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of
E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In
comparison, fungi thrive at slightly acidic pH values of 5.0–6.0.
C. Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-
oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are
extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Species of the archaean genus
Ferroplasma live in acid mine drainage at pH values of 0–2.9. Lactobacillus bacteria, which are an important
part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also
contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic
production of lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are
less tolerant of acidity.
D. At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and
10.5. Vibrio cholerae, the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can
survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH,
the bright pink archaean Natronobacterium, found in the soda lakes of the African Rift Valley, may hold the
record at a pH of 10.5
IV. 9.4 Temperature and Microbial Growth. Microbes can be roughly classified according to the range of
temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for
the organism. The lowest temperature at which the organism can survive, and replicate is its minimum growth
temperature. The highest temperature at which growth can occur is its maximum growth temperature. The
following ranges of permissive growth temperatures are approximate only and can vary according to other
environmental factors.
A. Organisms categorized as mesophiles (“middle loving”) are adapted to moderate temperatures, with
optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C. As would be
expected from the core temperature of the human body, 37 °C (98.6 °F), normal human microbiota and
pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles.
B. Organisms called psychrotrophs, aka psychrotolerant, prefer cooler environments, from a high
temperature of 25 °C to refrigeration temperature about 4 °C. They are found in many natural
environments in temperate climates. They are also responsible for the spoilage of refrigerated food. The
organisms retrieved from arctic lakes such as Lake Whillans are considered extreme psychrophiles (cold
loving). Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth
temperature close to 15 °C, and usually do not survive at temperatures above 20 °C. They are found in
permanently cold environments such as the deep waters of the oceans. Because they are active at low
temperature, psychrophiles and psychrotrophs are important decomposers in cold climates.
C. Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles
(“heat loving”). They do not multiply at room temperature. Thermophiles are widely distributed in hot
springs, geothermal soils, and manmade environments such as garden compost piles where the
microbes break down kitchen scraps and vegetal material. Examples of thermophiles include Thermus
aquaticus and Geobacillus spp.
 D. Higher up on the extreme temperature scale we find the hyperthermophiles, which are characterized by
growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples that survive
temperatures above 121 °C, the average temperature of an autoclave. The hydrothermal vents at the
bottom of the ocean are a prime example of extreme environments, with temperatures reaching an
estimated 340 °C. Microbes isolated from the vents achieve optimal growth at temperatures higher than
100 °C. Noteworthy examples are Pyrobolus and Pyrodictium, archaea that grow at 105 °C and survive
autoclaving.

V. 9.5 Other Environmental Conditions That Effect Growth

A. Osmotic and Barometric Pressure
1. Most natural environments tend to have lower solute concentrations than the cytoplasm of most
microorganisms. Rigid cell walls protect the cells from bursting in a dilute environment. Not much
protection is available against high osmotic pressure. In this case, water, following its concentration
gradient, flows out of the cell. This results in plasmolysis (the shrinking of the protoplasm away from
the intact cell wall) and cell death. This fact explains why brines and layering meat and fish in salt are
time-honored methods of preserving food. Microorganisms called halophiles (“salt loving”) actually
require high salt concentrations for growth. These organisms are found in marine environments
where salt concentrations hover at 3.5%. Extreme halophilic microorganisms, such as the red alga
Dunaliella salina and the archaeal species Halobacterium grow in hypersaline lakes such as the Great
Salt Lake, which is 3.5–8 times saltier than the ocean, and the Dead Sea, which is 10 times saltier
than the ocean.
2. Microorganisms that require high atmospheric pressure for growth are called barophiles. The
bacteria that live at the bottom of the ocean must be able to withstand great pressures. Because it is
difficult to retrieve intact specimens and reproduce such growth conditions in the laboratory, the
characteristics of these microorganisms are largely unknown.
B. Light.
1. Photoautotrophs, such as cyanobacteria or green sulfur bacteria, and photoheterotrophs, such as
purple nonsulfur bacteria, depend on sufficient light intensity at the wavelengths absorbed by their
pigments to grow and multiply. Energy from light is captured by pigments and converted into
chemical energy that drives carbon fixation and other metabolic processes. The portion of the
electromagnetic spectrum that is absorbed by these organisms is defined as photosynthetically
active radiation (PAR). It lies within the visible light spectrum ranging from 400 to 700 nanometers
(nm) and extends in the near infrared for some photosynthetic bacteria. A number of accessory
pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful
range of wavelengths for photosynthesis and compensate for the low light levels available at greater
depths of water.
2. Other microorganisms, such as the archaea of the class Halobacteria, use light energy to drive their
proton and sodium pumps. The light is absorbed by a pigment protein complex called
bacteriorhodopsin, which is similar to the eye pigment rhodopsin.
3. Photosynthetic bacteria are present not only in aquatic environments but also in soil and in
symbiosis with fungi in lichens. The peculiar watermelon snow is caused by a microalga
Chlamydomonas nivalis, a green alga rich in a secondary red carotenoid pigment (astaxanthin) which
gives the pink hue to the snow where the alga grows.
VI. 9.6 Other Environmental Conditions That Effect Growth
A. Some media are considered general all-purpose media and support growth of a large variety of organisms. A
prime example of an all-purpose medium is tryptic soy broth (TSB). Specialized media are used in the
identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics.
1. Enriched media contains growth factors, vitamins, and other essential nutrients to promote the
growth of fastidious organisms, organisms that cannot make certain nutrients and require them to
be added to the medium.
2. When the complete chemical composition of a medium is known, it is called a chemically defined
medium. For example, in EZ medium, all individual chemical components are identified and the
exact amounts of each is known.
 3. In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise
chemical composition of the medium is not known.
B. Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of
interest by supplying nutrients and reducing competition are called selective media. An example of a
selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth
of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the
Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to
the presence of bile salts. The enrichment cultures foster the preferential growth of a desired
microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want
to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a
medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria.
C. The differential media make it easy to distinguish colonies of different bacteria by a change in the color of
the colonies or the color of the medium. Color changes are the result of end products created by interaction
of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the
lysis of red blood cells in the medium. The differential fermentation of lactose can be observed on
MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong
fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink
at low pH.