Lecture 2
The physiology of
microorganisms. Growth and reproduction of bacteria.
1.
Types of microbial
nutrition
2.
Respiration of
bacteria
3.
Growth and
reproduction of microbes
4.
Bacterial transport
systems
5.
Bacterial enzymes
6.
Nutrient media
7.
Isolated colonies obtaining
A heterotroph is an organism that cannot fix carbon and uses organic carbon for growth. This contrasts with autotrophs, such as plants and algae, which can use energy from sunlight (photoautotrophs) or inorganic compounds (lithoautotrophs
) to produce organic compounds such as carbohydrates,
fats, and proteins
from inorganic carbon dioxide. These reduced carbon compounds can be used as
an energy source by the autotroph and provide the energy in food consumed by
heterotrophs.
Heterotrophs can
be divided into two broad classes: photoheterotrophs
and chemoheterotrophs.
Photoheterotrophs, including most purple
bacteria and green bacteria, produce ATP from light and use organic compounds to
build structures. They consume little or none of the energy produced during photosynthesis
to reduce NADP+ to
NADPH for use in the Calvin cycle, as they do not need to use the Calvin
cycle if carbohydrates are available in their diets. Chemoheterotrophs produce
ATP by oxidizing chemical substances. There are two types of chemoheterotrophs:
chemoorganoheterotrophs and chemolithoheterotrophs.
Chemoorganoheterotrophs
(or simply organotrophs) exploit reduced carbon compounds as energy
sources, such as carbohydrates, fats, and proteins from plants and animals.
Chemolithoheterotrophs (or lithotrophic heterotrophs) such as colorless sulfur
bacteria (e.g., Beggiatoa and Thiobacillus) and sulfate-reducing bacteria utilize
inorganic substances to produce ATP, including hydrogen
sulfide, elemental sulfur, thiosulfate, and molecular hydrogen.
They use organic compounds to build structures. They do not fix carbon dioxide and
apparently do not have the Calvin cycle. Chemolithoheterotrophs can be
distinguished from mixotrophs (or facultative chemolithotroph), which can
utilize either carbon dioxide or organic carbon as the carbon source.
Heterotrophs, by
consuming reduced carbon compounds, are able to use all the energy that they
obtain from food for growth and reproduction, unlike autotrophs, which must use
some of their energy for carbon fixation. Heterotrophs are unable to make their
own food, however, and whether using organic or inorganic energy sources, they
can die from a lack of food. This applies not only to animals and fungi but
also to bacteria.
An autotroph,(self-feeding)
or producer, is an organism
that produces complex organic compounds (such as carbohydrates,
fats, and proteins)
from simple inorganic molecules using energy from light (by photosynthesis)
or inorganic chemical reactions (chemosynthesis).
They are the producers in a food chain,
such as plants
on land or algae
in water.
They are able to make their own food and can fix carbon.
Therefore, they do not use organic compounds as an energy source or a carbon source.
Autotrophs can reduce carbon dioxide (add hydrogen to it) to make organic
compounds. The reduction of carbon dioxide, a low-energy compound, creates a
store of chemical energy. Most autotrophs use water as the reducing
agent, but some can use other hydrogen compounds such as hydrogen
sulfide. An autotroph converts physical energy from sun light (in case of green
plants) into chemical energy in the form of reduced carbon.
Autotrophs
can be phototrophs or lithotrophs (chemoautotrophs).
Phototrophs use light as an energy source, while lithotrophs oxidize inorganic
compounds, such as hydrogen sulfide, elemental sulfur, ammonium
and ferrous iron.
Phototrophs and lithotrophs use a portion of the ATP
produced during photosynthesis or the oxidation of inorganic compounds to
reduce NADP+ to NADPH in order to
form organic compounds.
Autotrophs are
fundamental to the food chains of all ecosystems in the world. They take energy from the environment
in the form of sunlight or inorganic chemicals and use it to create energy-rich
molecules such as carbohydrates. This mechanism is called primary production. Other organisms, called heterotrophs,
take in autotrophs
as food
to carry out functions necessary for their life. Thus, heterotrophs — all animals, almost
all fungi,
as well as
most bacteria
and protozoa
— depend on autotrophs for the energy and raw materials they need. Heterotrophs
obtain energy by breaking down organic molecules (carbohydrates, fats, and
proteins) obtained in food. Carnivorous organisms ultimately rely on autotrophs
because the nutrients
obtained from their heterotroph prey come from autotrophs they consumed.
Most
ecosystems are supported by the autotrophic primary production of plants that capture photons
initially released by nuclear fusion reactions in the sun. The process of photosynthesis
splits a
water molecule (H2O), releasing oxygen (O2) into the atmosphere, and
reducing
carbon dioxide (CO2) to release the hydrogen
atoms that fuel the metabolic process of primary production. Plants convert and store
the energy of the photon into the chemical bonds of simple sugars
during photosynthesis. These plant sugars are polymerized
for storage as long-chain carbohydrates, including other sugars, starch,
and cellulose; glucose is also used to make fats and proteins.
When autotrophs are eaten by heterotrophs, i.e., consumers such as animals,
the carbohydrates,
fats, and proteins
contained in them become energy sources for the heterotrophs. Proteins can be
made using nitrates,
sulfates,
and phosphates
in the soil.
Some organisms rely on organic
compounds as a source of carbon, but are
able to use light
or inorganic compounds as a source of energy. Such
organisms are not defined as autotrophic, but rather as heterotrophic. An organism that obtains
carbon from organic compounds but obtains energy from light is called a photoheterotroph,
while an organism that obtains carbon from organic compounds but obtains energy
from the oxidation of inorganic compounds is termed a chemoheterotroph
or chemolithoheterotroph.
Evidence
suggests that some fungi may also obtain energy from radiation. Such radiotrophic fungi were found growing inside a
reactor of
the Chernobyl nuclear power plant.
·
Autotroph
o
Chemoautotroph
o
Photoautotroph
o
Chemoheterotroph
o
Photoheterotroph
An aerobic
organism or aerobe is an organism that can survive and grow in an oxygenated environment.
Faculitative anaerobes grow and survive in an oxygenated environment and so do
aerotolerant anaerobes.
A microaerophile
is a microorganism
that requires oxygen
to survive, but requires environments containing lower levels of oxygen than
are present in the atmosphere (~20% concentration). Many
microphiles are also capnophiles, as they require an elevated concentration of
carbon dioxide. In the laboratory they can be easily cultivated in a candle
jar. A candle jar is a container into which a lit candle is introduced before
sealing the container's airtight lid. The candle's flame burns until
extinguished by oxygen deprivation, which creates a carbon dioxide-rich,
oxygen-poor atmosphere in the jar.
Examples
include:
·
Borrelia burgdorferi, a species of spirochaete
bacteria that causes Lyme disease in humans.
·
Helicobacter pylori, a species of proteobacteria
that has been linked to peptic ulcers and some types of gastritis.
Some don't consider it a true obligate microaerophile.
·
Campylobacter has been described as microaerophilic.
·
Streptococcus intermedius has also been described
as microaerophilic.
·
Streptococcus pyogenes, a well known
microaerophile that causes streptococcal pharyngitis.
A facultative
anaerobic organism is an organism, usually a bacterium, that makes ATP by aerobic respiration if oxygen is present
but is also capable of switching to fermentation. In contrast, obligate
anaerobes die in the presence of oxygen.
Some examples of
facultative anaerobic bacteria are Staphylococcus
(Gram
positive), Escherichia coli and Shewanella oneidensis (Gram
negative), and Listeria (Gram positive). Certain eukaryote phyla are also
facultative anaerobes, including fungi such as yeasts and many aquatic invertebrates
such as Nereid (worm) polychaetes,
for example. There are also circulating white
blood cells that are classified as facultative anaerobes. These include neutrophils,
monocytes
and tissue macrophages.
The
concentrations of oxygen and fermentable material in the environment influence
the organism's use of aerobic respiration vs. fermentation to derive energy.
In brewer's yeast, the Pasteur shift
is the observed cessation of oxygen consumption when fermentable sugar is
supplied. In a growing culture, the energy "economics" disfavors
respiration due to the "overhead cost" of producing the apparatus, as
long as sufficient fermentable substrate is available, even though the energy
output per mole of fermented material is far less than
from respiration's complete oxidation of the same substrate. However, the rate
of production
of ATP can be up to a 100 times faster than that of oxidative phosphorylation .
Therefore, tissues and organisms that require fast consumption of ATP
preferentially use anaerobic glycolysis.
Obligate
anaerobes are microorganisms that live and grow in the absence of molecular oxygen;
some of these are killed by oxygen. (adj., anaero´bic., adj.)
Historically, it
was widely accepted that obligate (strict) anaerobes die in presence of oxygen
due to the absence of the enzymes superoxide dismutase and catalase,
which would convert the lethal superoxide formed in their cells due to the presence of
oxygen. While this is true in some cases, these enzyme activities have been
identified in some obligate anaerobes, and genes for these enzymes and related
proteins have been found in their genomes, such as Clostridium butyricum and Methanosarcina
barkeri, among others. However, these organisms are still incapable of
growing in the presence of oxygen. There are several hypotheses addressing why
strict anaerobes are sensitive to oxygen:
1. Dissolved oxygen
increases the redox potential of a solution, and
high redox potential inhibits the growth of some strict anaerobes. For example,
methanogens grow at a redox potential lower than -0.3 V.
2. Sulfide is an essential
component of some enzymes, and molecular oxygen oxidizes this to form
disulfide, thus inactivating certain enzymes. Organisms may not be able to grow
without these deactivated enzymes.
3. Growth may be inhibited
due to a lack of reducing equivalents for biosynthesis, because electrons are
exhausted in reducing oxygen.
It is most
likely a combination of these mechanisms that accounts for oxygen sensitivity
in obligate anaerobes.
Instead of
oxygen, obligate anaerobes use alternate electron
acceptors for cellular respiration such as sulfate,
nitrate,
iron, manganese,
mercury, and carbon
monoxide. The energy yield of these respiratory processes is less than oxygen respiration, and
not all of these electron acceptors are created equal.
·
In marine sediments large amounts of sulfate-reducing bacteria causes the rotten egg smell and
black material that can be found just a few centimeters below the sediment
surface.
Bacteroides and Clostridium
species are examples of non-spore forming and spore-forming strict anaerobes,
respectively.
Other obligate
anaerobes include Peptostreptococcus, Veillonella,
and Actinomyces.
Capnophiles are microorganisms which
thrive in the presence of high concentrations of carbon dioxide.
Some capnophiles
may have a metabolic requirement for carbon dioxide, while others merely
compete more successfully for resources under these conditions. The term is a
generally a descriptive one and has less relevance as a means of establishing a
taxonomic or evolutionary relationship among organisms with this characteristic
.
For example, the
ability of capnophiles to tolerate (or utilize) the amount of oxygen that is
also in their environment may vary widely and may be far more critical to their
survival. Species of Campylobacter are bacterial capnophiles that are
more easily identified because they are also microaerophiles,
organisms that can grow in high carbon dioxide as long as a small amount of
free oxygen is present, but at a dramatically reduced concentration . (In the earth’s atmosphere carbon dioxide levels are
approximately five hundred times lower than that of oxygen, 0.04% and 21% of
the total, respectively.) Obligate
anaerobes are microbes that will die in the presence of oxygen
without respect to the concentration of carbon dioxide in their environment,
and typically acquire energy through anaerobic respiration, or fermentation (biochemistry).
In 2004, a
capnophilic bacterium was characterized that appears to require carbon dioxide.
This organism, Mannheimia succiniciproducens, has a unique metabolism
involving carbon fixation . While carbon fixation is
common to most plant life on earth since it is the key initial step in the
biosynthesis of complex carbon compounds during photosynthesis (the Calvin cycle),
it is found in relatively few microorganisms and not found in animals.
Mannheimia succiniciproducens can attach carbon dioxide to the three-carbon
backbone of phosphoenolpyruvate, an endproduct in glycolysis,
to generate the four-carbon compound, oxaloacetic
acid, an intermediate in the Krebs cycle.
Although Mannheimia succiniciproducens has most of the intermediates in the
Krebs cycle, it is not capable of oxidative phosphorylation, the final step
in the electron transport chain that would allow
it to carry out the highly efficient process of harvesting energy by aerobic respiration .
Pathogenicity
There are
currently at least two relatively well characterized capnophilic groups of
microorganisms that include human pathogens. Campylobacter
species can cause intestinal disorders . Other capnophilic pathogens occur in
the Gram-negative Aggregatibacter spp. found in the mouth (Actinobacillus
actinomycetemcomitans | Aggregatibacter actinomycetemcomitans). These are a
cause of aggressive juvenile periodontitis .
However, capnophiles
are also normal flora in some ruminants. Mannheimia succiniciproducens,
in particular, was isolated from a bovine rumen. Its unusual biochemistry and
benign characteristics have attracted commercial interest .
Aerobic and anaerobic bacteria can be identified by growing them in a
liquid culture:
1: Obligate aerobic bacteria gather at the top of the test tube in order to
absorb maximal amount of oxygen.
2: Obligate anaerobic bacteria gather at the bottom to avoid oxygen.
3: Facultative bacteria gather mostly at the top, since aerobic respiration is
the most beneficial one; but as lack of oxygen does not hurt them, they can be
found all along the test tube.
4: Microaerophiles gather at the upper part of the test tube but not at the
top. They require oxygen but at a low concentration.
5: Aerotolerant bacteria are not affected at all by oxygen, and they are evenly
spread along the test tube.
Psychrophiles or cryophiles
(adj. cryophilic) are extremophilic organisms that
are capable of growth and reproduction in cold temperatures, ranging
from −15°C to +10°C. Temperatures as low as −15°C are found in
pockets of very salty water (brine) surrounded by sea ice. They can be
contrasted with thermophiles, which thrive at unusually hot temperatures.
The environments they inhabit are ubiquitous on Earth, as a large fraction of our
planetary surface experiences temperatures lower than 15°C. They are present in alpine
and arctic
soils, high-latitude
and deep ocean
waters, polar ice,
glaciers,
and snowfields.
They are of particular interest to astrobiology,
the field dedicated to the formulation of theory about the possibility of
extraterrestrial life, and to geomicrobiology,
the study of microbes active in geochemical processes. In experimental work at University of Alaska Fairbanks,
a 1000 litre biogas
digester
using psychrophiles harvested from "mud from a frozen lake in Alaska"
has produced 200–300 litres of methane per day, about 20–30 % of the
output from digesters in warmer climates.
Psychrophiles
use a wide variety of metabolic pathways, including photosynthesis,
chemoautotrophy (also sometimes known as lithotrophy),
and heterotrophy,
and form robust, diverse communities. Most psychrophiles are bacteria
or archaea,
and psychrophily is present in widely diverse microbial lineages within those
broad groups. Additionally, recent research has discovered novel groups of
psychrophilic fungi
living in oxygen-poor areas under alpine snowfields. A further group of eukaryotic
cold-adapted organisms are snow algae, which can cause watermelon
snow. Psychrophiles are characterized by lipid cell membranes
chemically resistant to the stiffening caused by extreme cold, and often create
protein 'antifreezes' to keep their internal space liquid and protect their DNA even in temperatures below
water's freezing point.
Examples are Arthrobacter
sp., Psychrobacter
sp. and members of the genera Halomonas,
Pseudomonas,
Hyphomonas
and Sphingomonas.
A mesophile
is an organism
that grows best in moderate temperature, neither too hot nor too cold,
typically between 25 and 40 °C (77 and 104 °F).
The term is mainly applied to microorganisms.
The habitats of
these organisms include especially cheese, yogurt and mesophile organisms are often included in the
process of beer
and wine
making.
Organisms
that prefer
cold environments are termed psychrophilic, those preferring warmer temperatures are
termed thermophilic
and those thriving in extremely hot environments are hyperthermophilic.
All bacteria
have their own optimum environmental surroundings and temperatures in which
they thrive the most.
A thermophile
is an organism — a type of extremophile — that thrives at relatively high
temperatures, between 45 and 122 °C
(113 and 252 °F). Many thermophiles are archaea.
Thermophilic eubacteria are suggested to be among the earliest
bacteria.
Thermophiles are
found
in various geothermally heated regions of the Earth, such as hot springs
like those in Yellowstone National Park (see image) and deep sea
hydrothermal vents, as well as decaying plant
matter, such as peat bogs and compost.
As a
prerequisite for their survival, thermophiles contain enzymes that can
function at high temperatures. Some of these enzymes are used in molecular
biology (for example, heat-stable DNA
polymerases for PCR), and in washing agents.
"Thermophile"
is derived from the Greek:
θερμότητα (thermotita), meaning
heat, and Greek: φίλια (philia), love.
A scientific
conference for those who study thermophiles has been held since 1990 at
locations throughout the world, including
Thermophiles are
classified into obligate and facultative thermophiles: Obligate thermophiles
(also called extreme thermophiles) require such high temperatures for growth,
whereas facultative thermophiles (also called moderate thermophiles) can thrive
at high temperatures, but also at lower temperatures (below 50°C). Hyperthermophiles
are particularly extreme thermophiles for which the optimal temperatures are
above 80°C.
Bacteria within
the Alicyclobacillus genus are acidophilic thermophiles, which can cause
contamination in fruit juice drinks.
Thermophiles, meaning heat-loving, are organisms with an optimum growth temperature of 50°C or more, a maximum of up to 70°C or more, and a minimum of about 40°C, but these are only approximate. Some extreme thermophiles (hyperthermophiles
) require a very high temperature (80°C to 105°C) for growth. Their
membranes and proteins are unusually stable at these extremely high
temperatures. Thus, many important biotechnological
processes
use thermophilic enzymes
because of their ability to withstand intense heat.
Many of the hyperthermophiles
Archea require elemental sulfur for growth. Some are anaerobes
that use the sulfur instead of oxygen as an electron
acceptor during cellular respiration. Some are lithotrophs
that oxidize sulfur to sulfuric acid as an energy source, thus
requiring the microorganism to be adapted to very low pH (i.e., it is an acidophile as well as thermophile). These
organisms are inhabitants of hot, sulfur-rich environments usually associated
with volcanism,
such as hot springs,
geysers,
and fumaroles.
In these places, especially in
Bacterial
growth
is the division of one bacterium
into two daughter cells in a process called binary
fission. Providing no mutational event occurs the resulting daughter cells
are genetically identical to the original cell. Hence, "local
doubling" of the bacterial population occurs. Both daughter cells from the
division do not necessarily survive. However, if the number surviving exceeds
unity on average, the bacterial population undergoes exponential growth. The measurement of an
exponential bacterial growth curve in batch culture was traditionally a part of
the training of all microbiologists; the basic means requires bacterial
enumeration (cell counting) by direct and individual (microscopic, flow
cytometry), direct and bulk (biomass), indirect and individual (colony
counting), or indirect and bulk (most probable number, turbidity, nutrient
uptake) methods. Models reconcile theory with the measurements.
In autecological studies, bacterial growth in batch
culture can be modeled with four different phases: lag phase (A), exponential
or log phase (B), stationary phase (C), and death phase (D). IN
the book "black" the bacterial growth phase classified 07 stages
like-(A)lag phase (B)early log phase (C) log/exponential Phase (D)Early
Stationery phase (E)stationary phase (f) Early Death phase (G)Death phase..
1.
During lag phase, bacteria
adapt themselves to growth conditions. It is the period where the individual bacteria
are maturing and not yet able to divide. During the lag phase of the bacterial
growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2.
Exponential phase (sometimes called the log phase
or the logarithmic phase) is a period characterized by cell doubling. The
number of new bacteria appearing per unit time is proportional to the present
population. If growth is not limited, doubling will continue at a constant rate
so both the number of cells and the rate of population increase doubles with
each consecutive time period. For this type of exponential growth, plotting the
natural logarithm of cell number against time produces a straight line. The
slope of this line is the specific growth rate of the organism, which is a
measure of the number of divisions per cell per unit time. The actual rate of
this growth (i.e. the slope of the line in the figure) depends upon the growth
conditions, which affect the frequency of cell division events and the
probability of both daughter cells surviving. Under controlled conditions, cyanobacteria
can double their population four times a day. Exponential growth cannot
continue indefinitely, however, because the medium is soon depleted of
nutrients and enriched with wastes.
3.
During stationary phase, the growth rate slows as
a result of nutrient depletion and accumulation of toxic products. This phase
is reached as the bacteria begin to exhaust the resources that are available to
them. This phase is a constant value as the rate of bacterial growth is equal
to the rate of bacterial death.
4.
At death phase, bacteria run out of nutrients and
die.
This basic batch
culture growth model draws out and emphasizes aspects of bacterial growth which
may differ from the growth of macrofauna. It emphasizes clonality, asexual
binary division, the short development time relative to replication itself, the
seemingly low death rate, the need to move from a dormant state to a
reproductive state or to condition the media, and finally, the tendency of lab
adapted strains to exhaust their nutrients.
In reality, even
in batch culture, the four phases are not well defined. The cells do not
reproduce in synchrony without explicit and continual prompting (as in
experiments with stalked bacteria ) and their exponential phase growth is often
not ever a constant rate, but instead a slowly decaying rate, a constant
stochastic response to pressures both to reproduce and to go dormant in the
face of declining nutrient concentrations and increasing waste concentrations.
Batch culture is
the most common laboratory growth method in which bacterial growth is studied,
but it is only one of many. It is ideally spatially unstructured and temporally
structured. The bacterial culture is incubated in a closed vessel with a single
batch of medium. In some experimental regimes, some of the bacterial culture is
periodically removed and added to fresh sterile medium. In the extreme case,
this leads to the continual renewal of the nutrients. This is a chemostat,
also known as continuous culture. It is ideally spatially unstructured and
temporally unstructured, in a steady state defined by the rates of nutrient
supply and bacterial growth. In comparison to batch culture, bacteria are
maintained in exponential growth phase, and the growth rate of the bacteria is
known. Related devices include turbidostats
and auxostats.
Bacterial growth
can be suppressed
with bacteriostats,
without necessarily killing the bacteria. In a synecological,
true-to-nature situation in which more than one bacterial species is present, the
growth of microbes is more dynamic and continual.
Liquid is not
the only laboratory environment for bacterial growth. Spatially structured
environments such as biofilms or agar surfaces present additional complex growth models.
Passive
transport means
moving
biochemicals
and other atomic
or molecular
substances across membranes. Unlike active
transport, this process does not involve chemical
energy,
because, unlike in an active transport, the transport across membrane is always
coupled with the growth of entropy of the system. So passive transport is dependent on
the permeability of the cell membrane, which,
in turn, is dependent on the organization and characteristics of the membrane lipids and proteins. The four main kinds of
passive transport are diffusion, facilitated diffusion, filtration
and osmosis.
Diffusion is the
net movement of material from an area of high concentration to an area with lower
concentration. The difference of concentration between the two areas is often
termed as the concentration gradient, and diffusion will continue until
this gradient has been eliminated. Since diffusion moves materials from an area
of higher concentration to the lower, it is described as moving solutes
"down the concentration gradient" (compared with active
transport, which often moves material from area of low concentration to
area of higher concentration, and therefore referred to as moving the material
"against the concentration gradient"). And that is why diffusion and
osmosis are the same. passive transport requires energy just unlike active
transport.
Facilitated
diffusion, also called carrier-mediated diffusion, is the movement of molecules
across the cell membrane via special transport
proteins that are embedded within the cellular membrane. Many large
molecules, such as glucose, are insoluble in lipids and too large
to fit through the membrane pores. Therefore, it will bind with its specific
carrier proteins, and the complex will then be bonded to a receptor site and moved through the
cellular membrane. Bear in mind, however, that facilitated diffusion is a
passive process, and the solutes still move down the concentration gradient.
Filtration is
movement of water and solute molecules across the cell membrane due to
hydrostatic pressure
generated by the cardiovascular system. Depending on the size of
the membrane pores, only solutes of a certain size may pass through it. For
example, the membrane pores of the Bowman's
capsule in the kidneys are very small, and only albumins,
the smallest of the proteins, have any chance of being filtered through. On the
other hand, the membrane pores of liver cells are extremely large, to allow a variety of solutes to
pass through and be metabolized.
Osmosis is the diffusion of water molecules across a selectively
permeable membrane. The net movement of water molecules through a partially
permeable membrane from a solution of high water potential to an area of low
water potential. A cell with a less negative water potential will draw in water
but this depends on other factors as well such as solute potential (pressure in
the cell e.g. solute molecules) and pressure potential (external pressure e.g.
cell wall). Because the cell wall is a wall around the wall of a cell of a cell
like a cell membrane.
Active
transport is the movement of a substance against its concentration gradient (from
low to high concentration). In all cells, this is usually concerned with
accumulating high concentrations of molecules that the cell needs, such as
ions, glucose, and amino acids. If the process uses chemical energy, such as from adenosine triphosphate (ATP), it is termed
primary active transport. Secondary active transport involves the
use of an electrochemical gradient. Active transport
uses energy, unlike passive transport, which does not use any type
of energy. Active transport is a good example of a process for which cells
require energy. Examples of active transport include the uptake of glucose in
the intestines in humans and the uptake of mineral ions into root hair cells of plants.
Specialized
trans-membrane proteins recognize the substance and
allows it access (or, in the case of secondary transport, expend energy on
forcing it) to cross the membrane when it otherwise would not, either because
it is one to which the phospholipid bilayer of the membrane is
impermeable or because it is moved in the direction of the concentration gradient. The last case, known
as primary active transport, and the proteins involved in it as pumps, normally
uses the chemical energy of ATP. The other cases, which usually derive their
energy through exploitation of an electrochemical gradient, are known as secondary
active transport and involve pore-forming proteins that form channels through
the cell membrane.
Sometimes the
system transports one substance in one direction at the same time as cotransporting
another substance in the other direction. This is called antiport. Symport is the
name if two substrates are being transported in the same direction across the
membrane. Antiport and symport are associated with secondary active transport, meaning that
one of the two substances are transported in the direction of their
concentration gradient utilizing the energy derived from the transport of
second substance (mostly Na+, K+ or H+) down its concentration gradient.
Particles moving
from areas of low concentration to areas of high concentration (i.e., in the
opposite direction as the concentration gradient) require specific
trans-membrane carrier proteins. These proteins have receptors that bind to
specific molecules (e.g., glucose) and thus transport them into
the cell. Because energy is required for this process, it is known as 'active'
transport. Examples of active transport include the transportation of sodium
out of the cell and potassium into the cell by the sodium-potassium pump.
Active transport often takes place in the internal lining of the small
intestine.
Plants need to
absorb mineral salts from the soil or other sources, but these salts exist in
very dilute solution. Active transport enables these cells to take up salts
from this dilute solution against the direction of the concentration gradient.
Primary active
transport, also called direct active transport, directly uses energy to
transport molecules across a membrane.
Most of the enzymes that
perform this type of transport are transmembrane ATPases. A primary ATPase universal
to all life is the sodium-potassium pump, which helps to maintain the cell
potential. Other sources of energy for Primary active transport are redox energy and photon energy (light). An example of
primary active transport using Redox energy is the mitochondrial electron transport chain that uses the
reduction energy of NADH
to move protons across the inner mitochondrial membrane against their
concentration gradient. An example of primary active transport using light
energy are the proteins involved in photosynthesis
that use the energy of photons to create a proton gradient across the thylakoid membrane and also to create reduction
power in the form of NADPH.
1. P-type
ATPase: sodium potassium pump, calcium
pump, proton
pump
2. F-ATPase:
mitochondrial ATP synthase, chloroplast ATP synthase
3. V-ATPase:
vacuolar ATPase
4. ABC (ATP binding cassette) transporter: MDR, CFTR,
etc.
In secondary
active transport
or co-transport,
energy is used to transport molecules across a membrane; however, in contrast
to primary active transport, there is no
direct coupling of ATP; instead, the electrochemical potential difference
created by
pumping ions out of the cell is used.
The two main
forms of this are antiport and symport.
In antiport two
species of ion or other solutes are pumped in opposite directions across a
membrane. One of these species is allowed to flow from high to low concentration
which yields the entropic
energy to drive the transport of the other solute from a low concentration
region to a high one. An example is the sodium-calcium exchanger or antiporter,
which allows three sodium ions into the cell to transport one calcium out.
Many cells
also possess a calcium ATPase, which can operate at lower
intracellular concentrations of calcium and sets the normal or resting
concentration of this important second
messenger. But the ATPase exports calcium ions more slowly: only 30 per
second versus 2000 per second by the exchanger. The exchanger comes into
service when the calcium concentration rises steeply or "spikes" and
enables rapid recovery. This shows that a single type of ion can be transported
by several enzymes, which need not be active all the time (constitutively), but
may exist to meet specific, intermittent needs.
Symport uses the
downhill movement of one solute species from high to low concentration to move another
molecule uphill from low concentration to high concentration (against its electrochemical gradient).
An example
is the glucose symporter SGLT1, which co-transports
one glucose
(or galactose)
molecule into the cell for every two sodium ions it imports into the cell. This
symporter
is located in the small intestines, trachea, heart, brain, testis, and
prostate. It is also located in the S3 segment of the proximal
tubule in each nephron in the kidneys. Its mechanism is exploited in glucose rehydration therapy and defects in
SGLT1 prevent effective reabsorption of glucose, causing familial renal
glucosuria.
·
Water,
ethanol,
and chloroform
exemplify simple molecules that do not require active transport to cross a
membrane.
·
Metal ions, such as Na+,
K+,
Mg2+,
or Ca2+,
require ion pumps or ion channels
to cross membranes and distribute through the body
·
The pump for sodium and potassium is called sodium-potassium pump or Na +/K+-ATPase
·
In the epithelial
cells of the stomach, gastric acid
is produced by hydrogen potassium ATPase, an electrogenic pump
Further information: Endocytosis
Endocytosis is
the process by which cells take in materials. The cellular membrane folds
around the desired materials outside the cell. The ingested particle becomes
trapped within a pouch, vacuole or inside the cytoplasm.
Often enzymes from lysosomes are then used to digest the molecules absorbed by
this process.
Biologists
distinguish two main types of endocyctosis: pinocytosis
and phagocytosis.
·
In pinocytosis, cells engulf liquid particles (in humans
this process occurs in the small intestine, cells there engulf fat droplets).
·
In phagocytosis, cells engulf solid particles.
An endoenzyme,
or intracellular enzyme, is an enzyme that
functions within the cell in which it was produced. Because the
majority of enzymes fall within this category, the term is used primarily to
differentiate a specific enzyme from an exoenzyme. It
is possible for a single enzyme to have both endoenzymatic and exoenzymatic
functions.
An exoenzyme,
or extracellular enzyme, is an enzyme that is
secreted by a cell and that works outside
of that cell. It is usually used for breaking up large molecules that would
not be able to enter the cell otherwise.
This term is
also often used to refer to the hydrolytic digestive enzymes secreted by fungi.
Constitutive
enzymes are produced in constant amounts without regard to the physiological
demand or the
concentration of the substrate. They are continuously synthesized because their
role in maintaining cell processes or structure is indispensable.
·
Ligase stubs
·
Lyase stubs
A growth
medium or culture medium is a liquid or gel designed to support the
growth of microorganisms or cells,
or small plants
like the moss
Physcomitrella patens. There are different types of
media for growing different types of cells.
There are two
major types
of growth media: those used for cell culture,
which use specific cell types derived from plants or animals, and microbiological culture, which are used
for growing microorganisms, such as bacteria
or yeast.
The most common growth media for microorganisms are nutrient broths and agar plates;
specialized media are sometimes required for microorganism and cell culture
growth. Some organisms, termed fastidious organisms, require specialized
environments due to complex nutritional requirements. Viruses, for example,
are obligate intracellular parasites and require a growth medium containing living
cells.
The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth
). Liquid media are often mixed with agar and poured into Petri dishes
to solidify. These agar plates provide a solid medium on which microbes
may be cultured. They remain solid, as very few bacteria are able to decompose
agar. Bacteria grown in liquid cultures often form colloidal
suspensions.
The difference
between growth media used for cell culture and those used for microbiological
culture is that cells derived from whole organisms and grown in culture
often cannot grow without the addition of, for instance, hormones
or growth
factors which usually occur in vivo.
In the case of animal cells, this difficulty is often addressed by the addition
of blood serum
or a synthetic serum replacement to the medium. In the case of microorganisms,
there are no such limitations, as they are often unicellular organisms. One other major difference
is that animal cells in culture are often grown on a flat surface to which they
attach, and the medium is provided in a liquid form, which covers the cells. In
contrast, bacteria such as Escherichia
coli may be grown on solid media or in liquid media.
An important
distinction between growth media types is that of defined versus undefined media. A
defined medium will have known quantities of all ingredients. For
microorganisms, they consist of providing trace elements and vitamins required
by the microbe and especially a defined carbon source
and nitrogen
source. Glucose or glycerol are often used as carbon sources, and ammonium
salts or nitrates
as inorganic
nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract
or casein
hydrolysate, which consist of a mixture of many, many chemical species in
unknown proportions. Undefined media are sometimes chosen based on price and
sometimes by necessity - some microorganisms have never been cultured on
defined media.
A good example of a growth medium
is the wort used to make beer. The wort contains
all the nutrients required for yeast growth, and under anaerobic
conditions, alcohol is produced. When the fermentation process is complete, the
combination of medium and dormant microbes, now beer, is ready for consumption.
·
a source of amino acids and nitrogen (e.g., beef, yeast
extract)
This is an
undefined medium because the amino acid source contains a variety of compounds
with the exact composition being unknown. Nutrient media contain all the
elements that most bacteria need for growth and are non-selective, so they are
used for the general cultivation and maintenance of bacteria kept in laboratory
culture collections.
An undefined
medium (also known as a basal or complex medium) is a medium
that contains:
·
a carbon source such as glucose for
bacterial growth
·
water
·
various salts needed for bacterial growth
A defined
medium (also known as chemically defined medium or synthetic
medium) is a medium in which
·
all the chemicals used are known
·
no yeast, animal or plant tissue is present
A differential
medium is a medium that includes
·
some sort of added indicator that allows for the
differentiation of particular chemical reactions occurring during growth.
Minimal media are those that contain
the minimum nutrients possible for colony growth, generally without the presence
of amino acids, and are often used by microbiologists and geneticists to grow
"wild type" microorganisms. Minimal media can also be used to select for or
against recombinants or exconjugants.
Minimal medium
typically contains:
·
a carbon source for bacterial growth, which may be a
sugar such as glucose,
or a less energy-rich source like succinate
·
various salts, which may vary among bacteria species and growing
conditions; these generally provide essential elements such as magnesium,
nitrogen,
phosphorus,
and sulfur
to allow the bacteria to synthesize protein
and nucleic acid
·
water
Supplementary
minimal media are a type of minimal media that also contains a single selected agent, usually an amino acid
or a sugar.
This supplementation allows for the culturing of specific lines of auxotrophic
recombinants.
Blood agar plates are often used to diagnose infection. On the right is a
positive Streptococcus culture; on the left a positive Staphylococcus
culture.
Selective
media are used for the
growth of only selected microorganisms. For example, if a microorganism is
resistant to a certain antibiotic, such as ampicillin
or tetracycline,
then that antibiotic can be added to the medium in order to prevent other
cells, which do not possess the resistance, from growing. Media lacking an amino acid
such as proline
in conjunction with E. coli unable to synthesize it were commonly
used by geneticists before the emergence of genomics
to map bacterial chromosomes.
Selective growth
media are also used in cell culture to ensure the survival or
proliferation of cells with certain properties, such as antibiotic resistance or the ability to
synthesize a certain metabolite. Normally, the presence of a specific gene or an allele of a
gene confers upon the cell the ability to grow in the selective medium. In such
cases, the gene is termed a marker.
Selective
growth media for eukaryotic cells commonly contain neomycin
to select cells that have been successfully transfected
with a plasmid carrying the neomycin resistance gene as a marker. Gancyclovir
is an exception to the rule as it is used to specifically kill cells that carry
its respective marker, the Herpes simplex virus thymidine kinase (HSV TK).
Four types of agar plates demonstrating differential growth depending on
bacterial metabolism.
Some examples of
selective media include:
·
eosin methylene blue (EMB) that contains methylene
blue – toxic to Gram-positive bacteria, allowing only the growth of Gram
negative bacteria
·
YM (yeast and mold) which has a low pH, deterring bacterial
growth
·
MacConkey agar for Gram-negative
bacteria
·
Hektoen enteric agar (HE) which is selective
for Gram-negative bacteria
·
mannitol salt agar (MSA) which is selective for
Gram-positive bacteria and differential for mannitol
·
Terrific Broth (TB) is used with glycerol in cultivating
recombinant strains of Escherichia coli.
·
xylose lysine desoxyscholate (XLD), which is selective for
Gram-negative bacteria
·
buffered charcoal yeast extract
agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila
Differential
media or
indicator media distinguish one microorganism type from another growing on
the same media. This type of media uses the biochemical characteristics of a
microorganism growing in the presence of specific nutrients or indicators (such
as neutral
red, phenol
red, eosin y,
or methylene
blue) added to the medium to visibly indicate the defining characteristics
of a microorganism. This type of media is used for the detection of
microorganisms and by molecular biologists to detect recombinant strains of
bacteria.
Examples of
differential media include:
·
blood agar (used in strep tests), which contains bovine
heart blood that becomes transparent in the presence of hemolytic Streptococcus
·
eosin methylene blue (EMB), which is
differential for lactose and sucrose fermentation
·
MacConkey (MCK), which is differential for lactose
fermentation
·
mannitol salt agar (MSA), which is differential
for mannitol fermentation
·
X-gal
plates, which are differential for lac operon
mutants
Transport media
should fulfill the following criteria:
·
temporary storage of specimens being transported to the
laboratory for cultivation.
·
maintain the viability of all organisms in the specimen
without altering their concentration.
·
contain only buffers and salt.
·
lack of carbon, nitrogen, and organic growth factors so
as to prevent microbial multiplication.
·
transport media used in the isolation of anaerobes must
be free of molecular oxygen.
Examples of
transport media include:
·
Thioglycolate broth for strict anaerobes.
·
Stuart transport medium - a non-nutrient soft agar gel containing
a reducing agent to prevent oxidation, and charcoal to neutralise
·
Certain bacterial inhibitors- for gonococci, and buffered
glycerol saline for enteric bacilli.
·
Venkat-Ramakrishnan(VR) medium for v. cholerae.
Enriched media
contain the nutrients required to support the growth of a wide variety of
organisms, including some of the more fastidious ones. They are commonly used
to harvest as many different types of microbes as are present in the specimen. Blood agar
is an enriched medium in which nutritionally rich whole blood supplements the
basic nutrients. Chocolate agar is enriched with heat-treated blood
(40-45°C), which turns brown and gives the medium the color for which it is
named.
A bacterial
colony is defined as a visible cluster of bacteria
growing on the surface of or within a solid medium, presumably cultured from a
single cell. Because all organisms within the colony descend from a single
ancestor, they are genetically identical (except for mutations that
occur at a low, unavoidable frequency, as well as the more likely possibility
of contamination).
Obtaining such genetically identical organisms (or pure strains)
can be useful in many cases; this is done by spreading bacteria on a culture
plate and starting a new stock of bacteria from a single colony.
A biofilm is a
colony of microorganisms often comprising several species, with
properties and capabilities greater than the aggregate of capabilities of the
individual organisms.
A microbiological
culture, or microbial culture, is a method of multiplying microbial
organisms by letting them reproduce in predetermined culture media under
controlled laboratory conditions. Microbial cultures are used to determine
the type of organism, its abundance in the sample being tested, or both. It is
one of the primary diagnostic methods of microbiology
and used as a tool to determine the cause of infectious disease by letting the agent
multiply in a predetermined medium. For example, a throat culture is taken by
scraping the lining of tissue in the back of the throat and blotting the sample
into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative
agent of strep throat. Furthermore, the term culture is more generally used
informally to refer to "selectively growing" a specific kind of
microorganism in the lab.
Microbial
cultures are foundational and basic diagnostic methods used extensively as a
research tool in molecular biology. It is often essential to
isolate a pure culture of microorganisms. A pure (or axenic) culture is a
population of cells or multicellular organisms growing in the
absence of other species
or types. A pure culture may originate from a single cell or single organism,
in which case the cells are genetic clones of one another.
For the purpose
of gelling the microbial culture, the medium of agarose gel (agar) is used.
Agar is a gelatinous substance derived from seaweed. A cheap substitute for
agar is guar gum, which can be used for the isolation and maintenance of
thermophiles.
Microbiological
cultures can be grown in petri dishes of differing sizes that have a thin layer
of agar-based growth medium. Once the growth medium in the petri dish is
inoculated with the desired bacteria, the plates are incubated at the best
temperature for the growing of the selected bacteria (for example, usually at
37 degrees Celsius for cultures from humans or animals, or lower for
environmental cultures).
Another method
of bacterial culture is liquid culture, in which the desired bacteria are
suspended in liquid broth, a nutrient medium. These are ideal for preparation
of an antimicrobial assay. The experimenter would inoculate liquid broth with
bacteria and let it grow overnight (they may use a shaker for uniform growth).
Then they would take aliquots of the sample to test for the antimicrobial
activity of a specific drug or protein (antimicrobial peptides).
As an
alternative, the microbiologist may decide to use static liquid cultures. These
cultures are not shaken and they provide the microbes with an oxygen gradient.
The main ones
being:
|
Collection Acronym |
Name |
Location |
|
Collection
d'Institut Pasteur |
||
|
Netherlands Culture
Collection of Bacteria |
||
|
National Collection
of industrial, Marine and food bacteria |
Virus or phage cultures
require host cells in which the virus or phage multiply. For bacteriophages,
cultures are grown by infecting bacterial cells. The phage can then be isolated
from the resulting plaques in a lawn of bacteria on a plate. Virus cultures are
obtained from their appropriate eukaryotic host cells.
Main article: Cell culture
Main article: Axenic
For
single-celled eukaryotes, such as yeast, the isolation of pure cultures uses
the same techniques as for bacterial cultures. Pure cultures of multicellular
organisms are often more easily isolated by simply picking out a single
individual to initiate a culture. This is a useful technique for pure culture
of fungi,
multicellular algae,
and small metazoa,
for example. m/M;
Developing pure culture
techniques is crucial to the observation of the specimen in question. The most
common method to isolate individual cells and produce a pure culture is to
prepare a streak plate. The streak plate method is a way to physically separate
the microbial population, and is done by spreading the inoculate back and forth
with an inoculating loop over the solid agar plate. Upon incubation, colonies
will arise and single cells will have been isolated from the biomass.