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.)
Metabolism
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.
Examples
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
Classification
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.
Phases
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 (Simple)
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
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
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
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.
Details
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
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.
ATP using primary active transport types
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.
Secondary active transport
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.
Antiport
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
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.
Examples
· 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
Endocytosis
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.
Subcategories
· 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.
Types of growth media
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.
Nutrient media
· 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
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
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
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
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.
Bacterial culture
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 and phage culture
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.
Eukaryotic cell culture
Main article: Cell culture
Isolation of pure cultures
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.
