Microbiology Lecture: Chapter 4 PDF

MICROBIOLOGY LECTURE CHAPTER 4 - MICROBIAL AND GROWTH AND ITS CONTROL → Cells are comprised of macromolecules CULTURING MICROBES AND MEASURING (proteins, lipids, polysaccharides, THEIR GROWTH lipopolysaccha...

MICROBIOLOGY LECTURE CHAPTER 4 - MICROBIAL AND GROWTH AND ITS CONTROL → Cells are comprised of macromolecules CULTURING MICROBES AND MEASURING (proteins, lipids, polysaccharides, THEIR GROWTH lipopolysaccharides, and nucleic acids) Comprise more than 96% of dry FEEDING THE MICROBE: CELL NUTRITION weight in E. coli → Metabolic capacities of microbes differ. → Microbes require a core set of nutrients. Macronutrients are required in large amounts. Micronutrients are required in minute amounts. CHEMICAL MAKEUP OF A CELL → Vast majority of proteins are RNA → A single Escherichia coli: DNA contributes small percentage of dry weight. Weighs 10-12 g (75% of this is water) Dry weighs about 184 x 10-15g (184 fg) → Chemical elements that dominate in living systems. Account about 96% of the dry weight of an average bacterial cell: Carbon (C) Oxygen (O) Nitrogen (N) Hydrogen (H) Phosphorus (P) Sulfur (S) → Some other elements are required by most but not all mcgs. 3.7% of a cell’s mass is composed of: CARBON, NITROGEN, AND OTHER Potassium (K) MICRONUTRIENTS Sodium (Na) Calcium (Ca) Magnesium (Mg) → Carbon and nitrogen are present in large Chlorine (Cl) amounts in all cells. Iron (Fe) → Heterotrophs: organisms that require organic carbon  Obtain carbon from the breakdown of Acts as a cofactor to catalyze specific organic polymers. reactions. Or from the direct uptake of their monomeric constituents: amino acids, → Growth o of microorganisms requires fatty acids, organic acids, sugars, various metals. nitrogen bases, and other organic Iron (Fe) is popular. It is present in compounds cytochromes and several other enzymes that play major roles in → Autotrophs: can synthesize organic cellular respiration or oxidation- compounds from carbon dioxide (CO2) reduction reactions. → Bulk of nitrogen in nature exists in → The metals are called Trace metals Proteins, Ammonia (NH3), Nitrate (NO3-), because they are required in small Nitrogen gas (N2) amounts. All microorganisms can use NH3 as their nitrogen source (can also use → Enzymes with a trace metal requirement NO3-) may be synthesized by the cell. Some microbes can use organic If starved for these metals, cell will nitrogen sources such as amino acids. not function properly. Few can use N2 (the nitrogen fixing bacteria → Growth factors: are organic micronutrients rather than metals. → Macronutrients are also needed but in Vitamins are the most frequently smaller amounts. required growth factors that function Phosphorus for nucleic acids and as coenzymes. phospholipids - Usually assimilated as inorganic → Some microbes are able to biosynthesize phosphate. all of their growth factors. Sulfur present in amino acids Many need to assimilate diverse cysteine and methionine also some growth factors from the environment. vitamins. - Can assimilate inorganic forms → Growth factor requirements vary widely such as sulfate or sulfide. among microorganisms. - Other assimilate from organic sulfur compounds. Cyanobacteria Lactic Acid Potassium is required for enzymes. Bacteria Magnesium stabilizes ribosomes, Autotrophic Streptococcus, membranes, and nucleic acids. Also microbes that Lactobacillus, required for enzyme activity. inhabit aquatic Leuconostoc Calcium and Sodium are essential environments inhabit guts of for only few organisms. animals and foods Can synthesize all Get their growth their growth factors from the factors and need place they inhabit MICRONUTRIENTS: TRACE METALS AND little if any like the animal gut GROWTH FACTORS supplementations. or food. → Many enzymes require a metal ion or → Cells need to uptake macronutrients and small organic molecule. micronutrients to grow and divide.  GROWTH MEDIA AND LABORATORY Media used by Pasteur was a CULTURE complex media with yeast extract. → Culture media can be prepared selective → Laboratory cultures of microorganisms or differential (or both): are grown in culture media. → Selective medium: contains compounds → Culture media: nutrient solutions tailored that inhibit the growth or some but not to the particular organism to be grown. others. Commercially available for the → Culture media must be sterilized before isolation of certain common gut use and sterilization is typically achieved pathogens such as Salmonella or by heating the medium under pressure in those strains of E. coli that cause an autoclave. foodborne illness. Example: Bile salts are added for selective isolation of these bacteria because bile salts kill many bacteria CLASSES OF CULTURE MEDIA unable to grow in the gut. → Differential medium: an indicator → Two broad classes of culture media: (typically a dye) is added, which reveals by a color change whether a particular 1. Defined media: prepared by adding metabolic reaction has occurred during precise amounts or pure inorganic or growth. organic chemical to distilled water. Useful for distinguishing bacteria and are widely used in clinical diagnostic. → Exact composition of a defined medium Example: Incorporating a pH- is known. sensitive dye facilitates detection of lactic acid bacteria by indicating → Carbon source is a major importance acidification through color change. in any culture medium because cells need large amounts or carbon to make new cell material. NUTRITIONAL REQUIREMENTS AND → Particular carbon source and BIOSYNTETIC CAPACITY concentration depends on the organism to be cultured. → Complex → Some defined media are considered media for “simple” because they only contain a Escherichia coli single carbon source. (common enteric bacterium) and 2. Complex media: made from digests of Leuconostoc microbial, animal, or plant products such mesenteroides as protein (casein), beef (beef extract), (lactic acid soybeans (tryptic soy broth), yeast cells bacterium) (yeast extract) or any number of other Rich in nutrients highly nutritious substances. Easy to prepare → Example:  → Simple defined → Main takeaway: different microorganisms medium for E.coli have vastly different nutritional Needs fewer requirements. nutritional requirements than L. → For successful cultivation, it is necessary mesenteroides to understand an organism’s physiology and nutritional requirements. LABORATORY CULTURE → Defined medium → Laboratory media can be either liquid or for L. solid. mesenteroides Has more → Agar: solidifies culture media. Is an algal nutritional polysaccharide used in the studies of requirements Robert Koch. (requires more growth factors) → Solid media immobilize cells so that as Can be satisfied they grow, they accumulate in a pile to by preparing a highly form visible isolated cell masses. supplemented Colonies: are the visible isolated cell defined medium or a masses. complex medium. Has individual → Microbial colonies vary in: nutrients to be added. Shape Color → Defined culture Texture medium for Size Thiobacillus thioparus → But it will depend on: Bacterium is an Organism itself aerobic sulfur- oxidizing Culture conditions chemolithotroph. Nutrient supply Derives all carbon Physiological parameters from CO2 and conserves energy → Colony appearance is consistent for a from oxidation of given organism on a particular type of sulfur compounds. medium. Common in low nutrient sulfur rich environments that only has access to → Colony morphology: visible inorganic nutrients. characteristics of a colony. T. thioparus has evolved to Can sometimes be used to identify biosynthesize all its needed growth microorganisms. factors and does not require organic Used routinely to determine if a nutrients. culture is pure, contaminated, or mixed. → Plates inoculated from a mixed culture Dried sample can be stained to or from a contaminated pure culture increase contrast between cells and will typically contain more than one background. colony type. With liquid samples, counting → Propagation of microbial cultures requires chambers consisting of a grid with aseptic technique. squares of known area etched on the surface of a glass slide are used. → Aseptic technique: series of steps by Coverslip is placed on the chamber, which microbes are transferred between each square on the grid has a precise growth media without contamination. volume. Number of cells per unit area of grid → Contamination can be introduced from can be counted. microbes in the air, liquid droplets, or on Gives a measure of the number of surfaces. cells per small chamber volume. Cell count per mL uses a chamber → Goal in liquid medium is to transfer a volume conversion factor. microbial culture while protecting the culture vessel from air currents or contact with nonsterile surfaces. → Same with agar surfaces but with greater emphasis on keeping the surface of the agar protected from aerosols or particulate matter in air. → Mastery of aseptic technique is required for maintaining pure cultures. → Streak plate technique: primary method for obtaining pure cultures and of verifying culture purity. Inoculating loop is used to spread a sample. MICROSCOPIC COUNTS OF MICROBIAL CELL NUMBERS → Microscopic counting is a quick and easy way of estimating microbial cell numbers. TOTAL CELL COUNT → But microscopic counting has limitations that restricts its usefulness: Total counts of microbial numbers can Without special staining techniques, be done by observing ad enumerating dead cells cannot be distinguished the cells present by a microscopic from living. cell count. Precision is difficult to achieve even Can be performed on samples dried when replicate counts are made. on slides or on liquid samples. Small cells are difficult to see which leads to erroneous counts.  Cells suspensions of low density will have few if any cells in microscope → When cell numbers are low (e.g., ocean filed unless the sample is first water), filtering concentrates them before concentrated and resuspended in a staining for easier counting. small volume. Motile cells must be killed or immobilized before counting. Debris may be easily mistaken for microbial cells. VIABLE COUNTING OF MICROBIAL NUMBERS MICROSCOPIC CELL COUNTS IN → Viable cell: one that is alive and able to MICROBIAL ECOLOGY grow. → Viable count: performed by spreading → Ecologists use microscopic cell counts on microbes on a solid media and counting natural samples. colonies. → Do this by using stains to visualize the → Often called plate count because agar cells. plates are required. → Stains yield phylogenetic or other info about the cell like their metabolic → Viable counts estimate cell numbers by property. assuming each live cell grows into a single colony on a plate. → Many fluorescing stains can be used in a general way → Example: DAPI stains all cells because it binds to DNA METHODS FOR VIABLE COUNTS → Other stains can differentiate live from dead cells by detecting whether a cell’s cytoplasmic membrane is intact or not. → There are two ways of performing a plate count: → Fluorescent stains that are highly specific → Spread plate method: a volume of a for certain organisms or groups can be diluted culture is spread over the surface prepared by attaching fluorescent dyes to of an agar plate using a sterile glass specific nucleic acid probes. spreader. → Example: Phylogenetic stains, unique to bacteria or archaea, are combined with → Pour plate method: a known volume of nonspecific stains to determine the culture is pipetted into an empty sterile proportion of each domain in a sample. petri plate. Molten agar medium is then added and gently mixed before allowing → Other fluorescent probes target genes the agar to solidify. encoding enzymes that catalyze specific metabolic processes. → Important that the number of colonies → If cell is stained by one of these probes, a developing on or in the medium not be metabolism can be inferred that may too many or few. reveal the cell’s ecological role → On crowded plates, some cells might not form colonies, some may also fuse, leading to erroneous measurements.  → In this way, one milliliter of a 1000-fold → If number of colonies is too small, dilution contains 0.001 ml (10-3 ml) of statistical significance of calculated count sample and 0.999 ml of diluent. will be low. → Most valid statistically, is to count → To estimate cell number, we divide colony colonies only if it contains between 30 count by the plated sample volume. If we and 300 colonies. plate 1ml of a 1000x dilution, this effectively analyzes 0.001ml of the → Though viable counts assume one cell original sample. leads to one colony, some bacteria clump If 159 colonies form from this sample, or form chains, skewing the cell count the original sample must have estimate. contained 159 x 103 (which is Example: a bacterial filament might equivalent to 1.59 x 105) CFU contain 10 or more cells, but since the cells are firmly attached together, the filaments will form only one colony. → Viable counts use colony forming units (CFU) instead of actual cell count due to cell clumps. DILUTING A SAMPLE → High bacterial concentrations (thousands to billions) necessitate sample dilution to achieve a countable number of colonies on culture plates. → Series of 10-fold dilutions are commonly used prior to performing viable plate counts. → A 10-fold dilution is achieved by mixing 1 mL of sample with 9 mL of diluent (water,salt solution, or growth APPLICATIONS OF THE PLATE COUNT medium). This final solution contains 0.1 mL (10%) of the original sample and 0.9 mL of diluent per 1 mL volume. → In food, dairy, medical, and aquatic microbiology viable counts are employed → Several serial dilutions are needed to routinely. yield a countable number of colonies. → Example: If a sample is suspected to → High sensitivity is a key advantage of this contain more than 105 cells then the method, as it can detect as few as one sample can be diluted 1000-fold by viable cell per plated sample. This makes performing three successive 10-fold it valuable for sensitive detection of dilutions (1/10 * 1/10 * 1/10 = 1/1000)  microbial contamination in foods and → Why do plate counts reveal lower other materials. number of cells than direct microscopic counts? → Highly selective culture media and growth Diverse microbes within small conditions enable plate counts to target samples have vastly different nutrient particular species within a diverse sample and growth needs in lab by promoting the growth of desired cultures. Therefore, one medium and bacteria while inhibiting others. set of conditions can only support a Example: Complex media containing limited subset of the entire microbial 10% NaCl are highly useful for community. isolating potentially pathogenic Example: If a sample has 109 viable Staphylococcus species from skin cells but only 106 are capable of samples due to their selective growing on the medium, then the inhibition of most other skin bacteria. viable count will miss 99.9% of the cell population. → In the food industry, viable counting performed using both a complex medium → Plate counts targeted to specific and a selective medium on the same organisms using highly selective media sample allows for simultaneous can often yield quite reliable data. quantitative and qualitative assessments Since the physiology of the targeted of food quality and safety. organisms is known and so the Complex medium yields total cell recovery of the viable cells is near count (indicates freshness and shelf 100% life) Selective medium indicates presence → Total cell counts from single-condition or absence of particular pathogen that experiments (medium and growth may be transmitted through food conditions) underestimate actual numbers by 1-1000 magnitudes, as different cells → Viable counting is also common in wastewater and other water analyses. require different environments to thrive. Example: Enteric bacteria, like E. coli from poop, are easily detected in water using special media. Finding TURBIDIMETRIC MEASURES OF them in swimming water suggests MICROBIAL CELL NUMBERS poop contamination, making it unsafe. → Microbial cells are large enough to scatter light. Amount of light scattered in cell CAVEATS TO PLATE COUNTING METHODS suspension is proportional to number of cells present. → Direct microscopic counts of natural → The cloudy appearance of cell samples often show many more microbes suspensions arises from light scattering than single culture medium can capture, by individual cells, which act as making plate counts inaccurate for microscopic reflectors. estimating total cell numbers in environments like soil and water. → More cells present – more light scattered This is referred to as the great plat – more turbid suspension. count anomaly. → Turbidity measurements offer a rapid and → For unicellular organisms: OD is widely used approach to estimating cell proportional within certain limits, to cell numbers in solution. number. → Turbidity readings can be used as a substitute for total or viable counting methods. → A standard curve must be prepared that relates cell number to turbidity. At high cell densities, light scattering gets complex, affecting the accuracy of converting turbidity to cell number. However, with a pre-made standard, turbidity measurements can still provide useful estimates of bacterial numbers. OPTICAL DENSITY AND ITS RELATIONSHIP TO CELL NUMBERS → Spectrophotometer: measures turbidity. Is an instrument that measures the unscattered light that passes through a sample. Employs a prism or diffraction grating that shines light at specific wavelengths (like blue 480nm, green 540 nm, and red 660 nm) through a sample to measure its cloudiness (turbidity). This technique is useful for studying bacteria. OTHER ISSUES WITH TURBIDIMETRIC → Sensitivity is best a shorter wavelength, GROWTH ESTIMATES but measurements of dense cell suspensions are more accurate at longer wavelengths. → Turbidity measurements are quick and → Optical Density (OD): unit of the easy to perform and can be made without disturbing a sample. turbidity at the wavelength specified. Example: OD540 for measurements of 540 nm → With turbidimetric assays, the same sample can be checked repeatedly over time and the measurements plotted on a semilogarithmic plot versus time to measure growth of a microorganism over time.  DYNAMICS OF MICROBIAL GROWTH BINARY FISSION AND THE MICROBIAL GROWTH CYCLE → Growth is the result of cell division. → Growth is defined as an increase in the number if cells. → Example: In rod-shaped bacteria like E. coli, cells elongate to twice their original length and then form a partition that → Turbidity measurements can also be constructs the cell into two identical problematic. daughter cells That process is called binary fission. → Although many microorganisms grow “Binary” indicate that two cells have evenly distributed in suspensions in liquid arisen from one. medium, many do not. Some bacteria routinely form small to → Septum: partition that forms between large clumps, and OD measurements dividing cells. may be quite inaccurate as a Results from inward growth of the cell measure of total microbial mass. envelope from opposing directions. Continues until two daughter cells are → Biofilms and clumps formed by many pinched off. bacteria interfere with accurate cell mass estimation (and hence cell number) through optical density (OD) → There are some different variations in measurements in liquid septum formation. For example: cultures. Therefore, minimizing these is Bacillus subtilis: septum forms w/o crucial for reliable results. cell wall constriction. Mixing the culture prevents bacteria Caulobacter: constriction occurs but from clumping together or attaching to no septum is formed. surfaces during growth. → Planktonic bacteria stay suspended, allowing accurate cell number estimation by measuring the liquid's cloudiness. However, this method fails for bacteria that form static biofilms on surfaces, as these clumps distort the measurement. → Cell separates to form two cells = one generation occurred.  Time required for this to happen is generation time (doubling time). → Microbes are grown in batch culture – growth of microbes in a fixed volume of → Each daughter cell is identical, having liquid enclosed within a container such as received a copy of the chromosomes and a test tube or flask. sufficient copies of all cellular materials required to begin life as an independent → Nutrients in a culture flask are finite and entity. cannot support growth indefinitely. → Microbes differ in their doubling times. → In batch culture microbes typically exhibit Doubling time varies on their growth a growth cycle called the microbial conditions. growth curve. → Under optimal conditions, the generation → Growth cure is composed of 4 phases: time of E. coli is about 20 min. Lag phase Exponential phase → The fastest growing known microbes can Stationary phase double in less than 10 minutes. Decline phase. → Slowest can have a generation time of several months or even longer. LAG AND EXPONENTIAL PHASE → Lag phase: period between inoculation and the onset of growth. When microbial culture is inoculated into fresh growth media, there is typically an initial pause during which the cells do not grow. THE MICROBIAL GROWTH CYCLE → Microbes exhibit a lag phase because transfer to fresh medium represents new → Optimal conditions can cause increase environmental conditions for the cells, (numbers of cells) in growth by binary which need to alter their metabolic state fission. to respond to these new conditions. But conditions are rarely optimal, even in laboratory culture. → Lag phase may be brief or extended → Exponential growth continues until depending on the previous history of the conditions in the batch culture can no cells, choice of growth medium, and longer sustain growth. growth conditions. → Example: If an actively growing culture is transferred into a new flask containing the STATIONARY AND DEATH PHASES same type of medium under the same growth conditions, there will be little lag. Because cell needs to make very few → Exponential growth cannot continue metabolic adjustments to this new forever. condition This is impossible because the carrying capacity of an environment → Example: If inoculum is from an old will always limit growth of the culture, there is usually longer lag. organisms within it. Because cell viability may be low, or the cells may need to synthesize → In batch cultures, growth is limited many enzymes and macromolecules because of their nutrient depletion or the before they can begin growing. accumulation of microbial waste products. → A long lag is observed when a microbial → Stationary phase: there is no net culture is transferred from nutrient rich increase or decrease in cell number and to nutrient poor culture medium. thus the growth rate of the population is zero. → To grow cell must have a complete set of When exponential growth ceases the enzymes to biosynthesize the essential population, the population enters metabolites absent from the nutrient poor stationary phase. medium. The more enzymes and molecules → During stationary phase, cellular that a cell must manufacture to grow metabolism shifts away from growth as in a new environment, the longer the the cell prepares for maintenance and lag. survival. → Exponential phase: period when the → Despite growth arrest, energy metabolism growing cell population doubles at regular and biosynthetic processes in stationary intervals. phase cell may continue, but at a greatly Also called balanced growth reduced rate. because cells are as close to be metabolically identical throughout this → Decline phase: total number of cells period as they can be. decreases due to cell death. → To minimize experimental variation, most → Cell division may still occur for some cells experiments are performed with microbes in the population during stationary and taken from cultures in the exponential decline phases, but this meager increase phase of growth. is balanced by death of other cells. → Rates of exponential growth vary greatly → Cryptic growth: subpopulations of cells and are influenced by choice of media, adapt to cannibalize and reuse resources growth conditions, and the organism released from dying cells. itself. → If a culture is prevented from dying, some since generation times can be inferred number of cells may remain for month or directly from the graph. even years. → Example: The generation time can be determined by finding the time it takes for the number of cells to double. QUANTITATIVE ASPECTS OF MICROBIAL → Generation time (g) is the time (t) per GROWTH generation (n), g = t/n PLOTTING GROWTH DATA → Example: population that takes six hours (t= 6 hours) to double once (n= 1) has a → Exponential growth of a population occurs generation time of 6 hours. during a period when cell number doubles 6 = 6/1 at regular intervals. → When we plot the change in cell number of over time on arithmetic (linear) MATHEMATICS OF BACTERIAL GROWTH coordinates, we obtain an upwardly sweeping curve with a continuously increasing slope. → When the cell number is plotted on a logarithmic (log10) scale as a function of time (semilogarithmic graph) the points fall on a straight line. → This straight-line function reflects the fact that the cells are growing exponentially, and that the population is doubling in a constant time interval → Starting with a single cell and because the cell number of an exponentially growing culture doubles every generation, the number of cells present at generation n can be expressed as 2n. → Example: 3 generations there will be 23 = 8 cells 10 generations there will be 210 = 1024 cells → If exponential growth began with two cells instead of one, the number of cells at any generation would be doubled. 2 x 210 = 2048 cells → Semilogarithmic graphs are also convenient for estimating the generation time of a culture from actual growth data → If we started with three cells, the number of cells at any generation would be tripled. 3 x 210 = 2048 cells → It is possible to calculate the number of cells (N) in an exponentially growing culture at any point in time from the following expression: SPECIFIC GROWTH RATES N t = N 02 n Nt = cell number at time (t) N0 = initial cell number at time 0 → Specific growth rate (k): expresses the n = number of generations during the rate at which the population at any period of exponential growth. instant. k is expressed in units of reciprocal → n (number of generation) can be solved hours (h-1) by taking the logarithms of both sides and using algebra to yield → In contrast to (g) which is the mean time n = [(log Nt - log N0)/log2] required for the cell population to double. → Example: → The specific growth rate is a function of Nt = 108 the change in cell number over times and N0 = 5 x 107 is expressed as dN/dt = kN t=2 The specific growth rate (k) indicates the rate at which the cell number is increasing at any point of time. → Integration of this equation using natural logarithms gives the expression: Nt = N0kt → Thus, in this example n = 1. We can use the equation g = t/n to → To estimate or express k: determine that g = 2 hours (2 = 2/1) k = 0.693/g  → Limitations of those constant changes can be circumvented in a continuous culture device. → The most common type of continuous culture device is the chemostat. → Batch culture is a closed system unlike CONSEQUENCES OF EXPONENTIAL the chemostat which is an open system. GROWTH → In a chemostat, a known volume of sterile medium is added at a constant rate while → During exponential growth, increase in an equal volume of spent culture medium cell number is slow at first but will is removed at the same rate. increase at very fast rate. Leads to explosive increase in cell → Once in equilibrium, the culture volume, number in later stages. cell number, and nutrient/waste product status remain constant, and the culture → Example: attains steady state. Rate of cell production is first 30 minutes is 1 cell per 30 minutes. However, between 4 and 4.5 h, the rate of cell production is 256 cells THE CHEMOSTAT AND THE CONCEPT OF per 30 minutes. STEADY STATE Between 5.5 and 6 hours of growth it is 2048 cells per 30 min. → Chemostat: enables control over both → Exponential growth can have implications the specific growth rate and growth yield in everyday life. of a microbial culture. Example: Spoilage of milk. The lactic acid bacteria contaminate → Fresh sterile medium is added to a the milk during its collection and exist culture vessel and spend media washed in fresh, pasteurized milk in low out at equal rates resulting in a culture numbers. that maintains a fixed volume. These organisms grow slowly at refrigerator temp but faster at room temp overnight. If week-old milk which contains week’s worth of slow bacterial growth is left standing, a huge amount of lactic acid is made and spoilage results. CONTINUOUS CULTURE → The environment in a batch culture is constantly changing because of nutrient consumption and waster production.  → Changing the dilution rate (D) does not affect the growth yield (biomass produced per unit of substrate consumed) until washout occurs (when all the substrate is consumed). However, increasing the nutrient concentration in the medium allows for more cells to be produced but does not change the growth rate (except at very low nutrient concentrations). Therefore, by adjusting the dilution rate or the nutrient concentration, one can cultivate cultures that grow exponentially at a desired growth rate and cell density. → The supply of medium is defined by the dilution rate (D) which is expressed as F/V. F is the flow rate V is the culture volume. → In the chemostat: specific growth rate is controlled by the dilution rate (D) growth yield is controlled by the concentration of a limiting nutrient EXPERIMENTAL USES OF THE in the fresh medium added to the CHEMOSTAT vessel. → At a steady state, cells grow at the same → Practical advantage of chemostat is that time they are removed by outflow from cell population can be maintained in the the system. exponential growth phase for long periods. → A large number of cells are competing for a limiting nutrient. → Exponential phase cells are usually most The nutrient added in the fresh desirable for physiological experiments. medium is consumed rapidly by the Such cells are available at any time in cells thereby limiting their growth rate. the chemostat, and the vessel can be sampled repeatedly. → In a chemostat, cells compete for a limiting nutrient. As the flow of nutrients → Chemostat cultures have been used increases, the cells can grow faster until extensively to study bacterial physiology the dilution rate is too high, and they are and have also been used in studies of washed out. bacterial ecology and evolution. → Example: Chemostats replicate nature's low nutrient conditions, enabling  researchers to identify "champion → Colonization starts when microbes begin competitors" within diverse communities to grow and produce sticky extracellular by testing growth rates across different polysaccharides (EPS) nutrient limitations. Colonization and growth causes changes in the biofilm that lead to → Chemostats selectively enrich and isolate development. bacteria from environments by gradually increasing "dilution rate" (D), essentially → During, development cells in the biofilm weeding out slower-growing species. This begin to change their metabolism. Cause biofilms to develop complex allowed isolation of a soil bacterium with an systems of mushroom like columns incredibly fast doubling time of 6 minutes, and channels that trigger metabolic the quickest known. differentiation of microbes at the surface of the biofilm from those at its base. BIOFILM GROWTH → Dispersal of cells from a mature biofilm allows microbes to colonize new sites. Usually prompted by changes in the → Microbial growth in liquid suspensions environment, such as nutrient such as growth of free-floating or free limitation or other forms of stress. swimming cells is called planktonic growth. → Sessile growth is growth while attached to a surface and is quite common in the microbial world. → Microbes that are attached to surfaces → Biofilms can be studied in a flow often develop into biofilms. chamber in which liquid media flows continuously between two layers of glass. BIOFILM FORMATION → Flow chambers are designed so that biofilm growth can be monitored microscopically over time. → Biofilm: population of cells enmeshed in a polysaccharide matrix that is → Pseudomonas aeruginosa is a model attached to a surface. organism that has been used to study They form in stages. microorganisms. Stages: Relevant model because it forms - Attachment biofilms in the lungs of humans with - Colonization genetic disease cystic fibrosis. - Development Biofilm formation allows cells of P. - Dispersal aeruginosa persist and resist removal. → Begins with the attachment of planktonic cells to a surface. → Biofilms tend to make bacteria more Is often mediated by flagella, resistant to drugs because they provide a fimbriae, or pili. penetration barrier and promote metabolic differentiation.  BIOFILMS AND HUMANS → Formation of biofilms also happens on the hulls of ships is a major problem → Biofilms are common growth form for because they decrease speed and bacteria in nature. energy efficiency and thereby increase Because the intensely interwoven shipping times and shipping costs. nature of the structure prevents harmful chemicals from reaching cells deep within the biofilm structure. ALTERNATIVES TO BINARY FISSION → Biofilms also provide a physical barrier that protects cells from grazing by protists and allows then to remain in a safe a → Planktonic cells that grow by binary favorable habitat. fission undergo balanced growth during exponential phase. → Some biofilms form multilayered sheets with different organisms present in the individual layers, these biofilms are called microbial mats. → Mats composed of various phototrophic and chemotrophic bacteria are common → All cells in such a culture are nearly in the outflows of hot springs and identical, genetically and metabolically. marine intertidal regions and can form Uniformity of such cultures makes crusty mat like structures quickly in them desirable as experimental puddles of water that stay moist for as systems. little as a few days. → Cells within biofilms fo not exhibit → Biofilms affect many aspects of our lives, balanced growth because their including human health. metabolic characteristics and growth rates vary depending on their position on Biofilms have been implicated in: the biofilm. - Difficult to treat infections of human joints. - Implanted medical devices such as artificial heart valves and joints. - Indwelling devices such as BUDDING CELL DIVISION catheters. → Biofilms are also responsible for the → Budding bacteria exhibit unequal cell formation of dental caries (cavities) growth and produce daughter cells that and are a cause of gum disease. have different characteristics. → In industrial and municipal settings, → Budding division: a new cell emerges biofilms can cause fouling, plugging, and buds off from a mother cell, the and corrosion in pipes used to transport latter of which retains its original identity. liquids. → Some budding bacteria form → Biofilms can even form in fuel storage cytoplasmic extensions such as stalks tanks where they contaminate fuel by or hyphae. producing corrosive chemicals such as Examples are Caulobacter and H2S. Hyphomicrobium  binary fission because growth of the cell → Caulobacter – the mother cell is attached is not linked directly to cell division. to a surface by its stalk. The daughter cell that buds off is motile. It lacks a stalk and → Cells elongate and replicate DNA as has a flagellum that allows it to disperse they grow but do not produce septa. by swimming motility. Instead, hyphae form cross-walls away from the point of cell growth. → Hyphomicrobium – forms a long stalk These cross-walls do not define from which a new cell emerges and buds. independent cells but instead allows transport to occur between adjacent → Ancalomicrobium – produce multiple compartments in the hyphal filament. appendages that resemble arms extending away from the cell. The → Hyphae often weave together to form appendages increase the surface to mycelia which form complex hyphal volume ratio of the cell which increases filaments, and mature mycelia often form its ability to extract nutrients from arthrospores. oligotrophic (very dilute) habitats. → Arthrospores are survival structures. Differ from endospores in their mechanism of development and their lack of resistance to heat and harsh chemicals. → Arthrospores develop by multiple fission, in which a single hyphal filament forms many septa simultaneously along its length. Cause many cells to form all at once along the filament, each of which ultimately differentiates into a mature arthrospore. → Multiple fission is also seen in certain cyanobacteria. Example: Cells of cyanobacteria Stanieria begin about 1um in diameter and grow to as large as 30um in diameter before undergoing multiple fission HYPHAL GROWTH AND MULTIPLE FISSION a process in which the large cell subdivides into tens of smaller cells, each of which begins the → Actinomycetes are gram positive replication cycle anew. filamentous bacteria that are common in soils and grow as long thin filaments → Some bacteria even form intracellular called hyphae. offspring. Species of the genus Streptomyces Bacteria such as the giant called are typical actinomycetes. Epulopiscium grow by forming multiple daughter cells within → Hyphal growth: occurs only at the tip of cytoplasm of the mother cell. an elongating filament and is unlike  Once the daughter cells are mature, range for any given organism is they burst out of the mother cell typically less than 40C. and begin a new round of daughter cell production. → The maximum growth temperature of an organism reflects the temperature above which denaturation of one or ENVIRONMENTAL EFFECTS ON GROWTH: more essential component such as a key TEMPERATURE enzyme occurs. TEMPERATURE CLASSES OF → The factors controlling an organism’s MICROORGANISMS minimum growth temperature are not as clear. CARDINAL TEMPERATURES However, the cytoplasmic membrane must remain in a semifluid state for nutrient to → Temperatures affects microorganisms transport and bioenergetic functions to in two opposing ways. take place. As temperatures rise, the rate of If an organism’s cytoplasmic enzymatic reactions increases, and membrane stiffens the organism growth becomes faster. cannot grow. However, above a certain temperature, proteins or other → The growth temperature optimum critical cell component may be reflects a state in which all or most denatured or otherwise irreversibly cellular components are functioning at damaged. their maximum rate. Typically lies close to maximum than → For every microorganism there is a: minimum. Minimum temperature below which growth is not possible. Optimum temperature at which growth is most rapid. Maximum temperature above which growth is not possible. → The three temperatures are called the cardinal temperature which are characteristic of any given microorganism and can differ dramatically between different species. → Example: some organisms have growth temperature optima near 0C, whereas the optima for others can be higher than 100C. The temperature range throughout TEMPERATURE CLASSES OF ORGANISMS which microbial growth is possible is even wider than this, from as low as -15C to at least 122C. → It is possible to distinguish 4 broad However no organism can grow over classes of microorganisms in relation to this whole temperature range, as the their growth temperature optima:  Psychrophiles – low temp optima → These cold environments support diverse Mesophiles – midrange temp optima microbial life. Thermophile – high temp optima As do glaciers, where the networks of Hyperthermophiles – very high temp liquid water channels that run through optima and under the glacier are teeming with microbes. Even in solidly frozen materials there remain small pockets of liquid water where solutes have concentrated and microorganisms can metabolize and grow, albeit very slowly. → In considering cold environments as microbial habitats, it is important to → Mesophiles are widespread in nature distinguish between environments that and the most commonly studies. are constantly cold and seasonally Found in intestines of endothermic cold. (warm-blooded) animals in terrestrial and aquatic environments in → Seasonally cold of temp climates may temperature and tropical latitudes. have summer temps as high as 40C Example: a temperate lake may have → Escherichia coli is a typical mesophile ice cover in the winter but the water and its cardinal temperatures have been may remain 0C for only a brief time. precisely defined: Minimum temp: 8C → In contrast, Antarctic lakes contain a Optimum temp: 39C permanent ice cover several meter thick Maximum temp: 48C and the water column below the ice Temperature range for E. coli is about remains 0C or colder year round. 40C. → Marine sediments and glaciers are also → Psychrophiles and thermophiles are constantly cold, as are subglacial lakes— found in unusually cold and hot lakes deep beneath the glacier surface— environments. and all of these are teeming with microbial life. → Hyperthermophiles are found in extremely hot habitats such as hot springs, where temps can be as hot as 100C, and deep sea hydrothermal vents which can PSYCHROPHILIC AND PSYCHOTOLERANT exceed 100C. → Psychrophile: a microbe with an optimal MICROBIAL LIFE IN THE COLD growth temperature of 15C or lower, a maximum growth temp below 20C and COLD ENVIRONMENTS minimum growth temp of 0C or lower. If it has an optima of 20-40C it is psychrotolerant. → Oceans (half of Earth) average 5°C, with depths at 1-3°C constant. Arctic and → Cold-loving psychrophiles thrive in Antarctic regions are mostly frozen. frigid environments and die at moderate temperatures, demanding  meticulous handling in labs to prevent → Psychrotolerant microbes survive at 0°C warming during study. but grow slowly, taking weeks for visible growth in labs. At 30°C, they thrive like → Psychrophilic algae and bacteria grow in most mesophiles. These include diverse dense masses under sea ice. bacteria, archaea, and even some Can also be found on surface of eukaryotes. permanent snowfield and glaciers where they put a distinctive coloration to the surface. MOLECULAR ADAPTATIONS TO LIFE IN → Chlamydomonas nivalis example of an THE COLD algae that leaves coloration on snow. The carotenoid pigment astaxanthin in its spores is → Psychrophiles produce enzymes thrive in responsible for the brilliant red color of cold yet denature and deactivate at the snow surface. moderate temperatures. Grows within the snow as a green Molecular basis is not entirely pigmented vegetative cell and the understood but is linked to protein sporulates. structure. Spores concentrate on the surface as snow melts and evaporates. → Cold-active enzymes have more flexible α- Related species contain different helices (allows proteins greater flexibility pigments. for catalyzing reactions) and less rigid β- sheets compared to inactive counterparts, → Several psychrophilic Bacteria and few allowing for better function at low psychrophilic Archaea have been isolated and shows very low growth temp optima. temperatures. Planococcus halocryophilus – permafrost bacterium grows slowly at → Additionally, they possess fewer weak -15°C, the lowest growth temp bonds and more polar and lesser documented. hydrophobic amino acids, further contributing to their cold-adapted → Bacteria might metabolize at much colder flexibility. temperatures than previously thought. Microbial respiration has been → Psychrophile’s cytoplasmic membranes observed in -40°C tundra soil, and have more unsaturated, short-chain enzymes from cold-adapted bacteria fatty acids, keeping them semifluid for function at -20°C. efficient transport and energy functions at While growth would be slow, it could low temperatures. allow populations to persist in Some even have polyunsaturated extremely cold environments. fatty acids that remain flexible at cold temps. → Psychrotolerant microorganisms are Unlike monounsaturated or fully more widely distributed in nature than saturated fatty acids that tend to psychrophiles. stiffen at low temps. Can be isolated from soils and water in temperate climates. → Another molecular adaptation includes As well as from meat, dairy products, “cold shock” proteins and cider, vegetables, and fruits stored at cryoprotectants (these are not limited to standard refrigeration temp. psychrophiles)  Cold shock proteins: molecular Hydrothermal vents at the bottom of chaperons that maintains cold the ocean can have temps of 350°C. sensitive proteins in an active form or binds specific mRNAs and translation → Hot springs are found all over the world under cold conditions. but abundant in Western US, New Cryoprotectants: includes antifreeze Zealand, Iceland, Japan, Italy, Indonesia, proteins or specific solutes (glycerol Central America, and Central Africa. and certain sugars) that help prevent formation of ice crystals that puncture → Largest concentration of hot springs: they cytoplasmic membrane. Yellowstone National Park, Wyoming Exopolysaccharide (EPS) cell surface (USA) slime confer cryoprotection as well. → Some hot springs vary widely in → Freezing temperatures stall microbial temperature, many have constant high growth, but not always kill them. This temps, varying less than a degree or two is used for long-term storage in culture over many years. collections, where cells with cryoprotectants survive in frozen states (- → Different springs have different chemical 80°C or -196°C) for years. compositions and pH values. → In habitats hotter than 65°C only prokaryotic cells can thrive. MICROBIAL LIFE AT HIGH TEMPERATURES THERMAL ENVIRONMENTS HYPERTHERMOPHILES AND THERMOPHILES → Thermophiles: organisms whose growth temp optimum exceeds 45°C → Variety of hyperthermophiles → Hyperthermophiles: optimum temp (chemoorganotrophic and exceed 80°C. chemolithotrophic) inhabit boiling hot springs. → Surface of soils subject to full sunlight can be heated to above 50°C at midday, and → Scientists study growth rates of heat- some may warm to as high as 70°C. loving microbes in springs by placing a slide in the water and tracking tiny → Fermenting materials such as compost colonies over time. piles and silage can also reach The slide is an excellent surface for temperatures of 70°C. microbial attachment and subsequent growth. → Thermophiles abound in such Small microbial colonies form, and environments. growth rates can be calculated from Most extreme high-temp cell number data. environments in nature are hot springs and these are home to huge → Ecological studies have shown that diversity of thermophiles and growth rates in boiling springs are hyperthermophiles. often quite high, with generation times (g) as short as 1h not uncommon. → Many terrestrial hot springs have temps at or near boiling. → Cultures of diverse have been obtained and a variety of morphological and physiological types of both Bacteria and Archaea are known. → Some hyperthermophilic Archaea have growth temperature optima above 100°C. → No species of Bacteria have yet been discovered that grow above 95 °C. → Growing lab cultures of organisms with optima above boiling point requires pressurized vessels that permit temperatures in the growth medium to rise above 100°C without boiling. → Most heat-tolerant organisms known inhabit hydrothermal vents. Most thermophilic example is Methanopyrus, a methane producing genus of Archaea capable of growth at up to 122°C. PROTEIN AND MEMBRANE STABILITY AT HIGH TEMPERATURES → Thermophiles inhabit moderately hot or intermittently hot environments. → The enzymes and proteins of → As boiling water leaves a hot spring, it thermophiles and hyperthermophiles gradually cools setting up a thermal are more heat stable than and function gradient. optimally at high temps. Along this gradient, microorganisms become established, with different → The heat stability of an enzyme is often species growing in the different temp due to subtle changes in amino acid ranges. sequence from the corresponding By studying species distribution, it has enzyme of a mesophile. been possible to determine the upper These changes affect protein temperature limits for various classes structure and function to resist heat of microbes. denaturation. → Thermophilic Bacteria and Archaea have → Heat stable proteins also show also been found in artificial thermal increased ionic bonding between basic environments such as hot water heaters. and acidic amino acids and have highly hydrophobic interiors. These are factors that prevent unfolding. → Solutes such as di-inositol phosphate, diglycerol phosphate, and mannosylglycerate are produced at high levels.  These helps stabilize their proteins against thermal denaturation. ENVIRONMENTAL EFFECTS ON GROWTH: PH, OSMOLARITY AND OXYGEN → Enzymes from thermophiles and hyperthermophiles have significant EFFECTS OF PH ON MICROBIAL GROWTH commercial uses. → Heat stable enzymes can catalyze → Acidity or alkalinity of a solution is biochemical reactions at high temps. expressed in its pH on a logarithmic scale Also more stable than enzymes from in which neutrality is pH 7. mesophiles, prolonging shelf life of enzyme preparations. → pH values less than 7 are acidic and Example: DNA polymerase from those greater than 7 are alkaline. Thermus aquaticus - Taq-polymerase” which is used to → Every microorganism gas a pH range automate the repetitive steps in about 2-3 pH units, within which growth the polymerase chain, a technique is possible. for amplifying DNA and a major tool of modern biology. → Each organism shows a well-defined pH optimum, where growth occurs best. → The cytoplasmic membranes of thermophiles and hyperthermophiles → Most natural environments have a pH must be heat stable. between 3 and 9. Heat naturally works to peel apart the organisms with pH growth optima in lipid bilayer in the cytoplasmic this range are common. membrane. The cytoplasmic membrane has a higher content of long-chain and saturated fatty acids and a lower content of unsaturated fatty acids. The saturated fatty acids form stronger hydrophobic environment and longer chain fatty acids have higher melting point that increase membrane stability. → Hyperthermophiles (most Archaea) do not contain fatty acids in their membranes. Instead have C40 hydrocarbons composed of repeating units of isoprene bonded by ether linkage to glycerol phosphate. Will form a lipid monolayer than bilayer. The monolayer covalently links both halves of the membrane and prevents it from melting at high growth temperatures.  ACIDOPHILES → Example: Most acidophilic microbes is Picrophilus oshimae, species of Archaea that grows optimally at pH 0.7 and 60°C Above pH 4 cells will lyse. ALKALIPHILES → Few extremophiles have a very high pH optima which can be as high as 10 pH. → Neutrophiles: organisms that grow → Alkaliphiles: microorganisms showing optimally at pH value in the range themed pH optima of 8 or higher. circumneutral (5.5 – 7.9) Example: E. coli is a neutrophile, → Best studied alkaliphilic bacteria are Bacillus species, such as Bacillus → Acidophile: organisms that grow best firmus. below pH 5.5. This organism is alkaliphilic but has an unusually broad range for growth from → There are different classes of acidophiles, pH 7.5 to 11. some growing best at moderately acidic pH and others at very low pH. → Some extremely alkaliphilic microbes are halophilic (salt-loving), and most of → Many fungi and bacteria grow best at pH these are Archaea. 5 or below. A more restricted number grow is best below pH 3. → Some phototrophic purple bacteria are also strongly alkaliphilic. → An even more restricted group grow best below pH 2 and those with pH optima → Certain alkaliphiles have commercial below 1 are rare. uses because they excrete hydrolytic enzymes such as proteases and lipases → Most acidophiles cannot grow at pH 7 that maintain their activities at alkaline and many cannot grow at pH values more pH. than 2 units above their optimum. These enzymes are added to laundry detergents to remove protein and fat → Critical factor governing acidophily: stains. stability of the cytoplasmic membrane. → A problem for alkaliphiles is managing → pH is neutral: cytoplasmic membrane of membrane bioenergetics. strongly acidophilic bacteria is destroyed B. firmus uses sodium (Na+) rather then cell lyses. than H+ to drive transport reactions Indicates that these organisms are not and rotate its flagellum. just acid-tolerant but that high Forms a sodium motive force instead concentrations of protons are required of proton motive force. for cytoplasmic membrane stability.  B. firmus uses a proton motive force → Water availability depends not only on to drive ATP synthesis even though how moist or dry an environment is but the external membrane surface is also on the concentration of solutes highly alkaline. (salts, sugars, or substances) → Solutes bind water, making it less available to organisms. Hence for CYTOPLASMIC PH AND BUFFERS organisms to thrive in high solute environments, physiological adjustments are necessary. → The optimal pH for growth of an organism refers to the extracellular → Water availability is expressed in terms of environment only. water activity (aw), the ratio of the vapor pressure of air in equilibrium with a → The intracellular pH must be substance or solution to the vapor maintained at a value consistent with the pressure of pure water. stability of macromolecules, a range of about 4 pH units from pH 5 to 9 → Values of aw vary between 0 (no free water) and 1 (pure water). → Despite pH in habitats, extreme acidophiles and alkaliphiles maintain cytoplasmic values neared to neutrality. → To prevent major shift in pH during microbial growth, buffers are along with the nutrients required for growth. However, any given buffer works over only a relatively narrow pH range. → For neutrophilic species, potassium phosphate (KH2PO4) or sodium bicarbonate (NaHCO3) is often employed. → Buffer keeps the enzyme solution at optimal pH during the assay thus ensuring that the enzyme remains → Water diffuses from regions of higher catalytically active and unaffected by any water concentrations to regions of lower protons or hydroxyl ions generated in the concertation in a process of osmosis. enzymatic reaction. → The cytoplasm of a cell typically has a higher solute concentration than environment, so water has the tendency OSMOLARITY AND MICROBIAL GROWTH to diffuse into the cell. → Cell is said to be in positive water → Water is the solvent of life and its balance, which is the normal state of the availability is an important factor affecting cell. the growth of microorganisms. However, when a cells is placed in a environment where solute  concentration exceeds that of the → Extreme halophiles: halophiles capable cytoplasm, water will flow out. pf growth in very salty environments. If a cell has no strategy to These organisms require very high counteract this, it will become levels of NaCl, typically 15-30% dehydrated and unable to grow. → Osmophiles: organisms able to live in environments high in sugar. HALOPHILES AND RELATED ORGANISMS → Xerophiles: organisms able to grow in very dry environments. → Osmotic effects happen mainly in habitats with high concentration of salts. → Seawater contains about 3% of NaCl plus small amounts other minerals and elements. Microorganisms that inhabit marine environments always show an NaCl requirement and grow optimally at the aw of seawater, 0.98. Such organisms are called halophiles → The requirement for NaCl by halophiles is absolute and cannot be replaced by other salts such as KCl, CaCl2, MgCl2. → The lower water activity limit for living → Although halophiles require at least some organisms is 0.61. NaCl for growth, the optimum amount This is likely set by the varies with the organisms and is habitat physiochemical constraints on dependents. obtaining water in osmotic environments of aw less than 0.6 that → Example: cannot overcome through biochemical Marine microorganisms typically adaptation by the cell. grow best with 1-4% NaCl. Organisms from hypersaline → Matric water activity: measure of water environments grow best at 3-12% bound to a surface, is measured in the NaCl. same way as osmotic water activity but Organisms from extremely can drop to lower than 0.6 and still hypersaline environments require contain viable microbial communities. higher levels of NaCl. → Example: hyper arid hot desert soils can → Organisms isolated from brackish waters have matric aw values as low as 0.1 may or may not be halophilic. during daylight hours. But these environments absorb → Halotolerant: can tolerate some level of moisture at night and during rain dissolved solutes but grow best in the events. absence of the added solute.  It increases water activity to above 0.6 halophilic, or extremely halophilic are making conditions suitable for to some extent a reflection of their microbial metabolism and growth. genetic capacity to produce or accumulate compatible solutes. COMPATIBLE SOLUTES OXYGEN AND MICROBIAL GROWTH → In low-water environments, organisms raise their internal solute levels to hold → Oxygen (O2) is an essential nutrient for onto water. They do this by either many microbes. They are unable to importing solutes or making their own. metabolize growth without it. In either case, the solute must not inhibit biochemical processes in the → Others cannot grow in the presence of O2 cell and is thus called a compatible and may even be killed by it. solute. → Compatible solutes are highly water- OXYGEN CLASSES OF MICROORGANISMS soluble organic molecules and include sugars, alcohols, and amino acid derivatives. → Microorganisms can be grouped according to their relationship with O2. → Glycine betaine – an analog of the amino acid glycine, is widely distributed among → Aerobes: can grow at full oxygen halophilic bacteria. tensions and respire O2 in their metabolism. → Other common compatible solutes include: → Microaerophiles: aerobes that can use sugars such as sucrose and trehalose only O2 when it is present at levels dimethyl sulfoniopropionate – reduced from that in air. produced by marine algae. This is because of the limited capacity Glycerol – common solute in xerophilic of these organisms to respires or fungi. because they contain some O2 sensitive molecule such as O2 labile → KCl is the compatible solute of extremely enzyme. halophilic bacteria such as Halobacterium. → Facultative aerobes: under appropriate nutrient and culture conditions they can → Cells adjust compatible solute levels grow in the absence of O2. based on their environment in response to the challenge of external solutes. → Anaerobes: organisms that cannot The maximal level of compatible respire oxygen. There are two types: solute tolerated is a genetically coded Aerotolerant anaerobes: can tolerate characteristic. O2 and grow in its presence even As a result, different organisms have though they cannot respire. evolved to thrive in habitats of Obligate anaerobes: inhibited or different salinities. killed by O2. → Organisms designated as → Anoxic (O2 free) microbial habitats are nonhalotolerant, halotolerant, common in nature and include muds and  other sediments, bogs, marshes, water- the medium through a fine glass tube or logged soils, intestinal tracts of animals, porous glass disk. sewage sludge, the deep subsurface of earth. → For culturing anaerobes, the problem is not to provide O2 but to exclude it. → Bottles or tubes filled completely to the top with culture medium and fitted with leakproof closures provide suitably anoxic conditions for organisms that are not overly sensitive to small amounts of O2. → A chemical called a reducing agent may → Obligate anaerobiosis is characteristic of be added to such vessels to remove three groups: traces of O2 by reducing it to water. Bacteria Example: thioglycolate, which is Archaea present in thioglycolate broth. Few fungi and protozoa Thioglycolate broth is a complex medium containing a small amount of → Some of the best known prokaryotic agar, making the medium viscous but anaerobes are Clostridium – a gnus of still fluid. gram positive endospore forming Bacteria and the methanogens – a group of → After thioglycolate reacts with O2 methane producing Archaea. throughout the tube. O2 can penetrate only near the top of the tube where the → Among obligate anaerobes, the sensitivity medium contacts air. to O2 varies. Many clostridia although require anoxic conditions for growth, can tolerate traces of O2, or even full exposure to air. Others, such as the methanogens are killed rapidly by O2 exposure. CULTURE TECHNIQUES FOR AEROBES AND ANAEROBES → Extensive care is needed when growing aerobes because O2 that is consumed by the organisms during growth is not replaced fast enough by diffusion from the air. → Facultative organisms grow throughout the tube but grow best near the top. → Forced aeration of liquid cultures is needed and can be achieved by either → Microaerophiles grow near the top but vigorously shaking the flask on a not right at the top. shaker or by bubbling sterilized air into → Anaerobes grow only near the bottom of → Electron carriers found in cells like the tube where O2 cannot penetrate. flavoproteins, quinones, and iron sulfur proteins also catalyze some of the → Aerotolerant anaerobes grow byproducts. throughout the tube. Thus regardless of whether it can respire O2, an organisms exposed → The redox indicator dye resazurin is to O2 will experience toxic forms present in thioglycolate broth to signal of oxygen and it not destroyed can oxic regions. wreak havoc to cells. Dye is pink when oxidized. Colorless when reduced. Gives a visual assessment of the → Example: Superoxide anion and OH are degree of penetration of O2 into the strong oxidizing agents that can oxidize medium. macromolecules and other organic compounds in the cell. → To remove all traces of O2 for culture of strict anaerobes, one can incubate in a → Example: Peroxides such as H2O2 can glass jar flushed with an O2-free gas or also damage cell components but are not fitted with an O2 consumption system. as toxic as O2- or OH → For manipulating cultures in an anoxic atmosphere, special enclosures called anoxic glove bags permit work with open SUPEROXIDE DIMUTASE AND OTHER cultures in completely anoxic ENZYMES THAT DESTROY TOXIC OXYGEN atmospheres. → To keep toxic oxygen molecules under control, microbes have enzymes that destroy the compounds. WHY IS OXYGEN TOXIC → Superoxide anion and H2O2 are the most toxic oxygen species. → Oxygen is not toxic, but it can be converted to toxic oxygen by products. → The enzymes catalase and peroxidase These byproducts can harm or kill attack H2O2 forming O2 and H2O cells not able to deal with them respectively. These include superoxide anion (O2-), hydrogen peroxide (H2O2) and → Superoxide anion is destroyed by the hydroxyl radical (OH ) enzyme superoxide dismutase, an enzyme that generates H2O2 and O2 from two molecules of O2- → Superoxide dismutase and catalase (or peroxidase) thus work in series to convert O2- to harmless products. → Aerobes and facultative aerobes typically contain both superoxide dismutase and catalase. → Some aerotolerant anaerobes lack them and use protein-free manganese complexes instead to carry out the dismutation of O2- to H2O2 and O2. Is not as efficient as superoxide dismutase but is sufficient to protect cells from damage. → In some strictly anaerobic Archaea and Bacteria, superoxide dismutase is absent. Instead, the enzyme superoxide reductase functions to remove O2- Superoxide reductase reduces remove O2- to H2O2 w/o the production of O2