Microbial Growth - University of Lethbridge



Microbial GrowthWhy study growth?Important to understanding biology of an organism – growth is essential to any organism's existenceInformation on growth is required for control microoganismsDefinitions of GrowthSteady increase in all the chemical components of an organism that may result in an increase cell size, cell number or bothIncrease in biomass as measured by changes inDry weight increase Increase in absorbance Increase in cellular constituents Protein Nucleic acids other constituents e.g., peptidoglycan and chitinGrowth results in increased cell size and frequently cell divisionParticularly relevant to unicellular organisms:In unicellular organisms cell growth results in increase in numbersIn multicellular organisms cell growth results in an increase in organism sizeI.Factors that Affect GrowthA.Chemical factors Nutrients are substances used in biosynthesis and energy release and are therefore required for growthOne must define nutritional requirements in order to cultivate the microbe in the laboratoryChemical factors are supplied by i) the culture medium (pl. - media) that contains substrates required for growth and ii) culture conditions (i.e., aerobic vs anaerobic conditions).1. Macroelements (major elements - C, O, H, N, S, P, K, Ca, Mg, and Fe)required in large amounts by the cell – >95% of cells are composed of macroelements (sometimes call macronutrients)C, O, H, N, S, P are components of macromoleculesCarbonLife on earth is carbon basedHalf of the dry weight of a typical cell is carbonNitrogenNitrogen makes up approximately 14% of the dry weight of a typical cellMajor constituent of protein and nucleic acids, some carbohydrates and lipidsNH3, NO3-, N2 (nitrogen fixation) and organic N compounds (e.g., amino acids) from the environment. Some bacteria use atmospheric nitrogen (N2) as a nitrogen sourcePhosphoruscomponent of phospholipids and nucleic acids, nucleotides such as ATP, some proteinsavailable as organic and inorganic forms in the environmentSulfurstructural role in methionine and cysteine as well as a number of vitamins (thiamine, biotin), coenzyme A and some carbohydratesavailable usually from inorganic sources SO42- or H2S and organic sulfur compounds such as cysteineK, Ca, Mg, and Fe are cations in cells and required for a variety of roles e.g., - cofactors (K+, Ca2+, Mg2+, and Fe2+ or Fe3+)- stabilize membranes and ribosomes (Mg2+)- contribute to heat resistance of endospores (Ca2+) - components of biomolecules such as cytochromes (Fe2+ and Fe3+) 2. Trace elements or Micronutrientsrequired in lesser or trace amounts.Critical to cell functionMany are metals – structural role with many enzymes - cofactorsoften trace elements present in medium components or water provide all that is required for growthCo, Cu, Mn, Mo, Ni, and Zn are needed by most cells. Some cells require Cr, Se, W, and V3. Oxygena) Aerobic organisms growth at full atmospheric O2 tensions (21% O2 in the atmosphere)facultative organisms (under appropriate nutrient and culture conditions) can grow under either aerobic or anaerobic condition obligate aerobes - require O2 for growthO2 is poorly soluble - forced aeration is often used in culture systems to provide O2b) Anaerobic organismsobligate (strict) anaerobes - grow only in the absence of O2; sensitive to O2 and brief exposure will kill these organisms; perhaps because these organisms are unable to detoxify some of the products of O2 metabolismlack a respiratory system and can’t use oxygen as a terminal electron acceptorThese organisms do use oxygen found in cellular materialsObligate anaerobiosis - prokaryotes, and a few groups of fungi and protozoaToxic forms of oxygenOxygen itself is not toxic to anaerobic organisms – rather it is certain derivatives that are toxicreduction of O2 in respiration produces several toxic products singlet oxygen (1O2-) – produced photochemically and biochemically (peroxidase activity). Outer shell electrons become highly reactive; carry out spontaneous and undesirable oxidations in the cellhydrogen peroxide (H2O2) – Produced during aerobic respiration; damage cell components but not as toxic as O2.-, or OH·superoxide (O2.-) – Formed in small amounts during aerobic respiration; highly reactive and can oxidize any organic compound in the cell hydroxyl radical (OH·) - most reactive, instantly oxidize any organic substance in the cell.All cells contain flavoproteins, quinines, thiols, and iron-sulfur proteins that can react with O2 and produce superoxideIonizing radiation is the major source of hydroxyl radicals. Small amounts of hydroxyl radicals can be produced from H2O2.A number of enzymes have evolved to detoxify oxygen speciesCatalasedestroys H2O2 H2O2 + H2O2 2 H2O + O2Catalase test - 30% H2O2 place on cells. Cells with catalase activity produces vigourous bubbling as O2 is releasedPeroxidase destroys H2O2 but does not produce O2. May require a reductant such as NADHH2O2 + 2H+ 2 H2OSuperoxide dismutase (SOD)Destroys superoxideIndispensable to aerobic cellsO2.- + O2.- + 2H+ H2O2 + O2Generally works in tandem with catalase: 4O2- + 4H+ 2H2O + 3O2Superoxide reductaseFound in some obligately anaerobic prokaryotesO2.- + 2H+ + cytochrome creduced H2O2 + cytochrome coxidizedAvoids production of O2 as found with SODH2O2 may then be removed by peroxidase activityAerobes and facultative anaerobes usually produce superoxide dismutase and catalasec) Aerotolerant anaerobes tolerate O2 and grow in its presence even though they can’t use oxygen. Aerotolerant organisms can tolerate oxygen because they produce SOD or equivalent system that neutralizes toxic oxygen species. Usually lack catalase activityd) Microaerophiles grow only at reduced O2 concentrations (2 to 10%)These organisms have limited capacity to respire or have some oxygen-labile molecules; sensitivity to oxygen may also be due to the sensitivity superoxide radicals and peroxidesO2 usually excluded from culture systems by one or a combination of the following mechanismsFill container to the top and sealBoil medium to drive out O2Use reducing agents that react with O2; reduces it to H2O (e.g., thioglycolate, cysteine, H2S)Seal containers under O2 free gasUse redox indicators such as resazurin to indicate the presence of O2.Use O2 consuming devices (catalyst)Work under a stream of O2 free gas or in an anoxic glove box/anaerobic chamber4. Other required elementsSome microbes may have particular requirements that reflect their specific environment (Halophiles require Na+) and morphology (Diatoms and Silicon dioxide based cell walls)5. Growth FactorsSome microbes have the enzymes and biochemical pathways needed to synthesize all cellular components using minerals and sources of energy, carbon, nitrogen, phosphorus and sulfur.Other microbes lack one or more enzymes necessary to synthesize essential constituents – they get these constituents or precursors from the environmentGrowth factors are organic compounds that are essential cellular components or precursors of these components but cannot be synthesized by the organismMajor Classes of Growth factorsamino acidspurine and pyrimidinesvitamins (e.g., thiamine, biotin, cobalamin, pyridoxine)Other growth factors include heme (nonprotein component of many cytochromes) or cholesterolUnderstanding growth factor requirements has practical implicationsBioassays using microbes to detect the specific growth factor that they need. Growth-response assay – uses this approach to detect the amount of a growth factor in solution. These assays can be specific, sensitive, simple and quantitativeManufacture of growth factors by specific microorganisms (e.g., Vitamin D by Saccharomyces) in industrial fermentationsB.Physical (or environmental) Factors1. The Effect of Temperature on Growth Cardinal temperatures (Fig 6.1)Depend on environmental factors such as pH and available nutrientsa) Minimum temperature - below which cells are inactive reduced membrane fluidity – perhaps affects nutrient transport or proton gradient formationb) Optimum temperaturehighest rate of growth and reproduction, always nearer maximum temperaturec) Maximum temperature - above which growth is not possibleGrowth stops because of inactivation of one or more key proteins, damages transport carriers or other proteins, or thermal disruption of membraneCardinal temperatures vary for different organismsMedium composition can have a slight affectTemperature optima usually vary from 0C to 75C Pyrolobus fumarii (archaeon) - maximum temperature = 113CGrowth temperature range for a particular organisms usually spans 30 to 40CDistinguish five groups of microbes based on temperature optimai) PsychrophilesGrow well at 0C and have an optimum temperature 15C and a maximum temperature around 20Cheat sensitive and unable to survive temperate climatesAdaptations to PsychrophilyEnzymes, transport systems and protein synthetic apparatus work well at low temperaturesenzymes with low temperature optima greater amounts of -helix and lesser amounts of sheet secondary structuregreater amounts of polar amino acids and lesser amounts of hydrophobic amino acidsmembranes contain higher amounts of unsaturated fatty acidssome psychrophiles have membranes higher in polyunsaturated fatty acidsii) Psychrotolerant (psychrotrophs, facultative psychrophiles) grow at 0C but have optima of 20 - 30Ciii) MesophilesOptimum temperature between 25 and 40CMinimum temperature between 15 and 20CMaximum temperature 45CMost common type of microbee.g., E. coli Optimum temperature < 39CMaximum temperature < 48CMinimum temperature 8Civ) Thermophiles Optimum temperature between 50 and 60CMinimum temperature around 45CMaximum temperature 45COnly prokaryotes grow above 60C The most thermophilic organisms are ArchaeaNonphototrophic organisms are able to grow at higher temperatures than phototrophic formsv) HyperthermophilesOptimum temperature > 80CExtreme thermophiles are usually Archaea The highest growth temperatures for an archaeon is 113C (Pyrolobus fumarii)Adaptations to Thermophilyi) Enzymes and other proteins are heat stableSubtle amino acid substitutionsIncreased number of salt bridges Densely packed hydrophobic interiorsThe presence of certain solutes such as di-inositol phosphate and diglycerol phosphateii) Macromolecules function optimally at high temperaturesiii) Membrane is heat stableMembrane lipids are more branched, rich in saturated fatty acids and of higher molecular weightIn some cases they have lipid monolayers (diglycerol tetraethers)iv) DNA is stabilized by special histone – like proteinsReview cell membrane structure – Chapter 4Why don’t eukaryotes grow above 60C?Applications of ThermophilyHigh temperature enzymese.g., feed pelleting processPCR – Taq DNA polymerase from Thermus aquaticus 2. The Effect of pH on Growth All organisms have a characteristic pH range within which growth is possible. The range is usually 2 – 3 pH units.In nature, environmental pH ranges from 5 to 9Few organisms can growth at pH < 2 and > 10pH is a great influence on growth ratepH is important because of its effect on proteins (charge is important to protein conformation) as well as the plasma membranea) neutrophiles - pH optimum between 5.5 and 8Most bacteria grow well within the pH range of 6 - 9 b) alkaliphiles - prefer growth under alkaline conditions (pH 8.0 to 11.5)many produce enzymes that work well at high pH – useful for the detergent industryc) acidophiles - restricted to growth at low pH values – between 0 and 5.5Fungi are generally more acid tolerant than bacteria – many grow at pH 4 to 6Some Bacteria and Archaea are obligate acidophilese.g., Bacteria - Thiobacillus Archaea - SulfolobuspH has an important effect on stability of acidophile plasma membraneIntracellular pHIntracellular pH is usually between pH 6 to 8 but internal pH as low as 4.6 and as high as 9.5 have been measuredMaintained by pumping H+ across the membrane, internal buffering and synthesizing new proteins (e.g., acid shock proteins and heat shock proteins) that function by pumping protons or acting as chaperones3. Osmotic Effects on GrowthMicrobes require water to grow – their cells are 80 – 90% waterWater availability depends not only on amount of water present in any environment but also the concentration of solutes present (e.g., salts, sugars,…). Water activity (aw) - amount of water that is free to react = availability of water in a substanceaw = a ratio of the vapour pressure of the air in equilibrium with a substance or solution to the vapour pressure of pure water (1/100 the relative humidity of a solution)aw ranges between 0 and 1Most bacteria require an aw of 0.9 for active metabolismMost organisms are adversely affected by very low water activity (They suffer from plasmolysis)In nature osmotic effects are of interest mainly in habitats with high salt concentrationa) Halophilic bacteriaA organism requiring salt (NaCl) for growthmicrobes found in the sea (which is 3% NaCl) usually have a growth requirement for saltMild halophile – salt requirements between 1 and 6%Moderate halophile - salt requirements between 7 and 15%Extreme halophiles - salt requirements between 15 and 30% (e.g., Archaebacteria such as Halobacterium species)Halotolerant organisms can withstand some reduction in aw but generally grow best without added soluteOsmotolerant – grow over a wide range of water activityOsmophiles - require high solute (e.g., sugar) concentration for growthXerophiles – able to grow in very dry environments (i.e., made dry by lack of water)How does an organism grow under low aw?Increases internal solute concentration Pumps inorganic ions (e.g., K+) into the cellSynthesize or concentrate an organic solute (e.g., proline, glycine betaine, sucrose, trehalose, mannitol)These substances must not inhibit biological processes; they are usually highly water solubleHow does an organism grow under high aw?II.Microbial Growth in Natural EnvironmentsMost natural ecosystems are complex and constantly changingLow concentrations of usable nutrients (Oligotrophic)CompetitionGrowth in an environment depends on the nutrient supply and the microbes tolerance for the environment.Liebig’s law - the total biomass of an organism will be determined by the nutrient present in the lowest concentration relative to the organism’s requirementsShelford’s law – there are limits to environmental factors below and above which a microorganism cannot survive and grow regardless of the nutrient supplyMost bacteria are likely to experience starvation. How do they deal with nutrient limitation?Reduction in cell sizeChange in morphology – increase surface area and ability to absorb nutrientsShutdown of metabolism except for housekeeping maintenance genesBiofilmsMost microbes are typically found in biofilms in natureBiofilms consist of cells embedded in EPS (Chapter 4)Microbes in biofilms share nutrients, communicate (e.g., quorum sensing), exchange genetic information and are sheltered from adverse environmental factors (i.e., desiccation, antibiotics, host immune response)Microbes in biofilms can be 1000X more resistant to antimicobial compoundsMicrobes in biofilms can carry out complex chemical processes (i.e., breakdown of plant cell walls such as occurs in the rumen)IIICulture MediaA culture medium (pl = media) is a nutrient solution used to grow microorganisms in the laboratory. The growth medium is the most important factor when culturing microbesThere are vast differences in the biosynthetic capacities of microorganisms and thus a need for a variety of culture media. Knowledge of the microoganism’s normal habitat is useful in selecting an appropriate mediumSpecialized media are used for a variety of purposes, including isolation and identification of microorganisms, testing antibiotic sensitivities, water and food analysis, industrial microbiology Factors like temperature, pH, Oxygen and pressure must also be considered when culturing micoroganisms Inoculum (pl. = inocula) = microbes introduced into a culture medium to initiate growth. These cells multiply and are referred to as the culture.Fastidious microorganisms - have very rigorous or complex requirements (e.g., for vitamins, amino acids...)A.Chemical and Physical Types of Culture Media1. Chemically defined (synthetic) media the exact chemical composition of the medium is known measured amounts of highly purified inorganic and organic chemicals are added to distilled waterBM+G (chemically defined medium)Ingredientg/L in dH2OGlucose2.0(NH4)2SO4 2.0K2HPO40.5Monosodium glutamate5.0MgSO4.7H2O0.3MnSO4.H2O0.05CaCl20.08ZnSO4.7H2O0.005CuSO4.5H2O0.005FeSO4.7H2O0.00052. Complex mediacertain components are of unknown composition and these components may change from batch to batch.Use of this type of medium results in the loss of control of nutrient compositionLuria Burtani (LB; Chemically undefined or Complex medium)Ingredientg/L in dH2OYeast Extract5.0Tryptone10.0NaCl5.0Tryptic Soy Broth (TSB Chemically undefined or Complex medium)Ingredientg/L in dH2OTryptone17.0Peptone3.0Glucose2.5NaCl5.0Dipotassium phosphate2.5Refer to appendix 8 of lab manual for other examples of complex media3. Liquid or solidified mediaBoth liquid and solidified media are routinely used in microbiologySolidified media is particularly important for the establishment of pure cultures as well as determination of cell number. It is often desirable to have cells produce colonies (visible, isolated masses of cells) - Colonies come in different shapes, sizes, textures and colors, and colonial morphology may be useful in identifying a microorganismAgar is the most commonly used solidifying agent. It is extracted from red algae and is a sulfated heteropolymer of D-galactose, 3,6-anhydro-L-galactose and D-glucuronic acid. Agar is added to a final concentration between 1 and 2% with 1.5% w/v being the most commonly used concentration.Agar is particularly well suited for this application because it melts at a relatively high temperature (90C) but does not solidify until it reaches 45C. Moreover, very few microorganisms can hydrolyze agar.Agar is melted during sterilization and the molten medium is poured into Petri dishes and allowed to solidifyB.Functional Types of Culture MediaComplex media such as tryptic soy broth are called general purpose media or supportive media because they sustain the growth of many microorganismsFor some particularly fastidious organisms additional components such as whole blood or serum must be added. These media are referred to as enriched media and designed to better mimic natural conditions (i.e., host for pathogens)Selective mediumA medium with a composition favoring growth of certain types of microorganisms while inhibiting growth of any other microorganisms that may be present.ExamplesDifferential mediumA medium that contains substance(s) that permits for the differentiation of particular metabolic activities during growth. Useful in distinguishing particular groups of microbes and may provide information useful in identificationExamplesSelective and differential characteristics may be combined in a single mediumExamplesC.Enrichment techniqueDeveloped by BeijerinckThe use of culture media or conditions that favour growth of one type or group of physiologically related microorganisms over all other microorganisms present in the sampleD.Notes on culturing microbesnot all microbes can be cultured in the laboratory General usage media generally permit the growth of a wide variety of microbes.At times it is desirable to use environmental or nutritional factors to selectively cultivate a certain group or kind of microorganism.Aseptic TechniqueSeries of steps used to minimize contamination during the manipulations of cultures and sterile culture mediaSterilize all media and implements for handling materials of interestClean working areaLimit exposure to potential sources of contaminationPreparation of Pure CulturesStreak plate techniqueDilution Deposition of individual cells or clumps of cells (known as colony forming units or CFU) on agar mediumCell growth multiplication resulting in the production of colonies (visible mass of cells)each isolated colony on the streak plate is assumed to have originated from a single CFU (It is unknown whether the cells in the colony came from a single cell or a clump of cells)Preserving Bacterial Cultures1. Refrigeration at 4Cshort term solution - several weeks to several monthsduration depends on type of medium2. Glycerol stocksSterile glycerol is added to liquid cultures to a final concentration of 15 – 25%The stocks are placed in small plastic tubes with tight fitting lids (i.e., preferably screw cap tubes with gaskets in the lids)The glycerol stocks are stored at -20C (1 to 2 years) or -80C (up to 10 years or more)3. LyophilizationFreeze dryingCulture is quick-frozen at temperatures ranging from -50 to -90C and then dried under vacuum on a lyophilizer; freeze dried cultures are stored in sealed glass ampules for extended periods of time.Microbial Culture CollectionsSources of microbial culturesCultures are distributed for a fee or free depending on the culture collectionATCCAmerican Type Culture CollectionDSMZDeutsche Sammlung von Mikrooganismen und ZellkulturenNCTCNational Collections of Type Cultures and Pathogenic FungiNCIMBNational Collections of Industrial and Marine BacteriaEGSCE. coli Genetic Stock CentreBGSCBacillus Genetic Stock CentreFGSCFungal Genetic Stock CentreIV.Growth of Microbial Culturesi) Eukaryotic Cell Cycle – review Biol 1010 notesii) Prokaryotic cell cycle most often is accomplished by Binary Fission but budding, fragmentation and other processes may occurMother cell two daughter cells …Generation time (g)Binary fission in E. coli takes 20 minutes under optimal conditionsRequired as many as 2000 chemical reactions Length of time depends on a number of factors, including nutrition, genetics and environmentRapidly Growing CellsIn E. coli, the cell cycle takes 60 min to complete: 40 minutes for DNA replication and partitioning and 20 min for septum formation and CytokinesisBut E. coli can complete this entire process in 20 min under optimal conditionsThis is possible because E. coli starts a second round of DNA replication (and sometimes a third and a fourth round) before the first round of replication is completed. A.Population GrowthGrowth rate change in cell number or cell mass per unit timeGenerationinterval for the formation of two cells from one cellGeneration time (doubling time) time it takes for one cell to become two cellstime it takes for the population to doubledepends on growth medium and conditions1.The Mathematics of Growth (Growth Equations)Growth by binary fission results in exponential growth of the population (Figure 6.13 & 6.14)Geometric progression of the number 221222324 (1) Nt = N02nNt = final number of cells at time tN0 = initial number of cellsn = number of generations that have occurred during period of exponential growthSolving for n (where all logarithms are to the base 10)log Nt = log N0 + n log 2 and(2)n = log Nt - log N0 = log Nt - log N0 log 2 0.301Growth rate can also be expressed as the mean growth rate constant (k). The specific growth rate is a measure of the number of generations that occur per unit time(3) k = n/t = log Nt - log N0 0.301tCan now calculate the mean generation time (g) or mean doubling time.When the population doubles t = g and Nt = 2N0; substitute 2N0 into (3)(4)k = log (2N0) - log N0 = log 2 + log N0 – log N0 = 1/g 0.301g0.301gTherefore(5)g = 1/k Generation time can also be calculated from the slope of a line obtained in a semi-log plot of exponential growth(6) slope = 0.301/g ; g = 0.301/slopeHow can we use growth rate information?2.Culture Systems"Fermentation" - cultivation of microorganisms in a controlled, enclosed systemi.Batch CultureA fixed volume of liquid medium is inoculated and incubated for an appropriate period of time with no further addition of microorganisms or growth substratesclosed environmentmost common method of microbial cultivationnutrient concentration is a determinant of growth rate and cell yieldThe batch culture has a continually changing environment nutrients are depletedproducts producedcells changeUltimately the culture quits growing due to nutrient limitation or product accumulatione.g., test tube to flask to 100,000 L fermenterii.Fed BatchA nutrient stock (limiting nutrient) is added at intervals or continuously to a batch cultureiii.Continuous CultureSpent culture is replaced by fresh medium allowing continual growth of the culture.Open system system can be manipulated to reach an equilibrium or steady state where the cell density and nutrient status remain constantCan control culture growth rate as well as yield of cells by manipulating dilution rate and the level of the limiting nutrient, respectivelyMore sophisticated apparatus requiredSuperior productivity possible because of reduced downtime.e.g., Chemostatuses dilution rate and nutrient concentration to control growth and population densitygrowth rate (adjust dilution rate) and yield (adjust limiting nutrient) can be controlled independently of each other Compared to batch culture – the chemostat allows:experimenter to vary growth rate and population density independently of each othercan maintain population in exponential phase at a known growth rate for long periods of timeCan study microbial growth at very low nutrient concentrations – close to those present in nature3.Bacterial Growth CurveGrowth of a batch culture population of cells can be monitored and plotted as a growth curveA typical batch culture growth curve can be divided into 4 phases (Fig 6.15)i) Lag PhaseInitial phase during which time cells are adjusting their metabolism to prepare for a new cycle of growth.There is no increase in cell number - increase in cell sizeThe cells are transporting nutrients, synthesizing RNA and subsequently enzymes needed for growth; replicating DNAThe length of this phase depends on the history of the culture and growth conditionsExamples:ii) Exponential Phase (Log phase)Cell are growing and dividing at the maximum growth rate possible given their genetic potential, the nature of the medium and incubation conditions.One cell gives rise to two and so on: Cell number is increasing as an exponential function of time Log transformation of data results in a linear curveDuring this phase the resulting cell population is most uniform with respect to chemical and physiological properties; cells in this phase are most often used in biochemical and physiological studiesExponential growth is said to be balanced growth because all cellular components are made at constant rates relative to each other. If the nutrient levels or some other environmental parameter changes then unbalance growth results: growth during which the rates of synthesis of the various cellular constituents vary relative to one another until a new balanced state is reached. Shift-up (culture is transferred from a nutritionally poor medium to a richer medium) and shift-down (culture is moved to from a nutritionally rich medium to a poor medium) experiments produce unbalanced growth. In the shift-up experiment there is a lag in while the cells first produce more ribosomes to enhance protein synthesis. There is then an increase in protein and DNA synthesis followed by the rise in productivity.In the shift down experiment:Determinants of growth rate Different nutrients and nutrient concentration allow for different growth rates. Growth rate increases with increasing nutrient concentration. At some point nutrient transport systems are saturated and growth rate can increase no furtherTemperature, pH, Oxygen and other physical parametersGenetic determinantsSmall cells generally grow faster than larger cells (surface area to volume ratio)Nutrient concentration affects maximum cell yieldiii) Stationary PhaseClosed system - cells can’t grow indefinitelyNo further net increase in cell numberTotal number of viable cells remains unchanged because i) growth rate = death rate (i.e., some cells in the population grow while others die. This is known as cryptic growth) or ii) the population may not be dividing but remain metabolically activeStationary phase is entered because 1) nutrient limitation, 2) oxygen limitation, 3) build up of toxic wastes (e.g., organic acids), 4) a critical population level is reached, or 5) several of these factors acting togetherCellular composition and activity changes Prokaryotes have evolved a number of strategies to deal with starvation. A few genera will produce endospores but most will reduce cell size, which is often accompanied by protoplast shrinkages and nucleoid condensation. Morphological changes can also occure.g., Arthrobacter - log cells - rods - stationary cells - coccoidThe most important changes are in gene expression and physiology. Different genes are turned on (e.g., catalase, exonuclease and acid phosphatases; survival genes (sur) have been identified for E. coli)Most starving cells produced starvation proteins that make the cell more resistant to environmental stresses (e.g., elevated temperature, osmotic pressure and toxic chemicals such as hydrogen peroxide and chlorine) and harder to kill. The cells increase peptidoglycan crosslinking and cell wall strength, produce proteins to protect their DNA (DNA binding protein from starved cells – Dps) and to prevent protein denaturation and renature damaged proteins (Chaperone proteins).vi) Death Phase (Senescence phase)Exponential decline in viable cell numbers. Typically the rate of exponential decline is much slower than that of exponential growthIn many instances this phase can be reversed if modify the environmental parametersIn many cases the decline is cell number is associated with a loss of intact cells. In other cases this is not the caseA decline in viable cell numbers may be explained by simple cell death associated with starvation or build up of toxins. But two other hypotheses have been proposedi) Not all cells are culturable = Viable but nonculturable (VBNC) cells.Cells are viable as demonstrated by the presence of metabolic activities but can't be cultivated in the lab - detected by discrepancies between indirect and direct counts. VBNC cells are genetically programmed to become dormant (genetic response triggered in starving stationary phase cells) and when appropriate conditions become available (e.g., change in temperature, passage through animals), the cells begin growing again.ii) Programmed cell death. A fraction of the microbial population is genetically programmed to commit suicide – nonculturable cells are dead and the nutrients that they leak enable eventual growth of those cells in the population that did not commit suicide.4.Measurement of GrowthEnumeration of microbial populations or measuring massi) Measurement of Cell Numbersa) Direct Counting (counts all cells - viable and dead)Direct microscopic counts with counting chambers (Fig 6.20)Use a chamber (e.g., Petroff-Hausser counting chamber) of defined volumes. Count cells the aid of a microscopecan also use samples dried onto slidesAdvantages rapidcounts all cells in a sample (can often count individual cells in clumps)can acquire cell morphology information with these methodsDisadvantages can't determine which cells are viable unless they are treated in a special manner (e.g.,fluorescent live/dead cell stains).small cells are difficult to seeaffected by debris in samplesnot suitable for cell suspensions of low density (< 106/mL); precision difficult to achievemotile cells are difficult to countphase contrast microscopy required if sample not stainedmay require expensive pieces of equipmentunable to perform further studies on the observed microbes without further cultivationFiltrationknown volume of a suspension filtered onto a black polycarbonate filter membrane.cells are stained with fluorescent dyes and counted under the microscopeCoulter Counterautomated method of counting cell.as cell pass through a aperture they disturb an electric field perturbations are transformed into number and size data.Most useful for larger cellsFluorescence Activated Cell Sorter (FACS)b) Viable Counts (counts viable cells that can be cultured)Viable Plate Countcounts viable cultivable bacteriaViable count methods assume that each viable cell can grow and divide to yield one colonySerial dilutions of cultures are prepared and these suspensions of bacteria are plated onto agar mediumuse spread plate or pour plate techniqueFollowing incubation - count number of colonies in order to determine the number of colony forming units (CFUs) per unit volume.limit counting to plates with between 30 and 300 coloniesplates containing less than 30 colonies are not acceptable for statistical reasonsplates containing greater than 300 (TNTC) - plates are crowded and it becomes hard to distinguish and count colonies.Problems with culturability of particular microbes on the medium - may be selective!!!!Spread Plate (Fig 6.17)suspension of microbes is spread over the surface of agar medium.spreading separates cells that grow and give rise to isolated coloniesassumes each colony arises from a single cell or clump of cells (CFU).suspension of cells must be dilute enough otherwise the plate will be overgrown - too many cell get confluent growth or a lawn of cells with no discrete colonies.Usually spreading 0.1 mL of less on the platePour Plate (Fig 6.17)suspensions of cells (0.1 to 1.0 mL) are added to molten agar (42 to 45C)Note - agar begins solidifies at approx. 42C.molten agar is poured into a petri dish, allowed to solidify and incubated; the hot agar may kill or injure sensitive cells Advantages of viable plate countsCounts only viable cells – widely used in food, dairy, medical industries and researchVery sensitive – detect presence of very few cellsUse of selective and/or differential media can restrict counts to a particular cell typethe techniques require inexpensive materialsonce counts are completed you have viable cultures to use in subsequent experimentsDisadvantages of plate countsthese methods are selective and count only viable cells or cells that can be grown with the culture techniques used (i.e., they underestimate actual cell number) they do not distinguish between an individual cell and a cluster of cells and therefore underestimate cell numberstakes time for data acquisition (i.e., Cells must grow for >12 h to be counted with the viable count methodssize of colonies vary and it is easy to miss small coloniessubject to large errors if not done carefully – require adequate replicationMost Probable Number (MPN)another technique for counting viable CFUdilute to extinction - such that not all aliquots transferred to tubes of growth medium will contain a cellfollowing incubation one checks for growth and compares results to a table of statistical probability for obtaining the observed results.Membrane filtrationAquatic samples are filtered through a membrane – trapping cells on the membraneThe membrane is placed on an agar medium and incubated until each cell forms a colonyUseful for analyzing water samples especially when the populations are lowc. Indirect estimation of Bacterial NumbersMicrobial Dry WeightCells growing in liquid medium are collected by centrifugation or filtration, washed, dried in a vacuum oven and weighedTime consuming, not very sensitive but good for filamentous fungiTurbidity (Spectophotometry)rapid and sensitive method for obtaining estimate of culture densityThe more cells that are present the more light that is scattered by a suspensioncan measure transmittance of light and determine the optical density (OD) of a suspension using a spectrophotometergrowth results in increased turbidity and OD proportional to cell number for unicellular organismsCan generate a standard curve to relate OD to CFU's/unit volume or some other measure of growth (e.g., dry weight)Metabolic ActivityMeasures a metabolic product and assumes there is a direct relationship between the amount of the metabolic product and the cell number.Measurement of CO2 evolution ................
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