Microbial Growth Why study growth? Important to understanding biology of an organism growth is essential to any organism's existence Information on growth is required for control microoganisms Definitions of Growth Steady increase in all the chemical components of an organism that may result in an increase cell size, cell number or both Increase in biomass as measured by changes in Dry weight increase Increase in absorbance Increase in cellular constituents Protein Nucleic acids other constituents e.g., peptidoglycan and chitin Growth results in increased cell size and frequently cell division Particularly relevant to unicellular organisms: o In unicellular organisms cell growth results in increase in numbers o In multicellular organisms cell growth results in an increase in organism size I. Factors that Affect Growth A. Chemical factors Nutrients are substances used in biosynthesis and energy release and are therefore required for growth One must define nutritional requirements in order to cultivate the microbe in the laboratory Chemical 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 macromolecules Carbon o Life on earth is carbon based o Half of the dry weight of a typical cell is carbon 1
Nitrogen o Nitrogen makes up approximately 14% of the dry weight of a typical cell o Major constituent of protein and nucleic acids, some carbohydrates and lipids o NH 3, NO 3 -, N 2 (nitrogen fixation) and organic N compounds (e.g., amino acids) from the environment. Some bacteria use atmospheric nitrogen (N 2 ) as a nitrogen source Phosphorus o component of phospholipids and nucleic acids, nucleotides such as ATP, some proteins o available as organic and inorganic forms in the environment Sulfur o structural role in methionine and cysteine as well as a number of vitamins (thiamine, biotin), coenzyme A and some carbohydrates o available usually from inorganic sources SO 4 2- or H 2 S and organic sulfur compounds such as cysteine K, Ca, Mg, and Fe are cations in cells and required for a variety of roles e.g., - cofactors (K +, Ca 2+, Mg 2+, and Fe 2+ or Fe 3+ ) - stabilize membranes and ribosomes (Mg 2+ ) - contribute to heat resistance of endospores (Ca 2+ ) - components of biomolecules such as cytochromes (Fe 2+ and Fe 3+ ) 2. Trace elements or Micronutrients required in lesser or trace amounts. Critical to cell function Many are metals structural role with many enzymes - cofactors often trace elements present in medium components or water provide all that is required for growth Co, Cu, Mn, Mo, Ni, and Zn are needed by most cells. Some cells require Cr, Se, W, and V 3. Oxygen a) Aerobic organisms growth at full atmospheric O 2 tensions (21% O 2 in the atmosphere) facultative organisms (under appropriate nutrient and culture conditions) can grow under either aerobic or anaerobic condition obligate aerobes - require O 2 for growth O 2 is poorly soluble - forced aeration is often used in culture systems to provide O 2 2
b) Anaerobic organisms obligate (strict) anaerobes - grow only in the absence of O 2 ; sensitive to O 2 and brief exposure will kill these organisms; perhaps because these organisms are unable to detoxify some of the products of O 2 metabolism lack a respiratory system and can t use oxygen as a terminal electron acceptor These organisms do use oxygen found in cellular materials Obligate anaerobiosis - prokaryotes, and a few groups of fungi and protozoa Toxic forms of oxygen Oxygen itself is not toxic to anaerobic organisms rather it is certain derivatives that are toxic reduction of O 2 in respiration produces several toxic products singlet oxygen ( 1 O 2 - ) produced photochemically and biochemically (peroxidase activity). Outer shell electrons become highly reactive; carry out spontaneous and undesirable oxidations in the cell hydrogen peroxide (H 2 O 2 ) Produced during aerobic respiration; damage cell components but not as toxic as O 2.-, or OH superoxide (O 2.- ) 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 O 2 and produce superoxide Ionizing radiation is the major source of hydroxyl radicals. Small amounts of hydroxyl radicals can be produced from H 2 O 2. A number of enzymes have evolved to detoxify oxygen species Catalase o destroys H 2 O 2 o H 2 O 2 + H 2 O 2 2 H 2 O + O 2 o Catalase test - 30% H 2 O 2 place on cells. Cells with catalase activity produces vigourous bubbling as O 2 is released Peroxidase o destroys H 2 O 2 but does not produce O 2. May require a reductant such as NADH o H 2 O 2 + 2H + 2 H 2 O Superoxide dismutase (SOD) o Destroys superoxide o Indispensable to aerobic cells o O 2.- + O 2.- + 2H + H 2 O 2 + O 2 o Generally works in tandem with catalase: 4O 2 - + 4H + 2H 2 O + 3O 2 Superoxide reductase o Found in some obligately anaerobic prokaryotes o O 2.- + 2H + + cytochrome c reduced H 2 O 2 + cytochrome c oxidized o Avoids production of O 2 as found with SOD o H 2 O 2 may then be removed by peroxidase activity 3
Aerobes and facultative anaerobes usually produce superoxide dismutase and catalase c) Aerotolerant anaerobes tolerate O 2 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 activity d) Microaerophiles grow only at reduced O 2 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 peroxides O 2 usually excluded from culture systems by one or a combination of the following mechanisms Fill container to the top and seal Boil medium to drive out O 2 Use reducing agents that react with O 2 ; reduces it to H 2 O (e.g., thioglycolate, cysteine, H 2 S) Seal containers under O 2 free gas Use redox indicators such as resazurin to indicate the presence of O 2. Use O 2 consuming devices (catalyst) Work under a stream of O 2 free gas or in an anoxic glove box/anaerobic chamber 4. Other required elements Some microbes may have particular requirements that reflect their specific environment (Halophiles require Na + ) and morphology (Diatoms and Silicon dioxide based cell walls) 5. Growth Factors Some 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 environment Growth factors are organic compounds that are essential cellular components or precursors of these components but cannot be synthesized by the organism Major Classes of Growth factors 1. amino acids 2. purine and pyrimidines 3. vitamins (e.g., thiamine, biotin, cobalamin, pyridoxine) Other growth factors include heme (nonprotein component of many cytochromes) or cholesterol Understanding growth factor requirements has practical implications o Bioassays using microbes to detect the specific growth factor that they need. Growthresponse assay uses this approach to detect the amount of a growth factor in solution. These assays can be specific, sensitive, simple and quantitative o Manufacture of growth factors by specific microorganisms (e.g., Vitamin D by Saccharomyces) in industrial fermentations 4
B. Physical (or environmental) Factors 1. The Effect of Temperature on Growth Cardinal temperatures (Fig 6.1) Depend on environmental factors such as ph and available nutrients a) Minimum temperature - below which cells are inactive reduced membrane fluidity perhaps affects nutrient transport or proton gradient formation b) Optimum temperature highest rate of growth and reproduction, always nearer maximum temperature c) Maximum temperature - above which growth is not possible Growth stops because of inactivation of one or more key proteins, damages transport carriers or other proteins, or thermal disruption of membrane Cardinal temperatures vary for different organisms Medium composition can have a slight affect Temperature optima usually vary from 0 C to 75 C Pyrolobus fumarii (archaeon) - maximum temperature = 113 C Growth temperature range for a particular organisms usually spans 30 to 40 C Distinguish five groups of microbes based on temperature optima i) Psychrophiles Grow well at 0 C and have an optimum temperature 15 C and a maximum temperature around 20 C heat sensitive and unable to survive temperate climates Adaptations to Psychrophily o Enzymes, transport systems and protein synthetic apparatus work well at low temperatures enzymes with low temperature optima o greater amounts of α-helix and lesser amounts of β sheet secondary structure o greater amounts of polar amino acids and lesser amounts of hydrophobic amino acids membranes contain higher amounts of unsaturated fatty acids o some psychrophiles have membranes higher in polyunsaturated fatty acids 5
ii) Psychrotolerant (psychrotrophs, facultative psychrophiles) grow at 0 C but have optima of 20-30 C iii) Mesophiles Optimum temperature between 25 and 40 C Minimum temperature between 15 and 20 C Maximum temperature 45 C Most common type of microbe e.g., E. coli Optimum temperature < 39 C Maximum temperature < 48 C Minimum temperature 8 C iv) Thermophiles Optimum temperature between 50 and 60 C Minimum temperature around 45 C Maximum temperature 45 C Only prokaryotes grow above 60 C The most thermophilic organisms are Archaea Nonphototrophic organisms are able to grow at higher temperatures than phototrophic forms v) Hyperthermophiles Optimum temperature > 80 C Extreme thermophiles are usually Archaea The highest growth temperatures for an archaeon is 113 C (Pyrolobus fumarii) Adaptations to Thermophily i) Enzymes and other proteins are heat stable Subtle amino acid substitutions Increased number of salt bridges Densely packed hydrophobic interiors The presence of certain solutes such as di-inositol phosphate and diglycerol phosphate ii) Macromolecules function optimally at high temperatures iii) Membrane is heat stable Membrane lipids are more branched, rich in saturated fatty acids and of higher molecular weight In some cases they have lipid monolayers (diglycerol tetraethers) iv) DNA is stabilized by special histone like proteins 6
Review cell membrane structure Chapter 4 Why don t eukaryotes grow above 60 C? Applications of Thermophily High temperature enzymes e.g., feed pelleting process PCR 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 9 Few organisms can growth at ph < 2 and > 10 ph is a great influence on growth rate ph is important because of its effect on proteins (charge is important to protein conformation) as well as the plasma membrane a) neutrophiles - ph optimum between 5.5 and 8 Most 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 industry c) acidophiles - restricted to growth at low ph values between 0 and 5.5 Fungi are generally more acid tolerant than bacteria many grow at ph 4 to 6 Some Bacteria and Archaea are obligate acidophiles e.g., Bacteria - Thiobacillus Archaea - Sulfolobus ph has an important effect on stability of acidophile plasma membrane Intracellular ph o Intracellular ph is usually between ph 6 to 8 but internal ph as low as 4.6 and as high as 9.5 have been measured o Maintained 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 chaperones 7
3. Osmotic Effects on Growth Microbes require water to grow their cells are 80 90% water Water 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 (a w ) - amount of water that is free to react = availability of water in a substance a w = 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) a w ranges between 0 and 1 Most bacteria require an a w of 0.9 for active metabolism Most 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 concentration a) Halophilic bacteria A organism requiring salt (NaCl) for growth microbes found in the sea (which is 3% NaCl) usually have a growth requirement for salt Mild 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 a w but generally grow best without added solute Osmotolerant grow over a wide range of water activity Osmophiles - require high solute (e.g., sugar) concentration for growth Xerophiles able to grow in very dry environments (i.e., made dry by lack of water) How does an organism grow under low a w? Increases internal solute concentration Pumps inorganic ions (e.g., K + ) into the cell Synthesize 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 soluble How does an organism grow under high a w? II. Microbial Growth in Natural Environments Most natural ecosystems are complex and constantly changing Low concentrations of usable nutrients (Oligotrophic) Competition 8
Growth 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 requirements Shelford s law there are limits to environmental factors below and above which a microorganism cannot survive and grow regardless of the nutrient supply Most bacteria are likely to experience starvation. How do they deal with nutrient limitation? Reduction in cell size Change in morphology increase surface area and ability to absorb nutrients Shutdown of metabolism except for housekeeping maintenance genes Biofilms Most microbes are typically found in biofilms in nature Biofilms 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 compounds Microbes in biofilms can carry out complex chemical processes (i.e., breakdown of plant cell walls such as occurs in the rumen) III Culture Media A 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 microbes There 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 medium Specialized 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...) 9
A. Chemical and Physical Types of Culture Media 1. 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 water BM+G (chemically defined medium) Ingredient g/l in dh 2 O Glucose 2.0 (NH 4 ) 2 SO 4 2.0 K 2 HPO 4 0.5 Monosodium glutamate 5.0 MgSO 4.7H 2 O 0.3 MnSO 4.H2O 0.05 CaCl 2 0.08 ZnSO 4.7H 2 O 0.005 CuSO 4.5H 2 O 0.005 FeSO 4.7H 2 O 0.0005 2. Complex media certain 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 composition Luria Burtani (LB; Chemically undefined or Complex medium) Ingredient g/l in dh2o Yeast Extract 5.0 Tryptone 10.0 NaCl 5.0 Tryptic Soy Broth (TSB Chemically undefined or Complex medium) Ingredient g/l in dh2o Tryptone 17.0 Peptone 3.0 Glucose 2.5 NaCl 5.0 Dipotassium phosphate 2.5 Refer to appendix 8 of lab manual for other examples of complex media 10
3. Liquid or solidified media Both liquid and solidified media are routinely used in microbiology Solidified 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 microorganism Agar 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 (90 C) but does not solidify until it reaches 45 C. Moreover, very few microorganisms can hydrolyze agar. Agar is melted during sterilization and the molten medium is poured into Petri dishes and allowed to solidify B. Functional Types of Culture Media Complex media such as tryptic soy broth are called general purpose media or supportive media because they sustain the growth of many microorganisms For 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 medium A medium with a composition favoring growth of certain types of microorganisms while inhibiting growth of any other microorganisms that may be present. Examples Differential medium A 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 identification Examples Selective and differential characteristics may be combined in a single medium Examples 11
C. Enrichment technique Developed by Beijerinck The 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 sample D. Notes on culturing microbes not 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 Technique Series of steps used to minimize contamination during the manipulations of cultures and sterile culture media Sterilize all media and implements for handling materials of interest Clean working area Limit exposure to potential sources of contamination Preparation of Pure Cultures Streak plate technique Dilution Deposition of individual cells or clumps of cells (known as colony forming units or CFU) on agar medium Cell 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 Cultures 1. Refrigeration at 4 C short term solution - several weeks to several months duration depends on type of medium 2. Glycerol stocks Sterile 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 -20 C (1 to 2 years) or -80 C (up to 10 years or more) 3. Lyophilization Freeze drying Culture is quick-frozen at temperatures ranging from -50 to -90 C and then dried under vacuum on a lyophilizer; freeze dried cultures are stored in sealed glass ampules for extended periods of time. 12
Microbial Culture Collections Sources of microbial cultures Cultures are distributed for a fee or free depending on the culture collection ATCC DSMZ NCTC NCIMB EGSC BGSC FGSC American Type Culture Collection Deutsche Sammlung von Mikrooganismen und Zellkulturen National Collections of Type Cultures and Pathogenic Fungi National Collections of Industrial and Marine Bacteria E. coli Genetic Stock Centre Bacillus Genetic Stock Centre Fungal Genetic Stock Centre IV. Growth of Microbial Cultures i) Eukaryotic Cell Cycle review Biol 1010 notes ii) Prokaryotic cell cycle most often is accomplished by Binary Fission but budding, fragmentation and other processes may occur Mother cell two daughter cells Generation time (g) Binary fission in E. coli takes 20 minutes under optimal conditions Required as many as 2000 chemical reactions Length of time depends on a number of factors, including nutrition, genetics and environment Rapidly Growing Cells In E. coli, the cell cycle takes 60 min to complete: 40 minutes for DNA replication and partitioning and 20 min for septum formation and Cytokinesis But E. coli can complete this entire process in 20 min under optimal conditions This 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 Growth Growth rate change in cell number or cell mass per unit time Generation interval for the formation of two cells from one cell 13
Generation time (doubling time) time it takes for one cell to become two cells time it takes for the population to double depends on growth medium and conditions 1. 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 2 2 1 2 2 2 3 2 4 (1) N t = N 0 2 n N t = final number of cells at time t N 0 = initial number of cells n = number of generations that have occurred during period of exponential growth Solving for n (where all logarithms are to the base 10) log N t = log N 0 + n log 2 and (2) n = log N t - log N 0 = log N t - log N 0 log 2 0.301 Growth 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 N t - log N 0 0.301t Can now calculate the mean generation time (g) or mean doubling time. When the population doubles t = g and N t = 2N 0 ; substitute 2N 0 into (3) (4) k = log (2N 0 ) - log N 0 = log 2 + log N 0 log N 0 = 1/g 0.301g 0.301g Therefore (5) g = 1/k 14
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/slope How can we use growth rate information? 2. Culture Systems "Fermentation" - cultivation of microorganisms in a controlled, enclosed system i. Batch Culture A fixed volume of liquid medium is inoculated and incubated for an appropriate period of time with no further addition of microorganisms or growth substrates closed environment most common method of microbial cultivation nutrient concentration is a determinant of growth rate and cell yield The batch culture has a continually changing environment o nutrients are depleted o products produced o cells change Ultimately the culture quits growing due to nutrient limitation or product accumulation e.g., test tube to flask to 100,000 L fermenter ii. Fed Batch A nutrient stock (limiting nutrient) is added at intervals or continuously to a batch culture iii. Continuous Culture Spent 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 constant Can control culture growth rate as well as yield of cells by manipulating dilution rate and the level of the limiting nutrient, respectively More sophisticated apparatus required Superior productivity possible because of reduced downtime. 15
e.g., Chemostat uses dilution rate and nutrient concentration to control growth and population density growth 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 other can maintain population in exponential phase at a known growth rate for long periods of time Can study microbial growth at very low nutrient concentrations close to those present in nature 3. Bacterial Growth Curve Growth of a batch culture population of cells can be monitored and plotted as a growth curve A typical batch culture growth curve can be divided into 4 phases (Fig 6.15) 16
i) Lag Phase Initial 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 size The cells are transporting nutrients, synthesizing RNA and subsequently enzymes needed for growth; replicating DNA The length of this phase depends on the history of the culture and growth conditions Examples: 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 curve During 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 studies Exponential 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 further Temperature, ph, Oxygen and other physical parameters Genetic determinants Small cells generally grow faster than larger cells (surface area to volume ratio) Nutrient concentration affects maximum cell yield iii) Stationary Phase Closed system - cells can t grow indefinitely No further net increase in cell number Total 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 active 17
Stationary 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 together Cellular 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 occur e.g., Arthrobacter - log cells - rods - stationary cells - coccoid The most important changes are in gene expression and physiology. o Different genes are turned on (e.g., catalase, exonuclease and acid phosphatases; survival genes (sur) have been identified for E. coli) o 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 growth In many instances this phase can be reversed if modify the environmental parameters In many cases the decline is cell number is associated with a loss of intact cells. In other cases this is not the case A 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 proposed i) 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. 18
4. Measurement of Growth Enumeration of microbial populations or measuring mass i) Measurement of Cell Numbers a) 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 microscope can also use samples dried onto slides Advantages rapid counts all cells in a sample (can often count individual cells in clumps) can acquire cell morphology information with these methods Disadvantages 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 see affected by debris in samples not suitable for cell suspensions of low density (< 106/mL); precision difficult to achieve motile cells are difficult to count phase contrast microscopy required if sample not stained may require expensive pieces of equipment unable to perform further studies on the observed microbes without further cultivation Filtration known volume of a suspension filtered onto a black polycarbonate filter membrane. cells are stained with fluorescent dyes and counted under the microscope Coulter Counter automated 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 cells Fluorescence Activated Cell Sorter (FACS) 19
b) Viable Counts (counts viable cells that can be cultured) Viable Plate Count counts viable cultivable bacteria Viable count methods assume that each viable cell can grow and divide to yield one colony Serial dilutions of cultures are prepared and these suspensions of bacteria are plated onto agar medium use spread plate or pour plate technique Following 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 colonies plates containing less than 30 colonies are not acceptable for statistical reasons plates 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 colonies assumes 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 plate Pour Plate (Fig 6.17) suspensions of cells (0.1 to 1.0 ml) are added to molten agar (42 to 45 C) Note - agar begins solidifies at approx. 42 C. 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 counts Counts only viable cells widely used in food, dairy, medical industries and research Very sensitive detect presence of very few cells Use of selective and/or differential media can restrict counts to a particular cell type the techniques require inexpensive materials once counts are completed you have viable cultures to use in subsequent experiments Disadvantages of plate counts these 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 numbers takes time for data acquisition (i.e., Cells must grow for >12 h to be counted with the viable count methods size of colonies vary and it is easy to miss small colonies subject to large errors if not done carefully require adequate replication 20
Most Probable Number (MPN) another technique for counting viable CFU dilute to extinction - such that not all aliquots transferred to tubes of growth medium will contain a cell following incubation one checks for growth and compares results to a table of statistical probability for obtaining the observed results. Membrane filtration Aquatic samples are filtered through a membrane trapping cells on the membrane The membrane is placed on an agar medium and incubated until each cell forms a colony Useful for analyzing water samples especially when the populations are low c. Indirect estimation of Bacterial Numbers Microbial Dry Weight Cells growing in liquid medium are collected by centrifugation or filtration, washed, dried in a vacuum oven and weighed Time consuming, not very sensitive but good for filamentous fungi Turbidity (Spectophotometry) rapid and sensitive method for obtaining estimate of culture density The more cells that are present the more light that is scattered by a suspension can measure transmittance of light and determine the optical density (OD) of a suspension using a spectrophotometer growth results in increased turbidity and OD proportional to cell number for unicellular organisms Can generate a standard curve to relate OD to CFU's/unit volume or some other measure of growth (e.g., dry weight) Metabolic Activity Measures a metabolic product and assumes there is a direct relationship between the amount of the metabolic product and the cell number. Measurement of CO 2 evolution 21