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Microbial Growth

Binary fission The growth cycle Measurements of microbial growth Environmental effects on microbial growth: temperature, pH, water activity, oxygen

C. Vetriani and V. Starovoytov

The general process of binary fission in a rod-shaped prokaryote. For simplicity, the nucleoid is depicted as a single circle in green.

C. Vetriani and V. Starovoytov

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FtsI activity is blocked by penicillin

The divisome is responsible for the synthesis of new cytoplasmic membrane and cell wall material

Phase contrast

Nucleoid stain

FtsZ stain

FtsZ stain and nucleoid stain

The FtsZ ring and cell division. Cell division and chromosome replication are coordinately regulated, and the Fts proteins are the keys to these processes. Fts proteins interact to form a division apparatus in the cell called the divisome.

New cell wall is synthesized during bacterial growth by inserting new glycan units into preexisting wall material

Autolysin activity creates small openings in the cell wall New peptidoglycan is synthesized Transpeptidation: reaction that leads to the final crossing of two peptidoglycan chains. It is inhibited by the antibiotic penicillin. Synthesis of new cell wall stops and the continued action of autolysins leads to cell lysis

Cell wall synthesis in gram-positive Bacteria. (a) Localization of new cell wall synthesis during cell division. In cocci, new cell wall synthesis (shown in green) is localized at only one point. The FtsZ ring defines the cell division plane.

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Typical growth curve for a bacterial population in a batch culture (closed system). There is usually a lag phase, then exponential growth commences. As essential nutrients are depleted or toxic products build up, growth ceases, and the population enters the stationary phase. If incubation continues, cells may begin to die (the death phase).

Compare the rate of cell production between 0-0.5 h and 4-4.5 h

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Microbial populations show a characteristic type of growth pattern called exponential growth, which is best seen by plotting the number of cells over time on a semilogarithmic graph (# of cells is plotted on a logarithmic scale and time is plotted arithmetically).

N = N02n

N = Final cell number N0 = Initial cell number n = Number of generations

g = t/n

g = Generation time (doubling time) t = Time of exponential growth

The rate of growth of a microbial culture. (a) Data for a population that doubles every 30 min. (b) Data plotted on an arithmetic (left ordinate) and a logarithmic (right ordinate) scale.

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The Mathematics of Exponential Growth

To express the equation N = N 02n in terms of n: n = (log N - log N0)/log 2 n = [log 10 8 - log (5 x 10 7)]/0.301 = 1 g = t/n g = 2/1 = 2 h

Method of estimating the generation times (g) of exponentially growing populations with generation times of (a) 6 h and (b) 2 h from data plotted on semilogarithmic graphs. The slope of each line is equal to 0.301/g and n equals the number of generations that have occurred in the time, t.

Growth kinetics

Growth rate constant - number of generations that occur per unit time - (expressed as k or µ; h -1 ): (1) k = (ln N 2 - ln N1)/(t2 - t 1) where N2 and N 1 = cells ml -1 at time t2 and t1 (in h) (2) Convert (1) to log: k = (log N2 - log N1 ) (2.303)/(t2 - t 1) Generation time - time required for the cell population to double - (expressed as g; h): (3) g = (ln 2)/k

Example I: A bacterial culture in exponential growth contained 10 4 cells/ml at time zero and 10 8 cells/ml at time 4 h. What is the growth rate constant (k)? From (1) k = (ln 10 8 - ln 104)/(4 - 0) = 2.3 h -1

What is the generation time? From (3) g = ln 2/k = 0.693/2.3 = 0.30 h = 18 min

Example II: E. coli grew exponentially from 5 x 10 5 cells/ml to 3.5 x 107 cells/ml in 5 h. Its generation time was 40 min. Was there a lag phase? From (1) and (3) (t2 - t 1) = (ln N2 - ln N1)/k = g (ln N2 - ln N1)/ln 2 = 40 [ln (3.5 x 107) - ln (5 x 105)]/0.693 = 245 min

Total growth period = 5 h = 300 min Lag = 300 - 245 = 55 min

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Direct microscopic counting procedure using the Petroff-Hausser counting chamber.

Method not suitable for cell suspensions of low density or motile cells Total cell counts (done microscopically) measure the total number of cells in a population

Counting living cells

Count plates that have between 30-300 colonies

Two methods of performing a viable count (plate count). In either case the sample must usually be diluted before plating. Cell counts (plate counts) measure only the living, reproducing population.

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Procedure for viable counting using serial dilutions of the sample and the pour plate method.

The " Great Plate Count Anomaly"

Plate counts reveal lower numbers of cells that direct microscopic counts Why? 1. Microscopic methods count dead cells 2. Different organisms in a given sample may have physiological requirements that are not met by the specific culture conditions used

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What if an organism does not grow on solid medium?

Turbidity measurements of microbial growth. (a) Measurements of turbidity are made in a spectrophotometer or photometer. The photocell measures incident light unscattered by cells in suspension and gives readings in optical density or photometer units.

Turbidity measurements of microbial growth. (b) Typical growth curve data obtained in Klett units or optical density (OD) for two organisms growing at different growth rates. For practice, calculate the generation time ( g) of the two cultures using the formula n = 3.3 (log N ­ log N0) where N and N 0 are two different Klett values taken between a time interval t . Which organism is growing faster, A or B? (c) Relationship between cell number or dry weight and turbidity readings. Note that the one-to-one correspondence between these relationships breaks down at high turbidities.

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Batch cultures vs. continuous cultures

Batch culture

µ= 0.48 h -1 tg= 1.44 h (86 min) Cells ml-1

Time (h)

Schematic for a continuous culture device (chemostat). In such a device, the population density is controlled by the concentration of limiting nutrient in the reservoir, and the growth rate is controlled by the flow rate. Both parameters can be set by the experimenter. Chemostats are a means of maintaining cell populations in exponential growth for long periods.

Relationship between nutrient concentration, growth rate (green curve), and growth yield (red curve) in a batch culture (closed system). At low nutrient concentrations both growth rate and growth yield are affected.

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Steady-state relationships in the chemostat. The dilution rate is determined from the flow rate and the volume of the culture vessel.

Narrow range

Wide range

Relation of temperature to growth rates of a typical psychrophile, a typical mesophile, a typical thermophile, and two different hyperthermophiles.

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Effect of temperature on growth rate and the molecular consequences for the cell. The three cardinal temperatures vary by organism.

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Adaptations to High Temperatures

DNA stability: reverse DNA gyrase - positive coils archaeal histones, Sac7d Lipid stability: monolayers (hydrophobic chains linked together)

Stability of monomers: ATP and NAD+ have half-life < 30 min at 120°C Protein stability: Increased flexibility

The pH scale. Note that although some microorganisms can live at very low or very high pH, the cell's internal pH remains near neutrality.

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aw: ratio of the vapor pressure of the air in equilibrium with a substance to the vapor pressure of pure water

Effect of sodium ion concentration on growth of microorganisms of different salt tolerances or requirements. The optimum NaCl concentration for marine microorganisms such as V. fischeri is about 3%; for extreme halophiles, it is between 15 and 30%, depending on the organism.

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Water activity becomes limiting to an organism when the dissolved solute concentration in its environment increases. To counteract this situation, organisms produce or accumulate intracellular compatible solutes that maintain the cell in positive water balance.

Derivative of the aa aspartate

Structures of some common compatible solutes in microorganisms.

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Aerobic (a), anaerobic (b), facultative (c), microaerophilic (d), and aerotolerant (e) anaerobe growth, as revealed by the position of microbial colonies (depicted here as black dots) within tubes of thioglycolate broth culture medium.

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Several toxic forms of oxygen can be formed in the cell...

Four-electron reduction of O 2 to water by stepwise addition of electrons. All the intermediates formed are reactive and toxic to cells except for water, of course.

...but enzymes are present that can neutralize most of them

Enzymes that destroy toxic oxygen species. (a) Catalases and (b) peroxidases are porphyrin-containing proteins, although some flavoproteins may consume toxic oxygen species as well. (c) Superoxide dismutases are metal-containing proteins, the metals being copper and zinc, manganese, or iron. (d) Combined reaction of superoxide dismutase and catalase. (e) Superoxide reductase catalyzes the one electron reduction of O 2- to H2O2 using reduced cytochrome c as the electron donor.

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Method for testing a microbial culture for the presence of catalase. A heavy loopful of cells from an agar culture was mixed on a slide with a drop of 30% hydrogen peroxide. The immediate appearance of bubbles is indicative of the presence of catalase. The bubbles are O 2 produced by the reaction H2O2 + H2O2 2 H 2O + O2.

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