Microbial growth

Microbial growth
Ha Mai Dung M.D., PhD.
OUTLINE
BACTERIAL CELL DIVISION

GROWTH OF BACTERIAL POPULATIONS

MEASURING MICROBIAL GROWTH

TEMPERATURE AND MICROBIAL GROWTH

OTHER ENVIRONMENTAL FACTORS AFFECTING GROWTH
BACTERIAL CELL DIVISION
Cell Growth and Binary Fission
In microbiology, growth is defined as an increase in the number of cells.

Bacterial cell growth depends upon a large number of chemical reactions of a wide variety of types.

The main reactions of cell synthesis are the polymerization reactions that make macromolecules from monomers. As macromolecules accumulate in the cytoplasm of a cell, they are assembled into new structures, such as the cell wall, cytoplasmic membrane, flagella, ribosomes, inclusion bodies, enzyme complexes, and so on, eventually leading to cell division.
Binary Fission
Cell division following enlargement of a cell to twice its minimum size

There is partition called a septum separating the cell into two daughter cells

The time required for this process is called the generation time. The time required for a generation in a given bacterial species is highly variable and is dependent on both nutritional and genetic factors. Under the best nutritional conditions the generation time of a laboratory culture of E. coli is about 20 min.
Binary fission in a rod-shaped prokaryote.  Cell numbers double every generation.
Binary fission under microscope view
GROWTH OF BACTERIAL POPULATIONS
Exponential Growth
Growth of a microbial population in which cell numbers double within a specific time interval

During exponential growth, the increase in cell number is initially rather slow but increases at an ever faster rate. In the later stages of growth, this results in an explosive increase in cell numbers
The rate of growth of a microbial culture.  
(a) Data for a population that doubles every 30 min. (b) Data plotted on arithmetic (left ordinate) and logarithmic (right ordinate) scales.
The Microbial Growth Cycle
For a culture growing in an enclosed vessel, such as a tube or a flask, a condition called a batch culturea (closed-system microbial culture of fixed volume) , exponential growth cannot continue indefinitely. Instead, a typical growth curve for a population of cells is obtained

The growth curve describes an entire growth cycle, including the lag phase, exponential phase, stationary phase, and death phase.
Typical growth curve for a bacterial population.  A viable count measures the cells in the culture that are capable of reproducing. Optical density (turbidity), a quantitative measure of light scattering by a liquid culture, increases with the increase in cell number.
Lag Phase
When a microbial population is inoculated into a fresh medium, growth usually begins only after a period of time called the lag phase.

This interval may be brief or extended, depending on the history of the inoculum and the growth conditions.

A lag is also observed when a microbial population is transferred from a rich culture medium to a poorer one; for example, from a complex medium to a defined medium
Exponential Phase
During the exponential phase of growth each cell divides to form two cells, each of which also divides to form two more cells, and so on

Cells in exponential growth are typically in their healthiest state and hence are most desirable for studies of their enzymes or other cell components.

The rate of exponential growth is influenced by environmental conditions (temperature, composition of the culture medium), as well as by genetic characteristics of the organism itself. In general, prokaryotes grow faster than eukaryotic microorganisms, and small eukaryotes grow faster than large ones.
Stationary Phase
In a batch culture, such as in a tube or a flask, exponential growth is limited.

Typically, either or both of two things limit growth: (1) an essential nutrient of the culture medium is used up, or (2) a waste product of the organism accumulates in the medium and inhibits growth. Either way, exponential growth ceases and the population reaches the stationary phase.

In the stationary phase, there is no net increase or decrease in cell number and thus the growth rate of the population is zero. Some cells in the population grow, whereas others die, the two processes balancing each other out. This is a phenomenon called cryptic growth.
Death Phase
If incubation continues after a population reaches the stationary phase, the cells may remain alive and continue to metabolize, but they will eventually die. When this occurs, the population enters the death phase of the growth cycle.

In some cases death is accompanied by actual cell lysis.

The rate of cell death is much slower than the rate of exponential growth.
Continuous Culture
Unlike a batch culture, a continuous culture is an open system.

The continuous culture vessel maintains a constant volume to which fresh medium is added at a constant rate, and an equal volume of spent culture medium is removed at the same rate.

Once such a system is in equilibrium, the chemostat volume, cell number, and nutrient status remain constant, and the system is said to be in steady state.
Chemostat
A device that allows for the continuous culture of microorganisms with independent control of both growth rate and cell number

Two factors are important in such control:
(1) The dilution rate, the rate at which fresh medium is pumped in and spent medium is removed; and

(2) The concentration of a limiting nutrient, such as a carbon or nitrogen source, present in the sterile medium entering the chemostat vessel.
Schematic for a continuous culture device (chemostat).  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.
The effect of nutrients on growth.  Relationship between nutrient concentration, growth rate (green curve), and growth yield (red curve) in a batch culture (closed system). Only at low nutrient concentrations are both growth rate and growth yield affected.
Steady-state relationships in the chemostat.  The dilution rate is determined from the flow rate and the volume of the culture vessel. Thus, with a vessel of 1,000 ml and a flow rate through the vessel of 500 ml/h, the dilution rate would be 0.5 h−1. Note that at high dilution rates, growth cannot balance dilution, and the population washes out. Note also that although the population density remains constant during steady state, the growth rate (doubling time) can vary over a wide range.
Experimental Uses of the Chemostat
A practical advantage to the chemostat is that a cell population may be maintained in the exponential growth phase for long periods, days and even weeks.

The chemostat has been used in microbial ecology as well as in microbial physiology.

 Chemostats have also been used for enrichment and isolation of bacteria.
MEASURING MICROBIAL GROWTH
Measurements of Total Cell Numbers: Microscopic Counts
The most common total count method is the microscopic cell count. Microscopic counts can be done on either samples dried on slides or on samples in liquid. Dried samples can be stained to increase contrast between cells and their background. With liquid samples, specially designed counting chambers are used. In such a counting chamber, a grid with squares of known area is marked on the surface of a glass slide

A second method of enumerating cells in liquid samples is with a flow cytometer. This is a machine that employs a laser beam and complex electronics to count individual cells.
Microscopic Count
Microscopic counting is a quick and easy way of estimating microbial cell number. However, it has several limitations: (1) without special staining techniques dead cells cannot be distinguished from live cells; (2) small cells are difficult to see under the microscope, and some cells are inevitably missed; (3) precision is difficult to achieve; (4) a phase-contrast microscope is required if the sample is not stained; (5) cell suspensions of low density (less than about 106 cells/milliliter) have few if any bacteria in the microscope field unless a sample is first concentrated and resuspended in a small volume; (6) motile cells must be immobilized before counting; (7) debris in the sample may be mistaken for microbial cells.

In microbial ecology, total cell counts are often performed on natural samples using stains to visualize the cells. The stain DAPI stains all cells in a sample because it reacts with DNA.
Direct microscopic counting procedure using the Petroff–Hausser counting chamber.  A phase-contrast microscope is typically used to count the cells to avoid the necessity for staining.
Viable Cell Counting
A viable cell is one that is able to divide and form offspring. Usually, we determine the number of cells in the sample capable of forming colonies on a agar medium  also called a plate count.

There are at least two ways of performing a plate count: the spread-plate method and the pour-plate method
Two methods for the viable count.  In the pour-plate method, colonies form within the agar as well as on the agar surface. In the fourth column is shown a photo of colonies of Escherichia coli formed from cells plated by the spread-plate method (top) or the pour-plate method (bottom).
Diluting Cell Suspensions before Plating
The number of colonies developing on the plate must not be too many or too few. It is statistically valid to count colonies only on plates that have between 30 and 300 colonies.

To obtain the appropriate colony number, the sample to be counted must almost always be diluted. Several 10-fold dilutions of the sample are commonly used.
Procedure for viable counting using serial dilutions of the sample and the pour-plate method.  The sterile liquid used for making dilutions can simply be water, but a balanced salt solution or growth medium may yield a higher recovery. The dilution factor is the reciprocal of the dilution.
Measurements of Microbial Mass: Turbidimetric Methods
During exponential growth, all cellular components increase in proportion to the increase in cell numbers. Thus, instead of measuring changes in cell number over time, we could instead measure the increase in protein, DNA, or dry weight of a culture as a barometer of growth.

Because cell mass is proportional to cell number, we can use turbidity as a measure of cell numbers in a growing culture.
Turbidity measurements of microbial growth.  (a) Measurements of turbidity are made in a spectrophotometer. (b) Typical growth curve data for two organisms growing at different growth rates. (c) Relationship between cell number or dry weight and turbidity readings.
TEMPERATURE AND MICROBIAL GROWTH
Effect of Temperature on Microbial Growth
Temperature is probably the most important environmental factor affecting the growth and survival of microorganisms.

The minimum and maximum temperatures for growth vary greatly among different microorganisms and usually reflect the temperature range and average temperature of their habitats.
Cardinal Temperatures
Be the minimum, maximum, and optimum growth temperatures for a given organism  characteristic for any given microorganism.


The cardinal temperatures of different microorganisms differ widely; from below freezing to well above the boiling point of water. The range for any given organism is typically 25–40 degrees.
The cardinal temperatures: Minimum, optimum, and maximum.   The actual values may vary greatly for different organisms
Temperature Classes of Organisms
It is possible to distinguish at least four groups of microorganisms in relation to their growth temperature optima:
Psychrophile: An organism with a growth temperature optimum of 15°C or lower and a maximum growth temperature below 20°C
Mesophile: An organism that grows best at temperatures between 20 and 45°C
Thermophile: An organism whose growth temperature optimum lies between 45 and 80°C
Hyperthermophile: A prokaryote that has a growth temperature optimum of 80°C or greater
Mesophiles are widespread in nature.
Temperature and growth relations in different temperature classes of microorganisms.  The temperature optimum of each example organism is shown on the graph.
Microbial Growth at Cold Temperatures
Extremophile: An organism that grows optimally under one or more chemical or physical extremes, such as high or low temperature or pH

Even in frozen materials there are often small pockets of liquid water where solutes have concentrated and microorganisms can metabolize and grow.

Psychrophiles are found in environments that are constantly cold and may be rapidly killed by warming, even to as little as 20°C.
Antarctic microbial habitats and microorganisms.  (a) A core of frozen seawater from McMurdo Sound, Antarctica. The core is about 8 cm wide. Note the dense coloration due to pigmented microorganisms. (b) Phase-contrast micrograph of phototrophic microorganisms from the core shown in (a). Most organisms are either diatoms or green algae (both eukaryotic phototrophs). (c) Transmission electron micrograph of Polaromonas, a gas vesiculate bacterium that lives in sea ice and grows optimally at 4°C. (d) Photo of the surface of Lake Bonney, McMurdo Dry Valleys, Antarctica.
Snow algae.  (a) Snow bank in the Sierra Nevada, California, with red coloration caused by the presence of snow algae. Pink snow such as this is common on summer snow banks at high altitudes throughout the world. (b) Photomicrograph of red-pigmented spores of the snow alga Chlamydomonas nivalis. The spores germinate to yield motile green algal cells. Some strains of snow algae are true psychrophiles but many are psychrotolerant, growing best at temperatures above 20°C.
Psychrotolerant Microorganisms
Capable of growing at low temperatures but having an optimum above 20°C

Psychrotolerant microorganisms are more widely distributed in nature than are psychrophiles and can be isolated from soils and water in temperate climates as well as from meat, milk and other dairy products, cider, vegetables, and fruit stored at refrigeration temperatures (∼4°C).

Various Bacteria, Archaea, and microbial eukaryotes are psychrotolerant.
Microbial Growth at High Temperatures
Thermophiles and hyperthermophiles can survive at high temperature:
First, their enzymes and other proteins are much more heat-stable than are those of mesophiles and actually function optimally at high temperatures
Second, the cytoplasmic membranes of thermophiles and hyperthermophiles must be heat-stable.

A classic example of a heat-stable enzyme of great importance to biology is the DNA polymerase isolated from Thermus aquaticus. Taq polymerase, as this enzyme is known, has been used to automate the repetitive steps in the polymerase chain reaction (PCR) technique
Growth of hyperthermophiles in boiling water.  
(a) Boulder Spring, a small boiling spring in Yellowstone National Park. This spring is superheated, having a temperature 1–2°C above the boiling point. The mineral deposits around the spring consist mainly of silica and sulfur.
(b) Photomicrograph of a microcolony of prokaryotes that developed on a microscope slide immersed in a boiling spring such as that shown in (a).
Growth of thermophilic cyanobacteria in a hot spring in Yellowstone National Park.  Characteristic V-shaped pattern (shown by the dashed red lines) formed by cyanobacteria at the upper temperature for phototrophic life, 70–74°C, in the thermal gradient formed from a boiling hot spring. The pattern develops because the water cools more rapidly at the edges than in the center of the channel. The spring flows from the back of the picture toward the foreground. The light-green color is from a high-temperature strain of the cyanobacterium Synechococcus. As water flows down the gradient, the density of cells increases, less thermophilic strains enter, and the color becomes more intensely green.
OTHER ENVIRONMENTAL FACTORS AFFECTING GROWTH
Microbial Growth at Low or High pH
Most organisms show a growth pH range of 2–3 units. Most natural environments have pH values between 4 and 9, and organisms with optima in this range are most commonly encountered.

Only a few species can grow at pH values of lower than 3 or greater than 9.
The pH scale.  Although some microorganisms can live at very low or very high pH, the cell’s internal pH remains near neutrality.
Acidophiles
Organisms that grow optimally at low pH, typically below pH 6, are called acidophiles.

Fungi as a group tend to be more acid tolerant than bacteria. Many fungi grow best at pH 5 or below, and a few grow well at pH values as low as 2.

A critical factor governing acidophily is the stability of the cytoplasmic membrane. When the pH is raised to neutrality, the cytoplasmic membranes of strongly acidophilic bacteria are destroyed and the cells lyse.
Alkaliphiles
A few extremophiles have very high pH optima for growth, sometimes as high as pH 11. Microorganisms showing growth pH optima of 9 or higher are called alkaliphiles.

Alkaliphilic microorganisms are typically found in highly alkaline habitats, such as soda lakes and high-carbonate soils

The most well-studied alkaliphilic prokaryotes have been Bacillus species, such as Bacillus firmus.
Internal Cell pH
The optimal pH for growth of any organism is a measure of the pH of the extracellular environment only. The intracellular pH must remain relatively close to neutrality to prevent destruction of macromolecules in the cell.

In acidophiles and alkaliphiles the internal pH can vary from neutrality. For example, the internal pH has been measured at pH 4.6 and 9.5, they are extremely close to the limits . This is because DNA is acid-labile and RNA is alkaline-labile; if a cell cannot maintain these key macromolecules in a stable state, it obviously cannot survive.
Buffers
In a batch culture, the pH can change during growth as the result of metabolic reactions of microorganisms that consume or produce acidic or basic substances. Thus, buffers are frequently added to microbial culture media to keep the pH relatively constant.

For near-neutral pH ranges, potassium phosphate (KH2PO4) and calcium carbonate (CaCO3) are good buffers.

The best buffering system for one organism or enzyme may be considerably different from that for another. Thus, the optimal buffer for use in a particular situation must usually be determined empirically.
Osmotic Effects on Microbial Growth
Water is the solvent of life, and water availability is an important factor affecting the growth of microorganisms.

Water availability not only depends on the absolute water content of an environment, that is, how moist or dry it is, but it is also a function of the concentration of solutes such as salts, sugars, or other substances that are dissolved in the water.
Water Activity and Osmosis

Water activity, abbreviated aw, is defined as the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water. Thus, values of aw vary between 0 and 1

Water activities in agricultural soils generally range between 0.90 and 1.
Halophiles and Related Organisms
Halophile is a microorganism that requires NaCl for growth

Halotolerant: Not requiring NaCl for growth but able to grow in the presence of salt, in some cases, substantial levels of salt

Extreme halophile: A microorganism that requires very large amounts of salt (NaCl), usually greater than 10% and in some cases near to saturation, for growth

The terms mild halophile and moderate halophile are used to describe halophiles with low (1–6%) and moderate (7–15%) NaCl requirements, respectively
Effect of sodium chloride 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.
Compatible Solutes
When an organism grows in a medium with a low water activity, it can obtain water from its environment only by increasing its internal solute concentration.

Compatible solute: A molecule that is accumulated in the cytoplasm of a cell for adjustment of water activity but that does not inhibit biochemical processes

Osmophile: An organism that grows best in the presence of high levels of solute, typically a sugar

Xerophile: An organism that is able to live, or that lives best, in very dry environments

Oxygen Classes of Microorganisms
Aerobe: An organism that can use oxygen (O2) in respiration; some require oxygen

Microaerophile: An aerobic organism that can grow only when oxygen tensions are reduced from that present in air

Facultative with respect to oxygen, an organism that can grow in either its presence or absence

Anaerobe: An organism that cannot use O2 in respiration and whose growth is typically inhibited by O2

Obligate anaerobe: An organism that cannot grow in the presence of O2
Culture Techniques for Aerobes and Anaerobes
For the growth of many aerobes, it is necessary to provide extensive aeration. This is because the oxygen that is consumed by the organisms during growth is not replaced fast enough by simple diffusion from the air. Aerobes typically grow better with forced aeration than with oxygen supplied from simple diffusion.

For the culture of anaerobes, the problem is not to provide air, but to exclude it. Obligate anaerobes vary in their sensitivity to oxygen, and procedures are available for reducing the oxygen content of cultures.

Growth versus oxygen concentration.  (a–e) Aerobic, anaerobic, facultative, microaerophilic, and aerotolerant anaerobe growth, as revealed by the position of microbial colonies (depicted here as black dots) within tubes of thioglycolate broth culture medium. A small amount of agar has been added to keep the liquid from becoming disturbed, and the redox dye, resazurin, which is pink when oxidized and colorless when reduced, is added as a redox indicator.
Incubation under anoxic conditions.  (a) Anoxic jar. A chemical reaction in the envelope in the jar generates H2 + CO2. The H2 reacts with O2 in the jar on the surface of a palladium catalyst to yield H2O; the final atmosphere contains N2, H2, and CO2. (b) Anoxic glove bag for manipulating and incubating cultures under anoxic conditions. The airlock on the right, which can be evacuated and filled with O2-free gas, serves as a port for adding and removing materials to and from the glove bag.
Toxic Forms of Oxygen
Oxygen is a powerful oxidant and the best electron acceptor for respiration.

But oxygen can also be a poison to obligate anaerobes because the toxic derivatives of oxygen can damage cells
Oxygen Chemistry
Oxygen in its ground state is called triplet oxygen (3O2). However, other electronic configurations of oxygen are possible, and most are toxic to cells.

Singlet oxygen is produced both photochemically and biochemically, the latter through the activity of various peroxidase enzymes

Organisms that frequently encounter singlet oxygen, such as airborne bacteria and phototrophic microorganisms, often contain pigments called carotenoids, which function to convert singlet oxygen to nontoxic forms.
Superoxide and Other Toxic Oxygen Species
Besides singlet oxygen, many other toxic forms of oxygen exist, including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH•). All of these are produced as by-products of the reduction of O2 to H2O in respiration

With so many toxic oxygen derivatives to deal with, organisms have evolved enzymes that destroy these compounds
Four-electron reduction of O2 to H2O by stepwise addition of electrons.  All the intermediates formed are reactive and toxic to cells
Enzymes that destroy toxic oxygen species. 
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 (right) with a drop of 30% hydrogen peroxide. The immediate appearance of bubbles is indicative of the presence of catalase. The bubbles are O2 produced by the reaction H2O + H2O2 → 2 H2O + O2.