Microbial Growth and Environmental Factors

Biofilms

Biofilms are thin layers of microorganisms enmeshed in a matrix material.
They are organized ecosystems where cells interact through signaling and coordinated responses.
Biofilms provide a protected environment and aid colonization.
They have clinical importance, forming on medical devices and causing health-acquired infections.
Biofilms form on teeth as plaque, in the lungs of patients with cystic fibrosis, and on the cardiac tissue of patients with endocarditis.
They also grow on International Space Station objects and within systems.
A method to prevent biofilm growth is through surface preparation, reducing the ability of microorganisms to adhere to materials.
Studying biofilms requires new approaches due to the cells’ adhesion properties.

Binary Fission

The bacterial cell cycle involves DNA replication and partitioning of cellular components into two daughter cells.
Reproduction in prokaryotes is asexual, with genetic recombination occurring through horizontal gene transfer.
Most bacteria have a single circular chromosome.
Binary fission is the most common mechanism of cell replication in bacteria.
Before dividing, the cell grows and increases its number of cellular components.
DNA replication starts at the origin of replication, where the chromosome is attached to the inner cell membrane.
Replication continues in opposite directions until the terminus is reached.
The center of the enlarged cell constricts until two daughter cells are formed, each receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis).
Cytokinesis and cell division are directed by the protein FtsZ.
FtsZ assembles into a Z ring on the cytoplasmic membrane.
The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells.
Additional proteins are added to the Z ring to form the divisome.
The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells.
Specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Generation Time

In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations.
In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time.
Doubling time is defined as the time it takes for the population to double through one round of binary fission.
Bacterial doubling times vary enormously.
$N<em>n = N</em>02^n$
- $N_n$ is the number of cells at any generation n.
- $N_0$ is the initial number of cells.
- $n$ is the number of generations.

The Growth Curve

Microorganisms grown in closed culture (batch culture) follow a reproducible growth pattern referred to as the growth curve.
Culture density is the number of cells per unit volume.
Distinct phases can be observed in the curve when the number of live cells is plotted against time.

The Lag Phase

The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium.
The initial phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of growth.
The number of cells does not change during the lag phase.
Cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium.
If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase.
The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.

The Log Phase

In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission, and their number increases exponentially.
For any given bacterial species, the generation time under specific growth conditions is genetically determined, and this generation time is called the intrinsic growth rate.
During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is often plotted on a semilogarithmic graph.
Cells in the log phase show constant growth rate and uniform metabolic activity.
Cells in the log phase are preferentially used for industrial applications and research work.
The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.

Stationary Phase

As the number of cells increases through the log phase, waste products accumulate, and nutrients are gradually used up.
Gradual depletion of oxygen begins to limit aerobic cell growth.
This combination of unfavorable conditions slows and finally stalls population growth.
The total number of live cells reaches a plateau referred to as the stationary phase.
In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant.
The culture density in a stationary culture is constant.
The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the same conditions.
During the stationary phase, cells switch to a survival mode of metabolism.
As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes.
In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase.
Secondary metabolites, including antibiotics, are synthesized in the stationary phase.
In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism.
Quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.

The Death Phase

As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers.
The number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells.
This is the aptly named death phase, sometimes called the decline phase.
Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores.
A few cells, the so-called persisters, are characterized by a slow metabolic rate.
Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.

Sustaining Microbial Growth

It is advantageous to maintain cells in the logarithmic phase of growth.
A chemostat is used to maintain a continuous culture in which nutrients are supplied at a steady rate.
A controlled amount of air is mixed in for aerobic processes.
Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.

Measurement of Bacterial Growth

Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists.
The number of bacteria in a clinical sample serves as an indication of the extent of an infection.
Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease.
Two major approaches are used to measure cell number: direct methods involve counting cells, whereas indirect methods depend on the measurement of cell presence or activity without actually counting individual cells.
Both direct and indirect methods have advantages and disadvantages for specific applications.

Direct Cell Count

Direct cell count refers to counting the cells in a liquid culture or colonies on a plate.
It is a direct way of estimating how many organisms are present in a sample.
The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope.
The calibrated slide is called a Petroff-Hausser chamber and is similar to a hemocytometer used to count red blood cells.
By counting individual cells in a number of squares and determining the volume of the sample observed, it is possible to estimate the concentration of cells.
The concentration must be corrected for dilution if the sample was diluted before enumeration.
*Newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria.
Viability stains (or live stains) bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane.
The primary stain, which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells.
The secondary stain, which fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged.
Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids.
Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance in a saline solution.
Direct counts provide an estimate of the total number of cells in a sample.
However, in many situations, it is important to know the number of live, or viable, cells.
Counts of live cells are needed when assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water.

Plate Count

The viable plate count, or simply plate count, is a count of viable or live cells.
It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen.
The results are usually expressed as colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony.
Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells.
Some cells are described as viable but nonculturable and will not form colonies on solid media.
The viable plate count is considered a low estimate of the actual number of live cells.
Microbiologists typically count plates with 30–300 colonies.
There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods.

Serial Dilution

The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method.
The goal of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10 to simplify calculation.
The number of serial dilutions is chosen according to a preliminary estimate of the culture density.
A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth.
This step represents a dilution factor of 10, or 1:10, compared with the original culture.
From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution.
The dilution factor is now 1:100 compared with the original culture.
This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting.
From each tube, a sample is plated on solid medium using either the pour plate method or the spread plate method.
The plates are incubated until colonies appear.
Two to three plates are usually prepared from each dilution, and the numbers of colonies counted on each plate are averaged.
In all cases, thorough mixing of samples with the dilution medium is paramount to obtaining reliable results.
A very dilute sample may not contain enough organisms to use either of the plate count methods described.
In such cases, the original sample must be concentrated rather than diluted before plating.
This can be accomplished using a modification of the plate count technique called the membrane filtration technique.
Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms.
The membrane is transferred to a Petri plate containing an appropriate growth medium.
Colonies are counted after incubation.
Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid.

The Most Probable Number

The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far.
For these specimens, microbiologists routinely use the most probable number (MPN) method, a statistical procedure for estimating of the number of viable microorganisms in a sample.
Often used for water and food samples, the MPN method evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.
A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water.
Coliforms are gram-negative rod bacteria that ferment lactose.
The presence of coliforms in water is considered a sign of contamination by fecal matter.

Indirect Cell Counts

Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to estimate and compare cell densities in a culture.
The foremost approach is to measure the turbidity (cloudiness) of a sample of bacteria in a liquid suspension.
The laboratory instrument used to measure turbidity is called a spectrophotometer.
In a spectrophotometer, a light beam is transmitted through a bacterial suspension, the light passing through the suspension is measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent transmission or a logarithmic value called absorbance (optical density).
As the numbers of bacteria in a suspension increase, the turbidity also increases and causes less light to reach the detector.
Measuring dry weight of a culture sample is another indirect method of evaluating culture density without directly measuring cell counts.
Recently, indirect ways of measuring live cells have been developed that are both fast and easy to implement. These methods measure cell activity by following the production of metabolic products or disappearance of reactants.
Adenosine triphosphate (ATP) formation, biosynthesis of proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of cells.

Alternative Patterns of Cell Division

Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one.
Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments.
In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of many new cells at once.
The new cells often split from the parent filament and float away in a process called fragmentation.
Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant bacterium Epulopiscium, where several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment.
Other species may form a long narrow extension at one pole in a process called budding, where the tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell.

Biofilms

In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a variety of environmental surfaces.
Biofilms are not random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.
Environmental conditions influence the overall structure of biofilms: filamentous biofilms called streamers form in rapidly flowing water, while in still or slow-moving water, biofilms mainly assume a mushroom-like shape.
Detailed observations of biofilms reveal clusters of microorganisms embedded in a matrix interspersed with open water channels.
The extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the organisms in the biofilm, and it plays a key role in maintaining the integrity and function of the biofilm.
Free-floating microbial cells that live in an aquatic environment are called planktonic cells.
The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile.
In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites; this stage is referred to as dispersal.
Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another.
The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing.
Signaling molecules in quorum sensing belong to two major classes: N-acylated homoserine lactones in gram-negative bacteria and small peptides in gram-positive bacteria.
The human body harbors many types of biofilms, some beneficial and some harmful.
Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts.
Researchers have treated surfaces with a layer of lubricant, which was infused into the tiny crevices of the surface and held there by capillary action, which was effective at preventing biofilm growth because it eliminated microorganism adhesion.

Oxygen Requirements of Microorganisms

Molecular oxygen ($O_2$) is not always needed for life.
Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis did oxygen levels increase in the atmosphere, causing a massive extinction.
Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS).
Many ecosystems are still free of molecular oxygen.
We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube cultures.
Diagram of bacterial cell distribution in thioglycolate tubes:
- Tube A: obligate (strict) aerobes that cannot grow without an abundant supply of oxygen.
- Tube B: obligate anaerobes, which are killed by oxygen.
- Tube C: Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence.
- Tube D: Aerotolerant anaerobes are indifferent to the presence of oxygen.
- Tube E: Microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere.
Examples of obligate aerobes include Mycobacterium tuberculosis and Micrococcus luteus.
Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life.
Prolonged use of antibiotics for other infections increases the probability of a patient developing a secondary C. difficile infection.
Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by Clostridioides difficile, causing a significant inflammation of the colon.
The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen.
Staphylococci and Enterobacteriaceae are examples of facultative anaerobes.
Examples of aerotolerant anaerobes include lactobacilli and streptococci.
Campylobacter jejuni, which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.

Detoxification of Reactive Oxygen Species

Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified.
Even organisms that do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen.
Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase.
- $X−(2H+)+H2O2→oxidized−X+2H_2O$
  - Reactions of type seen in Reaction 1 are catalyzed by peroxidases
- $2O2−+2H+→H2O2+O2$
  - Reaction 2 is mediated by the enzyme superoxide dismutase (SOD)
- $2H2O2→2H2O+O2$
  - The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
Obligate anaerobes usually lack all three enzymes.
Aerotolerant anaerobes do have SOD but no catalase.
Reaction 3 is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes.
Bacteria that grow best in a higher concentration of $CO_2$ and a lower concentration of oxygen than present in the atmosphere are called capnophiles.
Common approach to grow capnophiles is to use a candle jar.

The Effects of pH on Microbial Growth

Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic.
The optimum growth pH is the most favorable pH for the growth of an organism. The lowest and highest pH values that an organism can tolerate are called, respectively, the minimum growth pH and the maximum growth pH.
Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7.
Microorganisms that grow optimally at pH less than 5.55 are called acidophiles.
At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5.
Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining, and the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori.
H. pylori creates a microenvironment in which the pH is nearly neutral by producing large amounts of the enzyme urease, which breaks down urea to form $NH4^+$ and $CO2$.

Temperature and Microbial Growth

Microbes can be roughly classified according to the range of temperature at which they can grow.
Growth rates are the highest at the optimum growth temperature for the organism.
The lowest temperature at which the organism can survive and replicate is its minimum growth temperature.
The highest temperature at which growth can occur is its maximum growth temperature.
Mesophiles (“middle loving”) are adapted to moderate temperatures, with optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C.
Organisms called psychrotrophs, also known as psychrotolerant, prefer cooler environments, from a high temperature of 25 °C to refrigeration temperature about 4 °C.
Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth temperature close to 15 °C, and usually do not survive at temperatures above 20 °C.
Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles (“heat loving”).
The hyperthermophiles grow optimally from 80 °C to a maximum of 110 °C.
Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes.
Very low temperatures affect cells in many ways, such as membranes losing their fluidity and proteins becoming too rigid to catalyze reactions.
Proteins in psychrophiles are, in general, rich in hydrophobic residues, display an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from mesophiles.
Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the mesophiles.
The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA sequences show a higher proportion of guanine–cytosine nitrogenous bases.

Other Environmental Conditions that Affect Growth

Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels.
Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms, and rigid cell walls protect the cells from bursting in a dilute environment.
Microorganisms called halophiles (“salt loving”) actually require high salt concentrations for growth.
Water activity ($aw$) is the ratio of the vapor pressure of the medium of interest to the vapor pressure of pure distilled water ($aw$ of water = 1.0).
*Microorganisms that require high atmospheric pressure for growth are called barophiles.
Photoautotrophs and photoheterotrophs depend on sufficient light intensity at the wavelengths absorbed by their pigments to grow and multiply.

Eye on Ethics: Feeding the World…and the World’s Algae

Artificial fertilizers provide nitrogen and phosphorus, key limiting nutrients, to crop plants, removing the normal barriers that would otherwise limit the rate of growth.
However, careless use and overuse of artificial fertilizers have been demonstrated to have significant negative impacts on aquatic ecosystems, both freshwater and marine.
Fertilizers that are applied at inappropriate times or in too-large quantities allow nitrogen and phosphorus compounds to escape use by crop plants and enter drainage systems.
Heavy rains cause runoff of fertilizers into Lake Erie, triggering extensive algal blooms.

Media Used for Bacterial Growth

The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions.
Some media are considered general all-purpose media and support growth of a large variety of organisms (e.g., tryptic soy broth).
Enriched media contain growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms.
When the complete chemical composition of a medium is known, it is called a chemically defined medium.
In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known.
Non-selective media support the growth of all microorganisms without any specific inhibition.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest are called selective media (e.g., MacConkey agar).
The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum.
The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium.
Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

Case in Point: The End-of-Year Picnic

Bacteria can cause gastroenteritis either by colonizing and replicating in the host (infection) or by secreting toxins (intoxication).
Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples.
Samples from the salad showed the presence of gram-positive cocci bacteria in clusters.
The toxin secreted by S. aureus is known to cause severe gastroenteritis.
The organism was probably introduced into the salad during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the speeches.