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LEARNING OBJECTIVES

Define the generation time for growth based on binary fission

Identify and describe the activities of microorganisms undergoing typical phases of binary
fission (simple cell division) in a growth curve
Explain several laboratory methods used to determine viable and total cell counts in
populations undergoing exponential growth
Describe examples of cell division not involving binary fission, such as budding or
fragmentation
Describe the formation and characteristics of biofilms
Identify health risks associated with biofilms and how they are addressed
Describe quorum sensing and its role in cell-to-cell communication and coordination of
cellular activities

CLINICAL FOCUS

Part 1
Jeni, a 24-year-old pregnant woman in her second trimester, visits a clinic with complaints of
high fever, 38.9 °C (102 °F), fatigue, and muscle aches—typical flu-like signs and symptoms.
Jeni exercises regularly and follows a nutritious diet with emphasis on organic foods, including
raw milk that she purchases from a local farmer’s market. All of her immunizations are up to
date. However, the health-care provider who sees Jeni is concerned and orders a blood
sample to be sent for testing by the microbiology laboratory.

Why is the health-care provider concerned about Jeni’s signs and symptoms?

Jump to the next Clinical Focus box

The bacterial cell cycle involves the formation of new cells through the replication of DNA and
partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always
asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as
will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some
exceptions exist. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear
chromosome.

Binary Fission
The most common mechanism of cell replication in bacteria is a process called binary fission, which is
depicted in Figure 9.2. Before dividing, the cell grows and increases its number of cellular components.
Next, the replication of DNA starts at a location on the circular chromosome called the origin of
replication, where the chromosome is attached to the inner cell membrane. Replication continues in
opposite directions along the chromosome until the terminus is reached.

The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a
complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of
cytokinesis and cell division is directed by a protein called FtsZ. FtsZ assembles into a Z ring on the
cytoplasmic membrane (Figure 9.3). The Z ring is anchored by FtsZ-binding proteins and defines the
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division plane between the two daughter cells. Additional proteins required for cell division are added to
the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan
cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the
division septum, where all of the cells’ outer layers (the cell wall and outer membranes, if present) must
be remodeled to complete division. For example, we know that specific enzymes break bonds between
the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Figure 9.2 (a) The electron micrograph depicts two cells of Salmonella typhimurium after a binary fission event. (b) Binary
fission in bacteria starts with the replication of DNA as the cell elongates. A division septum forms in the center of the cell.
Two daughter cells of similar size form and separate, each receiving a copy of the original chromosome. (credit a:
modification of work by Centers for Disease Control and Prevention)

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Figure 9.3 FtsZ proteins assemble to form a Z ring that is anchored to the plasma membrane. The Z ring pinches the cell
envelope to separate the cytoplasm of the new cells.

CHECK YOUR UNDERSTANDING

What is the name of the protein that assembles into a Z ring to initiate cytokinesis and
cell division?

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Generation Time
In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two
successive generations. For example, the typical generation time for the human population is 25 years.
This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for
thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the
doubling time and is defined as the time it takes for the population to double through one round of
binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little
as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need
several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but
there are exceptions. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, has
a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s
disease (leprosy), grows much more slowly, with a doubling time of 14 days.

MICRO CONNECTIONS

Calculating Number of Cells

It is possible to predict the number of cells in a population when they divide by binary fission
at a constant rate. As an example, consider what happens if a single cell divides every 30
minutes for 24 hours. The diagram in Figure 9.4 shows the increase in cell numbers for the
first three generations.

The number of cells increases exponentially and can be expressed as 2n, where n is the
number of generations. If cells divide every 30 minutes, after 24 hours, 48 divisions would
have taken place. If we apply the formula 2n, where n is equal to 48, the single cell would give
rise to 248 or 281,474,976,710,656 cells at 48 generations (24 hours). When dealing with such
huge numbers, it is more practical to use scientific notation. Therefore, we express the
number of cells as 2.8 × 1014 cells.

In our example, we used one cell as the initial number of cells. For any number of starting
cells, the formula is adapted as follows:
n
Nn = N0 2

Nn is the number of cells at any generation n, N0 is the initial number of cells, and n is the
number of generations.

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Figure 9.4 The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn,
divides, giving a total of four cells in the second generation and eight cells in the third generation. Each
division doubles the number of cells.

CHECK YOUR UNDERSTANDING

With a doubling time of 30 minutes and a starting population size of 1 × 105 cells, how
many cells will be present after 2 hours, assuming no cell death?

The Growth Curve

Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added
and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve.
An example of a batch culture in nature is a pond in which a small number of cells grow in a closed
environment. The culture density is defined as the number of cells per unit volume. In a closed
environment, the culture density is also a measure of the number of cells in the population. Infections of
the body do not always follow the growth curve, but correlations can exist depending upon the site and
type of infection. When the number of live cells is plotted against time, distinct phases can be observed
in the curve (Figure 9.5).

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Figure 9.5 The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a
function of time. The graph can be divided into four phases according to the slope, each of which matches events in the
cell. The four phases are lag, log, stationary, and death.

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, a nutritional broth that supports growth. 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; however, 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 (nutrients, temperature, pH, and so forth)
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, as shown in Figure 9.6, which gives the
appearance of a linear relationship.

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Figure 9.6 Both graphs illustrate population growth during the log phase for a bacterial sample with an initial population
of one cell and a doubling time of 1 hour. (a) When plotted on an arithmetic scale, the growth rate resembles a curve. (b)
When plotted on a semilogarithmic scale (meaning the values on the y-axis are logarithmic), the growth rate appears
linear.

Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, 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, several factors contribute to a slowing of the
growth rate. Waste products accumulate and nutrients are gradually used up. In addition, 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 (Figure 9.5). 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. For example, 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. Soon, the number of dying cells exceeds the number of dividing cells, leading to an
exponential decrease in the number of cells (Figure 9.5). This is the aptly named death phase,
sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing
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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

The growth pattern shown in Figure 9.5 takes place in a closed environment; nutrients are not added
and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells
in the logarithmic phase of growth. One example is in industries that harvest microbial products. A
chemostat (Figure 9.7) 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.

Figure 9.7 A chemostat is a culture vessel fitted with an

opening to add nutrients (feed) and an outlet to remove
contents (effluent), effectively diluting toxic wastes and dead
cells. The addition and removal of fluids is adjusted to
maintain the culture in the logarithmic phase of growth. If
aerobic bacteria are grown, suitable oxygen levels are
maintained.

CHECK YOUR UNDERSTANDING

During which phase does growth occur at the fastest rate?

Name two factors that limit microbial growth.

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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. The direct methods involve counting cells, whereas the
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. Let’s look first at a simple and fast method that
requires only a specialized slide and a compound microscope.

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 (Figure 9.8) and is similar to a
hemocytometer used to count red blood cells. The central area of the counting chamber is etched into
squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip
that is placed at a specific height from the surface of the grid. It is possible to estimate the
concentration of cells in the original sample by counting individual cells in a number of squares and
determining the volume of the sample observed. The area of the squares and the height at which the
coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if
the sample was diluted before enumeration.

Figure 9.8 (a) A Petroff-Hausser chamber is a special slide designed for counting the bacterial cells in a measured volume
of a sample. A grid is etched on the slide to facilitate precision in counting. (b) This diagram illustrates the grid of a
Petroff-Hausser chamber, which is made up of squares of known areas. The enlarged view shows the square within which
bacteria (red cells) are counted. If the coverslip is 0.2 mm above the grid and the square has an area of 0.04 mm2, then
the volume is 0.008 mm3, or 0.000008 mL. Since there are 10 cells inside the square, the density of bacteria is 10
cells/0.000008 mL, which equates to 1,250,000 cells/mL. (credit a: modification of work by Jeffrey M. Vinocur)

Cells in several small squares must be counted and the average taken to obtain a reliable measurement.
The advantages of the chamber are that the method is easy to use, relatively fast, and inexpensive. On
the downside, the counting chamber does not work well with dilute cultures because there may not be
enough cells to count.

Using a counting chamber does not necessarily yield an accurate count of the number of live cells
because it is not always possible to distinguish between live cells, dead cells, and debris of the same
size under the microscope. However, newly developed fluorescence staining techniques make it

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possible to distinguish viable and dead bacteria. These 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 (Figure 9.9).

Figure 9.9 Fluorescence staining can be used to differentiate between viable and dead
bacterial cells in a sample for purposes of counting. Viable cells are stained green, whereas
dead cells are stained red. (credit: modification of work by Panseri S, Cunha C, D’Alessandro
T, Sandri M, Giavaresi G, Maracci M, Hung CT, Tampieri A)

Another technique uses an electronic cell counting device (Coulter counter) to detect and count the
changes in electrical resistance in a saline solution. A glass tube with a small opening is immersed in an
electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located
outside of the tube. As cells are drawn through the small aperture in the glass tube, they briefly change
the resistance measured between the two electrodes and the change is recorded by an electronic
sensor (Figure 9.10); each resistance change represents a cell. The method is rapid and accurate within
a range of concentrations; however, if the culture is too concentrated, more than one cell may pass
through the aperture at any given time and skew the results. This method also does not differentiate
between live and dead cells.

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.

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Figure 9.10 A Coulter counter is an electronic device that counts cells. It measures the change in resistance in an
electrolyte solution that takes place when a cell passes through a small opening in the inside container wall. A detector
automatically counts the number of cells passing through the opening. (credit b: modification of work by National
Institutes of Health)

CHECK YOUR UNDERSTANDING

Why would you count the number of cells in more than one square in the Petroff-
Hausser chamber to estimate cell numbers?
In the viability staining method, why do dead cells appear red?

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. For all these reasons, the viable plate count
is considered a low estimate of the actual number of live cells. These limitations do not detract from the
usefulness of the method, which provides estimates of live bacterial numbers.

Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not
give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately
count individual colonies. Also, counts in this range minimize occurrences of more than one bacterial
cell forming a single colony. Thus, the calculated CFU is closer to the true number of live bacteria in the
population.

There are two common approaches to inoculating plates for viable counts: the pour plate and the
spread plate methods. Although the final inoculation procedure differs between these two methods, they
both start with a serial dilution of the culture.

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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. Figure
9.11 illustrates the serial dilution method.

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 (Figure 9.12) or the spread plate method (Figure 9.13). 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 (to ensure the cell distribution in the tube is random) is paramount to obtaining
reliable results.

Figure 9.11 Serial dilution involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution
as an inoculum. The result is dilution of the original culture by an exponentially growing factor. (credit: modification of
work by “Leberechtc”/Wikimedia Commons)

The dilution factor is used to calculate the number of cells in the original cell culture. In our example, an
average of 50 colonies was counted on the plates obtained from the 1:10,000 dilution. Because only 0.1
mL of suspension was pipetted on the plate, the multiplier required to reconstitute the original
concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 10 × 10,000 = 5,000,000. The
number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the
1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights
the issue of inaccuracy when colony counts are greater than 300 and more than one bacterial cell grows
into a single colony.

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Figure 9.12 In the pour plate method of cell counting, the sample is mixed in liquid warm agar (45–50 °C) poured into a
sterile Petri dish and further mixed by swirling. This process is repeated for each serial dilution prepared. The resulting
colonies are counted and provide an estimate of the number of cells in the original volume sampled.

Figure 9.13 In the spread plate method of cell counting, the sample is poured onto solid agar and then spread using a
sterile spreader. This process is repeated for each serial dilution prepared. The resulting colonies are counted and provide
an estimate of the number of cells in the original volume samples.

A very dilute sample—drinking water, for example—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.

LINK TO LEARNING

Watch this video for demonstrations of serial dilutions and spread plate techniques.

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

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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. For the method illustrated in Figure 9.14, a series
of three dilutions of the water sample is tested by inoculating five lactose broth tubes with 10 mL of
sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of
sample. The lactose broth tubes contain a pH indicator that changes color from red to yellow when the
lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of
coliform growth by a color change in media from red to yellow. The first set of tubes (10-mL sample)
showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of five;
in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2,
and 0 are compared with Figure B1 in Appendix B, which has been constructed using a probability
model of the sampling procedure. From our reading of the table, we conclude that 49 is the most
probable number of bacteria per 100 mL of pond water.no lo

Figure 9.14 In the most probable number method, sets of five lactose broth tubes are inoculated with three different
volumes of pond water: 10 mL, 1 mL, and 0.1mL. Bacterial growth is assessed through a change in the color of the broth
from red to yellow as lactose is fermented.

CHECK YOUR UNDERSTANDING

What is a colony-forming unit?

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What two methods are frequently used to estimate bacterial numbers in water
samples?

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 (Figure 9.15). 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. The decrease in light passing through the sample
and reaching the detector is associated with a decrease in percent transmission and increase in
absorbance measured by the spectrophotometer.

Measuring turbidity is a fast method to estimate cell density as long as there are enough cells in a
sample to produce turbidity. It is possible to correlate turbidity readings to the actual number of cells by
performing a viable plate count of samples taken from cultures having a range of absorbance values.
Using these values, a calibration curve is generated by plotting turbidity as a function of cell density.
Once the calibration curve has been produced, it can be used to estimate cell counts for all samples
obtained or cultured under similar conditions and with densities within the range of values used to
construct the curve.

Figure 9.15 (a) A spectrophotometer is commonly used to measure the turbidity of a bacterial cell suspension as an
indirect measure of cell density. (b) A spectrophotometer works by splitting white light from a source into a spectrum. The
spectrophotometer allows choice of the wavelength of light to use for the measurement. The optical density (turbidity) of
the sample will depend on the wavelength, so once one wavelength is chosen, it must be used consistently. The filtered
light passes through the sample (or a control with only medium) and the light intensity is measured by a detector. The light
passing into a suspension of bacteria is scattered by the cells in such a way that some fraction of it never reaches the
detector. This scattering happens to a far lesser degree in the control tube with only the medium. (credit a: modification of
work by Hwang HS, Kim MS; credit b “test tube photos”: modification of work by Suzanne Wakim)

Measuring dry weight of a culture sample is another indirect method of evaluating culture density
without directly measuring cell counts. The cell suspension used for weighing must be concentrated by
filtration or centrifugation, washed, and then dried before the measurements are taken. The degree of

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drying must be standardized to account for residual water content. This method is especially useful for
filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.

As we have seen, methods to estimate viable cell numbers can be labor intensive and take time
because cells must be grown. 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.

CHECK YOUR UNDERSTANDING

What is the purpose of a calibration curve when estimating cell count from turbidity
measurements?
What are the newer indirect methods of counting live 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 (Figure 9.16). Fragmentation is commonly observed in
the Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil. Another
curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the
giant bacterium Epulopiscium. 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. The tip of the extension swells and forms a smaller
cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast (Figure
9.16), but it is also observed in prosthecate bacteria and some cyanobacteria.

Figure 9.16 (a) Filamentous cyanobacteria, like those pictured here, replicate by fragmentation. (b) In this electron
micrograph, cells of the bacterium Gemmata obscuriglobus are budding. The larger cell is the mother cell. Labels indicate
the nucleoids (N) and the still-forming nuclear envelope (NE) of the daughter cell. (credit a: modification of work by
CSIRO; credit b: modification of work by Kuo-Chang Lee, Rick I Webb and John A Fuerst)

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The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in
fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low
nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments, elongated cells
divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise
to new colonies.

CHECK YOUR UNDERSTANDING

Identify at least one difference between fragmentation and budding.

Biofilms
In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a
variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river
beds. Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid
environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that
float on water, for example, are biofilms that contain large populations of photosynthetic
microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species.
Regardless of the environment where they occur, biofilms are not random collections of microorganisms;
rather, they are highly structured communities that provide a selective advantage to their constituent
microorganisms.

Biofilm Structure
Observations using confocal microscopy have shown that environmental conditions influence the overall
structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as
freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions
in fast-moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats
downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like
shape. The structure of biofilms may also change with other environmental conditions such as nutrient
availability.

Detailed observations of biofilms under confocal laser and scanning electron microscopes 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. The extracellular matrix represents a large fraction of the biofilm, accounting for 50%–90%
of the total dry mass. The properties of the EPS vary according to the resident organisms and
environmental conditions.

EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules
such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of
the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm.
This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from
predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).

Biofilm Formation
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 (attached to a surface). This occurs in stages, as depicted in Figure 9.17. The first stage

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involves the attachment of planktonic cells to a surface coated with a conditioning film of organic
material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes
that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm
develops characteristic structures, including an extensive matrix and water channels. Appendages such
as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that
such structures are required for the establishment of a mature biofilm. 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.

Figure 9.17 Stages in the formation and life cycle of a biofilm. (credit: modification of work by Public Library of Science
and American Society for Microbiology)

Within a biofilm, different species of microorganisms establish metabolic collaborations in which the
waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms
consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in
many polymicrobial infections that involve both aerobic and anaerobic pathogens.

The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli
is called quorum sensing. Quorum sensing—which can occur between cells of different species within
a biofilm—enables microorganisms to detect their cell density through the release and binding of small,
diffusible molecules called autoinducers. When the cell population reaches a critical threshold (a
quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular
functions that are beneficial only when the population reaches a critical density. For example, in some
pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the
immune defenses of the host. Although mostly studied in bacterial populations, quorum sensing takes
place between bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida
albicans, a common member of the human microbiota that can cause infections in
immunocompromised individuals.

The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria
communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use
small peptides (Figure 9.18). In all cases, the first step in quorum sensing consists of the binding of the
autoinducer to its specific receptor only when a threshold concentration of signaling molecules is

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reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in
gene expression. The result is the activation of biological responses linked to quorum sensing, notably
an increase in the production of signaling molecules themselves, hence the term autoinducer.

Figure 9.18 Short peptides in gram-positive bacteria and N-acetylated homoserine lactones in gram-negative bacteria
act as autoinducers in quorum sensing and mediate the coordinated response of bacterial cells. The R side chain of
the N-acetylated homoserine lactone is specific for the species of gram-negative bacteria. Some secreted homoserine
lactones are recognized by more than one species.

Biofilms and Human Health

The human body harbors many types of biofilms, some beneficial and some harmful. For example, the
layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off
infections by pathogens. However, other biofilms in the body can have a detrimental effect on health.
For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal
disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The
bacterium Pseudomonas aeruginosa often colonizes biofilms in the airways of patients with cystic
fibrosis, causing chronic and sometimes fatal infections of the lungs. Biofilms can also form on medical
devices used in or on the body, causing infections in patients with in-dwelling catheters, artificial joints,
or contact lenses.

Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating
counterparts. Several hypotheses have been proposed to explain why. Cells in the deep layers of a
biofilm are metabolically inactive and may be less susceptible to the action of antibiotics that disrupt
metabolic activities. The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them
from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the
increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of
efflux pumps, membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have
been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria.
Finally, biofilms provide an ideal environment for the exchange of extrachromosomal DNA, which often
includes genes that confer antibiotic resistance.

CHECK YOUR UNDERSTANDING

What is the matrix of a biofilm composed of?

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What is the role of quorum sensing in a biofilm?

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