Growth and Enumeration of Bacteria
Modes of Replication
All of the eubacteria replicate via asexual means.
The most common form of replication is binary fission, a process which
is analogous to mitosis in eukaryotic cells. In binary fission, a
cell first replicates its chromosome, then invaginates both cell membrane
and wall to form a septum between the two replicated rings of DNA.
Once the septum has formed, the two new offspring cells can either separate
from one another or remain linked together. Over time, linked cells
can form a chain, as is seen in streptococci such as Streptococcus pneumoniae,
and streptobacilli such as Bacillus subtilis. It is important to
note that all progeny cells produced by binary fission are genetically
identical to the original parent cell, since the original parent DNA was
Alternative forms of asexual replication in
microorganisms include hyphal fragmentation
, as in members of the
genus Streptomyces, and budding, found in yeasts such as Saccharomyces
cerevisiae, common brewer's yeast which is used in the production of
bread and alcohol, and Candida albicans, the opportunistic
pathogen which causes yeast infections in humans. As previously stated,
these processes result in the creation of progeny which are genetically
identical to the parent, however, in these, the progeny arise from an unequal
division of cytoplasm, unlike binary fission. Offspring will ultimately
undergo growth processes to reach the nominal size dictated by the genetic
code supplied by the parental cell. To estimate bacterial numbers,
one can utilize the simple formula:
Nf = Ni
Where Nf is the final
number of bacteria, Ni is the initial number of bacteria,
2 represents the number of new bacteria formed asexually when a
parental cell divides, and n represents the number of generations.
The Bacterial Colony Growth Curve
Bacteria, like other forms of life, must have
proper environmental conditions, including temperature, sufficient space
and nutrients in order to survive. Given the relatively simple nature
of the replicative process of bacteria, under optimal conditions their
numbers will increase geometrically in a very short period of time.
This means that unless governed by some environmental pressure, bacteria
would overwealm the earth, regardless of their microscopic size, within
a few days. However, such an outcome cannot occur. All environments
are limited in space and nutrients, thus continued growth above this limit,
called carrying capacity, results in competition, reduction in nutrients,
and a decline in the number of organisms.
In simple mathematical terms, the growth rate
is equal to the number of new cells (births) minus the number of cells
which undergo involution (autolysis) and die, and the generation (doubling)
time is the time necessary for a single cell or population of cells to
double. By raising bacteria in a batch culture which has no
means of adding additional nutrients or removing waste, such as a culture
dish or container of broth, it is possible to examine the pattern of population
1. When bacteria are first added to a new closed culture, there
is a period of adjustment called
the Lag Phase, where the number of
births and deaths are approximately equal. During this
time, bacteria are adapting to the new conditions
and preparing for growth and replication.
2. After they have adjusted to the environmental conditions, the
bacteria now enter a Log Phase
or phase of exponential growth, wherein the
number of new cells created by binary fission far
exceeds the number of cells undergoing involution.
This pattern will continue until the organisms
reach the carrying capacity of their environment.
3. Upon reaching the limits placed on them by food and available
space for growth, the bacteria
will begin a Stationary Phase, wherein
the numbers of births and deaths are once again
approximately equal. This stage will
continue until the level of available nutrients declines, and the
amount of metabolic wastes created by the
cells reaches toxic levels.
4. Ultimately, as nutrients decline, toxic waste levels increase,
and older cells begin to outnumber
young cells, the culture will enter into a
Decline (death) Phase, wherein the number of deaths
exceeds the number of newly produced cells.
While not all of the bacteria in the culture will
die immediately, their numbers will drop rapidly
and further growth will be inhibited.
This pattern of population growth and decline
occurs any time bacteria are cultured in a closed system. However,
it is possible to maintain bacterial populations at the stationary level
through the use of a chemostat , a device which adds fresh nutrients
to a continuously growing batch of bacteria while removing dead cells and
metabolic wastes. A continuous culture also can be used to
raise bacteria by continuously adding fresh media, to keep the population
at the exponetial phase of growth. Through the use of devices such as the
chemostat and continuous cultures, large, pure cultures can be maintained
for long periods of time.
The Enumeration of Bacteria
Direct Counting Methods
When it becomes necessary to count numbers of
bacteria, one can do so either directly to get accurate, quantitative numbers,
or indirectly, to produce a qualitative estimate of population size.
Given the small size of bacteria and speed at which they reproduce, most
laboratories perform indirect counting methods. But it is possible
to obtain exact numbers directly by utilizing techniques such as the Petroff-Hauser
slide (also known as a hemocytometer, which is used to count numbers
of blood cells), and the Coulter counter. The Petroff-Hauser slide
is a microscope slide which has on its surface an etched grid. A
fluid sample of known volume is placed on the slide, and the numbers of
bacteria are directly counted microscopically. It is impossible,
however, to determine if cells are alive (viable), and motile cells may
move from one graid to another, which could result in an inaccurate count.
A Coulter counter is a mechanical device originally designed to
count the formed elements of human blood which uses optics to count bacterial
cells in a known volume of fluid as they pass a light sensor. As
with the Petroff-Hauser technique, it is impossible to determine viability
of the cells which are counted.
Indirect Counting Methods
When groups of cells grow on the surface
of an agar culture plate, they form a visible structure called a colony.
Since it can be reasonably assumed that each new cell in a colony has arisen
from one original parent via binary fission or another asexual means, and
that the cells which form the colony are viable, such aggregations of cells
can be referred to as colony forming units or CFUs. By counting
CFUs, it is possible to produce a reasonable estimate of bacterial
numbers present in food or water samples. This method is much quicker
than making a direct count such as through the use of the Petroff-Hauser
slide, but the exact number of bacteria found in each colony is unknown,
therefore the results produced are only estimates. Through a process
of counting large numbers of samples, however, reasonably accurate, statistically
valid conclusions can be reached.
Examples of indirect counting include filtration,
the spread- and pour-plate, and the most probable number
(MPN) techniques. In filtration, a known volume of fluid containing
bacteria is passed through a nitrocellulose filter which has a pore (opening)
size ranging from about 0.2 mm to about 4.5 mm, which is just small enough
to trap bacteria while allowing the fluid to pass. The filter is
then placed on the agar surface of a culture plate, incubated, and colonies
counted. In the spread-plate, a volume of fluid is placed on an agar
plate and spread with a sterile rod, incubated and colonies counted, and
in the pour-plate method known dilutions of a sample are dispensed into
plates and covered with melted agar which allowed to solidify before incubation
and counting. Both spread- and pour-plate techniques produce viable
In the most probable number technique, fluid
samples are prepared through serial dilution, then each dilution is incubated.
After incubation, the samples are placed in a device called a spectrophotometer,
which measures the transmittance, or amount of light which passes through
a sample. By examining the level of transmittance of many serial
dilutions which have been replicated many times, then comparining the results
to a statistical table of bacterial numbers, it is possible to obtain a
reasonably valid estimate of population size.
Factors Which Influence Bacterial Growth
All species of bacteria grow at different rates
under different environmental conditions. Each species has its own
set of optimal conditions for growth, including temperature, oxygen level,
osmotic potential, pH, pressure, and light.
Temperature has a direct influence on the rate
of enzyme activity which occurs within the bacterial cell. If the
temperature becomes too low, enzymatic activity declines, and if it becomes
too high, enzymes denature(lose the shape necessary for their proper activity),
thus are unable to participate in metabolic pathways. The optimal
temperature is one at which enzyme activity is the greatest, and generation
time, thus growth rate, is maximal. Organisms can be classified on
the basis of their optimal temperature range as follows:
1. Psychrophiles grow best at a temperature range below 20o
C. Examples inclube Bacillus
globisporus and Vibrio marinus.
2. Mesophiles have an optimum range bewteen 20o
and 45o C. Examples include Staphylococcus
aureus, Bacillus subtilis, and
Escherichia coli. Since the average human body temperature is
approximately 37o C, all of the
pathogenic bacteria could be considered to be mesophiles.
3. Thermophiles grow at temperatures at or above 45o
C. Examples include Bacillus
stearothermophilus and Beggatoia sp.
Some bacteria are thermoduric, meaning that
they are capable of tolerating higher temperatures than optimal for growth.
Generally, gram positive cells tend to be more thermoduric than gram negative
cells. Also, organisms such as members of the genus Bacillus and
the genus Clostridium can also survive excessive heat through the production
of resistant endospores. For example, the spores of Clostridium botulinum
can remain in boiling water for up to five hours and still remain viable,
and the spores of Bacillus stearothermophilus are so heat resistant that
they are used to test the effectiveness of autoclaves.
Though we are most familiar with molecular
oxygen (O2), this gas can also occur in forms which are damaging
to bacteria, including free radicals (ions which attack biological
molecules such as DNA) such as superoxide (O2-; inactivated
by the enzyme superoxide dismutase), singlet oxygen (O) which is
produced by some phagocytes, and hydrogen peroxide (H2O2;
inactivated by the enzyme catalase). Organisms are grouped
according to their oxygen requires as:
1. Obligate aerobes which require molecular oxygen. Pseudomonas
aeruginosa is an example.
2. Obligate anaerobes which grow only in the absence of oxygen
and lack enzymes such as
superoxide dismutase and catalase. Clostridium
botulinum and C. tetani are examples.
3. Microaerophiles such as capenic microorganisms (capnophiles)
which grow best
under conditions which include CO2, such
as Nessieria gonorrhoea and Campylobacter jejuni.
4. Aerotolerant species which survive in the presence of oxygen,
but do not need it
for growth, such as Lactobacillus acidophilus.
5. Facultative anaerobes which do not need molecular oxygen,
but will grow equally well in its
presence. Examples include Staphylococcus
aureus, Bacillus subtilis, and Micrococcus luteus.
The amount of specific solutes dissolved in the
surrounding environment can limit the ability of many microorganisms
to survive. If the solution surrounding the cell is hypotonic, water
will rush in and could cause lysis. If the solution is hypertonic,
water will rush out of the cell, causing it to plasmolyze. Knowledge
of this has led to the preservation of foods through the use of salt or
sugar, but some organisms withstand or thrive under harsh osmotic conditions.
Halotolerant organisms, such as Staphylococcus aureus and
S. epidermidis do not utilize salt, but can withstand concentrations
as high as 7.5%. Halophiles, such as Halobacterium sp.,
can only grow where the salt concentration is 3% or greater. Some
fungi grow best in media which contain high concentrations of sugar, such
as Saboraud's agar, which can exclude most microbes.
Most bacteria grow best at a pH range from 6.0
to 7.1. These can be classified as neutralophiles (neutrophiles).
Acidophiles, such as Lactobacillus acidophilus, Thiobacillus
thioxidans, and Helicobacter pylori grow best at a pH range
of 0.8-6.6. Alkalophiles (alkophiles), such as Bacillus
alkalophilus grow best at pH levels above 7.
All living cells require moisture to carry out their
normal metabolic processes. While multicellular organisms have evolved
protective outer coverings and water retention strategies, unicellular
microorganisms must be constantly bathed in water to survive. No
single cell can live for an extended period of time out of a watery environment
in its vegetative form. Treponema pallidum, for example, will
die within twenty seconds on the surface of a dry fomite. However,
those microorganisms which have the ability to form resting stages such
as spores and cysts can remain viable for long periods of time in a dessicating
The force exerted by a column of water on
an object is called hydrostatic pressure. Organisms which have adapted
to high levels of hydrostatic pressure are called barophiles.
One example, Beggatoia sp., is a bacterium found living near deep
ocean hydrothermal vents called fumeroles. To overcome these
extreme conditions, Beggatoia has evolved enzyme systems which are maintain
their proper activity only in the presence of raised temperatures and pressures.
Barotolerant organisms can withstand high hydrostatic pressure, but
are not dependent upon it for the proper activity of their enzyme systems.
A. Calculate the number of bacteria present in a culture after a given
number of generations
and initial number of cells:
1. Ni = 150, n = 7
2. Ni = 2000, n = 10
3. Ni = 1, n = 20
B. Match the technique with its use. Some letters may be used
more than once.
a. reduces bacterial numbers
_____ Petroff-Hauser slide
b. indirect count
c. gives actual bacterial numbers
_____ serial dilution
d. direct count
_____ Coulter counter
e. estimates bacterial numbers
Test Yourself- Use this to quiz yourself about
aspects of bacterial growth.