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 copied exactly.

     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  X 2n

     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 growth:

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 plate counts.

     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
     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.
 

Oxygen
      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.
 
 

Osmotic Potential
     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.
 
Acidity
     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.
 
Moisture
    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 environment.
 
Hydrostatic Pressure
      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.
 
 

Cooperative Activities

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.

_____  pour-plate                                                         a. reduces bacterial numbers
_____  Petroff-Hauser slide                                          b. indirect count
_____ MPN                                                                 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.
 
 
 

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