Microbial Growth

Requirements for Growth

1. Physical requirements
2. Chemical requirements
Temperature
Microorganisms are classified into three primary groups on the basis of their preferred
range of temperature:
1. Psychrophiles (cold-loving microbes),
2. mesophiles (moderate-temperature– loving microbes),
3. thermophiles (heat-loving microbes).
Hyperthermophiles (extreme thermophiles) – favor temperatures
above 100 degrees Celsius
Pyrolobus fumarii – lives at 113 degrees Celsius
Psychotrophs - group of Psychrophiles that love refrigerator
temperature (4 degrees Celsius)
Psychroduric organisms – prefer warmer temperatures but can
tolerate or endure very cold temperatures, can be preserved in the
frozen state
 Refrigeration is the most common method of
preserving household food supplies. It is based
on the principle that microbial reproductive
rates decrease at low temperatures.
 Microbes usually survive even subfreezing
temperatures (they might become entirely
dormant), they gradually decline in number.
 Psychrotrophs do not grow well at low
temperatures, except in comparison with other
organisms; given time, however, they
are able to slowly degrade food.
 pH - refers to the acidity or alkalinity of a solution. Most bacteria grow best in
a narrow pH range near neutrality, between pH 6.5 and 7.5.

 Most microorganisms prefer a neutral or slight alkaline medium

- Fungi prefer acidic environments.
Acidophiles – prefer acidic environment (live in the stomach; thrive in
pickled foods)
Alkaliphiles – prefer an alkaline environment (live in the intestines)
- Vibrio cholera = only human pathogen that grows well above pH8
 One type of chemoautotrophic bacteria, which is found in the drainage water from
coal mines and oxidizes sulfur to form sulfuric acid, can survive at a pH 1.
 Molds and yeasts will grow over a greater pH range than bacteria will, but the
optimum pH of molds and yeasts is generally below that of bacteria, usually about
pH 5 to 6.
 Alkalinity also inhibits microbial growth but is rarely used to preserve foods.
When bacteria are cultured in the laboratory, they often produce acids that
eventually interfere with their own growth.
 To neutralize the acids and maintain the proper pH, chemical buffers are included
in the growth medium. The peptones and amino acids in some media act as buffers,
and many media also contain phosphate salts. Phosphate salts have the advantage
of exhibiting their buffering effect in the pH growth range of most bacteria.
 Osmotic Pressure
Microorganisms obtain almost all their nutrients in
solution from the surrounding water. Thus, they require
water for growth, and their composition is 80–90%
water.
 High osmotic pressures have the effect of removing
necessary water from a cell.
Types of osmotic solutions:
1. hypertonic – concentration of solute in the environment
outside of a cell is higher than inside a cell
- Cells lose water and shrink in a hypertonic solution (RBC
– crenation; bacterial cell – plasmolysis)
2. Hypotonic – concentration of solute outside a cell is lower
than the inside of the cell (RBC – hemolysis; Bacterial cell –
plasmoptysis)
3. Isotonic – equal concentration of solute inside and outside
of the cell
 Halophilic organisms – prefer salty environments - thrive in
ocean waters. Extreme halophiles, have adapted so well to high salt
concentrations that they actually require them for growth. In this case,
they may be termed obligate halophiles
V. cholerae and other Vibrio species, are halophilic.
 Haloduric organisms - do not prefer to live in salty
environments but are capable of surviving there
(Staphylococcus aureus)
 The growth of the cell is inhibited as the plasma membrane pulls away from
the cell wall. Thus, the addition of salts (or other solutes) to a solution, and
the resulting increase in osmotic pressure, can be used to preserve foods.
 Salted fish, honey, and sweetened condensed milk are preserved largely by
this mechanism; the high salt or sugar concentrations draw water out of any
microbial cells that are present and thus prevent their growth.
 Facultative halophiles, do not require high salt concentrations but are able
to grow at salt concentrations up to 2%, a concentration that inhibits the
growth of many other organisms. A few species of facultative halophiles
can tolerate even 15% salt.
 Barometric pressure

Most bacteria are not affected by minor changes in

barometric pressure.

Barometric Some thrive at normal atmospheric pressure (about 14.7

pressure
pounds per square inch [psi]).

Others, known as piezophiles, thrive deep in the ocean and

in oil wells, where the atmospheric pressure is very high.

Some archaea, for example, are piezophiles, capable of

living in the deepest parts of the ocean.
  Some microbes (obligate aerobes) prefer the
same atmosphere that humans do (i.e., about
20%–21% oxygen and 78%–79% nitrogen).
 Although microaerophiles also require oxygen,
they require reduced concentrations of oxygen
Gaseous (around 5%).
 Obligate anaerobes are killed by the presence
Atmosphere of oxygen.
 To grow a particular microorganism in the
laboratory, it is necessary to provide the
atmosphere that it requires. For example, to
obtain maximum growth in the laboratory,
capnophiles require increased concentrations
of carbon dioxide (usually from 5% to 10%).
- capnophiles – 55 to 10% carbondioxide
Chemical Requirements
 Carbon
Carbon is the structural backbone of living matter; it is needed for all the
organic compounds that make up a living cell. Half the dry weight of a
typical bacterial cell is carbon.
 Chemoheterotrophs get most of their carbon from the source of their energy
—organic materials such as proteins, carbohydrates, and lipids.
 Chemoautotrophs and photoautotrophs derive their carbon from carbon
dioxide.
 Nitrogen, Sulfur, and Phosphorus
Microorganisms need other elements to synthesize cellular material. For
example, protein synthesis requires considerable amounts of nitrogen as
well as some sulfur.
 The syntheses of DNA and RNA also require nitrogen and some
phosphorus, as does the synthesis of ATP, the molecule so important for
the storage and transfer of chemical energy within the cell.
 Nitrogen makes up about 14% of the dry weight of a bacterial cell, and
sulfur and phosphorus together constitute about another 4.
 Nitrogen - forms the amino group of the amino acids of proteins. Other
bacteria use nitrogen from ammonium ions (NH4+), which are already in the
reduced form and are usually found in organic cellular material. Still other
bacteria are able to derive nitrogen from nitrates (compounds that dissociate
to give the nitrate ion, NO3-, in solution). Some important bacteria, including
many of the photosynthesizing cyanobacteria, use gaseous nitrogen (N2)
directly from the atmosphere. This process is called nitrogen fixation.
 Sulfur is used to synthesize sulfur-containing amino acids and vitamins such
as thiamine and biotin.
 Phosphorus is essential for the synthesis of nucleic acids and the
phospholipids of cell membranes. It is also found in the energy bonds of ATP.
A source of phosphorus is
the phosphate ion (PO43-).
 Potassium, magnesium, and calcium are also elements that microorganisms
require, often as cofactors for enzymes.
 Trace Elements
Microbes require very small amounts of other mineral
elements, such as iron, copper, molybdenum, and zinc;
these are referred to as trace elements.
 Most are essential for the functions of certain enzymes,
usually as cofactors. Although these elements are
sometimes added to a laboratory medium, they are usually
assumed to be naturally present in tap water and other
components of media. Even most distilled waters contain
adequate amounts, but tap water is sometimes specified to
ensure that these trace minerals will be present in culture
media
Oxygen
 Microbes that use molecular oxygen (aerobes) extract more energy from
nutrients than microbes that do not use oxygen (anaerobes). Organisms
that require oxygen to live are called obligate aerobes
 Obligate aerobes are at a disadvantage because oxygen is poorly soluble
in the water of their environment. Therefore, many of the aerobic bacteria
have developed, or retained, the ability to continue growing in the
absence of oxygen. Such organisms are called facultative anaerobes.
Facultative anaerobes can use oxygen when it is present but are able to
continue growth by using fermentation or anaerobic respiration when
oxygen is not available. An example of facultative anaerobes is the
familiar Escherichia coli that are found in the human intestinal tract.
 Many yeasts are also facultative anaerobes.
 Anaerobes are bacteria that are unable to use molecular oxygen for energy-
yielding reactions. The genus Clostridium which contains the species that cause
tetanus and botulism, is the most familiar example. Clostridium obtains energy
by
anaerobic respiration.
 Obligate anaerobes usually produce neither superoxide dismutase nor catalase.
Obligate anaerobes are extremely sensitive to oxygen.
 Aerotolerant anaerobes are fermentative and cannot use oxygen for growth, but
they tolerate it fairly well. Common examples of lactic acid–producing
aerotolerant anaerobes are the lactobacilli used in the production of many acidic
fermented foods, such as pickles and cheese.
 Microaerophiles are aerobic; they do require oxygen. However, they grow only
in oxygen concentrations lower than those in air. In a test tube of solid nutrient
medium, they grow only at a depth where small amounts of oxygen have
diffused into the medium; they do not grow near the oxygen-rich surface or
below the narrow zone of adequate oxygen.
 Organic Growth Factors - essential organic compounds an
organism is unable to synthesize; they must be directly obtained from
the environment.
 One group of organic growth factors is vitamins. Most vitamins
function as coenzymes.
 Many bacteria can synthesize all their own vitamins and do not
depend on outside sources. Other organic growth factors required by
some bacteria are amino acids, purines, and pyrimidines
Culture Media
 A nutrient material prepared for the growth of microorganisms in a laboratory is
called a culture medium. Some bacteria can grow well on just about any culture
medium; others require special media
 Microbes that are introduced into a culture medium to initiate growth are called an
inoculum. The microbes that grow and multiply in or on a culture medium are
referred to as a culture.
 When it is desirable to grow bacteria on a solid medium, a solidifying agent such as
agar is added to the medium. A complex polysaccharide derived from a marine alga,
agar has long been used as a thickener in foods. Also, agar liquefies at about 100°C
(the boiling point of water) and at sea level remains liquid until the temperature drops
to
about 40°C.
 Agar media are usually contained in test tubes or Petri dishes. The test tubes are
called slants when their contents are allowed to solidify with the tube held at an angle
so that a large surface area for growth is available. When the agar solidifies in a
vertical tube, it is called a deep.
 A chemically defined
medium is one whose
exact chemical
composition is known
 Organisms that require
many growth factors are
described as fastidious.
Organisms of this type,
such as Lactobacillus is
sometimes used in tests
that determine the
concentration of a
particular vitamin in a
substance.
 Complex media are made up of nutrients
including extracts from yeasts, meat, or
plants, or digests of proteins from these and
other sources. The exact chemical
composition varies slightly from batch to
batch.
 In complex media, the energy, carbon,
nitrogen, and sulfur requirements of the
growing microorganisms are provided
primarily by protein.
 Vitamins and other organic growth factors
are provided by meat extracts or yeast
extracts.
 If a complex medium is in liquid form, it is
called nutrient broth.
 When agar is added, it is called nutrient
agar.
Special culture techniques
 Many bacteria have never been successfully grown on artificial laboratory media.
 Mycobacterium leprae, the leprosy bacillus, is now usually grown in armadillos, which have a
relatively body temperature that matches the requirements of the microbe.
 Another example is the syphilis spirochete, although certain nonpathogenic strains of this
microbe have been grown on laboratory media.
 The obligate intracellular bacteria, such as the rickettsias and the chlamydias, do not grow on
artificial media. They can reproduce only
in a living host cell.
 Many clinical laboratories have special carbon dioxide incubators in which to grow aerobic
bacteria that require concentrations of CO2 higher or lower than that found in the atmosphere.
 High CO2 levels are also obtained with simple candle jars. Cultures are placed in a large
sealed jar containing a lighted candle, which consumes oxygen. Microbes that grow better at
high CO2 concentrations are called capnophiles. The low-oxygen, high-CO2 conditions
resemble those found in the intestinal tract, respiratory tract, and other body tissues where
pathogenic bacteria grow.
Selective and Differential Media - detect the presence of specific microorganisms
associated with disease or poor sanitation.
 Selective media are designed to suppress the growth of unwanted bacteria and
encourage the growth of the desired microbes. For example, bismuth sulfite agar is one
medium used to isolate the typhoid bacterium, the
gram-negative Salmonella Typhi, from feces.
 Bismuth sulfite inhibits gram-positive bacteria and most gram-negative intestinal
bacteria (other than Salmonella Typhi), as well. Sabouraud’s dextrose agar, which has a
pH of 5.6, is used to isolate fungi that outgrow most bacteria at this pH.
 Differential media make it easier to distinguish colonies of the desired organism from
other colonies growing on the same plate. Blood agar (which contains red blood cells)
is a medium that microbiologists often use to identify bacterial species that destroy red
blood cells. These species, such as Streptococcus pyogenes, the bacterium that causes
strep throat, show a clear ring around their colonies where they have lysed the
surrounding blood cells.
 Sometimes, selective and differential characteristics are combined in a single medium.
 Example: We want to isolate the common bacterium Staphylococcus aureus, found in
the nasal passages. This organism has a tolerance for high concentrations of sodium
chloride; it can also ferment the carbohydrate mannitol to form acid.
 Mannitol salt agar contains 7.5% sodium chloride, which will discourage the growth
of competing organisms and thus select for (favor the growth of) S. aureus. This salty
medium also contains a pH indicator that changes color if the mannitol in the medium
is fermented to acid; the mannitol-fermenting colonies of S. aureus are thus
differentiated from colonies of bacteria that do not ferment mannitol.
 Bacteria that grow at the high salt concentration and ferment mannitol to acid can be
readily identified by the color change
Enrichment Culture is used to encourage the growth of a
particular microorganism in a mixed culture.

 Because bacteria present in small numbers can be missed, especially

if other bacteria are present in much larger numbers, it is sometimes
necessary to use an enrichment culture.
 This is often the case for soil or fecal samples. The medium
(enrichment medium) for an enrichment culture is usually liquid and
provides nutrients and environmental conditions that favor the growth
of a particular microbe but not others. In this sense, it is also a
selective medium, but it is designed to increase very small numbers of
the desired type of organism to detectable levels.
Obtaining Pure Cultures
 If infectious materials are plated out onto the surface of a
solid medium, colonies will form that are exact copies of the
original organism.
 A visible colony arises from a single spore or vegetative cell
or from a group of the same microorganisms attached to one
another in clumps or chains.
 Microbial colonies often have a distinctive appearance that
distinguishes one microbe from another
 Most bacteriological work requires pure cultures, or clones
of bacteria.
 The isolation method most commonly used to get pure
cultures is the streak plate method . A sterile inoculating
loop is dipped into a mixed culture that contains more than
one type of microbe and is streaked in a pattern over the
surface of the nutrient medium
Preserving Bacterial Cultures
 Refrigeration can be used for the short-term storage of bacterial cultures.
 Two common methods of preserving microbial cultures for long periods
are deep-freezing and lyophilization.
 Deep-freezing is a process in which a pure culture of microbes is placed in
a suspending liquid and quick-frozen at temperatures ranging from -50°C
to -95°C. The culture can usually be thawed and cultured even several
years later.
 During lyophilization (freeze-drying), a suspension of microbes is
quickly frozen at temperatures ranging from -54°C to -72°C, and the water
is removed by a high vacuum (sublimation).
While under vacuum, the container is sealed by melting the glass with a
high-temperature torch. The remaining powder-like residue that contains
the surviving microbes can be stored for years. The organisms can be
revived at any time by hydration with a suitable liquid nutrient medium.
Bacterial Growth
 Bacterial growth refers to an increase in bacterial numbers, not an
increase in the size of the individual cells.
 Bacteria normally reproduce by binary fission
 A few bacterial species reproduce by budding; they form
a small initial outgrowth (a bud) that enlarges until its size
approaches that of the parent cell, and then it separates.
 Some filamentous bacteria (certain actinomycetes) reproduce by
producing chains of conidiospores (an asexual spore) carried
externally at the tips of the filaments.
 The time required for a cell to divide (and its population to double) is called
the generation time. It varies considerably among organisms and with
environmental conditions, such temperature. Most bacteria have a generation
time of 1 to 3 hours; others require more than 24 hours per generation.
 If a doubling occurred every 20 minutes—which is the case for E. coli under
favorable
conditions—after 20 generations a single initial cell would increase to over 1
million cells. This would require a little less than 7 hours.
 In 30 generations, or 10 hours, the population would be 1 billion, and in 24
hours it would be a number trailed by 21 zeros.
 Phases of bacterial growth
 Lag Phase
For a while, the number of cells changes very little because the cells do not
immediately reproduce in a new medium. This period of little or no cell division is
called the lag phase, and it can last for 1 hour or several days. During this time,
however, the cells are not dormant. The microbial population is undergoing a
period of intense metabolic activity involving, in particular, synthesis of enzymes
and various molecules.
 The Log Phase
The cells begin to divide and enter a period of growth, or logarithmic increase,
called the log phase, or exponential growth phase. Cellular reproduction is most
active during this
period, and generation time (intervals during which the population doubles)
reaches a constant minimum. Because the generation time is constant, a
logarithmic plot of growth during the log phase is a straight line. The log phase is
the time when cells are most active metabolically and is preferred for industrial
purposes.
 Stationary phase
 The growth rate slows, the number of microbial deaths balances the number of new
cells, and the population stabilizes. This period of equilibrium is called the
stationary phase. Exponential growth stops because the bacteria approach the
carrying capacity, the number of organisms that an environment can support.
Carrying capacity is controlled by available nutrients, accumulation of wastes, and
space. When a population exceeds the carrying capacity, it will run out of nutrients
and accumulation of wastes, and space. When a population exceeds the carrying
capacity, it will run out of nutrients and space

 Death phase
 The number of deaths eventually exceeds the number of new cells formed, and the
population enters the death phase, or logarithmic decline phase. This phase
continues until the population is diminished to a tiny fraction of the number of cells
in the previous phase or until the population dies out entirely
Direct measurement of microbial growth
 Growth of microbial populations can be measured in a number of ways.
Some methods measure cell numbers; other methods measure the
population’s total mass, which is often directly proportional to cell numbers.
Population numbers are
usually recorded as the number of cells in a milliliter of liquid or in a gram
of solid material. Because bacterial populations are usually very large, most
methods of counting them are based on direct or indirect counts of very
small samples; calculations then determine the size of the total population.
 Assume, for example, that a millionth of a milliliter (10-6 ml) of sour
milk is found to contain 70 bacterial cells. Then there must be 70 times 1
million, or 70 million, cells per milliliter
 It is not practical to measure out a millionth of a milliliter of liquid or a
millionth of a gram of food. Therefore, the procedure is done indirectly,
in a series of dilutions. For example, if we add 1 ml of milk to 99 ml of
water, each milliliter of this dilution now has one-hundredth as many
bacteria as each milliliter of the original sample had. By making a series
of such dilutions, we can readily estimate the number of bacteria in our
original sample.
 A heterotrophic plate count
reflects the number of viable
microbes and assumes that
each bacterium grows into a
single colony; plate counts
are reported as number of
colony-forming units (CFU).
 A plate count may be done by
either the pour plate method
or the spread plate method.
Pour Plates and Spread Plates A plate count
is done by either the pour plate method or the
spread plate method.
The pour plate method
Either 1 ml or 0.1 ml of dilutions of the
bacterial suspension is introduced into a Petri
dish. The nutrient medium, in which the agar is
kept liquid by holding it in a water bath at
about 50°C, is poured over the sample, which
is then mixed into the medium by gentle
agitation of the plate. When the agar solidifies,
the plate is incubated. With the pour plate
technique, colonies will grow within the
nutrient agar (from cells suspended in the
nutrient medium as the agar solidifies) as well
as on the surface of the agar plate
 Filtration
When the quantity of bacteria is very small, as in lakes or relatively pure
streams, bacteria can be counted by filtration methods. At least 100 ml
of water are passed through a thin membrane filter whose pores are too
small to allow bacteria to pass. Bacteria are filtered out and retained on
the surface of the filter. This filter is then transferred to a Petri dish
containing nutrient medium, where colonies arise from the bacteria on
the filter’s surface. This method is applied frequently to detection and
enumeration of coliform bacteria, which are indicators of fecal
contamination of food or water. The colonies formed by these bacteria
are distinctive when a differential nutrient medium is used.
 Most Probable Number (MPN) Method
Another method for determining the number of bacteria in a sample is the most probable
number (MPN) method.
 This technique is based on the fact that the greater the number of bacteria in a
sample, the more dilution is needed to reduce the density to the point at which no bacteria
are left to grow in the tubes in a dilution series. The MPN method is most useful when the
microbes being counted will not grow on solid media. It is also useful
when the growth of bacteria in a liquid differential medium is used to identify the microbes
(such as coliform bacteria, which selectively ferment lactose to acid, in water testing).
 The MPN is only a statement that there is a 95% chance that the bacterial
population falls within a certain range and that the MPN is statistically the most probable
number.
In filtration, bacteria are retained on the surface of a membrane
filter and then transferred to a culture medium to grow and
subsequently be counted.
12. The most probable number (MPN) method can be used for
microbes that will grow in a liquid medium; it is a statistical
estimation.
13. In a direct microscopic count, the microbes in a measured
volume
of a bacterial suspension are counted with the use of a specially
designed slid