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NUTRITION OF MICROORGANISMS
INTRODUCTION
Microbial cells are structurally complex and carryout numerous functions
...
Nutrients are substances used in biosynthesis and energy production and therefore are required for
microbial growth
...

These are called macro elements or macronutrients
...
The first six elements (C, O, H, N, S, and P) are components of carbohydrates, lipids, proteins, and
nucleic acids
...

For example:
➢ Potassium (K+) is required for activity by a number of enzymes, including some of those involved in
protein synthesis
...

➢ Magnesium (Mg2+) serves as a cofactor for many enzymes, complexes with ATP, and stabilizes
ribosomes and cell membranes
...

In addition to macro elements all microorganisms require several nutrients in small amounts
...
The micronutrients are —manganese, zinc, cobalt, molybdenum, nickel, and
copper
...
However, cells require such elements in small amounts that contaminants
in water, glassware, and regular media components often are adequate for growth
...
These are normally a part of enzymes and cofactors, and
they aid in the catalysis of reactions and maintenance of protein structure
...
coli aspartate carbamoyltransferase
...

➢ Molybdenum (Mo2+) is required for nitrogen fixation, and
➢ Cobalt (Co2+) is a component of vitamin B12
...
Diatoms need silicic acid (H4SiO4) to construct
their beautiful cell walls of silica [(SiO2)n]
...
Finally, it must be emphasized that microorganisms require a balanced mixture of nutrients
...


NUTRITIONAL CLASSES OF MICROORGANISMS
In addition to the need for carbon, hydrogen, and oxygen, all organisms require sources of energy and electrons
for growth to take place
...
1)
...


There are only two sources of energy available to organisms: (1) light energy, and (2) the energy derived from
oxidizing organic or inorganic molecules
...

Microorganisms also have only two sources for electrons
...
e
...


Despite the great metabolic diversity seen in microorganisms, most may be placed in one of four nutritional
classes based on their primary sources of carbon, energy, and electrons (table 5
...


The large majority of microorganisms thus far studied are either photolithotrophic autotrophs or
chemoorganotrophic heterotrophs
...
Eucaryotic algae and cyanobacteria employ water as the electron donor and
release oxygen
...

➢ Chemoorganotrophic heterotrophs (often called chemoheterotrophs, chemoorganoheterotrophs, or even
heterotrophs) use organic compounds as sources of energy, hydrogen, electrons, and carbon
...
It should be noted that essentially all pathogenic
microorganisms are chemoheterotrophs
...
Some
purple and green bacteria are photosynthetic and use organic matter as their electron donor and carbon source
...
Some of these bacteria also can grow as photoautotrophs with molecular hydrogen as an
electron donor
...

Carbon dioxide is the carbon source
...
Chemolithotrophs contribute greatly to the chemical transformations of elements
(e
...
, the conversion of ammonia to nitrate or sulfur to sulfate) that continually occur in the ecosystem
...

For example, many purple nonsulfur bacteria act as photoorganotrophic heterotrophs in the absence of oxygen
but oxidize organic molecules and function chemotrophically at normal oxygen levels
...

Another example is provided by bacteria such as Beggiatoa that rely on inorganic energy sources and organic
(or sometimes CO2) carbon sources
...
This sort of flexibility seems complex and
confusing, yet it gives its possessor a definite advantage if environmental conditions frequently change
...
Most saprobes, notably bacteria and fungi, have a rigid cell wall and cannot engulf large particles of
food
...
Obligate saprobes exist strictly on organic matter in soil and
water and are unable to adapt to the body of a live host
...
When a saprobe infects a host, it is considered a facultative parasite
...
For example,
although its natural habitat is soil and water, Pseudomonas aeruginosa frequently causes infections in hospitalized
patients
...

Parasitic Microorganisms Parasites live in or on the body of a host, which they usually harm to some degree
...
Parasites range from
viruses to helminth worms, and they can live on the body (ectoparasites), in the organs and tissues (endoparasites)
or even within cells (intracellular parasites, the most extreme type)
...
Obligate parasites (for example, the leprosy bacillus and the syphilis spirochete) are
unable to grow outside of a living host
...
Bacteria such as Streptococcus pyogenes (the cause of strep
throat) and Staphylococcus aureus can grow on artificial media as saprobes
...
Intracellular parasites obtain different substances
from the host cell, depending on the group
...
Rickettsias are primarily energy parasites, and the malaria protozoan is a hemoglobin
parasite
...
All cells require carbon and most
prokaryotes require organic compound as their source of carbon
...
Hydrogen and oxygen are also important elements in
organic molecules
...
These are also needed to reduce molecules during biosynthesis
...
Carbon sources normally
contribute both oxygen and hydrogen atoms also
...
They also serve as energy
sources
...
g
...
Thus carbon
sources frequently serve as energy sources
...
On dry weight basis, a typical cell is about 50% carbon and carbon is the major element in
all classes of macromolecules
...
One important carbon source that does not supply hydrogen or energy is carbon dioxide (CO2)
...
The energy needed for
this process is obtained from either light or inorganic chemicals
...
Thus many microorganisms cannot use CO2 as their sole carbon source but must rely on the presence of
more reduced, complex molecules such as glucose for a supply of carbon
...
They use reduced organic compounds as sources of both
carbon and energy
...

Among the common organic molecules that can satisfy this requirement are proteins, carbohydrates, lipids and
nucleic acids
...
Some are restricted to
a few substrates where as others are so versatile that they can metabolize more than 100 different substrates
(pseudomonas)
...
Parasitic members of the
genus Leptospira use only long-chain fatty acids as their major source of carbon and energy
...
It plays
an important role in the structural and enzymatic functions of the cell
...
A typical cell accounts 20% O2 on dry weight
basis
...
The oxygen is supplied in the form of H 2O, O2
and organic compounds
...

Hydrogen: Hydrogen is a major element in all organic compounds and several inorganic ones, including water
(H2O), salts (Ca(OH)2), and certain naturally occurring gases (H2S, CH4, and H2)
...
Hydrogen performs the following overlapping roles in the biochemistry of cells:
(1) Maintaining pH,
(2) Forming hydrogen bonds between molecules, and (3) serving as the source of free energy in oxidationreduction reactions of respiration
...

Nitrogen: This is the most abundant element in the cell after carbon
...
Nitrogen is needed for the synthesis of amino acids, purines, pyrimidines, some
carbohydrates and lipids, enzyme cofactors, and other substances
...
In nature nitrogen is available in both organic and inorganic forms
...
Most of the bacteria are capable of using NH3 as the sole N2
source and many can also use nitrate
...
Many
microorganisms can use the nitrogen in amino acids, and ammonia often is directly incorporated through the
action of such enzymes as glutamate dehydrogenase or glutamine synthetase and glutamate synthase
...

Ex: many Cyanobacteria and the symbiotic bacterium Rhizobium) can reduce and assimilate atmospheric nitrogen
using the nitrogenase system into NH4+
...

Phosphorus:
Phosphorus is present in nucleic acids, phospholipids, nucleotides like ATP, several cofactors, some proteins, and
other cell components
...
Almost all microorganisms use inorganic
phosphate as their phosphorus source and incorporate it directly
...
Ex: E
...
Some
organophosphates such as hexose 6-phosphates can be taken up directly by transport proteins of the cell
...

Sulfur: Sulfur is needed for the synthesis of substances like the amino acids cysteine and methionine, some
carbohydrates, biotin, and thiamine
...
Sulfur is available to organisms in a variety of forms and undergoes a
number of chemical transformations in nature, many of which are carried out exclusively by microorganisms
...
Cysteine determines shape and structural stability of proteins by forming unique
linkages called disulfide bonds
...
A variety of enzymes including some of those involved in
protein synthesis, specifically require potassium for activity
...

Magnesium: Magnesium is a component of chlorophyll
...

It is required for the activity of many enzymes
...
It helps for stabilization
of cell wall and plays a key role in the stability of endospores of bacteria
...

Sodium: Sodium is important for some types of cell transport
...
Under anoxic conditions, iron is generally in the +2 oxidation state (Fe2+) and
soluble
...
To obtain iron from minerals, cells produce iron binding agents called siderophores that bind iron and
transport it into the cell
...


Micronutrients:
They required in less amounts for cell functions
...
They play a role as components of various enzymes
...
If a culture medium contains highly purified
chemicals dissolved in high purity distilled water a trace element deficiency can occur
...


Zinc is an essential regulatory element for eucaryotic genetics
...
Copper, cobalt, nickel, molybdenum, manganese,
silicon, iodine, and boron are needed in small amounts by some microbes but not others
...
For example, the bacteria that cause gonorrhea and meningitis grow more rapidly in the presence
of iron ions
...
Also found in poly ketide
antibiotics
...

Iron (Fe)
Cytochromes, catalases, peroxidases, iron sulfur proteins, oxygenases and all
nitrogenases
Manganese (Mn)
Activator of many enzymes, present in certain superoxide dismutases and in the water
splitting enzyme photosystem II
Molybdenum (Mo) Certain flavin containing enzymes, some nitrogenases, nitrate reductases, sulfite
oxidases, formate dehydrogenases
Nickel (Ni)
Most dehydrogenases, Coenzyme F430 of methanogens, carbonmonoxide
dehydrogenase, urease
Selenium (Se)
formate dehydrogenases, some hydrogenases, aminoacid seleno cysteine
Tungsten (W)
Some formate dehydrogenases, oxotransferases of hyper thermophiles
...
These organisms have the enzymes and pathways necessary to synthesize all cell
components required for their wellbeing
...
Therefore they cannot manufacture all indispensable constituents but must obtain them or their
precursors from the environment
...

There are three major classes of growth factors:
➢ Amino acids,
➢ Purines and pyrimidines, and
➢ Vitamins
...
Vitamins are
small organic molecules that usually make up all or part of enzyme cofactors, and are needed in only very small
amounts to sustain growth
...
Some microorganisms require many vitamins
...
Other growth factors are also seen
...

➢ Mycoplasmas need cholesterol
...

The vitamins most commonly required by microorganisms are thiamine, biotin, pyridoxine and cobalamin
...

Knowledge of the specific growth factor requirements of many microorganisms makes possible quantitative
growth response assays for a variety of substances
...
These assays are based on the observation that the amount
of growth in a culture is related to the amount of growth factor present
...
If the growth factor concentration doubles, the amount of microbial
growth doubles
...
The appropriate bacterium is grown in a series of culture vessels, each
containing medium with an excess amount of all required components except the growth factor to be assayed
...
The standard curve is prepared by plotting the growth
factor quantity or concentration against the total extent of bacterial growth
...
The quantity of the growth factor in a test sample is determined by
comparing the extent of growth caused by the unknown sample with that resulting from the standards
...
They still are used in the assay of substances like
vitamin B12 and biotin, despite advances in chemical assay techniques
...
Several water-soluble and fat-soluble vitamins are produced partly or
completely using industrial fermentations
...


CULTIVATION OF MICROORGANISMS

CULTURE MEDIA
Much of the study of microbiology depends on the ability to grow and maintain microorganisms in the laboratory,
and this is possible only if suitable culture media are available
...
To be effective, the medium must contain all the nutrients the
microorganism requires for growth
...
Although all microorganisms
need sources of energy, carbon, nitrogen, phosphorus, sulfur, and various minerals, the precise composition of a
satisfactory medium will depend on the species one is trying to cultivate because nutritional requirements vary so
greatly
...
Frequently a medium is used to select
and grow specific microorganisms or to help identify a particular species
...

Types of media
Culture media can be classified on the basis of several parameters:
➢ Physical nature: Liquid, Semi solid and Solid
➢ Chemical composition: Defined or Synthetic media, Complex media
➢ Functional type: Supportive, Enriched, Selective, Differential
Physical nature:
Liquid media: Broth media are used as liquid media
...
Examples are Nutrient broth, trypticase soy broth, lactose broth
etc
...

Culture media some times prepared in a semisolid form by the addition of a gelling agent to liquid media, such
solid media immobilize cells, allowing them to grow and form visible, isolated masses called colonies
...
This is prepared with agar at concentrations of 0
...

Solid media: Solid media are prepared in the same way as for liquid media except that before sterilization, agar
is added as a gelling agent usually at a concentration of 1
...
The agar melts during the sterilization process and
the molten medium is then poured into sterile glass or plastic plates and allowed to solidify before use
...
Both defined
and complex media can be solidified with the addition of 1
...
0% agar, most commonly 1
...
Agar
is a sulfated polymer composed mainly of D-galactose, 3, 6-anhydro L-galactose and D-glucuronic acid
...
Agar is well suited as a solidifying agent for several reasons:
➢ It melts at about 90°C but once melted does not harden until it reaches about 45°C
...

➢ Agar is an excellent hardening agent because most microorganisms cannot degrade it
...

A medium in which all chemical components are known is a defined or synthetic medium
...

Media that contain some ingredients of unknown chemical composition are called as complex media
...
A defined medium that has just enough ingredients to support growth is called as minimal medium
...
E
...
It must contain only an organic
source of carbon (for example: glucose) and a few inorganic salts
...

They can be grown on relatively simple media containing CO 2 as a carbon source (often added as sodium
carbonate or bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate, and a variety of minerals
...
Not all defined media are as simple as the examples in table 5
...
Defined media are used widely in research, as it is often desirable to
know what the experimental microorganism is metabolizing
...
Such media are very
useful as a single complex medium
...
Complex media are needed because the nutritional requirements of a particular
microorganism are unknown, and thus a defined medium cannot be constructed
...
A liquid complex medium is called as broth
...

Complex media contain undefined components like peptones, meat extract, and yeast extract
...
They serve as sources of carbon, energy, and nitrogen
...
Beef extract contains amino acids, peptides, nucleotides, organic
acids, vitamins, and minerals
...

Partial hydrolysis breaks proteins into peptides
...
Partial
hydrolyzed casein is called as peptone
...
Complete hydrolyzed casein is called as casein hydrolysate
...
Three commonly used
complex media are (1) nutrient broth, (2) tryptic soy broth, and (3) Mac Conkey agar
...
0 to 2
...
5%
is used
...

Media such as tryptic soy broth and tryptic soy agar are called general purpose media because they support the
growth of many microorganisms
...
This media is used to isolate or
detect the favored species in a complex mixture of other microorganisms
...

Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting
the growth of gram-positive bacteria without affecting gram-negative organisms
...
coli and related bacteria in water
supplies and elsewhere
...
Mac Conkey
agar also contains bile salts
...
For example SPS agar contains sulfadiazine and polymyxin
sulfate
...
botulinum
...
Bacteria also may be selected by incubation with nutrients that they
specifically can use
...
The possibilities for selection are endless, and there are dozens of special
selective media in use
...
This media is used to
identify microorganisms by the appearance of their colonies
...
It distinguishes between hemolytic and non
hemolytic bacteria
...
g
...

Mac Conkey agar is both differential and selective
...


Selective and Differential media
Some media are both selective and differential
...

It is used to detect strains of salmonella and shigella
...

Mac conkey agar is differential because it contains neutral red (pH indicator) and lactose (milk sugar)
...
Neutral red dye that is yellow when neutral and pink or red when acidic
...
colonies of salmonella and shigella are
easily distinguished because their colonies are uncolored
...
These specially fortified media (e
...
, blood agar) are called enriched media
...
An enriched medium contains complex organic substances such as blood, serum, hemoglobin, or
special growth factors (specific vitamins, amino acids) that certain species must have in order to grow
...

➢ Pathogenic Neisseria (one species causes gonorrhea) are grown on Thayer-Martin medium or chocolate
agar, which is made by heating blood agar
...

➢ Nitrogen fixing bacteria can be isolated by culturing soil inoculums in a nitrogen freee medium
...

Environmental microbiologists use enrichment cultures to find a microorganism that can break down a particular
toxic chemical
...
Plating methods can be combined with the use of selective or differential media to enrich and isolate rare
microorganisms
...

Bacteria able to metabolize 2,4-D can be obtained with a liquid medium containing 2,4-D as its sole carbon source
and the required nitrogen, phosphorus, sulfur, and mineral components
...
After incubation, a sample of the original culture is transferred to
a fresh flask of selective medium for further enrichment of 2,4-D metabolizing bacteria
...
Pure cultures can be obtained by plating this
mixture on agar containing 2,4-D as the sole carbon source
...
This same general approach is used to isolate and purify a variety of bacteria by
selecting for specific physiological characteristics
...


SOLIDIFYING AGENTS
Liquefiable solid media, sometimes called reversible solid media, contain a solidifying agent that is
thermoplastic: Its physical properties change in response to temperature
...
Agar is a sulfated polymer

composed mainly of D-galactose, 3,6-anhydro-L- alactose, and D-glucuronic acid
...
It is solid at room temperature, and it melts (liquefies) at the boiling temperature of water (100°C)
...
Agar is flexible and moldable, and it
provides a basic frame work to hold moisture and nutrients, though it is not itself a digestible nutrient for most
microorganisms
...
Nutrient agar is
a common one
...
5% agar by weight
...

Other solidifying agents are sometimes employed
...


PURE CULTURE METHODS
Isolation of Pure Cultures:
In natural habitats microorganisms usually grow in complex, mixed populations containing several species
...
These microbes may be present in extremely large numbers
...
A pure culture is a
population of cells arising from a single cell, to characterize an individual species
...
Within about 20 years after the development of pure culture techniques most pathogens responsible
for the major human bacterial diseases had been isolated
...

➢ One single cell is isolated and cultivated to produce a clone
...
They are streak plate method, pour plate method and spread
plate method
...
To isolate a single cell the population must be diluted
...

Streak Plate Technique: The easiest and most commonly used method of diluting a microbial population is the
streak plate method
...
A population of microorganisms is picked up with a
selective wire inoculating loop
...
As the loop is streaked back and forth, fewer and fewer microorganisms are deposited on
the surface
...
This time the streaks overlap the first set of streaks so that some microbial cells
are dragged on to afresh, sterile region of the surface
...
Thus this is essentially a dilution process
...
Each colony is probably a clone derived from a single cell and represents a pure culture
...
The upper illustration shows a petri dish of agar being streaked with an
inoculating loop
...

The Pour Plate
In the pour plate and spread plate methods, dilutions are made before samples are put on the plate
...
Usually serial 10 fold or 100 fold dilutions are made
...
Then the mixture is shaken thoroughly
and the process is repeated
...

After six, it has been diluted a million fold (106)
...
Then small volumes of several diluted samples are mixed with liquid agar that has been
cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes
...
After the agar has hardened, each cell is fixed in place and
forms an individual colony
...
The total number of
colonies equals the number of viable microorganisms in the diluted sample
...


Figure: The Pour-Plate Technique
...
The most diluted samples are then mixed with warm agar and poured into petri dishes
...
The surface colonies are circular; subsurface
colonies would be lenticular or lens shaped
...
The practical solution is to
make serial dilutions
...
For 10 fold dilutions, 1 ml of cells is
added to 9ml of sterile culture medium or saline solution
...
In the spread plate method, the diluted sample is poured onto the surface of
an agar plate and spread evenly over the surface with a sterile bent-glass rod
...
The dispersed cells develop into isolated colonies
...


Figure: Spread-Plate Technique
...
(2) Dip a
glass spreader into a beaker of ethanol
...
(4)
Spread the sample evenly over the agar surface with the sterilized spreader and incubate
...
Anaerobic techniques make use of deaerated boiled nutrient media, free of air bubbles in sealed bottles,
O2 free gaseous atmosphere in desiccators or anaerobic jars, use of O2 absorbing substances (alkaline pyrogallol,
dithionite) and other means
...

O2 must be excluded by several ways, which includes:
(1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used
...

The reducing agents will eliminate any dissolved O2 remaining within the medium so that anaerobes can grow
beneath its surface
...
Often CO2 as well as nitrogen is added to the chamber since many anaerobes
require a small amount of CO2 for best growth
...
In this
procedure the environment is made anaerobic by using hydrogen and a palladium catalyst to remove O2 through
the formation of water
...


Figure: The Gas Pak Anaerobic System
...

The palladium catalyst in the chamber lid catalyzes the formation of water from hydrogen and oxygen, there by
removing oxygen from the sealed chamber
...
These have a catalyst and calcium carbonate to produce an anaerobic, carbon-dioxide rich

atmosphere
...

(5) Even very O2 sensitive bacteria can be transferred in the open atmosphere, provided that continuous stream of
O2 free nitrogen through the culture vessel prevents contact of the medium with air
...
As an indicator for anaerobic
conditions, resazurin can be added to media
...

(6) Also a beaker with alkaline glucose-methylene blue solution, which is decolorized under anaerobic conditions,
can be added to the incubation jar
...

A reducing agent, ex: Cysteine is added to further lower the O2 content
...
The medium is then dispensed into tubes which are being flushed with O2 free N2,
stoppered tightly, and sterilized by autoclaving
...

During inoculation, the tubes are continuously flushed with O2 free CO2 by means of canula, restoppered and
incubated
...
It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission
...
The daughter cells are identical except for the
occasional mutation
...

Microbial growth is usually studied as a population not an individual
...
If the microorganism is coenocytic—that is, a multinucleate organism in which nuclear
divisions are not accompanied by cell divisions— growth results in an increase in cell size but not cell number
...
Therefore, when studying growth, microbiologists normally follow changes in the total
population number
...
When microorganisms are
cultivated in liquid medium, they usually are grown in a batch culture or closed system— that is, they are
incubated in a closed culture vessel with a single batch of medium
...
The growth of microorganisms
reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus the incubation
time
...


Figure: Microbial Growth Curve in a Closed System
...
Although cell division does not take place right away
and there is no net increase in mass, the cell is synthesizing new components
...

Some of the factors influence the growth in lag phase those are:
➢ The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized
before growth can begin
...
Here new
enzymes would be needed to use different nutrients
...
Whatever the causes,
eventually the cells retool, replicate their DNA, begin to increase in mass, and finally divide
...
This phase may be quite long if the inoculum is from an old culture or one that has been
refrigerated
...

➢ On the other hand, when a young, vigorously growing exponential phase culture is transferred to fresh
medium of the same composition, the lag phase will be short or absent
...

The rate of growth is constant during the exponential phase i
...
the microorganisms are dividing and doubling in
number at regular intervals
...
The population is most uniform in terms of chemical and physiological
properties during this phase
...

Balanced and Unbalanced growth:
Exponential growth is a balanced growth
...
If nutrient levels or other environmental conditions are change, it results in unbalanced
growth
...
This response is readily observed in a shift-up experiment in which bacteria are
transferred from a nutritionally poor medium to a richer one
...
This is followed by increases in protein and DNA synthesis
...
Unbalanced growth also results when a bacterial population is
shifted down from a rich medium to a poor one
...
When shifted to a nutritionally inadequate medium, they need time to
make the enzymes required for the biosynthesis of unavailable nutrients
...
The cells become smaller and
reorganize themselves metabolically until they are able to grow again
...

These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated
control and responds quickly to changes in environmental conditions
...
This stationary phase usually is
attained by bacteria at a population level of around 109 cells per ml
...
Of course final population size depends on nutrient availability and other factors, as well as the type
of microorganism being cultured
...


This may result from a balance between cell division and cell death, or the population may simply cease to divide
though remaining metabolically active
...

➢ One obvious factor is nutrient limitation - if an essential nutrient is severely depleted, population growth
will slow
...
Oxygen is not very soluble and may be
depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth
...

➢ Population growth also may cease due to the accumulation of toxic waste products
...
For example, streptococci can produce so much lactic acid
and other organic acids from sugar fermentation that their medium becomes acidic and growth is inhibited
...

➢ Finally, growth also may cease when a critical population level is reached
...
Bacteria in a batch culture may enter
stationary phase in response to starvation
...

Death Phase
Detrimental environmental changes like nutrient deprivation and the buildup of toxic wastes lead to the decline
in the number of viable cells characteristic of the death phase
...
Death
is defined to be the irreversible loss of the ability to reproduce
...
This
is due to the extended survival of particularly resistant cells
...


MATHEMATICS OF GROWTH
Growth rate studies contribute to basic physiological and ecological research and the solution of applied
problems in industry
...

During the exponential phase each microorganism is dividing at constant intervals
...
This situation
can be illustrated with a simple example
...
1)
...
Because the population is doubling every generation, the increase in population is always 2 n where n is
the number of generations
...


These observations can be expressed as equations for the generation time
...
1 will show that
Nt =N0 × 2n
...
log 2 = log Nt – log N0
n = log Nt – log N0 / log 2
= log Nt – log N0 / 0
...

This is the number of generations per unit time, often expressed as the generations per hour
...
301t
The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g),
can now be calculated
...

Substitute 2N0 into the mean growth rate equation and solve for k
...
301g
= log2 + logN0 – log N0 / 0
...
301g
= 0
...
301g
k = 1/g
The mean generation time is the reciprocal of the mean growth rate constant
...
The generation time also may be calculated
directly from the previous equations
...

k = log Nt – log N0 / 0
...
301) (10hrs)
= 9 – 3 / 3
...
99hr
k = 2 generations per / hr
g = 1/k
= 1/ 2 generations per / hr
= 0
...
They range
from less than 10 minutes (0
...

Generation times in nature are usually much longer than in culture
...
Either
population mass or number may be followed because growth leads to increases in both
...


Measurement of Cell Numbers
The most obvious way to determine microbial numbers is through direct counting
...
It also gives information about the size and morphology of
microorganisms
...

➢ Petroff-Hausser counting chambers: Prokaryotes are more easily counted in these chambers if they are
stained, or when a phase-contrast or a fluorescence microscope is employed
...
The number of microorganisms
in a sample can be calculated by taking into account the chamber’s volume and any sample dilutions
required
...
The microbial population must be fairly large for
accuracy because such a small volume is sampled
...


Figure: The Petroff-Hausser Counting Chamber
...
(b) A top view of the chamber
...
(c) An enlarged view of the grid
...
The average
number of bacteria in these squares is used to calculate the concentration of cells in the original sample
...

The chamber is 0
...

The number of bacteria per cm3 is 103 times this value
...
5 × 107
...
The microbial suspension is forced through a small hole or
orifice
...
Every time a microbial cell passes through the orifice, electrical resistance increases
(or the conductivity drops) and the cell is counted
...
It is not as useful in counting
bacteria because of interference by small debris particles, the formation of filaments, and other problems
...


➢ Plating techniques:
There are also several viable counting techniques, procedures specific for cells able to grow and reproduce
...
Each microorganism or group of microorganisms develops into a distinct colony
...
For example, if 1
...
5 ×108 cells per ml
...
In
this way the spread-plate and pour-plate techniques may be used to find the number of microorganisms in a
sample
...
Several problems, however, can lead to inaccurate counts
...
These
results are often expressed in terms of colony forming units (CFU) rather than the number of microorganisms
...
Of course the counts will also be low
if the agar medium employed cannot support growth of all the viable microorganisms present
...

➢ Membrane filter Technique:
Microbial numbers are frequently determined from counts of colonies growing on special membrane filters
having pores small enough to trap bacteria
...
The filter is then placed on an agar medium or on a pad soaked with liquid media
and incubated until each cell forms a separate colony
...
This technique is
especially useful in analyzing aquatic samples
...
Membranes with different pore sizes are used to trap different
microorganisms
...

Membrane filters also are used to count bacteria directly
...
The bacteria then are stained
with a fluorescent dye such as acridine orange or DAPI and observed microscopically
...
Usually the
counts obtained with this approach are much higher than those from culture techniques because some of the
bacteria are dead
...
This makes it possible to directly count the number of live and dead microorganisms in a sample
...
Therefore techniques
for measuring changes in cell mass can be used in following growth
...

➢ Dry weight:
Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed
...
It is time consuming, however, and not
very sensitive
...


➢ Turbidity measurement:
More rapid, sensitive techniques depend on the fact that microbial cells scatter light striking them
...
When the concentration of bacteria
reaches about 10 million cells (107) per ml, the medium appears slightly cloudy or turbid
...
The extent of light
scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at
low absorbance levels
...
If the amount of a substance in each cell is constant,
the total quantity of that cell constituent is directly related to the total microbial cell mass
...
An increase in the microbial population will be reflected in higher total protein levels
...


Figure: Turbidity and Microbial Mass Measurement
...
As the population and turbidity increase, more light is scattered and the absorbance reading given
by the spectrophotometer increases
...
The bottom scale displays
absorbance and the top scale, percent transmittance
...


EFFECT OF ENVIRONMENTAL FACTORS ON GROWTH
Microorganisms must be able to respond to variations in nutrient levels, and particularly to nutrient limitation
...

An understanding of environmental influences aids in the control of microbial growth and the study of the
ecological distribution of microorganisms
...

Prokaryotes are found everywhere life can exist
...
5
miles below the Earth’s surface, without oxygen and at temperatures above 60°C
...

Major factors are solutes and water activity, pH, temperature, oxygen level, pressure, and radiation
...


Solutes and Water Activity
Because a selectively permeable plasma membrane separates microorganisms from their environment, they can
be affected by changes in the osmotic concentration of their surroundings
...
The osmotic concentration of
the cytoplasm can be reduced by use of inclusion bodies
...

Most bacteria, algae, and fungi have rigid cell walls that maintain the shape and integrity of the cell
...
This dehydrates the cell and may
damage the plasma membrane; the cell usually becomes metabolically inactive and ceases to grow
...

Compatible solutes are solutes that are compatible with metabolism and growth at high intracellular
concentrations
...
Elevated levels
of potassium ions are also involved to some extent
...
Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme
structure and function
...
Halobacterium’s enzymes have
been altered so that they actually require high salt concentrations for normal activity
...

Water activity:

The amount of water available to microorganisms can be reduced by interaction with solute molecules (the
osmotic effect) or by adsorption to the surfaces of solids (the matric effect)
...
Microbiologists generally use water activity (aw) for this purpose (water availability also may be
expressed as water potential, which is related to aw)
...
It is also equivalent to the ratio of the solution’s vapor pressure
(Psoln) to that of pure water (Pwater)
...
Suppose after a sample is treated in this way, the air above it
is 95% saturated—that is, the air contains 95% of the moisture it would have when equilibrated at the same
temperature with a sample of pure water
...
95
...

➢ Osmotolerant: Microorganisms differ greatly in their ability to adapt to habitats with low water activity
...
Some microorganisms can do this and are osmotolerant;
they will grow over wide ranges of water activity or osmotic concentration
...
It is well
adapted for growth on the skin
...
8 M and saturation (about 6
...
The archaeon Halobacterium can be isolated from the Dead Sea (a salt lake between Israel and
Jordan and the lowest lake in the world), the Great Salt Lake in Utah, and other aquatic habitats with salt
concentrations approaching saturation
...
These
extreme halophiles accumulate enormous quantities of potassium in order to remain hypertonic to their
environment; the internal potassium concentration may reach 4 to 7 M
...

pH= –log [H+] = log(1/[H+])
The pH scale extends from pH 0
...
0 M H+) to pH 14
...
0 ×10–14 M H+), and each pH unit represents a tenfold
change in hydrogen ion concentration
...
Each species has a definite pH
growth range and pH growth optimum
...
5
...
5 and 8
...
5 to 11
...

➢ Extreme alkalophiles have growth optima at pH 10 or higher
...

Most fungi prefer slightly acid surroundings, about pH 4 to 6; algae also seem to favor slight acidity
...

Drastic variations in cytoplasmic pH can harm microorganisms by disrupting the plasma membrane or inhibiting
the activity of enzymes and membrane transport proteins
...
0 to 5
...
Changes in the external pH also might alter the ionization of nutrient molecules and thus reduce their
availability to the organism
...

➢ The plasma membrane may be relatively impermeable to protons
...

➢ Internal buffering also may contribute to pH homeostasis
...
Phosphate is a commonly
used buffer and a good example of buffering effect
...

Temperature
Environmental temperature profoundly affects microorganisms, like all other organisms
...
For these reasons, microbial cell temperature directly reflects that of the cell’s
surroundings
...

➢ At low temperatures a temperature rise increases the growth rate because the velocity of an enzymecatalyzed reaction, like that of any chemical reaction, will roughly double for every 10°C rise in
temperature
...
Beyond a certain point further increases actually slow
growth, and sufficiently high temperatures are lethal
...

Microbial membranes are also disrupted by temperature extremes; the lipid bilayer simply melts and
disintegrates
...
At very low
temperatures, membranes solidify and enzymes don’t work rapidly
...


Figure: The effect of temperature on growth rate
...
The cardinal temperatures for a particular species are not rigidly fixed but
often depend to some extent on other environmental factors such as pH and the available nutrients
...


Figure: Temperature Ranges for Microbial Growth
...
They are readily isolated from Arctic and Antarctic habitats; because 90% of the ocean is 5°C or
colder
...

Psychrophiles are widespread among bacterial taxa and found in such genera as Pseudomonas, Vibrio,
Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium, and Shewanella
...

Psychrophilic microorganisms have adapted to their environment in several ways
...

➢ The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and
remain semi fluid when cold
...

Psychrotrophs: Many species can grow at 0 to 7°C even though they have optima between 20 and 30°C, and
maxima at about 35°C
...

Psychrotrophic bacteria and fungi are major factors in the spoilage of refrigerated foods
...
Their maximum is about 45°C or lower
...
Almost all human pathogens are mesophiles, as might be expected since their environment is a fairly
constant 37°C
...
Their
growth minimum is usually around 45°C and they often have optima between 55 and 65°C
...

➢ These organisms flourish in many habitats including composts, self-heating hay stacks, hot water lines,
and hot springs
...

➢ Their membrane lipids are also more saturated than those of mesophiles and have higher melting points;
therefore thermophile membranes remain intact at higher temperatures
...

Procaryotes that have growth optima between 80°C and about 113°C are called hyperthermophiles
...
Pyrococcus abyssi and Pyrodictium occultum are examples of marine
hyperthermophiles found in hot areas of the seafloor
...
An illustration of the growth of bacteria with varying responses to
oxygen
...
The surface, which
is directly exposed to atmospheric oxygen, will be aerobic
...

An organism able to grow in the presence of atmospheric O 2 is an aerobe, whereas one that can grow in its
absence is an anaerobe
...


Facultative anaerobes: They do not require O2 for growth but do grow better in its presence
...

Aero tolerant anaerobes: They simply ignore O2 and grow equally well whether it is present or not
...

Aero tolerant and strict anaerobes cannot generate energy through respiration and must employ fermentation or
anaerobic respiration pathways for this purpose
...

The nature of bacterial O2 responses can be readily determined by growing the bacteria in culture tubes filled with
a solid culture medium or a special medium like thioglycollate broth, which contains a reducing agent to lower
O2 levels
All five types are found among the procaryotes and protozoa
...
Algae are almost always obligate aerobes
...
Yet the deep sea (ocean of 1,000 m or more in depth) is
75% of the total ocean volume
...

Despite these extremes, bacteria survive and adapt
...
Some bacteria in the gut of deep-sea invertebrates
such as amphipods and holothurians are truly barophilic—they grow more rapidly at high pressures
...
One barophile has been recovered from
the Mariana trench near the Philippines (depth about 10,500 m) that is actually unable to grow at pressures below
about 400 to 500 atm when incubated at 2°C
...
g
...
Some members of the Archaea are thermobarophiles (e
...
,
Pyrococcus spp
...

Radiation
Our world is bombarded with electromagnetic radiation of various types
...
The distance between two wave
crests or troughs is the wavelength
...


Figure: The Electromagnetic Spectrum
...
Sunlight is the major source of radiation
on the Earth
...

Visible light is a most conspicuous and important aspect of our environment: all life is dependent on the ability
of
photosynthetic organisms to trap the light energy of the sun
...
Infrared is the major source of the Earth’s heat
...
This is particularly true of ionizing
radiation, radiation of very short wavelength or high energy, which can cause atoms to lose electrons or ionize
...
Low levels of ionizing radiation will produce mutations and may indirectly
result in death, whereas higher levels are directly lethal
...

Ionizing radiation can be used to sterilize items
...
g
...

A variety of changes in cells are due to ionizing radiation; it breaks hydrogen bonds, oxidizes double bonds,
destroys ring structures, and polymerizes some molecules
...
Although many types of constituents can be affected, it is
reasonable to suppose that destruction of DNA is the most important cause of death
...
The most lethal UV radiation has a wavelength of 260 nm,
the wavelength most effectively absorbed by DNA
...
Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and
function
...
Exposure to near-UV radiation induces tryptophan breakdown to
toxic photoproducts
...

Visible light is immensely beneficial because it is the source of energy for photosynthesis
...
Usually pigments called photosensitizers
and O2are required
...
The excited
photosensitizer (P) transfers its energy to O2 generating singlet oxygen (1O2)
...
It is probably the
major agent employed by phagocytes to destroy engulfed bacteria
...
Carotenoids effectively
quench singlet oxygen—that is, they absorb energy from singlet oxygen and convert it back into the unexcited
ground state
...


CONTINUOUS CULTURE OF MICROORGANISMS
In closed systems such as batch cultures the nutrient supplies are not renewed and wastes are not removed
...
However, it is
possible to grow microorganisms in an open system, a system with constant environmental conditions maintained
through continual provision of nutrients and removal of wastes
...

A microbial population can be maintained in the exponential growth phase and at a constant biomass
concentration for extended periods in a continuous culture system
...

Chemostat
A chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media
containing microorganisms is removed
...
The fresh medium contains a limiting amount of an
essential nutrient
...

The culture medium for a chemostat possesses an essential nutrient (e
...
, an amino acid) in limiting quantities
...
The
rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture
vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml)
...
30 / hr
Both the microbial population level and the generation time are related to the dilution rate
...
The generation time decreases (i
...
,
the growth rate rises) as the dilution rate increases
...

If the dilution rate rises too high, the microorganisms can actually be washed out of the culture vessel before
reproducing because the dilution rate is greater than the maximum growth rate
...
At very low dilution rates, an
increase in D causes a rise in both cell density and the growth rate
...
Only a limited supply of nutrient is
available at low dilution rates
...
As the dilution rate increases, the amount of nutrients and the resulting cell density rise because
energy is available for both maintenance and growth
...

Turbidostat
The second type of continuous culture system, the turbidostat, has a photocell that measures the absorbance or
turbidity of the culture in the growth vessel
...
The turbidostat differs from the chemostat in several ways
...
The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower
dilution rates
...
They make possible the study of microbial growth at very low nutrient levels,
concentrations close to those present in natural environments
...
Continuous systems also are used in food and industrial
microbiology

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