General microbiology
CLASSIFICATION OF MICRO-ORGANISMS
·
Use of five point kingdom system developed
by Whittaker
·
Use of Bergy’s classification system
(Refer to general microbiology notes)
VIRUSES
Viruses are not classified by using the Binomial
naming system and do not belong to any of the five kingdoms.
Viruses are not
cellular and are dependent on host cells for their replication.
Viruses are classified in two different ways:
1) According to their structure - genetic (DNA or
RNA?) and physical (shape etc) – this scheme is favored by scientists doing
fundamental work.
2) According to the type of disease they cause, this
scheme is favored by medical workers who need to correlate given viruses with
given diseases.
General
Structure of Viruses
The basic structure of a virus is made up of a
genetic information molecule and a protein layer that protects that information
molecule.
The arrangement of the protein layer and the genetic
information comes in a variety of presentations. The core of the virus is made
up of nucleic acids, which then make up the genetic information in the form of
RNA or DNA.
The protein layer that surrounds and protects the
nucleic acids is called the capsid.
When a single virus is in its complete form and has
reached full infectivity outside of the cell, it is known as a virion.
A virus
structure can be one of the following: icosahedral, enveloped, complex or
helical.
When a virus infects a cell, and directs that cell
to make new copies of it. This is a very different process than cell
reproduction and is therefore NOT called reproduction - it is called virus
replication.
There are five stages in virus replication
1. Attachment- specific attachment of the virus to
the host cell, it attaches because it binds with specific molecules on the cell
wall surface of the bacterium, the same principle of specific recognition of a
binding or attachment site is found with viruses that infect animal and other
cells that do not have a cell wall, and in that case, it is molecules
projecting from the target cell membrane that act as recognition sites so that
the virus will attach to the cell.
2. Penetration - the virus (or in some cases - just
its genome) enters the cell. When a cell has a cell wall (bacteria, fungi,
plant cells, algal cells) the usual strategy is for insertion of just the
genome of the virus into the cell cytoplasm, cell walls are rigid and difficult
for entire virus particles to penetrate. In animal cells, the entire virus
enters.
3. Synthesis - viral genes are transcribed and then
direct the host cell membrane to make components of the virus. In many cases
the first step is the destruction of the host cell genome, followed by
transcription and translation of viral nucleic acid that results in generation
of virus components.
4. Maturation - the viral components assemble into
complete virus copies, this is a thermodynamic process, it is not directly
controlled, it is just that there is a thermodynamically favorable tendency for
the different virus particle components to associate in the correct way to form
a functioning virus particle.
5. Release - the new viruses are released, which may
or may not kill the host cell. In some
cases, as with cells that have cell walls, the virus has to direct lytic
processes that lyse or burst the cell by first damaging the cell walls. In
other cases, as with animal cells that lack a cell wall, there may be an
immediate release of virus particles that bursts and kills the host cell, or
the virus can
escape from the host cell by forming an envelope
derived from the host cell membrane, and this can be done in a way (essentially
exocytosis) that does not cause host cell destruction, at least not
immediately, so that many viruses leave the cell in this fashion.
The immediate viral infection, replication and
exiting of newly replicated viruses from cells, with the destruction of the
host cell is called the lytic cycle.
Differences
between Viruses and Bacteria
i. They
cannot be observed using a light microscope
ii. They
have no internal cellular structure
iii. They
contain either DNA or RNA, but not both*
iv. They
are incapable of replication unless occupying an appropriate living host cell
v. They
are incapable of metabolism
vi. Individuals
show no increase in size.
MICROBIAL GROWTH
GROWTH
CURVE
Lag phase:
§
When bacteria are inoculated into new fresh media, it does not divide
immediately. Bacteria take some time to adjust to the new environment. The time
period in which bacteria is metabolically active but do not divide is called as
lag phase.
§
Lag phase is characterized by the period during which there is no
increase in number of cell.
§
Size of bacteria increase continuously so the bacteria have largest size at
the end of lag phase.
§
In this phase, microorganism tries
to adopt in new environment. It is the phase of adjustment necessary for the
synthesis of enzymes and co-enzymes for physiological activities.
§
Time is required for adjustment in physical environment around each cell.
§
Duration of lag phase
varies according to conditions and species of bacteria.
§
If the culture organism is taken
from old culture, the duration will be longer but if the culture is fresh,
duration is short.
§
Similarly, if the culture media is
different from the previous culture then duration is long because bacteria
takes some more time to adjust in the new media.
§
At the end of lag phase, bacteria become fully prepared for cell division.
2. Log phase or exponential phase:
§
During this phase bacteria divides continuously at constant rate and the
number of bacteria increase exponentially.
§
In this phase all bacteria are in their rapid stage of cell division and
show balanced growth.
§
Due to rapid cell division, bacteria have smallest size in this phase.
§
Bacterial population is nearly uniform in terms of their metabolic
activities, chemical composition of cell and other physiological
characteristics.
§
Biochemical and physiological characteristics are commonly used for
identification of bacteria are manifested during log phase of growth.
§
Generation time of bacteria is usually determined during log phase.
However, it is not same for all bacteria in culture.
§
Generation time is shortest during log phase and is strongly dependent upon
growth factors present in the medium.
§
This phase lasts for several hours depending on the type of organism,
conditions of growth and density of organism.
3. Stationary phase:
§
The bacteria growth reaches a state during which there is no net increase
in bacterial population. This is called as stationary phase.
§
In this phase a constant bacterial population is maintained by balance
between cell division and cell death.
§
In some bacteria, complete cessation of cell division occurs hence there is
no net increase or decrease in number of bacteria.
§
Stationary phase is
induced by- increased bacterial cell density, depletion of nutrition in media
and accumulation of toxic secondary metabolic wastes.
§
Production of antibiotics
such as Penicillin, streptomycin etc. and enzymes by certain bacteria occur
during stationary phase of their growth.
§
In endospore forming bacteria, sporulation occurs as the bacteria enter
stationary phase.
4. Death phase or decline phase:
§
In this phase, number of bacteria decrease continuously and exponentially.
§
During this phase, total count of
bacteria may remain constant but the viable count decreases.
§
It is just inverse of log phase. But the death rate is slower than growth
rate.
§
Death phase is brought
about by various reasons, such as depletion of nutrition and accumulation of
toxic wastes.
§
Not all bacteria die at same rate, some die faster and some are more
resistant and remain viable for longer time. E.g. Spore forming bacteria.
FACTORS AFFECTING MICROBIAL GROWTH
The
ability of microorganisms (except viruses) to grow or multiply in a food is
determined by the food environment as well as the environment in which the food
is stored, designated as the intrinsic and extrinsic
environment of food, respectively.
Intrinsic factors of a food include nutrients, growth factors, and
inhibitors (or antimicrobials), water activity, pH, and oxidation–reduction
potential.
Nutrients and Growth
ü
Microbial growth is accomplished through the synthesis of cellular
components and energy.
ü
The necessary nutrients for this process are derived from the immediate
environment of a microbial cell and, if the cell is growing in a food, it
supplies the nutrients. These nutrients include carbohydrates, proteins,
lipids, minerals, and vitamins.
ü
Water is not considered a nutrient, but it is essential as a medium for the
biochemical reactions necessary for the synthesis of cell mass and energy.
ü
Microorganisms normally present in food vary greatly in nutrient
requirements, with bacteria requiring the most, followed by yeasts and molds.
ü
Microorganisms also differ greatly in their ability to utilize large and
complex carbohydrates (e.g., starch and cellulose), large proteins (e.g.,
casein in milk), and lipids.
ü
Microorganisms capable of using these molecules do so by producing specific
extracellular enzymes (or exoenzymes) and hydrolyzing the complex molecules to
simpler forms outside before transporting them inside the cell. Molds are the
most capable of doing this.
ü
However, this provides an opportunity for a species to grow in a mixed
population even when it is incapable of metabolizing the complex molecules.
ü
Microbial cells, following death and lysis, release intracellular enzymes
that can also catalyze breakdown of complex food nutrients to simpler forms,
which can then be utilized by other microorganisms.
Water
Activity and Growth
ü
Water activity (Aw)
is a measure of the availability of water for biological functions and relates
to water present in a food in free form. In a food system, total
water or moisture is present in free and bound forms.
ü
Bound water is the fraction used to hydrate hydrophilic molecules and to
dissolve solutes, and is not available for biological functions; thus, it does
not contribute to Aw. The Aw of food ranges from 0.1 to 0.99.
ü
The free water in a food is necessary for microbial growth.
ü
It is necessary to transport nutrients and remove waste materials, carry
out enzymatic reactions, synthesize cellular materials, and take part in other
biochemical reactions, such as hydrolysis of a polymer to monomers (proteins to
amino acids).
ü
Each microbial species (or group) has an optimum, maximum, and minimum Aw level for growth.
ü
In general, the minimum Aw values
for growth of microbial groups are as follows: most molds, 0.8, with xerophilic
molds as low as 0.6; most yeasts, 0.85, with osmophilic yeasts, 0.6 to 0.7;
most Gram-positive bacteria, 0.90; and Gram-negative bacteria, 0.93.
ü
Some exceptions are growth of Staphylococcus
aureus at 0.85 and halophilic bacteria at 0.75. The Aw need for spore-forming bacteria to
sporulate and the spores to germinate and the toxin-producing microorganisms to
produce toxins is generally higher than the minimum Aw for their growth.
ü
Also, the minimum Aw for growth
in an ideal condition is lower than that in a non-ideal condition.
ü
This information is used to control spoilage and pathogenic microorganisms
in food as well as enhance the growth of desirable types in food bioprocessing
(such as adding salt in processing of cured ham) and in laboratory detection of
microorganisms (adding salt to media to enumerate Sta. aureus).
pH and Growth
ü
pH indicates the hydrogen ion concentrations in a system and is expressed
as –log [H+], the negative logarithm of the hydrogen ion or proton
concentration.
ü
It ranges from 0 to 14, with 7.0 being neutral pH. [H+] concentrations can
differ in a system, depending on what acid is present.
ü
Some strong acids used in foods, such as HCl and phosphoric acid,
dissociate completely. Weak acids, such as acetic or lactic acids, remain in
equilibrium with the dissociated and undissociated forms:
ü
On the basis of pH, foods can be grouped as high-acid foods (pH below 4.6)
and low-acid foods (pH 4.6 and above).
ü
Most fruits, fruit juices, fermented foods (from fruits, vegetables, meat,
and milk), and salad dressings are high-acid (low-pH) foods, whereas most
vegetables, meat, fish, milk, and soups are low-acid (high-pH) foods.
ü
Tomato, however, is a high-acid vegetable (pH 4.1 to 4.4). The higher pH
limit of most low-acid foods remains below 7.0; only in a few foods, such as
clams (pH 7.1) and egg albumen (pH 8.5), does the pH exceed 7.0. Similarly, the
low pH limit of most high-acid foods remains above 3.0, except in some citrus
fruits (lemon, lime, grapefruit) and cranberry juice, in which the pH can be as
low as 2.2.
ü
The pH of a food has a profound effect on the growth and viability of
microbial cells. Each species has an optimum and a range of pH for growth.
ü
In general, molds and yeasts are able to grow at lower pH than do bacteria,
and Gram-negative bacteria are more sensitive to low pH than are Gram-positive
bacteria.
ü
The pH range of growth for molds is 1.5 to 9.0; for yeasts, 2.0 to 8.5; for
Gram-positive bacteria, 4.0 to 8.5; and for Gram-negative bacteria, 4.5 to 9.0. Individual species
differ greatly in lower pH limit for growth; for example, Pediococcus acidilactici can grow at pH 3.8 and Sta. aureus can grow at pH 4.5, but
normally Salmonella cannot.
ü
The lower pH limit of growth of a species can be a little higher if the pH
is adjusted with strong acid instead of a weak acid (due to its un-dissociated
molecules). Acid-resistant or tolerant strains can acquire resistance to lower
pH compared with the other strains of a species (e.g., acid-resistant Salmonella).
ü
When the pH in a food is reduced below the lower limit for growth of a
microbial species, the cells not only stop growing but also lose viability, the
rate of which depends on the extent of pH reduction.
Redox
Potential, Oxygen, and Growth
ü
The redox or oxidation–reduction (O–R) potential measures the potential
difference in a system generated by a coupled reaction in which one substance
is oxidized and a second substance is reduced simultaneously.
ü
The process involves the loss of electrons from a reduced substance (thus
it is oxidized) and the gain of electrons by an oxidized substance (thus it is
reduced). The electron donor, because it reduces an oxidized substance, is also
called a reducing agent. Similarly, the electron recipient is called an
oxidizing agent.
ü
In biological systems, the oxidation and reduction of substances are the
primary means of generating energy. If free oxygen is present in the system,
then it can act as an electron acceptor.
ü
The redox potential of a food is influenced by its chemical composition,
specific processing treatment given, and its storage condition (in relation to
air).
ü
Fresh foods of plant and animal origin are in a reduced state, because of
the presence of reducing substances such as ascorbic acid, reducing sugars, and
–SH group of proteins.
ü
Following stoppage of respiration of the cells in a food, oxygen diffuses
inside and changes the redox potential. Processing, such as heating, can
increase or decrease reducing compounds and alter the Eh.
ü
A food stored in air will have a higher Eh than when it is stored under
vacuum or in modified gas (such as CO2 or N2).
ü
Oxygen can be present in a food in the gaseous state (on the surface,
trapped inside) or in dissolved form.
ü
On the basis of their growth in the presence and absence of free oxygen,
microorganisms have been grouped as aerobes, anaerobes, facultative anaerobes, or
microaerophiles.
ü
Aerobes need free oxygen for
energy generation, as the free oxygen acts as the final electron acceptor
through aerobic respiration.
ü
Facultative anaerobes can generate energy if
free oxygen is available, or they can use bound oxygen in compounds such as NO3
or SO4 as final electron acceptors through anaerobic respiration. If oxygen is
not available, then other compounds are used to accept the electron (or
hydrogen) through (anaerobic) fermentation.
ü
Anaerobic and
facultative anaerobic microorganisms can only transfer electrons through fermentation. Many
anaerobes (obligate or strict anaerobes) cannot grow in the presence of even
small amounts of free oxygen as they lack the superoxide dismutase necessary to
scavenge the toxic oxygen free radicals.
ü
The Eh range at which different groups of microorganisms can grow are as
follows: aerobes, +500 to +300 mV; facultative anaerobes, +300 to +100 mV; and
anaerobes, +100 to –250 mV or lower. H
EXTRINSIC FACTORS
Extrinsic
factors important in microbial growth in a food include the environmental
conditions in which it is stored. These are temperature, relative humidity, and
gaseous environment. The relative humidity and gaseous condition of storage,
respectively, influence the Aw and Eh
of the food.
Temperature and Growth
ü
Microbial growth is accomplished through enzymatic reactions. It is well
known that within a certain range, with every 100C rise in
temperature, the catalytic rate of an enzyme doubles. Similarly, the enzymatic
reaction rate is reduced to half by decreasing the temperature by 100C.
This relationship changes beyond the growth range.
ü
Because temperature influences enzyme reactions, it has an important role
in microbial growth in food.
ü
Microorganisms important in foods are divided into three groups on the
basis of their temperature of growth, each group having an optimum temperature
and a temperature range of growth: (1) thermophiles
(grow at relatively high temperature), with optimum ca. 550C and
range 45 to 700C; (2) mesophiles (grow at ambient temperature), with
optimum at 350C and range 10 to 450C; and (3)
psychrophiles (grow at cold temperature), with optimum at 150C and
range –5 to 200C.
ü
Psychrotrophs are microorganisms that grow at refrigerated temperature (0
to 50C), irrespective of their optimum range of growth temperature.
They usually grow rapidly between 10 and 300C.
ü
Molds; yeasts; many Gramnegative bacteria from genera Pseudomonas, Achromobacter, Yersinia, Serratia, and Aeromonas; and Gram-positive bacteria
from genera Leuconostoc, Lactobacillus,
Bacillus, Clostridium, and Listeria are
included in this group.
ü
Microorganisms that survive pasteurization temperature are designated as
thermodurics. They include species from genera Micrococcus, Bacillus, Clostridium, Lactobacillus, Pediococcus, and
Enterococcus. Bacterial spores are
also included in this group. They have different growth temperatures and many
can grow at refrigerated temperature as well as thermophilic temperature.
ü
When the foods are exposed to temperatures beyond the maximum and minimum
temperatures of growth, microbial cells die rapidly at higher temperatures and
relatively slowly at lower temperatures.
RELATIVE HUMIDITY
ü
Relative humidity is a measure of water activity of the gas phase.
ü
High RH influence the water content of food and high water content promote
microbial growth.
ü
When food is stored in high relative humidity, dormant spores of bacteria
or fungi germinate.
ü
Once they are actively growing, they produce water as an end product of
respiration increasing water content and food spoilage.
ü
Therefore, dry conditions are considered better for food storage than moist
conditions.
GASES
ü
Presence or absence of gases affects type and number of microbial
populations.
ü
Carbon dioxide regulates cell growth of certain bacteria.
ü
If partial pressure of carbon dioxide increases over a critical level,
metabolic activity will be retarded.
ü
Delaying effect of Carbon dioxide increases with increase in
concentrations.
ü
Carbon dioxide is used in packaging of some food items in order to control
the growth of microbes.
CULTURE MEDIA AND CULTIVATION
OF MICROORGANISMS
The study of microorganisms
requires techniques for isolating cells from natural sources and
growing them in the laboratory
on synthetic media.
Media are used to
- isolate and identify micro-organisms,
- reveal their metabolic properties, and
- Allow long-term storage of pure cultures.
Following components are essential for the growth of microorganisms:
1. Carbon – Carbon is needed for the
skeleton of all organic molecules. Microorganisms acquire it either as inorganic carbon in the form of carbon
dioxide (autotrophs) or from organic nutrients (heterotrophs). Microorganisms,
in general, have extraordinary flexibility with respect to carbon sources.
There is no naturally occurring organic molecule that cannot be used by some
microorganisms. Some bacteria can use almost anything as carbon source while
others are fastidious and employ only few carbon compounds.
2. Nitrogen – Nitrogen is an
essential atom in certain cellular macromolecules, e.g. amino acids, purines,
pyrimidines, enzymes, cofactors etc. Some microbes use atmospheric nitrogen,
while others rely on inorganic nitrogen compounds as nitrate or ammonium salts.
Still some others can use only organic compounds like amino acids.
3. Sulphur – Sulphur is a part of some
amino acids, some carbohydrates, biotin and thiamine. It is obtained either as
elemental sulphur, inorganic sulphur e.g., as SO4 – or organic compounds like
amino acids.
4. Phosphorus – Phosphorus is used in the
form of phosphate salt by microorganisms. It is involved in formation of
nucleic acids, phospholipids, several cofactors and energy rich compound ATP
(adenosine triphosphate).
5. Potassium, Calcium, Iron and Magnesium – These are supplied by
inorganic salts and exist in the cell as cations. These perform various
functions in the cell like stabilizing ribosomes and cell membrane, needed for
enzyme activity, giving heat resistance to endospores, or a part of cytochromes
etc.
6. Vitamins – Vitamins are also essential
in small amounts for cellular activities and growth. These are also the
coenzymes for active enzyme systems. These usually make up all or part of the
enzyme cofactor. Some microbes can synthesize vitamins while others need it
from outside source.
7. Water – All cells require water for various cellular
activities.
Types of culture media
Media
can be classified according to three properties
- Physical state
a)
Liquid media: They are water-based solutions
generally termed as broths, milks, and infusions
b)
Solid media: They contain a high percentage
(1-5%) of agar, which enables the formation of discrete colonies. Advantages of
solid media: (a) Bacteria may be identified by studying the colony character;
(b) Mixed bacteria can be separated. Solid media is used for the isolation of
bacteria as pure culture (c) Organisms grow and stay in one play unlike in
broth medium where all the organisms spread out and it is therefore difficult
to see how many colonies there are
c)
Semi solid media: They contain a low percentage
(<1%) of agar, which can be used for motility testing
- Composition
a)
Synthetic media:
They contain pure organic and inorganic compounds that are chemically
defined (i.e. known molecular formula)
b)
Non-synthetic or complex media: complex or enriched media contain ingredients
that are not chemically defined or pure (i.e. animal extracts)
- Functional types
a)
Enriched media: They are used to grow fastidious
bacteria
b)
Selective media: Enables one type of bacteria to
grow. MSA is selective for Staphylococcus (has 7% NaCl)
c)
Differential
media: Allows bacteria to show different reactions (i.e colony color).
STAINING
a) Stains differentiate microorganisms from their surrounding environment
b) They allow detailed observation of microbial structures at high
magnification
c) Certain staining protocols can help to differentiate between different
types of micro-organisms
(d) Allows for determination of the viability of microbial cells.
Numerous staining techniques
are available for visualization, differentiation, and
separation of bacteria in terms of morphological characteristics and cellular
structures. Broadly, staining methods are simple staining and differential
staining.
• Simple staining is defined as the process of coloring bacteria or
other cells by applying a single solution of a stain to a fixed smear. It is
used for visualization of morphological shape (cocci, bacilli and spirilli) and
arrangement (chains, clusters, pairs and tetrads) of bacteria.
• Differential staining is the use of more than one staining reagent
to bring out differences in microbial cell types, or to differentiate
particular cellular components from the rest of the cell body.
COUNTING
The four general approaches used for estimating the
sizes of microbial populations are direct
and indirect counts of cells.
Direct
Count: Using a Counting Chamber, under the microscope
Indirect Count: Viable count,
Most Probable Number
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