Download Introduction to Microbiology

Introduction to Microbiology
 Microbiology is the study of microorganisms, a large and diverse group
of microscopic organisms which must be viewed with a microscope that
exist as single cells or cell clusters; it also includes viruses, which are
microscopic but not cellular .
Importance of microbiology
The importance of microbiology includes:
used in biomedical research, creation of medicines, environmental
applications and new research tools.
 Bacteria are important for fixing N2 in a usable form for plants.
 Bacteria and some fungi are important in decomposition and recycling
of materials.
 Industry applications of microbiology: waste management, food
industry, mining, medicine, research and biotechnology.
Anatomy of bacteria
The bacterial cell is a prokaryote cell which is simpler, and therefore
smaller, than a eukaryote cell, lacking a nucleus and most of the other
organelles of eukaryotes. Nuclear material of prokaryotic cell consist of a
single chromosome which is in direct contact with cytoplasm. Here the
undefined nuclear region in the cytoplasm is called nucleoid.
A
prokaryotic
cell
has
two
architectural
regions:
• On the outside, flagelig and pilli project from the cell’s surface. These
are structures (not present in all prokaryotes) made of proteins that
facilitate
movement
and
communication
between
cells;
• Enclosing the cell is the cell envelope — generally consisting of a cell
wall covering a plasma membrane though some bacteria also have a
further covering layer called a capsule. The envelope gives rigidity to the
cell and separates the interior of the cell from its environment, serving as
a protective filter. Though most prokaryotes have a cell wall, there are
exceptions such as Mycoplasma (bacteria) and Thermoplasma (archaea).
The cell wall consists of peptidoglycan in.bacteria, and acts as an
additional barrier against exterior forces. It also prevents the cell from
expanding and finally bursting (cytolysis) from osmotic pressure against
a hypotonic environment.
Extra cellular structures
*Capsule
The cell capsule is a very large structure this a gelatinous structure is
present in some bacteria outside the cell wall. It is considered a virulence
factor because it enhances the ability of bacteria to cause disease . The
capsule may be polysaccharide as in pneumococci , meningococci or
polypeptide as Bacillus anthracis or hyaluronic acid as in streptococci .
The capsule is antigenic. The capsule has antiphagocytic function so it
determines the virulence of many bacteria. It also plays a role in
attachment of the organism to mucous membranes
Demonstration of Capsule
1. India ink staining: the capsule appears as a clear halo around the
bacterium as the ink can't penetrate the capusule.
2. Serological methods: Capsular material is antigenic and can be
demonstrated by mixing it with a specific anticapsular serum. When
examined under the microscope, the capsule appears 'swollen' due to an
increase in its refractivity. This phenomenon is called as capsule swelling
reaction or Quellung phenomenon.
3. Special capsule staining: These techniques employ copper salts as
mordants for staining the capsule .
For Vaccination capsular material is effective against some organisms
(e.g., H. influenzae type b and S. pneumoniae).
*Flagella
Flagella are the organelles of cellular motility. They arise from
cytoplasm and extrude through the cell wall. They are long and thick
thread-like appendages, protein in nature called flagellin. Three types of
arrangement are known: monotrichous (single polar flagellum),
lophotrichous (multiple polar flagella), and peritrichous (flagella
distributed over the entire cell). staining with basic fuchsin makes the
flagella visible in the light microscope
*Fimbriae (pill)
They are short and thin hair like filaments, formed of protein called
pilin (antigenic). Fimbriae are responsible for attachment of bacteria to
specific receptors of human cell (adherence). There are special types of
pili called (sex pili) involved in conjunction.
*Cilia
Cilia are especially notable on the single-celled protozoans. They are
composed of extensions of the cell membrane that contain microtubules,
they move materials like these present in respiratory system that consists
of mucus-secreting cells lining the trachea and bronchi, and ciliated
epithelial cells that move the mucus ever-upward .
*Cell Wall
The bacterial cell wall is strength layer composed of a substance
variously referred to as murein, mucopeptide, or peptidoglycan (all are
synonyms). In addition to giving osmotic protection, the cell wall plays
an essential role in cell division as well as serving as a primer for its own
biosynthesis. Various layers of the wall are the sites of major antigenic
determinants of the cell surface, and one component—the
lipopolysaccharide of gram-negative cell walls—is responsible for the
nonspecific endotoxin activity of gram-negative bacteria.
The Peptidoglycan Layer
Peptidoglycan is a complex polymer consisting of three parts: a
backbone, composed of alternating N-acetylglucosamine and Nacetylmuramic acid; a set of identical tetrapeptide side chains attached to
N-acetylmuramic acid; and a set of identical peptide cross-bridges .
The tetrapeptide side chains of all species, however, have certain
important features in common. Most have L-alanine at position 1
(attached to N-acetylmuramic acid), D-glutamate or substituted Dglutamate at position 2, and D-alanine at position 4. Position 3 is the most
variable one: Most gram-negative bacteria have diaminopimelic acid at
this position, to which is linked the lipoprotein cell wall component.
Gram-positive bacteria usually have L-lysine at position 3; however,
some may have diaminopimelic acid or another amino acid at this
position.
Diaminopimelic acid is a unique element of bacterial cell walls. It is
never found in the cell walls of Archaea or eukaryotes. Diaminopimelic
acid is the immediate precursor of lysine in the bacterial biosynthesis of
that amino acid The fact that all peptidoglycan chains are cross-linked
means that each peptidoglycan layer is a single giant molecule. In grampositive bacteria, there are as many as 40 sheets of peptidoglycan,
comprising up to 50% of the cell wall material; in gram-negative bacteria,
there appears to be only one or two sheets, comprising 5–10% of the wall
material.
Special Components of Gram-Positive Cell Walls
Most gram-positive cell walls contain considerable amounts of 1teichoic acid containing glycerophosphate or ribitol phosphate
residues. These polyalcohols are connected by phosphodiester linkages
and usually have other sugars and D-alanine attached .There are two
types of teichoic acids: wall teichoic acid (WTA), covalently linked to
peptidoglycan, and membrane teichoic acid(MTA), covalently linked to
membrane glycolipid. Because the latter are intimately associated with
lipids, they have been called lipoteichoic acids (LTA) and 2teichuronic acid, which may account for up to 50% of the dry weight of
the wall and 10% of the dry weight of the total cell. In addition .The
teichuronic acids are similar polymers, but the repeat units include sugar
acids (such as N-acetylmannosuronic or D-glucosuronic acid) instead of
phosphoric acids. They are synthesized in place of teichoic acids when
phosphate is limiting. some gram-positive walls may contain
polysaccharide molecules.
Special Components of Gram-Negative Cell Walls
Outer Membrane
The outer membrane is chemically distinct from all other biological
membranes. It is a bilayered structure ; it has special channels, consisting
of protein molecules called porins, that permit the passive diffusion of
low-molecular-weight hydrophilic compounds like sugars, amino acids,
and certain ions.
Lipopolysaccharide (LPS)
The LPS of gram-negative cell walls consists of a complex glycolipid,
called lipid A, to which is attached a polysaccharide made up of a core
and a terminal series of repeat units .
Lipid A consists of phosphorylated glucosamine disaccharide units to
which are attached a number of long-chain fatty acids .Hydroxymyristic
acid, a 14 fatty acid, is always present and is unique to this lipid.
The polysaccharide cores similar in all gram-negative species that have
two characteristic sugars, ketodeoxyoctanoic acid (KDO) and a
heptose.
LPS, which is extremely toxic to animals, has been called the endotoxin
of gram-negative bacteria because it is firmly bound to the cell surface
and is released only when the cells are lysed. When LPS is split into lipid
A and polysaccharide, all of the toxicity is associated with the former.
The O antigen is highly immunogenic in a vertebrate animal.
Lipoprotein
Molecules of an unusual lipoprotein cross-link the outer membrane and
peptidoglycan layers .The lipoprotein contains 57 amino acids,
representing repeats of a 15-amino-acid sequence.
The Periplasmic Space
The space between the inner and outer membranes, called the
periplasmic space, contains the peptidoglycan layer and a gel-like
solution of proteins.
Cell Membranes
The boundary of the cell, sometimes called the plasma membrane,
separates internal metabolic events from the external environment and
controls the movement of materials into and out of the cell. This
membrane is very selective about what it allows to pass through; this
characteristic is referred to as “selective permeability.” For example, it
allows oxygen and nutrients to enter the cell while keeping toxins and
waste products out. The plasma membrane is a double phospholipid
membrane, or a lipid bilayer, with the nonpolar hydrophobic tails
pointing toward the inside of the membrane and the polar hydrophilic
heads forming the inner and outer surfaces of the
membrane.
Membrane transport
*Passive Transport Across the Cell Membrane
Passive transport describes the movement of substances down a
concentration gradient and does not require energy use the following .
• Bulk flow is the collective movement of substances in the same
direction in response to a force, such as pressure. Blood moving through
a vessel is an example of bulk flow.
• Simple diffusion is the net movement of substances from an area of
higher concentration to an area of lower concentration.
• Facilitated diffusion is the diffusion of solutes through channel
proteins in the plasma membrane. .
• Osmosis is the diffusion of water molecules across a selectively
permeable membrane.
• Dialysis is the diffusion of solutes across a selectively permeable
membrane.
*Active Transport Across the Cell Membrane
Active transport is the movement of solutes against a gradient and
requires the expenditure of energy, usually in the form of ATP. Active
transport is achieved through :
Protein Pumps
• Transport proteins in the plasma membrane transfer solutes such as
small ions (Na ,K), amino acids, and monosaccharides.
• The proteins involved with active transport are also known as ion
pumps.
* Protein pumps are catalyses in the splitting of ATP to ADP +
phosphate,
so
they
are
called
ATPase
enzyme.
* The sodium-potassium pump actively moves sodium out of the cell and
potassium into the cell. and are essential in transmission of nerve
impulses and in muscular contractions.
*Vesicular Transport
• Vesicles or other bodies in the cytoplasm move macromolecules or
large particles across the plasma membrane. Types of vesicular transport
include:
1. Exocytosis, which describes the process of vesicles fusing with the
plasma membrane and releasing their contents to the outside of the cell.
This process is common when a cell produces substances for export.
2. Endocytosis, which describes the capture of a substance outside the
cell when the plasma membrane merges to engulf it. The substance
subsequently enters the cytoplasm enclosed in a vesicle.
There are three kinds of endocytosis:
• Phagocytosis or cellular eating, occurs when the dissolved materials
enter the cell. The plasma membrane engulfs the solid material, forming a
phagocytic vesicle.
• Pinocytosis or cellular drinking occurs when the plasma membrane
folds inward to form a channel allowing dissolved substances to enter the
cell. When the channel is closed, the liquid is encircled within a pinocytic
vesicle.
• Receptor-mediated endocytosis occurs when specific molecules in the
fluid surrounding the cell bind to specialized receptors in the plasma
membrane. As in pinocytosis, the plasma membrane folds inward and the
formation of a vesicle follows.
Eubacteria Lacking Cell Walls
These are microorganisms that lack cell walls (commonly called
mycoplasmas and comprising the class Mollicutes) and do not synthesize
the precursors of peptidoglycan. They are enclosed by a unit membrane,
the plasma membrane They resemble the L forms that can be generated
from many species of bacteria (notably gram-positive eubacteria); unlike
L forms, however, mycoplasmas never revert to the walled state, and
there are no antigenic relationships between mycoplasmas and eubacterial
L forms.
Bacterial physiology
The biochemical reactions that together enable bacteria to live,
grow, and reproduce. Strictly speaking, metabolism describes the total
chemical reactions that take place in a cell, while physiology describes
the role of metabolic reactions in the life processes of a bacterium.
Cell Metabolism
Cell metabolism is the total energy released and consumed by a cell.
Metabolism describes all of the chemical reaction that are happening in
the cell. Some reactions, called anabolic reactions, create’ needed
products. Other reactions, called catabolic reactions, break down
products. Your body is, performing both anabolic. and catabolic reactions
at the same time and around the clock, twenty four hours a day, to keep
your body alive and functioning. Even while you ‘sleep, your cells are
busy metabolizing.
• Catabolism: The energy releasing process in which a chemical or food
is used (broken down) by degradation or decomposition, into smaller
pieces.
• Anabolism: Anabolism is just the opposite of catabolism. In this
portion of metabolism, the cell consumes energy to produce larger
molecules via smaller ones. ATP is the currency of the cell.
Physical and chemical growth determinate
Main Requirements
Many food-poisoning bacteria have to multiply to high numbers in
food before they are likely to cause illness. The four main requirements
for bacterial growth are nutrition, moisture, warmth and time.
Nutritional Requirements
1-Basic nutritional requirements for growth :
a-Carbon - building blocks of cell components
b-Nitrogen - production of proteins, nucleic acids
c-Hydrogen - occur in organic compounds
d-Oxygen - involved in the production of energy
e-Minerals, Trace Elements - required in small
amount.
2-Special metabolites ( growth factors )
a-Substances required for growth that the cell
cannot produce using the basic requirements already listed
( Ex. : vitamins, amino acids, carbohydrates, blood factors )
b- Organisms may be described as being fastidious
Two types organisms based on source of nutrients :
1-Autotrophs - utilize inorganic compounds
( C - CO2, carbonates; N - NH4, N2, NO3 )
2-Heterotrophs - utilize organic compounds
( C - CHO, lipids; N - proteins )
a- Saprophytes - nonliving organic material
b- Parasites - viable (living) organic material
Moisture
Most foods naturally contain sufficient moisture to provide bacteria
with the water they need in order to grow. Where moisture has been
deliberately removed (e.g. in dehydrated foods such as milk powder, soup
mixes, etc.), then bacteria will not grow whilst the food remains dry, but
once water is added then bacterial growth may occur once more.
Warmth / Temperature
Bacteria have varying requirements in terms of the range of
temperatures in which they will grow. Those which grow at low
temperatures (usually below 20°C) are called psychrophiles and at high
temperatures (above 45°C) are thermophiles. Most pathogens are known
as mesophiles. They will grow at temperatures between 5°C and 63°C,
commonly referred to as the growth or 'danger' zone and have an
optimum temperature for growth of about 37°C.
Time
In ideal conditions (i.e. in moist foods at 37°C) bacteria will grow and
multiply by dividing into two every 20 minutes.
Other Factors Affecting Growth
pH Level
The acidity or alkalinity of foods will affect bacterial growth. Most
bacteria like neutral conditions (pH value of 7) .
Concentration of H+, OH- ions
1 ----------------------------------- 7 ------------------------------14
Acid
Neutral
Basic
.
Divided into four groups according to pH range :
.
*Neutrophiles - 5 to 8
*Alkalinophiles - 8 to 12
*Optimum pH 7.0 - 7.2
*Acidophiles - 0 to 5
Oxygen
Pathogens vary in their oxygen requirements. Those which require oxygen
are called aerobes, e.g. Bacillus cereus. Those which do not need oxygen
are called anaerobes, e.g. Clostridium perfringens .Those which will
grow or survive with or without oxygen are known as facultative
anaerobes and include Salmonella species and Staphylococcus aureus.
Microaerophilic - require the presence of small amounts of oxygen (2% 10%) .
Osmotic pressure
a- Exerted by solutes in water
b- Increase o.p. outside cell - water leaves cell
( very high o.p. - dehydrates cell )
c- Decreased o.p. outside cell - water enters cell
( very low o.p. - lysis of cell )
d- Halophiles - require the presence of 3% NaCl
( extreme halophiles - 20 to 30% NaCl )
LIGHT ( RADIATION )
1-Very small group photosynthetic bacteria (cyanobacteria)
- require UV light
2- Nonphotosynthetic bacteria (eubacteria) - UV light is lethal
(causes mutations)
Competition
Where there are a number of different bacteria present in food, they
compete for the same nutrients. Pathogens are often not as competitive as
spoilage bacteria and unless present in high numbers, will usually die.
Bacterial growth
Bacterial growth is the division of one bacterium into two daughter cells
in a process called binary fission. Providing no mutational event occurs
the resulting daughter cells are genetically identical to the original cell.
Bacterial Reproduction
1- Occurs by binary fission (simple division) .
Process
*Slight enlargement in cell size due to :
1-Increase in metabolic activities
2-Production of energy and cell parts
3-DNA replicates (duplicated)
4-Cell wall and membrane grow inward separating DNA
5-Divides contents of cell and DNA molecules
6-Two daughter cells formed
GENERATION TIME
Time required for one cell to produce two new cells.
A-Varies with type organism and environmental conditions.
B-Average 15 - 20 min. (varies - 10 minutes to 24 hrs.)
MEASUREMENT OF BACTERIAL GROWTH
1-Optical density
2-Plate count
3-Direct microscopic count
Phases of growth
In autecological studies, bacterial growth in batch culture can be modeled
with four different phases: lag phase (A), exponential or log phase (B),
stationary phase (C), and death phase (D).
Growth is shown as L = log(numbers) where numbers is the number of
colony forming units per ml, versus T (time.)
1. During lag phase, bacteria adapt themselves to growth conditions. It is
the period where the individual bacteria are maturing and not yet able
to divide. During the lag phase of the bacterial growth cycle, synthesis
of enzymes and other molecules occurs, no increase in number of
cells.
2. Exponential phase (sometimes called the log phase or the logarithmic
phase) is a period characterized by cell doubling. The number of new
bacteria appearing per unit time is proportional to the present
population. The slope of this line is the specific growth rate of the
organism, which is a measure of the number of divisions per cell per
unit time.
3. The "stationary phase" is due to a growth-limiting factor; this is mostly
depletion of a nutrient, and/or the formation of inhibitory products such
as organic acids. Rate of growth influenced by environmental factors.
Stationary Phase - rate of reproduction = rate of death. Due to exhaustion
of nutrients, accumulation of wastes.
4- At death phase, bacteria run out of nutrients and die. rate of death 
rate of reproduction.Some species die quickly, others survive longer.
Sporulation
The sporulation process begins when nutritional conditions become
unfavorable, near depletion of the nitrogen or carbon source (or both)
Many environmental bacteria are able to produce stable dormant, or
resting, forms as a branch of their life cycle to enhance their survival
under adverse conditions. Such dormant forms are called endospores,
cysts, or heterocysts (primarily seen in cyanobacteria), depending on the
method of spore formation, which differs between groups of bacteria.
Sporulation involves the production of many new structures, enzymes,
and metabolites along with the disappearance of many vegetative cell
components. These changes represent a true process of differentiation: A
series of genes whose products determine the formation and final
composition of the spore are activated.
Germination
The germination process occurs in three stages: activation, initiation, and
outgrowth:
Activation
Most endospores cannot germinate immediately after they have formed.
But they can germinate after they have rested for several days or are first
activated, in a nutritionally rich medium, by one or another agent that
damages the spore coat. Among the agents that can overcome spore
dormancy are heat, abrasion, acidity, and compounds containing free
sulfhydryl groups.
Initiation
Once activated, a spore will initiate germination if the environmental
conditions are favorable. Different species have evolved receptors that
recognize different effectors as signaling a rich medium: Thus, initiation
is triggered by L-alanine in one species and by adenosine in another.
Outgrowth
Degradation of the cortex and outer layers results in the emergence of a
new vegetative cell consisting of the spore protoplast with its surrounding
wall. Outgrowth requires a supply of all nutrients essential for cell
growth.