Chair of Microbiology, Virology, and Immunology
The role of microbiology in the dentist’s
medical practice. The history of the
microbiology development.
Classification and morphology of the
bacteria. Physiology of microorganisms.
Growth and reproduction of the bacteria.
Lecturer Prof. S.I. Klymnyuk
The role of microbiology in the dentist’s
medical practice.
The history of the microbiology
development.
Classification and morphology of the
bacteria.
Microbiology is a science, which study most
shallow living creatures - microorganisms.
Before inventing of microscope humanity was
in dark about their existence. But during the
centuries people could make use of processes
vital activity of microbes for its needs. They
could
prepare a koumiss, alcohol, wine,
vinegar, bread, and other products. During
many centuries the nature of fermentations
remained incomprehensible.
Brueghel: The Triumph of Death (1560)
Microbiology learns morphology, physiology,
genetics and microorganisms systematization,
their ecology and the other life forms.
Specific Classes of Microorganisms
 Algae
 Protozoa
 Fungi (yeasts and molds)
 Bacteria
 Rickettsiae
 Viruses
 Prions
The Microorganisms are extraordinarily
widely spread in nature. They literally
ubiquitous forward us from birth to our death.
Daily, hourly we eat up thousands and
thousands of microbes together with air, water,
food. On our skin, in mouth and nasal cavities,
on mucous membranes and in bowels enormous
amount of microorganisms live and act. Many
of them are found in earth cortex and in the air,
and in the ocean’s, sea’s, river’s water, on all of
latitudes, mainlands and continents.
For the first time term “microbe" was
offered by French scientist Sh. Sedillot in
1878. It derives from Greek “microbe", that
means briefly living, or most shallow living
creature.
Science,
which
learns
the
microorganisms, was named by E. Duclaux
microbiology. For short development period
this science accumulated great factual
material. The separate microbiological
branches such as bacteriology, mycology,
protistology, virology quickly appeared.
Comparative sizes of Bacteria
Periods of microbiology
development
• Morphologic
• Physiologic
• Prophylactic
Development
of
microbiological science was
interlinked with art of glass
and diamonds grinding. This
brought to creation of the
first microscope by Hans
and Zacharian Jansen in
Holland in 1590.
The
discovery
of
microorganisms is associated
with the name of Antony van
Leeuwenhoek (1632-1723).
In 1683 Leeuwenhoek described the basic bacterium
forms. His scientific supervisions Leeuwenhoek described in
special letters and sent off them to the London Royal
Scientific Society. He sent away about 300 letters. The
Leeuwenhoek’s letters brought on enormous surprise among
English scientists. They opened a fantastic world of invisible
creatures. He named them “living animals" (animalcula
viva) and in one of letter wrote: “In my mouth there are
more animacula viva, than peoples in all United Kingdom".
These wonderful discovery of Dutch
naturalist were the embryo, with which science of
bacteria developed. Namely from these times
starts the so-called morphological period in
microbiology history (XVII middle of age). It is
also called micrographycal period, as the study of
microorganism came only to description of their
dimensions and forms. Biological properties and
their significances for man still a long time
remained incomprehensible.
However,
using
the
primitive
microscopes of that time it was difficult to
determine the difference between separate
bacteria species. Even celebrated founder of
scientific systematization of all of living
organisms Karl Linney renounced to classify
the bacteria. He gave them general name
“chaos".
In the second half of XIX century
microbiology strongly affirms as independent
science. Namely these sciences were fruitful soil, on
which Pasteur's talent evinced.
He studied wine "illness“,
fermentation,
made
Pasteurization method, offered to
grow microbes on artificial
nutrient media, he proved, that
on definite cultivation conditions
the pathogenic bacteria lose its
virulence, made vaccine against
anthrax, rabies.
Physiological period has began
Not less important are
scientific works of celebrated
German scientist R. Koch.
He performed classic researches on
etiology
of anthrax,
opened
tuberculosis bacilli, cholera vibrio,
proposed to isolate pure bacterial
cultures on solid nutrient media
(gelatin, potatoes), developed the
preparations staining methods by
aniline dye-stuffs, method of
hanging drop for examination of
bacteria motility, offered apparatus
for sterilization
The Patriarch of world and Ukrainian
microbiology - I. Metchnikov
He studied inflammation pathology,
phagocytosis, bases about antagonism of
bacteria.
From all microbes-antagonists
I.Metchnikov preferred the lactic
bacteria. On their base he offered three
medical
preparations
sour clotted milk, yogurt and
lactobacillin.
Now they are called by
eubiotics.
Classic
Metchnikov's
researches defined a prophylactic
period in microbiology history.
In 1892 D. Ivanovskiy described an virus of mosaic
tobacco illness – new class of infectious agents
Microorganisms constitute a very antique
group of living organisms which appeared
on the Earth's surface almost 3000 million
years ago.
There are natural
classifications system.
and
artificial
Bergey's Manual of Determinative
Bacteriology - the "bible" of bacterial
taxonomy.
Classifying Bacteria
• Bergey’s Manual of Systematic
Bacteriology
– Classifies bacteria via evolutionary or genetic
relationships.
• Bergey’s Manual of Determinative
Bacteriology
– Classifies bacteria by cell wall composition,
morphology, biochemical tests, differential
staining, etc.
The Three-Domain System
Figure 10.1
Prokaryotes
Procaryotae Kingdom has 4 Divisions according to the
structure of cell wall and Gram staining:
Gracilicutes (gracilis - thin, cutis - skin) – Gram-negative
bacteria,
Firmicutes (firmus - firm) – Gram-positive bacteria,
Tenericutes (tener – soft, tender) – microbes without cell
wall,
Mendosicutes (mendosus - mistaket) – microbes with
atipical peptidoglican
35 of the major groups of bacteria are distinguished
primarily on morphological characteristics, namely: cell
shapes (rods, cocci, curved, or filament forming); spore
production; staining reactions; motility.
Other groups are defined based on their metabolism, on
combinations of
morphological and physiological
characteristics.
Some of the Major Groups of Bacteria in Bergey's Manual
Spirochetes
Very slender rods that are helically
coiled around a central axial
filament; includes the bacteria that
cause syphilis and Lyme disease
Gram-positive cocci
Bacteria that have a cell wall
structure that results in their staining
blue-purple by the Gram stain
procedure and that are spherical;
include
the
streptococci
and
staphylococci
Endospore-forming
rods and cocci
Bacteria that form heat-resistant
bodies called endospores within
their cells; include the bacteria that
cause gas gangrene, botulism,
tetanus, and anthrax
Species is population of microbes, which
have the only source of origin, common
genotype, and during the present stage of
evolution are characterized by similar
morphological, biochemical, physiological
and other signs
If deviations from the typical species properties
are found on examination of the isolated bacteria, then
culture is considered a subspecies.
Infrasubspecies subdivisions
serovar (antigenic properties)
morphovar (morphological properties)
chemovar (chemical properties)
biovar (biochemical or physiological properties)
pathovar (pathogenic properties)
phagovar (relation to phages)
The term clone was applied to designate a
group of individuals arising from one cell
Population is an elementary evolutional
unit (structure) of a definite species
The term strain designates a microbial
culture obtained from the different sources or from
one source but in different time
Taxonomic Grades
Rank
Example
Kingdom
Procaryotae
Division
Gracilicutis
Class
Scotobacteria
Order
Spirochaetales
Family
Leptospiraceae
Genus
Leptospira
Species
L. interrogans
Bacterial Nomenclature
• Binomial naming system
– Two word naming system
• First word is genus name
– Always capitalized
• Escherichia
• Second word is species name
– Not capitalized
• coli
• When writing full name genus usually abbreviated
– E. coli
• Full name always italicized
– Or underlined
Bacteria (Gk. bakterion - small staff) are
unicellular organisms lacking chlorophyll.
Morphological Classification of Bacteria
Morphologically, bacteria possess four main forms:
spherical (cocci)
rod-shaped (bacteria, bacilli, and clostridia)
 spiral-shaped (vibrios, spirilla and spirochaetes)
thread-shaped (non-pathogenic)
Cocci groupings
Coccus
Diplococcus
Streptococcus
Tetrad
Sarcinae
Staphylococcus
Cocci (Gk. kokkos berry). These forms of bacteria are
spherical, ellipsoidal, bean-shaped, and lanceolate. Cocci are
subdivided into six groups according to cell arrangement, cell
division and biological properties
Micrococci (Micrococcus). The
cells are arranged singly or
irregularly.
They
are
saprophytes, and live in water
and in air ( M. roseus, M.
luteus, etc.).
Diplococci (Gk. diplos
double) divide in one
plane and remain attached
in pairs. These include:
Meningococcus
(causative
agent
of
epidemic
cerebrospinal
meningitis,
and
gonococcus,
causative
agent of gonorrhoea and
blennorrhoea)
Pneumococcus (causative
agents of pneumonia)
Streptococci (Gk. streptos curved, kokkos berry) divide in
one plane and are arranged in chains of different length.
Some streptococci are pathogenic for humans and are
responsible for various diseases.
Tetracocci (Gk. tetra four) divide in two planes at right
angles to one another and form groups of fours. They
very rarely produce diseases in humans.
Staphylococci (Gk. staphyle cluster of grapes) divide in
several planes resulting in irregular bunches of cells,
sometimes resembling clusters of grapes. Some species
of Staphylococci cause diseases in man and animals
Sarcinae (L. sarcio
to tie) divide in three
planes at right angles
to one another and
resemble packets of
8, 16 or more cells.
They are frequently
found in the air.
Virulent species have
not been encountered
Rods. Rod-shaped forms are subdivided into:
 bacteria,
bacilli,
clostridia
Bacteria include those microorganisms which, as a rule, do
not produce spores (colibacillus, and organisms responsible
for enteric fever, paratyphoids, dysentery, diphtheria,
tuberculosis, etc.).
Bacilli and clostridia include organisms the majority of
which produce spores (hay bacillus, bacilli responsible for
anthrax, tetanus, anaerobic infections, etc.)
According to their arrangement, cylindrical forms can be
subdivided into three groups:
monobacteria
monobacilli
C. tetani
E. coli
Y. pestis
C. botulinum
diplobacteria
K. pneumoniae
diplobacilli
streptobacteria
streptobacilli
Haemophilus ducreyi
Bacillus anthracis
(chancroid)
(anthrax)
Spiral-shaped bacteria
Vibriones (L. vibrio to
vibrate) are cells which
resemble a comma in
appearance.
Typical
representatives of this
group
are
Vibrio
cholerae, the causative
agent of cholera, and
aquatic vibriones which
are widely distributed in
fresh water reservoirs.
Spirilla (L. spira coil) are coiled forms of bacteria
exhibiting twists with one or more turns. Only one
pathogenic species is known {Spirillum minus} which
is responsible for a disease in humans transmitted
through the bite of rats and other rodents (rat-bite fever,
sodoku)
Spirochaetes (L. spira curve, Gk. chaite cock, mane)
differ from bacteria in structure with a corkscrew
spiral shape
Borrelia. Their cells have
large, obtuse-angled, irregular
spirals, the number of which
varies from 3 to 10. Pathogenic
for man are the causative
agents of relapsing fever
transmitted by lice (Borrelia
hispanica), and by ticks
(Borrelia persica, etc.). These
stain blue-violet with the
Romanowsky-Giemsa stain
Leptospira (Gk. leplos thin, speira coil) are characterized by
very thin cell structure. The leptospirae form 12 to 18 coils
wound close to each other, shaping small primary spirals. The
organisms have two paired axial filaments attached at
opposite ends (basal bodies) of the cell and directed toward
each other.
Leptospira
interrogans
which is pathogenic for
animals and man cause
leptospirosis
Treponema (Gk. trepein turn, nema thread) exhibits thin,
flexible cells with 6-14 twists. The micro-organisms do not
appear to have a visible axial filament or an axial crest
when viewed under the microscope
A typical representative is the
causative agent of syphilis
Treponema pallidum
Properties of prokaryotes and eukaryotes
Prokaryotes
Eukaryotes
The nucleoid has no membrane
separating it from the cytoplasm
Karyoplasm is separated from the
cytoplasm by membrane
Chromosome is a one ball of double Chromosome is more than one,
twisted DNA threads. Mitosis is There is a mitosis
absent
DNA of cytoplasm are represented DNA of cytoplasm are represented
in plasmids
in organelles
There aren’t cytoplasmic organelle There are cytoplasmic organelle
which is surrounded by membrane
which is surrounded by membrane
The respiratory system is localized The respiratory system is localized
in cytoplasmic membrane
mitochondrion
There are
cytoplasm
ribosome
70S
in There are ribosome 80S
cytoplasm
in
Peptidoglycan are included in cell’s Peptidoglycan aren’t included in
wall (Murein)
cell’s wall
The structure of procaryotes
Nucleus. The prokaryotic nucleus can
be seen with the light microscope in
stained material. It is Feulgenpositive, indicating the presence of
DNA. Histonelike proteins have
recently been discovered in bacteria
and presumably play a role similar to
that of histones in eukaryotic
chromatin
The DNA is seen to be a single,
continuous,
"giant"
circular
molecule with a molecular weight of
approximately 3 X 109. The
unfolded nuclear DNA would be
about 1-3 mm long (compared with
an average length of 1 to 2 µm for
bacterial cells)
Plasmids
 small circular, double-stranded DNA
 free or integrated into the chromosome
 duplicated and passed on to offspring
 not essential to bacterial growth & metabolism
 may encode antibiotic resistance, tolerance to toxic metals,
enzymes & toxins
 used in genetic engineering- readily manipulated & transferred from
cell to cell
 There may be several different plasmids in one cell and the
numbers of each may vary from only one to 100s in a cell
Plasmids:
R, Col, Hly, Ent, Sal
Prokaryotic Ribosome
 A ribosome (70 S) is a
combination of RNA and
protein, and is the site for
protein synthesis
 Composed of large (50S)
and small (30S) subunits
 S = Svedverg unit,
measures molecular size
The 80S ribosomes of
eukaryotes are made up of
40S and 60S subunits.
Inclusions, granules
• Storage granules
– Metachromatic
granules
– Polysaccharide
granules
– Lipid inclusions
– Sulfur granules
– Carboxyzomes
– Magnetosomes
• Gas vesicles
Volutin granules
Corynebacterium diphtheriae
Neisser's staining
Loeffler's technique
Cell Envelope
Composted of
A. The cytoplasmic
membrane
To act as a physical
barrier btw cytoplasm and
environments and
selectively controls the
movement of substaces
into and out of the cell
“Semipermeable”
B. Cell wall
The rigid layer that
protect the fragile
cytoplasmic membrane
from rupturing
To maintains cell’s shape
C. Capsule or slime
layer (glycocalyx)
Cell membrane
Bacterial plasma membrane are
composed of 40 percent
phospholipid and 60 percent
protein.
The phospholipids are amphoteric
molecules with a polar hydrophilic
glycerol "head" attached via an
ester bond to two nonpolar
hydrophobic fatty acid tails, which
naturally form a bilayer in aqueous
environments. Dispersed within the
bilayer are various structural and
enzymatic proteins which carry out
most membrane functions.
Peripheral
Membrane
Protein
Phospholipid
Integral
Membrane
Protein
Peripheral
Membrane
Protein
Mesosome
The predominant functions of bacterial membranes are:
1. Osmotic or permeability barrier;
2. Location of transport systems for specific solutes (nutrients and ions);
3. Energy generating functions, involving respiratory and photosynthetic
electron transport systems, establishment of proton motive force, and
transmembranous, ATP-synthesizing ATPase;
4. Synthesis of membrane lipids (including lipopolysaccharide in Gramnegative cells);
5. Synthesis of murein (cell wall peptidoglycan);
6. Assembly and secretion of extracytoplasmic proteins;
7. Coordination of DNA replication and segregation with septum formation
and cell division;
8. Chemotaxis (both motility per se and sensing functions);
9. Location of specialized enzyme system.
Cell wall
• Unique chemical structure
– Distinguishes Gram positive from Gram-negative
– bacteria and archaea bacterial species
• Rigidity of cell wall is due to peptidoglycan
(PTG)
– Compound found only in bacteria
– Archaea –psudomurein or other sugars, proteins,
glycoproteins
• Many antimicrobial interfere with synthesis of
PTG
• Penicillin; Lysozyme
Structure of peptidoglycan
•
Basic structure of peptidoglycan
– Alternating series of two
subunits
• N-acetylglucosamin (NAG)
• N-acetylmuramic acid (NAM)
– Joined subunits form glycan
chain
• Glycan chains held together
by string of four amino acids
– Tetrapeptide chain:
L-ala-D-glu-DAP-D-ala
L-ala-D-glu-Lys-D-ala
•
Interpeptide bridge
Differences of cell wall structure in Grampositive and Gram negative cells
Structures associated with gram-positive and gram-negative cell walls.
L Forms
Glycocalyx
 Capsule
Protects bacteria from phagocytic cells
 Slime layer
Enable attachment and aggregation of
bacterial cells
Capsules
Most prokaryotes contain some sort of a
polysaccharide layer outside of the cell wall polymer
Only capsule of B. anthracis consist of polypeptide
(polyglutamic acid)
Capsule
The capsule is covalently
bound to the cell wall.
Associated with virulence in
bacteria.
Example:
Streptococcus pneumoniae
Slime Layer
The slime layer is
loosely bound to the
cell.
Carbohydrate rich
material enhances
adherence of cells on
surfaces
Example:
Streptococcus mutans
and “plaque formation”
Biofilms
The slime layer is
associated with cell
aggregation and the
formation of biofilms
Example:
Staphylococcus
epidermidis biofilms
on catheter tips
General capsule function
•Adhesion
•Avoidance of immune response
•Protection from dehydration
•Protection of bacterial cells from engulfment
by protozoa or white blood cells (phagocytes), or
from attack by antimicrobial agents of plant or
animal origin.
•They provide virulent properties of bacteria
(S. pneumoniae, B. anthracis)
Flagella
• 3 parts
– filament – long, thin,
helical structure
composed of proteins
– hook- curved sheath
– basal body – stack of
rings firmly anchored
in cell wall
• rotates 360o
• 1-2 or many
distributed over entire
cell
• functions in motility
Flagellar arrangements
1. Monotrichous – single flagellum at
one end (cholera vibrio, blue pus
bacillus),
2. Lophotrichous – small bunches
arising from one end of cell (bluegreen milk bacillus,
Alcaligenes faecalis)
3. Amphitrichous – flagella at both
ends of cell (Spirillum volutans),
4. Peritrichous – flagella dispersed
over surface of cell, slowest E.
coli, salmonellae of enteric fever
and paratyphoids A and B
Bacterial Motility
Flagella are
important for:
Motility
(dispersal)
Antigenic
determinant
Number and
location species
specific
The rotation of the flagella
enables bacteria to be motile.
Pili and Fimbriae
• Short, hair-like structures on the surfaces of procaryotic cells
• Proteinaceuse filaments (~20 nm in diameter)
• Very common in Gram-negative bacteria
• Functions:
– Adherence to surface/ substrates: teeth, tissues
– Involved in transfer of genetic information btw cells
– Have nothing to do with bacterial movement (Except the twitching
movement of Pseudomonas)
Fimbriae are smaller than flagella and are important for attachment
Bacterial endospores
• Bacterial spores are often called “endospore” (since they are
formed within the vegetative cell)
• Produced in response to nutrient limitation or extreme
environments
• Highly resistant
• Highly dehydrated (15% water)
• Metabolically inactive
• Stable for years
• Not reproductive
• Functions: to survive under an extreme growth conditions such
as high temperature, drought, etc.
Bacillus, Clostridium, Sporolactobacillus, Thermoactinomyces,
Sporosarcina, Desulfotomaculum species sporulate
Spore
Spores
• Key compositions:
– Dipicolinic acid (DPA)
– Calcium (Ca2+)
• Structure
–
–
–
–
–
–
Core / Cytoplasm
Plasma membrane
Core wall/ spore wall
Cortex
Spore coat
Exosporium
Endospores
The sporulation process begins when nutritional conditions become
unfavorable, depletion of the nitrogen or carbon source (or both) being the most
significant factor. Sporulation involves the production of many new structures,
enzymes, and metabolites along with the disappearance of many vegetative
cell components.
Spores are located:
1) Centrally (B. anthracis);
2) Terminally (С. tetani);
3) Subterminally (C. botulinum, C. perfringens)
The spores of certain bacilli are capable of
withstanding
boiling
and
high
concentrations of disinfectants. They are
killed in an autoclave exposed to saturated
steam, at a temperature of 115-125 C, and
also at a temperature of 150-170 C in a
Pasteur hot-air oven.
Physiology of
microorganisms. Growth
and reproduction of the
bacteria
Metabolism refers to all the
biochemical
reactions
that
occur in a cell or organism.
The
study
of
bacterial
metabolism focuses on the
chemical diversity of substrate
oxidations and dissimilation
reactions (reactions by which
substrate molecules are broken
down), which normally function
in bacteria to generate energy.
Chemical composition of
bacteria
Protein
Total RNA
DNA
Phospholipid
Lipopolysaccharide
Murein
Inorganic ions
55 %
20.5 %
3.1 %
9.1 %
3.4 %
2.5 %
1.0 %
Bacterial cell consists of:
Water – 70-90 %
Dry weight – 10-30 %
Proteins – 55 %, 2,35 million of molecules, 1850 different types of
molecules
RNA – 20,5 %, 250000 molecules, 660 different types of molecules
DNA – 3,1 %, 2 molecules
Lipids – 9 %, 22 million of molecules
Lipopolysaccharides –3,4 %, 1,5 million of molecules
Peptidoglycan – 1 molecule
Microbial metabolism
1. Catabolism (Dissimilation)
- Pathways that breakdown
organic substrates
(carbohydrates, lipids, &
proteins) to yield metabolic
energy
for growth and maintenance.
2. Anabolism (Assimilation)
- Assimilatory pathways for
the formation of key
intermediates and then to
end products (cellular
components).
4. Intermediary metabolism Integrate two processes
Catabolism
Substrate-level
phosphorylation
Fermentation
Glycolysis (EMP
pathway)
Aerobic
respiration
Pyruvate: universal intermediate
The bacterial cell is a highly specialized energy
transformer. Chemical energy generated by
substrate oxidations is conserved by formation
of high-energy compounds such as adenosine
diphosphate (ADP) and adenosine triphosphate
(ATP) or compounds containing the thioester
bond
O
║
(R –C ~ S – R), such as acetyl ~ Scoenzyme A
Another form of energy
transmembrane potential - ΔμН+
Chemiosmosis
• Production of ATP in
Electron Transport
• Electrochemical Gradient
Formed between
membranes
• H+ (Protons) generated
from NADH
• Electrical Force (+) & pH
Force (Acid)
• Gradient formed
• ATPase enzyme that
channels H+ from High to
Low concentration
– 3 ATP/NADH
– 2 ATP/NADH
Sources of metabolic energy
Substrate-level phosphorylation
Fermentation: metabolic
process in which the final
electron acceptor is an organic
compound.
Respiration: chemical
reduction of an electron
acceptor through a specific
series of electron carriers in
the membrane. The electron
acceptor is commonly O2,
but CO2, SO42-, and NO3are employed by some
microorganisms.
Photosynthesis: similar to
respiration except that the
reductant and oxidant are
created by light energy.
Respiration can provide
photosynthetic organisms
with energy in the absence
of light.
The Krebs cycle intermediate compounds
serve as precursor molecules (building
blocks) for the energy-requiring biosynthesis
of complex organic compounds in bacteria.
Degradation reactions that simultaneously
produce energy and generate precursor
molecules for the biosynthesis of new
cellular constituents are called amphibolic.
Energy Requirements
Oxidation of organic compounds - Chemotrophs
Sunlight - Phototrophs
Metabolic Requirements
Carbon source
- Autotrophs (lithotrophs): use CO2 as the C source
Photosynthetic autotrophs: use light energy
Chemolithotrophs: use inorganics
- Heterotrophs (organotrophs): use organic carbon (eg.
glucose) for growth.
- Clinical Labs classify bacteria by the carbon sources
(eg. Lactose) & the end products (eg. Ethanol,…).
Nitrogen source
Ammonium (NH4+) is used as the sole N source by most
microorganisms. Ammonium could be produced from N2 by
nitrogen fixation, or from reduction of nitrate (NO3-)and
nitrite (NO2).
Physiologic types of bacterial existence
Energy Source
Oxidation of organic
compounds - Chemotrophs
Sunlight - Phototrophs
Carbon Source
Organic - Heterotrophs
Inorganic - Autotrophs
Electrone donor
Оrganic -Organotrophs
Inorganic - Lithotrophs
Chemoorganoheterotrophic bacteria
Metabolic Requirements
Sulfur source
A component of several coenzymes and amino acids.
Most microorganisms can use sulfate (SO42-) as the S source.
Phosphorus source
- A component of ATP, nucleic acids, coenzymes, phospholipids,
teichoic acid, capsular polysaccharides; also is required for
signal transduction.
- Phosphate (PO43-) is usually used as the P source.
Mineral source
- Required for enzyme function.
- For most microorganisms, it is necessary to provide sources
of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-.
- Many other minerals (eg., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)
can be provided in tap water or as contaminants of other
medium ingredients.
- Uptake of Fe is facilitated by production of siderophores
(Iron-chelating compound, eg. Enterobactin).
Growth factors: organic compounds (e.g., amino acids, sugars,
nucleotides, vitamines) a cell must contain in order to grow but
which it is unable to synthesize. Purines and pyrimidines: required for
synthesis of nucleic acids (DNA and RNA);
Amino acids: required for the synthesis of proteins;
Vitamins: needed as coenzymes and functional groups of certain
enzymes.
Transport systems
The proteins that mediate the passage of solutes
through membranes are referred to as transport
systems, carrier proteins, porters, and
permeases. Transport systems operate by one of
three transport processes.
In a uniport process, a solute passes through the
membrane unidirectionally. In symport processes
(cotransport) two solutes must be transported in the
same direction at the same time; in antiport
processes (exchange diffusion), one solute is
transported in one direction simultaneously as a
second solute is transported in the opposite
direction.
Transport systems
Diffusion systems
• passive diffusion
• facilitated diffusion
• ion-driven transport
• binding protein dependent transport
• group translocation
• Membrane is selectively permeable
– Few molecules pass through freely
– Movement involves both active and passive
processes
Passive processes
– no energy (ATP) required
– Along gradient
– simple diffusion, facilitated diffusion,
osmosis
• Simple diffusion
• Facilitated diffusion
Can
reduce concentration gradient
but can’t create one
Osmosis
• Osmotic pressure
Active processes
• energy (ATP)
required
– Active transport
– Group
translocation
Facilitated diffusion
Active transport
Transport systems
TEMPERATURE
• One of the most important
factors
• optimal growth
temperature
– temperature range at which
the highest rate of
reproduction occurs
• optimal growth
temperature for human
pathogens ????
TEMPERATURE
• Microorganisms can be categorized
based on their optimal temperature
requirements
– Psychrophiles
• 0 - 20 ºC
– Mesophiles
• 20 - 40 ºC
– Thermophiles
• 40 - 90 ºC
• Most bacteria are mesophiles
especially pathogens that require 37
ºC
BACTERIAL TEMPERATURE
REQUIREMENTS
100
% Max
Growt
h
Psychrophile
50
Thermophile
Mesophile
0
0 C
0
37 C
0
Variable
90 C
0
Effects of Temperature on
Growth
Thermophiles
70o-110o
Mesophiles
10o-50o
For lecture only
BC Yang
TEMPERATURE
• Psychrophiles
– some will exist below 0 oC if liquid water is
available
• oceans
• refrigerators
• freezers
Pigmented bacteria in
Antarctic ice
TEMPERATURE
• Mesophiles
– most human flora
and pathogens
TEMPERATURE
• Thermophiles
– hot springs
– effluents from
laundromat
– deep ocean thermal
vents
Respiration in Bacteria
Obligate Aerobe
Microaerophile
Obligate Anaerobe
Facultative Anaerobe (Facultative
Aerobe)
Aerotolerant Anaerobe
Capneic bacteria
Categories of Oxygen
Requirement
Aerobe – utilizes oxygen and can detoxify it
 obligate aerobe - cannot grow without oxygen
(Mycobacterium tuberculosis, Micrococcus spp.,
Bacillus spp., Pseudomonas spp.
 facultative anaerobe – utilizes oxygen but can
also grow in its absence (Echericihia spp.,
Salmonella spp., Sta[phylococcus spp.)
 microaerophylic – requires only a small
amount of oxygen (Helycobacter spp.,
Lactobacillus spp.)
124
Categories of Oxygen
Requirement
Anaerobe – does not utilize oxygen
• obligate anaerobe - lacks the
enzymes to detoxify oxygen so cannot
survive in an oxygen environment
(Clostridium spp., Bacteroides spp.)
• aerotolerance anaerobes – do no
utilize oxygen but can survive and grow
in its presence (Streptococcus
pyogenes)
125
Carbon Dioxide Requirement
All microbes require some carbon dioxide in
their metabolism.
• capneic – grows best at higher CO2
tensions than normally present in the
atmosphere (Brucella abortus)
126
OXYGEN
Obligate
Aerobe
Facultative
Anaerobe
Obligate
Anaerobe
Four Toxic Forms of Oxygen
Toxic Oxygen Forms
Are formed:
Singlet oxygen
during photosynthesis as molecular oxygen
with electrons are boosted to higher energy
state
Superoxide radicals
during incomplete reduction of oxygen in
aerobic and anaerobic respiration
Peroxide anion
during reactions that neutralizes superoxide
radicals
Hydroxyl radical
from ionizing radiation and from incomplete
reduction of hydrogen peroxide
Four Toxic Forms of Oxygen
Toxic Oxygen Forms
Are neutralized by:
Singlet oxygen
carotenoids that remove the excess energy of
singlet oxygen
Superoxide radicals
superoxide dismutases, enzymes that detoxify
them
Peroxide anion
catalase or peroxidase, enzymes that detoxify
peroxide anion
Hydroxyl radical
catalase, peroxidase, and antioxidants such as
vitamins C and E that protect against toxic
oxygen products
Enzymes and Their Role in Metabolism
Enzymes, organic catalysts of a highly molecular
structure, are produced by the living cell. They are of a
protein nature, are strictly specific in action, and play an
important part in the metabolism of micro-organisms. Their
specificity is associated with active centres formed by a
group of amino acids.
Some enzymes are excreted by the cell into the
environment (exoenzymes) for breaking down
complex colloid nutrient materials while other
enzymes are contained inside the cell
(endoenzymes).
Bacterial enzymes are subdivided into some groups:
1. Hydrolases which catalyse the breakdown of the link between the
carbon and nitrogen atoms, between the oxygen and sulphur atoms,
binding one molecule of water (esterases. glucosidases, proteases.
amilases, nucleases, etc.).
2. Transferases perform catalysis by transferring certain radicals from
one molecule to another (transglucosidases, transacylases.
transaminases).
3. Oxidative enzymes (oxyreductases) which catalyse the oxidationreduction processes (oxidases, dehydrogenases, peroxidases,
catalases).
4. Isomerases and racemases play an important part in carbohydrate
metabolism. Rearrangement atoms of a molecule.
5. Lyases (remove chemical groups from molecules without adding
water).
6. Lygases (join two molecules together and usually require energy
from ATP).
Enzymes
Significance of the enzymes
With the help of amylase produced by mould fungi starch
is saccharified and this is employed in beer making,
industrial alcohol production and bread making.
Proteinases produced by microbes are used
for
removing the hair from hides, tanning hides, liquefying the
gelatinous layer from films during regeneration, and for
dry cleaning.
Fibrinolysin produced by streptococci dissolves the
thrombi in human blood vessels. Enzymes which
hydrolyse cellulose aid in an easier assimilation of rough
fodder.
Due to the application of microbial enzymes, the medical industry has
been able to obtain alkaloids, polysaccharides, and steroids
(hydrocortisone, prednisone, prednisolone. etc.).
Bacteria play an important role in the treatment of caouichouc, coffee,
cocoa, and tobacco.
Enzymes permit some species of microorganisms to assimilate
methane. butane, and other hydrocarbons, and to synthesize complex
organic compounds from them.
With the help of the enzymatic ability of yeasts in special-type industrial
installations protein-vitamin concentrates (PVC) can be obtained from
waste products of petroleum (paraffin’s).
Metabolism Results in
Reproduction
• Microbial growth – an increase in a
population of microbes rather than an
increase in size of an individual
• Result of microbial growth is discrete
colony – an aggregation of cells arising
from single parent cell
• Reproduction results in growth
BINARY FISSION
• division exactly in half
• most common means of bacterial reproduction
– forming two equal size progeny
– genetically identical offspring
– cells divide in a geometric progression
doubling cell number
BINARY
FISSION
Doubling time is the unit
of measurement of
microbial growth
CULTURE GROWTH
• Growth of culture goes
through four phases with
time
• 1) Lag phase
• 2) Log or Logarithmic
phase
• 3) Stationary phase
• 4) Death or Decline
phase
BACTERIAL GROWTH CURVE
LAG PHASE
• Organisms are adjusting to the
environment
Mouse click for lag
phase adjustment
– little or no division
• synthesizing DNA, ribosomes and
enzymes
– in order to
breakdown
nutrients, and to
be used for growth
LOGARITHMIC PHASE
• Division is at a constant rate
(generation time)
• Cells are most susceptible to inhibitors
STATIONARY PHASE
• Dying and dividing organisms are at an
equilibrium
• Death is due to reduced nutrients, pH changes,
toxic waste and reduced oxygen
• Cells are smaller and have fewer ribosomes
• In some cases cells do not die but they are not
multiplying
STATIONARY PHASE
DEATH PHASE
In bioreactors
in 37oC, pH 5.1 ; in 45oC, pH 6.2
For lecture only
BC Yang
ENUMERATION OF
BACTERIA
• 1) viable plate count
• 2) direct count
• 3) most probable number (MPN)
4
1
2
3
5
9
8
6
7
10
VIABLE PLATE COUNT
• Most common procedure for assessing
bacterial numbers
– 1) serial dilutions of a suspension of bacteria
are plated and incubated
VIABLE PLATE COUNT
– 2) the number of colonies developing are then
counted
• it is assumed that each colony arises from an
individual bacterial cell
VIABLE PLATE COUNT
3) by counting the
colonies and taking into
account the dilution
factors the concentration
of bacteria in original
sample can be
determined
4) only plates having
between 30 and 300
colonies are used in the
calculations
See next slide for bigger
diagram
VIABLE PLATE COUNT
VIABLE PLATE COUNT
– 5) multiply the number of colonies times the dilution
factor to find the number of bacteria in the sample
– Example
• Plate count = 54
• Dilution factor = 1:10,000 ml
• Calculation
– 54 X 10,000 = 540,000 bacteria/ml
VIABLE PLATE COUNT
• “TNTC”
– if the number of colonies is too great (over
300) the sample is labeled “TNTC”
– Too Numerous To Count
• limitation of viable plate count
– selective as to the bacterial types that will
grow given the incubation temperature and
nutrient type
VIABLE PLATE COUNT
Dilution factor of
1/1,000 (10 -3)
417 colonies
Click to
incubate
“TNTC”
VIABLE PLATE COUNT
Dilution factor of
1/1,000,000 (10 -6)
22 colonies
Click to
incubate
Too few the count
is less than 30
VIABLE PLATE COUNT
Dilution factor of
1/100,000 (10 -5)
Click to
incubate
42 colonies
Calculate the number
of bacteria per ml
VIABLE PLATE COUNT
• Calculate:
– 42 colonies
– dilution factor of 100,000
• 42 X 100,000 = ???
• 4,200,000 bacteria/ml
Nutrient media
• Ordinary (simple) media
• Special media (serum agar, serum broth, coagulated
serum, potatoes, blood agar, blood broth, etc.).
• Elective media
• Enriched media
• Differential diagnostic media: (1) proteolytic action;
• (2) fermentation of carbohydrates (Hiss media);
• (3) haemolytic activity (blood agar);
• (4) reductive activity of micro-organisms;
• (5) media containing substances assimilated only by
certain microbes.
Biochemical properties
Colonies
Colonies
Colonies
Pure Cultures Isolation
Isolated colonies obtaining
Important Point: