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Chair of Microbiology, Virology, and Immunology
SIGNIFICANCE OF MICROBIOLOGY IN
PRACTICAL ACTIVITY OF DOCTORS. THE
HISTORY OF MICROBIOLOGY.
Lecture schedule
1. History of Microbiology.
2. Classification of bacteria.
3. Structure of bacterial cell
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.
Idea: Whitaker – five kingdoms (1969)
The Three-Domain System
Prokaryotes
Idea: Woese- domains
(1978)
Figure 10.1
Comparison of the three
domains
Characteristic Eubacteria
Archaea
Eucarya
Cell type
Prokaryote Prokaryote Eukaryote
Cell wall
Peptidoglycan
Varies
Varies
Membrane
lipids
Sensitive to
antibiotics?
Unbranched
Branched
Unbranched
Yes
No
No
Circular
chromosome?
Yes
Yes
No (except in
mitochondria and
chloroplasts)
Histones?
No
Yes
Yes
Prokaryotes
Classification Systems in the
Procaryotae
1. Microscopic morphology
2. Macroscopic morphology – colony
appearance
3. Physiological / biochemical characteristics
4. Chemical analysis
5. Serological analysis
6. Genetic and molecular analysis
•
•
•
G + C base composition
DNA analysis using genetic probes
Nucleic acid sequencing and rRNA analysis
Bacterial Taxonomy Based
on Bergey’s Manual
• Bergey’s Manual of Determinative
Bacteriology – five volume resource
covering all known procaryotes
– classification based on genetic information –
phylogenetic
– two domains: Archaea and Bacteria
– five major subgroups with 25 different phyla
Taxonomy
• Domain
• Kingdom
• Phylum
• Class
• Order
• Family
• Genus
• species
Major Taxonomic Groups of
Bacteria
• Vol 1A: Domain Archaea
– primitive, adapted to extreme habitats and
modes of nutrition
• Vol 1B: Domain Bacteria
• Vol 2-5:
– Phylum Proteobacteria – Gram-negative cell
walls
– Phylum Firmicutes – mainly Gram-positive
with low G + C content
– Phylum Actinobacteria – Gram-positive with
high G + C content
Microbial Phylogeny
• Phylogeny of domain Bacteria
– The 2nd edition of Bergey’s Manual of
Systematic Bacteriology divides domain
Bacteria into 23 phyla.
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Proteobacteria
– The largest group of gram-negative bacteria
– Extremely complex group, with over 400 genera and
1300 named species
– All major nutritional types are represented: phototrophy,
heterotrophy, and several types of chemolithotrophy
– Sometimes called the “purple bacteria,” although very
few are purple; the term refers to a hypothetical purple
photosynthetic bacterium from which the group is
believed to have evolved
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Proteobacteria (cont.)
– Divided into 5 classes: Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria,
Deltaproteobacteria, Epsilonproteobacteria
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Proteobacteria (cont.)
– Significant groups and genera include:
» The family Enterobacteriaceae, the “gram-negative
enteric bacteria,” which includes genera Escherichia,
Proteus, Enterobacter, Klebsiella, Salmonella,
Shigella, Serratia, and others
» The family Pseudomonadaceae, which includes
genus Pseudomonas and related genera
» Other medically important Proteobacteria include
genera Haemophilus, Vibrio, Camphylobacter,
Helicobacter, Rickessia, Brucella
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Firmicutes
– “Low G + C gram-positive” bacteria
– Divided into 3 classes
» Class I – Clostridia; includes genera Clostridium and
Desulfotomaculatum, and others
» Class II – Mollicutes; bacteria in this class cannot
make peptidoglycan and lack cell walls; includes
genera Mycoplasma, Ureaplasma, and others
» Class III – Bacilli; includes genera Bacillus,
Lactobacillus, Streptococcus, Lactococcus,
Geobacillus, Enterococcus, Listeria, Staphylococcus,
and others
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Actinobacteria
– “High G + C gram-positive” bacteria
– Includes genera Actinomyces, Streptomyces,
Corynebacterium, Micrococcus, Mycobacterium,
Propionibacterium
• Phylum Chlamidiae
– Small phylum containing the genus Chlamydia
Microbial Phylogeny
• Phylogeny of domain Bacteria (cont.)
• Phylum Spirochaetes
– The spirochaetes
– Characterized by flexible, helical cells with a modified
outer membrane (the outer sheath) and modified flagella
(axial filaments) located within the outer sheath
– Important pathogenic genera include Treponema,
Borrelia, and Leptospira
• Phylum Bacteroidetes
– Includes genera Bacteroides, Flavobacterium,
Flexibacter, and Cytophyga; Flexibacter and Cytophyga
are motile by means of “gliding motility”
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
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
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 population
of cells derived from a single 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
Or: A subgroup within a species with one or more
haracteristics that distinguish it from other subgroups
in the species
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.
Important Point: