Transcript Clinical Microbiology

INTRODUCTION TO
MICROBIOLOGY
MICROBIOLOGY
• The study of organisms too small to be seen
without magnification
– Bacteria
– Viruses
– Fungi
– Protozoa
– Helminths (worms)
– Algae
– Some multicellular parasites
Pioneers of Microbiology
• Robert Hooke, UK (1665)
– Proposed the Cell Theory
– Observed cork with crude microscope
– All living things are composed of cells
• Spontaneous generation
– Some forms of life could arise spontaneously from
non-living matter
• Francesco Redi, IT (1668)
– Redi’s experiments first to dispprove S.G.
Pioneers of Microbiology
• Antoni van Leeuwenhoek, DE (1673)
– First observed live microorganisms (animalcules)
• Schleiden and Schwann, DE
– Formulated Cell Theory: cells are the fundamental
units of life and carry out all the basic functions of
living things
• Pasteur, FR and Tyndall, UK (1861)
– Finally disproved S.G.
Pioneers of Microbiology
• Louis Pasteur (1822-1895), Chemist
– Fermentation (1857)
– Pasteurization: heat liquid enough to kill spoilage
bacteria (1864)
– Vaccine development – rabies
– Proposed the germ theory of disease
– Proposed aseptic techniques (prevent
contamination by unwanted microbes)
– Director of Pasteur Institute, Paris (1894)
Pioneers of Microbiology
• Joseph Lister, UK (1867)
– Used phenol (carbolic acid) to disinfect wounds
– First aseptic technique in surgery
• Robert Koch, DE (1876)
– Postulates – Germ theory (1876)
– Identified microbes that caused anthrax (1876),
tuberculosis (1882) and cholera (1883)
– Developed microbiological media & streak plates for
pure culture (1881)
Koch’s Postulates
• The specific causative agent must be found in
every case of the disease.
• The disease organism must be isolated from the
lesions of the infected case and maintained in
pure culture.
• The pure culture, inoculated into a susceptible or
experimental animal, should produce the
symptoms of the disease.
• The same bacterium should be re-isolated in
pure culture from the intentionally infected
animal.
Branches of Microbiology
• Bacteriology: study of bacteria
• Mycology: study of fungi
• Parasitology: study of protozoa and parasitic worms
• Virology: study of viruses
– Beijerinck, NE: discovered intracellular reproduction of
TMV; coined the term “virus” (1899)
Branches of Microbiology
• Chemotherapy
– Treatment of disease by using chemical means
– Antibiotics produced naturally
– Synthetic drugs
– Paul Ehrlich (1878) – used arsenic compounds to
fight disease – ‘magic bullet’
• Immunology: study of immunity
– Edward Jenner, UK: developed vaccination (1798)
– Metchnikoff, RU: discovered phagocytes (1884)
– Paul Ehrlich, DE: theory of immunity (1890)
Branches of Microbiology
• Chemotherapy
– Alexander Fleming, Scotland (1928)
discovered penicillin
– Selman Waksman, Ukraine (1944)
discovered streptomycin
• Problems
– Toxicity of drugs => Selective toxicity
– Resistance of bacteria to drugs
Branches of Microbiology
• Recombinant DNA Technology
–Recombinant DNA
–Genetic engineering/biotechnology
–Microbial genetics – mechanism by
which microbes inherit genes
–Molecular biology – structure and
function (expression) of genes
–Molecular epidemiology/diagnostics
MICROBES ARE INVOLVED IN
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Nutrient production & energy flow
Decomposition (bioremediation)
Production of foods
Production of drugs & vaccines
Genetic engineering
Causing disease
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MICROORGANISM CLASSIFICATION
• Microorganisms and all other living organisms
are classified as prokaryotes or eukaryotes.
• Prokaryotes are probably the smallest living
organisms, ranging in size from 0.15 um
(mycoplasmas) to about 2.0 um (many of the
bacteria).
• Viruses and subparticles such as prions are
considered neither prokaryotes nor eukaryotes
because they lack the characteristics of living
things, except the ability to replicate.
Prokaryotes
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Cell Wall
Teichoic Acids
LPS
Endospores
Circular DNA
Plasmids
Classification Schemes
• Traditionally these have been inferred on the basis of
morphologic or biochemical characteristics.
• Schemes have recently been revised based on the
degree of genetic (DNA, RNA) similarity between
different species.
• Genus and species are of primary importance in
designating a microorganism.
• The correct format for naming an organism is genus
(capitalized, italicized, or underlined), species
(lowercase, italicized, or underlined): Escherichia coli
(abbreviation, E. coli).
Size of Bacteria
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Average bacterial cell diameter is 0.5 - 2.0 um.
Surface Area ~12 square um
Volume is ~4 cubic um
Surface Area to Volume is 3:1
Typical Eukaryote Cell SA/ Vol. is 0.3:1
Food enters through SA, quickly reaches all
parts of bacteria
• Eukaryotes need structures & organelles
Shapes of Bacteria
• Spherical (Cocci)
– Chain = Streptococcus
– Cluster = Staphylococcus
• Rod Shape (Bacilli)
– Chain = Streptobacillus
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Coccobacilli
Comma shape (Vibrios)
Spirillum
Spirochete
Square
Star
Bacterial Structures
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Flagella
Pili
Capsule
Cell Wall
- Lipopolysaccharides
- Teichoic Acids
Plasma Membrane
Cytoplasm
- Genetic materials
- Ribosomes
Inclusions
Spores
Extracellular Polymeric Substance (EPS)
• Polysaccharide on external surface: Capsule,
Glycocalyx, or Slime (Antigen)
• EPS does not take ordinary stains, is not necessary
for survival of the cell, and may be lost upon
continuous cultivation
• Adherence of bacteria to surfaces (S. mutans and
enamel of teeth)
• Prevention of Phagocytosis (Complement cannot
penetrate sugars)
The Cell Wall
• The cell wall of bacteria is a complex, semi-rigid
structure that is made up of peptidoglycan
(mucopeptide or murein), responsible for the shape of
the cell.
• It differs between gram positive and gram negative
bacteria
• In most gram-positive bacteria the cell wall consists of
many layers of peptidoglycan forming a thick rigid
structure.
• By contrast, gram-negative cell walls contain only one
(or very few) layers of peptidoglycan.
Cell Wall
• Peptidoglycan Polymer (amino acids + sugars)
• Unique to bacteria
• Sugars
- N- acetylglucosamine (NAG)
- N- acetylmuramic acid (NAM)
• D form of Amino acids used not L form
– Hard to break down D form
• Amino acids cross link NAG & NAM
Cell Wall
• Gram positive Bacteria
- Peptidoglycan
- Teichoic ( ribitol or glycerol residues) and Teichuronic
(Sugar acids) acids; wall and membrane Teichoic
acids supply cell with magnesium.
- Polysaccharide
• Gram negative Bacteria
- Peptidoglycan
- Lipoprotein
- Outer membrane
- Periplasmic space
The Outer Membrane
• Gram-negative cells possess an outer
membrane that is composed of lipoproteins,
lipopolysaccharides, and phospholipids.
• The outer membrane helps some organisms
evade phagocytosis, provides a barrier to
certain antibiotics, and confers properties of
virulence (endotoxin).
Lipopolysaccharide (LPS)
• Endotoxin or Pyrogen
– Fever causing
– Toxin nomenclature
• Endo - part of bacteria
• Exo - excreted into environment
• Structure
– Lipid A
– Polysaccharide
• O Antigen
• Gram negative bacteria only
– Removed by Alcohol/Acetone
LPS (cont’d.)
• Appearance of Colonies
– Mucoid = Smooth (lots of LPS or capsule)
– Dry = Rough (little LPS or capsule)
• O Antigen of Salmonella and E. coli
– 2,000 different O Ags of Salmonella
– 100’s different O Ags of E. coli
• E. coli O157
• O Ags differ in Sugars, not Lipid A
LPS (cont’d)
• Functions
– Toxic; kills mice, pigs, humans
• G - ve septicemia; death due to LPS
– Pyrogen; causes fever
• DPT vaccination always causes fevers
– Adjuvant; stimulates immunity
• Heat Resistant; hard to remove
• Detection (all topical & IV products)
– Rabbits (measure fever)
– Horse shoe crab (Amoebocytes Lyse in presence of
LPS)
Cell Wall Summary
• Unique to bacteria
• 20-40% of bacterial cell weight
• Determines shape of bacteria
• Prevents osmotic rupture
• Target for some antibiotics (Penicillin)
Cell Membrane
• The plasma membrane encloses the cytoplasm of the
cell and provides selective permeability for nutrients to
enter.
• Phospholipid Bilayer
• Water can penetrate
• Flexible
• Not strong, ruptures easily
– Osmotic Pressure created by cytoplasm
Cytoplasmic Structures
• 80% Water {20% Salts-Proteins)
• DNA is a single long circular molecule of doublestranded DNA “bacterial chromosome”.
– More efficient; grows quicker
– Mutations allow adaptation to environment quicker
• Plasmids; small circular transferable, doublestranded DNA molecules
– Antibiotic Resistance
• Bacteria also contain transposons
• Ribosomes function as the site of protein synthesis.
• No organelles (Mitochondria, Golgi, etc.)
Appendages of Bacteria
• Some bacteria have flagella which are long
filamentous appendages that can propel the cell.
• Many gram-negative bacteria possess hair-like
appendages that are used for attachment rather than
for motility. These are divided into two types, fimbriae
and pili.
• Fimbriae enable a bacterial cell to adhere to surfaces
(including other cells) while pili join bacterial cells in
preparation for the transfer of DNA from one cell to
another.
Flagella
• Motility - movement
• Swarming occurs with some bacteria
– Spread across Petri Dish
– Proteus species most evident
• Arrangement basis for classification
– Monotrichous; 1 flagella
– Lophotrichous; tuft at one end
– Amphitrichous; both ends
– Peritrichous; all around bacteria
Mono- or Lophotrichorus
Pilli
• Short protein appendages
– smaller than flagella
• Adherence of bacteria to surfaces
– E. coli has numerous types
• K88, K99, F41, etc.
– Antibodies to will block adherence
• F- Pilus; used in conjugation
– Exchange of genetic information
F- Pilus for Conjugation
Endospores
• When essential nutrients are depleted, certain
gram positive bacteria (e.g., Clostridium and
Bacillus), form “resting” cells called endospores.
• These endospores contain condensed nuclear
material and protein and can survive extreme
heat, lack of water, and exposure to toxic
chemicals.
• When growth conditions permit, the cell will
germinate into a dividing bacterium.
Endospores
• Resistant structure
– Heat, irradiation, cold
– Boiling >1 hr
• Takes time and energy to make spores
• Location important in classification
– Central, Subterminal, Terminal
• Bacillus stearothermophilus -spores
– Used for quality control of heat sterilization
equipment
• Bacillus anthracis - spores
– Used in biological warfare