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Bio 411 Master Presentation Dr. Rowe SH 222 X2521, j.rowe@notes.udayton.edu Overview of Microbiology The study of organisms and agents to small to be seen clearly by the unaided eye Organisms involved are prions, viruses, bacteria, fungi, protozoa,and algae Historical Perspectives

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bio 411 master presentation

Bio 411 Master Presentation

Dr. Rowe

SH 222

X2521, j.rowe@notes.udayton.edu

overview of microbiology
Overview of Microbiology
  • The study of organisms and agents to small to be seen clearly by the unaided eye
  • Organisms involved are prions, viruses, bacteria, fungi, protozoa,and algae
historical perspectives
Historical Perspectives
  • Spontaneous Generation. Living organisms can develop from non living matter.
  • Until the 17th century this was the primary hypothesis to explain how life arises.
historical perspectives4
Historical Perspectives
  • Francesco Redi (1626-1697) Experimented with decaying meats and showed maggots wouldn’t develop in the meat spontaneously if covered in gauze but flies laid eggs on top of gauze which developed into maggots thus temporarily disproving spontaneous generation.
slide5
Antony van Leeuwenhoek (1623-1723) Dutch. First to observe and describe microoganisms under amicroscope.
historical perspectives6
Historical Perspectives
  • With the discovery of microorganisms the idea of Spontaneous Generation resurfaced for the origin of microbes.
  • Needham (Catholic Priest), Schwann and Pasteur all performed experiments between 1780 and 1870 which proved microbes did not originate by spontaneous generation.
historical perspectives8
Historical Perspectives
  • Joseph Lister -1867
    • Developed the concept of antiseptic surgery.
    • Instruments are heat sterilized and phenol is used afterward to eliminate infection.
  • Pasteur correlated process of wine making and beer making with yeast by- products
  • Winogradsky and Biejerinck showed importance of bacteria in biogeochemical cycling (Carbon, sulfur, nitrogen)
microbiology in the 20th century
Microbiology in the 20th Century
  • Robert Koch (1843-1910) Demonstrated the role of microbes in causing disease during his study of anthrax.
  • Koch established the relationship between Bacillus anthracis and anthrax by isolating the organisms from infected animals and injecting them into mice.
microbiology in the 20th century10
Microbiology in the 20th Century
  • Koch’s Postulates

1. Microbe must be present in every case of the disease but not in healthy animal

2.Suspected microbe must be isolated and grown in pure culture

3.Same disease must result when pure culture inoculated into healthy host

4. Same microbe isolated from infected host

germ theory
Germ Theory
  • Idea that microbes caused disease was not well accepted until the late 1800’s
  • Pasteur was attributed to be the father of germ theory although Koch and Lister played major roles
immunology
IMMUNOLOGY

Pasteur studied chicken cholera and found that if cultures were incubated for very long periods between transfers they would become

ATTENUATED=NO LONGER ABLE TO CAUSE DISEASE.

When the attenuated form was injected into healthy chickens they became resistant to infection

Pasteur developed other means to attenuate a bacteria via heat treatment or chemical treatment.

vaccine production
Vaccine Production
  • The idea of injecting attenuated strains to protect healthy animals or humans from microbial infection was termed vaccination
  • Pasteur developed the first vaccine against anthrax and the viral disease rabies.
vaccination
Vaccination
  • The acute contagious disease diphtheria caused by a toxin produced by the bacteria Corynebacterium diphtheriae was the first such disease identified
  • Von Behring and Kitasato responsible for identifying and creating a vaccine and antitioxin by injection of healthy animals with inaactivated toxin.
koch s molecular postulates
Koch’s Molecular Postulates
  • 1. The virulence trait under study should be associated much more with pathogenic strains of the species than non pathogenic strains
  • 2. Inactivation of the genes or genes (by mutation or knockout) associated with the suspected virulence trait should substantially decrease pathogenicity
  • 3. Replacement of the mutated gene (or knockout) with the normal wild-type gene should fully restore pathogenicity
koch s molecular postulates16
Koch’s Molecular Postulates
  • 4. The gene should be expressed at some point during the infection or disease process
  • 5. Antibodies or immune system cells directed against the identified gene products should protect the host.
microbiology in the 20th century17
Microbiology in the 20th Century
  • Diseases such as diphtheria,yellow fever, malaria, were shown to be infections of microorganisms
  • Penicillin - 1st antibiotic
    • discovered by Flemming in 1945
  • Waksman’s discovered streptomycin in 1952
story of louis pasteur
Story of Louis Pasteur
  • Struggle of science and pseudoscience
  • Pasteurization process for wine

3. Story of establishment of germ theory

4. Story of scientific development of first human/animal vaccines, rabies, anthrax

5. Story of virus disease and bacterial disease

6. Story of antiseptic surgery

7. Story of modern culture technique and microscopy and disease

microbiology in the 20th century19
Microbiology in the 20th Century

Microorganisms were used as experimental models for developing the area of genetics and molecular biology

  • 1962 - Watson and Crick discovered the genetic material DNA
  • 1965 - Jacob and Monod revealed how transcription of DNA is regulated
important techniques in microbiology
Important Techniques in Microbiology
  • Aseptic (sterile) techniques
  • Culture media
  • Solid Agar media for pure culture technique
  • Microscopy-light microscopy, scanning and transmission electron microscopy
the microbes
The microbes
  • Domain Eucaryea
    • contain microbes in three kingdoms
      • Plants
      • Fungi
      • Protozoa
the microbes27
The microbes
  • Domain Bacteria -includes all procaryotes with bacterial ribosomal(rRNA) RNA and cell membranes with traditional ester bonds between glycerol and fatty acids.
the microbes28
The Microbes
  • Domain Archaea- All Procaryotes with membranes containing glycerol and fatty acids linked by an ether bond and a special Archaeal rRNA
bright field microscope
Bright Field Microscope
  • Dark image against light background
  • Generally must stain bacteria to increase
  • visibility
  • Magnification is from 50X-1000X
preparation and staining
Preparation and Staining
  • Fixation-Process whereby the internal and external structures of the cell and the microorganism itself are fixed in place
  • Two types for bacteriology-heat fix and chemical fix.
simple stains
Simple Stains

Basic Dyes are positively charged and bind to negatively charged macromolecules like nucleic acids. Bacterial surfaces are negatively charged so it basic stains are commonly used.

  • Crystal violet
  • Malachite green
  • safranin
simple stains36
Simple Stains

Acid Dyes are negatively charged and thus bind to positively charged macromolecules such as calcium rich substances.

  • Acid fuchsin
  • eosin
differential staining
Differential staining

Gram stain

Acid-fast-Use harsh treatment to allow penetration of dye. Heat, basic fuchsin and phenol. A rinse with acid alchohol. Acid fast cells retain the red fuchsin because of the presence of positively charged molecules. Used to stain mycobacterium

transmission electron microscopy41
Transmission Electron Microscopy
  • Uses magnetic lenses
  • Uses electron beam in place of light
  • used for virus and cross sections of cells
  • specimen must be stained: osmium tetroxide or phosphotungstate are common stains.
scanning electron microscopy
Scanning Electron Microscopy
  • specimen scanned with primary electron beam and sample emits a shower of secondary electrons which can be detected
  • Visualizes surfaces or surface structures
parameters used in the phenotypic identification of microbes
Parameters used in the phenotypic identification of microbes
  • Cell shape
  • Cell size
  • Colony morphology
  • Staining behavior
  • Physiological and biochemical characteristics
cell shape
Cell Shape
  • Cocci (spherical)
  • Rod
  • spirochete
bacterial nomenclature
Bacterial Nomenclature
  • Bacterial species is defined differently for bacteria than for higher organism where it is based on interbreeding
  • Species for bacteria is a collection of strains that share many stable properties and differ significantly form other groups of strains
  • A strain is a population of organisms that descends from a single organism or pure culture. Strains only differ slightly from one another
identification of bacteria
Identification of Bacteria
  • Variety of tests are used
  • Categorization based on Bergey’s Manual of Determinative Bacteriology
microbe of the week
Mycobacterium tuberculosis-gram positive rod shaped actinomycete which is the infectious agent for tuberculosis. Does accept staining well.

Often characterized because it responds to acid fast stain

MICROBE OF THE WEEK
acid fast stain
Acid Fast Stain
  • Since Mycobacterium does not accept simple stains because of high lipid content of cell wall
  • it must be stained by harsh staining treatment called acid fast
acid fast stain56
ACID FAST STAIN
  • After bacteria heat fixed to slide a combination of basic fuchsin and phenol is applied and heated.
  • Once stain takes place it is not easily removed because of high lipid content of cell wall especially mycolic acid so does not destain with acid-alcohol wash
slide57

Acid Fast stain

Mycobacterium

tuberculosis
Tuberculosis

20% of the worlds population is infected

Most infections result in a latent disease which could resurface as a active infection

Such individuals are Skin test positive to a Purified Protein Derivative(PPD) from the organism

tuberculosis60
Tuberculosis
  • Incubation period 10-12 weeks and skin test sensitive
  • Usually becomes sequestered in the lungs and becomes latent
  • Active infection cough, weight loss, bloody sputum
new problems
NEW PROBLEMS
  • AIDS victims much more susceptible so number of cases dramatically increasing
treatment of tb
Treatment of TB
  • Antibiotics are effective-isonazid plus rifamin, very effective.
  • MDR-TB Multi-drug-resistant tuberculosis has arisen and a factor in treatment
procaryote structure
Procaryote Structure

Figure 3.4 p 41

plasma membrane
Plasma Membrane
  • biochemically composed of a phospholipid bilayer associated with proteins responsible for transport, respiration, receptor sites etc.

Fig.3.7 p 42

plasma membrane65
Plasma membrane
  • Fluid Mosaic Model for membranes-interspersed in the lipid bilayer are 20% peripheral proteins and 80% integral proteins with specific function.
  • Peripheral proteins are water soluble
  • Integral proteins have hydrophobic pockets and also polar groups
plasma membrane function
Plasma Membrane Function
  • The plasma membrane is a selectively permeable membrane which contains the cytoplasm and is also the site of key metabolic functions such as transport and respiration.
lipid bilayer biochem
Lipid Bilayer biochem

Eubacteria membrane and Eukaryl membranes mainly phospholipid

glycerol, fatty acids in position 1 and 2

phosphoethanolamine, phosphoserine or phosphocholine in position 3

archeabacteria
Archeabacteria
  • different biochemical structure from eucaryote and eubacterial membranes

fig.

microbe of the week lyme disease
Microbe of The WeekLyme disease
  • Significant disease in the US but was not discovered until 1975
  • The tick is the vector for the bacterial etiologicl agent
  • Cause of infection are the gram negative spirochetes Borrelia burgdorferi, B.garinii or B. afzelii
  • Deer and field mice are the reservoirs for the bacteria
lyme disease three stages
Lyme Disease-Three stages
  • 1. Localized stage-ring shaped with red boarder around bite accompanied by flu-like symptoms,(fever, headache,malaise)
  • Stage 2. Dissemination weeks or months later.Neurological abnormalities, heart inflamation, arthritis-like symptoms
  • Stage3 years later. Alzheimer like symptoms,or Multiple Sclerosis-like symptoms.
diagnosis of lyme disease
Diagnosis of lyme disease
  • 1. Isolation of the spriochete
  • 2. Identification or organisms DNA in blood by PCR
  • 3. Serological testing for Ab against organism
treatment of lyme disease
Treatment of Lyme Disease
  • Treatment with Amoxicillin or tetracycline early in disease (first stage) results in immediate recovery
  • At late second or third stage where blood brain barrier breeched use ceftriaxone long term therapy
archea
Archea
  • No peptidoglycan but do stain with gram stain either neg or pos
  • Cell wall composed of proteins or polysaccharides
removal of cell wall
Removal of Cell Wall
  • Lysozyme attacks peptidoglycan by hydrolysis of 1-4 glycosidic bond between NAMA and NAGA
  • Penicillin inhibits peptidoglycan synthesis. If bacteria are treated with penicillin in an isotonic solution they become protoplasts and continue dividing.
prevention and control of lyme disease
Prevention and Control of Lyme Disease
  • 1. Tick must stay attached for 24 hours therefore immediate removal of insect
  • 2. Awareness of seasonal times at which you are most at risk. May-July
  • 3. If outdoors use insect repellent effective against ticks and dress properly
  • 4 After outdoor experiences check yourself out carefully during shower etc
spheroplasts
Spheroplasts
  • Treatment of growing gram negative cells with penicillin leaves the outer membrane of the cell wall and these are call spheroplasts
  • Treatment of gram positive cells leaves only cytoplasmic membrane and these are called protoplasts
composition of cytoplasm
Composition of Cytoplasm
  • metabolic enzymes (proteins)
  • regulatory proteins and chaperones
  • ribosomes
  • inclusion bodies (organic, inorganic)
  • nucleoid (chromosomal DNA)
  • plasmid DNA
organic inclusion bodies
Organic Inclusion Bodies
  • Glycogen-polymer of glucose which is a source of stored carbon and energy
  • polybeta hydroxybutyrate-source of stored carbon and energy
  • Carboxysomes-Found in bluegreen algae. contain CO2fixation enzyme
inorganic inclusion bodies
Inorganic Inclusion Bodies
  • polyphosphate granules also called volutin or metachromatic granules. Source of energy and phosphate
  • Sulfur granules-inorganic sulfur.-
ribosomes
Ribosomes
  • 70S in procaryotes made up of 50S and 30S subunits
  • Composed of rRNA and protein
  • Site of protein synthesis
nucleoid
Nucleoid
  • Chromosomal DNA is circular DS DNA containing chromosomal genes
  • Usually one copy per cell, not surrounded by membrane
  • In contact with the plasma membrane
plasmid dna
Plasmid DNA
  • Extrachromosomal DNA which contains nonessential genes
  • self replication and copy number self contained ie separte from nucleoid DNA
  • Codes for such things as antibiotic resistance and chromosomal mobilization
flagella structure
Flagella Structure

Fig.3.37, p.61

flagella
Flagella
  • Composition is protein
  • Subunits called flagellin
  • Functions for motility
  • In some cases functions in attachment of cells to surfaces
flagella position
Flagella position
  • Monotrichous-one flagella located at a pole or end
  • Amphitrichous-single flagella at both ends
  • Lophotrichous-tuft of flagella at one or both ends
  • Peritrichous-around entire surface of bacteria
flagellar motility
Flagellar Motility

Fig. 3.39, p. 62

bacterial capsule composition
Bacterial Capsule Composition
  • Biochemically are usually carbohydrate
  • Often called the glycocalyx
  • Bacillus anthracis has capsule made of poly-D-Glutamic Acid
function of glycocalyx capsule
Function of Glycocalyx (capsule)
  • Helps bacteria resist phagocytosis
  • Stores lots of water thus makes cell resistant to drying
  • Aids in attachement to surfaces
  • Helps exclude antibiotics and viruses
pili or fimbriae composition and function
Pili or Fimbriae Composition and Function

Made of the protein pilin

Function in transfer of genetic material and these are specific pili called sex pili

Function in attachment to surfaces

bacterial endospores
Bacterial Endospores
  • Dormant bodies formed from vegetative cells during adversed conditions.
  • Highly resistant to heat, chemicals, drying, ultraviolet light and other physical treatment
  • Two genera used as paradigms: Bacillus and Clostridium
endospore location
Endospore Location

Fig. 3.44-3.45, p.67

sporogenesis
Sporogenesis

Fig. 3.47, p. 68

other genetic means for determining phylogeny
Other genetic means for determining phylogeny
  • Comparison of complete genome sequences

Comparison of nucleic acid composition

mol%GC = G+C_____

G+C+A+T

eucaryotic plasma membrane
Eucaryotic Plasma Membrane
  • Not the site of respiration
  • Lots of protein pumps transporters and receptors
  • lipid bilayer/fluid mosaic
  • Contains cholesterol for stability
mitochondria
Mitochondria

Site of respiration and ATP synthesis

Contains its own DNA

DNA is bacterial in nature: circular, haploid

Mitochondria multiplies separately from cell

malaria bug of the week
Malaria-bug of the week
  • Protozoan disease
  • Plasmodium vivax, P. malariae, P. ovale and P. falciparum
  • Requires the female mosquito (Anopheles)as an insect vector .
  • Most important of human parasites
epidemiology
Epidemiology

• 300 million people infected each year

• 1 million die in Africa alone

•Mainly a disease of the tropics where there are lots of mosquito vectors

pathological changes
Pathological changes
  • Symptoms are from synchronized release of merzoites from RBC
  • RBC lyse and release toxins etc causing fever, chills and sweating. Occurs at 48-72 hour intervals
  • Remission after a few weeks then repetition
treatment
Treatment
  • Usually a quinine such as chloroquine, amodiaquine or mefloquine which eradicate erythrocite stage
  • Drug resistant strains cropping up so there are new treatments
  • Prophylactic treatment effective
asides
Asides
  • Sickle Cell associated with resistance-individuals have modified Hb
  • Recently finished genome project of Anopheles and Plasmodium falciparum and combined with Human Genome project new molecular cures likely
nucleus
Nucleus
  • Double membrane-inner and outer
  • Contains Chromosomes (Cell genetic material) and nucleolus (dense region which is site of ribosome synthesis
lysozomes
Lysozomes
  • Intracellular Digestion and contain all enzymes necessary to degrade the 4 macromolecules
  • Important for cells that use endocytosis for obtaining nutrients
endoplasmic reticulum er
Endoplasmic Reticulum (ER)
  • Smooth ER-Main site of lipid synthesis
  • Rough ER-Protein Synthesis and packaging. Studded with ribosomes
golgi
Golgi
  • Packaging (targeting) and secretion of proteins for various purposes.
  • Formation of lysozomes
microfilaments microtubules
Microfilaments, Microtubules

Protein(actin)

Function in Cell Structure and movements, form the cytoskeleton

flagella and cilia
Flagella and Cilia

Composed of microtubules in a 9+2 array

flagella and cilia function
Flagella and Cilia Function

Motility

Beat rather than act like a propeller

chloroplast
Chloroplast

Site of photosynthesis

Membrane bound organelle

Bacterial circular DNA and ribosomes

Multiplies independently

Origin is a bacterial (endosymbiont)