Summary of Lectures to For First Exam in Microbiology

 

 

 

Origin of Universe – Current Evidence

 

Hubble Expansion - Galaxies appear to be moving away from our galaxy, initially observed by Edward Hubble in 1927; Galaxies have a red shift in light produced by the Doppler effect similar to sound (also used in radar); The movement of galaxies away from us corresponds to an equation called "Hubble's Law"; The time elements in Hubble's Law permit the estimation of the age of the universe – about 15 billion years (+ or – a few billion)

 

Nucleosynthesis Occurred during seconds 100 to 300 following big bang

Result of temperature of universe dropping below that required for nuclear fusion (4 H to He); Produced a large amount of Helium which is very stable and cannot be converted easily into heavier elements; Amount of Helium measured in universe is consistent with Big Bang Nucleosynthesis

 

The cosmic microwave background was predicted in 1948 by George Gamow and Ralph Alpher, and by Alpher and Robert Herman   1964-65Arno Penzias and Robert Woodrow Wilson measure the temperature to be approximately 3 K. Robert Dicke, P. J. E. Peebles, P. G. Roll and D. T. Wilkinson interpret this radiation as a signature of the big bang.

Penzias and Wilson received the 1978 Nobel Prize in Physics for their discovery.

Existence of this radiation inconsistent with steady state model.

 

Origin of Elements

 

Hydrogen fuses to form helium; Hydrogen & Helium most abundant; Sun’s energy drives life processes; Stars become red giants as hydrogen runs low (about 10 billion years); Helium fuses into other elements; Star goes nova (blows up) and heavier elements released into space

 

 

Origin of Sun and Planets

Sun formed about 5 billion years ago from gravitational attraction of gases; gravitational field becomes great enough to initiate fusion reactions of hydrogen into helium generating energy; accretion forms inner planets & moons;  process occurs quickly-earth & moon about same age; takes about 100 million years

 

Early Earth – Current Evidence

Earth 4.6 billion years old (U238 dating)

Early Atmosphere mostly non-oxidizing contains Nitrogen Carbon dioxide Water (as water vapor) and lesser amounts  of CO, H2, NH3, H2S and CH4  Compare to atmospheres of Venus and Mars

 

 

 

Characteristics of Life

Composed of  Cells

Reproduce in kind

Metabolism / Energy Transformations

Respond to stimuli

 

Abiogenesis  vs Biogensis

The process of life from the inanimate versus life from preexisting life abiogenesis includes:

Formation of organic monomers from inorganic molecules

Formation of organic polymers from organic monomers

Evolution of membranes

Evolution of DNA based reproduction

 

Chemical Evolution

Requires a non-oxidizing atmosphere - No oxygen initially

Requires a source of energy - Lightening, UV light, Volcanoes & Meteorites

Requires hydrogen, nitrogen, carbon, oxygen

Components of organic and biological compounds

water, ammonia, carbon dioxide/methane

Requires time

 

Molecular Clues to Origins

The following suggest common origin: Organisms use molecules based mostly on hydrogen, nitrogen and carbon present on early earth  Only L-amino acids found in proteins DNA & RNA are universal in all organisms ATP is energy intermediate in all organisms All organisms initiate carbohydrate metabolism with similar steps

–Genetic code is universal

 

Organic Monomers

Oparin & Haldane suggest organic molecules could form from precursors (1930)

Miller & Urey test using an apparatus which simulates early earth (~1950)

 

Organic Polymers

Major Groups - Nucleic acid, proteins, lipids, polysaccharides - have been formed synthetically Protenoids will form spontaneously on clay  D & L amino acids can be selected on calcite - a common crystalline mineral

Molecules in living things must have 3 capabilities:  Information vs. Structural vs. Catalytic  RNA - has all three capabilities suggesting first life like capability occurred in RNA

 

RNA “Life

Ribose, a component of RNA will form spontaneously from formaldehyde and HCN

Some RNA’s have been found to have catalytic activity - ribozymes

RNA has structural capability in ribosomes

RNA’s have an information carrying capacity in viruses & RNA’s have been induced to take on new traits

 

DNA Life

Separation of functional roles of molecules  occurs because molecules more suited to different roles and there is a constant input of energy

Separation of information carrying capacity from other roles of molecules in cells

RNA to DNA

RNA to Protein - catalytic capacity

Protein & polysacharides take on structural roles in cells

 

Membranes

Why cells?

Inside vs. outside

Concentration effect on reactions

Indications of process

Microspheres - hydrocarbons in water form microsperes which can contain other molecules

Liposomes - artificial lipid bilayers very similar to cell membranes but smaller - used for drug transport

 

Prokaryotic Cells

Appear about 3.5 billion years ago

Photosynthesis in blue-green algae begins to modify atmosphere

Oxygen in atmosphere begins to modify types of organisms

 

Eukaryotic Cells

Begin to appear in fossil record about 2.5 billion years ago

Considerable internal structure relative to prokaryotic cells

Precursors to multicellular organisms

 

Fossil Record

 

Dating

Stratographic analysis

–Radiometric dating

Geologic Time

–Precambrian - 4.6 to 0.57 billion years ago

Fossils all unicellular

Caambrian – 0.57 billion years ago to present

Multicellular organisms

Extinction Level Events

 

Evidence for Evolution

Physical methods – radiometric dating

Fossil record

Anatomical comparisons

DNA sequence analysis

Laboratory experiments showing selection

 

 

 

Microscopy

History

Compound Light Microscopes

Types of Microscopy

Measurement Systems

Measuring in a Microscope

Staining Procedures

 

Early History of Devices that Alter Light:

Claudius Ptolemy (2nd Century B.C.)

»Described refraction water

Seneca (1st Century A.D.)

»Described magnification by a globe of water

Alhazen (962-1038 A.D.)

»Described optical principles & anatomy of eye

Roger Bacon (1267 A.D.)

»Described simple magnification

 

Lenses derived need to improve eyesight

Pliny the Elder wrote of Nero’s use of emeralds to watch gladiators

Reinvention of spectacles occurred around 1280 to 1285 in Florence, Italy

Dutch spectacle maker Zaccharias Jansen was probably first to combine two lenses into compound microscope (1595)

 

Robert Hooke (1665)

Contemporary of Robert Boyle; Described cork with “cells” – first use of “cell” to describe structure of living organism; made & used a compound microscope

 

Antoni van Leeuwenhoek

Made his own simple single lens microscopes; First to describe bacteria, blood, protozoa & sperm; sent letters & drawings to Royal Society

 

Problems with early microscopes

Chromatic aberration Occurs when different wavelengths of light are refracted through the lenses at different angles; Corrected using glass of different types

 

Spherical aberration - Distortion because light hitting edge of lens does not have same focal length as middle corrected using small apertures or diaphragms; Solved by Joseph Jackson Lister in 1830

 

Microscope parts

Ocular

Objectives

Stage

Diaphragm

Condenser

Light Source

Course adjustment

Fine adjustment

 

Modern compound microscope

Diaphragm

Condenser

Oil Immersion

 

Compound Microscope

Total Magnification

»Ocular  X  Objective equals Total

Refractive Index

»A measure of the relative velocity of light passing through a substance

Oil immersion

»prevents light scattering between slide and objective – has same refractive index as slide

 

Compound Microscope – Resolution

The ability of a lens to distinguish between two points as separate objects

Depends on wavelength of light – usually maximum resolution is wavelength / 2

Maximum for light microscope is about 0.2 microns or about 2000x

 

Types of Modern Microscopy

Bright field

Dark field

Phase Contrast

Electron Microscopy

Scanning Electron Microscopy

Fluorescent (UV)

 

Measurement (Metric System)

Meter (m) 100

Centimeter (cm) 10-2

Millimeter (mm) 10-3

Micrometer (µm) 10-6

Nanometer (nm)          10-9

 

Staining Techniques

Preparation Smear Heat Fixation Stain/counter stainNegative stain

Simple stains Crystal violet Saffron Methylene blue  

Mordant Intensifies stain Iodine used in Gram stain

Differential Stains Stain one group of organisms/cells  different than another; includes Gram stain Acid Fast Stain

 Special Stains Capsule Endospore Flagella

 

 

 

History of Microbiology

Science is systematized knowledge developed through the application of the scientific method; Scientific method includes:

Observations (objective vs. subjective)

Formulate hypothesis

Test hypothesis with controlled experiments

Accept, revise or reject hypothesis

 

Early Observations & Experiments in Microbiology

Microscopes

van Leeuwenhoek & Hooke

Spontaneous Generation Controversy

Germ Theory of Disease & Robert Koch

 

Spontaneous Generation

 

Biogenesis vs. Abiogenesis

Biogenesis - development of life from preceding life forms

Abiogenesis - life arises from inorganic or non-living materials

Jan Baptista van Helmont
 (1580-1644)

Reported in late 1500’s that barley grains and old shirts left in a corner would spontaneously give rise to mice; Claimed as evidence that supported spontaneous generation or abiogenesis

 

Francisco Redi (1626-1697)

Set up controlled experiment to test idea of spontaneous generation with respect to maggots appearing on rotting meat: open jar with meat; screened jar with meat sealed jar with meat

 

John T. Needham (1713-1781)

Flies do not arise spontaneously but the “animalcules” described by van Leeuwenhoek must; In 1748 Needham boiled mutton broth, stoppered and noted that flask became turbid; Argued that the turbidity, which included many “animalcules” must have arisen spontaneously

 

Lazzaro Spallanzani (1729-1799)

Repeated Needham’s experiments used flasks that were sealed by melting the glass rather than with a cork; Found that if sealed properly, flasks boiled 45 minutes would remain sterile thus refuting Needham’s conclusions

 

Theodor Schwann (1810-1882)

An argument against Spallanzani experiments is that they excluded air; Constructed apparatus to sterilize air coming into flask; Results supported biogenesis

 

Louis Pasteur (1822-1895)

Looked at air which had been filtered; Developed swan neck flask to deal with heated air problem; Looked at frequency of occurrence of contaminated flasks; Settled controversy

Performed a large number of experiments under a variety of conditions

 

Germ Theory of Disease

Observation on causative agents of potato blight and diseases of silkworms led to hypothesis

Formalized through work of Pasteur and Koch (and others) led to theory that germs or microorganisms may cause disease

Germ Theory of Disease

Robert Koch first developed relationship between microorganisms and disease

Developed Koch’s Postulates for testing relationship

Discovered cause of anthrax and tuberculosis

 

Koch’s Postulates:

Same microorganism must be observed in every instance of disease

Organism must be isolated from diseased host and grown in pure culture

Specific disease must be reproduced when pure culture is reintroduced into host

 

 

 

History of Cells

Robert Hooke (1600’s) first described cells in thin sections of cork that he examined under microscope

Robert Brown (1820) first to describe that a nucleus seemed to be associated with all cells (at least eukaryotic cells)

Theodore Schwann & Matthias Schleiden (1839) advanced cell theory

»All organisms are composed of cells

»The cell is the basic unit of life

»All cells arise from preexisting cells

 

Cells

Cell Types Prokaryotic(beginning cells and eukaryotic (true cells)

Sizes  Prokaryotic (0.2 to 2.0 microns) Eukaryotic (      10 to 100 microns                  0

 

 

Size Determinants of Cells

Cell surface to volume relationships govern cell size

The smaller the cell the more efficiently materials can be transported into the cell

Cell must also be large enough to deal with information and metabolic requirements

 

Common Components to All Cells

Plasma membrane – phospholipid bilayer that controls movement of substances into and out of cells

Ribosomes – site of protein synthesis

Cytoplasm –matrix on interior of cell consisting of water soluble proteins and other materials

Nuclear material – DNA/Protein complex that stores information

»Prokaryotic – circular

»Eukaryotic – linear and in chromosomes

 

Eukaryotic Cells

Larger than prokaryotic More complex than prokaryotic All multicellular organisms composed of eukaryotic cells Eukaryotic cells composed of many internal structures called organelles

 

Structures in Eukaryotic cells

lNucleus

Regulates growth and reproduction of cell; Contains DNA and chromosomes

lNucleolus  Ribosomal RNA synthesis

lMitochondria

Energy production in cell; Contains its own DNA (circular)

lEndoplasmic reticulum (rough and smooth)

Site of protein synthesis in cells; Start of protein export process; Connected to nuclear pores and Golgi body

lGolgi body

Sorting center for proteins in cell; Produces vesicles which fuse with plasma membrane

lLysosome

Only in animal cells; Production of intracellular digestive enzymes; Involved with phagocytosis

lPeroxisomes

Peroxisomes are small rounded organelles found free floating in the cell cytoplasm; Contain at least 50 enzymes and are separated from the cytoplasm by a lipid bilayer single membrane barrier; Produce hydrogen peroxide which is toxic but is rapidly degraded by catalase

lFlagella & cilia

Involved with motility of cells; Composed of microtubules

lVacuoles

Found only in plants Large central organelle in plant cells; Regulates water in plant cells

lChloroplast

Site of photosynthesis in plant cells; Has own DNA (circular); Found only in plants

 

 

 

 

Prokaryotic Cells

Morphology & Specialized Structures & Ultrastructure

 

Size, Shape & Arrangement of Prokaryotic Cells

0.2 µm to 2.0 µm diameter

2 µm to 8 µm in length

Three basic shapes

cocci (spheres)

bacillus (rods)

spirochete (twisted/spiral)

Pleomorphic shapes

 

 

Characteristics of Prokaryotic Cells

Smaller than eukaryotic cells

No nuclei or chromosomes

No membrane-bound internal organelles

Circular genomic DNA

70S Ribosomes

Cell division by binary fission

No meiosis; recombination by transfer of DNA fragments

 

Shapes of Prokaryotic Cells

Cocci spheres Staphylo- clusters; Strepto- chains;

Bacilli  rods

Spirochete spirals

Specialized Structures

Endospores – a structure produce primarily by Clostridia and Bacillus species to resist desication

Acid Fast Bacteria – a waxy sheet or coat produced by Mycobacteria

Club shape – produced by Corneybacteria

 

Prokaryotic cells external structures

Flagella  Long filamentous protein; filament; hook; basal body

Rotates to propel cell in response to taxis (positive and negative)

Capsules Slime layer Polysaccharide or polypeptide Protection from phagocytosis by host; gives cells “Stickiness” permits adherence also prevents desiccation

Cell Envelope or Cell Wall

Gram positive Peptidoglycan (Multi layer) Teichoic acid

Gram negative Peptidoglycan (single layer) Outer membrane (lipopolysaccharide) Primary function is resist changes in osmotic pressure; also functions in recognition

Peptidoglycan  composed of polysaccharide of alternating N-acetylglucosamine & N-acetylmuramic acid; Peptide composed of D- and L- amino acids

Lipopolysaccharide of gram negative cells in outer membrane of Gram negative cells & composed of lipid and carbohydrate; Carbohydrate referred to as O-antigen; Associated with some endotoxins in pathogenic G- bacteria

Teichoic Acid - sugar alcohol plus phosphate found only in Gram positive cell walls

Fimbriae associated with the adhesiveness of cells to surfaces

Pili involved in the transfer of genetic material

 

 

 

Plasma Membrane

Basic molecular components of membrane

Phospholipids

Membrane proteins

Membranes must be fluid

Fluid Mosaic Model

 

Phospholipid bilayer

Formed by association of hydrophobic tails

Polar (hydrophillic) groups form inside and outside of membrane

 

Membrane Proteins

Proteins are integral or peripheral

Proteins involved in plasma membrane functions

Membrane must be fluid to function

Components are free (to a degree) to move across surface

 

Functions

Boundary between inside and outside

Recognition

Energy (respiration and photosynthesis)

Selective permeability of materials

Transport Processes Across Membranes

Diffusion

Active transport

Bulk transport

 

Diffusion Processes

Simple Diffusion – movement of small uncharged molecules

Osmosis – special case of diffusion; diffusion of water in response to solute concentration

Facilitated Diffusion – diffusion using a channel protein

Diffusion – the movement of molecules from area of high concentration to low concentration

Osmosis

 

Isotonic

Solute concentration same on both sides of plasma membrane

 

Hypotonic

Hypo- below

Solute concentration less outside cell than inside cell

 

Hypertonic

Hyper- above

Solute concentration greater outside cell than inside

 

Facilitated Diffusion

Diffusion that occurs through use of a channel protein

 

Active Transport

Movement against energy concentration gradient

Requires energy; usually in form of ATP

 

Bulk transport – An active transport process

Eukaryotic cells only

Endocytosis

Pinocytosis

Phagocytosis

Exocytosis & secretory processes