Fundamentals of Microbiology Unit 1: 7 days. January 7 th : Intro to Microbiology What are microbes?...

Fundamentals of Microbiology

Unit 1: 7 days

January 7th: Intro to Microbiology

• What are microbes?

• What do microbes do?

Microbes in our lives• Disease• Spoiled food• Food chain base• Decomposers• Digestion• Vitamin synthesis• Industrial synthesis of chemicals• Food production• Genetic engineering• Sewage treatment• Bioremediation

Microbes in our lives

• Only a small number of microorganisms are pathogenic

• Health care workers must still understand microbes in order to protect patients from normally harmless, but opportunistic organisms

Microbes in our lives

Historic Changes in Microbiology

• The field is only a few hundred years old, but bacterial DNA has been found in 3,000 year old Egyptian mummies

• Arguably the most important discovery in biology was in 1665 by Robert Hooke and a crude microscope

Historic Changes in Microbiology

• Hooke reported that the smallest structural unit of life was a “little box” or “cell”

• This launched cell theory, and all future research into cells was based on this first discovery

• Now cell theory states that “all living things are composed of cells”

Historic Changes in Microbiology

• A Dutch merchant and amateur scientist named Antoni Von Leeuwenhoek was the first to use stains and really see microorganisms

• He looked at rainwater, peppercorn water, and scrapings from his teeth under a magnifying lens and drew pictures of the ‘animalcules’ that he observed

Historic Changes in Microbiology

• How did these tiny organisms arise?• Were they born?• Or did they spontaneously generate?

Historic Changes in Microbiology

• This is not an unreasonable question 150 years ago

• People believed that toads were born out of wet soil, that flies came from manure, and that maggots could arise from decaying corpses

Historic Changes in Microbiology

• Italian physician Francesco Redi opposed this ‘spontaneous generation’ viewpoint

• He began trying to disprove that maggots came from rotting meat in 1668

Historic Changes in Microbiology

• First Redi took three jars and put decaying meat in them and then sealed them tightly

• Next he took three more jars, put more decaying meat in them, and left them open

• Maggots appeared in the open jars, because flies could land and lay their eggs

Historic Changes in Microbiology

• Critics said that fresh air was required for spontaneous generation

• Redi repeated the experiment, but this time instead of sealing the jars he spread a fine mesh across the tops

• Again, maggots only appeared in the open jars

Historic Changes in Microbiology

• Redi’s results were a serious blow to the belief that complex living material could be generated from non-living material

• However, people still believed that Van Leeuwenhoek’s animalcules were simple enough to spontaneously generate

Historic Changes in Microbiology

• The debate about microorganisms raged back and forth with many experiments being done to determine whether microbes could spontaneously arise from nutrient fluids

Historic Changes in Microbiology

• Sometimes heating and sealing prevented growth, but many argued that severe heating destroyed the ‘life force’ in the fluids

• Others argued that the was not enough oxygen (recently proven to be required by many life forms) in the sealed flask to allow for microbe generation

Historic Changes in Microbiology

• In 1858 German scientist Rudolf Virchow brought forth the concept of biogenesis – living cells can arise only from preexisting living cells

• In 1861 Louis Pasteur definitively answered the debate with a series of clever experiments– Pasteur’s discoveries form the basis of aseptic

technique today

The Golden Age of Microbiology• From 1857 to 1914• Rapid breakthroughs in the field:– Fermentation – using microbes to produce alcohol– Pasteurization – heating to kill most microbes– Germ Theory of Disease – microbes may cause

illness in both plants and animals– Koch’s Postulate – sequence of steps to relate a

microbe to a specific disease– Vaccination

The Golden Age of Microbiology

• People:• Edward Jenner – vaccination of smallpox• Paul Ehrlich – creator of synthetic drugs• Alexander Fleming – penicillin

New Areas of Research

• Immunology• Virology• Parasitology• Bacteriology• Mycology• Genetic engineering• Molecular biology

January 8th: Naming Microbes and Major Chemical Principles

• The system for scientific nomenclature was first developed in 1735 by Carolus Linnaeus

• They are Latinized and italicized• Each organism is given two names: – Genus – always capitalized– Species epithet

• Once the species name has been listed it can be abbreviated with the first letter of the genus and the species epithet

Naming Microbes

• Names can honor a researcher, describe the organism, or even denote the location where it is found

• Staphylococcus aureus = staph (clustered colonies), coccus (spherical cells), aureus (golden in color)

• Escherichia coli = Theodor Escherich, coli (found in the colon)

Naming Microbes

• In 1978 Carl Woese developed a classification system for bacteria

• Eubacteria – bacteria with peptidoglycan cell walls

• Archaea – bacteria lacking peptidoglycan cell walls

• Eukarya – all eukaryotes (protists, fungi, plants, and animals)

Bacteria

• Very small• Relatively simple• Single celled• Prokaryotic = pre-nucleus• Several shapes• Several arrangements• Binary fission asexual reproduction• Mostly heterotrophic, a few autotrophs

Fungi

• Eukaryotic • Nucleus contains DNA• Unicellular or multicellular• Heterotrophic• Cells walls made from chitin• Sexual or asexual• Unicellular species are oval in shape

Protozoa

• Eukaryotic• Unicellular• Classified based on locomotion– Amoebas – move extensions of their cytoplasm– Flagellates– Ciliates

• Sexual or asexual• Parasitic or free living

Algae

• Eukaryotic• Photosynthetic• Sexual or asexual• Cell walls – typically of cellulose• Abundant in fresh water, soil, and in

conjunction with plants

Viruses

• Very small - most are only visible with an electron microscope

• Only one type of nucleic acid – can be DNA or RNA

• Have a protein coat• Can have a lipid envelope• All parasitic• Reproduce by hijacking cellular machinery

Multicellular Animal Parasites

• Not strictly microorganisms• Medically important• Worms• Often have a microscopic stage of their life

cycle

Chemistry Review!

• Atomic structure• About 26 elements are commonly found in

living cells• Valence electrons and bonding– Ionic– Polar Covalent– Non Polar Covalent– Hydrogen Bonds

Chemistry Review!

• Molecular mass• Moles• Chemical reactions– Exothermic– Endothermic– Reversible

• Collisions required• Enzymes lower activation energy

Chemistry Review!

• Inorganic compounds:– Small– Ionically bonded– Water– Acids and bases• Hydrogen and Hydroxide ions

– Salts• pH• Buffers

Chemistry Review!

• Organic compounds:– Always contain carbon and hydrogen– Carbon atoms can bond 4 times– Mostly or entirely covalent– Many are large– Carbon skeleton– Functional groups

• Monomers and polymers

Chemistry Review!• Carbohydrates:– C H and O– Include sugars and starches– Isomers – same formula, different structure• Glucose and fructose

• Lipids:– Insolubility in water– Simple lipids – glycerol and 3 fatty acids– Saturated – no double bonds– Steroids – carbon ring group

Chemistry Review!

• Proteins:– Built by amino acids– C H O and N, sometimes S– 20 amino acids occur naturally

• Adenosine Triphosphate:– Stores chemical energy for various cellular activities– When the bond in the terminal phosphate group is

broken, energy is released– The energy from decomposition reactions is used to

regenerate ATP from ADP

Chemistry Review!

• Nucleic Acid:– Macromolecules– DNA and RNA– Pentose, phosphate, and a nitrogen containing

base– Right handed double helix– A,T,C, and G– RNA has ribose– Genes consist of sequences of nucleotides

January 9th: Observing Microbes and Microscopes

• Microorganisms are measured in micrometers (10 -6 m) and nanometers (10 -9 m)

Compound Light Microscope

• This is the most common instrument used in microbiology

• It is abbreviated LM

• Two sets of lenses– Ocular– Objective

Compound Light Microscope

• Total magnification is calculated by multiplying the magnification of the two lenses together

• A typical maximum magnification is 2000x• A typical maximum resolution is 0.2 μm

Compound Light Microscope

• Stains are used to add more definition to the image

• Immersion oil is used with the oil immersion lens to reduce light loss between the slide and the lens

Compound Light Microscope

• Brightfield illumination is used for stained smears

• Unstained cells are more productively observed using darkfield, phase-contrast, or DIC microscopy– These types of microscopy require modified

compound microscopes

Darkfield Microscopy

• Shows a light silhouette of an organism against a dark background

• This is the most useful technique for detecting extremely small organisms

Phase-Contrast Microscopy

• Brings direct and refracted light waves together in phase to form an image

• Allows for detailed observation of living organisms

Differential Interference Contrast

• Provides a colored image

• 3D

• Allows detailed observation of living cells

Fluorescence Microscopy

• First, specimens are stained

• Then they are viewed through a compound microscope using ultraviolet light

• Microorganisms appear as bright objects against a dark background

Confocal Microscopy

• A specimen is stained with a fluorescent dye and illuminated one plane at a time

• Using a computer to process the images, 2D and 3D images of cells can be produced

Electron Microscopy

• A beam of electrons is used, instead of light

• Electromagnets control the focus, illumination, and magnification

• Thin sections of organisms can be seen

Scanning Tunneling and Atomic Force

• STM and AFM produce 3D images of the surface of a molecule

Preparing Smears for Staining

• Staining means coloring a microorganism with a dye to make some structures more visible

• Fixing uses heat or alcohol to kill and attach microorganisms to a slide

• A smear is a thin film of material used for microscopic examination

Preparing Smears for Staining

• Bacteria are negatively charged

• Colored positive ions of a basic dye will stain bacterial cells

• Colored negative ions of an acidic dye will stain the background of a bacterial smear– This is called a negative stain

Simple Stains

• An aqueous or alcohol solution of a single basic dye

• Used to make cellular shapes and arrangements visible

• Mordant – used to improve bonding between the stain and the specimen

Differential Stain

• Differentiate bacteria according to their reaction to the stain– E.g. Gram Stain and Acid Fast Stain

Gram Stain

• Uses:– Crystal violet (purple stain)– Iodine (mordant)– Alcohol (decolorizer)– Red counter stain

• Gram –Positive bacteria retain the purple stain • Gram-Negative get decolorized and then

counter stained pink

Acid Fast Stain

• Acid-fast microbes retain carbolfuchsin after acid-alcohol decolorization – Appear red– E.g. genera Mycobacterium and Nocardia

• Non-acid-fast microbes take up the methylene blue counterstain – Appear blue

Special Stains

• The endospore stain and flagella stain are special stains that color and isolate certain parts of bacteria

• Negative staining is used to make microbial capsules visible

January 13th, 14th, 21st, and 22nd: Functional Anatomy of Prokaryotic Cells

• What is a prokaryote?

Prokaryote vs. Eukaryote

• Similar in their chemical composition and chemical reactions

• Prokaryotes lack membrane enclosed organelles– Including a nucleus

• Peptidoglycan is found in prokaryotic cell walls, but not in eukaryotic cell walls

The Prokaryotic Cell

• Bacteria are unicellular

• Most multiple by binary fission

• Species are differentiated by morphology, chemical composition, nutritional requirements, biochemical activities, and source of energy

Size, Shape, and Arrangement

• Most bacteria are 0.2 to 2 μm in diameter

• Most bacteria are 2 to 8 μm in length

Size, Shape, and Arrangement

• Three basic bacterial shapes are commonly found:– Coccus (sphere)– Bacillus (rods)– Spiral (twisted)

Size, Shape, and Arrangement

• Pleomorphic bacteria can assume several shapes

• Monomorphic bacteria have a shape determined by heredity– Most bacteria are monomorphic

Size, Shape, and Arrangement

• Diplococci – coccus that remain in pairs• Streptococci – coccus that form chains• Tetrads – remain in groups of four, divide in

two planes

Size, Shape, and Arrangement

• Sarcinae – form cubelike groups, divide in three planes

• Staphylococci – form grapelike clusters or broad sheets

Size, Shape, and Arrangement

• Diplobacilli – rods in pairs• Streptobacilli – chains of rods• Coccobacilli – oval shaped cells

Size, Shape, and Arrangement

• Vibrios – curved rods• Spirilla – helical or corkscrew shaped, fairly

rigid• Spirochetes – helical and flexible

External Structures

• Found outside of the cell wall

Glycocalyx

• Capsule, slime layer, or extracellular polysaccharide

• Gelatinous polysaccharide and/or polypeptide covering

Glycocalyx

• Enable adherence to surfaces

• Prevent desiccation

• May provide nutrients

Flagella

• Relatively long filamentous appendages

• Consist of:– Filament – Hook– Basal body

Flagella

• Prokaryotic flagella rotate to push the cell

• Motile bacteria exhibit taxis– Positive taxis is movement towards an attractant– Negative taxis is movement away from a repellant

Axial Filaments

• Also called an endoflagellum

• Spiral cells that move with an axial filament are called spirochetes

• Similar to flagella, but wrap completely around the cell

Fimbriae and Pili

• Short thin appendages

• Fimbriae – help cells adhere to surfaces

• Pili – join cells together for the transfer of DNA from one cell to another

The Cell Wall

• Outer protective layer

Composition and Characteristics

• Surrounds the cell membrane and protects the cell from changes in water pressure

• Consists of peptidoglycan (a polymer consisting of short chains of amino acids)

• Penicillin interferes with peptidoglycan synthesis

Composition and Characteristics

• Gram-positive cell walls contain many layers of peptidoglycan and also contain teichoic acid

• Gram-negative bacteria have a lipoprotein-lipopolysaccharide-phospholipid outer membrane surrounding a thin peptidoglycan layer

Composition and Characteristics

• The outer membrane protects the cell from phagocytosis and from penicillin, lysozyme, and other chemicals

Composition and Characteristics

• Porins – proteins that allow small molecules to pass through the outer membrane

Composition and Characteristics

• Channel proteins – allow specific other molecules to move through the outer membrane

Composition and Characteristics

• The lipopolysaccharide component of the outer membrane consists of sugars that function as antigens

• Also has lipid A, which is an endotoxin

Cell Walls and Gram Staining

• The crystal violet – iodine complex combines with peptidoglycan

• The decolorizer removes the lipid outer membrane of gram-negative bacteria and washes the crystal violet out

Atypical Cell Walls

• Mycoplasm is a bacterial genus that naturally lacks cell walls

• Archaea have pseudomurein (rather than peptidoglycan)

Damage to Cell Walls

• Lysozyme destroys gram-positive cell walls– The remaining cell contents are called a protoplast

• Lysozyme damages gram-negative cell walls, but does not destroy them– The remaining cell contents are called a

spheroplast

Damage to Cell Walls

• Protoplasts and spheroplasts are susceptible to osmotic lysis

• Antibiotics interfere with cell wall synthesis

Internal Structures

• Found within the cell wall

Plasma Membrane

• Encloses the cytoplasm• Is a phospholipid bilayer• Has protein imbedded (fluid mosaic model)• Selectively permeable

Plasma Membrane

• Carries enzymes for metabolic reactions– Nutrient breakdown– Energy production– Photosynthesis

• For photosynthesis these enzymes are located in infoldings of the plasma membrane that extend into the cytoplasm called thylakoids (or chromatophores)

Plasma Membrane

• Some plasma membranes contain one or more large irregular folds, called mesosomes

• The function of these is much debated, but it is currently thought that they are artifacts, not true cell structures

• Thought that they develop by the process used for preparing the specimen for electron microscopy

Plasma Membrane

• Can be destroyed by alcohols and polymixins– Disrupt the membrane’s phospholipid bilayer– Cause leakage of internal contents

Movement of Materials

• May be passive– From high concentration to low– No energy expended– Continues until equilibrium is reached

• May be facilitated diffusion– Molecules are carried across membranes by

carrier proteins (permeases)– Still from high to low concentration

Movement of Materials

• Osmosis– Movement of water – Selectively semipermeable membrane– Continues until equilibrium is reached

• Active transport– Materials move from low concentration to high– The cell must expend energy

Cytoplasm

• The fluid component inside the plasma membrane

• Contains: – Mostly water (80%)– Inorganic and organic molecules– DNA– Ribosomes– Inclusions (reserve deposits)

The Nuclear Area

• Contains the DNA of the bacterial chromosome

• Can also contain plasmids– Extra chromosomal DNA circles

Ribosomes

• The cytoplasm of a prokaryote contains many 70S ribosomes

• Consist of rRNA and protein

• Location of protein synthesis

• Can be inhibited by certain antibiotics

Inclusions

• Reserve deposits• Found in both prokaryotic and eukaryotic cells• Include: – Metachromatic granules (inorganic phosphate)– Polysaccharide granules (glycogen or starch)– Lipid inclusions– Sulfur granules– Carboxysomes (e.g. ribulose)– Magnetosomes (Fe3O4)– Gas vacuoles

Endospores

• Resting structures formed by some bacteria• Allow for survival during harsh environmental

conditions

• The formation process is called sporulation• Return process is called germination

Eukaryotic Cells

• Have membrane bound organelles• Some eukaryotic organisms can still be

unicellular

Flagella and Cilia

• Flagella:– Few and long in relation to cell size

• Cilia:– Numerous and short– Move substances along surface of the cell

• Used for motility• Consist of an arrangement of 9 pairs and 2

single microtubules

Cell Wall and Glycocalyx

• The cell walls of many algae and some fungi contain cellulose

• The main material of fungal cell walls is chitin• Yeast cell walls consist of glucan and mannan• Animal cells are surrounded by a glycocalyx – Strengthens the cell– Provides means of attachment to other cells

Plasma Membrane

• Similar to the prokaryotic membrane– Phospholipid bilayer with proteins

• Contain carbohydrates attached to the proteins

• Contain sterols• Can move molecules through endocytosis– Phagocytosis– Pinocytosis

Cytoplasm

• Includes everything inside the plasma membrane and external to the nucleus

• Resembles chemical composition of prokaryotic cytoplasm

• Has a cytoskeleton• Exhibits cytoplasmic streaming

Organelles

• Specialized membrane enclosed structures

• Located in the cytoplasm of eukaryotic cells

Organelles

• Nucleus – – Contains DNA– Most characteristic organelles

• Nuclear envelope – – Connected to a system of parallel membranes

called the endoplasmic reticulum

Organelles

• Endoplasmic reticulum – – Provides a surface for chemical reactions– Serves as a transporting network– Stores synthesized molecules– Location of lipid synthesis

• Rough endoplasmic reticulum – – Location of protein synthesis– Location of protein transport

Organelles

• Ribosomes – – 80S ribosomes found in the cytoplasm or attached

to the rough ER

• Golgi complex – – Flattened sacs (called cisterns)– Functions in membrane formation– Protein secretion

Organelles

• Mitochondria – – Primary sites of ATP production– Contain 70S ribosomes and DNA– Multiply by fission

• Chloroplasts – – Contain chlorophyll and enzymes– Contain 70S ribosomes and DNA– Multiply by fission

Organelles

• Lysosomes – – Formed from Golgi complexes– Store powerful digestive enzymes

• Centrioles – – Pair of cylindrical structures– Involved in cell division– Found near the nucleus

Organelles

• Vacuoles – – Membrane enclosed cavities– Derived from Golgi complex– Usually found in plant cells– Store various substances– Bring food into the cell– Increase cell size– Provide rigidity to leaves and stem

Evolution of Eukaryotes

• Endosymbiont theory• Organelles evolved from symbiotic

prokaryotes living inside other prokaryotic cells