• smallest: nanometers (viruses)
• normal: usually less than 1 mm
• largest: 200 micrometers

• most populous & diverse group
• recycle essential elements
• source of nutrients
• may cause disease
• can produce antibiotics

Prokaryotic

• lack true nucleus
• no membrane-bound organelles

Eukaryotic

• membrane-enclosed nucleus
• more morphologically complex
• larger than prokaryotic

1. Bacteria
2. Archaea
3. Eukarya

(described by Woese)

• single-celled
• cell wall w/ peptidoglycan
• found everywhere, including extreme environments
• cyanobacteria

• unique rRNA sequences
• no peptidoglycan in cell wall
• unique membrane lipids
• unusual metabolic characteristics
• extreme environments

• viruses
• viroids
• virusoids
• prions

• nucleic acid + protein
• smallest of microbes
• requires host to replicate
• disease-causing

• both made of nucleic acid
• infectious agents composed of RNA

• infectious proteins
• cause progessive degeneration of CNS
• normal brain proteins that misfold & aggregate

What were the earliest molecules?

• rRNA, tRNA, mRNA
• protein synthesis
• precursor to DNA
• ATP: ribonucleotide for energy storage
• regulate gene expression

• photosynthetic bacteria (stomatolites of cyanobacteria) producing O₂ leads to evolution of aerobic microbes
• 3.5 billion yrs ago

Who did all of the following:

• disproved spontaneous generation
• developed pasteurization
• studied fermentation
• proposed germ theory of disease
• developed vaccine for rabies

Who did the following:

• proved Bacillus anthracis caused anthrax
• proved germ theory of disease by use of 4 postulates

1. microbe present in every case of disease but absent from healthy ppl
2. microbe must be isolated & grown in pure culture
3. same disease must result when microbe inoculated into healthy host
4. same microbe isolated again from diseased host

• individual groups of microbes
• microbial physiology
• genetics
• molecular biology
• taxonomy

practice problems:

• disease
• water
• food
• industrial

Second Golden Age of Microbiology led to what discoveries?

• restriction of endonucleases
• recombinant DNA
• DNA sequencing
• bioinformatics

enzymes that catalyze biochemical reactions in cell nucleotide; ATP main reservoir for chemical energy

polypeptide = polymer of amino acids

measure of how greatly substance slows velocity of light

(light is refracted/bent when passing b/n mediums)

• creates dark image against bright background
• several objective lenses
• total magnification = ocular lens x objective lens

• converts differences in refractive index of components & cell density into variations of light intensity
• good way to observe living cells (i.e. motility & endospores)

• detects differences in refractive index & specimen thickness
• good way to observe living cells (live cells appear colored & 3D)

• specimens stained with fluorochromes that absorb light & reemit it
• bright image of object b/c of fluorescent light
• good for localizing specific proteins
• good for medical microbiology & microbial ecology

• confocal scanning laser microscopy (CSLM) creates sharp, composite 3D image
• uses laser beam & computer interface

ocular lens: 10X

objective lenses: 4X, 10X, 45X, 100X

uses beams of electrons focused by magnets; electron beam wavelength more shorter than light leading to higher resolution

2 types:

transmission - look at contents of cell

scanning - look at surface of cells

• electrons reflected from surface of specimen
• produces 3D image of surface features

• increases visibility
• accentuates morphological features
• preserves specimen

• single stain used
• can determine size, shape, & arrangement
• ionizable stains have charged groups

basic stains: + charge

acid stains: - charge

• microbes divided into groups based on staining properties (i.e. Gram or acid-fast)
• detects prescence or absence of structures

• preserves internal & external structures & fixes in position
• organisms killed & attached to slide

heat fixation: routine w/ bacteria & archaea (preserves morph, not internal structures)

chemical fixation: for larger, more delicate organisms (preserves fine cellular substructure & morph)

• most widely used differential stain
• bacteria divided into Gram + or Gram - based on cell wall structure
• crystal violet stains G + purple & G - pink

bacteria have thin cell walls & outer membrane containing LPS

(LPS = lipopolysaccharide: pyrogen that causes fever)

• carbol fuchsin stain mycobacteria hot pink & methylene blue stains other bacteria blue
• used when high lipid content in walls (mycolic acid)
• useful for diagnosis of tuberculosis and leprosy

• electons scatter when passed through thin sections of specimen
• transmitted electrons under vacuum; reduces scatter & clearer
• denser regions scatter more electrons; darker

• scanning tunneling microscope
• magnification 100 million X
• steady current b/n probe & specimen
• up & down movement creates image of specimen surface

Bacterial & Archaea Function & Structure:

• the two groups of prokaryotes
• differ in size, simplicity, shape, & arrangement

Cocci

(s. Coccus)

sheres

(1 of 2 most common)

Diplococci

(s. Diplococcus)

Bacilli

(s. Bacillus)

rods

(1 of 2 most common)

Spirilla

(s. Spirillum)

organisms that are variable in shape

(i.e. Archaea)

• requirement for living organism
• encompasses cytoplasm
• selectively permeable
• can detect/respond to chemicals in surroundings
• important for transport & metabolic processes

• composition of lipid bilayer
• polar hydrophilic end interacts w/ water
• nonpolar hydrophobic tails insoluble in water

rigid structure that lies just outside cell membrane

Gram + : thick peptidoglycan

Gram - : think peptidoglycan & outer membrane

1. maintains shape of bacterium
2. helps protect from osmotic lyses

• meshlike polymer of identical subunits forming long strands
• 2 alternating sugars of N-acetylglucosamine (NAG) & N-acetylmuramic acid
• alternating D- & L- amino acids

• composed primarily of peptidoglycan
• can contain teichoic acids
• periplamic space lies b/n plasma membrane & cell wall; smaller than gram -

• peptidoglycan 5-10% cell wall weight (thin layer thats surrounded by outer membrane)
• no teichoic acids
• more complex than gram +
• periplasmic space 20-40% of cell volume & contains many enzymes

1. Lipid A...embedded in outer membrane
2. core polysaccharide...extend out from cell
3. O side chain (O antigen)...extend out from cell

• stabilizes outer membrane structure
• contributes to attachment to surfaces & biofilm formation
• act as endtoxin

Mechanism of Gram Stain Reaction:

• shrinkage of pores of peptidoglycan of Gram + prevents loss of crystal violet during decolorization
• thinner peptidoglycan & larger pores does not prevent loss of crystal violet for Gram -

solute concentration outside cell less than inside cell; water moves into cell & cell swells

cell wall protects from lysis

Mycoplasma

• no cell wall
• plasma membrane more resistant to osmotic pressure

Components Outside of Cell Wall:

• outermost layer is cell envelope
• glycocalyx (capsules & slime layers)
• aid in attachment to solid surfaces

• usually made of polysaccharides
• well organized, not easily removed
• visible in light microscope
• protect from phagocytosis, dessication, & exclude viruses & detergents

• diffuse
• unorganized
• easily removed
• aid in motility

differ from bacterial in molecular makeup & organization

can lack cell wall & capsules/slime layers rare

• made of unique lipids (isoprene units & ether linkages instead of ester to glycerol)
• may have monolayer instead of bilayer

differ from bacterial

• lack peptidoglycan
• pseudomurein may be outermost layer

plasma membrane & everything w/n

structure of bacteria & archaea

material bounded by plasma membrane

structure of bacteria & archaea

cell divsion, protein localization, determination of cell shape

Eukaryotic homologs: 3 Bacteria & 2 Archaea

granules of organic or inorganic material that are stockpiled by cell for future use

can also be gas vacuoles found in aquatic, photosynthetic bacteria/archaea that provide buoyancy

• extrachromosomal DNA
• small, closed circular DNA found in bacteria, archaea, & fungi
• exist & replicate independently of chromosome
• contain few non-essential genes
• may exist in many copies
• inherited stably during cell division

protection, attachment to surfaces, horizontal gene transfer, cell movement

pili, fimbriae, flagella

• short, thin, proteinaceious, hair-like structures on the surface
• function in attachment
• Sex pilus functions in DNA transfer from donor cell to recipient; longer thicker & less numerous; found on plasmids; required for conjugation

• long motility structures; threadlike locomotor appendage
• motility & swaming
• attachment to surfaces 

• thin, rigid protein structures; cannot be seen w/ bright field unless stained
• rotate like a propeller (counterclockwise = fwd)
• bacterial much smaller/simpler than eukaryoticc; powered by ATP; different mechanisms
• eukaryotic cilia identical in cross-section to eukaryotic flagella

move toward chemical attractants such as nutrients; away from harmful substances

changing concentrations bind chemoreceptors of chemosensing system

• motility structures in spirochetes (G -)
• nucleus contains DNA organized into pairs of chromosomes
• surrounded by nuclear envelope which has pores for transport b/n nucleus & cytoplasm

• multiple flageela form axial fibril that winds around cell
• flagella in periplasmic space outside outer sheath
• corkscrew shape exhibits flexing & spinning

• complex, dormant structure
• thin spore covering is exosporium
• spore coat is thick layers of protein
• core has nucleoid & ribosomes
• various locations
• resistant to heat, radiation, chemicals, & dessication

• process of endospore formation in hours (max 10)
• stops when growth ceases due to lack of nutrients
• complex multistage process

protists

fungi

• plasma membrane (lipid bilayer) and all coverings external to it
• membrane lipids include phosphoglycerides &cholesterol
• cell walls of algae have cellulose, pectin, silica
• cell walls of fungi have cellulose, chitin, glucan

• complex of membranous organelles & vesicles that move material into cell, out of cell, & within
• involves Golgi apparatus, Endoplasmic Reticulum, & lysosomes

• made up of liquid (cytosol), organelles, & cytoskeleton
• filaments of cytoskeleton are microfilaments (actin), microtubules (tubulin), & intermediate filaments

• irregular network of branching & fusing membranous tubules & flattened sacs
• Rough: ribosomes attached, synthesis of secreted & membrane proteins
• Smooth: no ribosomes, synthesis of lipids

• modification, packaging, & secretion of materials (exocytosis)
• cis and trans faces

• brings materials into cell
• solutes/particles taken up & enclosed in vesicles from from plasma membrane (endosomes)
• endosomes fuse w/ lysosomes enabling hydrolysis of contents
• Eukarya only

• Phagocytosis - use of cell surface protrusions to surround & engulf particles; fuse w/ lysosomes
• Pinocytosis - cell "drinking"

• membrane-bound vesicles used for intracellular digestion ("cellular stomach")
• contain hydrolases; hydrolyze molecules & work best in acid
• maintain acidic environment by pumping protons into interior

• membrane-bound spherical structure that houses genetic material of eukaryote (double membrane)
• contains dense, fibrous chromatin (DNA, histones, & proteins...condenses into chromosomes during division)
• continuous with ER
• penetrated by nuclear pores

• not membrane-bound organelle
• important in ribosome synthesis
• directs synthesis/processing of rRNA & ribosomal subunits

• 80s in size (with 60S + 40S subunits)
• larger than 70S bacterial/archaeal ribosomes (50S + 30S)
• may be attached to RER (proteins secreted into ER) or free in cytoplasm (nonsecretory/nonmembrane proteins)

• "power house of the cell"
• site of tricarboxylic acid cycle activity
• where ATP is generated by electron transport & oxidative phosphorylation
• reproduce by binary fission like bacterial

• outer membrane (w/ porins)
• inner membrane (highly folded to form cristae)
• matrix enclosed by inner membrane contains ribosomes & circular mitochondrial DNA

• type of plasmid; pigment containing organelle in plants/algae
• site of photosynthetic rxns
• double membrane

• stroma w/n inner membrane that contains DNA, ribosomes, lipids, starch, & thylakoids)
• stroma = site of dark rxn of photosynthesis (carbs formed from water and CO₂)

• flattened, membrane-bound sacs
• stacks of thylakoidsare grana
• site of light rxns (traps light to generate ATP & O₂)

cilia: 5-20 micrometers; beat w/ 2 phases, work like oars

flagella: 100-2000 micrometers; move in undulating fashion

microtubules in 9+2 arrangement

• cytoplasm
• pellicle
• vacuoles
• energery conserving organelles (mitochondria, chloroplasts)
• cilia/flagella

• can be asexual or sexual
• micronucleus - genetic recombination
• macronucleus - trophic & regenerative activities

Characteristics of Fungal Structure:

• spore-bearing
• absorptive nutrition
• lacks chlorophyll
• cell encased in chitin
• single celled: yeast
• multicellular: macroscopic mold
• thread-like filaments called hyphae form mycelium

septate hyphae: discreet cells

nonseptate hyphae: multinucleated

• asexual : fungal spores help survival
• sexual : alternation of haploid & diploid stages; fusion of compatible nuclei

• major cause of disease
• important in evolution
•  model systems for molecular biology
• infects all cell types (bacterial viruses: bacteriophages; but most are eukaryotic)

• complete virus particle
• > 1 DNA or RNA enclosed in coat of protein
• cannot repoduce independent of living cell
• range from 10-400 m
• all contain nucleocapside of nucleic acid (DNA/RNA) 7 protein coat (capside)

• large macromolecular structures taht serve as protein coat of virus
• protect viral genetic material & aids in transfer b/n host cels
• made of protein subunits
• can be helical, icosahedral, or complex

Describe Viral Envelopes

• outer, flexible, membranous layer
• for animals is lipids & carbs
• proteins are viral encoded & can project from surface as spikes
• used for viral attachment to host, identification

• virus may have single or double stranded DNA/RNA
• size of nucleic acid and nature of genome varies
• can be segmented or circular

1. attachment to host cell
2. entry
3. uncoating of genome
4. synthesis
5. assembly
6. release

nonenveloped: lyse the host cell

enveloped: use budding; proteins incorporated into host, nucleocapsid bind to proteins

• virulent phage - 1 reproductive choice; multiplies immediately upon entry; lyses host
• temperate phage - 2 reproductive options; (1) reproduce lytically like virulent or (2) remain in cell w/o destroying...lysogeny

• bacteriophage/prophage - integrate genome into host
• lysogens/lysogenic bacteria - infected bacterial host appears normal but can switch from lysogenic to lytic

1. phage remains viable but cannot replicate
2. ensures survival of host cell

• cytocidal infections leads to cell death (lysis)
• persistent infections can last years
• transformation to malignant cell

growth or lump of tissue

benign tumors remain in place

• complex, multistep process that often involves oncogenes
• oncogenes = cancer causing genes that may come from virus or be transformed host protooncogenes

• cultivated in broth or agar for suitable, young actively growing bacteria
• broth loses turbidity as viruses reproduce
• plaques observed on agar

hole in monolayer of cells by 1 infectious virus particle; serial dilutions & plaque assays are used to measure # of infectious virus particles in preparation

localized area of celluar destruction & lysis that enlarges as virus replicates

PFU = plaque forming unit

• tissue (cell) cultures; cells infected w/ virus; viral plaques
• cytopathic effects
• embryonated eggs for animal viruses

• direct counting - count viral particles
• indirect counting by observable property of virus (like plaque assays)

• infectious agents; closed circular ssRNAs
• do not encode gene products
• needs host cell DNA-dependent RNA pol to replicate
• cause plant diseases

• formerly called satellite viruses
• covalently closed circular ssRNAs
• encode 1 or more gene products
• require  helper virus for replication

• degenerative diseases in humans & animals
• scarpie in sheep
• BSE or mad cow disease
• CJD or vCJD
• kuru

Difference b/n vCJD and CJD:

differ in origin

• variant creutzfeldt-jakob: eating meat from BSE infected cattle
• creutzfeldt-jakob: spontaneous mutation of gene that codes for prion protein

use organic molecules as carbon sources which often serve as energy source

can use variety of carbon sources

use carbon dioxide as sole or principal carbon source

must obtain energy from other sources

• needed for synthesis of important molecules like amino acids & nucleic acids
• nitrogen supplied in several ways
• phosphorus from inorganic phosphate
• sulfur from sulfate via reduction

• amino acids: protein synthesis
• purines & pyrimidines: nucleic acid synthesis
• vitamins: function as enzymes & cofactors

molecules move from region of higher concentration to one of lower concentration

water, oxygen, & carbon dioxide often move across membranes this way (simple diffusion)

• movement of molecules NOT energy dependent
• movement from high concentration to low concentration
• uses membrane bound carrier molecules (permeases)
• transports glycerol, sugars, & a.a.
• all microorganisms

• energy-dependent process (ATP or proton motive force used; pH gradient)
• move molecules against gradient
• concentrates molecules inside cell
• involves carrier proteins
• all microorganisms

• covalent modification of transported substance occurs simultaneously w/ transport
• energy dependent that chemically modifies molecule as its brought into cell
• PTS is example
• bacteria and archaea only

use ion gradients to cotransport substances

• symport - 2 substances both move in same direction
• antiport - 2 substances move in opposite direction

• solid or liquid preparation that has to contain all nutrients for organism to grow in lab
• dependent on chemical constituents, physical nautre, & function

supportive

enriched

selective

differential

Define supportive or general purpose media:

favor the growth of some microbes and inhibit growth of others

(i.e. MacConkey agar)

• population of cells arising from single cell (Koch)
• spread plate, streak plate, & pour plate

• involves spreading a mixture of cells on agar surface so individual cells are well separated from each other
• each cell reproduces to form colony
• isolate pure cultures of  bacteria from mixture

small volume of diluted mixture containing 30-300 cells is transferred & spread evenly over surface with sterile bent rod

count colonies to determine # of viable bacteria

sample serially diluted; mixed with liquid agar; mixture of cells & agar poured into sterile culture dishes

count colonies to determine # of viable bacteria

haploid only; asexual

binary fission, budding, filamentous

all must replicate and segregate the genome prior to division

icosahedrons

filamentous morphology

• bacterial chromosomes circular
• 1 origin of replication
• terminus (replication terminated) opposite of origin
• DNA replication proceeds in both directions
• replisome - group of proteins needed for DNA synthesis
• origins move to oppposite ends of cell

growth of population, not individual cells

inc in celluar constituents may cause inc in cell number & size

• cell synthesizing new components
• replenishes spent materials
• adapts to new medium
• varies in length

• also called log phase
• rate of growth & division is constant & maximal
• population most uniform in terms of chemical & physical properties

• closed system population growth eventually ceases
• total # of viable cells remains constant
• active cells stop reproducing or reproductive rate = death rate

• nutrient limitation
• limited oxygen available
• toxic waste accumulation
• critical population density reached

• time required for population to double in size
• varies depending on microbe & environment
• can be 10 min to several days
• population doubling every generation

1. counting chambers
2. electonic counters (flow cytometry)
3. membrane filters

• easy, inexpensive, quick
• can count eukaryotic & prokaryotic
• cannot distinguish b/n living & dead cells

• microbial suspension forced through small orifice w/ laser light beam
• impacts electric current
• instances of current disruption counted

• cells filtered through special membrane
• provides dark background to observe cells
• cells stained w/ fluorescent dyes
• can distinguish b/n living & dead cells w/ certain dyes
• bacteria from aquatic samples trapped on membranes w/ known pore size
• colony count determines # of bacteria in original sample

light scanning; used to measure cell mass

quick, easy, sensitive

• device for controlled, uniform growth of microbes for extended times
• rate of incoming medium = rate of removal of medium from vessel
•  essential nutrient is in limiting quantities

grow best in high concentrations of salt

grow optimally at >0.2M NaCl

microbes can't regulate internal temp

high temps may inhibit enzyme functioning

grows in presence of atomospheric oxygen which is 20%

obligate aerobe - requires oxygen

grows in absence of oxygen

obligate anaerobe - usually killed in presence of oxygen

• x rays & gamma
• mutation leads to death (sterilization)
• disrupts chemical structure & DNA
• can be repaired by DNA repair mechanism
• penetrates deep, destroys bacterial endospores but not effective against viruses

• mutations lead to death
• forms thymine dimers in DNA
• requires direct exposure on microbial surface
• can be repaired by DNA repair mechanism
• limited to surface sterilization; cannot penetrate many surfaces

Characteristics of Biofilms:

• complex, slime enclosed communities
• ubiquitous in nature in water
• most microbes grow attached to surfaces (sessile) than free floating (planktonic)

bacterial cells communicate in biofilms in density dependent manner

produce small proteins that inc in concentration as microbes replicate

killing, inhibition, or removal of disease causing organisms

disinfectants usually chemical agents used on inanimate objects

prevention of infection of living tissue by microorganisms

antiseptics are chemical agents taht kill/inhibit growth of microorganisms when applied to tissue

cidal agents kill

static agents inhibit growth

kills pathogens & many other nonpathogens but not necessarily endospores

-cide = agent kills

Conditions that influence antimicrobial activity:

• population size (larger take longer to kill)
• population composition (sensitivity differences)
• concentration/intensity of agent (higher conc. kills more rapidly; but not linear relationship)
• duration of exposure (longer exposure = more killed)
• temperature (higher temp = more killing)
• lcoal environment

• destroys viruses, fungi, & bacteria
• boiling doesn't destroy spores & doesn't sterilize
• degrades nucleic acids, denatures proteins, disrupts membranes

• above 100 C
• autoclave - steam + pressure kills everything
• effective against all types of microbes including endospores

• controlled heating at temps well below boiling
• used for milk, beer, & other beverages
• does not sterilize but kills pathogens & slows spoilage

• less effective than moist heat
• requires higher temp & longer exposure
• oxidizes cell constituents & denatures proteins

• reduces microbial population or sterilizes solns of heat sensitive material by removing microbes
• can be applied to liquids or air

porous membranes w/ define pore sizes; physical screening

replaced depth filters

Filtering Air

• surgical mask
• cotton plugs
• high-efficiency particulate air filter (HEPA) : used in laminar flow biological safety cabinets

• commonly used as lab & hospital disinfectants
• denature proteins & disrupt membranes

• most widely used disinfectant
• ethanol & isopropanol most common 
• bactericidal, fungicidal, not sporicidal
• inactivate some viruses

• skin antiseptic
• oxidizes cell constituents & iodinates proteins
• may kill spores

• oxidizes cell constituents
• disinfects water
• destroys vegetative bacteria & fungi
• gas is sporicidal
• can create carcinogenic compounds

• formaldehyde & glutaraldehyde
• reactive
• sporicidal