exam 1

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microbiology

study of organisms too small to be seen w the human eye

what are the sub disciplines of microbiology?

• bacteriology
• mycology
• food microbiology
• environmental microbiology
• forensic microbiology
• virology
• parasitology

when was microbiology born and by who?

• 1674
• anthony van leeuwenhoek saw bacteria/protozoa with his homemade microscope. called organisms “animacules”. used lens to peer into a drop of lake water.
• robert hooke was the first to see a microorganism. observed “microscopical mushroom”. later identified as common bread mold.

theory of spontaneous generation

• theory of how microorganisms originated
• “organisms can arise spontaneously from non living matter”

who were the 3 detractors of the theory of spontaneous generation?

• francesco redo
• louis pasteur
• john tyndall

louis pasteur

• developed the swan necked flask
• father of modern microbiology
• showed air is filled w/ microorganisms in 1861
  • proved by filtering air through a cotton plug, trapping microorganisms
  • identified organisms in cotton as same organisms contaminating infusions

ferdinand cohn

german botanist that discovered endospores in 1876

robert koch

established the role of endospores in disease transmission

what was anthrax caused by?

bacillus anthracis

microbes

• responsible for the production of oxygen and nitrogen
• key elements for all living organisms

why are microorganisms decomposers?

bc they are responsible for the breakdown of wide variety of materials

bioremediation

use organisms to degrade environmental waste

• clean up oil spills
• treat radioactive waste
• degrade PCBs, DDT

what products can bacteria synthesize?

• ethanol
• pesticides
• antibiotics
• dietary amino acids

genetic engineering

introduce genes of one organisms into an unrelated organism to confer new properties on the organism

golden age of microbiology

• 1854 - 1918
• time of great interest in the study of microorganisms
• lead to the initiation of prevention and treatment of disease

what are factors associated w emerging diseases?

• changing lifestyles
• genetic changes in organisms

reasons for resurgence of old diseases

• increased travel
• unvaccinated individuals susceptible to infection
• increase in immune compromised population

example of chronic disease caused by microbes

gastric ulcers (H.pylori)

why are host-bacterial interactions beneficial?

• simulate immune system
• outnumber cells in the body 10:1
• keep disease causing organisms from breaching hose defenses
• estimated 10k species of bacteria reside in and on the body

why are microorganisms a great model to study?

• metabolism same as higher forms of life
• genetic properties mimic other organisms
• building blocks of macromolecules same as other life forms

what is a domain?

a group in which all living things (organisms) can be classifies in

what are the 3 domains?

• bacteria
• archaea
• eukarya

characteristics of bacteria

• unicellular
• prokaryotic
• lacks nucleus and membrane-bound organelles
• has peptidoglycan

characteristics or archaea

• unicellular
• prokaryotic
• can live in extreme environmental conditions
• lacks peptidoglycan

characteristics of eukarya

• true nucleus and membrane bound organelles
• contains chromosomes
• algae can be unicellular or multicellular
• protozoa is unicellular (protists)
• fungi can be uni or multi
• helminths can be multi or parasitic

infectious agents (non-living)

• viruses
• viroids
• prions
• usually consist of only a few molecules found i living cells
• called agents not organisms

prokaryotes

• pre - nucleus
• “pro” “karyote”

bacteria and archaea microbial world

• both single celled organisms
• contain no membrane bound nucleus or organelles
• DNA stores in nucleoid (clump of DNA)
• cytoplasm is surrounded by rigid cell wall

eukaryote

true nucleus

“eu” “karyote”

eukarya microbial world

• organisms that contain membrane bound nucleus
• contain internal organelles
  • makes organism more complex
  • ex: mitochondria
• may be single/uni or multicellular

domain bacteria

• common in human infection, widely diverse
• prominent features:
  • specific shapes (rod, spherical, spiral)
  • rigid cell walls, responsible for cell shape and contain peptidoglycan
  • multiply by binary fission, one cell divides into two and cells are genetically identical to the first
  • some bacteria are motile and move by means of flagella

domain archaea

• cell wall lacks peptidoglycan
• same shapes as bacteria
• multiplies by binary fission
• moves by means of flagellum(archaellum)
• found in extreme temperatures and environmental conditions (ex:high conc. of salt)

domain eukarya - algae

• diverse group that includes single and multicellular organisms
• all contain chloroplasts
  • structure used to absorb light to be converted into energy
• usually found near surface waters
• contain rigid cell walls
  • distinct from bacterial cell walls (contain polysaccharides and glycoproteins)

domain eukarya - fungi

• diverse single and multicellular organisms
  • single/uni cellular = yeast
  • multicellular = molds
• gain energy from organic materials
• decomposers
• mostly found on land

domain eukarya - protozoa

• single/uni cellular organisms
• found in water and on land
• complex
• larger than prokaryotes
• lacks rigid cell wall
• gains energy from organic matter
• most are motile
  • means of motility is diverse and a feature of classification

domain eukarya - helminths

• multicellular parasites
• derive nutrients from host organisms
• include round and tapeworms

nomenclature

• binomial naming system
• first word is genus and always capitalized, often abbreviated
• second word is species, not capitalized
• ex: E. coli
• when writing out full name, its always italicized or underlined

viruses

• contain certain protein coat surrounding nucleic acid
  • essentially protein bag of nucleic acid
• viruses termed ‘obligate intracellular parasites”
  • must have host machinery to replicate
  • inactive outside of host
• all forms of life can be infected by viruses
• frequently kill hosts
  • some live harmoniously with host

viroids

• smaller and simpler than viruses but require host cell for replication
• consist of a single short piece of RNA
  • contain no protective protein coat
• generally cause diseases in plants

prions

• infectious proteins that have no nucleic acid
• responsible for 6 neurodegenerative diseases
• Animal disease
  • scrapie in sheep
  • mad cow disease in cattle
• Human disease
  • kuru (contaminated brain tissue, found amongst population that practices cannibalism)
  • creutzfeldt jakob (brain tissue)

size of microbial world

• large range
  • smalles virus approx. 1/1,000,000th size of largest eukaryotic cell
• basic unit of length is a meter (m) and all other units are fractions of a meter

light microscopy

• light passes through specimen, then through series of magnifying lenses
• most common and easiest to use, bright-field microscope

magnification of light microscopy

• microscope has 2 magnifying lenses
  • ocular lens and objective lens
  • called compound microscope
• lenses combine to enlarge objects
  • 10x(100x) = 1000x

resolution of light microscopy

• usefulness of microscope depends on its ability to resolve two objects that are very close together
• enhanced with lenses of higher magnification (100x) and by the use of immersion oil
  • oil reduces light refraction (bending or light)

contrast of light microscopy

• reflects the number of visible shades in a specimen
• higher contrast achieved for microscopy through specimen staining
• light microscopes that increase contrast
  • phase contrast
  • interference
  • dark-field
  • fluorescence
  • confocal scanning laser

phase contrast microscope

• amplifies differences between refractive indexes of cells and surrounding medium
• uses set of rings and diaphragms to achieve resolution

fluorescence microscope

• used to observe organisms that are naturally fluorescent or are tagged with fluorescent dye
  • fluorescent molecule absorbs ultraviolet light and emits visible light
  • image fluoresces on dark background

confocal scanning laser microscope

• constructs 3D image of thicker structures
• provides detailed sectional views of internal structures of an intact organism

electron microscope

• uses electromagnetic lenses, electrons and fluorescent screen to produce image
• resolution increased 1000 fold over brightfield microscope
• magnification increased to 100,000x
• 2 types of electron microscopes
  • transmission- internal structures
  • scanning- surface

transmission electron microscope (TEM)

• used to observe fine detail
• directs beam of electrons of specimen
  • electrons pass through or scatter at surface
  • shows dark and light areas
• specimen preparation through
  • thin sectioning
  • freeze fracturing or freeze etching

scanning electron microscope (SEM)

• used to observe surface detail
• beam of electrons scan surface of specimen
  • specimen coated with metal, usually gold
• electrons are released and reflected into viewing chamber
• some atomic microscopes capable of seeing single atoms

dyes and staining

• cells are frequently stained to observe organisms
• dyes carry + or - charge
  • molecules bind to certain cell structures
• dyes divided into basic or acidic based on charge

basic dyes

• carry a + charge and bond to cell structures that contain - charge
  • commonly stain the cell
• more commonly used than acidic dyes
• common basic dyes:
  • methylene blue
  • crystal violet
  • safranin
  • malachite green

acidic dyes

• carry a - charge and repelled by cell structures that contain - charge
• commonly stain the BACKGROUND

staining procedures

• simple stain uses one basic stain to stain the cell
  • allows for increased contrast btwn cell and background
  • all cells stained the same color
  • no differentiation btwn cell types

differential stains

• used to distinguish one bacterial group from another
• uses a series of reagents
• 2 most common differential stains:
  • gram stain
  • acid fast stain

gram stain

• most widely used procedure for staining bacteria
• developed over a century ago- Dr. Hans Christian Gram
• bacteria separated into 2 groups:
  • gram +, stained purple/blue
  • gram -, stained red/pink

4 reagents in gram stain

• primary stain
  • crystal violet: stains the cell
• mordant
  • grams iodine: holds primary dye to cell
• decolorizer
  • removes primary dye from gram - cell
• counter/secondary stain
  • recolors cells that lose stain through decolorization

acid fast stain

• stains organisms that resist conventional staining
• used to stain members of genus mycobacterium (TB, hansen’s disease)
  • high lipid concentration in cell wall prevents uptake of dye therefore harsh methods are needed to stain these organisms
  • once stained, these cells are very resistant against decolorizers

capsule stain

• example of negative stain: india ink
• allows capsule to stand out around organism

endospore stain

• staining enhances endospore
• uses heat to facilitate staining

flagella stain

• staining increases diameter of flagella
• makes it more visible

shapes of prokaryotic cells

• coccus
  • spherical
• bacillus
  • rod or cylinder, not to be confused with genus
• coccobacillus
  • short, round rod
• vibrio
  • curved rod
• spirillum
  • spiral
• spirochete
  • helical
• pleomorphic
  • ability to vary in shape

morphology of prokaryotic cells

• division along a single plane that may result in pairs or chains of cells
  • pairs: diplococci
  • chains: streptococii
• division along two or three perpendicular planes from cuboidal packets
• division along several random planes form clusters

multicellular associations

• ex: myxobacteria
• when conditions are favorable, these organisms secrete a slime layer that allows the formation of a swarm of cells
  • allows for release of enzymes which degrade organic material
  • in the absence of water or nutrients the cells come together to form a fruiting body

biofilms

• cells within biofilms alter their activities when a critical number is reached (staphylococcus and pseudomonas); dental plaque

cytoplasmic membrane

• delicate thin fluid structure surrounding cytoplasm of cell
• defines boundary
• serves as semipermeable barrier
  • barrier btwn internal and external environment

what is the structure of a cytoplasmic membrane

• a lipid bilayer w/ embedded proteins
• bilayers consists of two opposing leaflets
  • leaflets composed of phospholipids, each contain a hydrophilic phosphate head (- charge) and hydrophobic fatty acid tail

proteins embedded in a cytoplasmic membrane

• proteins fxn as receptors and transport gates
  • integral proteins- span membrane
  • peripheral proteins- on periphery either inside or outside of membrane
• provides mechanism to sense surroundings
• proteins are not stationary
  • constantly changing position, called fluid mosaic model

is the cytoplasmic membrane is selectively permeable? (T/F)

T

simple diffusion

• process by which molecules moved freely across the cytoplasmic membrane down a concentration gradient (high to low)
• water, certain gasses, small alcohols, small fatty acids and uncharged molecules pass through via simple diffusion

osmosis

• ability of water to flow freely across the semi permeable cytoplasmic membrane, usually through trans-membrane channels
• water flows to equalize solute concentrations inside and outside the cell

directed movement in a cytoplasmic membrane

• movement of many molecules directed by transport systems
  • transport systems employ highly selective proteins, transport proteins

transport protein- cytoplasmic membrane

• permeases or carriers
  • these proteins span membrane
  • single carrier gen transports specific type molecules
• most transport proteins are produced in a response to need

transport systems in cytoplasmic membrane

• facilitated diffusion
• active transport
• group translocation

facilitated diffusion

• rarely used by prokaryotes
• moves compounds across membrane, exploiting a concentration gradient
• via protein channel and carrier proteins

active transport

• moves compound against a concentration
• requires energy, "going up-hill”

2 primary mechanisms in active transport

• those that use proton motive force
  • MFS (major facilitator superfamily): as proton is brought in, another substance is either brought in or pumped out (ex: efflux pumps)
• those that use ATP
  • require ATP as energy source
  • binding proteins residing outside of the cytoplasmic membrane scavenge and deliver a given molecule to a specific transport complex within the membrane

proton motive force- cytoplasmic membrane

• transporters allow protons into cell
  • protons either bring in or expel other substances
• ex: efflux pumps used in antimicrobial resistance

ATP binding cassette system - CM

• use binding proteins to scavenge and deliver molecules to transport complex
• requires energy in form of ATP
• ex: maltose transport

group translocation- CM

• transport mechanism that chemically alters molecule during passage
  • uptake of molecules does not alter concentration gradient

phosphotransferase system- CM

• example of group transort system

• phosphorylates sugar (ex: glucose) molecule during transport

• phosphorylation changes molecule and therefore doesn’t change sugar balance across the membrane

• energy expended to phosphorylate the sugar is later regained (glycolysis)

secretion- CM

• primary mechanism used to secrete proteins synthesized by the cell
• recognizes “signal sequence”

signal sequence

• serves as a tag marking those proteins destines for secretion
  • signal sequence removed during process of secretion

bacterial cell wall

• rigid structure
• surrounds cytoplasmic membrane
• determines shape of bacteria
• holds cell together
• prevents cell from bursting
• unique chemical structure
  • distinguishes gram + from gram -

rigidity of cell wall

• due to peptidoglycan (PTG)
• only found in bacteria

basic structure of peptidoglycan

• alternating series of two subunits
  • NAG and NAM
• joined subunits form glycan chain
• glycan chain held together by string of four amino acids
• tetrapeptide chain

- joined directly in gram + bacteria

-joined indirectly by peptide interbridge in gram + bacteria

gram + cell wall

• relatively thick layer of PTG
  • PTG is permeable to numerous substances
• (lipo)teichoic acid component of PTG
  • gives cell - charge
  • antigenic and induces immune responses that are species specific
  • lipoteichoic acids are linked to cytoplasmic membrane

gram - cell wall

• more complex than gram +
• only contains thin layer of PTG
  • PTG sandwiched btwn outer membrane and cytoplasmic membrane
  • region btwn outer membrane and cytoplasmic membrane is called periplasm
• most secreted proteins contained here
• proteins of ABC transport system located here

outer membrane- gram -

• constructed of lipid bilayer
  • outer leaflet made of lipopolysaccharides
  • outer membrane called the lipopolysaccharide layer or LPS
• LPS serves as barrier to a larger # of molecules
  • small molecules or ions pass through channels called porins

what are the portions of LPS medically significant?

• o specific polysaccharide side chain
• lipid A

o specific polysaccharide side chain- outer membrane, gram -

• directed away from membrane
  • opposite location of lipid A
• used to identify certain species or strains

lipid A- outer membrane (gram -)

• portion that anchors LPS molecule in lipid bilayer
• plays role in recognition of infection (endotoxin)
  • molecule present w/ gram - infection of bloodstream

PTG as a target- cell wall

• many antimicrobials interfere w/ the synthesis of PTG
• examples include penicillin and lysozyme
• produced in many body fluids (tears/saliva)
• breaks bond linking NAG and NAM
• destroys structural integrity of cell wall

penicillin- cell wall

• binds proteins involved in cell wall synthesis
• bind to and inhibit enzymes involved in cell wall synthesis (cross linking of peptidoglycan)
• more effective against gram + bacterium
• outer membrane of gram - prevents medication from reaching site of action
• derivatives produced to protect against gram -

cell wall of gram +

cell wall of gram -

differences in cell wall

• gram + bacteria retain crystal violet iodine complex of gram stain
• gram - bacteria lose crystal violet iodine complex

what are the external layers to the cell wall?

• capsules and slime layer

capsules and slime layer

• capsule is a distinct gelatinous layer
• slime layer is irregular diffused layer
• chemical composition of capsules and slime layers varies depending on bacterial species
  • most are made of polysaccharides