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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