Exam 1 Flashcards
what is a microbe?
a microbe is a living organism that requires a microscope to be seen (most diverse group of science)
- viruses are grouped with microbes because they infect all forms of life
examples of microbes
bacteria, archea, and eukaryotes (fungi algae, protozoa)
Where are microbes found?
everywhere! hydrothermal vents, salines, arctic marine sediment
why is the human microbiome useful?
digestion, to see good from bad
why study microbes?
they have shaped human culture since our earliest civilizations
positively: microbes produce 50% of the world’s oxygen and all its fermented foods (bread, beer, cheese)
negatively: disease that causes death and suffering
how do we know about microbes?
most knowledge was accumulated after 1900, driven by advances in microscopy and molecular techniques
application of microbes in environmental health
knowledge: microbes are key players in most elemental cycles
manage: use of microbes in biodegradation of pollutants, wastewater treatment plants
application of microbes in health
knowledge: microbes supply essential nutrients to hosts, but can also cause disease
manage: fighting infectious disease managing microbiome for better health
application of microbes in industry
knowledge: microbial metabolism can enrich and spoil foods
manage: use/control microbes in food, medicine and biofuel production
louis pasteur (pasteurization)
broth was boiled to kill all microbes, after a year, none appeared.
the flask was tipped to allow the broth to reach the microbes, microbes quickly multiplied
(showed microbes were not appearing out of nowhere, disproved spontaneous generation)
culture independent approach: sequencing
data led to identify domains:
bacteria
archaea (first life on earth)
eukarya
bacteria + archaea = similar membrane composition
archaea + bacteria = no nucleas, gene expression machinery
germ theory of disease
many diseases are caused by microbes
central dogma: Koch’s postulates
ability to isolate and culture bacteria is essential
koch’s postulates
ordered set of criteria for establishing a causative link between an infectious agent and a disease
1. suspected microbe is always present in disease hosts and absent in healthy hosts
2. suspected microbe is grown in pure culture outside hosts - no other microbes present in culture
3. cultured microbe is introduced into healthy hosts- individuals become sick with same disease as original hosts
4. same microbial suspect is re-isolated from sick individuals
Edward jenner
typically credited with developing first vaccination approach: deliberately infected patients with material he collected from cowpox lesions
lady Mary Mantagu
introduced the practice of smallpox inoculation to Europe in 1717, learned from people in Turkey and Africa
Lynn Margulis
proposed that eukaryotic organelles, such as mitochondria and chloroplasts, evolved by endosymbiosis from prokaryotic cells engulfed by preeukaryotes (mitochondria)
why was the endosymbiosis theory highly controversial?
implied more complex ancestry of living species through lateral exchange of genetic material, instead of vertical decent with modification
microscopy has revealed:
- Earth is a microbial world. There are no unexpected places…
- We are just as microbial as we are human
- Bacteria are beautiful!
why bother observing microbial cells?
to understand why these organisms cause these phenomena, a closer look can help
to understand how microorganisms interact with each other
size at which objects can be distinguished depends on…
density of photoreceptors of observer’s eye
resolution
the smallest distance by which two objects can be separated and still distinguishes
human eye
about 100-200 µm resolution
detection
ability to determine the presence of an object
magnification
an increase in the apparent size of an image to resolve smaller separations between objects. we can detect microbes in a culture or environment, but only resolve single cells by magnification (see separated from one another)
microbial characteristics
- the shape of the bacteria gives you information about the type of bacteria it could be
- how they are aggregated gives you more information on what it may be
colony characteristics (describing colony morphology has become primary step in microbial identification)
size, color, texture, elevation, form, margin (edge of colony)
microscopy at different size scales
different microscopes are required to resolve various cells and sub cellular structures
why are we unable to look directly through an electron microscope to see the objects it can resolve?
our bare eyes cannot see electrons
size of object impacts…
what wavelength or EMR is needed
goal to resolve vs. detect impacts…
if the microscopy method uses stains, fluorescence or ligh scattering
alive vs. dead cells to visualize impacts…
does the microscopy method require fixation and/or staining/hybridization?
eukaryotic microbes
protozoa, algae, fungi (10-100µm) can be seen under a light microscope
prokaryotic microbes
bacteria, archaea (0.4-10µm) sub cellular structures are typically too small to resolve by light microscopy
phages and viruses
mostly in 5-200nm range, cannot be resolved by light microscopy
electromagnetic radiation conditions to resolve an object
contrast - cytoplasm absorbs light similarly to water (often undetectable with standard bright field microscopy)
wavelength - can be maximum 2x size of the object (visible light 0.4-0.75 µm, so smallest object = 0.2 µm)
magnification - light rays must spread far enough to match our eyes resolution (about 200µm) for visible light up to 1,000X magnification possible while maintaining resolution
interaction of light with matter
absorption is important in visualizing objects by microscopy
magnification relies on refraction (bend)
scattering of light is key to dark field microscopy
magnification requires the bending of light rays
refraction
wavefronts of light shift direction as they enter a substance of higher refractive index
what happens when light rays enter glass with parabolic curvature (a lens)
parallel rays bend such that all rays meet at a certain point, called the focal point (more curvature = more magnified)
bright field microscopy
generates a dark image over a light background
to increase resolution on bright field microscopy….
- use shorter-wavelength light
-increase contrast
to increase lens quality…
-use multiple lenses in compound microscopes
capture more light waves by…
-using a wider lens closer to specimen (wider angle of light captured)
-higher refraction by medium between specimen and objective: use immersion oil
fixation and staining
+ cells remain in a fixed position (stuck to slide), increase contrast, ability to do differential staining, staining specific parts of cell
- cells are typically killed
wet mount preparation
+ observation of cells in natural state
- little contrast; sample dries out quickly
simple stain
adds dark color specifically to cells, but not to the external medium or surrounding tissue (methylene blue)
differential stain
stains one type of cell but not another (gram stain)
fluorescence microscopy
specimen absorbs light of a defined wavelength and then emits light of lower energy, thus longer wavelength
- can use stains of specific molecules
- can be used to identify specific bacteria or proteins (too small to resolve but due to bright fluorescence, we can detect)
chemical imaging
- chemical imaging uses mass spectrometry to visualize distribution of chemicals within microbes or living cells
- allows us to detect activity of cells
dark field and phase contrast microscopy
advanced optical techniques allow the visualization of structures that are difficult or impossible to detect under a bright field microscope (either because their size is below the limit of resolution of light or because their cytoplasm is transparent)\
-uses light waves, light scattering and phase contrast
dark field microscopy
uses scattered light for detection (can detect very narrow cells)
electron microscopy
most forefront tool for observing shapes of macromolecular structures
-electrons behave like light waves, short wavelength allows great resolution, sample must absorb electrons: fixation + coated with heavy metal
-CryoEM allows for unfixed and unstained samples to be imaged
transmission EM
-electrons pass through the specimen
-reveals internal structures
scanning EM
-electrons scan the specimen
-reveals external features in 3D (image on right with post-imaging addition of color)
why study bacterial cell structure/function?
to understand the mechanism of pathogenicity and develop drug targets
also, environmentally: to understand membrane structure and how species respond to changing temperatures, and why certain species become abundant
fundamental bacterial traits
-thick, complex outer envelope
-compact genome
-tightly coordinated cell functions (for efficiency)
prokaryotes: use membrane and envelope sturctures
cytoplasm
consists of a gel-like network packed with DNA, RNA, proteins, solutes
cell membrane (plasma membrane)
encloses the cytoplasm
cell wall
rigid structure external to the membrane, limiting cell’s expansion
nucleoid
non-membrane-bound area of the cytoplasm that contains the chromosome in the form of looped coils
specialized structures
includes flagellum and chemosensors for motility
gram-negative bacteria
a lot packed inside of the plasma membrane!
-cell envelope is key to preserve cell function and its interaction with hosts and the environment
biochemical composition of bacteria
water, proteins, nucleic acids (C, P, N), lipids, peptidoglycan, essential ions
purpose of essential ions in bacteria
helps proteins function
how do you separate different components?
centrifugation at very high speeds
-remove Mg and Ca from membrane and allow sucrose to cross
(mild detergent can dissolve membranes without denaturing proteins)
cell membrane
a selectively permeable barrier that separates the internal cellular components from the external environment
- have about equal volumes of phospholipids and proteins
role of proteins in cell membrane
structural support, detection of environmental signals, secretion of virulence factors and communication signals, ion transport and energy storage
phospholipid
glycerol with ester links to two hydrophobic fatty acids and hydrophilic phosphoryl head group
palmitic acid
saturated (solid butter)
oleic acid
trans double bond
oleic acid
cis double bond
cyclopropane fatty acid
tricyclic carbon
impact on fluidity from saturation
cyclized makes it more rigid or solid, double bonds make it more fluid
extremophile membranes
evolution has resulted in ether links (bent COC) - more stable between glycerol and fatty acids (very rigid and solid to live in extreme environments)
how do O2 and CO2 permeate through the membrane?
passive diffusion
how does water enter the cell?
diffusion via osmosis - sped up by aquaporins (passive)
how do membrane-permanent weak acids and weak bases pass through the membrane?
pass uncharged through the membrane then become charged inside (passive)
purpose of membrane proteins
structural support, detection of environmental signals, ….
active transport
uses energy
passive transport
facilitated diffusion; requires protein; no energy (uses concentration gradient)
what structural element of a bacteria and virus is targeted when washing your hands with soap to kill potential pathogens?
soaps form micelles, water dissolves the polar part of micelles and soap dissolves the none polar part of the dirt or sickness
how do prokaryotes protect the cell membrane?
bacteria are unique in that they have a peptidoglycan cell wall (prime target for antibiotics), once that link is destroyed it cannot bind to the cell wall
gram stain
differentiates between two types of bacteria- gram positive retain crystal violent stain because of their thicker cell wall, while gram negative do not
mycobacterial cell envelope
(mycobacterium tuberculosis and M. leprae) - has less permeability, grows much slower
complex cell envelopes:
-unusual membrane lipids
-unusual sugars
S-layer
- an additional protective layer commonly found in bacteria & archaea
- crystalline layer of thick subunits consisting of protein or glycoprotein
-contributes to cell shape and help protect the cell from osmotic process
the capsule
polysaccharide within the protein layer on bacteria, protects from phagocytosis (being eaten by the immune system), sticky and helps the cell to not dry out
passive diffusion
only of gases and weak acids/bases
what happened to molecules too large for transport?
they are first degraded via secreted proteins
coupled transport
symport/antiport
one substrate goes up its concentration gradient and the other goes down
ABC transport
in Gr+; the solute binding protein is attached, uses ATP
active transport: siderophores
?
active transport: group translocation
?
rotary flagella
spiral filament that is rotated by a proton motive force; the motor possesses an axle and rotary parts, all composed of specific proteins
chemotaxis
flagella rotate and propel themselves forwards and then fall down (run and tumble), they run towards a source or away from one
lipopolysaccharides of Gr-
-main outward facing leaflet in Gr- bacteria
-act as endotoxins
-as long as the cell is intact, its harmless
-if released by a lysed cell, can overstimulate host defenses and induce lethal shock
periplasm
quality control of the proteins, works with the peptidoglycan
outer membrane protein
porin
cytoplasm
anything enclosed by the cytoplasmic membrane
-nucleic acids, enzymes, amino acids, carbohydrates, lipids, inorganic ions, and low weight molecular compounds
mutations in the cytoskeleton
wild type have a defined rod shape (bacillus subtilis) or curved
-mutants cause shape changes
shape influencing proteins
cell division requires FtsZ - like our tubulin (Z-ring)
rod-shaped cell uses MreB
both of these influence cell shape, if mutated, cell may have severe issues
bacterial chromosomes
-single circular strands of DNA
-aggregated in a dense area called the nucleoid
-DNA is tightly coiled around basic protein molecules to fit into the cell compartment
transcription and translation happen…
simultaneously!
DNA is organized into loops called….
domains
plasmids
bonus DNA! (that are smaller)
-separated double stranded circles of DNA
-duplicated and passed onto offspring during replication
-confer protective traits
-important in genetic engineering
genetically engineered bacteria
-initially used to produce insulin
-now used for a variety of purposes (diagnosis or cure disease, regulation of the immune system)
ribosomes
made of RNA and protein, dispersed throughout the cytoplasm
(different sections of ribosomes are conserved through the core)
S units (ribosomes)
measurement of the relative size of cell parts through sedimentation through centrifugation
bacterial ribosomes: 70S
eukaryotic ribosomes: 80S
inclusion bodies
-storage sites for nutrients during periods of abundance
granules
a type of inclusion body
-contain crystals of inorganic compounds
-are not enclosed by membranes
pili and stalks
favorable habitat (adherence and attachment structures)
bacterial endospores
-withstand hostile conditions and facilitate survival (not all can make endospores)
two phase cycle
vegetative cell: metabolically active
endospore: inert, resting condition
sporulation
triggered by depletion of nutrients, takes 6-8 hours in most species
endospores can resist….
heating, drying, freezing, radiation, chemicals
replisome
DNA with accessory proteins; where replication begins
