Microbiology Test 1
In epi-fluorescence microscopy, the light used to excite fluorescent molecules associated with the specimen comes from _____ the stage.
Above
Aseptic technique from a liquid culture: Prevents contamination
Flaming the loop sterilizes it Tube cap is removed Flaming tube tip sterilizes the surface Only sterilized portion of loop enters tube Tube is reflamed Organisms on loop used to inoculate tube/plate containing sterile medium Tube is recapped, loop is resterilized
Bacteria at hydrothermal vents
extract energy from reduced organic compounds
Cytoplasmic Membrane
25-40% Phospholipid 60-75% Protein 20% of cells protein is found in cytoplasmic membrane Hexagonal sugar molecule Integral: Hydrophobic and hydrophilic interaction holds it in membrane Peripheral Hopanoids
Microorganism Habitat
66% found on marine subsurface 26% found in terrestrial subsurface 4.8% on surface soil 2.2% Oceans 1.0% everywhere else 2.5 X 10^30 bacteria cells on Earth (less abundant than viruses)
Horizontal Gene Transfer
A process in which an organism incorporates genetic material from another organism without being the offspring of that organism (Genetic Exchange)
Scanning Electron Microscopy
Electrons bounce off specimen Would electrons normally bounce off a biological specimen? No, they are going to absorb electrons How must specimen be treated? Coat sample with metal: Sputter coating so electrons bounce off and hit detectors Lower resolution then TEM (0.2 nm), but better than light 3D image
Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS)(Unique to gram negative) -Lipid A (Hydrophobic, anchors it in outer membrane): Then core polysaccharide, then O-specific polysaccharide Net negative change Endotoxin: Toxic component of LPS (lipid A)(Only gram negative have LPS, so only they can release endotoxin)
Which of the following scientists disproved the theory of spontaneous generation?
Louis Pasteur
Magnification VS Contrast
Magnification: Increase in apparent size Contrast: Ability to distinguish objects from background
History of life on earth
Multicellular life arose about 400 million years ago Do we need to know the circle? Origin of cyanobacteria: about 3 billion years ago
NAG and NAM
N-acetylglucosamine (not unique) and N-acetylmuramic acid (Unique to bacteria) N-Acetylmuramic acid is unique to cells walls of bacteria
Strength of Peptidoglycan:
NAG and NAM: repeating units of the two, multiple layers (gram positive has more layers than gram negative) Covalent bonds holding together sugars: Glycocytic bonds (Horizontal between sugars/ covalent bonds within a layer) Amino acids form peptide bonds with each other (In Y direction, covalent bonds between layers (sheets), holding multiple sheets together) Very strong Peptide bonds in amino acids are only attached to NAM
Which of the following would provide the best resolving power when using brightfieqld microscopy? Green light (wavelength = 500 nm) ; objective with numerical aperture of 0.87 Green light (wavelength = 500 nm) ; objective with numerical aperture of 0.12 Red light (wavelength = 700 nm) ; objective with numerical aperture of 0.87
Green light (wavelength = 500 nm) ; objective with numerical aperture of 0.87 Lowest wavelength and highest numerical aperture
Atypical Bacterial Cell Walls
Mycobacterium species Acid fast cell wall with covalently attached mycolic acids (lipid) give waxy surface, hyrdrophobic consistency -No outer membrane Mycoplasma species NO cell wall (ie. NO peptidoglycan) but sterols and glycolipids in cytoplasmic membrane provide strength Planctomyces species Protein layer instead of peptidoglycan
Gram Positive Cell Wall
Thick peptideglycan layer Teichoic acids (negatively charged) only in gram positive cell wall -Binds to positive charges: Mechanism for gram positive to get calcium and magnesium and other positive things inside the cell (Binds small, positively charged molecules) Teichoic Acids covalently bound to NAM Lipoteichoic acids: Lipid attached, anchored to cytoplasmic membrane (anchored in hydrophobic region) Teichoic acid anchored in peptidoglycan
Gram negative cell wall
Thin peptidoglycan outer membrane Cell wall: Peptidoglycan layer and outer membrane (anything outside of cytoplasmic membrane) Outer membrane: Bi-layer (inner and outer leaflet): -Inner: primarily composed of phospholipid and lipoprotein (lipid portion anchors to membrane) -Outer (8nm): Phospholipid, Lipopolysaccharide (LPS)(Unique to gram negative) -Lipid A Anchors it in membrane, then core polysaccharide, then O-specific polysaccharide Cytoplasmic mebreane: Inner and outer leaflet
Phospholipid Bilayer
hydrophilic head (facing out) and hydrophobic tail Tail: Fatty acids (non-polar) Molecule is anthiopathic: Positive and negative end Selectively Permeable
Goal of streak plate:
is to obtain isolated colonies (we presume that colony arose from single bacterium); it doesn't matter which section has the isolated colonies
_____ is the only RNA that gets transferred into peptides
mRNA
Properties of all cells
metabolism, growth, evolution Cells take up nutrients, transform them and expel waste *All bacteria have the potential to adapt through mutations acquired during replication Additional properties of some cells: Differentiation (like spores), communication (chemical messengers), genetic exchange, motility Argument can be made that all cells use communication and genetic exchange
swan-necked flasks (Pasteur)
prevents contamination from outside sources, but allows for fresh air in the environment. -Non-sterile liquid poured into flask -Neck of flask drawn out so organisms can't fall in from air Sterilized by boiling Dust and organisms trapped in bend of tube, liquid remains sterile OR Flask tired such that microorganism laden dust contacts sterile liquid = liquid putrifies
Other Types of Stains
•Acid-fast stain: (differential technique)Used to identify mycobacterium species, which retain stain even after washing with acid alcohol due to waxy layer on cell wall (differential stain) •Developed by Koch, use for tuberculosis and leprosy/Hansens disease •Stain to vacili (rod shaped) •Waxy layer means they don't stain well -Image shown •Endospore stain(differential stain technique): green to stain endospores (endospores comes from single cell) •Flagella stain (special stain): Flagella is like 10 nanometers: these stains stain and make flagella bigger Negative stain(Indirect stain): Staining background instead of the cells (staining glass slide black so you can see clear cells
Brightfield Microscopy
objects are dark and the field is light Pigmented cells: Easy to see, high contrast What if cells are not pigmented? Not as easy to see with Brightfield, they don't really reflect light and provide high contrast Cells are mainly "bags of wáter": Dont absorb much light Therefore you must increase the contrast
Electron Microscopy
•Electromagnets focus electron beam •Two types: •Transmission EM (TEM): use to investigate detail of multiple layers of gram negative cell envelope •Ultra thin sections (100 nanometers in thickness) •Electrons pass through sample •Used to view cell structure •2D image •Very detailed image •Scanning EM (SEM) •Whole specimen, not sections •E- bounce off coated sample (electrons don't go through sample, like they do in TEM, they bounce off and are collected by detectors) External features (3D image) Very detailed Similar to light microscopy, just using electrons instead of light Electromagnets instead of lenses (focuseselctronbeam) Condensor= electromagnet Objective: Magnifies image 1000 nm in one micron (ultra thin sections are super small) Electrons go from condenser, to objective, to projector
Assuming optimal conditions for traditional light microscopy, what is the diameter of the smallest object that could be determined as separate and distinct from another close object?
0.2 micrometers
1 meter = _____ millimeters = _____ micrometers (microns) = ______ nanometers
1 meter = 1000 millimeters = 10^6 micrometers = 10^9 nanometers 10^3 (1000) micrometers in 1 millimeter 10^3 nanometers in one micrometer
Consider an epilopiscium fishelsoni cell that is 1 mm in length. Assuming a virus that infects this cell is 100 nm in diameter, how many viruses laid end-to-end would span the length of this cell?
1 mm = 1000 micrometers = 10^6 nm 10000
2 Important Bacteroidetes:
1) Porphyromonas gingivalis: Don't have gliding motility, use type 9 secretion for being able to cause disease (gingivitis), linked to Alzheimer's disease -Gingipains are toxins secreted by T9SS in P. gingivalis that break down host proteins 2) Flavobacterium johnsoniae (gliding motility) T9SS in P. Gingivalis homologous to F. Johnsoniae Gingipains are toxins secreted by T9SS in P. Gongivalis that ... T9SS moves secreted proteins (SprB) into outer membrane across sex system (required in gliding motility) Once SprB is in periplasm, type 9 secretion exports it across outer membrane Components of the T9SS have only been found in bacteroidetes
Molecular Techniques: Example 16S rRNA Sequencing
1. Isolate DNA from each organism 2. Make copies of rRNA gene by PCR (16S) 3. Sequence DNA 4. Analyze sequence 5. Generate Phylogenetic Tree
3 Primary Functions of the Cytoplasmic Membrane
1. Permeability Barrier: Prevents leakage (functions as a gateway for transport (Nutrients in, waste out)) -Phospholipid Bilayer is selectively permeable 2. Protein Anchor -Anchored proteins involved in transport, chemotaxis, bioenergetics (no organelles in prokaryotes, so electron transport chain proteins found in cycloplasmic membrane are important for ATP (As proteins move down chain, pumps proteins across membrane)), etc.... 3. Energy Conservation: Generation of the proton motive force -Change differential is between surface of inner and outer leaflets of cytoplasmic membrane -High concentration, protons want to flow into cell, driving ATP synthase
Assuming you could slice a 10 μm long E. coli cell from end to end for transmission electron microscopy, approximately how many slices could you make from one cell?
100 10 micrometers = 10,000 nm Sample for TEM = 100nm 10,000/100
Assuming a rod-shaped bacterial cell is 10μm in length and cells are laid end-to-end, how many cells would it take to span 1mm?
100 Convert 1mm to 1000 micro meters 1000 micrometers / 10 micrometers
Resolution
Ability to distinguish two objects as distinct from one another Determines the useful magnification level It is the limiting factor in our ability to see small objects *ABOUT 0.2 MICRONS for light microscope function of: The wavelength of light used AND The Numerical Aperture of the lens (light-gathering ability of the lens) *Wavelengths of electrons vs wavelengths of light: electron resolution allows resolution to be much higher resolution (able to distinguish between smaller things) Lenses with higher magnification have higher Numerical Aperture (capturing more light rays)
Laboratory Cultures: Advantages and disadvantages
Advantage: Large amount of target organsim Disadvantge: Most microorganisms are uncultrable We can culture less than 1% of bacteria
Advantages and Disadvantages of Stains
Advantages of Stains: Able to see cells with low contrast Certain techniques can differentiate cells types Disadvantages of Stains: Deforms/distorts cell features Kill cells How can you overcome these disadvantages? Use alternative light microscopy techniques
Which of the following is TRUE of fluorescent molecules? A) They absorb light at a specific wavelength B) They emit light at a specific wavelength C) They can be used to visualize either live or dead cells
All of the above
Anoxygenic v. oxygenic photosynthesis
Anoxygenic: Do not evolve/give off oxygen as part of metabolism (the first phototrophs on early earth)(do not evolve molecular oxygen during photosynthesis) Oxygenic: Cyanobacteria are oxygenic phototrophs, which was really important to oxygenate earths atmosphere
In which domain of life are microorganisms represented?
Archaea, bacteria, and eukaryotes
Which color of light would allow for the best resolution?
Blue Lowest wavelength Smallest numerator given by blue 0.5X400/0.87 = 229 NM (nanometers), or 1000 nm in micrometers, so 0.229 mm (microns) This is the smallest object you can see as distinct With red wavelength: 350/0.87 = 402 nm (larger)
Gram positive vs Gram negative
Both have cytoplasmic membrane and
Bacterial cytoplasmic membrane
By dry weight, membrane has higher percentage of protein than phospholipid The surface surrounding the outer leaflet has a net positive charge due to a high H+ concentration
Cell envelope vs cell wall
Cell envelope includes the cytoplasmic membrane and anything outside of it Gram Positive: cytoplasmic membrane and peptide glycan Gram Negative: cytoplasmic membrane and peptide glycan and outer membrane Cell Wall: Refers to everything outside the cytoplasmic membrane (does not include cytoplasmic membrane) Positive: Just thick peptidoglycan layer Negative: Peptidoglycan and outer membrane Function of cell wall: Peptideglcan unique to bacteria (good target) -Structure -Rigidity -Cell Shape -Protects assign osmotic lysis: Water wants to rush into cell because higher concentration in middle
Chemotaxis
Cell movement that occurs in response to chemical stimulus -Moving towards/away from chemical
Group Translocation (Active Transport) Common mechanism for transporting carbohydrates into a bacterial cell Phosphorylation of the transported substrate prevents it from being transported out of the cell The energy for transport comes from high-energy phosphorylated intermediate in glycolysis
Chemical modification of the transported substance driven by energy-rich compound phosphoenolpyruvate (PEP) (Intermediate in glycolysis) -Modifying glucose (gets glucose into cell) -Substance being transported in is chemically modified Common mechanism used by bacteria to move carbs into cell Molecule is modified, so it can't be transported back out of cell (locked in) PEP comes from phosphorelay, system involving multiple proteins (Phosphate originates donated by PEP Phosphate ends up attached to Glucose to form Glucose-6-Phosphate (first intermediate in glycolysis) First glycotic intermediate: Glucose-6-phsophate
Prokaryotic Cell Morphology
Coccus Rod (Bacillus) Spirillum Spirochete Budding and appended bacteria Hypha Filamentous Bacteria
Complex VS Defined Media
Complex: Don't know exact amount of nutrients in media (complex ingredients) -Easier -Can't define exact elemental composition -Nutrient broth, Agar Defined: Exact chemical composition known -You can calculate based on ingredients, exact amount of stuff on media -K2HPO4 -Exact chemical composition known
In phase contrast microscopy, which of the following puts all light rays in the same phase?
Condensor Annulus then the phase plate in the objective amplifies any differences after light passes through the specimen
Phase Contrast Microcopy
Condensor below specimen but above light souce: Focuses light on specimen Condenser Annulus: Puts all light rays in same phase (below condenser) light waves in phase (peaks and troughs line up) b/n condenser annulus and specimen Lamp: Light rays out of phase Light slowed slightly by specimen because cells have different refractive index from water Where does light go after it hits specimen: Objective, which magnifies it (inside objective is phase plate: amplifies subtle differences) - Above lens After light hits specimen its out of phase again, produces contrast you see when looking through microscope *Contrast is produced by direct and diffracted rays that are out of phase Result: Dark image on bright background So light is out of phase in the lamp, in phase between condenser and specimen, and out of phase after the phase plate
Role of peptide bond in structure of peptidoglycan
Cross links 2 N-acetylmuramic acid residues in overlapping sheets of peptidoglycan
Which group of microorganisms is primarily responsiblefor oxygenating the Earth's atmosphere?
Cyanobacteria Phototrophs that allowed for development of modern eukaryotes and aerobic metabolism
Structure of peptidoglycan
D-GlutamicGlycan tetrapeptide (Single unit has sugar portion and four peptides) Glycan portion: Repeating disaccharide of NAG and NAM (N-Acetylglucosamine and N-Acetylmuramic acid) -Linked by covalent (strong interaction) bond Gylcan unit same in all bacteria with peptidoglycan Found only in bacteria Made up of amino acids and sugars Peptide portion: 4 amino acids: L-Alanine (common, found in any), D-glutamic acid (Unique to peptidoglycan/bacteria), Diaminopimelic acid (unique to cell walls of bacteria), D-alanine (enantiomer, not common, Unique to peptidoglycan/bacteria) Any D amino acid is unique to bacteria peptiodglycan Peptide only attached to NAM D-Glutamic acid and N-acetylmuramic acid are unique to bacteria
When doing a simple stain, which of the following might a basic dye (such as methylene blue) bind to in a bacterial cell? DNA Polysaccharides The interior of the cytoplasmic membrane (i.e. space between lipid tails of the bilayer) AandB A, B and C
DNA Polysaccharides
What components are needed for a PCR reaction?
DNA template Primers (Bind to conserved regions) DNA polymerase (enzyme) dNTPs (nucleotides)
Passive Transport
DOES NOT REQUIRE AN ENERGY INPUT Two ways a substance can cross the cytoplasmic membrane: Passive and active Passive Transport (Diffusion): Solutes moving from high concentration to low (Down gradient) -No extra energy needed -High rate of permeability = good potential for diffusion into cell -Simple Diffusion: -Facilitated diffusion: Uniporter ( 1 thing moving): Solutes moved from an area of high concentration to an area of low concentration *Uniporters work by facilitated diffusion
Which of the following was an accomplishment of Louis Pasteur?
Disprovedthetheoryofspontaneousgeneration Developed an effective rabies vaccine
Numerical aperture
The amount of light entering the objective Increasing numerical aperture allows more light to enter the objective
Does the cell ever EXPORT macromolecules to the surface or outside the cell? What types of molecules do bacteria need to get outside the cell? Why?
Enzymes secreted outside cell sometimes -SprB Made inside cell Gram negative secretion systems must move proteins across 2 lipid bilayers Extracellullar Outer Membrane Periplasm Cytoplasmic membrane Cytoplasm 9 different systems: A single organism will NOT have all of these systems, but will have a select few T9SS: Linked to bacteroidete (Flavobacterium and Porphyromonas gingivalis) gliding motility and pathogenesis
If listing the following largest to smallest, which is correct? A) Flavobacteriumjohnsoniae;Epulopisciumfishelsoni;Paramecium;Influenzavirus B) Epulopiscium fishelsoni; Paramecium; Flavobacterium johnsoniae; Influenza virus C) Paramecium; Flavobacterium johnsoniae; Influenza virus; Epulopiscium fishelsoni D) Epulopisciumfishelsoni;Flavobacteriumjohnsoniae;Paramecium;Influenzavirus
Epulopiscium fishelsoni; Paramecium (50-330 micrometers); Flavobacterium johnsoniae; Influenza virus
Which of the following is an energy source for the bacteria that form the base of the food chain at hydrothermal vents?
H2S
Differential Stain/ Differential Gram Stain
Hans Christian Gram developed (Render different kinds of cells different colors) Example: Gram Stain: Distinguishes between gram positive (purple) and gram negative (pink) Both have cytoplasmic membrane. Outside of membrane the gram-positive has super thick layer of peptidoglycan Gram negative has another membrane outside of thin layer of peptide glycan After Step 1: Prepare smear, flood heat fixed smear with crystal violet for one minute -both gram positive and gram negative slides are going to be purple because both have negatively charged molecules, including both have peptidoglycan After step 2: Add iodine solution for 1 min: -Both will still be purple, iodine increases adherence (it's a mordant) of stain to peptidoglycan layer/improves adherence Step 3: (decolorize with alcohol for about 20 seconds)(most important step) -Alcohol decolorizes gram negative cells (pulls violet out of thin layer of gram negative cell by disrupting outer membrane) Gram positive doesn't have an outer membrane to disrupt so it stays purple (unless you decolorize for too long) Most important step, too much or too long with alcohol will decolorize both -If you look under microscope now, you wouldn't' be able to see gram-negative cells because they are clear Step 4: Counterstain with safranin for 1-2 minutes -Safranin (pink) stain added to dye clear gram negative pink (gram positive still purple) Gram Negative: Pink (WAY more common than gram positive) Gram positive: Purple
Hopanoids
Help Stabilize membranes Structurally similar to cholesterols in eukaryotes Present in membranes of many Bacteria (only found in bacteria)
How big is a colony? How big is a single cell? How many cells make up a colony? Colony is mass of bacterial cells on an agar surface that originated from a single bacterial cell mass
How big is a colony? About 2 mm How big is a single cell? .01 mm or 10 micrometers Virus: 0.01-0.3 Micrometers (um) or 10-300 nm Bacterial cell: 1-10 micrometers (1000-10,000 nm) Eukaryotic (algae, protists, fungi) cell: 0.8-300 micrometers (closer to 300)(800-300,000 nm)(about 10X larger than prokaryotes How many cells make up a colony? 1 million to 100 million
Oil Immersion Technique
Increases resolution by increasing numerical aperature THE SHORTER THE WAVELENGTH AND THE HIGHER THE NUMERICAL APERATURE (light gathering ability) THE BETTER THE RESOLUTION Place drop of oil on slide, examine with 100X objective lens
Confocal Scanning Laser Microscopy
Laser + Fluorescence Focus on Different Layers Application: Thick Specimens (Biofilms), Multi-Layer Image Laser (Excitation light = Green) (light source) Specimenemitshigherwavelengthlight (red) Fluorescence (emission= red folloiwngexcitation) Laserallowsyou to focuson veryparticular focal place of specimen/focuson different layers Dialscope/laserdownto gointonextfocal plane
Light Microscopy
Light Microscope •Magnification - 100-1500X •Resolution - 0.2 µm (200nm) = smallest thing that you can see Types Simple vs. Compound •Bright-Field, Phase-Contrast, Differential Interference Contrast (DIC), Dark-Field, Fluorescence Simple: Single lense Compound: 2 lens (objective lens (10X, 40X, 100X (10X-100X)) and ocular lens (10X-20X) Condenser focuses light onto specimen Then, light reflect into objective lens Total magnification = objective lens X ocular lens 10X * 100X = 1000X
Dark-Field Microscopy
Light reaches the specimen from the sides ONLY Requires 1) Darkfield stop 2) Abbe Condenser Only light scattered by specimen reaches the objective lens (if light doesn't hit cell it doesn't go into objective) Gives us bright image against dark background (type of cells shown are spiroceets, like the ones that cause lymedisease) This is useful technique because spiroceets are so much thinner and smaller, so you need something that makes them super obvious
Targeting Peptidoglycan
NAM-NAG Alexander Fleming discovered penicillin and lysozyme: these molecules perform functions through this Lysozyme cleaves glycosidic bond b/n NAM and NAG within a layer (sheets) L-Ala - D-Glu-DAP-D-Ala D-Ala binds to DAP If cell wall is weakened, causes osmotic lysis Lysozyme is an enzyme: Catalyzes breaking of NAG NAM bond Penicillin: Inhibitory molecule (not an enzyme) that prevents enzyme function/ prevents cross-linking between layers (sheets)
How does penicillin prevent cross-linking (not going to break it if its already there though)
New unit of peptide glycan needs to be added one at a time to make large sheets Glycocitic covalent bonds between M (NAM) and G (NAG) -Transglycosylase catalyzes the formation of the glycosidic bond (M-G bond) -Transpeptidase catalyses formation of the peptide bond (M-M between layers of sheets)(DAP of sheet 1 to D-Ala of sheet 2 to form peptide bond) Penicillin binds to active sight of Transpeptidases and inhibits it from performing its function (prevents peptide bond formation, does not break already made bonds) -When penicillin interacts with active sight, it becomes covalently linked and blocks active site
Will penicillin kill bacterial cells that are in a nutrient poor environment and are not actively growing?
No: Penicillin is not cleaving it. Penicillin has no effect on already formed peptide bond, only effects on formation of new peptide bonds Penicillin is not an enzyme It does not break bonds It bonds to transpeptidases and inhibits the cross linking function Therefore, penicillin only kills actively growing cells that are adding new units of peptidoglycan Transpeptidation form the peptide cross links Lysozyme on the other hand does break bonds and would have effect
*Microscopy gives you shape, not genus
Obtain detailed marine sediment core sample A molecular approach would be most appropriate to investigate the microbial diversity in this community. Take dirt with lots of different cells, break them open and (16S for prokaryotes, 18S for eukaryotes) Isolate DNA PCR amplify rRNA sequence from the isolated DNA (16S for prokaryotes and 18S for eukaryotes) Sequence the amplified DNA Align and compare DNA sequences Generate phylogenetic trees to observe evolutionary relationships Two potential confirmatory experiments include: A molecular approach to PCR amplify a different gene (e.g. cytochrome c) found in all organisms and build a new phylogeny. Then compare the new phylogeny with the one generated in (a) and look for similarities and differences. A culture and molecular based approach that involves culturing a subset of organisms (both prokaryotic and eukaryotic) and using similar molecular methods to confirm their presence in the phylogenies from Part A.
16S
Part of a small subunit (1500 base pairs in length) of ribosomal RNA that is highly conserved and used to establish evolutionary relationships/ used for molecular identification of bacteria -Analyze 16S by amplifying it with PCR -Single stranded nucleic acid, folds forming a tertiary structure -Has multiple conserved (same b/n species) and variable (different between species) region How do changes in 16S rRNA occur? Point mutations in variable region Why use ribosomal RNA gene? It has conserved regions that primers can amplify, and variable regions that we can analyze (Mutations in conserved region are generally lethal, mutations in variable region are usually neutral) *Note: Primers are complimentary to conserved region *Ribosomes/rRNA common. to ALL organisms (You can use stuff besides rRNA, but it has to be common)
ABC Transport (Active Transport)
Periplasmic binding proteins are involved and energy comes from ATP Driven by hydrolysis of ATP: When ATP is hydrolyzed: Changing configuration, now can bring molecules inside cell Transporter has 3 parts: 1. Organic compounds 2. Inorganic compounds 3. Trace models
Autoclave
Piece of equipment used to sterilize articles by way of steam under pressure and/or dry heat
Porins of Gram negative Cell envelope
Pores in the outer membrane that small molecules/ ions can get thorugh (permeable to small units) Some are selective (specific) and some are non-specific 3 identical subunits Unique to gram negative because gram positive dont have outer membrane Gram negative more resistant to antibiotics because of outer layer/porins, but depends on antibiotics)
Differential Interference Contrast (DIC) Microscopy
Principle Polarizer = puts light in single plane Condenser prism = results in TWO outgoing beams Objective prism = brings beams back together Application (use more foreukaryotes) View Cellular Structures Nucleus, Endospores, Vacuoles Similar to phase contrast, but gives more 3D image
Simple Stain
Putting stain on cells to increase contrast Flame passed through flame to fix the bacteria to slide (melts bacteria to slide), don't need to hold slide there for long What objective would you use for bacteria: Start with 10 or 40 to get slide in focus (10x eyepiece and three objectives of 4x, 10x & 40x to provide magnification levels of 40x, 100x and 400x.) Basic dyes (positively charged) -Crystal violet -Safranin (pink) -Methylene Blue Bind to negatively charged polysaccharides on surface, also nucleic acids and DNA are negative, and some lipids have negative attachment, some amino acids may be negatively charged as well
Active Transport
REQUIRES AN ENERGY INPUT Solutes ARE moved from low to high concentration (AGAINST gradient) 1. Simple Active Transport: Solutes are moved from low concentration to high concentration (against gradient) Requires Energy Proton concentration higher outside (Protons moving down concentration gradient) Driven by energy in the PROTON MOTIVE FORCE Symporter (transport molecules in same direction) and antiporter (protons going in, solutes going out) (Proton still flow down gradient, but kicks out something (Na) at the same time) *can be group as symporters or antiporters 2. Group Translocation: Chemical modification of the transported substance driven by phosphoenolpyruvate (results pyruvate) -How bacteria get sugars like glucose into cell (locks glucose into cell) -Phosphate given by phosphoenolpyruvate ends up on glucose 3. ABC Transporter: Periplasmic binding proteins are involved and energy comes from ATP
Robert Hooke
Robert Hooke: (around 1665?) Credited as first person to observe microorganism (Eukaryotic molds) -Published "Micrographia"
History of Microbiology
Robert Hooke: (around 1665?) Credited as first person to observe microorganism (Eukaryotic molds) -Published "Micrographia" 1687: Antonie Van Leeuwenhoek identified first bacteria using crude microscope Major advances in 19th century from two questions: Does spontaneous generation occur (where do microorganisms come from)? What is the nature of infectious disease? 1864: Louis Pasteur -Developed process of pasteurization in 1862 -Disproved spontaneous generation -Germ theory of disease (1880-1890) (Anthrax vaccine, fowl cholera vaccine, rabies vaccine (virus)) -Performed Swan-Necked Flask experiment 1895: Koch -Develoepd pure culture technique -Identified many disease causing microbes (Anthrax, tb, cholera) -Formulated Koch's postulates to demonstrate cause and effect Alexander Fleming (1881-1955): Discovered lysozyme (Enzyme that breaks down cell walls and is found in our secretions) -Accidentally discovered penicillin in 1928 (Bacteria didn't go near mold = zone of inhibition) Watson and Crick (1953): Determined structure of DNA, which began the genomic revolution 1977: Cloning became possible 1977: Discovery of archaea (Woese and Fox) -Woese also came up with using 16S 1988: Kary Mullis: Came up with Polymerase Chain Reaction (PCR) -Pulled apart double strands (held together by non-covalent H bonds) by heating, which denatures structure 1995: Venter: First bacterial genome 2010: Venter and Hutchinson Made first synthetic cell Now we have over 225,000 prokaryotic genomes, and bioinformatics is the future
The periplasm of Gram negative bacteria Outer membrane protects peptidoglycan
Space with Gell like liquid, spans outer leaflet of cytoplasmic membrane to inner leaflet of OM Hydrolytic enzymes, binding proteins, chemoreceptors ABC transporters: powered by ATP, binding protein for these found in periplasm in gram negative, brings solute to channel protein
Transmission Electron Microscopy (TEM)
Specimen Thinkness: 100 nm Magnification up to 1000000X Resoultionto 0.2 nm 2D image 1000X better than light microscopy Detailed 2D image (Sliced perpendicular to length of cell body
Methods that can be used to increase the contrast
Staining (used with brightfield microscopy) Phase-Contrast Microscopy Dark-Field Microscopy (lighter image on dark background) Fluorescence Microscopy
Functions of Outer Membrane
Structural Help pathogens evade phagocytosis due to negative charge Permeability Barrier
Functions of Outer Barrier
Structure... .
Advantages of Being Small
Surface Area: Supply of nutrients Volume: Demand for nutrients As size increases, surface/volume ratio decrease More efficient transport capabilities Faster metabolic rates and growth rates Rapid production of large populations of cells Greater adaptive potential *Exception to rule: Epulopiscium fishelsoni (600 micrometers in length, isolated by Angert)
Resolving Power
The diameter of the smallest object resolvable is equal to 0.5λ(wavelength) / numerical aperture Therefore, the SHORTER the λof lightAND the HIGHER the numerical aperture(ie. light gathering ability) the better the resolution Resolving Power Human eye = 0.2 mm (about 200 microns that you can see with human eye) Light microscope = 0.2 µm Electron microscope = 0.2 nm (0.0002 microns) Light microscopy would allow you to look at eukaryotes and prokaryotes but For viruses, you need electron microscopy
Endosymbiotic Theory
The endosymbiotic theory states that some of the organelles in today's eukaryotic cells were once prokaryotic microbes.
Microbiology
The study of microscopic organisms Basic Microbiology: Uses and develops tools for investigating the fundamental processes of life
Fluorescence Microscopy
Used to observe specimens that fluoresce Fluorescent molecules absorblight at one wavelength (lower) and emitlight at a different wavelength (higher)(less intensity) Barrier filter Specimen emits light at different wavelengths, goes back through Only emitted light goes through barrier filter (Filter optical clock turret) Excitation light source (allows fluorescent molecules to absorb light *Mercury Lamp To see specimen: filters eliminate all light except the emitted wavelength of the excited fluorescent molecule Alexa Fluor absorbs light at 550 and emits light at 555 Filter on scope takes out everything but emitted light (555) How does this light travel compared to traditional light microscope: Light source is above specimen (epi-flourescents= light from above) Light reflected down through objective, floursecent molecuels emit light at higher wavelength so all you see is that light *passes through objective before reaching specimen *Some cells autofluorescence We use fluorescent molecules to detect cell surface protein: Mostly used to label things Label SprB with fluorescent Anti-SprB antibody binds to SprBand used to label Howdo wemakeantibodies? Isolateprotein(SprB) and putproteinin animal model, get htemto makeantibodies, thenisolateantibodies.... Attachedto the antibodyisfluorescentlabel, so whateverthe antibdoybindsto, wecan see Nowwecan see whichcells haveSprB Counterstainwith Dapi,whichbindsto DNA (which all bacteria have) and stainsALL cells blue -DAPIbindsstronglyto A-T richregionsof DNA and emitsblue light Weuse DAPI to label all cells in fieldso wecan get a senseof howmanycells are expressingSprB(Mergeblue cells and Green highlightedcells to show whichcells haveSprB) FluorescneceMicroscopy canbe used to observe deadand livecells!
Symbiosis
Why is symbiosis beneficial to plant? Nitrogen! Bacteria can break atmospheric nitrogen into fixed nitrogen that can be used in DNA/Amino acids/building macromolecules Bacteria benefit from the sugars produced by plants Rhizobia induce the formation of a new organ in legume root Hydrothermal Vents: Reduced metals in water give microbes energy Undergo aerobic (carbs and sulfates) and anaerobic (carbs and methanes) chemosynthesis Take CO2 and build sugars/carbs -Symbiotic tube worms that live near vents -Trophosome _Microbes in tube worms generate food for tube worms through chemosynthesis (Eat H2S and fixed CO2 to produce Oxygen for worm) *ENERGY SOURCE FOR BACTERIA ON THERMAL VENTS = H2S -Microorganisms in termite gut: Bacteria digest cellulose (spirochete) so termite can eat wood
Virion
complete, fully developed viral particle 80 nm in diameter Head of Bacteriophage is 85 nm
