GEN MICROBIOLOGY BIOL 2051
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Date Created: 10/13/15
Parts of Chapter 17 and 18 Phylogeny study of evolutionary relationships of organisms Comparison of rRNA sequences can be used to determine the evolutionary relationships among organisms SSU small subunitrRNAsequencing of 168 or 188 subunits is common The Ribosomal Database Project RDP contains a large collection of rRNA sequences Phylogenetic trees based on rRNAhave been prepared for all the major prokaryotic and eukaryotic groups Comparative rRNA sequencing has de ned the 3 domains of life 1 Bacteria prokaryotes 2 Archaea prokaryotes 3 Eukarya Eukaryotes Algae protists Characteristics of the Domains of Life wall protein cell wall lack cell RNA 4 subunits 2 different ones 3 different ones polymerase with with 10 12 Protein 708 708 808 synthesis ribosomes ribosomes but ribosomes formylmethionine functionally similar to methionine eukaryotes methionine Taxonomic Heirarchy Domain Order Family Genus 7 168 rRNA sequence difference of less than 5 are same genus Species 7 168 rRNA sequence difference of less than 3 are same species 7029 prokaryotes recognized many more out there Nomenclature and Bergey39s Manual Binomial system of nomenclature descriptive genus name amp species epithet EX Bacillus subtilis The International Code of Nomenclature of Bacteria regulates naming of prokaryotes Bergey s manual of systematic bacteriology Formal recognition of a new prokaryotic species requires Deposition of a sample of the organism in 2 international culture collections Official publication of the new species name and description in International Journal of Systematic anal Evolutionary Microbiology IJSEAI Bergey39s Manual of Systematic Bacteriology and The Prokaryotes are major taxonomic compilations of Bacteria and Archaea Prokaryotic Diversity The Bacteria Undiscovered Bacteria 0 Many species uncharacterized 7 Many bacteria cannot yet be grown 7 Unclassified organisms Identified solely through rRNA sequence 7 Environmental samples rRNA is sequenced Many bacteria majority still unknown Bacterial Phyla Wellestudied phyla Thermophiles Cyanobacteria Gramepositives Proteobacteria 5 major branches Bacteroidetes Spirochetes Chlamydiae Over 50 other phyla u ullkn Plxnunmyunx ymtomltmblum vnuquotmantis l Deeply branching thermophiles 39m o o wth temp above 80 C found near marine hydrothermal vents and hot springs fastest growing cells known Aquifex Most ancientampthermophilic genus of known Bacteria grow up to 95 C T hermotoga Cells surrounded by protein covering toga that balloons over ends Chloro exi ex Chloroflexus Green nonsulfur bacteria lamentous anoxygenicphototrophs Most are therrnop 39les Microbial mats at Yellowstone Park Contain chlorosomes 7 bacteriochlorophyll containing structures that are attached to the cytoplasmic membrane Deinococcus not a thermophile 39 resistant to radiation due to DNA repair mechanisms First isolated from foods sterilized by gamma radiation 1000 rads ofionizing radiation kills humans 500000 rads does not kill Deinococcus Stains G due to thick peptidoglycan layer but has outer membrane like G T hermusaquaticus thermophile T aq DNA polymerase used in PCR 2 Cyanobacteria Large group of oxygenic phototrophs Fix C02 many also x N2 heterocysts Can cause nuisance blooms in freshwater Many have gas vesicles for buoyancy Many secrete neurotoxins animals ingesting water where bloom has occurred may be killed Have thylakoids Site of photosynthesis Similar to chloroplasts Many grow as laments 7 multiple cells growing in a line Others grow as colonies Many form akinetes Specialized spore cells Survive long periods of desiccation Germinate when conditions improve Prochlorophytes Prochlorococcus possibly the most abundant oxygenic phototroph on Earth 104105 cellsml in ocean water Gram positive bacteria Divided into 2 groups low GC Firmicutes amp high GC Actinobacteria based on guanine and cytosine in DNA GramPositive Firmicutes Rods and cocci 0 Many are pathogens GramPositive Endospore Forming Endospores are extremely heatresistant for millennia Toxin formers I Clostridium o Tetanus botox gangrene I Bacillus o Anthrax Bt Mollicutes Cell wallless small genomes Pleomorphic no distinct shape Classi ed with Gram due to phylogenic relatedness pathogenic M ycoplasma o Gram positive Actinobacteria o Actinomycetes I Branching laments called mycelia like lamentous fungi I Reproductive spores called conidia I Sporulation triggered by nutrient depletion I Streptomyces o earthy odor of soil 0 60 antibiotics o Mycobacteria I Acid fast due to mycolic acids Some human pathogens Tuberculosis leprosy Many are slowgrowers M ycobacterium 4 Gram Negative Proteobacteria and Nitrospirae 3 Ga Largest most metabolically diverse group of bacteria 5 major phylogenetic subdivisions 1 Alpha Proteobacteria Some endosymbionts Rhizobz39um Agrobacterium in plants Root nodules plant tumors Rickettsias in animals Obligate intracellular parasites Rocky Mountain spotted fever De cient in most metabolic functions must get metabolites from host Transmitted between animals by arthropods ProsthecateStalked bacteria Reproduce by budding Appendages used for attachment Aquatic environments 2 Beta Proteobacteria Lithotrophs Nitrate sulfur iron oxidizers Pathogens Neisseria gonorrhoeae Gram diplococci mma Proteobacteria Purple sulfur and non sulfur bacteria Found in mud and water Lithotrophs Can use iron or HZS as electron donor Some are anoxygenicphototrophs Vibrio and Photobacterium Rods and curved rods Most aquatic Some pathogenic Vibrio cholerae Some bioluminescent Enterics colonize human colon many are motile some in biofilms some pathogens Escherichia coli 4 Delta Proteobacteria M yxococcus Attacks other bacteria in packs Social movement Aggregates into fruiting bodies 0 Disperses myxospores 5 EpsilonProteobacteria Smallest group of proteobacteria H elicobacter pylori Cause of stomach ulcers Burrows below protective mucous layer Nitrospirae lstidentified by rRNA sequencing little is known about them spiral shape Nitrite oxidizers obligate aerobes Some species are found in microbial mats near hot springs 5 Bacteroidetes and Chlorobi Obligate anaerobes Bacteroz39des 30 of weight of human fecal material is made up of this Predominant microbe in lower digestive tract of humans amp other animalsican be pathogenic In intestine undigested food is fermented by Bacteroides Fermentation products are used by animal as carbon and energy source C hlorobi um 6 Spirochetes Green sulfur bacterium anoxygenicphotolithotroph Contain 39 39 to the cytoplasmic membrane Found at greatest depths of any phototrophic organism Use HzS hydrogen sulfide as electron donor oxidize it to sulfur Sulfur granules are deposited outside of the cells Autotrophs 7 No calvin cycle reverse citric acid cycle instead i39uac wi39 r39 yll 39 39 structures that are attached Motile 7 move like a corkscrew through media Endo agella located in the periplasm of the cell Treponemapallidum causes syphilis never been grown in laboratory culture Borreliaburgdorferi causes Lyme disease transmitted by ticks is a prokaryote with a linear chromosome 7 Chlamydiae Planctomycetes ampVerruc0micr0bia o Chlamydia Obligate intracellular parasites of animals Little metabolic capacity No peptidoglycan 3 species can cause human disease I Venereal disease chlamydia 7 most common sexually transmitted bacteria in US I Psittacosis epidemic in birds can cause pneumonia in humans I Conjunctivitis or trachoma leading cause of blindness in humans 2 body types I Larger reticulate body 0 Grows within cells 0 Does not survive outside host I Small elementary bodies 0 Survives outside of host 0 Similar in function to endospore o Planctomycetes S layer protein cell wall Reproduce by budding instead of binary ssion Some have protein stalks for attachment Multiple internal membranes Gemmata has membranebound nuclear material like a nucleus unique in prokaryotes o Verrucomicrobia wrinkled microbes Derived from Greek for warty Irregular shape Contains tubulin I Horizontal gene transfer from eukaryote could be possible in past From which group did mitochondria most likely evolve from Proteobacteria From which group did chloroplasts most likely evolve from Cyanobacteria No questions on exam 3 about these genera from chapter 18 Streptococcus Listeria Caulobacter Stell Burkhoderia Chormatium Salmonella Proteus Shigella Pseudomonas Bdellovibrio In library on first floor at reserve desk MICROBIOLOGY An Evolving Science call number is RX52 Circulates 2hrs BUILDING USE ONLY Early mxcroscope Chapter 2 Observing the Microbial Cell SIZE OF PROKARYOTES o Prokaryotes are generally smaller than eukaryotes I Not always true I I I quot L I This is important bc prokaryotes do not have in ernal membrane structures Prokaryotic cells can be as small as 02um The smallest eukaryotic cells are 2pm Cocci typically have diameter of 2 pm I Spherical shape Some microbiologists have proposed that bacteria smaller than 02 pm exist in nature cells referred to as nanobacteria If one considers the space needed to house all essential molecules of life it is unlikely they could exist within a volume available in a cell less than 01 pm Prokaryotic cells can have a wide variety of cellular morphologies which are often helpful in identification I Spherical cocci I Rod Shaped Bacilli I Sprial Bacterial Shapes 1 Simplest shape is sphere o singular coccus o plural cocci o Arrangement 0 Coccus single cells 0 Diplococcus 2 cells 0 Streptococcus chains of more than 2 cells I Skin and throat infections 0 Staphylococcus grapelike cluster of cells 0 Tetrads packets of 4 cells 2 Most common shape is rods 0 Singular bacillus o Plural bacilli 0 Can be very long amp thin or short amp fat 0 Occur singly or in chains 0 Can t have staphylobacillus because of the way it divides 3 Spirals 0 Rigid spiralshaped bacteria are called spirilla singularspirillum 0 Flexible spiralshaped bacteria are called spirochetes o Vibrio curved rods I Comma shaped Microscopy Simple microscopes 0 one lens o magnify 50 300X o Leeuwenhook saw prokaryotes with simple 0 Compound microscopes 0 multiple lenses 0 Much higher magnification I Ones in 2051 lab magnify 1000X o Hooke first to see eukaryotes with this Lens System of Compound Microscopes 0 Condenser lens 0 Located between light source and specimen 0 Focuses light rays up through specimen Does not magnify It focuses 0 Objective lens system 0 Closest to specimen just above specimen n revolving nose piece 0 Typical scope has 10 40 amp 100x objective lenses use 10x and 100x o In each objective are multiple lenses Increase magnification 0 Ocular lens system 0 Closest to the eye art you look through 0 Typically magnify 10X 0 Each ocular has multiple lenses I Co rrect impe rfectio ns Eye CT Ocular lens Ob ective lens 1 Specimen V Condenser lens l l Diaphragm El rim Light Magnification o A microscope with a 100X objective and 10X ocular has a total magnification of 1000x o This means an object will appear 1000x larger than it is 0 Can see bacteria at this magnification but not internal structures 0 Cannot see viruses 0 Only can see shape and arrangement 0 Sometimes can see endospores Resolution 0 ability to distinguish between 2 objects that are close together 0 be able to tell that 2 bacterial cells are different 0 Limits the usefulness of a microscope 0 Highest resolution of a typical light microscope is 02 micrometers 2 micro and above you ll be able to distinguish o This means that 2 objects closer than 02 pm to each other can t be distinguished 0 Resolution is determined by wavelength of light and numerical aperture 0 Increasing resolution 0 Multiple lenses Correct each other s aberrationsimperfections 0 Use shorter wavelength light Blue light quot400 nm is shortest wavelength so gives highest resolution 0 Increase numerical aperture measure of light gathering ability of objective lens More light gathered better resolution Wider lens closer to specimen Immersion oil same refractive index as glass I Light bends as it passes through specimen into air reduces resolving power I If space between specimen and objective is replaced with immersion oil resolution increases I Oil keeps light from scattering as much as in air I If no immersion oil light bends I Need to fill in air space 4 Increase contrast Contrast between bacteria amp background is small so bacteria are very hard to see Increase contrast by staining cells or using special type of microscope Many stains are basic dyes I Chromophore pigmented portion has a positive charge I Cell surface has a negative charge I Examples crystal violet methylene blue safranin I Reason it stains is charges 0 Simple Stains 0 Use one dye 0 Doesn t differentiate between types of cells or structures 0 Can tell shape arrangement size 0 Differential stains o Distinguish between different types of bacteria or different structures 0 Can see different colors 0 Uses more than one dyereagent Figure 222 o Gram stain distinguishes Gram positive bacteria from Gram negative bacteria I Stain different colors bc cell wall structure 0 Acidfast stain distinguishes Mycobacterium species from other bacteria I Can be diagnostic test takes 10 min Types of Microscopes Used To Increase Contrast o Microscope we typically use is called brightfield light microscope 0 See stained cells on bright white background 0 Instead of staining cells special microscopes can be used to increase contrast between cells amp medium Marni I Cells slow the speed of light passing through them so cells differ in refractive index from their surroundings 0 Cells changebend light as it passes through them O o This difference in quotphasequot is amplified by special ring in lens of phase contrast microscope 0 Liquid culture on slide I Not killing or staining it 0 Can see organellesinternal structures of eukaryotes 0 Cells appear bright against dark background 0 Light reaches specimen from 0 Only light scattered by sample reaches objective 0 Very good for narrow objects such as viewing a flagellum or seeing spirochete 0 Can observe living cells in wet mount 0 Excellent for studying bacterial motility and very narrow cells 0 See them moving and narrow structures 0 Problem dust particles also scatter light 0 Hard to tell if bacterial cells or dust l vl is r Polarized light passes through specimen I Light ray passes through filter 0 0 Sample boundaries bend light 0 Second polarized lens blocks light 0 Bent light result in 3D appearance l w 0 Some compounds absorb energy from invisible radiation like short wavelength UV light then radiate energy back as longer visible wavelength O OO O O O O O O O O O 0 Can use dyes to stain cell for fluorescence Used to view cells that make quot such as r have been stained with fluorescent dye quot or cells that These microscopes use a UV light source to fluoresce objects Can label molecules of interest in cell Resolving power of light microscopes is limited by wavelength of light used Electron microscopes use beams of electrons instead of visible light and electromagnets instead of lenses 0 This achieves a much higher resolution Can magnify over 100000x Can be used to view viruses proteins nucleic acids Sample must reflect electrons o Coated with heavy metal 0 Electron beam and sample are in a vacuum I So air will not interfere I Sample killed httpwwwyoutubecomwatchvfToTFijc5M Sample is sliced very thin I Microtome slices the cells very thinly Sample is stained with metal I Uranium I Osmium I These are typically used to coat sample Electrons travel through specimen I Go at different rates depending on density of sample High resolution 0 Used to view cell structures DNA amp some protein molecules 0 Sample is coated with heavy metal 0 Not sliced 0 Retains 3D structure 0 Gives 3D image 0 Image formed from electrons reflected from the specimen 0 Used to visualize surface cell structures What disease is caused by the organism on the last slide Mycrobacterium Ieprae I Causes leprosy 0 Normal flora E coli usually found in stomachintestines is ok 0 Break down food 0 However some E coli are very pathogenic Chapter 12 Biotechnology 0 Bacteria grow quickly 0 E coli genome relatively well understood 0 Small plasmids replicate quickly 0 Put gene of interest into a plasmid 0 Make huge amounts of protein 0 Easy to purify Applied Microbial Biotechnology 0 Use microbes to express genes 0 Produce eukaryotic proteins in large amounts I Human insulin 0 Use microbial gene products 0 Microbes have huge diversity of enzymes I PCR depends on polymerases from thermophiles o Polymerase tolerates high temps o Immunize against pathogens 0 Use genes from pathogens I Without exposure to pathogenic organism Insulin o Insulin is required for those with Type I diabetes amp many with Type II diabetes 0 Prior to genetic engineering insulin was harvested amp purified from pig or cow pancreas Nonhuman insulin is less effective 0 Human insulin was 1st human protein made commercially using engineered bacteria 0 Identical in every way to human produced insulin 0 Today engineered E colior yeast is used to make insulin Plant biotechnology o Glycophosphate Roundup herbicide resistant plants 0 Bacteria were found that were resistant to this herbicide o The gene for the herbicide resistance was put into some crop plants soybeans corn cotton Transgenic plants 0 Way for weeds to be controlled and plants protected from herbicides o Transgenic plants are also called roundup ready plants Insectresistant plants 0 Bacillus thuringensis produces chemical Bttoxin that is toxic to larvae of moths butterflies beetles flies 0 Gene for Bt toxin was put into cotton and potato plants 0 Expressed in the leaves of plant 0 Kills only insects that feed on those crops 0 Eliminates need to spray chemical pesticides I Chemicals are dangerous to use toxic to humans expensive Vaccines through Food Vaccines expose human to pathogen proteins 0 Expose immune system cells to proteins 0 We develop immune defenses to proteins 0 Protects us from later infection by pathogen I Flu shot inject dead flu virus so when exposed to alive flu virus we can fight it off 0 Proteins can be produced in plant absorbed via intestine o No longer need to grow pathogen to produce protein 0 Ex Bananas that have vaccine for cholera I Certain gene on organism in food is what makes bananas resistant to cholera Gene therapynonfunctional gene is replaced by functional gene 0 Clone desired gene into viral genome o Infect person with modified virus 0 Infected cells will express cloned gene I Make desired protein 0 Genes for many diseases have been located and the mutations in the defective genes have been identified Huntingtons cystic fibrosis SCID don t make antibodies Chapter 5 Environmental Influences and Control of Microbial Growth Environmental factors that affect microbial growth 0 Temperature 0 Pressure 0 Osmolarity 0 pH 0 Oxygen Temperature 0 T r is a major 39 39 factor quot39 g microbial growth 0 Cardinal temperatures minimum optimum amp maximum temps for an organism 0 Minimum temperature cellular processes slowcytoplasmic membranes stiffen Optimal temperature organism grows best between min amp max 0 Microorganisms can be grouped by the temperature ranges they require H U 6 n I I39I r foundIn cold 0 Example Chlamydomonas snow algae 0 Molecular adaptations of psychrophiles Membranes have a high content of unsaturated fatty acids 0 Semifluid at low temperatures 0 Proteins are more flexible compared to mesophiles or thermophiles Cryoprotectanis can be used to preserve microbial cultures at low temperatures 10 DMSO Dimethylsulfoxide amp10 glycerol are commonly used in laboratories to preserve microbial cultures for long time in freezers Mesophiles 0 Found in warm blooded animalsamp many terrestrial amp aquatic environments 0 Examples most organisms you are familiar with such as Escherichia coli found in the human intestine Thermophiles 0 Optimum temp above 40 degrees Celsius 0 Some archae have been found growing at temps above 110 degrees Celsius 0 Places thermophiles are found 0 soils subjected to full sunlight o fermenting materials Compost 0 Hot Springs 0 Thermusaquaticus is a common hot spring thermophile The heat stable DNA polymerase from this bacterium is mass produced and used in laboratories to replicate DNA in a test tube 0 Molecular adaptations of thermophiles o Membranes have a high content of saturated fatty acids 0 Stable and functional at high temperatures 0 Enzymes are heat stable proteins are more rigid compared to mesophiles or psychrophiles I Heat shock response 0 Occurs at high end of temperature range 0 quotEmergencyquot proteins from denaturing 0 Help keep proteins from denaturing o Induced by many stressful conditions 0 Heat 0 High salt concentrations 0 Arid conditions Pressure 0 Barophiles o Adapted to high pressure 0 Up to 1000 atm Barotolerantorganisms o Grow at high but not very high pressure 0 Prefer normal pressures Barosensative organisms 0 Die at high pressure 0 Most typica l bacteria all mammals Osmolarity 0 Water moves from areas of high water concentration to areas of lower water concentration 0 Water moves from areas of low solute concentration to areas of high solute concentration 0 The diffusion of water is osmosis In a environment water will move cell In a environment water will move a cell amp the cell will In a hypotonic environment water will move into a cell In a hypertonic environment water will move out of a cell and the cell will die from plasmolysis In a hypotonic environment the cell wall of most prokaryotes prevents too much water from entering cells even if equilibrium is never reached 0 Isotonic equal amount of soluteswater on inside amp outside of cells 0 However there is no physical barrier that prevents the cell from losing too much water if cell is in hypertonic environment 0 Some cells can increase solute concentrations in the cell to prevent too much water loss by microbes in salty environments pH 1 Pumping inorganic ions K into the cell 2 Making or concentrating an organic solute glycerol in the cell Osmophile organism that grows in high solute concentrations hypertonic environment Halophilesgrow best in salt habitats 7 Vibrio lives in ocean salt in ocean 3 salt Extreme halophiles require high levels 1530 of salts for growth 7 Halobacteriumsalinariumrequires 25 salt lives in very salty lakes Halotolerant cansurvive at higher salt concentrations but grow best in the absence of salt 7 Staph ylococcusaureus pH relative hydrogen ion concentration in a solution Scale is from 0 to 14 7 is neutral lt 7 is acidic gt 7 is basicalkaline Most bacteria grow at pH 68 Bacteria can be found to exist at almost any pH Most cells internal pH remains near 7 regardless of pH of their environment Most organisms have a pH range at which they can grow of 23 pH units Acidity or alkalinity of an environment can greatly affect microbial growth Weak acids can pass through cellular membranes 0 Good food preservatives Classification based on optimal pH 0 Some organisms have evolved to grow best at low or high pH or neutral pH but most organisms grow best between pH 6 and 8 are called neutralophiles Acidophiles grow best at lower pH I Stability of cytoplasmic membrane is critical since increases in pH can cause lysis I Ex Many fungi grow at pH 5 Thiobacillus produces sulfuric acid volcanic thermal soil archaeaPicrophiusoshimae grows optimally at pH 07 Oxygen 0 Double extremophile Sulfolobus thermophilic and acidophilic AlkaliphilesBasophile grow best at higher pH I found in soda lakes amp high carbonate soil I Many species of Bacillus live in very alkaline soils Bacillusfirmus has a pH range of 7511 Proteases and lipases made by alkaliphiles are mass producedamp used in household detergents I Flamingos eat a cyanobacteriaSpiroIina what makes them pink Microorganisms vary in their need or tolerance of oxygen 02 amp can be grouped based on their requirements for 02 Oxicenvironment Oxygen is present Anoxic environment Oxygen is not present Aerobes use oxygen to generate energy by respiration make ATP 39 ll w l suilll i L 0 210 microoxic environments Ex Streptococcus pneumonia use candle jar n lnilquot39tpli i1cjm m imzw lm m h ll 339 z i mm in tin 0 May die if even a minute amount of oxygen is present A reducing agent such as thioglycolate can be added to a medium to test an organism39s requirement for oxygen Thioglycolate reacts with oxygen reducing it to water In a culture medium thioglycolate will convert all Oz to water only top of culture is exposed to Oz in the air Special techniques are needed to grow aerobic amp anaerobic microorganisms in the laboratory Aerobes 0 culture medium must be oxygenated by shaking or bubbling air into the medium Anaerobes need Oz to be excluded 0 Bottles or tubes can be filled completely with media and sealed with a screw cap 0 Reducing agents thioglycolate can be added to convert all oxygen towater o Anoxic jars with a palladium catalyst convert oxygen to water 0 For obligate anaerobes that die if exposed to oxygen media must be boiled a reducing agent added then sealed under an oxygen free Hzor N2 gas 0 Work with these cultures must be done in an anoxic environment that can be provided by anoxic glove boxes Toxic Forms of Oxygen Several toxic forms of oxygen or molecules that contain oxygen can be formed in the cell during normal cellular processes Singlet oxygen 102 produced by peroxidases These are produced in last step of respiration Generated during the reduction of oxygen to water Superoxide anion 02 Hydrogen peroxide H202 9 Hydroxyl radical OH 0 Enzymes made by cells can neutralize toxic forms of oxygen 0 Catalase r 39 39 r 39 39 quot39 t r 39 reductase Controlling Microbial Growth Physical Agents Temperature o Pasteurization 63 C for 30 minutes 0 Flash pasteurization 72 C for 15 seconds 0 does NOT kill all cells 0 reduces microbial load number of viable organisms 0 kills most pathogens inhibits spoilage microbes o UHT Ultra high temperature 0 150 C for 3 seconds 0 Sterilizes all bacteria killed 0 creamer boxed milk Physical Agents Temperature Pressure 0 Autoclave 0 121 C 15 psi 20 min 0 Kills all bacteria sterilizes Destroys endospores Physical Agents Other Methods 0 Cold temperature 0 Refrigeration Slows growth of microbes Freezing Slows growth does not kill all bacteria o Irradiation Microwaves thermal effects 0 Heat kills organisms Ultraviolet radiation DNA damage Ionizing radiation X39raYS Nucleic acid and Gamma rays protein damage 0 Ultraviolet radiation 0 used to decontaminate surfaces and materials that don t absorb light air and water 0 Causes thymine dimers in DNA 0 Adjacent thymines will bond to each other 0 Messes up replication 0 UV Hood air is blown outward through a filter from the back and from edges of the hood so that the area inside the hood remains sterile once the UV light is turned off 0 Ionizing radiation 0 Gamma rays amp Xrays penetrates solid or lightabsorbing materials 0 widely used for sterilization amp decontaminationtreatment of an object or surface to make it safe to handle in medical and food industries 0 Causes breaks in DNAbreaks hydrogen bonds amp disulfide bridges in proteins 0 Filtration Filterdevice with pores too small for microorganisms to fit through but large enough for liquid or gas to pass through Filters remove microorganisms from air or liquids that are heat sensative 2 types depth amp membrane Depth filtersmost common fibrous sheets or mats made from a random array of overlapping paper asbestos or borosilicate traps large particles from liquid or air Examples HEPA filters Home airheat system Vacuumcleaner UV Hood Clean rooms and isolation rooms for quarantine Membrane filters thin sheets of polymers cellulose contain tiny holes of known size Act like sieves trap particles on membrane surface Antibioticsamp other pharmaceuticals Nucleation track Nucleopore filters used for concentrating a liquid sample for view on the scanning electron microscope Chemical Agents 0 Disinfectants used to reduce microbial numbers on nonliving material 0 bleach chlorine ethanol 0 Antiseptics 0 used to reduce microbial numbers on living tissues 0 Betadyne iodine H202 o Antibiotics Naturally occurring antimicrobial substances produced by microorganisms Fungi bacteria 0 Many known but less than 1 clinically useful because of poor uptake or toxicity Selectively kills microbes 0 May not work on all species 0 nterferes with bacterial specific enzymes 0 Cell wall synthesis 0 Bacterial ribosome 0 Many derivatives 0 Growing bacteria lyse 0 Slow growing bacteria take longer to die Biological Agents 0 Probiotics 0 quotGoodquot bacteria 0 Displace pathogens from tissues 0 Bacteriophage quotPhagequot Viruses that infect bacteria 0 Do not harm eukaryotes Chapter 3 Cell Structure and Function 3337 Basic cellular structures 1 Cytoplasm 2 Cytoplasmic Membrane Plasma Membrane 0 Holds cellular contents in cell 3 Nucleus or Nucleiod o Eukaryotes have nucleus 0 Contains all chromosomes bound by nuclear membrane 0 Prokaryotes have a nucleiod region 0 Not a membrane around it 4 Ribosomes 0 Protein synthesis 0 Not membrane bound organelles o Prokaryotes have these as well as eukaryotes 5 Cell wall found in plants amp most prokaryotes not in animals 0 Made of peptidoglycan or protein 0 Fungi have chitin cell walls Two types of cells 1 Prokaryote o simpler internal structure 0 lack membrane enclosed organelles I all components are in cytoplasm membrane or cytoplasm Bacteria amp Archaea Some have plasmid 2 Eukaryote larger amp more complex o Membrane enclosed organelles nucleus chloroplastphotosynthesis mitochondriapowerhouse etc o algae fungi protozoa plants animals Viruses 0 completely separate category 0 Noncellular o Reproduce only inside a host cell I Dormant outside of host cell in air on skinetc I Mutate inside host cell 0 Lack many characteristics of living things 0 Ex HIV virus Rhinoviruses colds The Bacterial Cell 0 Cytoplasm surrounded by envelope 0 Cytoplasm contains DNA in nucleiod o Envelope outer covering has lipid membrane boundary 0 Plus structural cell wall Bacterial cell structures il iLHv w a wr mm Mi lquot El and proteins quot3 lmlhm at r o Cytoplasmic membrane that form a bilayer with hydrophilic exteriors and a hydrophobic interior movement not fixed 0 CM is a phospholipid bilayer each layer is a leaflet o Phospholipid 0 Form bilayer o Glycerol with ester links to 2 fatty acids 0 Hydrophilic dissolve in water group faces cytoplasm or periplasm I Loves water 0 Hydrophobic doesn t dissolve in water fatty acids line up inside membrane I Doesn t like water I Tails that face each other 0 Attraction of nonpolar fatty acid portions of one phospholipid layer for other layer result in selective permeability of cell membrane 0 Integral membrane proteins span membrane 0 Transport nutrient across membrane 0 Peripheral membrane proteins are bound to the surface of the membrane don t go II the way across 0 Cytoplasmic Membrane Functions 1 Permeability barrier a Prevents leakage of 39 39 39 into the b Transport of substances nutrients amp waste products into amp out of cell 0 Due to hydrophilic outside and hydrophobic in the center of the membrane only water amp small uncharged molecules can freely diffuse through membrane 0 Other compounds must be transported active or passive by specific membrane proteins 2 Structural support site of many proteins involved in transport bioenergetics amp chemotaxis cellular movements 3 Energy conservation site of generation amp use of the proton motive force where ATP is made in Prokaryotes a Respiration o Eukaryotes have mitochondria to generate energy by respiration o In prokaryotes respiration occurs in the cytoplasmic membrane since they do not have mitochondria b Photosynthesis o Photosynthetic eukaryotes have chloroplasts to carry out photosynthesis o Photosynthetic prokaryotes carry out photosynthesis in the cytoplasmic membrane 0 To reinforce the membrane give stability Eukaryotes have sterols such as cholesterol Bacteria have hopanoids Archaea have terpenoids which increase stability at high temp amp low pH The Bacterial hi i mil l o Peptidoglycan murein a porous cagelike structure I It is porous so things can transfer across cell wall I Sugar chains wrapped in circles around cell 0 quotglycoquot quotsweetquot I Sugar chains linked to each other by short polymers of amino acids 0 amino acid quotpeptidequot 0 Structure of Peptidoglycan I Backbone made of 2 sugars o Nacetylglucosamine NAG o Nacetylmuramic acid NAM I Sugars are arranged in chains o MGMGMGMGMG I Chains held together by crosslinks short chains of amino acids between N acetylmuramic acids I Crosslinks differ among species I Gram negative bacteria have unusual amino acid mesodiaminopimelate m DAP in crosslink 0 Functions provides rigidity and shape to cell and prevent it from exploding due to high pressure inside cell 0 Peptidoglycan as a target for antibiotics I Since peptidoglycan is unique to bacteria it s a great target for antibiotics I Example Penicillin inhibits transpeptidase which crosslinks the peptides I However widespread use of antibiotics selects for resistant strains Example Many organisms produce twig 5 film 1 w 39 o Archaea I Lack peptidoglycan cell walls I Cell walls made of other polysaccharides I Some Archaea cell walls contain pseudopeptidoglycan I Other Archaea have a paracrystalline surface layer Slayer wall made of protein I Many bacteria also have S layers but it in addition to the cell wall 0 Gramnegative Bacteria I Only a few layers of peptidoglycan o Grampositive Bacteria 3 up to 40 0 Help give support and stability I Crosslinks within peptidoglycan are different often have Llysine instead of Meso diaminopimelate Mdap Lysozyme lti39 r2 0 Break beta 14 linkage between two suga s in peptidoglycan I Found in animal secretions amp thought to be a major line of defense against infection by bacteria 0 The Outer Membrane of GramNegative Bacteria 0 Found only in Gramnegative bacteria 0 Made of a lipid bilayer I Outer lipopolysaccharides LPS layer I nner phospholipid layer 0 Proteins porins within the membrane involved in transport across outer membrane 0 Endotoxin lipid Atoxic portion of LPS I Food poisoning diarrhea vomitting o Porins Transmembrane proteins allow for permeability through the outer membrane by creating channels that cross the membrane 0 Nonspecific porins waterfilled channels through which small substances can pass I Specific porins channels with binding sites for certain molecules that only allow those molecules to pass through 0 Usually nutrients o Periplasm I space between the outer and cytoplasmic membranes I contains proteins such as hydrolytic enzymes amp nutrient transporter binding proteins 0 The GramPositive Envelope 0 Capsule not all species I Polysaccharide I Slime layers 0 Thick sticky layer outside of bacterial cells 0 SLayer not all species I Made of protein 0 Thick cell wall I amino acid crosslinks in peptidoglycan I Teichoic acids for strength 0 Thin periplasm o Cytoplasmic membrane I Phospholipid bilayer and proteins that are embedded o The GramNegative Envelope 0 Capsule not all species I Polysaccharide I Sometimes polypeptide 0 Outer Membrane I Lipopolysaccharide 0 Thin cell wall I amino acid crosslinks in peptidoglycan 0 Thick periplasm o Cytoplasmic membrane Arrangement of DNA in Microbial Cells 0 Genome a cell39s complete set of genes 0 Most important 0 Chromosome and plasmid in prokaryotes more complex in eukaryotes 0 DNA is arranged to form chromosomes 0 Prokaryotes have a single circular chromosome and sometimes circular extrachromosal DNA plasmids o Plasmids are much smaller than chromosomes 0 Can be freely passed from one bacterial cell to another Eukaryotes several linear chromosomes Nucleus membraneenclosed structure that contains the chromosomes found in eukaryotes The Bacterial Nucleoid 0 Single loop of doublestranded DNA I 4x106 bp in many bacteria I Compacted via supercoiling 0 Attached to cell envelope I No membrane separates DNA from cytoplasm o Replicates once for each cell division Bacterial growth I One bacterial cell elongates and divides septum Cell Division 0 Cell elongates as it grows o Adds new wall at cell equator 0 DNA replicates to make 2 chromosomes 0 DNA replicates bidirectionally 0 Can begin next replication before the cell divides 0 Cell undergoes septation 0 Usually at equator 0 Each daughter has same shape 0 Fts Proteins the Cell Division Plane and Cell Morphology MFigure 337 o Divisome division apparatus in the cell formed by Fts proteins 0 Fts proteins required for cell division amp chromosome replication 0 FtsZ protein 0 Defines the division plane in prokaryotes o Polymerizes to form a ring where cell division will occur 0 FtsA protein 0 ATP hydrolyzing enzyme 0 Provides energy for assembly of other proteins to the ring 0 Activity is blocked by penicillin Helps define cell shape 0 Forms filamintous spiral shaped bonds around the inside of the cell under the cytoplasmic membrane CM 0 Defines cell shape by directionally exerting pressure against the CM 0 Coccus shaped bacteria lack MreB gene 0 Default bacterial shape sphere Peptidoglycan Synthesis and Cell Division 0 New cell wall is synthesized during bacterial growth by inserting new glycan units into pre existing cell wall units Autolysins create openings in existing cell walls to make space for new cell wall glycans to be inserted Autolysis spontaneous cell lysis may occur if there is an error in inserting new cell wall material 0 Bactoprenol Hydrophobic lipid alcohol 0 Can go across cytoplasmic membrane BindstheN 39U39 39 N 39 39acidl quot quot 39 precu rsors helps transport these new glycan units through the cytoplasmic membrane to become part of growing cell wall 0 Transpeptidation 0 Formation of peptide crosslinks between NAMs Bonds I quot U39 r into I quot gr r quot 039 layer 0 Bonding new cell wall to existing cell wall 0 Reaction is inhibited by penicillin cell lysis occurs Cell Inclusions o Prokaryotic cells often contain internal granules that function as storage materials or to orient the cell 0 Inclusion Granules 0 Not an organelle just a storage site 0 Densely compacted material in cytoplasm O Magnetosomes intracellular particles of magnetite Fe304 I Allow organisms to respond to a magnetic field 0 Glycogen Carbon and energy source I How bacteria store extra nutrients o Polyphosphate stores inorganic phosphate 0 PolyBhydroxybutyratecarbon amp energy source I Store extra nutrients o SulfurEnergy source I Some gramnegative prokaryotes can store elemental sulfur in globules in the periplasm Bacterial Cell Surface Structures 0 Pili amp Fimbriae o Bacteria may have 1 both or neither 0 Fimbriae I Nonmotile extensions that help bacteria attach to surfaces and other bacteria Neisseria biofilms biofim is community of organisms living together common in STDs I Shorter than flagella may have 100 s per cell 0 Look much like cilia in euk o Pili Singular pilus I aka conjugation pili I Hollow nonmotile tubes made of protein called pilin that connect some cells I Longer than fimbriae shorter than flagella may have 110 per cell o This is how bacteria have become antibiotic resistant bc transfer info to other bacteria I Purpose is to bring cells side by side Capsuleslime layer glycocalyx o Sticky polysaccharide or peptide layer surrounding cell 0 Protects cell from o Phagocytosis I Engulf and destroy I Streptococcus pneumonia has two types the one with capsule causes illness 0 Desiccation drying out 0 Help cells attach to objects such as teeth surface of other organisms medical equipment Cell Motility o Flagella 0 Long helical protein filaments O O O 0 Attached at ends or over whole cell Flagella rotate to propel the cell 0 Protein passage drives rotation I Clockwise or counterclockwise Eukaryotic flagella move in a whiplike motion Bacterial flagella rotate Arrangement of Flagella O Monotrichous 0 Single flagellum at 1 end O O Peritrichous 0 Several flagella around the cell Amphitrichous 1 on each end 0 ex spirochetes Structure of the flagella 3 parts 0 1 Basal body 2 Hook 3 Filament Basal Body 0 Motor part 0 lmbedded within the cell envelope 0 Made of either 2 or 4 protein rings connected by a central rod 0 C ring in Gram positive ampGram negative 0 MS ring in Gram positive amp Gram negative membrane E Gramnegative Bacterium Grampositive Bacterium C ring I In the cytoplasm I Attached to inner surface of cytoplasmic membrane 0 MS ring I In cytoplasmic membrane I End of central rod is attached to MS ring 0 P ring I In peptidoglycan layer 0 L ring I In LPS layer 0 Hook 0 Curved structure made of protein 0 Connects filament to basal body 0 Filament 0 Long rigid helical structures made of protein called flagellin o Prokaryotes such as filamentous cyanobacteria Myxococcus Cytophaga amp Flavobacterium move by gliding motility instead of flagella o Gliding can occur from slime secretions that moves cell along a solid surface 0 Motile bacteria can respond to chemical and physical gradients in the environment by moving toward or away from the signal molecule have receptors 0 Directed movements toward or away from a chemical or physical signal are known as o Chemotaxis directed movement of organisms in response to chemical signals 0 Phototaxis directed movement of organisms in response to light 0 Phototrophic bacterium Rhodosprilium moves toward light 0 Aerotaxis directed movement of organisms in response to oxygen 0 Osmotaxis directed movement of organisms in response to ionic strength 0 Organism that need salt move to places where can gain salt Figure 346 o Repellents cause clockwise rotation o Flagella fly apart 0 quotTumblequot change of direction 0 Receptors are located on cell envelope 0 Runs tumbles cause llrandom walkquot 0 Receptors detect attractant concentrations 0 Sugars amino acids 0 Attractant concentration increases and prolongs run 0 Net movement of bacteria toward attractants 0 Doesn t go in straight line to attractant tumble around Chapter 3 Cell Structure and Function Read 3337 Basic cellular structures Cytoplasm Cytoplasmic membrane Nucleus or nucleoid ribosomes protein synthesis cell wall found in plants amp most prokaryotes not in animals WPP E Jquot Two types of cells 1 Prokaryote 0 simpler internal structure 0 lack membraneenclosed organelles o Bacteria amp Archaea 2 Eukaryote 0 larger amp more complex 0 membrane enclosed organelles nucleus chloroplast mitochondria etc 0 algae fungi protozoa plants animals Viruses o Noncellular o Reproduce only inside a host cell 0 Lack many characteristics of living things 0 Ex HIV virus Rhinoviruses colds The Bacterial Cell 0 Cytoplasm surrounded by envelope 0 Cytoplasm contains DNA in nucleoid o Envelope has lipid membrane boundary 0 Plus structural cell wall Bacterial cell structures 0 cytoplasmic membrane quotfluidquot selective permeability barrier made of phospholipids and proteins that form a bilayer with hydrophilic exteriors and a hydrophobic interior 0 CM is a phospholipid bilayer each layer is a quotleafletquot o Phospholipid o Glycerol with ester links to 2 fatty acids 0 Hydrophilic dissolve in water group faces cytoplasm or periplasm o Hydrophobic don t dissolve in water fatty acids line up inside membrane 0 Attraction of nonpolar fatty acid portions of one phospholipid layer for other layer result in Selective permeability of cell Integral membrane proteins span membrane Peripheral membrane proteins are bound to surface of membrane Cytoplasmic Membrane Functions 1 Permeability barrier a Prevents leakage of 39 39 39 quot into b Transport of substances nutrients amp waste products into amp out of cell 000 Due toI outside and 39 center of only water amp small uncharged molecules can freely diffuse through membrane Other compounds must be transported active or passive by specific membrane proteins 2 Structural support site of many proteins involved in transport bioenergetics amp chemotaxis 3 Energy conservation site of generation amp use of the proton motive force a Respiration o Eukaryotes have mitochondria to generate n prokaryotes occurs in the b Photosynthesis o Photosynthetic eukaryotes have chloroplasts to carry out photosynthesis Photosynthetic prokaryotes carry out photosynthesis in the cytoplasmic membrane 0 To reinforce the membrane Eukaryotes have sterols such as cholesterol Bacteria have hopanoids Archaea have terpenoids which increase stability at high temp amp low pH energy by respiration The Bacterial Cell Wall sacculus O O O O 0 made of peptidoglycan murein a porous cagelike structure I sugar chains wrapped in circles around cell 0 quotglycoquot quotsweetquot I Sugar chains linked to each other by short polymers of amino acids 0 amino acid quotpeptidequot Structure of Peptidoglycan I Backbone made of 2 sugars N 39 39 NAG and N 39 39 acid NAM I Sugars are arranged in chains o MGMGMGMGMG I Chains held together by crosslinks short chains of amino acids between N acetylmuramic acids I Crosslinks differ among species I Gram negative bacteria have unusual amino acid mew quot 39 39 39 mDAP in crosslink Functions provides rigidity and shape to cell and prevents it from exploding due to high pressure inside cell Peptidoglycan as a target for antibiotics I Since peptidoglycan is unique to bacteria it s a great target for antibiotics I Example penicillin inhibits transpeptidase which crosslinks the peptides I However widespread use of antibiotics selects for resistant strain I Example Many organisms produce betalactamase which cleaves the Lactam ring of penicillin inactivating the penicillin Archaea I lack peptidoglycan I cell walls made of other polysaccharides I Some Archaea cell walls contain pseudopeptidoglycan I Other Archaea have a paracrystalline surface layer cell wall made of protein I Many bacteria also have S layers but it in addition to the cell wall 0 Gramnegative Bacteria I Only a few layers of peptidoglycan o Grampositive Bacteria I m layers of peptidoglycan up to E I 39 f 39 by negatively charged teichoic acids I Crosslinks within peptidoglycan are different often have Llysine instead of Meso diaminopimelate Lysozyme I Enzyme that destroys peptidoglycan leading to cell lysis I found in animal secretions amp thought to be a major line of defense against infection by Bacteria o The Outer Membrane of GramNegative Bacteria 0 Found only in Gramnegative bacteria 0 Made of a lipid bilayer I Outer layer quot 39 39 id LPS layer I nner phospholipid layer 0 Proteins porins within the membrane involved in transport 0 Endotoxin lipid A toxic portion of LPS 0 Porins transmembrane proteins allow for permeability through the outer membrane by creating channels that cross the membrane 0 Nonspecific porins waterfilled channels through which small substances can pass I Specific porins channels with binding sites for certain molecules that only allow those molecules to pass through 0 Periplasm I space between the outer and cytoplasmic membranes I contains proteins such as hydrolytic enzymes amp nutrient transporter binding proteins 0 The GramPositive Envelope 0 Capsule not all species I Polysaccharide o S layer not all species I Made of protein 0 Thick cell wall I amino acid crosslinks in peptidoglycan I Teichoic acids for strength 0 Thin periplasm o Cytoplasmic membrane 0 The GramNegative Envelope 0 Capsule not all species I Polysaccharide 0 Outer membrane I lipopolysaccharide 0 Thin cell wall I amino acid crosslinks in peptidoglycan 0 Thick periplasm o Cytoplasmic membrane Arrangement of DNA in Microbial Cells 0 genome a cell39s complete set of genes 0 DNA is arranged to form chromosomes 0 Prokaryotes have a single circular 39 and 39 circular 39 39 DNA plasmids o Euka ryotes several linear chromosomes 0 Nucleus membraneenclosed structure that contains the chromosomes found in eukaryotes o Nucleoid mass of DNA not bound by a 39 found in prokarvotes The Bacterial Nucleoid 0 Single loop of doublestranded DNA 0 4x106 bp in many bacteria 0 Compacted via supercoiling 0 Attached to cell evelope o No membrane separates DNA from cytoplasm o Replicates once for each cell division Bacterial growth 0 Growth of most microorganisms occurs by binary fission Cell Division 0 Cell elongates as it grows o Adds new wall at cell eguator 0 DNA replicates to make 2 chromosomes 0 DNA replicates bidirectionally 0 Can begin next replication before cell divides 0 Cell undergoes septation 0 Usually at eguator 0 Each daughter has same shape 0 Fts Proteins the Cell Division Plane and Cell Morphology o Divisome division apparatus in the cell formed by Fts proteins 0 Fts proteins required for cell division amp chromosome replication 0 FtsZ protein 0 defines the division plane in prokaryotes o polymerizes to form a ring where cell division will occur 0 FtsA protein 0 ATPhydrolyzingenzyme 0 provides energy for assembly of other proteins to the ring 0 Ftsl protein 0 involved in I I 39 39 U synthesis for the new cell wall activity is blocked by penicillin helps define cell shape forms filamentous spiral shaped bands around the inside of the cell under the cytoplasmic membrane CM defines cell shape by quot 39 quot exerting pressure against CM Coccus shaped bacteria lack MreB gene default bacterial shape sphere O 0 MreB O O OO O Peptidoglycan Synthesis and Cell Division I New cell wall is synthesized during bacterial growth by inserting new glycan units into preexisting wall material I Autolysins create openings in existing cell wall to make space for new cell wall glycans to be inserted Autolysis spontaneous cell lysis may occur if there is an error in inserting new cell wall material I bactoprenol hydrophobic lipid alcohol I binds the N 39 39 precursors helps transport these new glycan units through to become part of growing cell wall 0 Transpeptidation 0 formation ofpeptide cross links between NAMS 0 bonds I 39 39 I U I l r C I I 0 reaction is inhibited by penicillin cell lysis occurs N 39 39acidl quot quot 39 U layer Cell Inclusions o Prokaryotic cells often contain internal granules that function as storage materials or to orient the cell 0 Inclusion Granules densely compacted material in cytoplasm o quotquot U 39 quot 39 particles ofa5uiw Fegg 39allow organisms to respond to a magnetic field Glycogen carbon and energy source Polyphosphate stores inorganic phosphate PolyBhydroxybutyratecarbon amp energy source Sulfur energy source I Some gramnegative prokaryotes can store elemental sulfur in globules in the periplasm 0000 Bacterial Cell Surface Structures 0 Pili amp Fimbriae o Bacteria may have 1 both or neither 0 Fimbriae I Nonmotile extensions that help bacteria attach to surfaces and to other bacteria Neisseria biofilms I Shorter than flagella may have 100 s per cell 0 Pili I aka conjugation pili Hollow nonmotile tubes made of protein called pilin that connect some cells Longer than fimbriae shorter than flagella may have 110 per cell I Used to move DNA from 1 cell to another by 39 39 capsuleslime layer glycocalyx o Sticky polysaccharide or polypepetide layer surrounding cell 0 Protects cell from o Phagocytosis o Desiccation drying out 0 Help cells attach to objects such as teeth Cell Motility o Flagella 0 Long helical protein filaments Attached at ends or over whole cell Flagella rotate to propel cell Proton passage drives rotation Clockwise or counterclockwise o Arrangement of Flagella Monotrichous single flagellum at 1 ende Lophotrichous several flagella at 1 or both ends Peritrichous several flagella all around cell Amphitrichous 1 on each end 000 0 Structure of the flagella 3 parts 1 Basal body 2 Hook 3 Filament o Basal Body Imbedded within cell envelope 0 Made of 2 or 4 protein rings 39 by a central rod Q ring in 63 amp G o m ring in 63 amp G o 3 ring in 6 only 0 ring in 6 only C ring In cytoplasm Attached to inner surface of cytoplasmic membrane 0 MS ring In cytoplasmic membrane End of central rod is attached to MS ring 0 P ring In peptidoglycan layer 0 L ring In E layer 0 Hook 0 Curved structure made of protein connects filament to basal body 0 Filament 0 Long rigid helical structures made of protein called flagellin o Prokaryotes such as filamentous cyanobacteria Myxococcus Cytophaga amp Flavobacterium move by gliding motility instead of flagella o Gliding can occur from slime secretion that moves cell along solid surface 0 Motile bacteria can respond to chemical and physical gradients in environment by moving toward or away from the signal molecule 0 Directed movements toward or away from a chemical or physical signal are known as o Chemotaxis directed movement of organisms in response to chemical signals 0 Phototaxis directed movement of organisms in response to gm 0 Aerotaxis directed movement of organisms in response to oxygen o Osmotaxis directed movement of organisms in response to ionic strength 0 Attractants cause counterclockwise rotation o Flagella bundle together 0 Push cell forward 0 quotrunquot 0 Repellents cause clockwise rotation o Flagella fly apart 0 quottumblequot change of direction 0 Runs tumbles cause llrandom walkquot 0 Receptors detect attractant concentrations 0 Sugars amino acids 0 Attractant 39 increases and prolongs run 0 Net movement of bacteria toward attractants 39Chapter 7 Genomes and Chromosomes Genetics Molecule of heredity 7 Deoxyribonucleic acid DNA Gene 7 Sequence of nucleotides in DNA that codes for a protein 7 Nucleotide sequence corresponds to sequence of amino acids RNA serves as an intermediate The three key processes of macromolecular synthesis are 1 DNA replication making a copy of DNA 2 transcription synthesis of RNA from a DNA template 3 translation synthesis of proteins using messenger RNA as a template Central dogma of molecular biology 539 lt3 I w Jl ici llm 5 DNA 5 339 39 5 3 TRANSCRIPTION 0F BOTTOM OF STRAND RNA 5 m Ill 339 lTRANSLATION Protein Ilz l l39 Wquot Basic processes are the same in prokawotes amp eukawotes but the organization of genetic information is more complex in eukawotes Major dif39ferences Eukawotes chromosomes are linear introns DNA sequences within genes that are not part of coding sequence for protein contain huge amount of noncoding DNA between genes In humans over 90 of the total DNA is noncoding enhancer sequences promoters gene remnants pieces of viral genomes Prokaryotes Chromosomes are usually circular have little noncoding DNA less than 15 Operons are common cluster of genes controlled by 1 regulatory sequence genes are transcribed together into 1 mRNA strand then translated into separate proteins DNA is a double stranded helix twisted ladder The two strands in the double helix are antiparallel run in opposite directions The 2 DNA polynucleotide strands have base sequences that are complementary Adenine pairs with thymine by two hydrogen bonds Guanine pairs with cytosine by three hydrogen bonds ANTIPARALLEL STRANDS OF DNA 5 5 TV A L CEO 5 5 DNA is made of deoxyribonucleotides linked by phosphodiester bonds Deoxyribonucleotide sugar deoxyribose nitrogenous base phosphate Size of DNA Expressed as number of thousands of nucleotide bases or base pairs per molecule 1000 bases 1 kb kilobase or 1kbpkilobase pairs Mycoplasma has smallest cellular genome known at 580 kbp Escherichia coli 4640 kbp Humans over 32 million kbp Nucleoid of E coli circle of dsDNA 1500x the size of the cell Can be packaged into the cell because it is supercoiled Topoisomerases assist in supercoiling u 2i vm g39iizigl U tiiil Cwi aunt quot lijj v jml l n i 3 ilf 39g il u lilvl39 Quinolone antibiotics such as ciprofloxacin target bacterial type II topoisomerases In addition to the chromosome several other genetic elements exist in cells 39 v 39F M s i39mIL Nil 3 Ni 391 7 itquot l39l l Mitochondria and chloroplasts contain their own DNA chromosomes Transposable elements exist as a part of other genetic elements Effect of Temperature on DNA Structure Hydrogen bonds hold 2 strands of DNA together 7 Individual hydrogen bonds are weak Covalent phosphodiester bonds hold backbone of DNA strand together Apply heat to DNA 7 Break hydrogen bonds but not covalent bonds 7 Called quotdenaturingquot DNA Temperature required to denaturemelt DNA depends on GCAT ratio More energy is required to break the 3 hydrogen bonds of GC so DNA with high content of GC requires higher denaturing temperature Lowering the temperature allows DNA strands to reunite htt video 00 lecom video la docid8469441567821436217 Major enzymes involved in DNA replication in bacteria 7 Helicase unwinds helix at replication fork 7 Single stranded binding protein prevents open helix from annealing 7 Primase primes new strands of DNA 7 DNA Polymerase major polymerizing enzyme 7 DNA Polymerase excises RNA primer and fills gaps 7 Ligase seals nicks in DNA 3 phases of DNA replication 1 Initiation melting unwinding DNA helix and binding of DNA polymerase complex 2 Elongation addition of 39 then r g 3 Termination DNA is duplicated supercoils are restored DNA is methylated 39 Most prokaryotes have a single circular chromosome 39 DNA replication begins at a unique site a specific sequence of nucleotides called the origin of replication or oriC Eukaryotes have multiple origins of replication 39 Replication proceeds in both directions bidirectional replication from the origin and ends at a site halfway around the chromosome called the terminus tersite Formation of the replication bubble 39 For chromosome to be replicated it must first be unwound by enzyme called helicase 39 Helicase breaks the phosphodiester in one strand a nick unwinds the strands a small amount and seals the nick 39 The unwound area is called a replication bubble 39 Singlestranded binding protein ssb prevents strands from coming back together DNA polymerization m c quot 2 lll 1th him Can only add to the 3 end not the 5 end 39 DNA polymerase cannot begin a new chain from scratch 39 To begin DNA replication there must be a primer site at which DNA polymerase can attach the first deoxyribonucleotide 39 Primer short stretch of RNA ribonucleotides made by the enzyme primase Deoxyribonucleotides can then be added to the at the end of the RNA primer ading amp lagging strands V V lam H in 39il 39 b i if i leading strand Y replication fork N 5 5y lagging 239 strand What bonds are formed when DNA is synthesized 0 Hydrogen bonds then form between complementary bases on opposite strand Extending the leading amp lagging strands 39 As replication fork opens leading strand continues to be made toward the replication fork 5 to 3 39 Lagging strand can t be made toward the fork so a gap is formed lagging 39 strand 39 Primase and DNA polymerase fill this gap by starting a new chain at fork and extending it to the lagging strand 39 These fragments are Okazaki fragments 1000 nucleotides long 39 RNA primer and stretch of DNA Process continues until terminus is reached Primers are then removed by RNase H and replaced with DNA by DNA Polymerase Okazaki fragment lagging slrand Sealing the nicks Once both strands have been copied there are still nicks between the Okasaki fragments nick gt gt These nicks are sealed by DNA ligase Copying entire E coli chromosome takes about 40 minutes Cell division takes about 20 minutes Partially replicated chromosome can start new rounds of replication even before the first round is complete Accuracy of DNA replication Few errors are made 7 About one per 10 9 nucleotides 1 Complementary basepairing 2 Proofreading by DNA polymerase III 7 Remove incorrect nucleotide and replace with correct one exonuclease activity htt www outubecom watchvteV622rm2P0 http AIAInnrtoncomcolle ebiologymquot Plasmids I ainaspchnoch07a01 Extrachromosomal pieces of DNA 0 Replicate independently of the chromosome 0 Lowcopynumber plasmids 7 One or two copies per cell 0 Highcopynumber plasmids 7 Up to 50 copies per cell 7 Divide continuously Plasmid Replication o Bidirectionalreplication 7 Similar to chromosomal replication 0 Unidirectional quotrolling circlequot replication 7 Starts at nick 7 Helicase moves around plasmid repeatedly 7 Complementary strand synthesized 7 Used by many bacteriophage 7 Concatemer I Many copies of plasmid binded together 7 Cos sites Plasmid Genes Advantageous under special conditions 0 Antibioticresistance genes Genetic Making Genes encoding resistance to toxic metals Genes encoding proteins to metabolize rare food sources Virulence genes to allow pathogenesis Organism may produce toxins Genes to allow symbiosis engineering new DNA molecules Agarose resulting DNA fragments have either blunt ends or staggeredstickycohesive ends can be separated using agarose gel electrophoresis gel electrophoresis Used to separate and determine sizes of DNA fragments DNA samples are loaded into slots at one end of an agarose gel An electrical current is passed through the gel DNA has negative charge so moves toward positive electrode smaller DNA fragments move farther than larger fragment Using a size standard size of each band is known size of other fragments can be determined Stain DNA with methylene blue or flourescent dye bc DNA is colorless I I I I I I http 39 39 39 hfml Negative electrode lquotgt0quot39 l39JONIquotI I HZHNNEO Positive electrode Recombinant DNA DNA fragments from different DNA molecules can be cut with the same restriction enzyme then joined together using ligase o Polymerase chain reaction method used to make many copies ofa specific DNA sequence in vitro using primers and DNA polymerase First described in 1985 Nobel Prize for Kary Mullis in 1993 materialsreagents used in PCR DNA nucleotides building blocks for the new DNA Template DNA the DNA sequence that you want to amplify copy Primers singlestranded DNAs between 20 and 50 nucleotides long quot that are 39 y to region on either side of template DNA u I DNA polymerase a heat stable enzyme that catalyzes the synthesis of new DNA 3 major steps in PCR repeated for 20 to 40 cycles in automated Thermal Cycler which can heat and cool reaction tubes quickly 3 steps is one cycle Denaturation quot 94 C double stranded DNA melts to single stranded DNA Annealing quot 54 C Primers hydrogen bond to complementary sequences of DNA template Extension quot 72 C Deoxyribonucleotides complementary to the template are added to the primer on the 339 end http 39 39 39 39 39 ntpcrhtml httD flmlrnrgddnalcresou 39 39 html Metagenomics use of genomic techniques such as DNA sequencing and PCR to study mixtures of microbes directly from environmental samples no need to grow organisms in lab DNA sequences determine which species are present and which known species they are closely related to 1 How do complex and defined media differ 7 ln ue me chemical do me a n is unknown N What are 3 forms of solid medias How is each used a Plates deeps and slants r Plates are u eu lUl Slants are used for the maintenance of stock cultures 3 Define Sterilization How does this differ from pasteurization ueep areu eu lo 11 39 to killdi ea er chemosterilizers EXP SQUES 0N5 HANDWAS NG The least effective a dial 2 incerno tor 39 s i enters the bloodstream ortissues a 39 39 39 39 39 no way Ba Frnm enviro 4 Why could there be more bacteria present after hand washing washingthe old ora off ofthe hands 5 what ismeant bytheterm mannaflora a 39 39 n nth hd under normal conditions EX 4 UES 0N5 OlONVMORPHOlOGV microorganism mar in han I t39 pigmentation r lllellleuiulll a Study the chart on pg 14 EXP SQUES ONS PURE CUlTURE 1 why must the loop be amed between each duadrant a llluluuplllulu 39 39 39 me 2 what is the difference between a mixed culture and a pure culture 7 A pure culture is a culture containing a single organism r A mixed culture is a culture containing more than one organism a what is meant bythe term colony II in the colony should be genetically identical 4 After examining your 39 39 me TA says only 39 me mixed broth 39 this a A contamination may have caused the fourth colonytype if the lid is otrotthe agar plate tortoo 39 39 39rnayna au u 39 39 39 ln39theprpra Vii technique is not used contamination may occur r in orderto obtain a pure culture the colonies must be isolated Exsu 1 whyarethestainsperrormedtodaycalledsimplestains r y morphology can be seen clearly Do some of a Streptococcus chained round cells a Staphylococcus round grape like clusters 7 Bacillus rod shaped e Spirosoma twisted resemble corkscrews a what are the three principal cell shapes 7 The three shapes are cocci round bacilli rodrshaped and spiral 4 Explain why staphylobacilli do not exist never form clusters of rods EX 10 QUEST 0N5 TH BROTH CUlTUREA PURE CUlTURE plate 39 What does this indicate about the number ofmicroorganisms in the broth culture 2 mixed 2 Considerthe results oi your simple stain again Did youranswertothe duestion quotlsthis broth a pure culture change what did your conclusion change 0r staythe same a same 3 I 39 r 39 pp pure on th imnl tain 39 when diiierent colonies where observed on the streak plate y y m lllh39ll39 arn characteristics to differentiate one cellfrom another 4 39 morphology present but there are two why a contamination EX 11 QUEST 0N5 GRAM STA 1 39 39 moment rt nlnri r a Primary stain Crystal violet rdant a Decolorizer Ethanol a Counterstain a Sairanin 5 m the mordant li h t would then be the Gram reaction oi all the cells Why a 39 39 l the a mec u r r r rauu u result iithe smear were overrdecolorized e lithe decolorizer is not added then all oithe cells would appear purple a lithe smear 39 39 39 39 mey really are not 4 whyis a counterstain added 39 39 me llliuu Lupe A g 39 on Monday quot me rairr 39 39 39 ror mi incorrect Gram reaction a lithe oacreria 39 39 au ly rorera meir primary rairr g 39 rarrrreaction in Gramrpositive and Gramrnegative bacteria 7 Both gramrpositive and gramrnegative cells have cell walls made ofpeptidoglycan Gramrpositive tuiLlluiL Liu EX 12 QUEST 0N5 ENDOSPORE STA N ofthese bacteria 7 The gram positive rods of Clostridium and Bacillus commonly form Endospores 2 Are Endospores reproductive structures why or why not a No 39 produced under stressful environmental conditions 3 Describe the process of sporulation when will sporulation occur at ueuume will occ 4 inenignie e to piayaroie in the heat resistance of Endospores 5 39 39 39 H 39 How does the presence Ul lluu pure n 39 39 u 39 39 7 Some common diseases are botulism anthrax gasgangrene and tetanus The ability forthese bacteria 39 39 quot t ia would normal die under 5 39 39 39 d nlnri r 39 39 39 39 lluu pure r The primary stain is malachite green the decolorizer is water the counterstain is safranin EXP 13 QUEST 0N5 A FAST STA 1 Vyy r irycoricaciu are 39 39 to p p 2 what was the appearance of Mycobpctarium smapmptis on the Myco plate Why a 39 39 This looks this way because ofthe hydrophobic nature ofthe cell wall a whywas heat used with the stain 7 Drive primary stain into cell wall 4 N nlnri r r 39 39 The decolorizer39 methylene blue 5 what important genus ofbacteria isthis stain used toidentify e The stain is used to identifythe genus Mycobacterl um a Are acidrfast bacteria Gram positive or Gram negative 4 in h i a Tuberculosis and Hansen s disease are caused by members of Mycobpcrerium 8 Why is this stain so useful for clinical diagnosis 7 M a nearinenrio lllepuliulll irn EXP 14 QUEST ONS CAPSULE STA N 1 wnal is me main componenl ofthe capsule wnal are other componenls r w y the cell wall ofbacteria 2 Whywasthesmearnotheatfixed a thesmearisheat xedthecapsulewillbe deslroyed a wnal are four possible functions ofthe capsule r rum I 39 39 lh r 39 39 nhan 39 quot all ii a n r source 39 her1 i W Hill Ulla rm usul 39 quot 39 possesses a capsule 5 Whyis il imporlanllo slain lne background with this capsule slain 7 iii nece aiy 39 appear a a clear EX 15 QUEST ONS EACTE Al POPULATION COUNTS 1 List three melnods ofenumerating bacteria 7 Direct Microscope counl Standard Plate Counl Turbidimetic Assay Z ruludulul llldllllullluu a Standard I A r V I L u E in 3 Why are only me plales with 307300 colonies considered counlable a 39 39 39 39 39 quot 5 manJuu I in all cells lo grow lo form colonies 4 Why musl a slerile pipellip be used lo make each dilulion a 39 39 rne dilulions 5 39 nr 7 Spread Plate and Four Plate EX 17AQUEST NS EFFECTOFTEMPERATURE Lllly lulul r Escherichia coli 5 whydces spoilage still cccurin milkthat has been pasteurized 39 39 quot Th r F a later germinate irheid at room temperature g a All microbes have a range oftemperatures over which they grow with the optimum temperature being that temperature at which they grcwn most rapidly 5 5 1 men 7 Because as temperatures drop from the optimum the rate or cellular enzyme activity diminishes and why does excessively high heat kill mcst organisms or cellular structures such as nucleic acids cccurs EXPt17B QUESTIONS EFFECT OF PH aLiu Laiiueu eu iuuu to which acids are added as a preservative Eth 17c QUESTIONS EFFECT OF UV 1 g lauiaiiuli Why n 39 39 p39 meiiu damaged cells 39 temperature I I lllE El 0 crganisms a 39 39 39 ruii ueauiiieiiii ir In inaipiuieu inein nuinu radiaticn Whatpanui damagech does this influence cell survival 39 39 39 the DNA These dimers can cause harmful mutations or death ofthe cells EX 17DQUEST NS EFFECTOFOSMOT PRESSURE 1 which organism showed the greatest range ofosmotic tolerance a vibrio natriegens and Staphylococcus aureus z a V p 39 39 us usa39 ioou pie e eu tni ay 4 39 poisoning 7 it inati39 FECTANTSANDANT EFT S EX 18 UESTONS D 1 xplain 39 properly used harsh to be appiied 39 39 39 illge liull 2 quot quot i i each rat a nt 1 a r l 39 uni llillleitisle on the surface 4 39 pluLE llluLLalE for the sick 7 Resistant to antibiotics endospores so harderto kill 5 39 39 39 39 39 llle same amount or 39 me again How could this be explained a endospores EXP 1 MEDIA PREPARATION AND STER De ned medium exact chemical composition is known llulll llluuupllyla Solid media in Petri dishes are called plates Bees mm usuallyusedforanaerobicgrowth rant 39 g me surface ofthe agar is used for maintenance of stock cultures ofmicroorganisms llleil team unue pie uie rkills endospores 121C at 15psi for 15 min Dry neat sterilization kills by oxidation eriects Flaming simplest metnod ofdryheat sterilization Htuil quot 39 39 quot 39 lu 39 HUI glassware Filtration heat sensitive liquids or gases can be sterilized by this rpaper leather wood metal and rubber products NASA l ammara UV light only useful to sterilize exposed substances degrade or alter biopolymers such as DNA and proteins Kw Pathogenic disease causing most microorganisms are NOTthis Nosocomialrhospital acduired infections EXP 3 HAND WASHING this Normalul 39 39 39 39 inemseie onoi39 normal condilions survive and mulliply iiinlroduced inlo bloodslream or lissue can be palnogenic me uuuy p 39 unable lo mulliply here and will die pH of skin is acidicrangingfrom 4 lo 5 EXP COlONV MORPHOlOGV luml NYE il NYE LEII u Eu are Separated from each other on the Surface OHhe medium Colony morphology snape onne colony Form onne colony margin elevalion and pigmenlalion ofthe colony are visible lo lne naked eye lo me cuuuie Medium the colony morphology ofthe organism is dependenl on this Calegories Whole colonysnape can be round irregular or rhizoid Margin shape can be smooln lobale orfilamentous Elevalion can be convex umbonale or llal Qplical properties opadue orlranslucenl Surface characteristics dull or sniny Pigmentation color Page 14 ExPs ASEPT TECHN QUE lul Tubes ul 39 39 lube Page 20 EXP 6 PURE CUlTURE Pure culture contains a single kind otorganism genetically identical EXP 7 SMEAR PREPARATION 39 1000 X Staining makes cells more visible Smear first step in staining neat SIMPlE STAIN i Staining allows the morphology ottne cell to be determined Basic shapes of bacteria coccus bacillus and spiral 39 39 nain l tr ntnl Spiral one ormore twists Vibrio look like curved rods Spirilla helicalshape thatis rigid Spirochetes helical and exible and resemble a corkscrew nutrients into the cell iypna iormamycen atranin and r talviolet USE or THE M CROSCOP m mag l a cell such that details of its structure are apparent Comgound microscoger consists of ocular and objective lenses 10 Binocular microscope for depends on wavelength and numerical aperture nine on en Egg where thesmearor wet mount is placed tight source should be set on 5 Condenser 39 39 the stage me loge me e are 1040 and mm x F e focusr to refocus the speciman EXP 10 TH BROTH CULTURE A PURE CUlTURE 39 ullly one type of microorganism is present A broth culture appears cloudy when growth has occurred u t atingtrom culture was mixed not pure nome ay ul Determine if sample is pure or mixed by doing both simple stains and streak plates th 11 GRAM STAIN tile elements of a cell or specimen linnnrnt in Stains y ii u u u x Di th 12 ENDOSPORE STAIN structures Vegetative Cel can grow and divide under optimal conditions L Sporulation a process of Endospore formation within a vegetative cell PartsoFEndospore nnr lat mr p terminal or subterminal d Germination when an Endospore returns to a vegetative state eutgrowtn visible swelling due to water uptake EXPt 13 ACIDFAST STAIN quot 39 39 39 39 39 we 5mm ul m Carbol fuchsin heating acidralcohol methylene blue 4 y th 14 CAPSUlE ST psule 39 p p p39 quot 39 39 i lllecellwallofbacteria lumleu ill llle virulence Factor enhances the organism s abilityto cause disease th 15 EACrERIAl POPUlA110N COUNTS rapid no incubation required no selection against organisms Viable Count one living cell will give rise to one oolonv live cells I nair hain l Sgread Plate u eu lUl 39 gm in and mu El necessary simple llvpe of standard plate count Pour Plate no alcohol or ames involved type of standard plate count Countable Plate one that yields 307300 colonies cmml eo y organi n EX 1 MOST PROBABlE NUMBER MPN TECH Potable Water waterthat is safe todrink Ente d eases diseases that affect the gastrointestinal tract lactose within 48 hours of incubation at 35 c lndioalor g ismra pathogens Escherichia coli Total coliform count used to indicate sewage pollution quotni ue 39 lllleelel 39 39 ga piooooioniioin lucoliforms nr Ilmnli enrirm d i Lola m rne gio rnoi undesirable competitors o mioroorganisrn or rm i a 39 39 i r between mannitol and nonrmannitol fermenters Eosin I I differentiates lactose fermenters from nonrlactose fermenters nat eieo ioi39 39 39 39 39 quot other t organism s and lactose is a dirierenlial component Broth medium 39 39 test Eosin contains differenlial componenl Fishrexe Colonie pale pink colonies E aemgenes ExPc 17 IIIILI Mun Ilnulunl h 39 39 nor pring 5 nornan g s 39 inArclic regions Range of Temgerature all microorganisms have a range oflemps over which they grow oblimum LH the acidilv or alkalinilv of a soluiion basic 7714 acidic 17 neutral 7 Ogtimum pH lne pH al which the microorganism grows besl Acidopnil grow al pH values below a Neulropniles a preferpH close lo 7 Basopnile also called alkalinophiles prefer pH values above 7 Tnvmine 39 f d 39L39l 39 39 39 dealn of cells Mulalion may provide beneficial new cnaraclerislics for me organism i 7L 7 E Solution composed of a subslance solute dissolved in a solvenl ale H area ul equilibrium HU uul iu L the cell Plasmolysi plasma membrane pulls away from the cell wall L lllallla alu el 39 llleillleliulul llle LI oell water will ow intothe cell o greater than 3 Extreme Haloghile members of Archaea thrive in solutions of 15730 NaCI concentrations ExPi 18 NFECrANTS AND ANTISEPTICS fungicides kill fungi 39 the growth oi surfaces ex Ammonia ethanol and bleach 39 39 illge liull ex ethanol and iodine Zone of inhibition il 39 39 39 39 organism 39 39 39 39 oi mi unei a lulu Hui quot nimi inn ml and effectiveness of the chemical agent ExPl 19 ANTIMICROBIC SENSITIVITY TESTING papei usul Zone ofln nLleal aiea in nien 39 39 in pay oi i line zone isa lonelionoi oom 39 39 me drug and 39 39 D39 39 Minimum 39 39 39 edge ofthe zone oFinhibition Kirb rBauer L a 5 lo laboratories a X 5 39 39 39 39 39 39 pathogens damages gram positive cells Fall 2012 BIOL 2051 General Microbiology Laboratory Midterm Study Guide There will be four pans to the midterm Section A a you will have to do a streak plate and perform a Gram stain Vou will do amp view your Gram stain ouringthe next iab nlate eti on 39 front of you Section c 7 questions about images you have seen through the microscope These images will be shown to you by PowerPoint presentation Section D 7 short answer questions chemical stained Morphology Reds Veg fuchsin NoanIue Capsule Around Congo 7 Mendeval s RedBrown Capsule Red stain II cu39ve and 39 39 39 39 e MPN Medium Selective agent Selective for DiHeremial Differential for visible indication of component differential component lSl broth Lauryl Sulfate lntestinal lactose Lactose Bubbles in the Bacteria fermentation inverted Durham tubes BGLB Brilliant Green Coliforms lactose Lactose Gas formation in broth fermentation durham tubes Bile EMB agar Eosin Methylene Gramhlegative fermentable Lactose fermenters Lactose Fermenters Blue lnhibit growth of gram carbohydrate lactose from nonrlacmse drop in pH uptake fermentors of dyes nonrlactose fermentersr pale or colorless colonies on EMB agar s E Gram stain must be both pink and purple 111 General rips Things to study Questions at end ofexperimenls Quizzes PPN 5 Vou will have to perform the streak plate technique properly Vou will be graded on whether you have isolated colonies of each bacterium in the mixed culture 5 Review any drawings you made Part of the exam is practical images ofmany of the materials used in lab can be seen in g by teoone and Pierce or online IV Dilution problem There WlLL be at least one dilution problem on the midterm iou I ha e I No calculators will be allowed v Evenbody s favorite question 7 what organisms do we need to knowquot Be sure you can spell the names correctly tunderiine Genus and species separately and capitalize rst letter or Genus SteptococcusEntemcoccus Gram positive coccus in chains Dei39nococcus radiodumns Gram coccus 439 39 39 4 39 meuiaiii m Mycobacteri39um Acidfast mycolic acid cell wall Aerobic rods nonendospore forming non motile Escherichia Coi39 Gram negative rod Coliform indicator organism forfecal contamination in us 39 Li ioagai Not salt tolerant Found in large number of sewage survives longer in water 4 ii Entembacter aemgenes Gram negative rod motile Coliform Fisheye colonies on EMB moderate lactose fermenter Sacchammyces cerevi39si39ae Eukaryote yeast acidophile Alcah39genes faecah39s Alkalinophile Vi39bri39o natri39egens Halophile Haobacteri39um sali39mm39um Archaea extreme halophile requires 25 salt t ferment lactose Hyphomonas neptunl um Vl brl o thoeras Shl gela dysenterl a cell surface area and help transport nutrients into the cell that form mycelium resistant animals Cholera pH 9 Dysentery Propl onl bacterl um acnes Development of acne part ofnormal mierobiota of skin pH o 4 A grobacterl um Grow in soils pH of 12 Alkalinophile Vl brl ofl sch erl Halophile inhabits marine waters grows optimally in salt concentrations ofEWa 20 than 1NaCI used by bacteria to adhere to enamel to cause pneumonia r Colony Morphology r Solated Colonies E m FM EZ mi Hummiimm some Oblum un mm km Vampwu aw Diop ev4 insc tie pmuai A iquot wveum adiusimes iing rimming awe swim mg Ohlecliv a Me77 imam mien Avemire W5 mammgrr knob Mgr imansm39 mm CondemsriA enlerma screws gt5 Fm mus armament nah x9235 car udgusl mam knnb Fiem ms dlnphmgm nrg x axis mm was mu DurhamTubes the right is positive for tuibidity l left is negative fish eyes E coli cpmpleted MPNLesL Suns m 5mm 5m nf anquot swam Cwsmv uhal Eabmulvmm pnmry 51mm m2 karma 5st mm mm n amnnn ms anm mm mm m cumn39wzu mum mm Bramn emu sayaw brawnosmvs eus mamuh m mmimwrv a 6m wmm W apmm Nd Spinosom39q linguae G farmrnegativ39espira Is Sfdphylo bt w ullre w Gramrpibsitive st abhylococ i 39 Eh QSRQre rv 39 439 quot v quotA w quotvf a l 9 7 1 194quot gtltL xquot 39l KR F l yquot 39 i 9 quot a quotk i in m gkd IKH E1kz gt 5 i G uh 1 17 Q L k h illkk In I Arg M an A V 77 we agfi39v q vm MJ g quot 1 13921 gm L q f 1i I nLV rNonacidfastExoli Zone of inhibition and Con uent Lawn of bacteria What causes strep throat Streptococcus pyogenes m ni gsmmlji rig h iniwjmliawe Wall iri ll ill i i if l39iillaliiquot E lg lalzn l cell line 5p l li hillyi Know what defensin peptides that destroy cell membranes of invading microbes effective against G G fungi enveloped viruses interferon interferes with viral replication made by infected host cells specie speci c virus non speci c type antiviral bind to unaffected cells amp make them resistant to viral infection type II stimulate immune cells become more sensitive vi it Vi prevents complement binding amp antibody mediating killing capsule antigen these are the choices forgot the question hehe Man M l 3i liigu 1 r l zrvL39i39l n quot 39gt 4339 l r if my my Amati ul g jl 1v wgtlll 39 y llq f i Frill lm 39 quot39lliUllzl clclgi fhost pathogenicity something about if39w 39 quot l Hui il la h w i l l39uallil39 lquot39ill3 polymorphonuclear leukocytes type ofWBC neutrophil engulfs phagosome basophil eosinophil at lrmwr i r toxoid tatenus diphtheria killed bacterial cell cholera inactivated virus influenze salk polio live cellvirus tuberculosis chicken pox purified Superantigens proteins that cause very strong immune response activate more T cells than normal bypass normal route of antigen processing by simultaneously binding TCR amp MHC without undergoing antigen processing results in cytokine production amp systematic in ammation TSS Scarlet fever forgot the question but the answer is vector insects that carry the diseases or something like that Phagosome vacuole formed around microorganism as it is engulfed by host cell What antibody is present in breast milk lgA quot i i m fil i i llCllij l lg Which ofthe following is a surface polysaccharide that prevents phagocytosis 0 antigen of Salmonella following organisms produces a sticky polys ll li39i 1 ll gtl l What would cause natural killer cells or macrophages I forget t forgot the question but know that V a7 i ll emu M pr 3 u AM 5 g 1 question says that a student is studying for his finals and then lists all of these things found in his body and asks which one will he likely get because he is stressed o Streptococcus pyogenes was in the list and strep throat is an option all ofthe other things listed were things that we have vaccines for 2 which is false 0 the stomach doesn t allow any pathogens to grow in it because of its low pH 0 l marked this one as false because helicobacter pylori can grow in the stomach wall but that s not technically the stomach so I don t know what to tell ya 0 All ofthe above are true 3 which has the most and diverse am of bacteria in it o duodenum o jejunum o ileum 10 What is the region called that binds the antigen 0 I knew the answer was the light chain variable region 11 which ofthe following is not a part ofthe complement system increase inflammation increase activation of phagocytes lysis of gram another option I don t remember all of the above are part of the complement system 15 which makes the antibody specific for the antigen 0 plasma cell 16 if you re exposed a second time and there s a fast response its bc of 0 memory cell 20 this is the number needed to infect half the experimental group IDso 21 Which of the following need mbriae to infect the host 0 Neissen39a and E coli 25 which is false 0 cold and flu virus are not deadly 29 which is infects the genital area 0 herpes virus 2 30 Which can lead to pelvic inflammatory disease if left untreated o Neissen39a gonorrhoeae a r Cliff oi ill r iiJl lbw il39wig Chapter 1 Microbial Life Origin and Discovery What Is a Microbe Organism that requires microscope to be seen 0 Microbial cells size 02 micrometer to mm viruses much smaller 0 Contradictions 7 Supersize cells Thiomargarita namibiensis sulfur pearl is size of fruit fly head 7 Microbial communities in biofilms microbes have specialized functions amp act as multicellular organism 7 Viruses are noncellular considered to be microbes but are not fully functional cells 0 6 major groups studied by microbiologists 7 Prokaryotes 1 Bacteria most common we know of 2 Archaea found in extreme environments 7 Eukaryotes 1 Algae 2 Protists 3 Fungi 7 Viruses Differentclassification 0 Non cellular Microbes Shape Human History 0 Microbes affect food availability some destroy crops corn rust late blightIrish famine some make foods beer bread cheese Microbial diseases change history 7 Black plague in Europe killed 13 population in 14 h century 7 Smallpox in Americas 7 Europeans brought over since Indians had no immunity they died 7 Sometimes Americans purposely used this as tactic to kill indians 7 More soldiers have died from infections than battle wounds Florence Nightingale convinced British govt to improve army living conditions amp upgrade army hospital standards 7 TB in 19 h century 7 AIDS today Discovery of Microbes 0 Light microscope invented in 1600s 0 Mid1600s Robert Hooke observes small eukaryotes fungi with compound microscope 7 1st to use term cells mitqm i v 1ml 39zr 7 Built simple microscopes 7 Described llwee animalcules H bliiy h39 u 7 Microscope for each specimen 0 One lens and magnify 300x Microbes Are Living Organisms Spontaneous generationidea that living organisms originate from nonliving matter 18005 0 Microbes arise only from other microbes 7 No spontaneous generation 7 1861 Pasteur shows that microbes do not grow in liquid until introduced from outside living organisms only arise from other living organisms 0 He disproved this using broth in swannecked flasks Sterilized broth by boiling L Open to air a 5 curve excludes dust and microbes Growth medium led to development of methods for controlling growth of microorganisms Pasteurization use of moderate heat to reduce number of microorganisms in foods milk m oysters Worked with wine industry amp studied fermentation Medical Microbiology Germ Theory of Disease Observations 7 Germs can infect and grow on food Hypothesis 7 Can germs infect and grow on people 7 That is do germs cause disease Hypothesisistestable 7 Are germs found in infected tissue 7 Can transmission of germs cause disease Koch s Postulates Developed from his study of anthrax Provides scientific basis for determining that a specific microbe causes a specific disease 7 quotDoes this germ cause that disease 7 Organism must meet 4 criteria 1 The microbe is found in all cases of disease but absent from healthy individuals 3 When the microbe is introduced into a healthy susceptible host the same disease occurs 4 The same strain of microbe is obtained from the newly diseased host Koch s postulates are only a guide 0 People are carriers 0 S aureus 0 M tuberculosis only 10 of people infected have symptoms 0 Organisms which can t be grown in a lab 0 Treponema palladium Syphillis found at extremely low concentrations in bloodstream No quottestquot animals for this Disease caused by more than one organism 39 u 1 1881 17 of all deaths caused by TB Thought it was contagious but had never seen organism Mycobacterium tuberculosis grows very slowly hard to stain due to lipid content Koch developed acidfast staining procedure patient enough to get pure culture 1905 Nobel Prize was awarded to Koch for his won t m m History ofimmunization Stimulation ofan immune response by deliberate 39 with an pathogen Lady Montagu early 1700 s brought practice of inoculation with smallpox variolation to England from Turkey Variolation Collect scabs of small pox grind them and put them into nostrils for mild case of smallpox so that they were immune to more serious cases sometimes didn t work very well 0 Would vaccinate people with a pustular material Edward Jenner 7 1778 found that milkmaids exposed to cowpox were immune to smallpox 7 Vaccinated people including his son with cowpox they were resistant to smallpox Pasteur 7 in 1879 studied fowl cholera 7 Isolated and grew bacteria which causes it then left for vacation 7 After vacation the aged bacteria no longer caused disease in chickens 7 Inoculated same chickens with fresh bacteria from new outbreak amp they still did not get sick 7 Attenuated weakened bacteria gave chickens immunity from disease 7 Developed vaccines for anthrax fowl cholera rabies from dried rabbit spinal cord quot 39 for quot39 g pathogens gnas Semmelweis mid 1800 s didn t know about germs at this time Puerperal feverchildbed fever 0 Illness caused by streptococcus didn t know at the time Medical students carried llcadaver particles from autopsy room to delivery room 0 Noticed that med students didn t clean hands and went straight to work on patients realized they must carry something bc death rate was higher with med students than with nursesmidwives Caused by Streptococcus 0 Had med students wash hands in chlorinated lime water reduced mortality from 18 to 1 0 Cut down on bad smells 0 Worked well reduced mortality rate People thought he was ridiculous for thinking this even though percentages showed it to work and he ended up going crazy 0 Put in asylum and died of streptococcus Joseph Lister 0 1864 British surgeon found that survival rate of surgical patients increased if surgeons washed hands sterilized instruments amp used disinfectants carbolic acidphenol during surgery 0 dea came after germ theory ofdisease had been proven Paul Ehrlich early 1900 s 0 Idea for llmagic bulletquot that would kill pathogens but not harm humans 0 found arsenic compounds used to destroy trypanosomes quotcompound 418quot cured sleeping sickness too much can kill patients Discovered Salvarsan quotcompound 606quot to treat syphilis 0 Too much can kill patient Used term quotchemotherapyquot using chemicals to kill pathogens without harming patient Arsenic turned out to be fairly toxic to humans 0 now know arsenic is fairly toxic and this was an option then if dosage wasn t harmful y Lil produced by penicillin notatum fungus on Staphylococcus o noticed mold didn t have straphylococcus I purified substance but not pure enough to take in body 1945 Nobel Prize in Medicine Fleming for discovery Florey amp Chain for determining mode of action of penicillin amp developing method of mass producing the drug 0 Florery and Chain purified so could be taken in body lli ll I Can produce in mass o 1945 penicillin became available for generalpublic use 0 used on battle wounds 0 mortality rate dropped Growth of microbes in pure culture Colony group of identical cells that arose from one culture Koch amp solid culture media 0 Potato slices 7 streaked bacteria onto potatoes 7 Problem too much moisture many contaminants 0 Beef broth solidified with gelatin 7 Problems Gelatin liquefies above 28 C 0 Body pathogens grow at 37 C Many microbes produce enzyme called gelatinase liquefies gelatin 0 Agar 7 polysaccharide derived from red algae She used it to make jelly Agar is derived from red seaweed thickens 7 Solid until 100 C amp most microbes cannot digest it Few microbes can produce agarase which breaks down agar but not many can do it and ones that can do it in small amounts Petri Dish 0 Richard Petri Koch s assistant invented them in 1887 0 Before this they used a bell jar or glass box now we use plastic so we can discard 0 1677 Observed llwee animalculesquot Antony Leeuwenhoek 1796 All quot quot rl 0 1850 Advocated washing hands to stop the spread of disease Ignaz Semmelweis 0 1861 Disproved spontaneous generation Louis Pasteur 0 1862 Supported Germ Theory of Disease Louis Pasteur 0 1867 Practiced antiseptic surgery Joseph Lister 0 1881 Growth of Bacteria on solid media Robert Koch 0 1882 Outlined Kochs postulates Robert och 1885 First Rabies vaccination Louis Pasteur 0 1887 Invented Petri Dish RJ Petri 1892 Discovered viruses Dmitri losifovich Ivanovskil filterable agents 0 1910 Discovered cure for syphilisSavarsan 606 Paul Ehrlich 0 1928 Discovered Penicillin Alexander Fleming Microbial Ecology 0 Most microbes don t grow on typical medium 7 Very few microorganisms can be cultured quot 01 of microbes in our biosphere can be cultured in the lab 0 Many live in varied conditions 7 Anaerobic no 02 0 Bottom of swamp mud ocean floor 7 High pressure 0 Bottom of ocean 7 Hot or cold temperatures 0 Below 0 C to 113 C 0 Strain 121 can grow at 121 degrees Cels 7 No organic carbon 0 Use light for energy photosynthetic C02 for carbon source make it into sugar 0 Can culture some microbes in natural mud environment 7 Winogradsky column 0 Layers grow different species reflecting different conditions they require 0 If need 02 they grow at top 0 If need sunlight grow toward side facing light 7 Can see variations in nature 0 Yellowstone geyser runoff 0 Colors reflect different species 0 Different growth temperatures 0 Microbes cycle most elements on earth very involved in nutrient cycle 7 Nitrogen cycle 0 Bacteria fix NZ to NH4 0 Most air we breath is nitrogen that bacteria convert for plants to use 7 Carbon cycle 0 Photosynthetic microbes fix most carbon 7 Sulfur cycle 7 Phosphorus cycle The Microbial Family Tree Microbial species are difficult to classify Carolus Linnaeus 7 Binomial nomenclature Categorize all microbes into group called chaos 7 Difficult to distinguish by shape 7 Pass DNA to each other without reproduction even if not closely related at all Use biochemical properties to classify 7 Gram stain Helps divide organisms into 2 categories based on cell wall structure 7 Ability to metabolize different substrates Use nucleic acid sequencing to classify 7 Bacterial genomes are relatively small so easy to sequence 7 2 distinct species share no more than 95 similarity of DNA sequence 0 if not 95 similar it is in other species Several lineages exist in the domains Bacteria and Archaea huge diversity of cell morphologies and physiologies Phylogenetic tree of life Fig 15 amp 127 3 domainsdetermined by rRNA sequencing ammo Algae and plum 5mm Fungl and mlqu mum Prokaryotic Microorganisms Bacteria most prokaryotes that we encounter soil bacteria diseasecausing bacteria etc 7 Exp Handri 39l39 coli quot 7 The ones we mostly know of I Archaea prokaryotes that live in extreme environments hot springs glaciers salt lakes swam as and Halobacterium salt environments 25 salt Pg Y Archaea are not bacteria 7 Similar size shape 7 Very different biochemistry 7 Different membranes 7 Archaeal ribosomes similar to eukaryotic ribosomes not bacterial ribosomes 7 Many but not all archaea live in harsh unusual environments Eukaryotic Microorganisms includes fungi algae amp protists Fungi nonphotosynthetic most have cell walls 0 Some are microorganisms and some are not 0 Cell walls made of chitin 7 Ex Yeasts unicellular and molds multicellular hyphae 0 Yeast budding is uneven division of yeast cell Saccharomyces yeast is used to make beer and bread 7 Penicillium mold produces penicillin o Algae 7 f contain 39 r39quothavecellwalls 39 have quot r39 7 Ex Diatoms amp seaweed o Protozoa Protists o Unicellular motile no cell walls 0 Different methods of motility Flagella cilia pseudopods 0 Some can cause very serious illnesses 0 Ex Amoeba Paramecium amp Giardia Eukaryotes evolved through endosymbiosis o Endosymbiosis 7 theory that eukaryotic organelles evolved through a symbiotic relationship between two prokaryotes 7 one cell engulfed a second cell and a symbiotic relationship developed 7 mitochondria and chloroplast are thought to have evolved this way 7 mitochondria from respiring bacteria similar to E coli 7 chloroplasts from phototroph related to cyanobacteria Evidence 7 Mitochondria and chloroplasts Many bacteria have 2 membranes 0 Inner and outer possess their own DNA amp ribosomes 0 similar to bacteria are about the size of a prokaryotic cell divide by a process similar to bacteria Chapter 24 The Adaptive Immune Response Recommended reading Chapter 241244 246 248 lmmunity ability ofan organism to resist infection All cells involved in immunity originate from common stem cells in bone marrow 39 Immune system protects against foreign cells amp macromolecules ex toxins 39 Antigenlmmunogen foreign cells or macromolecules that induce immune system Types of Immunity 39 l Naturally acquired immunity 39 2 Artificially acquired immunity Naturally acquired immunity 39 Can be classified as active or passive Naturally acquired active immunity 39 Host produces antibodies amp T cells in response to an infection 39 Immunity can last for years or lifetime Naturally acquired passive immunity 39 Occurs when antibodies are passed from one host to another mother to baby 39 Ex Antibodies pass through placenta from mother to fetus These circulate in infants system for several months after birth 39 Lasts few weeks to months Artificially acquired immunity 39 Can be classified as active or passive immunity Artificially acquired active immunity 39 Result of vaccination 39 Host makes antibodies that can last for years VaccinationsImmunizations To reduce risks vaccines contain inactivated pathogens or their products Toxoid chemically modified exotoxin retains antigenicity but loses toxicity Tetanus Diphtheria Killed bacteria cell killed by formaldehyde or heat Cholera lnactivated virus formaldehyde treatments inactivate Salk polio vaccine Influenza Live cellsvirus Most effective Attenuated weakened by a chemical has lost its virulence Tuberculosis chickenpox Purified polysaccharide M e ni ngitis Artificially acquired passive immunity 39 Host receives antibodies antiserum from another host that has formed antibodies against a specific antigen 39 Ex Snakebite victim receives antivenom 39 Lasts only a few weeks 39 Artificial and passive Innate or Nonspecific Immunity First line of defense Phagocytic leukocytes Engqu amp destroy pathogens and stimulate adaptive immune response Contain ysosomes 7 Inclusions containing HZOZ ysozyme proteases phosphatases nuclease and lipases that destroy invader Phagocytic leukocytes l Neutrophils 7 PMNs polymorphonuclear leukocytes 7 Large numbers in blood or inflammation site indicate active infection 2 39 39 I 39 a antig p ting cellsand 39 39 7 called monocyte when circulating differentiates into macrophage when enters tissues 7 Antigenpresenting cells Macrophage present peptide antigens to T cells to activate a specific immune response NORTON VIDEO IN CLASS Macrophages are phagocytic first to meet pathogens and take them in resuting vesical called phagosome where they cleave macromolecules to destroy pathogens antigenpresenting cells MHC brings antigen to surface Adaptive Immunity Immune system reacts to specific antigens 7 Parts of foreign proteins sugars chemicals Retains memory of those antigens 7 Faster response if exposed a second time 2 parts to adaptive immunity Humoral immunity 7 Production of antibody proteins Each binds a different antigen only ever one Cellular immunity 7 T cells that bind different antigens Kill pathogens control antibody production lmmunogenicity ability ofantigen to elicit immune response Immune system reacts to antigens 7 Proteins are good antigens very immunogenic Fixed nonrepetitive shape unlike lipids nucleic acids Immune reaction is specific 7 Response to one antigen usually does not protect against a different antigen 7 Attack only antigens from nonhost Blood types type A attacks type B blood cell antigen AB blood has both antigens won t make antibodies 0 has antigens to both look up to see if correct Antigenpresenting cell APC phagocytic cells that present processed antigens to T cells Antibody Immunoglobulin a soluble protein produced by B cells interacts with antigen neutralizes antigen Y shape structure Following ingestion of pathogen phagocytes and B cell lymphocytes present antigens on their surface to T cell lymphocytes resulting in Cellmediated immunity T cells Antibodymediatedhumoral immunity antibodies amp B cells Cell mediated immunity Involves T cell lymphocytes Mainly protects against tumor cells amp virusinfected cells Synthesis of T cells T cells are derived from bone marrow stem cells These stem cells migrate to the thymus amp differentiate into T lymphocytes B cells don t have to go to thymus and mature Presentation of the antigen When phagocytes digest pathogens polypeptides from pathogen are bound to surface of phagocyte Antigen Presenting Cell APC When an APC binds a T cell it causes the T cell to differentiate Each T cell type can recognize antigen that was presented to the original T cell Ma39or classes of T cells HelperTcells TH THl activate TC cells cytotoxic T cells THZ bind B cells amp activates them to divide into plasma cells amp memory cells THO undifferentiated T cells Cytotoxic T cells TC Destroy tumor cells amp infected cells by releasing cytotoxin and creating pores porifin Also recognize grafted tissues amp organs as foreign ex kidney transplant Memory T cells TM Allows host to respond to 2quotd exposure to same antigen quickly Tcell receptors TCR Receptor attached to Tcell surface membrane Binds antigen specifically Structure 7 2 peptides alpha amp beta each with a constant amp variable domain Variable domains bind antigen Major Histocompatibility Complex MHC proteins Self pr 39 on surface of cells mark cells to know they belong Bind to anti en E AntigenMHC complex represented on outside of cell is recognized by Ticell receptor Class MHC 39 all nucleated 39 Class II MHr F blood stream Trcells become activated when antigenM HC bindsto Trcell receptor look up figure 2420 mm Wquot nllligell U TC cells antigen u TH Mllm MumMail mmnlex rumin mmquot Antigm DiasMinqu Cellrmediated immunity 0 TC cells destroy the antigenrpresentingcellor o THl cells recruit macrophages 0 destroy antigenrbearing cell 0 Tumor cells transplanted organs amp tissues are attacked by TH 1 cell activated macrophages Antibodyr mediated immunity 39 Antibodies bind to antigensi inactivate them Most active apinst bacteria bacterial toxins amp viruses in bloodstream Also called humoral immunity Role of antibodies 39 Antibodiesdon t recognize entire pathogen Recognize regions ofantigens called epitopes Binding ofantibody to a foreign cell does not directly killthe cell Antibody binding lookfor figure with antibodies and bacteria 39 activatesthe complement system 39 enhances phagocytosisiopsonization 39 causes agglutination 39 neutralizes antigens The complement system main job is to form holes in cells 39 Group of proteins that act together to enhance immune system 39 Complement can Increase inflammation attract and activate phagocytes cause lysis Opsonization Process ofenhancing phagocytosis Antibodies can bind both to a microbe amp phagocyte simultaneously amp bringthem in closer proximity to each other Antibody bridge increases ability of phagocyte to ingest microbe Antibody Structure and Diversity Hinge regio allows each fragment to bind antigen independently Light chain yellow heavy chain blue constant region bound by immune cellsrsame for each class ofantibody variable region bindsantigenidifferent region produced by each B cell binds different antigens constant region bounds by immune cells7 same for each class of antibody Antibody Structure Yrshaped proteins lgG consists of4 polypeptide chains The 2 largeheavy chains are identical amp held together by 2 disulfide bonds The 2 smalllight chains are also identical amp held together by ldisulfide bond Constant and variable antigen binding site domains The constant region ofall lgG molecules isthe same Variable regionsrwhere antigens bind Specific for the antigen that the lgG molecule was made against Each lgG can bind 2 antigens IgG Anugan binding site Havy mam Constant regun Vanableregun 5 classes of antibodiesimmunoglobulins look up pics of each I Heavy chain constant domains define class IgG Most abundant 80 of Ig in serum is IgG Only antibody that can be transferred through placenta Gamma heavy chains IgA Predominant antibody in secretions saliva tears milk mucus etc Dimer looks like 2 IgG molecules held together Alpha heavy chains IgM 2quotd most abundant g liclass of antibody made in response to infection lSt on the scene Aggregate of 5 IgG molecules attached by disulfide bonds ampJ chain protein Can bind up to 10 antigens at once wonderful for glutination Mu heavy chains Low concentration responsible for allergies Binding ofantigen to IgE causes release of histamine allergies Epsilon heavy chains gD Found on the surface of B cells Binding ofantigen to these antibodies stimulates the B cells to produce antibody mostly IgG Delta heavy chains Synthesis of Antibodies B cells Type of lymphocyte makes antibodies B cells are made and mature in bone marrow then disperse through blood amp lymph B cells collect in lymph nodes amp spleen Presentation of the antigen 1st step in making antibodies is phagocytosis of foreign cell by B cell Foreign cell is digested amp polypeptides from that cell are bound to surface of B cell Activation of the B cell look for figure Next a TH cell binds polypeptide on B cell Binding of the TH cell triggers B cell to divide Differentiation of the B cell B cells differentiate into plasma cells life span up to 100 days or memory cells continue to divide amp circulate for many years Plasma cells make antibody specific for antigen that was presented to B cell If memory cells are exposed to same antigen again will differentiate into plasma cells amp produce antibody Primary immune response After initial exposure to an antigen it takes several days lag time before much antibody is produced 39 During this lag time new B cells are made that are specific for this antigen 39 IgM is made lit then IgG sotype switchingclass switching predominant antibody produced becomes IgG instead of IgM Secondary immune response 39 When host is exposed to same antigen again lag time much shorter 39 Much more IgG made 39 Faster response is due to memory B cells which quickly form large numbers of plasma cells I Over time antibody titer decreases but subsequent antigen exposure causes another secondary response quotbooster shot 39 Watch WW Norton video about T cell Immune Response Diseases I Hypersensitivities inappropriate immune response resulting in host damage I Superantigens pathogen proteins that cause widespread stimulation of immune cells result massive inflammatory response tissue damage Hypersensitivities Immediate hypersensitivity Type 1 39 Mediated by B cells 39 Results from 2 or more exposures to same allergen 39 Allergen normally harmless substance that causes immune response Response to allergen exposure 39 1st exposure B cells produce IgE 39 IgE binds mast cells amp basophils type of white blood cell 39 2quotd exposure mast cells amp basophils release histamine 39 Histamine binds cell Hl receptors produce prostaglandin leukotrienes cause allergy symptoms 39 Range from sneezing and watery eyes to anaphylactic shock constriction of airways amp drop in blood pressure 39 Occurs within minutes after exposure I Examples Hay fever bee stings cats I Treatment depends on severity 7 Antihistamines to neutralize histamine 7 Steroids to reduce inflammation 7 Adrenalin to counter the effects of histamine 7 Desensitization quotallergy shotsquot shift antibody production from IgE to IgG 7 Look for video on youtube Type II Hypersensitivity antibodies bind to foreign cell surface antigen 7 Blood transfusion recipients Type A responds to Type B blood and vice versa Each has IgG to respond to other type 7 Rh39 mother has antibodies to Rhl fetus 7 Can occur within hours of exposure Type III Hypersensitivity antibodies bind to soluble antigen cases it to become insoluble 7 Large amounts of antigen antibody complex are formed f can t be cleared quickly trigger complement cascade Also stimulate mast cells This can occur in response to certain antibiotics Can take weeks to occur Delayedtype hypersensitivity Type IV 39 Result of sensitivity to chemical or microbes T cell mediated THl cells release cytokines that activate macrophages NK cells and TC cells Can take a few days to occur Typical antigens microorganisms such as M tuberculosis TB skin test amp chemicals that covalently bind to skin creating new antigens poison ivy latex I Symptoms hardening swelling reddening pain and localized heating Autoimmune Response Can lead to autoimmune disease T or B cells respond to normal cells as if they are foreign 39 Host cells are destroyed 39 Are types of hypersensitivities overreaction Type II hypersensitivity autoimmune diseases Autoantibodies antibodies that interact with selfantigens destroying selfmolecules Rheumatic fever autoantibodies to cardiac cells damages heart valves caused by similarity between epitope in M protein of Streptococcus pyogenes amp cardiac tissue antigenic mimicry Hemolytic anemia autoantibodies to Rh antigen on red blood group destroys RBC9 causing anemia Graves disease autoantibodies to thyroid stimulating hormone receptor causes hyperthyroidism bulging eyes and thyroid 39 gravis receptor causes progressive muscle weakness Type III hypersensitivity autoimmune diseases 39 Antibodies bind soluble proteins create insoluble complexes leading to complement activation and inflammation 39 Rheumatoid arthritis complexes deposited in joints inflammation amp destruction of cartilage 39 Systemic lupus erythematosis complexes deposited in kidney lungs amp spleen 39 Butterfly rash on face Type IV hypersensitivity autoimmune diseases T cells respond to self antigens Multiple sclerosis T cells attack myelin protein destroys covering of neurons Type I diabetes T cells attack insulinproducing cells in pancreas prevents insulin production Superantigens 39 Proteins that can cause very strong immune response activate more T cells than normal Bypass normal route of antigen processing by simultaneously binding TCR amp MHC without undergoing antigen processing Results in cytokine production systemic inflammation 39 Symptoms fever diarrhea vomiting mucus production and systemic shock 39 Ex Toxic shock syndrome Staphylococcus aureus superantigen exotoxin 39 Ex Scarlet fever Streptococcus pyogenes superantigen erythrogenic toxin Chapter 1 Microbial Life Origin and Discovery Study oforganisms too small to be seen with the naked eye theV re everywhere bodies foods environment industry some are pathogens but most are harmless or beneficial some are used for medicine make antibiotics control disease agricuture nitrogen xing bacteria genetically engineered crops food beer wine bread yogurt pickles chocolate genetic engineering insulin growth hormone vitamins crops resistant to pests heat pesticides herbicides etc keep us alive recycle nutrients produce vitamins cause diseases HIV colds STDs food poisoning u What Is a Microbe Organism that requires a microscope to be seen Microbial cells size 02 micrometer to several hundred mm viruses are much smaller 0 Contradictions 7 Super size cells Thiomargarita namibiensis is size of a fruit y head 7 Microbial communities in biofilms microbes have specialized functions amp act as multicellular organism 7 Viruses are noncellular considered to be microbes but are not fully functional cells 6 major groups studied by microbiologists 7 Prokaryotes 1 Bacteria 2 Archaea 7 Eukaryotes 1 Algae 2 Protists 3 Fungi 7 Viruses Microbes Shape Human History Microbes affect food availability some destroy crops corn rust late blight thought to cause Irish potato famine some make food beer bread cheese 0 Microbial diseases change history 7 Black Plague in Europe killed 13 population in 14 h century 7 Smallpox in Americas 7 More soldiers have died from infections than from battle wounds lorence Nightingale convinced British govt to improve army living conditions amp upgrade army hospital standards 7 E in 19 h century 7 AIDS today Discovery of Microbes 0 Light microscope invented in 16005 Mid16005 Robert Hooke observes small eukaryotes with compound microscope more than one Iens Published a book Micrographia in 1665 1st to use term quotcellsquot Antoni van Leeuwenhoek 1676 First to see prokaryotes 7 Built simple microscopes one lens magni ed about 300x Described quotwee animalcules 7 Published first drawings of bacteria prokaryotes 7 Wasn t a scientist but a tailor originally made microscope to examine to quality of the fabric never told anyone how he made his lenses for his scopes Microbes Are Living Organisms Spontaneous generationidea that living organisms originate form nonliving matter Microbes arise only from other microbes 7 No spontaneous generation 1861 Pasteur shows that microbes do not grow in liquid until introduced from outside He disproved this using broth in swanneck asks Sterilized broth by boiling bent necks of flasks so that air could enter but microbes would be caught in neck Open to air 5 urve excludes dust and mkrnbes 7a Growth medium 0 led to development of methods for controlling growth of microorganisms Pasteurization use of moderate heat to reduce number of microorganisms in foods milk wine oysters doesn39t kill all of the bacteria 0 Work with wine industry amp studied fermentation Others tried Pasteur s expts but didn t use vegetable broth the bacteria in other mediums had spores to protect them from the high temperature when boiled Medical Microbiology Germ Theory of Disease 0 Observations 7 Germs can infect and grow on food 0 Hypothesis 7 Can germs infect and grow on food 7 That is do germs cause disease 0 Hypothesis is testable 7 Are germs found in infected tissue 7 Can transmission of germs cause disease Koch s Postulates Developed from his study of anthrax deadly bacterial disease found in very high number in blood of infected animals Provides scientific basis for determining that a specific microbe causes a specific disease 7 quotDoes this germ cause that diseasequot 7 Organism must meet 4 criteria 1 The microbe is found in all cases of disease but absent from healthy rabbit 2 The microbe is isolated from the diseased host and grown in pure culture 3 When the microbe is introduced into a healthy susceptible host the same disease has to occur 4 The same strain of microbe is obtained from the newly diseased host and cultured to make sure it s the same thing Exceptions to the postulates HIV streptococcus staph Some microbes don39t grow on pure media syphilis Same symptoms 2 difforganisms 2 organisms working together Koch amp Tuberculosis 0 1881 17 15 of all deaths caused by TB 0 Thought it was contagious but had never seen the organism Mycobacterium tuberculosis grows very slowly takes weeks in a lab hard to stain due to lipid content Koch developed acidfast staining procedure uses heat to stain patient enough to get pure culture 0 1905 Nobel Prize was awarded to Koch for his work on tuberculosis Facts about TB someone is newly infected with TB every second leading killer ofwomen creates more orphans than any other infectious disease leading cause of death among HV individuals not on the decline History of immunization Stimulation of an immune response by deliberate inoculation with an attenuated weakened pathogen any microbe that causes disease 0 Lady Montagu early 1700 s brought practice of inoculation with smallpox variolation to England from Turkey 0 Edward Jenner first scientific attempt at immunization 7 1778 found that milkmaids exposed to cowpox were immune to smallpox 7 Vaccinated people including his son with cowpox they were resistant to smallpox Pasteur 7 in 1879 studied fowl cholera 7 Isolated and grew bacteria which causes it then left for vacation 7 After vacation the aged bacteria no longer caused disease in chickens 7 Inoculated same chickens with fresh bacteria from new outbreaks and they still did not get sick 7 Attenuated weakened bacteria gave chickens immunity from disease 7 Developed vaccines for anthrax fowl cholera rabies from dried rabbit spinal cords p 39 and for g pathogens lgnas Semmelweis mid 1800 s Golden Age of Microbiology Puerperal fever childbed fever 0 Medical students carried lcadaver particles from autopsy room to delivery room Caused by streptococcus Had med students wash hands in chlorinated lime water reduced mortality from 18 to 1 Died from in infection due to streptococcus in an insane asylum Joseph Lister 0 1864 British surgeon found that survival rate of surgical patients increased if surgeons washed hands sterilized instrument and used disinfectant carbolic acidphenol during surgery Paul Ehrlich early 1900 s Idea for 39magic bullet that would kill pathogens but nor harm humans 0 found arsenic compounds used to destroy trypanosomes quotcompound 418quot cured sleeping sickness Discovered Salvarsan quotcompound 606quot to treat syphilis Used term quotchemotherapyquot using chemicals to kill pathogens without harming patient Arsenic turned out to be fairly toxic to humans Alexander Fleming 1929 o discovered antimicrobial effects of penicillin G produced by Penicillium notatum fungus on Staphylococcus o 1945 Nobel Prize in Medicine Fleming for discovery Florey amp Chain for determining mode of action of penicillin and developing method of mass producing the drug Inhibits cell wall synthesis 0 1945 penicillin became available for general use Growth of microbes in pure culture Koch amp solid culture media 0 Potato slices 7 streaked bacteria onto potatoes 7 Problem too much moisture many contaminants Beef broth solidified with gelatin 7 Problems Gelatin lique es above 28 degrees Celsius Many microbes produce enzyme called gelatinase liquefies gelatin 0 Agar 7 polysaccharide derived from red algae 7 1882 Fannie Hesse wife of Koch s coworker suggested agar She used it to make jelly 7 Solid until 100 degrees Celsius and most microbes cannot digest it 7 Few microbes can produce agarase that can digest agar Petri Dish 0 Richard Petri Koch s assistant invented them in 1887 0 Before this they used a bell jar or glass box 0 1677 Observed llwee animalculesquot Antony Leeuwenhoek 0 1796 First scientific Small pox vaccination Edward Jenner 0 1850 Advocated washing hands to stop the spread of disease Ignaz Semmelweis 0 1861 Disproved spontaneous generation Louis Pasteur 0 1862 Supported Germ Theory of Disease Louis Pasteur 0 1867 Practiced antiseptic surgery Joseph Lister 0 1881 Growth of Bacteria on solid media Robert Koch 0 1882 Outlined Kochs postulates Robert Koch 0 1885 First Rabies vaccination Louis Pasteur 0 1887 Invented Petri Dish RJ Petri 1892 Discovered viruses Dmitri losifovich Ivanovskil 0 1910 Discovered cure for syphilisSalvarsan 606 Paul Ehrlich 0 1928 Discovered Penicillin Alexander Fleming Microbial Ecology Most microbes don t grow on typical medium 0 quot 01 of microbes in our biosphere can be cultured in the lab 0 Many live in varied conditions 7 Anaerobic Bottom ofswamp 7 High pressure 0 Bottom of ocean 7 Hot or cold temperatures 0 Below 0 degrees Celsius to 113 degrees Celsius 7 No organic carbon 0 Use light for energy C02 for carbon carbon dioxide fixing 0 Can culture some microbes in natural mud environment 7 Winogradsky column 0 Layers grow different species reflecting different conditions 7 Can see variations in nature 0 Yellowstone geyser runoff The Microbi Mic Use and Phy Colors reflect different species Different growth temperatures Microbes cycle most elements on earth Nitrogen cycle Bacteria fix NZ to NH4 7 Carbon cycle Photosynthetic microbes x most carbon Sulfur cycle Phosphorus cycle a Family Tree robial species are difficult to classify Carolus Linnaeus 7 Difficult to distinguish by shape Often reproduce asexually Pass DNA to each other without reproduction one species to another don t have to be closely realated biochemical properties to classify 7 Gram stain 7 Ability to metabolize different substrates DNA sequence to classify Bacterial genomes are relatively small so easy to sequence 2 distinct species share no more than 95 similarity of DNA sequence Several lineages exist in the domains Bacteria and Archaea huge diversity of cell morphologies physiologies logenetic tree of life Fig 15 amp 127 Divided by nucleic acid sequencing no linger use kingdoms but domains amnion nmm Algae um Films 5mm Fungi an Animal mum Prokaryotic Microorganisms I Bacteria most prukaryotes soil bacteria quot39 39 bacteria etc 7 Ex Salmonella and Escherichia call I Archaear prokaryotes that live in extreme environments hot springs glaciers salt lakes 7 Ex Methanococcus and Halobacterium Archaea are not acteria 7 Similarsizeshape 7 Very different biochemistry 7 Different membranes 7 Archaeal ribosomes similarto eukaryotic ribosomes 7 Many archaea live in harsh environments Eukaryotic Microorganisms7 includes fungi algae amp protists 0 Fungi7 nonphotosynthetic most have cell walls 7 Ex1easts unicellular and molds multicellular hyphae 7 Saccharomyces yeast is used to make beer and bread 7 Penicillium mold produces penicillin o ae 7 Photosynthetic contain chlorophyll have cell walls 7 Ex Diatoms amp seaweed 0 Protozoa 0 Unicellular motile no cell walls 0 Ex Amoeba Paramecium amp Giardia Eukaryotes evolved through endosymbiosis o Endosymbiosis 7 theory that eukaryotic organelles evolved through a symbiotic relationship 7 one cell engulfed a second cell and a symbiotic relationship developed 7 mitochondria and chloroplast are thought to have evolved this way 7 mitochondria from respiring bacteria similar to E Coli 7 chloroplasts from phototroph related to cyanobacteria 7 Mitochondria and chloroplasts have 2 membranes possess their own DNA and ribosomes are about the size of a prokaryotic cell divide by a process similar to bacteria Chapter 25 Microbial Pathogenesis HostPathogen Interactions Infection entry of pathogen or parasite Infection doesn t always cause disease Most infections removed by immune system Primary pathogens Frank pathogen Have ability to penetrate host defenses Opportunistic pathogens Cause disease only in compromised hosts Immune system defective Break in tissue allows organism access to new site Loss of other micro ora allows organism to bloom Pathogenicity of organism Measure of ability to cause disease Determined by genetic makeup of organism Infectious dose ID50 Number of organisms to colonize 50 of hosts Virulence Rate of lethal infections Lethal dose LD50 Number of organisms to kill 50 of hosts Infection Cycle Spread by direct contact Spread by indirect contact Contact with fomites o Inanimate object or substance that can carry infectious microbes 0 Computer elevator buttons Horizontal transmission via vectors MosquitoesiYellow fever malaria Reservoir for disease organism May not show disease symptoms Mode of entry depends on pathogen Mucosal surfaces wounds insect bites Virulence Factors Virulence genes Encode factors allowing pathogen to invade host Toxins attachment proteins capsules 39 Pathogenicity islands 39 Section of genome 39 Contain multiple virulence genes 39 O en encode related functions Eg protein secretion system toxin production 39 Transferred as a block from other organisms EXPOSIIRE 9 to palnngens 39 is ADHEREIICE to skin or rrluKosa A Further 9 exposure v Funhur 3 39 a39 INVASION WW s es thmugh epilhelium T i communion and iv linovml Prndunion of virulence anan quot W a roxlclrv IIIVASWEIIESS mxln effects are local further growth at original av systemic site and disrant sites J l TISSUE DAMAGE DISEASE Virulence Factors 0 Structures amp characteristics of pathogens that allow them to cause disease Adhesion ability to adhere to mucus membranes Virulence factors affecting adhesion 39 Capsule 7 outer polysaccharide layer helps bacteria in attachment protects them from phagocytosis 39 Fimbriaepili 7 necessary for some organisms to attach Neisseria ampE coli 39 Adhesins 7 surface proteins bind host cells Invasion amp Colonization Invasion 39 Pathogens must be able to penetrate the outer layer of skin cells epithelium 39 They can reach inner layers of skin cells through lesions or mucus membranes Colonization 39 Once it has invaded a host it can establish itself and grow Examples of virulence factors affecting invasion amp growth The O amp Vi antigens of Salmonella O 7 cell surface polysaccharide 7 prevents phagocytosis Vi 7 capsule antigen 7 prevents complement binding amp antibody mediated killing Siderophores Compounds that bind iron amp make it available to the bacterial cell Leukosidin Staphylococcus aureus produces this compound which lyses white blood cells decreasing host resistance Localized infection Organisms grow locally at site of invasion most infections Systemic infection Organisms spread through body using blood or lymphatic systems Dissemination spreading of the pathogen Many pathogens make virulence factors which help them spread from the initial infection site Many of these are enzymes which break down tissues Examples of virulence factors which break down tissues Hyaluronidase Destroys polysaccharide that holds animal cells together Produced by Staphylococcus amp Streptococcus Collagenase Breaks down collagen Produced by Clostrz39alz39um Streptokinase Breaks down fibrin clots Clots are made by host to wall off bacterial infections amp prevent their spread to healthy tissues Made by Streptococcus pyogenes Hemolysins Disrupt cytoplasmic membrane of animal cells 7 lyse red blood cells Toxins Compounds produced by pathogens that damage host tissues 3 major groups of toxins Exotoxins worst Enterotoxins Endotoxins Exotoxins Proteins secreted by bacteria that can damage tissues far from infection site Examples of exotoxins Diphtheria toxin Inhibits protein synthesis Produced by Corynebacterium aliphtheriae causes diphtheria Toxin is produced by bacteria that are lysogenized by phage beta which carries tox genes Tetanus toxin Acts on motor neurons causes continual contraction of muscles can lead to lockj aw Produced by Closm39alz39um tetam39 which infects deep wounds Botulinum toxin intoxication Prevents muscle contraction 7 results in respiratory or cardiac failure Produced by Clostridium botulinum Organism grows in improperly stored foods where it produces toxin Botulism is caused by ingesting toxin not by infection with C botulz39num Children 2 and under should not have honey bc endospores and not enough normal ora to fight it off See Fig 2618 Enterotoxins Proteins that act on small intestine cause diarrhea Produced by food poisoning organisms such as Closm39alz39um E coli Salmonella Cholera toxin Produced by Virbrio cholera lives in marine environment Causes secretion of massive amount of water into small intestine diarrhea 7 death by dehydration Endotoxins When Gramnegative bacteria lyse lipid A portion of LPS is released toxic portion LPS from many organisms such as E coli Salmonella Shigella is toxic Cause diarrhea fever vomiting 7 rarely fatal Toxins Subvert Host Function AB Toxins B subunit binds to host cell Delivers A subunit to cell Often 5 B subunits form a pore for A entry A subunit has toxic activity ADPRibosyltransferase Diphtheria toxin Cholera toxin ADPRibosylating Toxins Cholera toxin Ribosylates to overactivate adenylate cyclase cAMP activates ion transport water follows Uncontrollable diarrhea Diphtheria toxin Ribosylates elongation factor 2 translation Blocks ribosome function cell dies Forms pseudomembrane over trachea Surviving within the Host Once inside host cell how to avoid death Cell ingests pathogens in phagosome Some pathogens use hemolysin to break out Shigella dysenteriae Listeria monocytogenes Phagosome fuses with acidic lysosome Some pathogens secrete proteins to prevent fusion Salmonella Chlamydia Some pathogens mature in acidic environment Coxiella burneting fever Viral Pathogenesis In uenza In uenza virus antigenic drift 39 RNA Polymerase does not correct replication errors Frequent mutations in hemagglutinin gene If mutation allows virus to avoid immune system virus spreads New u vaccine needed every year In uenza antigenic shift Two strains infect host at same time Chicken or swine Recombined viruses have mix of chromosomes Immediate alteration of several genes Cause of 1918 in uenza outbreak Largest singleyear loss of life in recorded history HIV HIV7Avoids host immunity by infecting immune cells Binds multiple receptors for entry CD4 chemokine receptor CCRS T cells Infected cells secrete viral protein Misregulates other immune system cells Chapter 26 Microbial Diseases Skin and Soft Tissue Infections Staphylococcus aureus Boils pimples7walled off from body with brin Impetigo skin infection common on children and bright red sore on face Scalded skin syndrome due to exfoliative toxin skin pealing off Can produce hemolysins superantigen enterotoxin A food poisoning coagulase leukocidin superantigen TSST toxic shock syndrome Can also cause pneumonia amp meningitis Treatment with penicillins MRSA7methicillinresistant S aureus Horizontal gene transfer of drugresistant genes Evolved in environment with high levels of antibiotic Hospitals7nosocomial infections Resistant to all drugs except vancomycin Streptococcus pyogenes 60 strains Strep throat pharyngitis 7 510 of sore throats diagnosis blood agar slow antibody test rapid infections of the inner ear impetigo skin infection Scarlet fever 7 exotoxin superantigen can follow pharyngitis pink rash amp strawberry tongue Rheumatic fever 7 autoimmune antibodies against S pyogenes react with host tissue 7 damages heart valves necrotizing fasciitis esh eating Many viruses can cause skin rashes Many Respi Strept Usually infect through respiratory tract Chickenpox caused by Varicella zoster virus VZV a herpesvirus VZV can lay dormant in nerve cells for long periods of time Shingles is caused by the migration of VZV to the skin painful blisters Measlesrubeola caused by a paramyxovirus Symptoms fever cough rash Mumps in ammation of salivary glands RubellaGerman measles Caused by a togavirus symptoms similar to measles but milder Can be prevented with MMR vaccine all three measles mumps rubella fungal skin infections ratory Tract Infections Most respiratory pathogens are transferred from person to person via respiratory aerosols 7 coughing sneezing talking breathing Transmission usually occurs over short distances because microorganisms survive poorly in air Pathogens that can survive dry conditions such as many Gram amp Mycobacteria are easier to transmit by air ococcus pneumoniae lower respiratory diseases can also cause meningitis 39 r i Kr In ammatory reaction in alveoli air sacs in lungs Usually a secondary infection Capsule protects organism from phagocytosis untreated 30 mortality rate Alveoli become filled With blood bacteria amp phagocytic cells Symptoms sudden onset of chills labored breathing pleural chest pain Pneumonia can also be caused by viruses other bacteria or fungi S pneumoniae is found in respiratory ora in about 40 of healthy people Corynebacterium diphtheriae Childhood upper respiratory infection diphtheria Pathogen strains of Corynebacterium diphtheriae produce exotoxin that inhibits protein synthesis Formation of pseudomembrane 7 damaged host cells amp bacterial cells can cause hemorrhaging if torn Causes constriction of throat suffocation Early childhood immunization with diphtheria toxoid part of DTP is very effective for prevention of diptheria Bordetella pertussis 39 Childhood upper respiratory infection pertussis hooping cough Pertussis exotoxin causes tissue damage endotoxin causes cough Violent cough that lasts up to 6 weeks Pathogen binds to ciliated epithelial cells of bronchi and trachea Early childhood immunization with proteins gacellular pertussis 1 derived from B pertussis part of DTP is very effective M ycobacterium tuberculosis acidfast 39 Tuberculosis As much as l 3 world population infected 11 of deaths due to infectious disease caused by tuberculosis Tuberculin test for hypersensitivity acidfast stain for active infection Treatment with isoniazid amp rifampin 7 9 months Immunization with an attenuated strain of M bovis BCG effectively prevents tuberculosis Primary initial infection Inhalation of M tuberculosis Bacteria settle in the lungs Host immune response macrophage ingestion of bacteria results in a delayedtype hypersensitivity reaction aggregates of macrophages 7 tubercles full of immune cells brin Mycobacterium The bacteria survive and grow within the macrophages Most cases infection remains localized Reinfection or Postprimary infection Reinfection from outside sources or reactivation of dormant bacteria Aging malnutrition overcrowding stress can reduce effective immunity and allow reactivation of dormant bacteria Colds and in uenza u are the most common infectious diseases Common cold about 150 different cold viruses exist Rhinovirus 5075 ofcolds Coronavirus N 15 Adenovirus and others N 10 Specific local neutralizing IgA immune response Symptoms last about 1 weeki sneezing runny nose In uenza Caused by orthomyxoviruses In uenza A In uenza B and In uenza C Flu symptoms 7 fever headache body aches Can last from 1 to few weeks Worldwide epidemics can occur 19181919 20 7 40 million people died Immunity due to IgA Early treatment drugs that inhibit viral replication Immunization is effective but choice of vaccines is complicated by the of strains Why do Ineed a new u shot each year Antigenic shift 7 genome divided into 8 pieces which can recombine when multiple strains infect same cell changes in capsid protein In uenza virus Strain 1 In uenza virus Strain 2 Host cell In uenza virus New strain In uenza virus New strain Antigenic drift 7 high mutation rates cause a more gradual change In uenza outbreaks occur annually and more serious epidemics amp pandemics occur Gastrointestinal Tract Infections H elico bacter pylori Secretes urease converts urea to NH4 o Neutralizes stomach acid Burrows into protective mucous layer Gastric ulcers amp cancers Antibody immune response not sufficient Treatment 7 antibiotics plus antacids Foodbome Diseases Foodbome infections Caused by organisms that are transmitted in food amp grow in intestinal tract Can take several days for symptoms Salmonella 7 causes headache fever chills vomiting diarrhea Salmonella can be found in meat poultry eggs dairy Escherichia call can be found in beef causes bloody diarrhea amp kidney failure Also responsible for Traveler s diarrhea Causes 7250 deathsyear Other bacterial foodbome infections Campylobacterjejuni 7 Most frequent bacterial cause of diarrhea Vibrio cholerae cholera Shigella Enteroinvasive E coli EIEC 7 EIEC and Shigella produce Shiga toxin EnteroHemorrhagic E coli EHEC 0157H7 7 0157serotype of LPS H7serotype of agella Food poisoning Occurs when food is contaminated with a toxin producing microbe Toxin causes symptoms which can appear within 1 hour of eating Most common cause is S aureus vomiting amp diarrhea very salt tolerant organism Clostridium perfringens causes diarrhea amp intestinal cramps Clostridium botulinum causes botulism most severe type of food poisoning Produces neurotoxin often fatal m gastroenteritis caused by viruses Rotaviruses dsRNA Reovirus 7 Highly infectious 7 Can cause lethal dehydration Antibiotics ineffective on viruses 7 kill competing bacteria allows C d cile to grow can occur at low levels Causes gastrointestinal disease Protozoal infections Entamoeba C ryptosporz39alium Giardia lamblia Genitourinary Tract Infections Urinary tract infections 7 Many gram negative bacteria grow in urine Uropathogenic E coli UPEC Klebsz39ella Pseudomonas Enterobacter Pili 7 cells adhere against urine stream Sexually Transmitted Infections Most sexually transmitted infections are curable or controllable with timely appropriate medical intervention Prevention by abstinence or barriers condoms Neisseria gonorrhoeae 39 Gonorrhea Gram negative diplococcus One of most common human diseases Enters body through mucous membranes Infants 7 eye infections during birth Female 7 in ammation of vaginal mucosa easily unnoticeable can lead to pelvic in ammatory disease PID Male 7 painful urethra infection discharge Untreated can result in sterility damage to heart valves amp joints Prophylactic 7 erythromycin eye ointment for newborns Treponema pallidum Syphilis Newborns can contract it from mothercongenital syphilis Primary stage Initial site of infection is usually genitals lesion called chancre forms Chancre usually heals but bacteria spread Secondary stage Generalized rash Tertiary stage infections of skin bone CNS cardiovascular effects dementia death Most of last symptoms due to delayed hypersensitivity reactions Chlamydia trachomatis Most common STD 75 of women and 50 of men 7 no symptoms If symptoms do occur they are similar to gonorrhea Newborns can contract C u 39 7 39 quot quotis J Nongonococcal urethritis not caused by N gonorrhea Can cause PID amp fallopian tube damage Trichomonas vaginalis Protozoan responsible for trichomoniasis Can survive up to 24 hours in urine or semen therefore transmission can occur by contact with uids toilet seats sauna benches Symptoms none in males vaginal discharge in ammation of vaginal mucosa painful urination in females Diagnosis by microscopic observation of protozoan in discharge Viruses Human papillomaviruses Genital warts cervical cancer Vaccine Gardisil to 4 most virulent viruses available Herpes simplex viruses Herpes simplex virus type 1 Cold soresfever blisters Latent infection virus persists in nerve tissue Herpes simplex virus type 2 39 Painful blisters in genital region Spread by contact with blisters Correlation between genital herpes amp cervical cancer Can be transmitted to newborn by mother Csection advised No cure but blister treatment with oral and topical antiviral drug acyclovir to inhibit viral replication and limit shed of active virus Human Immunode ciency Virus HIV 39 Retrovirus that causes AIDS Acquired Immunode ciency Syndrome one of the most prevalent infectious diseases HIV destroys the immune system by infecting macrophages amp CD4 Tcells Opportunistic J quot kill the r 39 A host Worldwide estimated 33 million people infected highest percentage in college age women Highly effective antiretroviral therapy treatment 7 multiple drugs given at once First stage7AIDSrelated complex 7 Fever headache rash Second stage7AIDS 7 Depletion of T cells 7 Opportunistic infections Third stage7AIDSrelated dementia 0 Fourth stage7rare cancers 7 Kaposi s sarcoma via Herpes virus type 8 infection Central Nervous System Infections West Nile Virus West Nile fever Transmitted by mosquito from birds to humans Symptoms most inapparent 720 fever headache nausea muscle aches rash swollen lymph nodes malaise ltl encephalitis or meningitis No effective antiviral drugs for treatment No human vaccine yet Prevention by mosquito extermination Neisseria meningitidis Bacterial meningococcal meningitis in ammation of meninges membranes that line CNS Occur in epidemics in closed populations like dorms amp military 30 are carriers Initial upper respiratory and blood infections Meningitis can also occur characterized by sudden onset of rash headache vomiting stiff neck within hours coma amp death Vaccination made of polysaccharides from most prevalent strains Others bacteria and viruses can also cause meningitis Clostridium toxins C botulinum7botulinum toxin Botox 7 Anaerobe grows in canned food 7 Spores survive unless autoclaved 7 Toxin blocks release of acetylcholine Prevents muscle movement C tetam397tetanus toxin 7 Anaerobe grows in puncture wounds Blood ow interrupted tissue becomes anaerobic 7 Toxin blocks release of inhibitory transmitter Muscles contract uncontrollably lethal spasms 7 Immunization with tetanus toxoid vaccination DTP is effective for prevention Rabies Caused by a member of the Rhabdovirus family Transmitted from infected animal raccoon skunk coyote to human through infected saliva Infects cells of CNS causing fever excessive salivation anxiety throat muscle spasms death from respiratory paralysis Treatment with immunoglobulin or antibodies Effective immunization for domestic animals amp highrisk humans Cardiovascular System Infections Malaria o Caused by protozoan Plasmodium which is spread by mosquitoes 4 Plasmodium species 0 173 million deathsyear 39 Infects liver RBCs New merozoites released every 48772 hours lysis of RBCs results in high fever Organism remains in liver can cause relapses years later Treatment with chloroquin organisms are developing resistance Vaccination expensive and shortlived Prevention by mosquito extermination Systemic Infections Plague 39 Caused by Y ersim39a pestis Transmitted by eas from rats to humans Untreated death occurs within 5 days Types differ by place of infection Bubonic plague 7 lymph node swelling Bubos Pneumonic plague 7 inhaled shortness of breath chest pain cough death within 2 days Septicemic plague 7 rapid spread through bloodstream death before diagnosis Prevention by control of rodent populations Borrelia burgdorferi Lyme disease Transmitted by deer tick bites Symptoms headache backache chills fatigue large rash at bite site left untreated 7 arthritis neurological problems heart damage Lyme disease vaccines are available for veterinary use Prevention by reducing exposure to ticks Other bacterial infections Salmonella typhz397typhoid fever contaminated food or water Mary Mallon F rancisella tularensz39s7tularemia 7 rabbit fever transmitted from rodents or rabbits by ticks amp ies to humans Rickettsial diseases 0 Epidemic Rickettsial prowazekiz39 transmitted by head louse fecal matter singular for lice 0 Rocky Mountain spotted fever transmitted by dog amp wood ticks fecal matter Symptoms fever headache weakness rash Bloodbome viruses 7 HAV7Hepatitis A7transmitted by contaminated food amp water Disease resolves 7 HBV7Hepatitis B7 transmitted by contaminated body uids 7 HCV7Hepatitis C7 transmitted by contaminated blood Hepatitis symptoms fever amp jaundice yellowing of skin from excess bilirubin due to destruction of liver cells liver in ammation destruction of cells in acute infections cirrhosis liver cancer 7 Treatment is mainly supportive to allow liver to repair 7 Immunization for HAV and HBV is effective All schoolage children are required to have HBV vaccination BIOL 2051 Lecture Exam 4 2326 The bladder is not sterile The colon has the most diverse and highest quantity ofbacteria in the body UT infections caused by fac Anaerobes Escherichia Staphylococcus Proteus Neisseria Correct order of phagocytosis Interferon Defensin Mother passes immunity to child naturally acquired passive immunity Snakebite victim is an example of artificially acquired passive immunity Pathogens inside ofphagocytes can avoid death by preventing phagosomes from fusing with lysosomes use hemolysins to break out What is the region called that binds the antigen hypervariable region Which of the following is not part of a complement system Which is the first to respond to infection IgM Which is the most abundant IgG Which is found in breast milk IgA Which makes the antibody specific for the antigen Plasma Cell Secondtime exposure results in faster response because ofmemory cell Mother making antibodies for baby is what type of hypersensitivity 11 Which type of hypersensitivity involves T cells IV Which is a delayed hypersensitivity Mycobacterium tuberculosis This is the number needed to infect half the experimental group ID50 Which of the following need fimbriae to infect the host Neisseria and E coli Which would break down a clot in a stroke patient Streptococcus pyogenes Vibrio cholera is an example of an enterotoxin Which of the following is not an exotoxin Cholera Several questions about exo entero and endo toxins Which is false The cold and u virus are not deadly Which is associated with Streptococcus All of the above tonsillitis impetigo scarlet fever Pathogen that binds to ciliated epithelial cells of brochi and trachea Bordetella pertussis Most common STD Chlamydia Which infects the genital area Herpes virus 11 Which can lead to pelvic in ammatory disease if untreated Neisseria gonorrhea Which is associated with Clostridium botulinum Blocks the release of acetylcholine Question similar to the previous but about Clostridium tetenani Which of the following is the cause of typhoid fever Deer tick dog tick other options What causes strep throat Streptococcus pyogenes What should not have microbes Kidney Least to most microbes Lower resp stomach upper resp small intestine large intestine What organism can be found in the genitourinary Plasma B cells make specific antibodies Know what defensin interferon O and Vi are Question about iron Siderophores What organism produces leukosidin lyses white blood cells Know about pili Put these in order exposure adhesion invasion colonization Know immunogenicity Know L50 and 150 Some question about polymorphonuclear leukocytes Answer is Neutrophils An example of artificially acquired active immunity Toxoid Compare hypersensitivities Superantigens What causes the toxic in gram negatives Lipid A What happened in 1918 In uenza What causes impetigo Staph aureus and strep pyogenes Pathogen that binds to cili epithelia cells of bronchi and trachea Bordetella pertussis Transmitted by ticks Borreliaburgdorferi Which plaque is most life threatening Septicemic plaque Something about insects carrying diseases Vector What organism secretes urease to neutralize acid in stomach Helicobacter pylori Which is the best immune response to Staph aureus infection in blood Options are specific immune response and nonspecific Which of the following is a surface polysaccharide that prevents phagocytosis The 0 antigen of Salmonella What disease is caused by Ricksettia Rocky Mountain Spotted Fever What disease is caused by orthomyxoviruses Flu Which is used to make diphtheria vaccine Toxoid Which describes what streptokinase does Helps invade the host tissue further by breaking down clots Which organism produces dextran Strep mutans What would cause NK cells to destroy a host cell Cell stops producing MHC I Superantigens cause a very strong immune response and can cause systemic shock Type II hypersensitivity would cause a mothers immune system to attach her fetus s blood Mast cells release histamine Group ofproteins that cause enhanced immune function and increased phagocytosis Complement What happens on the second exposure to an antigen IgE causes histamine release Which is used to describe the number of organisms needed to kill 50 LD50 Malaria caused by Plasmodium Thc binds to antigen presented by B cell after phagocytosis of foreign object Which of the following releases antibodies upon second exposure to pathogens Memory B cells Which is the mode ofaction of tetanus toxins Inhibits glycine Which org produces toxin affecting nervous system Clostridium botulinum Which organism sticks to teeth after producing polysacc from sucrose Streptococcus mutans Septic shockis due to what Extravasation deals with what type of cell Macrophage Is poison ivy an autoimmune disease Receiving anteriserum is an example of what Artificially acquired passive T cell lymphocytes originate from Bone marrow What is the most dominant immunoglobin IgG Difference bt hypersensitivity type 2 and type 3 What is an example of delayed hypersensitivity TB skin test Which hypersensitivity involves T cellS Type 4 What virulence factor binds iron Siderophore What portion of gram neg bacteria is toxic Lipid A Gram pos can be transmitted through the air What microbe inhibits protein synthesis Corynebacterium diphtheria Not a symptom ofin uenza Sore throat Travelers diarrhea E coli Disease that form chacres Syphilis Erythromycin is eye ointment for infants with gonorrhea September 30 2010 Chapter 9 Gene Transfer Mutations and Genome Evolution DNA sequence is not static 7 Horizontal gene transfer Transformation Conjugation Transduction Mutations Deletions removed Insertions inserted Effects of Gene Transfer Spreads useful genes among bacteria Antibiotic resistance genes Spread wherever antibiotics are overused Hospitals farms Pathogenicity islands Encode genes for cell to act as pathogen Difference between typical E coli in gut and pathogenic E coli 0157 H7 can be shared or transferred from one organism to another can be Virulence factor Genes to degrade special metabolites oil spills Mechanisms of horizontal gene transfer Transformation Transduction Conjugation Some cells are naturally competent Bacillus Some cells can be made competent in a lab if we want to get new DNA info into them 7 Salt treatments Neutralizes charges in cell more likely to get new DNA INSIDE 7 Electronoration electrical shock opens pores in cell membrane DNA enters through pores If free DNA isn t incorporated into LII it will be degraded Free DNA can t replicate on its own a Normally viral DNA is packaged b Rarely bacterial DNA is packaged by mistake 3 DNA is transferred to new host 4 Can bring new bacterial genes to host Two t es of transduction 5W rt Hquot i lb 1 Donor DNA is either incorporated into recipient chromosome or degraded Tmnsduclmn gt genevahzed transduch Darncte Sometimes lambda DNA excises incorrectly and brings adjacent host genes gal or bio with it 1 cluber 5 1mm Contact between bacteria is rst made with pilus Ceu M11pole Cannesquot hm39gth m1 een 112mm Censue hm 39awnmgnhznafmnamz ngpair lrznuzlmns rufDNA HWQPIMTMMW e Om type afplmxd39hnt canbe angled amp transferred m new een Rxm39 anupla md camesamhimic ns39slznzegms m 2 ms mF puma reeen aaesmmme F puma F een CAN farm apdussemix15 cwpytnnzw eem mm s enaup ssh Type TVsenerm sysm e pilus lrnuI39nn 125ml m pdus bung Amen A InrenplenzcdlumsfmedDNAfmnsanzwFplzgnid a Once an mums umsrmea thznyalymenszswdl capy39hz VIth 511 an quotAgobm mwn writ1mm T max mmang memHmm gall asesse m plm39sji rns T plamxdxs Wm seeemgs use fax geneqee mgneexed plants m kansfex D Fig jvm Fumzw39nnufan 39 F plamndun memes ingztzimn Illzlmsl chmmnmm Hf hagm equencyxecmnhmms 1min phgnid has Tmegmea imn nee chmmnmm Cmugnhm can39hmleadm kansf uflilgng39nnsu hzlmslchmnmmm lm nn The F plmxdcmbeexc39nd nmdlz chmmusnme Samanmesxhszxusedmtact T O39hzxumeS me uf39hzchmmummiszxcigd almigwizhdle phsnil G FameJphmmmsemMmmunmshwAmn Theses am wny39hatnzwplamnds aremade Recombination Mobile Genetic Elements In all these processes conjugation transformation transduction a piece of DNA enters a bacterial cell New DNA can replace portion of chromosome this is recombination Donor DNA must be very similar 1 homologous to recipient DNA for recombination to occur Requires speci c recombination proteins RecA RecB RuVAB some proteins necessary for recombination EX If donor DNA has 16102 gene it would replace the 16102 gene in the recipient chromosome they are homologous Ethe new lacZ gene is mutated lacZ it could be badIf a cell had a mutated and acquired a functional one it could be good Ii they were both functional then it could be neutral Recipient Donor I I Chromosome sequence is not xed always changing 39 Transposable elements insert into chromosome 39 Found in all species 39 can jump from one site to another jumping genes 39 Nonreplicative transposition 39 Can copy itself to a new site 39 Replicative transposition Mutations amp Mutants Mutation change in DNA sequence Mutant organism that has a mutation lacZ can make enzyme betagalactosidase Mutant lacZ means there is a change in DNA sequence inthat gene Phenotype observable properties of a strain it Phenotype of a lacZ strain is LacZ Even if genotype of amutant is different from the parental strain they can have the same phenotype Mutations Point mutation change in a single base pair can cause a single amino acid change in a polypeptide or no change at all depending on the codon involved 39 Transition purine gt purine pyrimidine gt pyrimidine 39 Transversion purine lt gt pyrimidine M transposons Deletions amp insertions of bases cause more serious changes in the DNA including frameshift mutations and often result in loss of gene function 1 or 2 not multiple of three disrupts reading frame 3 nucleotides are read at one time Mutagens Cause Mutations Electromagnetic radiation Xrays gamma rays UV light gt skin cancer Spontaneous change in N base structure Chemicals Analogs of N bases similar in structure to N base but not same function Basemodifying chemicals EX deamination Intercalators insert between N bases Cause frameshift mutations DNA Repair Mismatch repair Mispaired base cut out of strand Strand without methyl groups is assumed to be newer strand Assumed to be in error ThymidineThvmine dimers Induced by UV 39 DNA is unable to replicate too many thymines can cause death to cell Cut out by UvrABC complex nucleotide excision repair second option is photoreactivation using photolyase light reverses bond that causes thymine dimer repairs bases without excision Damaged bases excised by speci c enzymes base excision repair Replaced by DNA Polymerase I Recombinational repair Occurs just after strand has replicated Undamaged strand is copied Replaces damaged strand with new nucleotides Catalyzed by RecA recombinase SOS repair Extensive DNA damage inactivates LexA Activation of many repair genes and enzymes to be made Rapid polymerization of DNA Chapter 19 Archaeal Diversity Archaeal Traits and Diversity Widest temperature range 7 2 C7121 C Widest range of environments 7 pH 0 high pressure anaerobic Unique biochemistry 7 Methane production 7 No peptidoglycan 7 Differences in glycolytic pathways 7 Differences in lipids and membranes 4 Phyla 0f Archaea 2 major 2 minor Phylum l Euryarchaeota 4 groups of Euryarchaeota largest group extreme halophiles Methanogens Thermoplasmatales Hyperthermophiles Phylum Euryarchaeota extreme halophiles Require at least 9 NaCl for growth 7 most require 1223 Common in salt lakes and saltems ponds used to prepare solar salt by evaporation of sea water Give red color to water EX Halobacteriumsalmarium To prevent water loss in hypertonic environment pump inorganic ions K into cells equalizing concentration in and out make or concentrate an organic solute in cell Lightmediated energy production 39 Some extreme halophiles use light to produce energy Don t have chlorophyll have protein called bacteriorhodopsin red color Light energy is used to pump protons to outside surface of cell membrane and generate a H or Na gradient ATPase uses this gradient to form ATP V Methanogens Release 100 million tons of methane into atmosphere each year Found in anerobic environments Methanogenic habitats 39 Freshwater sediments 7 rice paddies marshes ets Gastrointestinal tract of animals 7 human large intestine Rumen 7 1020 of total methane emitted to atmosphere originates in rumen Termite gut 2 Thermoplasmatales Lack cell walls Thermophilic and acidophilic pH 2 Most strains have been isolated from selfheating coal refuse piles Thermoplasma 3 Hyperthermophiles Optimum growth at 1000 C Pyrococcus 7 reball Phylum 2Crenarchae0ta Often irregular in shape Many have no cell wall Always have unique lipid 7 Crenarchaeol Most are hyperthermophiles hot springs some are psychrophiles sea ice Often very acidic and anaerobic Most use sulfur as electron acceptor Example Sulfolobus Phylum 3Nan0archae0ta Nanoarchaeum cells are symbionts or parasites of Ignicoccus crenarcheote none freeliving Tiny cells1 ofthe volume of Ecoli Small genome Lacks genes for most metabolic functions depends on host hydrothermal vents and hot springs Phylum 4K0rarchae0ta 39 Found in Obsidian Pool at Yellowstone Hyperthermophiles 85 C Originally detected from 16s rRNA sequence No pure cultures exist Chapter 1 Microbial Life Origin and Discovery Read Sections 1115 What is Microbiology The study of organisma too small to be seen with the naked eye 0 Microorganisms are everywhere 7 Our bodies 7 Foods 7 Environment 0 Majority of soil is microbes 7 industg 0 Some are pathogens but most are harmless or beneficial helpful or neutral Importance 0 Medicine 0 Making antibiotics o Controlling disease 0 Agriculture 0 Nitrogenfixing bacteria I Plants cannot do this but the soil can 0 Genetically engineered crops 0 Food industry 0 Beer wine bread yogurt pickles chocolate 0 Genetic engineering 0 Insulin growth hormone vitamins o Crops resistant to pests heat pesticides herbicides etc 0 Keep us alive 0 Recycle nutrients 0 Produce vitamins in our intestine o Degrade organic matter 0 Cause diseases most microbes are not pathogenic 0 HIV colds STD s food poisoning flu Microbiologists study 0 Bacteria Viruses noncellular Protozoans fungi and algae eukaryotes Diatoms and more Chapter 1 Microbial Lifequot Origin and Discovery What Is a Microbe Organism that requires a microscope to be seen 0 Microbial cells size 2 39 to mm viruses much smaller 0 Contradictions 7 Supersize cells Thiomargarita namibiensis is size of fruit fly head 7 Microbial communities in biofilms microbes have specialized functions amp act as multicellular organism 7 Viruses are noncellular considered to be microbes but are not fully functional cells 0 6 major groups studied by microbiologists 7 Prokaryotes freeliving 39 with no nucleus or 39 bound organelles 1 Bacteria 2 Archaea metabolism and environments are different 7 Eukaryotes 1 Algae photosynthetic 2 Protists 3 Fungi 7 Viruses Microbes Shape Human History 0 Microbes affect food availability Some destroy crops corn rust late blight said to be cause of Irish potato famine Some make foods beer bread cheese 0 Microbial diseases change history 7 Black plague Bubonic Plague in Europe killed 13 population in 14 h century 7 Smallpox in Americas 7 Settlers using biological warfare on Native Americans 7 More soldiers have died from infections that are from battle wounds 7 Florence Nightingale convinced British government to improve army living conditions amp upgrade army hospital standards 7 Tuberculosis TB in 19 h century 7 AIDS today Discovery of Microbes 0 Light microscope invented in 1600s 0 Mid1600s Robert Hooke observes small eukaryotes with 7 Saw fungi in shoe leather 7 1st to use term quotcellsquot 0 Antoni van Leeuwenhoek 1676 7 Built simple microscopes 7 Described wee animalcules or beastiesquot 7 Published first drawings of bacteria prokaryotes Microbes Are Living Organisms Spontaneous generationidea that living organisms originate from nonliving matter 7 Van Helmont s mice recipe place grain cheese and old rags in a container and store in a quiet dark place After a while mice will appear This is how they thought mice were made 0 Microbes arise only from other microbes 7 No spontaneous generation 7 1861 Pasteur shows that microbes do not grow in liquid until introduced from outside 0 He disproved this using broth in swannecked flasks Sterilized broth by boiling bent necks of flasks so that air could enter but microbes would be caught in the neck Open 7 air 5 urve exdudes n dust and microbes Gmwth medium led quot 39 memuu nu quot39 L u 39 39 I Pasteurization use of moderate heat to reduce number of microorganisms in foods milk wine oysters I Worked with wine industw and studied fermentation Others tried Pasteur s experiments didn39t have same results as he didwh ecause some bacteria can form endospores which can survive boiling 9bacteria will grow again Medical Microbiology Ge rm Theow of Disease I Observations Germs can infect and grow on food I Hypothesis an germs infect and grow on people 7 That is do germs cause disease I Hypothesis is testable germs found in infected tissue Can transmission of germs cause disease Koch39s Postulates Developed from his study of anthrax I Provides scientific basis for determiningthat a specific microbe causes a specific disease 7 quotD this germ cause that disease Organism must meet 4 criteria 1 The microbe is found in all cases of disease but absent from healthy individuals 2 The microbe is isolated from the diseased host and grown in culture 3 When the microbe is introduced into a healthy susceptible host the same disease occurs 4 The same strain of microbe is obtained from the newly diseased host 0 Koch39s postulates are only a guide Can you think of instances when Koch39s Postulates could not be used 0 People who are carriers S aureus I M tuberculosis only 10 of people who are infected have symptoms I Organisms which can39t be grown in a lab 0 Treponema pallidum 0 Virus I HIV 0 Found at extremely low concentrations in bloodstream o No quottestquot animal I Diseases caused by more than one organism Koch amp Tuberculosis 0 1881 17 of all deaths caused by E 0 Thought it was U39 but had never seen organism 39 39 39 39 grows very slowly hard to stain due to lipid content Koch developed acidfast staining procedure patient enough to get pure culture 0 1905 Nobel Prize was awarded to Koch for his work on tuberculosis History of immunization Stimulation ofan immune response by deliberate 39 39 39 with an 39 pathogen 0 Lady Montagu early 1700 s brought practice of39 39 39 with smallpox variolation to England from Turkey 0 Edward Jenner 7 1778 found that milkmaids exposed to cowpox were immune to smallpox Vaccinated people including his son with cowpox they were resistant to smallpox Pasteur 7 in 1879 studied fowl cholera disease of chickens 7 Isolated and grew bacteria which causes it then left for vacation 7 After vacation the aged bacteria no longer cause disease in chickens 7 Inoculated same chickens with fresh bacteria from new outbreak and they still did not get sick 7 Attenuated weakened bacteria Gave chickens immunity from disease 7 Developed vaccines for anthrax fowl cholera rabies from dried rabbit spinal cord p and for Ignas Semmelweis mid 1800 s 0 Puerperal feverchildbed fever 0 Medical students carried llcadaver particles from autopsy room to deliver room 0 Caused by Streptococcus 0 Had med students wash hands in chlorinated lime water reduced mortality from 18 to 1 39 g pathogens Joseph Lister 0 1864 British surgeon found that survival rate of surgical patients increased if surgeons washed hands sterilized 39 and used quot 39 carbolic acidphenol during surgery Paul Ehrlich early 1900 s 0 Idea for llmagic bulletquot that would kill pathogens but not harm humans found arsenic compounds used to destroy trypanosomes Discovered Salvarsan to treat syphilis 0 Used term quotchemotherapyquot using chemicals to kill pathogens without harming patient 0 Arsenic turned out to be fairly toxic to humans Alexander Fleming 1929 o Discovered antimicrobial effects of penicillin G produced by Penicillium notatum fungus on Staphylococcus o 1945 Nobel Prize in Medicine Fleming for discovery Florey amp Chain for determining mode of action of penicillin and developing method of mass producing the drug 0 1945 penicillin became available for general us Growth of microbes in pure culture Koch amp solid culture media 0 Potato slices 7 Strea ked bacteria onto potatoes 7 Problem too much moisture39 many contaminants 0 Beef broth solidified with gelatin 7 Problems Gelatin liguefies above 28quotC Many microbes produce enzyme called gelantinase liguefies gelatin Agar 7 polysaccharide derived from red algae 7 1882 Fannie Hesse wife of Koch s coworker suggested agar She used it to make jelly 7 Solid until 100uC and m ostm 39 39 d past it Petri Dish 0 Richard Petri Koch s assistant invented them in 1887 0 Before this they used a bell 39ar or glass box Discovery of viruses 0 Dmitrii vanowski cause tobacco mosaic disease could pass through a filter 0 Matinus Beijerinck llfilterable agent was too small to be bacterium o Wendell Stanley purified and crystallized tobacco mosaic virus 0 1677 Observed llwee animalculesquot Antony Leeuwenhoek 0 1796 First scientific Small pox vaccination Edward Jenner 0 1850 Advocated washing hands to stop the spread of disease Ignaz Semmelweis 0 1861 Disproved spontaneous generation Louis Pasteur 0 1862 Supported Germ Theory of Disease Louis Pasteur 0 1867 Practiced antiseptic surgery Joseph Lister 0 1881 Growth of Bacteria on solid media Robert Koch 0 1882 Outlined Kochs postulates Robert och 0 1885 First Rabies vaccination Louis Pasteur 0 1887 Invented Petri Dish RJ Petri 1892 Discovered viruses Dmitri losifovich vanovskil 0 1910 Discovered cure for syphilisSalvarsan 606 Paul Ehrlich 0 1928 Discovered Penicillin Alexander Fleming Microbial Ecology 0 Most microbes don t grow on typical medium 0 quot 01 of microbes in our biosphere can be cultured in the lab 0 Many live in varied conditions 7 Anaerobic 0 Bottom of swamp 7 High pressure 0 Bottom of ocean 7 Hot or cold temperatures 0 Below OGC to 113GC 7 No organic carbon 0 Use light for energy 2 for carbon 0 Can culture some microbes in natural mud environment 7 Winogradsky column enrichment Layers grow different species reflecting different conditions 7 Can see variations in nature 0 Yellowstone geyser runoff 0 Colors reflect different species 0 Different growth temperatures 0 Microbes cycle most elements on earth 7 Nitrogen cycle 0 Bacteria fix N to NHA 7 Carbon cycle 0 Photosynthetic microbes fix most carbon 7 Sulfur cycle 7 Phosphorus cycle The Microbial Family Tree 0 Microbial species are difficult to classi Carolus Linnaeus 7 Difficult to distinguish by shape 7 Often reproduce asexually 7 Pass DNA to each other without reproduction 0 Use biochemical properties to classify 7 Gram stain 7 Ability to metabolize different substrates 0 Use DNA sequence to classify 7 Bacterial genomes are relatively small 7 2 distinct species share no more than 95 similarity of DNA seguence 0 Several lineages exist in the domains Bacteria and Archaea huge diversity of cell morphologies and physiologies Phylogenetic tree of life Fig 15 amp 127 latterly Proka Common I EHKESKOY Auhaa Algaeand planks Eulmrya Fungi and animals Prorlsrs Prokawotic Microorganisms n t piukawotes um soil bacteria quot39 bacteria etc EX Salmonella and Escherichia coli I Archaea live in extreme hot springs glaciers salt lakes EX Methanococcus and Halobacterium 7 Archaea are not bacteria Similar size shape Vew dif39ferent biochemistm Dif39ferent membranes Archaeal ribosomes similarto eukamotic ribosomes Many archaea live in harsh environments Eukawotic Microorganisms includes fungi algae amp protists 0 Fungi nonphotsynthetic most have cell walls 7 X easts unicellular and molds multicellular hyphae 7 Sacc aromyces yeast is used to make beer and bread 7 Penicillium mold produces penicillin 0 Algae 7 Photosynthetic contain chlorophyll have cellwalls 7 EX Diatoms amp seaweed 0 Protozoa Protists 0 Unicellular motile no cellwalls 0 EX Amoeba Paramecium amp Giardia Eukawotes evolved through endosymbiosis 0 Endosymbiosis 7 t eow L 39 quot through asvmbiotic relationship one cell engulfed a second cell and a symbiotic relationship developed mitochondria and chloroplasts are thought to have evolved this way mitochondria from respiring bacteria similarto E coli chloroplasts from phototroph related to cynobacteria 7 Mitochondria and chloroplasts membranes possesstheir own DNA and ribosomes are about the size of a prokamotic cell divide bya process similarto bacteria Chapter 23 Human Microflora and Nonspecific Host Defenses Highly recommended that you read this chapter in the text Human Microflora Humans colonized by many microbes 7 Bacteria fungi viruses Normal flora live in and on us don t usually cause disease 7 quotcommensalquot mutualistic organisms one benefits and other isn t effected we both benefit more mutualistic Microbe populations change constantly throughout the year based on diet etc 7 Vary with type of tissue and conditions bacteroidslive in intestine pH moisture other microbes present Intestinal flora varies with food eaten lot s of meat vs vegetarian 7 Can cause disease if reach abnormal location Metagenome collective genome of all microbes in and on body Normal Human Flora There are billions of microbial cells living in amp on the human body Areas that should be sterile Internal organs Blood Cerebrospinal fluid Sites where bacteria are normally found Skin Oral cavity Gastrointestinal tract Respiratory tract Urogenital tract See Fig 231 parts of the body Human Microflora Skin Skin is difficult to colonize 7 Dry salty acidic lactic acid protective oils sebaceous glands lysozyme breaks down peptidoglycan inhibits some G organisms not many lysozymes so some G present Microbes grow mostly in moist areas underarms genitals etc Associated with sebaceous oil glands and apocrine sweat glands pH 46 Factors that affect types of normal flora weather summer creates more moisture age personal hygiene Microbes commonly associated with the skin Most are G bacteria Staphylococcus Streptococcus many are nonpathogenic Propionibacterium rod shaped 7 Gram anaerobic rods 7 Propionibacterium acnes can cause acne Bacillus Some yeasts Candida Human Microflora Eye not many organisms in our eyes tears blinking lysozymes Most inhibited by lysozyme and continual rinsing of conjunctiva eye surface Sometimes S epidermidis and some Gram rods can temporarily reside in eye without causing infection Pinkeyeconjunctivitis can be caused by several types of bacteria and viruses Human Microflora Mouth large population of normal flora Bacteria in the mouth prior to tooth formation babies are mainly aerotolerant anaerobes such as Streptococcus and Lactobacillus as well as some yeast After teeth emerge anaerobic areas between teeth and gums support anaerobes such as Prevotella and Fusobacterium Teeth and gingival crevices colonized by over 500 species of bacteria Straight from the dentist with clean teeth Acidic glycoproteins in saliva form a layer on teeth providing a firm attachment site for microbes usuay Streptococcus species Extensive growth of these microbes results in thick bacterial layers called dental plaque Mixture of microorganisms and polysaccharides Dental procedures can cause organisms to enter the bloodstream and cause bacteremia which is quickly cleared by immune system In patients with mitral valve prolapse heart murmur bacteria can be trapped in defective valve and form vegetations bacterial cells glycocalyx fibrinclot caled subacute bacterial endocarditis microflora of dental plaques make acids that decalcify tooth enamel 7 results in dental caries tooth decay cavities Tooth surfaces in and near the gingival crevice where food particles remain are common sites of tooth decay Floss Diets high in sucrose table sugar are cariogenic cavity causing Lactic acid bacteria ferment the sucrose to lactic acid 5treptococcus mutans produces dextran extremely sticky polysaccharide used for attachment to tooth surface only when sucrose is present main bacteria causing cavities Diets containing fluoride small amounttoxic if high amount during formation of tooth enamel make teeth more resistant to tooth decay Incorporation of fluoride into the calcium phosphate crystal matrix increases resistance to decalcification Human Microflora Respiratory Tract upper and lower Upper respiratory tractnasopharynx oral cavity and throat Most bacteria that are breathed in are trapped in hairs in nose and mucus Are many opportunistic pathogens in nasopharynx Opportunistic pathogens which can be found in the nasopharynx Staphylococcus aureus Causes sinus and ear infections First and most opportunistic pathogen Streptococcuspneumonia Causes sinus infections and bronchitis Streptococcus pyogenes Causes sinus infections and quotstrepquot throat Corynebacterium diphtheriae Causes diptheria severe childhood illness Lower respiratory tract trachea bronchi and lungs Bacteria are not normally found in lower respiratory tract Lower respiratory tract is lined with cilia which push bacteria and dust particles up where they are caught in mucus and saliva mucociliary elevator pushed back up Normal flora of the gastrointestinal tract esophagus stomach small intestine super long large intestine Human Microflora Stomach pH NZ don t take antiacids we need stomach acids Only acidtolerant bacteria can colonize stomach wall mush attach to the wall ex Helicobacterpyori peptic ulcers Very few bacteria are found in stomach fluids should be killed Human Microflora Small Intestine ii of bacteria increases down through small intestine due to rising pH Different parts starting with closest to the stomach Duodenum few bacteria due to stomach acids pancreatic secretions and bile mainly Gram cocci and bacilli Je39unum En terococcus 6 Lactobacilus leum microbiota reflects that of large intestine larger ii of microbes bc of neutral pH large number of Bacteroides and facultative aerobes like E Coli Human Microflora Large Intestine largest diversity of organisms Fermentation vessel many bacteria gt300 species mostly anaerobes most have never been cultured Commonly lOquotll bacterial cellsgram of intestinal contents Small number of facultative aerobes E coli consume oxygen which allows obligate anaerobes to grow Bacteria ferment ingested food that made it this far into products we can absorb we absorb 15 of caloric intake this way studies on sets of twins where one was obese and one lean showed drastic differences in their gut microbiomes Provide nutrients inhibit pathogens Common intestinal flora E coli Bacteroides Bacteroidetes predominate organism in intestines Costridium Firmicutes quot quot looks like but found in intestines Many adults also have methanogens which convert hydrogen and carbon dioxide to methane gas Diet affects intestinal flora even on daily basis ex More meat in diet more Bacteroides intestinal flora can determine whether you gain weight easily or not Some vitamins are produced in intestine by bacteria Vit B12 K riboflavin thiamine Antibiotics can reduce number of normal intestinal flora can lead to digestive problems if overdone 7 Probiotics supply bacteria yogurt activia etc take up space in intestines while normal flora recover I LactobacilusBifidobacterium Bacteria in the intestinal tract are constantly being displaced downward by the flow of material and replaced by new microbes The growth rate of bacteria in the lumen of the intestinal tract is 12 doublings per day generation time 1224 hours Human Genitourinary Microflora Kidney is sterile used to think bladder was also Several types of facultative anaerobes mostly Gram rods are associated with urethra Escherichia bc it s close to anus Staphylococcus gra m Enterococcus Neisseria G ram cocci Many of these are opportunistic pathogens can cause urinary tract infections UTI most common in UTI is Ecoi Lactobacilus acidophius is often found in vagina of adult women Acidic secretions prevent pathogens Probiotic treatment if on antibiotics should take probiotics so you don t get a yeast infection Risks Benefits of Microflora Commensal flora benefits human host 7 Makes vitamins 7 Digests food 7 Prevents colonization by pathogens Opportunisticpathogens 7 Surface breach surgical wound allows bacterial entry 7 Bacteroides abscess or gangrene can get into abdomen or other places they don t belong 7 Immunocompromised hosts Costridium dlficie enterocolitits in intestines when on antibiotics for extended periods normal flora can die and C dif can take over diarrhea and other severe problems occurs in elderly or sick mostly treatment transposions most common in nursing home patients people who have been in hospital for a while Gnotobiotic animals 7 Either germfree or all microbial species are known 7 Poorly developed immune systems 7 Low cardiac output 7 Thin intestinal walls 7 Very susceptible to infection by pathogens no normal flora to protect them Overview of the Immune System InnatenonadaptiveImmunity 7 Physical barriers to infection skin 7 Nonspecific reaction to destroy invading cells lysozyme Adaptive Immunity 7 Specific Reaction to specific antigens Parts of foreign proteins sugars chemicals 7 Body reacts to antigens when exposed Retains quotmemoryquot of those antigens Faster response if exposed a second time Cells of the Immune System quotWhite blood cells 7 NeutrophilsPMN and monocytes both are phagocytic Engqu and destroy microbes 7 Monocytes macrophages and dendritic cells differentiate into those when they move into tissues 7 Basophils and Eosinophils have granules with chemicals contents to kill pathogensinfected body cell Release toxins to poison microbes 7 Lymphocytes involved in specific immune response T cells regulate specific immune response B cells produce antibodies to bind antigens Innate Host Defenses nonadaptive Physical barriers to infection 7 Skin many layers Keratin oil cells that phagocytize microbes Dead skin cells washing remove attached cells 7 Mucous Trap destroy pathogens Mucous layers slough off removed 7 Cilia remove microbes from lungs constantly moving 7 Tolllike receptor proteins on tissues first found in fruit flies Gut M cells Microfoldtake up bacteria by endocytosis where there are phagocytes waiting Skin Langerhans cells similar to M cells Chemical barriers to infection 7 Acidic pH stomach skin vagina 7 Lysozymetears skin Destroys peptidoglycan 7 Defensins Peptides which disrupt pathogens cytoplasmic membrane Effective against 6 6 fungi enveloped viruses Inflammation when bacteria gets through initial barrier Nonspecific Redness swelling pain heat at inflammation site Fluids containing WBC s leak into wound Clot forms to localize pathogen Fibrin produced to quotwalloffquot bacteria Septic shock infection and inflammation spread through body can cause hospitalization andor death Fever nonspecific Some products of pathogens endotoxin are pyrogenic feverinducing Cause hypothalamus to reset body temp Endogenous pyrogens fever causing are produced by leukocytes Higher temp 7 Increases phagocytosis and antibody production 7 Slows bacterial growth 7 However very high temp damages host tissue Acute Inflammatory Response Infection releases microbes to tissue Resident already in the area macrophage engulf bacteria Macrophage release vasoactive factors and cytokines which cause capillaries to dilate and signal more macrophages into the area Fluids containing WBC s leak into wound Macrophage squeeze between capillary cells extravasation 7 Leave capillary 7 Attack bacteria Damaged tissue secretes bradykinin 7 Stimulates mast cells Mast cells degranulate release histamine Histamine stimulates vessels to open further 7 Blood plasma platelets released into area 7 Prostaglandin released Prostaglandin stimulates nerve cells 7 Signal itching or pain 7 Cyclooxygenase COX enzyme involved in prostaglandin synthesis Aspirin Vioxx Celebrex are COX inhibitors prevent synthesis of prostaglandins reduce pain Chronic Inflammation Some pathogens resist host defenses Mycobacterium 7 Remain in body 7 Body walls off site in granuloma trying to contain the pathogens Fibrotic lesion around bacteria Autoimmune response 7 Immune system responds to body cells as if they were foreign Lupus erythematosus rheumatoid arthritis Crohn s disease 7 Destruction of host tissue Phagocytosis Phagocytes kill ampdigest engulfed bacteria 7 Produce lysozyme destroys peptidoglycan and antimicrobial peptides to kill bacteria 7 Kill with reactive oxidative burst Increase in oxygen consumption during phagocytosis results in production of reactive oxygen species such as superoxide ions H202 Innate Defense by Interferon Interferons quotinterfere with viral replication 7 Produced by infected host cells 7 Species specific mouse interferon will not work on humans humanhuman 7 Virus nonspecific human interferon helps protect against influenza and cold viruses Type 1 are antiviral 7 Bind to uninfected cells amp make them resistant to viral infection One type cleaves viral RNA another prevents translation of viral RNA Type 2 stimulates immune cells 7 Nearby immune cells become more sensitive 7 Activates macrophage natural killer cells T cells Natural Killer Cells Destroy infected amp cancerous host cells Healthy cells make surface MHC I major histocompatibility complex protein 7 Cancerous amp infected cells stop making MHC I Antibodies bind to viral proteins on infected cell surface NK cells recognize those proteins shown on the surface NK kills antibodybound and cells lacking MHC I 7 Secretes perforin protein into target cell 7 Perforin creates membrane pores to lyse cell Role of Complement 20 proteins in blood serum Several are proteases which sequentially cleave each other Activation of complement cascade results in pores in target microbial membranes membrane attack complex MAC Works well against gram bacteria Cell contents leak out cell dies Look at chart of Nonspecific and specific defenseswhat comes first 1 and 2 are nonspecific 3 is specific skin mucous membranes chemicals 2 fever phagocytosis 3 lymphocytesantibodies The host39s ability to resist infection depends on Age young amp old more susceptible Stress high stress increases susceptibility Diet low protein amp calories increases susceptibility low in nutrients General health and lifestyle smoking excess alcohol consumption drug use amp lack of sleep increase susceptibility Prior or concurrent disease increases susceptibility Many hospital patients with noninfectious diseases ex Cancer or heart disease acquire nosocomial infections because they are compromised hosts Genetic conditions especially those affecting immune system In libraw on first floor at reserve desk MICROBIOLOGY An Evolving Science 1St edition call number is RXSZ Circulates 2hrs BUILDING USE ONLY Chapter 2 Observingthe Microbial Cell Read Section 2126 SIZE OF PROKARYOTES 0 Prokawotes are generally smallerthan eukamotes 0 Small size allows nutrients to reach all parts of cell guickly I u L n 2pm The n I Cocci typically have diameter of 2 um Some microbiologists have proposed that bacteria smallerthan 02 um exist in nature cells referred to as nanobacteria L 7um If one considers the space needed to house all essential molecules of life it is unlikelythey could existwithin a volume available in a cell less than 01 Prokawotic cells can have a wide variety of cell morphologies which are often helpful in identification Bacterial Shapes 1 Simplest shape is sphere singularcoccus o plural cocci o Arrangement Coccus single cells Diplococcus 2 cells Streptococcus chains of more than 2 cells Staphylococcus gra pelike cluster of cells Tetrads packets of 4 cells 2 Most common shape is o Singular bacillus o Plural bacilli 0 Can be vew long amp thin or short amp fat 0 Occur singly or in chains 3 Spirals O Eye Rigid spiralshaped bacteria are called spirilla singular spirllum m Flexible spiralshaped bacteria are called spirochetes o Vibrio curved rods 0 Ocular lens Microscopy 0 Simple microscopes o magnify 50 300gtlt 0 Compound microscopes I o mu tiple lenses OblEltthelen5 0 Much higher magnifications Specimen Ones in 2051 lab magnify 1000gtlt Condenser lens I l Diaphragm 239 mm Light Lens System of Compound Microscopes o Condenser lens 0 located between light source and specimen 0 Focuses light rays up through specimen 0 Objective lens system 0 Closest to specimen 0 Typical scope has 10 40 amp 100X objective lenses 0 In each objective are multiple lenses 0 Ocular lens system 0 Closest to the eye 0 Typically magnify amp 0 Each ocular has multiple lenses Magnification o A microscope with a 100X objective and 10X ocular has a total magnification of o This means an object will appear 1000x larger than it is 0 Can see bacteria at this magnification but not internal structures 0 Cannot see viruses Resolution 0 ability to quot 39 U 39 39 between 2 obiects that are close together 0 Limits the usefulness of a microscope 0 Highest resolution of a typical light microscope is 02 micrometers o This means that 2 objects closer than 02 pm to each other can t be distinguished of light and numerical aperture 0 Resolution is determined by 0 Increasing resolution 1 Multiple lenses Correct each other s aberrations 2 Use shorter wavelength light lue li ht 400nm is shortest wavelength so gives highest resolution 3 Increase numerical aperture measure of light gathering ability of obiective lens Wider lens closer to specimen Immersion oil ight bends as it passes through specimen into air reduces resolving power If space between specimen and obiective is replaced with 39 oil resolution increases Oil keeps light from scattering as much as in air 4 Increase contrast Contrast between bacteria amp background is small so bacteria are very hard to see Increase contrast by staining cells or using special type of Many stains are basic dyes I Chromophore pigmented portion has positive 1 charge I Cell surface has negative charge I Examples crystal violet methylene blue safranin 0 Simple Stains 0 Use one dye 0 Doesn t differentiate between types of cells or structures 0 Differential stains o Distinguish between different types of bacteria or different structures 0 Uses more than on dye o Gram stain distinguishes Gram positive bacteria from Gram negative bacteria 0 Acidfast stain distinguishes Mycobacterium species from other bacteria Types of Microscopes Used To Increase Contrast o Microscope we typically use is called brightfield light microscope 0 See stained cells on bright background 0 Instead of staining cells special microscopes can be used to increase contrast between cells amp medium 0 PhaseContrastMicroscopy 0 Cells slow the speed of light passing through them so cells differ in refractive index from their surroundings o This difference in quotphasequot is amplified by special ring in lens of phase contrast microscope 0 Can observe living cells in wet mount 0 Can see organelles of eukaryotes o DarkField Microscopy 0 Cells appear bright against dark background Darkfield stop in condenser doesn t let light pass directly through the specimen Only light scattered by sample reaches ob39ective Can observe living cells in wet mount Excellent for studying bacterial motility and very narrow cells 0 39 39 cell is too narrow to resolve by light 39 0 Problem dust particles also scatter light 0 Differential Interference Contrast DIC Microscopy o Polarized light passes through specimen I Sample boundaries bend light I Second polarized lens blocks light I Bent light results in 3D appearance 0 Fluorescence Microscopy 0 Some compounds absorb energy from invisible radiation like short wavelength UV light then radiate energy back as a longer visible wavelength I Fluorophores absorb hi hener li ht short wavelen th then emit lower energy light longer wavelength 0 Used to view cells that make fluorescent compounds such as chlorophyll or cells that have been stained with florescent dyes 0 These microscopes use a UV light source to fluoresce objects 0 Can label molecules of interest in cell 0 Electron Microscopy o Resolving power of light microscopes is limited by wavelength of light used 0 Electron 39 r use beams of electrons instead of visible light and 39 instead of lenses 0 O O O 0 Can magnify over 100 000x 0 Can be used to view viruses proteins nucleic acids 0 Sample must reflect electrons o Coated with heavy meta 0 Electron beam and sample are in a vacuum htt www outubecom watchvfToTF39ch5M 0 Transmission electron microscope TEM 0 Sample is sliced very thin I Microtome 0 Sample is stained with I Uranium I Osmium o Electrons travel through specimen 0 High resolution 0 Used to view cell structures DNA amp some protein molecules 0 Scanning electron microscope SEM 0 Sample is coated with heavy metal 0 Not sliced 0 Retains 3D structure 0 Gives 3D image 0 Image formed from electrons reflected from the specimen 0 Used to visualize surface cell structures What disease is caused by the organism on the last slide 0 Mycobacterium leprae 9 leprosy Chapter 23 Human Microflora and Nonspecific Host Defenses Highly recommended that you read this chapter in the text Human Microflora Humans colonized by many microbes Normal flora mutualistic organisms 7 Commensalism one organism benefits the other is unaffected Microbe populations change constantly 7 Vary with type of tissue and conditions pH moisture wetter more microbes other microbes present Intestinal flora varies with food eaten 7 Can cause disease if reach wirwmm magi l m miiiiw Normal Human Flora There are billions of microbial cells living in amp on the human body Areas which should be sterile See Fig 231 Human Microflora Skin Skin is difficult to colonize pflii H39i39 HI VH S39I young children Microbes c Most are Some yeasts Human Microflora 39 Most inhibited by Sometimes 1 1 infection After teeth 1 and Teeth and gingival crevices colonized by over 5 of bacteria Acidic glycoproteins in saliva form a layer on teeth providing a firm attachment site for microbes usually species Extensive growth ofthese microbes results in thick bacterial layers called Dental procedures can cause organisms to enter the bloodstream and cause i is quickly cleared by immune system In patients with m Hirel P I heart murmur bacteria can be tr bacterial cells glycocalyx 39h39 i 75 quot u valve and form 3 tooth d l Li39 W39 3 a m gm the sucrose to l Lactic acid bacteria 1quot only when decay 2 increases resistance Most bacteria that are breathed in are trapped in mucus Are many opportunistic pathogens in nasopharynx Opportunistic pathogens which can be found in the f throat Causes ill Lower respiratory tract Bacteria are not normally found in lower respiratory tract Lower respiratory tract is lined with are caught in and Normal flora of the gastrointestinal tract Human Microflora Stomach pH E Only iciaiaviai 2w bacteria can colonize stomach wall ex m peptic ulcers Very few bacteria are found in stomach fluids most are killed 39 which push bacteria and dust particles up where they v 1 f ilLi Lif vlfllllEl W l Human Microflora Small Intestine be acidophile Cr anaerobe and can surive Human Microflora Large Intestine most have never been n which allows obligate anaerobes Small number of is mi wi 1 ram l consume 39 to grow into products we can absorb we absorb m of L quot sulfa Tail Ll wj Nil e 3m il ii i h39iiz ii 5i can lead to digestive problems Several types of facultative anaerobes mostly 3 1 are associated with urethra 1 pathogens can cause um mm mar in r s often found in vagina of adult women Acidic secretions prevent pathogens Probiotic treatment Risks Benefits of Microflora Commensal flora benefits human host 39 wild l i siiii min w y 1 Opportunistic pathogens six M Li i v39ll Gnotobiotic animals 7 Either germ free or all microbial species are known 7 Poorly developed immune systems 7 Low cardiac output 7 Thin intestinal walls 7 Very susceptible to infection by pathogens Overview of the Immune System Adaptive Immunity Reaction to r Faster response if exposed a 5 f i39wl lawn Cells of the Immune System regulate specific immune response N produce antibodies to bind antigens Innate Host Defenses Physical barriers to infection Keratin oil cells that phagocytize microbes Dead skin cells washing remove attached cells Trap destroy pathogens Chemical barriers to infection tears skin Destroys peptidoglycan Peptides which disrupt pathogens cytoplasmic membrane Effective against 6 6 fungi enveloped viruses Inflammation Redness swelling pain heat infection and inflammation spread through body endotoxin are fever inducing 1 to reset body temp are produced by I 2m I rrr39 V Higher temp 7 Increases i2 7 Slows I7 r i i il g 7 However very high temp damages hm Mg m Acute Inflammatory Response Infection releases m w Resident nn engulf bacteria Macrophage release Fluids containing 7 Leave capillary 7 Attackbacteria o Damaged tissue secretes Aspirin Vioxx Celebrex are reduce i Chronic Inflammation Some pathogens resist host defenses u prevent synthesis of 9 7 Remain in body 7 Body walls off site in gigging lemma lEi l wi iilt lew39mm bacteria Autoimmune response disease 7 Destruction of hi Phagocytosis Phagocytesamp engulfed bacteria 7 Produce p m e and antimicrobial peptides to kill bacteria during phagocytosis results in production of it is in creates we m i Role of Complement to lyse cell 20 proteins in blood serum Several are proteases which sequentially cleave each other Activation of complement cascade results in pores in target microbial membranes cell contents leak out cell dies The host39s ability to resist infection depends on Age young amp old more susceptible Stress high stress increases susceptibility Diet low protein amp calories increases susceptibility General health and lifestyle smoking excess alcohol consumption drug use amp lack of sleep increase susceptibility Prior or concurrent disease increases susceptibility Many hospital patients with noninfectious diseases ex Cancer or heart disease acquire nosocomial infections because they are compromised hosts Genetic conditions especially those affecting immune system Chapter 20 Eukaryotic Diversity Phylogeny of Eukaryotes o Eukaryotes have greatest diversity of size 0 Groups containing microscopic eukaryotes 7 Fungi 7 Algae 7 Protozoa Fungi 7 Cell walls contain chitin 7 Nonmotile 7 Most grow hyphae Mycelium branched mass of hyphae 7 Absorptive heterotrophs o Fungi7Yeasts 7 Unicellular fungi 7 Reproduce Via budding o Fungi7Chytrids 7 Only motile agella reproductive zoospores 7 Associations with animals Symbiont in bovine rumen Frog pathogen o Fungi7Zygomycetes 7 Nonmotilesporangiospores gametes Spread Via air or water currents fuse to form zygote called zygospore 7 Rhizopus7 bread mold 7 Arbuscularmycorrhizae Mutualistic association with plant roots Increase root absorption 0 Fungi7Ascomycetes 7 Fruiting bodies form asci containing ascospores 7 Penicillium 7 Candida 7 Saccharomyces 7 Morels amp truf es o Fungi7Basidiomycetes 7 Produces spores called basidiospores 7 True mushrooms 7 Crytococcus Opportunistic pathogen Algae o Phytoplankton 0 Common features 7 All have chloroplasts 7 Many have paired agella 7 Cell wall made of glycoprotein or cellulose 7 Contractile vacuole removes excess water 7 Stores energy as starch o Algae7Chlorophyta 7 Green algae Chlorophylla 7 Grow near top of water 7 Multiple life forms 7 Unicellular forms have agella Filaments Individual cells Sheets Colonies o Algae7Rhodophyta 7 Red algae Phycoerythrin gives the red color Allows growth in deeper waters Sulfated sugar polymers agar agarose carrageenan Unicellular laments or sheets Porphyra used for wrapping sushi 0 Secondary Endosymbionts 7 Algae engulfed by a protist Mixotrophs combine phototrophy and heterotrophy 7 Diatoms Cell secrete Si02 quartz shell frustule 7 Brown algae Energy storage lipid Kelp Protists o Amoebozoa 7 Amorphous shape constantly changing shape Move using pseudopods Actin pushes cytoplasmic streams ahead Cell rolls over membrane Engulf food with pseudopods Entamoebahistolytica 7 causes severe stomach illness 7 Slime molds Feed mate as individual cells Aggregate to form fruiting body Spores released from fruiting body 0 Cercozoa7Shelled Amoebas 7 Live in marine habitats 7 Radiolarians Needlelike pseudopods Shells tests have holes for pseudopods to project from Pseudopods stabilized with microtubules 7 Foraminiferans Shells made of calcium carbonate Indicators of petroleum deposits 0 Alveolates 7 Alveola Flattened vacuoles at outer cortex 7 Extrusomes Secrete enzymes toxins 7 Microtubules Stabilize structure Wellstructured cell shape 7 Multiple cilia or agella o Alveolates Ciliates 7 Many cilia for motility Also for feeding 7 Contractile vacuole to maintain osmotic balance 7 Stalked ciliates attach to surface 0 Alveolates Dino agellates 7 some bioluminescent if water moves 7 Phytoplankton 7 Carotenoids in some red color Red tide bloom or overgrowth of dino agellates 7 Two long agella One wrapped around cell groove 7 Extrusome secretes toxins Neurotoxins 7 Some are endosymbionts Essential for coral survival Coral bleaching when endosymbionts leave the coral o AlveolatesApicomplexans 7 Apical complex invades host cells 7 No cilia 7 Obligate parasites Toxoplasma gondii Carried by cats Dangerous to pregnant women Plasmodium7 Malaria Most important parasitic disease of humans worldwide Transmitted to humans by Anopheles mosquito Infects liver amp red blood cells in human Other Eukaryotic Groups 0 Includes important parasites 7 Trypanosomes Similar to parasitic Leishmania 0 T rypanosomabrucei carried by tsete y causes African sleeping sickness T cruzi carried by kissing bug causes Chaga s Disease 7 Excavates Lack mitochondria Obligate parasites Anaerobes Giardialamblia Chapter 2 Observing the Microbial Cell SIZE OF PROKARYOTES o Prokaryotes are generally smaller then eukaryotes 0 Small size allows nutrients to reach all parts of cell quickly 0 Prokaryotic cells can be as small as 02um The smallest eukaryotic cells are 2pm 0 M typically has diameter of 2 pm 0 Some microbiologists have proposed that bacteria smaller than 02 pm exist in nature cells referred to as nanobacteria o If one considers the space needed to house all essential molecules of life it is unlikely they could exist within a volume available in a cell less than 01 pm 0 Prokaryotic cells can have a wide variety of cellular morphology shape which are often helpful in identification Bacterial Shapes 1 Simplest shape is a sphere a round ball 0 singularcoccus o pluralcocci o Arrangement 0 Coccussingle cells 0 Diplococcus 2 cells 0 Streptococcus chains of more than two cells of more than 2 cells 0 Staphylococcus grapelike cluster of cells 0 Tetrads packets of4 cells dinacoccus is a tetrad 2 Most common shape is rods 0 Singular bacillus o Plural bacilli 0 Can be very long amp thin or short amp fat 0 Occur singly or in chains 3 Spirals 0 Rigid spiralshaped bacteria are called spirilla singularspirillum 0 Flexible spiralshaped bacteria are called spirocheter 0 Vibrio curved rod Microscopy 0 Simple microscopes 0 one lens 0 magnify 50 300 X 0 Compound microscopes 0 multiple lenses 0 Much higher I Ones in 2051 lab magnify 1000X Lens System of Compound Microscopes 0 Condenser lens Eye 0 located between light source and specimen 16gt o Focuses light rays up through specimen Ocular lens 77 0 Does not magnify 0 Objective lens system 39 Ob ective lens 0 Closest to specimen 1 I 7 Speclmen 0 Typical scope has 10 40 amp 100x objective lenses Candenser lens f Diaphragm rm El o In each objective are multiple lenses 0 Ocular lens system Light 0 Closest to your eye 0 Typically magnify 10X 0 Each ocular has multiple lenses Magnification A microscope with a lOOX objective and 10X ocular has a total magnification of lOOOX This means an object will appear lOOOX larger than it is Can see bacteria at this magnification but not internal structures Cannot see viruses Can only see the basic shape and arrangement of bacteria Resolution ability to distinguish between 2 objects that are close together Limits the usefulness of a microscope Highest resolution of a typical light microscope is 02 micrometers This means that 2 objects closer than 02 pm to each other can t be distinguished Resolution is determined by wavelength of light and numerical aperture Increasing resolution 0 Use shorter wavelengths of light 0 Wider lens closer to specimen I Higher numerical aperture 0 lmmersion oil 0 Increase contrast 0 Multiple lenses I Correct each other s aberrations imperfections Wavelength of light 0 Shorter wavelength produces higher resolution 0 Blue Light 400nm is shortest wavelength so gives highest resolution Numerical aperture 0 Measure of light gathering of the object lenses how well it gathers light 0 Light bends as it passes through specimen into air reduces resolving power o If space between specimen and ob39ective oil immersion objective only is replaced with immersion oil resolution increases I Oil keeps light from scattering as much as in air 0 Contrast 0 Contrast between bacteria amp background is small so bacteria are very hard to see 0 Increase contrast by staining the cells or by using special types of microscopes that are used to increase contrast 0 Many stains are basic dyes I Chromophore pigmented portion has a positive charge I Cell surface has a negative charge 0 Positive and negative attract I Examples crystal violet methylene blue safranin 0 Simple Stains 0 Use one dye 0 Doesn t differentiate between types of cells or structures no spores or flagella 0 Differential stains o Distinguish between different types of bacteria or different types of structures 0 Uses more than one dye o Gram stain distinguishes Gram positive bacteria from Gram negative bacteria 0 Acidfast stain distinguishes Mycobacterium species from other bacteria I Useful medically Types of Microscopes Used To Increase Contrast Microscope we typically use is called bright filed light microscope 0 See stained cells on bright background have to kill microbacteria and stick them on a slide and duye them 0 Instead of staining cells special microscopes can be used to increase contrast between cells amp medium 0 PhaseContrast Microscopy 0 Cells slow the speed of light passing through them so cells differ in Refractive index from their surroundings glass slides air o This difference in phase is amplified by special ring in lens of phase contrast microscope 0 Can observe living cells in wet mount 0 DarkField Microscopy 0 Cells appear bright against dark background starry night type look 0 Darkfield stop in condenser doesn t let light pass directly thought the specimen 0 Light reaches specimen from sides only 0 Only light scattered by sample reaches objective 0 Can observe living cells in wet mount don t have to kill them or stain them 0 Differential Interference Contrast DIC Microscopy o Polarized light passes through specimen I Sample boundaries bend light I Second polarized lens blocks light I Bent light results in 3D appearance 0 Fluorescence Microscopy 0 Some compounds absorb energy from invisible radiation like short wavelength UV light wavelength UV light then radiate energy back as a longer visible wavelength 0 Fluorophores absorb highenergy light Short wavelength then emit lower energy lightonger wavelength 0 Used to view cells that make fluorescent compounds such as chlorophyll or cells that have been stained with florescent dyes 0 These microscopes use a UV light to fluoresce objects 0 Can label molecules of interest in cell Fluorescently Labeling Molecules 0 Attach directly to some molecules 0 DAPI binds DNA 0 Attach labeled antibody to molecules 0 Antibody binds specific molecules they are very specific I Fluor is covalently bound to antibody 0 Electron Microscopy O O Resolving power of light microscopes is limited by wavelength of light used 0 We cannot change the wavelengths of light Electron microscopes use beams of electrons instead of visible light and electromagnets instead of lenses Can magnify 100000X Can be used to view viruses proteins nucleic acidsvery very small molecules Sample must reflect electrons o Coated with heavy metal god 0 Electron beam and sample are in a vacuum bc electrons would be bounced around by air molecules if not in vacuum 0 Transmission electron microscope TEM O O O 0 Sample is sliced very thin I Microtomeused to slice the thingcan potentially slice a bacterial cell into 20 pieces Sample is stained with metal I Uranium I Osmium High resolution Used to view cell structures DNA amp some protein molecules 0 Scanning electron microscope SEM 0 Sample is coated with heavy metal 0 Not sliced whole sample placed on slide 0 Retains 3D structure 0 Gives 3D image Image formed from the specimen electrons bounce off specimen and they are used to generate a picture of the cell Used to visualize surface cell structures Selected topics from chapter 10 and 12 Chapter 10 Molecular Regulation Regulating Gene Expression 0 Microbes respond to changing environment 0 Alter growth rate 0 Alter proteins produced 0 Must sense their environment 0 Receptors on cell surface 0 Must transmit information to chromosome 0 Alter gene expression 0 Change transcription rate 0 Change translation rate Sensing the Environment 0 Twocomponent signal transduction 1 Sensor kinase protein in CM Binds to signal 0 Food 0 Chemical cue 39 Activates itself via phosphorylation 2 Cytoplasmic response regulator 39 Takes phosphate from sensor 39 Binds chromosome 0 Alters transcription rate of multiple genes Altering Transcription Rates 0 0 factor guides RNA polymerase to initiate transcription at promoter I Proteins can help guide 0 factor to promoter I Activator o Binds to DNA sites next to promoter I Proteins can block 0 factor from binding 0 Repressor o Binds to DNA sites next to promoter The amount of an enzyme in the cell can be controlled by decreasing repression or increasing induction the amount of mRNA that encodes the enzyme For negative control of transcription the regulatory molecule is called a repressor protein and it functions by inhibiting mRNA synthesis The E coli lac Operon Lactose milk sugar is used as nutrient source 0 Cannot pass through cell membrane 0 Lactose permease allows entry 0 Disaccharide lactose must be cleaved into monosaccharides to be digested o Bgalactosidase cleaves lactose lactose 0 People also make bgalactosidase o If not person is lactoseintolerant o The 062 gene encodes Bgalactosidase o The IacY gene encodes lactose permease 0 Need both proteins to digest lactose 0 Operon 0 Multiple genes transcribed from 1 promoter 0 Both genes are transcribed together 0 Repressor protein Lac blocks transcription 0 Repressor binds to operator 0 Blocks ofactorfrom binding promoter 0 Repressor responds to presence of lactose 0 Binds inducer allolactose or DNA not both 0 Add lactose gt repressorfalls of39f operator The lac operon is an example ofnegative control oftranscription a o l 391 r uLWWM IacYqugch I39rnnsu39iption blotked Repressor 39 Repressor lb Inducer allolactose For positive control oftranscription an activator protein binds to activatorbinding sites on the DNA to stimulate transcription 0 RNA polymerase can then proceed With transcrIp Ion The 39 example of quot uan ulpllull Anivamr ma b 39ngsite Promoter maIE muIF MJ maIG Ly lW39WMWM 1139 WWW No transcription 1Maltose activator protein a ma Promoter mulE maIF maIG lrnnstrip on proreeds Activator binding site W quot 39 Maltose activator protein b lnducer maltose Catobolite Repression 0 GLucose is easiest sugarto digest catabolize If glucose is present lac operon not transcribed I nor other sugar operons 0 Presence of glucose af fects signal inside cell I Lowers amount of CAMP 0 High glucose gt low cAMP 0 CRP protein is an activator protein 0 Binds nextto promoter 0 Increases transcription of Iacand other operons 0 CRP responds to presence of cAMP o Acts as activator only when bound to cAMP I Does not bind operons gt low level of Iactranscription 0 The lac operon is underthe control of catabolite repression as well as its own specific negative regulatow system Quorum Sensing 0 Cells work together at high cell density 0 V fischeri becomes bioluminescent 0 Many bacteria form biofilms 0 Send chemical signal to other cells 0 Chemical accumulation high cell density I Autoinducer chemical o Homoserine lactone for V fishcheri o Binds to sensor in cell I Sensor activates transcription http ted mm talksbonnie bassler on how bacteria 39 html Chapter 12 Biotechnology Bacteria grow quickly E coli genome relatively well understood Small plasmids replicate quickly Put gene of interest into a plasmid 0 Make huge amounts of protein 0 Easy to purify Applied Microbial Biotechnology Use microbes to express genes 0 Produce eukaryotic proteins in large amounts I Human insulin Use microbial o Microbes have huge diversity of enzymes I PCR depends on polymerases from thermophiles Immunize against pathogens 0 Use genes from pathogens I Without exposure to pathogenic organism Insulin Insulin is required for those with Type I diabetes amp many with Type II diabetes Prior to genetic engineering insulin was harvested amp purified from pig or cow pancreas Nonhuman insulin is less effective Human insulin was 15thuman protein made 39 quot using 39 quot using 039 bacteria 0 Identical in every way to human produced insulin Today engineered E colior yeast is used to make insulin Plant biotechnology Glycophosphate Roundup herbicide resistant plants Bacteria were found that were resistant to this herbicide The gene for the herbicide resistance was put into some crop plants soybeans corn cotton Transgenic plants Insectresistant plants Bacillus thuringensis produces chemical Bttoxin that is toxic to larvae of moths butterflies beetles flies Gene for Bt toxin was put into cotton and potato plants 0 Expressed in the leaves of plant 0 Kills only insects that feed on those crops 0 Eliminates need to spray chemical pesticides I Chemicals are dangerous to use toxic to humans expensive Vaccines through Food Vaccines expose human to pathogen proteins 0 Expose immune system cells to proteins 0 We develop immune defenses to proteins 0 Protects us from later infection by pathogen 0 Proteins can be produced in plant absorbed via intestine o No longer need to grow pathogen to produce protein 0 Ex Bananas that have vaccine for cholera Gene therapynonfunctional gene is replaced by functional gene Clone desired gene into viral genome Infect person with modified virus 0 Infected cells will express cloned gene I Make desired protein Genes for many diseases have been located and the mutations in the defective genes have been identified Huntingtons cystic fibrosis SCID Gene Therapy and Parkinson s Disease Currently 4 clinical trials using gene therapy noninfectious virus shuttles a gene into part of brain Gene leads to production of an enzyme which helps manage symptoms or protects brain from further damage No adverse effects in these trials but clinical usefulness still unknown Chapter 133 137 Energetics amp Catabolism Chapter 14 Respiration Lithotrophy amp Photolysis Metabolism Catabolism 7 Breaking down molecules for energy 7 Catabolic pathways breakdown large molecules in a series of steps coupled to reactions that store energy in small carriers such as ATPamp NADH Has to happen in a series of steps and many enzymes involved in breaking down energy Anabolism 7 Using energy to build cell components Metabolism 7 Balance between catabolism amp anabolism 7 Central biochemical pathways used for both amphibolic TCA cycle glycolysis pentose phosphate shunt Julia iii In uniLLJIIF 4 Q 1 ii E 1 4 915 aitQi s E i I Metabolism Reduced geological com pounds rocks Major Energy inorganic com pounds etoday Sunlight Reduced biological macromolecules Engrgysome starch fats formula 5 ohm First energy Phototrophy Lithotrophy Organotrophy CATABOLISRN ANABOLISM Longterm Energy energy storage I Shortterm Biosynthesis ener stora e 9y y Carbon nitrogen ATP water Catalysis and Enzymes Activation energy energy required to bring all molecules in a chemical reaction into the reactive state Takes certain amount of energy to bring all molecules together Enzymes Catalytic poteins speed up biochemical reaction rates lower activation energy by bringing substrates into proximity to each other amp correctly orienting them Activecatalytic site portion of an enzyme to which substrate binds enzyme Substrate quot9 products aldolase enzyme Fructose16bisphosphate 9 g1yceraldehyde3 phosphate dihydroxyacetone phosphate Very ggecific for their substrate Cellulose vs starch example Enzymes can have small non protein molecules that help in catalysis but aren t part of the enzyme or the substrate Two types 1 Prosthetic groups bound tightly to their enzyme usually covalenty amp permanently Example heme group in cytochromes enzymes in electric transport chain involved in making ATP 2 Coenzymes loosely bound to their enzyme may associate with different enzymes usually derivatives of Vitamins Example NAD derivative of niacin Enzymes are named either for the substrate they bind or for the chemical reaction they catalyze with the addition of the suf x ase For example cellulase breaks down cellulose into glucose hydrolases break various chemical bonds with the addition of water Catabolism The Microbial Buffet Microbes have great catabolic diversity 7 Electron donors Lithotrophy inorganic molecules Iron as electron donor Organotrophy organic molecules Glucose carbon hydrogen bonds Phototrophy use light energy to reduce compounds then use these as electron donor 7 Electron acceptors Respiration inorganic molecules Aerobic use 02 Anaerobic no 02 used Fermentation organic molecules OxidationReduction o Oxidationreduction redox reactions involve the transfer of electrons from electron donor to electron acceptor Electrons can t exist alone so for every oxidation there must also be a reduction In a redox reaction the substance oxidized is the electron donor the substance reduced is the electron acceptor OIL RIG Oxidation involves loss and reduction involves gain NAD as a Redox Electron Carrier In a cell the transfer of electrons from donor to acceptor typically involves one or more electron carriers Electron carriers can be membrane bound prosthetic groups like cytochrome c freely diffusible coenzyme like NAD nicotinamide adenine dinucleotide or NADP NADphosphate NADH carries electrons NADNADH 7 involved in energy generating catabolic reactions And hydrogens NADPNADPH involved in biosynthetic anabolic reactions Coenzymes like NAD and NADPH act as intermediaries between a primary electron donor and terminal electron acceptor that would not normally be redox couples gure EnergyRich Compounds and Energy Storage The energy released in redox reactions is conserved in the formation of compounds with energy rich bonds The most common of these is adenosine triphosphate ATP main energy carrier in the cell Especially for short term energy resources Cleavage of each phosphoanhydride bond releases free energy ATP 9 ADP 9 AMP ATP has two chances to release energy from cell ADP and AMP See Fig 136 Phosphate added to ADP via dehydration to make ATP 39 See Fig 136 Hydrolysis ofATP to ADP yields energy ADP phosphorylalinn io mp 9mm mummyanymsum mmmww vnunlhmpmwnn ATP can transfer energy to cell processes by 39 Hydrolysis releasingl or 2 phosphates 39 Phosphorylation of an organic molecule such as glucose in the phophotransferase system phosphate gets transferred to glucose Derivatives of coenzyme A have thioester bonds that release free energy upon hydrolysis 39 Longterm storage of energy involves polymers such as glycogen andpolyB hydroxybutyrate which can be consumed to yield ATP Major Catabolic Pathways Electron Transport and the Proton Motive Force Energy Conservation Options 1 Fermentation partial breakdown of organic substance without transfer of electrons to an inorganic terminal electron acceptor can occur Without ogygen 2 Respiration combines breakdown of organic molecule with electrontransfer to terminal electron acceptor such as o gen or nitrate yields more energy than fermentation 3 Photophosphorylation light absorption by chlorophyll drives photolysis of an organic molecule 39 In both fermentation amp respiration synthesis of ATP is driven by energy released in oxidationreduction reactions but the reactions and mechanisms for each differ 39 Cheese beer wine Sauerkraut coco beans In fermentation there are no exogenous electron acceptors 39 electron acceptor must be derived from electron donor In respiration molecular oxygen or other terminal electron acceptor is present In fermentation ATP is produced by substratelevel Phosphorylation during catabolism of an organic compound Substrate level phosphorylation a phosphate group is added to an intermediate in a biochemical pathway and is eventually transferred to ADP to form ATP A lntermediatesinthe biolthemicalpathway B P r l BP CP ADP D ATP SubslrnleIevel phosphnrylnlion In respiration ATP is produced by both substrate level Phosphorylation amp oxidative Phosphorylation Oxidative phosphorylation ATP is synthesized by a proton motive force generated by redox reactions Requires enzyme ATP synthase Which uses proton motive force to produce ATP from 39 Main way energy is used in aerobic reactions Energlzed xx x membrane X quot ADP P ATP 1 Less energlzed x membrane X X Oxidulive phosphorylalion Photophosphorylation method of producing ATP in photosynthetic organisms Similar to oxidative phosphorylation except light rather than a Chemical compound drives the redox reactions that generate the proton motive force Bacteria and archaea use 3 main routes to convert glucose to pyruvate l GlycolisisEmbdenMeyerhof Pamas EMP pathway Generates 2 ATP and 2 NADH 2 EnterDoudoroff ED pathway Glucose or sugar acids are converted to pyruvate generating 1 ATP lNADH and l NADPH Common in enterics o Organisms that live in intestine 3pentose phosphate shunt PPS Glucose is converted to sugars with 3 to 7 carbons which are precursors for biosynthesis or to pyruvate generating 1 ATP and 2 NADPH Glycolysis Fig 1317 Major pathway of glucose metabolism Series of reactions in which each molecule of glucose is oxidized to 2 molecules of pyruvate with small amount of energy ATP being generated 1 glucose92 pyruvate 2 ATP 2 NADH See Fig 133 Many carbon sources can be broken down to glucose and then enter glycolysis Starch is bunch of glucose amylase breaks it down Glumse 33 M 39ADP Glumse Evnhosnhate 4 0E5 Fructose 16 hisnhnsnhale some nihydmxyazemne phasphm mm Glyeraldehyde 3phasphate GZP 2 I mu 2Q 2 NA GGG 0 9 1Eisphosphoglyzeric add 2 SAD Lit 2 111 GOGG GOG B 3Phosphnglyc2ricacld 9 210 kHJ lt3 0 2 l Phasphosnnpymvic acid PEP up 9 Pyruvi acid EN ERGYINVESTMENT STAG E Step 1 Glucose is phosphorylated by Steps 2 and 3 The atoms ofglucose 6phosphate are rearranged to form fructose 6phosp ate Fructose 6phosphate is phosphorylated by LYSIS STAGE S ep 4 Fructose 16biphosphate is cleaved to form glyceraldehyde 3phosphate G3P and dihydroxyacetone phosp ate Step 5 DHAP is rearranged to form another G3P ENERGYCONSERVING STAGE Step 6 Inorganic phosphates are added to the 2 ed Oxidation of glyceraldehyde3 phosphate Oxidation removal of electrons Reduction addition of electrons 2 G3P are oxidized 2 NAD are reduced 2 13bPHA are produced Step 7 Two ADP are phosphorylated by substratelevel phosphorylation to form 2 ATP Hig energy phosphate group is transferred from substratelevel phosphorylation Step 10 Two ADP are phophorylated by substratelevel phosphorylation to form 2 ATP 150 blsplmsphuglvuelalew JyILware ATP S were used earlier ir the pathway 4 are 39H t n nitVi Precursor metabolites made in glycolysis 39 glucose6phosphate fructose6phosphate glyceraldehyde3phosphate 3phosphoglycerate phosphoenolpyruvate Pyruvate Precursor metabolites compounds used to make all the macromolecules in the cell Glycolysis summary Glucose is oxidized to 2 molecules of Pyruvate Net gain of 2 ATP 6 precursor metabolites are made NADH is formed which will be converted back to NAD in the electron transport system or in fermentation reactions Way they are converted back depends on way organism grows respiration or fermentation EntnerDudouroff Pathway Originally studied for its role in production of Mexican drink pulque or cactus beer by Zymomonas fermentation of the blue agave plant ED pathway allows E coli and other enterics to feed on mucus secreted by intestinal epithelium prevents colonization of pathogens Glucose to pyruvate using different enzymes than glycolysis Net 1 ATPglucose amp precursor metabolites Used by many enterics and Enterococcus faecalis Pentosephosphate pathway Named for phosphorylated pentose 5 carbon sugars ribulose xylulose and ribose that are formed from G6P Used for production of precursor metabolites needed in anabolic reactions Net 1 ATPglucose Ribulose 15 bisphosphate used in Calvin cycle is made by phosphorylating a precursor from this pathway Stain Structure Primary Mordant Decolorizer Counter Result Org Chemical stain stain stained Simple Highlight Methylene water stain Cocci entire blue Bacilli microorg So Crystal spiral that cellular violet shapes and Safranin basic structures are visible Gram Differentiates Crystal iodine ethanol safranin Gram E coli between gram violet is purple amp gram Gram Staphylococcus bacteria is red aurea Spirosoma linguale Endospore Endospore Malachite heat water safranin Endospore is Clostridium stained by green green and Bacillus primary and Vegetative cell vegetative cell is pinkred Bacillus is stained by megaterium counter stain Bacillus sphaericus Acidfast Cells that are Carbol Heat Acid Methylene Acidfast or Myobacterium not acid fast fuschin Phenol alcohol blue myobacteri lose the pink not acid primary stain fast or non during mycobacterium decolorization blue egg albumin used to make the smear mycobacterium from non myobacterium Capsule Stains the Congo water Maneval s Background Azobacter background red stain Dark purple vinelandii and cell but red cells are polysacc leaves the reddish brown capsule capsule clear capsules are capsulated clear Bacillus from uncapsulated anthra cis p olyp ep t cap sule Answer the following questions based on the video How the Earth Was Madequot from the History Channel Who is considered to be the Father of Geology James Hutton Where is he from Scotland Who was Kelvin A British expert in thermodynamics believed earth was slowly cooling down Believed earth used to be completely molten 21 million yrs to cool to current temp but incorrect about Earth s age Unaware of radioactivity Deteremined age of earth by thermodynamics Who was Holmes Used radiation to revolutionize our understanding of earth history Uranium decayed to lead used this to calculate age radiometn39c dating Estimated age of earth by radiometn39c dating What is the scienti cally accepted age of Earth 45 billion yrs is accepted age of Earth What is pillowlava Lava found under water and Rounded pillow shape is result of lava solidifying under deep water What type of rock were the continents made of 9 Meteorite Contains 5 of water What are stromatdites Orqanism would live off sunlight and fill atmosphere with oxygen orqanism can still be found today Formed by very thin layers of 39 39 bacterial alqa 39 39 fossilized year waste product is rock that build on each other year by How is oxygen thought to have appeared in the atmosphere Reaction of stromatdites 02 cleared out C02 and diluted oceans why sky and water is blue When is life thought to have arisen 35 billion yrs Explain plate tectonicsquot Study of continental movement Driven by destruction of old and creation of new recycle of ocean crust As ocean plate moves so does continent Like conveyer belt move slowly over time What is the rate of continental drift 25 cm per yr 1 inchyr What is Rodinia A super continent Canada and USA formed the heart with other continents bunched around Lifeless place barren desertlike What is thought to have happened 700 million years ago Snowball Earth Rodinia blocked current of warm water going up to north froze surface temp 40 F degrees disasterwhole planet was dying man39ne organisms only lived sheet of ice covered earth 1 mile thick Alga trapped in earth under mile thick of ice maybe not alga but some microorganisms Heat and C02 from eruptions split Rodinia What is the Cambrian explosion High 02 levels life blooms animals seen like never before What is the importance of the Burgess shale quary Window of world most important fossils in World Charles Doolittle Walcott 8311909 discovered fossil Extracted 60 000 fossils and 100 000 have now been extracted 500 million yrs ago life exploded with diversity When is the ozone layer thought to have formed 400 million yrs ago Free life forms from ocean Acts as UV shield Chapter 6 Virus Structure and Function General Properties of Viruses virions o Obligate intracellular parasite made of nucleic acid surrounded by a protein coat 0 Only replicate inside host No nucleus organelles cytoplasm Viral genomes contain 0 information for taking over host cell 0 information for making viral proteins 0 Capsid genome replication proteins Virus Structure Capsids o Capsid protein coat surrounding nucleic acid 0 Capsid proteins are arranged to give virion symmetry I cosahedral capsids o Roughly spherical o 20 equilateral triangles or faces I Filamentous capsids M13 Ebola 0 Long tube of protein with genome inside 0 Tube made up of 100 s of identical protein subunits 0 Tube length reflects size of viral genome I Complex capsids o Mixture of icosahedral amp filamentous shapes 0 Many bacteriophage viruses that infect bacteria I Asymmetrical irregular shapes 0 Tend to be larger viruses 0 Poxviruses Virus Structure Envelopes o Envelope lipid bilayer around capsid of some viruses 0 Allows fusion to host cell membrane 0 Only occurs if host cell not covered by cell wall 0 Bacteriophages are NONenveloped o Envelope lipids come from host 0 Not encoded by viral genome 0 Proteins embedded in envelope may be encoded by virus 0 Coats viral capsid as virus leaves cell Virus Structure Genome 0 DNA or RNA 0 Includes genes encoding viral proteins 0 Capsid o Envelope proteins if virus is enveloped 0 Any polymerase not found in host cell International Committee on Taxonomy of Viruses classification system based on 1 Genome composition DNA or RNA Single or double stranded Linear or circular Whole or segmented 2 Capsid symmetry helical or icosahedral 3 Envelope Presence or absence of host derived envelope 4 Size of virus particle Related viruses are similar in size 5 Host range Related viruses infect same or related hosts Baltimore virus classification based on genome structure Group double stranded DNA herpesvirus group cold sores venereal disease chicken pox poxvirus group smallpox vaccinia cowpox remain latent in the body for years becoming active Group II single stranded DNA parvovirusred cheeks in kids Group III double stranded RNA Rotavirus member of reovirus family most common cause of infant diarrhea Group IV sense single stranded RNA RNA genome acts directly as mRNA for making proteins West Nile virus hepatitis C poliovirus rhinovirus colds Group V sense single stranded RNA genomic RNA does not serve directly as mRNA but is transcribed into a complement that functions as mRNA Often segmented Influenza rhabdovirus rabies Ebola measles Groule Retroviruses Lentiviruses such as HIV feline leukemia virus Enzyme reverse transcriptase copies RNA DNA integrates into like a temperatelysogenic virus Viral DNA is transcribed into mRNA amp RNA for new viruses New viruses are assembled and released Viral RNA AZT I reverse transcriptaseRT 1 Viral DNA Host cell DNA Retroviru 5 DNA provirus http 39 39 videohiv quot 39 and lifecvcle Group V Pa ra retroviruses Double stranded DNA genome which is copied in to RNA then reverse transcribed into progeny DNA Hepatitis B cauliflower mosaic virus Emmi 9 Hi9 1 Attachment to host cell receptor proteins 0 Receptors proteins with important functions for host cell 0 Examples sugar uptake iron uptake conjugation 0 Virus takes advantage of host proteins 2 Injects genome through cell wall to cytoplasm 0 Force of injection is very strong 3 Replicate genome lytic vs lysogenic phage o Lytic cycle T4 0 Phage quickly replicates kills host cell 0 Lysogenic cycle Lambda 0 LysogenicTemperate phage enter a state called lysogeny where the viral genome integrates into host chromosome and is replicated with host chromosome Don t immediately make new phage The virus is called a prophage when its genome exists as part of host genome 0 Induction of prophage gt lytic cycle gt host cell lysis Eventually new phage are made lytic cycle occurs amp cells lyse I http AIAInnrtoncomcolle ebiologym39 39 ainaspchnoch06a01 4 Use cell components to synthesize capsids 5 Assemble progeny phages 6 Exit from cell 0 Lysis 0 Makes protein to depolymerize peptidoglycan o Bursts host cell to release progeny phage 0 Slow release 0 Filamentous phages can extrude individual progeny through cell envelope Quantification methods Plaque assay 0 Used to count bacterial amp some animal viruses 0 When an infected cell lyses and releases phage those phage infect neighboring cells which causes them to lyse This cycle continues 0 This causes a clearing in a lawn of bacteria called a plaque 0 Each plaque results from infection by a single virus particle Viral Restriction amp Modification by the Host 0 Some viruses modify their DNA to avoid digestion by the host s restriction endonucleases o Methylation 0 Host cells have similar means of protecting their DNA from degradation by viral RE of by their own RE Animal Viruses 4 Types of infection by animal viruses 1 Lytic o Destruction of host cells 2 Persistent 0 New virions leave host by budding 0 Cell does not die but remains infected and will produce virions indefinitely 3 Latent 0 Virus is not actively replicating dormant 0 Symptoms appear only when virus emerges from latency 4 Transformation 0 Virus can change normal cell into cancer cell 0 Genetic changes that regulate growth 0 Benign or malignant tumors Eukaryotic Virus Life Cycles 0 Attachment to host cell receptor 0 Entry into cell 0 Injects genome directly 0 Taken up via endocytosis o Fuses envelope to plasma membrane then releases capsid into cytoplasm o Genome replication 0 DNA viruses must go to cell nucleus to use host polymerase or replicate in cytoplasm with viral polymerase If it is RNA virus or has its own polymerase 0 RNA viruses must encode a viral polymerase 0 Host cells cannot copy RNA into more RNA I All viruses make proteins with host ribosomes 0 Translation occurs in cytoplasm I Assembly of new viruses 0 Capsid and genome 0 Assembly may occur in cytoplasm or in nucleus Capsid proteins must move into nucleus Envelope proteins are inserted in host membrane 0 Release of progeny viruses from host cell 0 Lysis of cell 0 Similar to bacteria 0 Budding 0 Virus passes through membrane 0 Membrane lipids surround capsid to form envelope 0 All enveloped viruses bud from a membrane Culturing Viruses on Plates 0 Viruses grown with host cells as food 0 Bacteria in culture 0 Animal cells in tissue culture 0 Host cells form confluent lawn o Viruses form plaques where host cells killed 0 Plaque is a colony of viruses Viroids and Prions Viroids out capsid viruses without capsids M RNA with Prions Proteinonly infectious agent BSE mad cow scrapie sheep CreutzfeldJakob kuru humans chronic wasting disease deer elk Normal protein takes on abnormal shape loses normal function Viral Ecology Viruses present in all environments 0 Often at very high densities 107ml in ocean Viruses limit population host density 0 Half of all bacteria killed every day myxovirus in rabbits Viruses increase host diversity 0 Strong selection for virusresistant strains prevents dominance of 1 species People susceptible if exposed to new virus 0 West Nile Virus 0 Smallpox virus in New World Stephen Davis Biol 2051 Final Study Guide General The Final exam is worth 100 points Format 3 sections 1 Section A You will perform a test that is used to distinguish between different bacteria You have done this test at least once since midterm 2 Section B There will be a set of materials on the bench top in front of you that you will have to answer questions about This is similar in format to the midterm 3 Section C Short answer questions No microscope observations Covers experiments 2137 Look over all charts and tables in results and at the end of the experiments Review how you went about determining your unknowns Names of 39 to know tests to each from the others what is unique about each etc Name Details Staphylococcus aureus Gram cocci in clusters Exp 21 34 Catalase Part of normal microbiota Can be isolated from healthy individuals but is capable of causing severe illness especially nosocomially Can produce virulence factors and is considered a pathogen Produces a beta hemolysin and an enterotoxin responsible for gastroenteritis staph food poisoning Produces a toxin causing toxic shock syndrome Produces Coagulase causing plasma to clot Catalase fibrin clot helps organism protect itself Common infections are styes pimples boils and wound infections Responsible for high of nosocomial infections Ferments mannitol Salt tolerant Yellow on MSA plate Staphylococcus epidermidis Gram staphylococci Exp 21 25 34 35 Catalase Found on most skin and produces few virulence factors Can cause disease if given the opportunity for entry to the body Frequent cause of nosocomial infections following cardiac surgery Facultative anaerobe Pale on MSA plate Does not ferment mannitol Does not produce coagulase Commensalistic relationship with Clostridium sporogenes Salt tolerant Glucose growth acid production no gas production Sucrose growth acid production no gas production Lactose growth acid production no gas production Micrococcus Catalase Exp 21 Use oxygen for respiration Lactobacilus Gram Exp 21 31 Catalase Produce lactic acid in fermentation form sugars and is used commercially to produce yogurt and cottage cheese from mi Aerotolerant anaerobes can grow in the presence of oxygen but don t use it for respiration The colony morphology on MRS agar is irregular filamentous flat opaque shiny and white Grows with pH lt 60 Lactococcusactis Exp 25 Produce lactic acid ram Homofermentative metabolism Important for manufacturing of dairy products Saccharomyces cerevisiae Exp 25 Yeast that produces carbon dioxide and ethyl alcohol from sugar beerwine Bacillus cereus Exp 25 Glucose growth acid production no gas production Lactose growth no acid production no gas production Sucrose growth acid production no gas production Streptococcus mutans Exp 21 30 Catalase Gram normal microbiota of humans usually in chains In the mucous membranes and the intestine of humans Some are pathogens which causes strep throat scarlet fever and several other serious illnesses Facultative anaerobe Produces lactic acid in Fermentation from sugars and is used commercially to produce yogurt and cottage cheese from milk Produces alpha hemolysin partial clearing Streptococcus pyogenes Exp 21 30 Catalase Gram usually in chains In the mucous membranes and the intestine of humans Some are pathogens which causes strep throat scarlet fever and several other serious illnesses Produces a beta hemolysinwhich causes complete destruction of red blood cells Enterococcus Catalase Exp 21 Pseudomonas putida Oxidase Exp 22 Escherichia coli Oxidase Exp 22 23 24 25 26 27 28 Amalyse 33 Catalase Gelatinase Cysteine desulfhydride Indicator of fecal contamination Positive for indole and methyl red in lMVic Carbohydrate results Glucose no growth acid production and gas production Lactose no growth acid production and gas production Sucrose growth no acid production no gas production lMVic Results lndole MR VP Citrate NOZ39 present so it reduced N03 Bacillus subtilis Exp 23 24 Amalyse Makes a yelloworange zone of hydrolysis when iodine is added Caseinase Makes a clear transparent area around colony Gelatinase Makes a clear transparent area around colony when acid is added rest of the agar is cloudy Cytophagahutchinsonii Isolated originally from sugarcane piles Exp 23 Breaks down the cellulosecontaining sugarcane to use the simiple sugars as carbon and energy sources Degrades cellulose aerobically Citrobacterfreundii Cysteine desulfhyrase Exp 25 26 Black precipitate FeS formed with cysteine desulfhydrase enzyme Glucose growth acid production gas production Sucrose growth acid production gas production Lactose growth acid production gas production Enterobacteraerogenes Exp 27 Can be found in feces but may also be present in the environment and in decaying vegetation that has not been contaminated by feces Fish eyes on EMB agar lMViC results lndole MR VP Citrate Butanediol fermentation Production of acetyl methyl carbinol Paracoccusdenitrificans Exp 28 Used to test for nitrate reduction and proved to reduce nitrate to N2 and N20 by making gas in the Durham tube after incubated it did not turn red after Zn was added Azotobactervineandii Exp 29 Fix nitrogen aerobically Uses a third protein to reversibly complex to the nitrogenase enzyme complex to conformationally protect it from oxygen inactivation Produces an Ozretarding alginate capsule Can readily use mannitol as a carbon source Aerobic Gramnegative encapsulated bacillusshaped cells Have cysts which are ovoidshaped dormant droughtresistant structures produced Yellowgreen fluorescent pigment under UV Clostridiurn sporogenes Exp 35 Gram obligate anaerobe so it must be grown in an oxygen free medium thioglycollate broth Forms endospores Commensalistic relationship with Staphylococcus epidermis facultative anaerobe this organism removes the 02 from the environment so it can grow Media to knowselective ingredients differential ingredients test it was used for reagents to add organisms to know significant ingredients what results look like Medium Details Trypticase Soy Agar TSA xp 22 ma 3 nutritious medium for many bacteria starch agar Exp 22 For amylase test Amylase breaks down starch bonds yelloworange zone of hydrolysis around amylase producing organism Starch a macromolecule composed of glucose molecules connected by alpha 14 glycosidic bonds Dubos agarbroth Exp 23 Gelatin agar Hydrolysis of proteins Exp 24 Gelatinase production Addition of acid to the plate results is precipitation of the gelatin proteins causing a faint white precipitate clear transparent area around gelatinase producing organism Gelatin extracted from the connective tissue of bones of animals milk agar Hydrolysis of proteins Exp 24 Used for evidence of caseinase production clearing around caseinaseproducing colony Casein is the main protein of milk which gives it its opaque nature fermentation tubes with phenol red Exp 25 37 Phenol red is the ph indicator in nutrient broths containing non fermentable nutrients to support growth When neutral broths it is orangepink f acid present turns bright yellow Carbohydrate broth results Gas production in Durham tubes are present when fermentation occurs nitrate broth Exp 28 Tests for the reduction of nitrate Gas production shows nitrate reduction Nfree brothagar Exp 29 peptone iron agar Exp 26 37 Effective medium for demonstrating the production of hydrogen sulfide Medium contains peptone a digest of animal proteins that is rich in cysteine and ferric citrate which serves as the indicator Produces a black insoluble precipitate hydrogen sulfide reacts with FeS tryptone broth Used in the indole test Exp 27 37 Tryptophanaseenzyme breaks down the tryptophan to make indole MRVP broth Methyl Red and VogesProskauer glucose is the main ingredient Exp 27 37 For mixed acid fermentation and butanediol fermentation Red is positive and yellow is neutral Simmon s citrate agar Exp 27 37 Medium used to determine whether an organism can utilize citrate as its sole carbon source Turn blue as a result alkaline products are formed rise in pH green at neutrality blood agar Exp 30 A nutritious medium to which 5 sterile sheep blood has been added after the sterile medium has been tempered cooled to just above the temperature at which agar solidifies Demonstrates hemolysin production lf organism produces a hemolysin it causes hemolysis d hemolysis green darkening 3 hemolysis clearin v hemolysis no clearing no darkening MRS broth and agar Exp 31 Used to isolateLactobacilusfrom yogurt Selective due to low pH mannitol salt agar Exp 34 Developed for isolating staphylococci from food samples Contains 5 NaCl which inhibits the growth of many microorganisms and makes it selective for those that tolerate this concentration of salt Selective as well as differential in that it contains a carbohydrate mannitol and the pH indicator phenol red Neutral pH is orangered Low pH Bright redyellow is positive Alkaline pH it turns fuchsia pink negative Plasma Exp 34 The liquid portion of anticoagulated blood and contains clotting factors One clotting factor is fibrinogen which is present in plasma as a soluble protein eosin methylene blue agar Exp 37 17 Selective and differential Contains dyes which inhibit the growth of Gram bacteria and select for Gram bacteria also contains lactose as a differential component lactose fermentors vs non lactose fermentors Non lactose fermenters are colorless on this and coliforms G produce darkened colonies E coli metallic green and Enterobacteraerogenes fisheye phenyl ethyl alcohol agar Exp 37 17 Selective only for 6 organisms ENZYME SUBSTRATE PRODUCT REAGENT RESULT MEDIA ORGANISM INDICATOR Catalase Hydrogen Water H20 and Hydrogen Production of None Staphylococcus peroxide molecular oxygen peroxide bubbles OJ epidermidls H202 Oszubbles H202 Oxidase Oxygen Water Oxidase reagent Turns TSA used but Pseudomonas putida Ex tetramethylp purpleblue nothing specific Cytochrome phenylenediamine where reagent oxidase dihydrochloride is added Amylase Glycosidic bonds Oligosaccharides useiodine to make if starch is Starch agar Bacillus subtillus between Glucse of glucose a brown blue plate subunits in starch color yelloworange hydrolysis is present Cellulase Cellulose Oligosaccharides None Growth of Dubos agar of glucose yellow pigment platebroth Cytophagahutchinsonii on paper or paper degradation Caseinase Casein Peptides None A clear area is Milk agar plate Bacillus subtillus evident around and under the caseinase producing colony Gelatinase Gelatin Peptides Hydrochloric acid Clear Gelatin agar Bacillus subtillus transparent plate Cysteine Cysteine amino Hydrogen sulfide Ferric citrate Black precipitate Deeps of Citrobacterfreundii desul 1ydrase acid w sul 1ydryl H2S FeS peptone iron group Ferrous sul de agar inoculated edle Tryptophanase Tryptophan lndole Kovac s Red layer on Tryptone broth Escherichia coli reagent top of broth Citrate Citrate carbon Alkaline products Bromthymol blue Blue when pH Simmon sCitrate permease built into agar goes up Agar Slant Enterobacteraerogenes slantalkaline indicator Nitrate Noa39 nitrate NOg nitrite umaphthylamine Dense deep Nitrate Broth Escherichia coli reductase acetate Red color sulfanilic acid Nitrite NO 2 and N2 None Gas bubbles in Nitrate Broth reductase nitrogen gas durham tube Paracoccusdenilrificans Nitrogenase Dinitrogen N2 Ammonia NHa Ability to grow in Nfree Azotobactervinelandii Nune nitrogenfree brothagar brothagar Coagulase Fibrinogen Fibrin None Fibrin clot Plasma Staphylococcus plasma Solidi cation of plasma thickening Methyl Red Glucose Mixed acid Methyl Red Broth turns red Glucose MRVP Escherichia coli broth Vogues Glucose Butanediol Alpha naphthol Red not instant Glucose MRVP Enterobacter Proskaur acetoin and 40 falls from broth Potassium oxygen Hydroxide interface Exp 21 Catalase Production Metabolism the sum of the reactions that are associated with the life of a cell Enzymes proteins that are biological catalysts can only react with a specific substrate they are mandatory for living cells to exist because without them reactions wouldn t go fast enough can catalyze only one reaction Extracellular enzymes enzymes that act outside of the cell in which they are produced they degrade large molecules into units that are small enough to be transported across the cell membrane into the cell where they are useful Endoenzymes act inside the cell to degrade molecules to simpler molecule releasing energy or synthesizing molecules that are needed by the cell Inducibleadaptive enzymes produced only if the 39 substrate is available39 this is an energy conserving mechanism Constitutive enzymes they are essential to the cell and are produced at all times Macromolecules large molecules composed of a number of subunits generallyjoined by dehydration synthesis may also be degraded into the constituent subunits reactions are reversible and each is catalyzed by a specific enzyme Ph iology of organism an organisms metabolic capabilities and how it functions in the environment which includes an examination ofthe enzymes it produces and how it reacts biochemically this is what is looked at as opposed to cell morphology Catalase enzyme that breaks H202 into water and molecular oxygen H202 is toxic but is a product of aerobic respiration and for organisms to survive aerobically they must be able to detoxify it oxygen is seen by production of bubbles H2O2 is the substrate in testing for production of molecular oxygen produced by essentially all organisms that use 02 for respiration lactic acid bacteria used in the production of fermented dairy products utilize only fermentative metabolism and don t produce catalase Hydrogen Peroxide a byproduct of aerobic metabolism is toxic so it must be broken down Catalase Positive Catalase Neqative Staphylococcus Streptococcus M icrococcusLactococcus Enterococcus Questions What is the reagent that serves as the substrate in the Catalase test Hydrogen Peroxide What type of metabolism is associated with catalase production Respiration organisms use oxygen for respiration aerobic metabolism For what product is one testing when using the catalase test Oxygen Would you expect anaerobes to produce catalase No because they do not use oxygen to respirate so they would not need catalase to break down hydrogen peroxide Describe appearance of positive and negative test Look at picture has bubblesgas production and does not have bubblesgas production What does a positive or negative test indicate about the metabolism ofthe cells tested Positive test indicates that the cells produce catalase and therefore use oxygen for respiration Negative test indicates that the cell does not produce catalase and uses fermentative metabolism If your catalase test was positive to what 2 possible genera could the microorganism belong Staphylococcus or Micrococcus What is the equation catalyzed by the enzyme catalase 2H2O29 2H2O 0 Would you expect your cells to produce catalase Explain your answer Yes I would expect my cells to produce catalase because my cells aerobically respirate 9amp88 9 95 Procedure Add drop of H202 to each side of a glass slideMix loop of Streptococcus mutans with one drop and Staphylococcus epidermidis with the other drop look for bubbles oxygen which indicates positive catalase test result Experiment 22 Oxidase Production quot2 e l I r t W cytochrome oxidase is used to differentiate between differe p 394 t t t 4 t t Proced 1 A 5 8 8 Exp 23 The Hvdrolvsis of J to understand its function this is used because nt genera of bacteria rele am genera from 39 39 like o r Salmonella oxidase Vibrio and ne ative o Neisseria and Moraxe a oxidase positive from AcinetobacteKoxidase negative Oxidase enzymes are made by cells that do respira ion Cytochrome oxidase transfers electrons to 02 to form H20 or H20 as the nal electron transfer in electron transport of aerobic respiration There are different types of cyt oxidases ofwhich cyt c oxidase is one ure Use your inoculating loop inoculate plate with a line of Pseudomonas putida and a line of Escherichia coli on separate sides of the late A er incubating transfer oxidase reagent to line of each bacteria Positive result is indicated bythe organism that produces cytochrome oxidase which Is the organism that turns bluepurple but if exposed to air too long Questions What is the function of cytochrome oxidase Catalyzes the oxidation of a cytochrome a member ofthe m reduction of molecular oxygen to either H20 or H20 HZOZ g cata ases and idases to remove this byproduct Aerobes facultative anaerobes and microaerophiles can be identi ed using the oxidase enzyme activity Why are strict aerobes oxidase positive Because oxidase enzymes function in aerobic electron transport systems organisms that use oxygen can be indenti ed using the oxidase enzyme activit The oxidase test is used to differentiate among which groups of bacteria embrane electron transport chain and the subsequent t t u L a Used to differentiate Neisseria and Moraxella oxidase from Acinetobac teKoxidase Pseudomonads and Vibrionaceae oxidase from Enterobacteriaceae oxidase What is the function of the test reagent in this procedure Tetramethylp phenylenediaminedihydrochloride can be used to test an organism s ability to produce cytochrome oxidase Serves as an arti cial substrate to be oxidized by cytochrome oxidase in the presence of oxygen Ec Amvlase and Cellulase Carbohydrates compounds composed of carbon hydrogen and oxygen in a 121 ratio CH20n is the formula for a carbohydrate indicating there are twice as many hydrogen atoms as there are carbon and oxygen atoms Monosaccharides small carbohydrates which are simple sugars usually with ve or six carbons Ex ribose glucose Oligosaccharides few sugars two or more monosaccharidesEx maltose Polysaccharides larger polymers of monosaccharides serving as structural components or as important reserve carbon and energy sources Ex cellulose glycogen Cellulose structural polysaccharide in plants and is a carbohydrate polymer composed of glucose unitsjoined few mamma s can digest it Cellulose is found in eukaryotic plants and algae cell walls Cellulose decomposition is part of the natural cycling of carbon Breakdown of cellulose by Bacteria can occur aerobically by members of Cytophaga and Sporocytophaga and anaerobically by members of Clostridium Cellulase enzyme that hydrolyzes cellulose into glucose to be transported into the cell for metabolism fungi and bacteria in soil and rotting wood break down cellulose and produce cellulases Glycogen reserve energy polysaccharide of animal cells and human cells and is a polymer of glucose Starch macromolecule of glucose produced by plants serving as a reserve food source 0 Can be dissolved into a liquid growth medium into which agar is added Amylase extracellular enzyme that can break the glycosidic bonds between glucose subunits in starch o rolyses starch into the smaller subunits that can then be transported into the bacterial cell to provide energy or cellular componen s Microbially produced amylases have commercial and industrial uses food fermentation brewing distilling textiles paper e c enzyme that hydrolyzes starch into glucose to be transported into a cell for metabolism Iodine added to a plated medium to make starch evident because the iodine reacts with the starch to produce a brownblue color lfa bacteria that produce amylase is inoculated and incubated a yelloworange zone of hydrolysis will be evident around the colony a er iodine is a de 0 Starch agar plate is used to perform this test Zone onydrolysis clear area that will be evident around an amylaseproducing colony after the plate is ooded with iodine Procedure 1 To test for microbes ability to produce amylase Inoculate organism onto starch agar nutrient agar plus starch and incubate at room emp a DAY 2 1Food plates with iodine 2odine starch dark color 3ook for zone of hydrolysis around organism which is amylase positive 2 To test for microbes ability to produce cellulase Group A right side 10btain a Dubos agar plate Label with name and section 2nocuate Dubos agar plate with 4 drops 20uL of Cytophagahufchinsonii evenly spaced around a 5m drop In the middle control 3Using sterile forceps to place 4 different pieces of paper on top of the drops 1 on each drop leave entral drop without paper Gently press down on papers to ensure good contact with the bacteria er re Which strips of paper Will Cytophagabe able to degrade 62 labs aterLook at your Dubos agar plate 7s there bacterial growth on any of the strips ofpaper 8Are any of the strips of paper degraded 9Are the strips of paper on the control plate still intact b Group B left side 10btain 5 tubes of Dubos broth one of each cap color 4 of which each have a different piece ofpaper Label the to of each cap section bench only so you can identify yourtubes next wee 2Using the micropipettors inoculate each lube with 100uL of Cytophagahutchinsoniibroth You do not need use a new pipet each time 3 Place yourtubes in the rack on the cart The tubes will be ta en to a room temperature shaker berause Cytophaga needs aeration to degrade cellulose aerobically 4Which strips of paper will Cytophagabe able to degrade gamimm 52 labs later Look at your Dubos broths 6s here bacterial growth at the liquidair interface 7Has the strip of paper been sheared by the bacteria 8 Does the control tube look clear not turbid Questions 1 Which organism produced amylase Bacillus subfilis
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