GEN MICROBIOLOGY BIOL 2051
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This 109 page Class Notes was uploaded by Charles Kohler on Tuesday October 13, 2015. The Class Notes belongs to BIOL 2051 at Louisiana State University taught by G. Brininstool in Fall. Since its upload, it has received 16 views. For similar materials see /class/222790/biol-2051-louisiana-state-university in Biological Sciences at Louisiana State University.
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Date Created: 10/13/15
0 Whatever the cell needs it will take it in It goes through some processes known as catabolic processes to break it down 0 Metabolism 0 Sum of all Catabolic and Anabolism reactions I Catabolism 0 Breaking down the Molecules 0 Energy producing source organic and inorganic compounds light 0 Energy from Catabolism I Used for biosynthesis I Used for motility transport of nutrients 0 Waste products I Fermentation products acids alcohols gases reduced electron acceptors I Anabolism o Biosynthetic energy using 0 Energy used in catbolic reaction can be used in anabolic reaction to make new molecules 0 Building molecules which require energy 0 Macromolecules and other cell components 0 Oxidation Reduction Reactions Redox Reactions 0 Oxidation I Loss of electrons less negative 0 Reduction I Gains electrons more negative 0 LEO Loss of electron oxidation the Lion goes GER gains electrons reduction 0 Redox reactions are coupled I Electron donor transfers electrons to acceptor I Donor is oxidized and acceptor is reduced 0 Electron Carriers 0 Help transfer electrons from donor to acceptor o Membrane bound 0 Free diffusible coenzymes NAD NADH o EXAMPLES OF REDOX 0 Reaction One I An enzyme reacts with substrate electron donor and oxidized form of coenzyme NAD I Dehydrogenase is an enzyme that oxidizes a substrate by transferring a pair of electrons to an acceptor I NAD is wating to receive an electron to become NADH I Enzyme binds to the substrate and substrate donates an electron to NAD to reduce it to make NADH I Result NADH received an electron 0 Reaction Two I Need reduced form of NADH as its substrate I NADH is happy to give substrate an electron o Substrate is being reduced since its gaining an electron I NADH gives electron to substrate Catabolism I Energy is released from redox reaction I Energy is stored in molecules and transported where needed I Energy storage compounds 0 Short term I Adenosine Triphosphate ATP 0 2 phosphoanhydride bonds high energy phosphate bonds 0 Primary energy carrier in the cell 0 NO ATP would be made if redox reaction wouldn t occur I Derivatives of coenzyme A thioester bonds 0 Long Term I Pack energy also but might not be as obvious that they do 0 Glycogen if a cell stores glycogen It can break glucose off and feed it right into glycolysis to form ATP PolyBhydroxybutrate Elemental Sulfer some bacteria store it in the periplasmreduce it at the end of ETC if cell can use it at end of ETC then it can get energy from the cell I Energy comes from breaking bonds ofphosphate and oxygen 0 When you break one of those bonds a lot of energy is released enough energy to form a covalent bond Energy and Electrons 0 Energy and Electrons o Chemoorganotroph I Source Organic Compound I Carbon based molecule 0 Glucose lactosesugars 0 As long as its carbon based the odds are good that a molecule can use it as an energy source I Process fermentation aerobic respiration or anaerobic respiration o Chemolitthotroph I Source Inorganic Compound 0 Hydrogen Sulfide o Sulfate o Nitrateite I Process Anerobic Respiration Types of Phosphoralation o Substrate level phosphorylation o A Phosphate group PO4 3 is removed from a substrate and added to ADP to make ATP 0 In fermentation ATP is produced by this substrate level phosphoralation Referring to the fact that there is a substrate that s carbon based ad has a phosphate group that it is willing to give up 0 Phosphate group on energy rich organic intermediate is transferred directly to ADP to produce ATP 0 Oxidative Phosphorylation 0 Inorganic phosphate free in the cytoplasm is added onto ADP to make ATP by ATP synthase 0 ATP is produced at the expense of the proton motive force 0 No substrate involved 0 Fermentation vs Respiration 0 Both use energy released from redox reaction to make ATP 0 Electron acceptor present Respiration o No exogenous electron acceptorFermenatation o Fermentation I Anaerobic means of generating ATP I Organic compounds serve as electron acceptors and donors I Substrate level phosphoralation I An example of fermentation is Glycolysis o Glycolysis 0 Major pathway of glucose metabolism Converting glucose to pruvate Organisms that do respiration do glycolysis 2 ATP and 2 pruvate generate per glucose Occurs in the cytoplasm of cells capable of using glucose 0 If glucose if fermented its broken down to pruvate by gloycolysis which generates a net 2ATP and then pruvate is converted into various molecules to regenerate NAD 0 Molecule of Glucose o 6 carbons pruvate has 3 carbons I Lactic Acid Bacteria Fermentation of Glucose o Homofermentative 2 pruvate converted to lactate O O O O o Lactate product 0 Produce only lactate acid 0 Gram catalase negative Streptococcus pathogenic and Lactococcus salt and elevated temp tolerant o Heterofermentative o Lactate and other products like ethanols C02 0 Gram catalase negative Lactobaccilus yogurt and Leuconostoc buttery avor I Summary of Fermentation 0 From Glycolosis o Oxidation of glucose to pruvate 2 ATP by substrate level phosphoralation O 0 Reduction of Pyruvate o Regenerates NAD o Fermentation products 0 Syntrophy cooperative relationship in which the microorganisms work together to degrade a compound 0 Syntrophomas can ferment butyrate to acetate for energy if a methanogen is present to use the H2 generated What is Microbiology The study of cells and organisms too small to be seen with the naked eye Microbiologists tend to study 0 Bacteria Viruses Protozoans Fungi Algae Diatoms And much more A lot of microorganisms are eukaryotes 0 Note Bacteria is prokaryotic ALL prokaryotes are microorganisms and a HUGE percentage of eukaryotes are microorganisms o A lot of fungi are microbes 00000 0 Diversity There are three domains of life BACTERIA ARCHAEA and EUKARYA Prokaryotes o Bacteria I Grampositive bacteria I Proteobacteria I Mitochondrion I Cyanobacteria I Chloroplast I Hyperthermophiles o Archaea I Methanogens I Hyperthermophiles I Extreme Halophiles Eukaryotes o Eukarya I Giardia I Flagellates I Slime Molds I Animals 0 Eukaryotic 39Crown Species I Fungi o A lot are microbes I Plants Characteristics Both single cell and multicellular Multicellular o Algae fungi more advanced eukarya domain Notes Characteristics of life include Metabolism reproduction differentiation communication movement evolution There are 5x1030 bacteria on earth The earth is completely covered in microbes We have more microbes on our body than our body has cells Microorganisms are important in the agricultural industry For example legumes which live in close association with bacteria that form structures called nodules on their roots convert atmospheric nitrogen into fixed nitrogen that the plants use for growth The activities of the bacteria reduce the need for costly and polluting plant fertilizer Microorganisms are important in energy production including the production of methane natural gas energy stored in organisms biomass and ethanol Various microorganisms can be used to consume spilled oil solvents pesticides and other environmentally toxic pollutants Control ofinfectious diseases with improved sanitation and the discovery and use of antimicrobial agents Pathogens are evolving to survive in the presence of our antimicrobial agents Drug resistant microbes are becoming more common Foods that benefit from the effects of microorganisms include cheese yogurt buttermilk sauerkraut pickles sausages baked goods and alcoholic beverages Microorganisms also play important roles in the food industry both harmful and beneficial Because food fit for human consumption can support the growth of many microorganisms it must be properly prepared and monitored to avoid transmission of disease Biotechnology is the use of microorganisms in industrial biosynthesis typically by microorganisms that have been genetically modified to synthesize products of high commercial value Important People in Microbiology Robert Hooke was the first to describe microorganisms in 1665 0 He was able to magnify the cells that he was looking at in a way he designed the first microscope 0 He was the first to describe microorganisms using a microscope I He was looking at fungi In 1676 Antoni van Leeuwenhoek was the first to describe bacteria It was most likely microbes bacteria from his cheek or oor I Most microbes are very small yet you also have some very big ones I He would not have been able to see small microbes with the equipment he was using 0 He looked at staphylococci I Round shaped cell grouped together 0 Note A lot of microorganisms are agellated I e ning they move Li ht Mircrosco 4t es 1 Brightfield a Simplest form b The specimen is illuminated and magnified 1000x c Specimen is stained to increase contrast i Bacteria cells are made mostly ofwater 70 so we stain the bacteria but most stains kill the specimen so they no longer will move if agellated Note When you are using 10x objective it is 100x total magni cation Hence 1 0 0x objective 10 0 0x total magnification MJ Midigan M1Madigan MJ Madigan Bright eld Phase Contrast Dark eld 5quot Phase Contrast a Visualize live samples b No staining required c Image contrast derived from cell structures d Casts a shadowiyou can see the outline ofthe cell Darkfield a Same as phase constrast b Background is completely black and the specimen is illuminatedi only certain light is let through 5 Note Both Phase Contrast and Dark eld have there advantages because you don39t have to kill the cells by staining them Fluorescent q a 39 of molecule nnre rent tain b You hit the sample with the right wavelength of light and the specimen will turn a certain color c Used with specimens with special pigments i You can also dye the specimen 1 DAPI is the most common this type ofdying is used to count cells in the specimen If you are using DAPI you are hitting it with UVikills the cell 5quot Compound Light Microscopes Types 0 Brightfield 0 Phase Contrast o Darkfield o Fluorescent They optimize image RESOLUTION by using lenses with high lightgathering characteristics The limit of resolution is 2 microns o The smallest living cell that we know ofis 2 microns I EColi is very small 10 X 2 microns so we stain it with crystal violet to be able to see it more clearly Electron Microscopes Limit of resolution is 2 to 4 nm Two types of electron microscopy 0 Transmission Electron Microscope TEM I Used for observing internal cell structure I 2 resolution usually used for TEMs 0 Scanning Electron Microscope I Used for 3Dimensional imaging and viewing surfaces Note The main difference between the two scanning and transmission is the transmission image gives you details You have to section cut open very tedious process material that way you can see the inside of the cell The scanning image gives you surface information only Note In the TEM process when the cell divides the DNAis the first thing to get pushed towards the outside Electron microscopes can magnify much greater Leparsy Mycrobacterium leprae Carried by armadillos Summary of Notes SizeZStructure Microorganisms are as small as 2 microns Size affects physiology growth rate and ecology 2 microns is the size of the smallest freeliving pathogenic bacterium Mycoplasma pneumoniae 750 microns is the largest prokaryote 9 Thiomargarita namibiensis It is a spherical sulfur chemolithotroph High surface areatovolume ratio aids in nutrient and waste exchange with the environment 0 Ifa cells radius is 1 micron then its surfacevolume 3 o Ifa cells radius is 2 microns then its surfacevolume 15 If something is made in the middle of the cell it doesn t have to travel that far to get out of cell if the cell is small If the cell needs something to produce then what it needs doesn t have to travel as far either It is good to be small because it has quicker access to nutrients and also gets rid of waste quicker If something is small it can fit into all sort of different spaces Nanobacteria Major question Is it possible to contail all essential molecules oflife within a volume ofa cell less than 2 microns o 2 microns smallest free living bacterium I Mycoplasma pneumoniae very small and takes on many forms and shapes When you separate them from their host you can not get them to grow on their own 0 They have nutrient requirements that we don t know yet therefore we do not know how to get them to grow on their own 0 We need to use an electron microscope ad go through the process of figuring out how to culture microorganisms one day there might be such thing as nanobacteria Is it just coincidence that 2 microns is the maX magnification for a compound microscope Structure Basic components of all microbial cells 0 Cytoplasm o Cytoplasmic membrane I Liquid aqueous base I Ribosome I DNA 0 Nucleic Acid 0 Things get made from DNA such as RNA 0 RNA is also in the cell There are two structural types of cells that are recognized o Prokaryote I Makes up largest percentage I Bacteria and Archaea o Eukaryote I Eukarya 0 Animals 0 Fungi 0 Plants Prokaryotic Cell Made up of o Cytoplasm Nucleoid space not a membrane enclosed structure The SPACE in the cell where the DNA is found It is a locationspace NOT A MEMBRANE Ribosomes Plasmid allows bacterial cells to survive the affects of antibiotics Helps resist against antibiotics Cytoplasmic Membrane I Also called the cell membrane Cell wall 5 micrometers I Not only plant cells have cell walls bacterial cells also have cell walls Eukaryotic Cell All eukaryotic cells have the same structural components They a O O O 0000 000 O Typica re both plant and animal cells Plant cells have chloroplasts 9 how we tell the difference Eukaryotic cells have a Nucleus This is where DNA is DNA makes RNA hence RNA is in the nucleus also Sometimes RNA gets out and goes into rough ER Components of the cell include Cytoplasmic Membrane Endoplasmic Reticulum I Protein made here Ribosomes Nucleus Nucleolus Nuclear Membrane I Nuclear envelope forms out to the rough ER which is where protein is made from there protein goes straight to golgi Golgi I Tell the cell where the enzymes are and then tells the protein where to go The messenger system Cytoplasm Mitochondrion Chloroplasts I Plant cellssome photosynthetic cells Cell Wall 10 micrometers lly a eukaryotic cell is HUGE Note The biggest prokaryotic cells are bigger than the smallest eukaryotic cells The range is sizes is broad Note A virus is not living lacks characteristics ofliving cells Structures of the Cell Cytoplasmic Membrane for bacterial cell Highly selective permeability barrieri uid Made up of lipids and proteins The barrier between the cell and outside world Protein in cytoplasm are used to transportthings to and from the cell 0 Some are receptors used for sense9 senses oxygen sugar light etc o Relays information in the cel Sometimes there are enzymes embedded in cytoplasmic membrane The membrane is a phospholipid bilayeriit peals when it gets too hot 0 Heating it up and peeling makes it wea Lipids in cytoplasmic membrane 0 Phospholipids o Sterols I In eukarya o Hopanoids I In bacteria u g 7 r 1 mm 95quot I 39 K 39 Phospholipids in cytoplasmic membrane a lipid bilayer with polar exteriors and nonpolar interiors Ex Ester linkage of fatty acids to glycerol 0 If this structure is heated it will peel it wont break longitudinally 0 Fatty acids are connectedto a glycerol backbone Nonpolar Hydrophobic likes oil Polar9 Hydrophilic loves water Not hard to break hydrophobic interactions 0 Ex slap the top of oil and water to mix it together Hydraphilic r region 39 Hydrophobic region Hydrophilic region 39 39 Note llypothermophiles need it to be hot to survive but still has other structures in order to prevent peeling and to support the structure Archaea Lipid monolayer More resistantto peeling apart than bilayers There are no gaps 9 hence no fatty acids Nolipidbilayer The double bond to oxygen is lacking9 etherlinkage llas all covalent bonds 0 Takes a lot to break it apart Many archaea are thermophiles or hypothermophiles 0 Warm loving microbes 0 Have the structure to let them live in warm environments Function ofthe Cytoplasmic Membrane for archaeaic cell H d D a E a 5 mg 7 cr D a E D vents leakage and functions as a getaway for transport or nutrients into and out of cell The embedded protein allows for the transportation to and from This is where the ATP is made inbacterial prokaryotic cells 0 Because it doesn39thave mitochondria to make it in eukaryotic cells This causes charge difference between cells 2 Protein Anchor site of many proteins involved in transport bioe nergetics and chemotaXis 3 Energy Conservation site ofgeneration and sue ofthe protein motive force I 39 suu N f I 2 2 2 on Biphytanyl Note The main difference betweenbacteria and archaea is the one different sugar DON39T NEED TO KNOW THE DIFFERENT CELL WALLSlll Eukaryotic Cell Structures Can contain several membrane enclosed organelles o Nucleus I Has holes pores 0 Join the outer and inner nuclear membranes to allow for import and export ofproteins and nucleic acids I Contains the nucleolus 0 RNA made here I DNA cannot pass through but RNA can pass through since it is single stranded nucleotide I Outer layer forms to allow for rough ER to attach o Endoplasmic reticulum I Rough 0 Continuous with nuclear outer membrane 0 Ribosomes are there to do one thing only make protein site ofprotein synthesis 0 Ribosomes are from RNA 0 Makes glycoproteins and new membrane material I Smooth 0 Continuous with rough ER but has no ribosome s o No translation occurs here 0 Lipid synthesis occurs here along with carbohydrate metabolism 0 Golgi complex Chemically modifies carbohydrates on ER glycoproteins Looks and molecules and decides where it should go gives molecule a sequence or label sometimes may put molecule in a vesicle 0 Not all vesicles are equal some are called lysosomes Takes proteins from rough ER 0 Sorts molecules form the ER that are secreted from the cell and used within the cell 0 Lysosome I They are vesicles produced by budding of the golgi complex I Enzymes are part of these vesicles 0 Lipases o Nucleases o Proteases I pH is low here because enzymes have specific pHs at which they work best 0 They work best at a pH of 5 0 Cells have a pH of7 0 So ifa lysosome is opened in the cell it would be digested by all the enzymes worst case scenario o ReausucauywhenanmzymmamesapIIm me cellw ltrytu gemduflt Enzymesaresaferinmeveside lysusume Recydemacmmulecu Theyarevm39ylmpmantfunmmunemspunse o K111 fumgn baaena gt apupmsls call made Fusion arms secundary o Pam same I Slteufmdauun mdasemzymesbrmdawnhpxdsandmer muleculesfurenergy Betaro dadunufhpids an down a Zrcarbun acetyl unit atadme accursmpemmume yeruAmentranspumdmmcymsul Cuntams me mzymes mm andsupmxlde msmucase EamhelpdegadeIIz zand z These enzymes take cam uftuxlc molecules 0 Mltumundmn urh dm musume im dmdmn comes mmyummamermmtmmme nudearmembrane I Milnchnnrlrinn IS campused cf Oule mmne gdandsumewhatpm39meable mall urgamcmulecules andmns can pass uugh lnnerMemDrzne 0 Where a ncakesplace o Moremembrane9MumATPpmdudmn o Lessngdandfulded o Lesspermeab Transpmmruughspm cpmmns o sueufalecmmcrmspmandATPpmdumun mm o The verymiddle Ime mitud39mndrmn NO CYTOPLASM o NO ATP made here all made in inner membrane space 0 O Aqueous Contains enzymes of citric acid cycle 0 I Hydmgenosome replaces some mitochondrion in some ANAERO BIC eukaryotes O rganis ms that have hydrogenosomes DO NOT have Mitochondrionll o No electron transport 0 Site ofATP production but no electron transport 9 ATP made byprocess of fermentation No citric acid cycle 0 Very simple structure 0 Chloroplast I Found in phototrophic eukaryotes I Surrounded by double membrane Stroma o Aqueous inside of chloroplast 0 Has grana oating inside I Stacks of flattened membrane discs I Contains light harvesting pigments 9 chlorophyll I Site ofphotosynthetic electron transport ATP production 0 Site of CalvinBenson Cycle nzyme involved9 Ribulose B isphosphate CarboxylaseOxygenase RuBisCO I Fixes C02 into organic compounds llas abilityto form C02 from atmosphere I Has 3 membranes more membrane more ATP 0 0 Inner o Thylokoid o StromaSite of Grana o Granum9 Stacks of Thylokoids I A lot ofeukaryotic cells contain chloroplast I Chloroplast is a lot like mitochondria murmuran Spice Granum Stack a 1hylakol s Cymskeleton Skeleton ofthe cell Components Microtubules I Make up agella and cilia ofeukaryotic cells Polymers oftu ulin Maintains cells sha e Aids in cell motility by forming agella and cilia Aids in movement of organelle s and chromosomes Like the highway system ofthe cell Its how proteins get transferred 0 Myotic Spindle 0 Made oftubulin 9 Tubules make up tubulin Plays important role in moving chromosomes 0 Microfilaments Polymers of actin I De nes maintains and changes cell shape 0 In changing the cell shape the actin tells the cell Where t c ange I Aids in cell motility with cytoplasmic streaming ProkaryoticCytoskeIeton o FtsZ I Protein structure similar to tubulin I Determines Where cell division will occur o Fts A and MreB I Protein structures similar to actin I Fts A proteins hold Fts Z proteins in place I MreB proteins determine where new cell wall will be inserted in expandingdividing cell Note Coccus shaped bacteria lack MreB gene Cell Wall Structure composed of 0 Proteins o Glycoproteins o Polysaccharides Protozoans lack cell walls Many microbes have cell walls A lot ofvariability when looking at cell walls Function 0 Protection 0 Maintains cell shape 0 Prevents osmotic lysis o Interacts with the environment Eukaryotic Cell walls 0 Cellulose Algae o Chitin Fungi I Very similar to cytoplasm it is made up of a polymer of glucose I Exoskeleton also made of chitin o Glycoprotein is found throughout I Found in the extracellular matrix of fungi slime molds and yeast Bacterial Cell walls 0 Mycoplasm and Chlamydia groups lack cell wall I Chlamydia loves to live in our eyes 0 Leading cause of blindness 0 Human Pathogen 0 Hosts in vagina and penis also I Mycoplasm comes from hair and nail salon foot baths 0 All of species are very small 0 Absolute smallest genome 0 Human pathogen Note It is not a disadvantage to lack a cell wall for the cell itself The cell continues to live because antibiotic are designed to attack the cell wall since there is not cell wall to attack with these bacteria you have to make a special antibiotic that is designed to attack bacteria without a cell wall 0 Planctomyces group have a protein cell wall I Have organelle type species inside their cell wall I Lots of prokaryote characteristics yet some organelles make it seem eukaryotic o Peptidoglycan is found in the cell wall of all other groups of bacteria I Very common I Glycan Sugar I Peptido a few amino acids yet not a full protein I Has two main groups solely based on thickness of peptidoglycan layer 0 GramNegativeBacteria o The whole domain have only a few layers of peptidoglycan only 10 of cell wall 0 Thin layer of peptidoglycan outer membrane wavyrough and uid like outer membrane made up oflipopolysaccharide and protein I Lipopolysaccharide LPS 0 Lipid A toxic 0 When gramnegative bacteria embedded in large cell it is not a problem and doesn t cause infection 0 Lipid A is infectious when gramnegative bacteria cell is small 0 Core Polysaccharide o 0 Specific Polysaccharide o GramNegative Bacteria cell has three layers I Cytoplasmic Membrane Bottom Thin Peptidogylcan layer and the outer wavy membrane HUGE 0 Outer membrane is a phospholipid bilayer and has many molecules of LPS This is the major difference between grampositivenegative cells arid Cuepulyszharldn Dipolysacch on c mummu wall Perlplusm w v izhmpnonpm cymplusmn membrane GramPositive Bacteria llas several layers ofpeptidoglycan up to 90 of cell wall 0 Thicklayer very smooth membrane 0 Contains Teichoic and Lipoteichoic Acids I Teichoic Negatively charged polymers containing glycerophosphate or ribitol residues Bings Ca and Mg Calcium and Magnesium Calcium is needed for stucture Magnesium needed for very important enzymes such as DNApolymerase Combines cations I Lipoteichoic Teichoic acids covalentlylinked ot mamb rane lipids 0 Ex ofBacteria UTI and Kidney infectionbacteria are gramnegative I Causes fever 0 Wallassodated mum Teidmiz acid Lipateichoir add DGlumse 707 Fe um I can DAlanine n c P g y D Alanine u c ytaplasmir membyan u Rihiial a b I Repeating subunit that forms peptidoglycan layer 0 2 sugars both in acetyl glucose sugars acetylglucosamine G and acetlymuramic acid M in acetyl glucose and other in acetyl acid Has a beta linkage Has 4 amino acids tetrapeptide To form thick peptidoglycan layer gram positive9 layer afterlayer the 8 amino acids the bars in between M and G which are formed by a peptide bondhold the two sugars together I ALL LAYERS HELD TOGETHER BY TETRAPEPTIDE CROSS LINK 0 OOO Archaea Cell walls of Pseudomurein very similar to peptidoglyca n protein glycop rotein or polysaccharide Another domain ofprokaryotic cells Archaea cells have been around for as long as any other cells but were just recently introduced to humans so little study and research has been conducted on them thus far Pseudomurein I Very similar to peptidoglycan but know the difference l l I Archaea contains itbut bacteria do not 00 O I Has exactly the same sugar in acetyl glucosamine NAG as peptidoglycan but a different sugar in acetyl muramic acid 0 The different sugar is acetylalosaminuronic acid made up differently NAT I Some archaea like methanobacterium have a pseudomurein cell wall I It is lysozyme insensitive 0 Your tears cannot destroy archaea 0 Odds of coming in contact with this species is very small though I Some archaea like Halococcus have polysaccharide cells walls 0 Halococcous is an extreme halophile that has a rep eating 3part polysaccharide cell wall structure with sulfate groups on uronic acid 0 Sulfate group binds to Na o Slayer cell wall is made ofprotein and glycoprotein 0 Found in most archaea usually in addition to polysaccharide Note The main difference between archaea and bacteria is the one different sugar Lysozyme Enzyme that destroys peptidoglycan by cleaving Beta1 the 4 glycosidic bonds between the Nacetyl glucose sugars Can be found in animal secretions humans FIRST line of defense against bacteria 0 Produced in our tears and secretions A major line of defense against infections against bacteria It will bind to peptidoglycan and will cut cell wall 0 9 Weakens bacteria s cell wall Too much water will enter cell Cell becomes too weak to hold back pressure of all the water build up Cell busts Does very good job with defense ofbacteria in our eyes 0 Like a natural antibiotic Prokaryotic Cell Surface Structures Fimbriae Plural Fimbrial 0 Short protein filaments I They are sticky since protein is sticky I Much smaller than agella o Aid in attachment 0 Note that the longer structures extending from cell are agella PiliusZPilil Plural 0 Made of protein I Longer protein filaments than Fimbriae 0 Primary purpose Conjuction I Not necessarily sex 0 It is a hollow thin narrow tube connecting one cell to another 0 Carry and transfer genetic information Slayer 0 Two dimensional array of protein 0 Function selective sieve Capsule 0 Made ofpolysaccharide sugar or polypeptide protein layer 0 Function attachment I Helps cell stick to surfaces since the capsule is sticky o Surround the cell 0 Made by bacterial cells mainly pathogenic Note Difference between bacteria and pathogenic bacteria is BAD Slime Layer Loose layer ofpolysaccharide Used for gliding Like a capsule but much more uid Increase polysaccharide amount Moves better Not that much in tact pieces will start to fall off edge of cell OOOOO Gliding Non agellated prokaryotes Slow smooth movement of cell across a surface 0 Slime secretion 0 Ratchetprotein mechanism I Back and forth motion Eukaryotic Cell Flagella and Cilia 0 Only difference is the length structurally the same 0 Flagella are long and there are few I Move back and forth I The structure of archaea agella is not yet known 0 They are thinner but rotate like bacterial agella I In bacteria agella agellum is made up of o Filament 0 Made up ofprotein Flagellin 0 Hook 0 The rings that make up the motor have a hook 0 Motor 0 L ring P ring MS ring and C ring I Flagella is the one thing in which prokaryotes are more compleX than eukaryotes in o Cilia are short and there are many I Move back and forth by dynein motor proteins I Made of microtubules I 9 pair surrounding 2 central Flagella in Bacteria Made up of lament hook and motor The proton motor force drives the agellar motor to rotate the agellum filament o 60 cell lengthssecond can be achieved 0 1000 H per rotation The motor is like a revolving door 0 The electrostatic forces from the motor proteins large positive charge on the rings of the basal body are what causes the basal body to rotate 0 Only prokaryotic agella rotate I Eukaryotic agella go back and forth The filament is like at boat motor propeller The ring in the cytoplasmic membrane 0 Space between the outer membrane and the cytoplasmic membrane called periplasmic I H goes through the motor protein causing the motor to turn Fastest Human Swimmer Eamon Sullivan 0 2128 sec 50 meter freestlye o 189m 6 ft 2 in tall 0 124 human lengths per second Australian Swimmer Happened in March 2008 On April 26 2009 Frederick Bousquet broke under 21 seconds with 2094 seconds FRA BUT he was wearing a banned performance enhancing suit Location of Flagella Polar o Flagella are attached at one or both ends of the cell 0 The movement ofpolar agellum tends to be more rapid and it spins around from place to place 0 REMEMBER Flagellum located on ends NOT middle Movement with polar agella can be either reversible or unidirectional I Reversible Flagella Go from a CCW rotation to and CW rotation to the right and back to CCW rotation from the CW rotation to the left 0 I Unidirectional Flagella Rotates CW then stops momentarily to reorient and moves in the new direction still in a CW rotation Peritrichous o Flagella are inserted at many locations around the cells surface 0 Movement is typically slow and in a straight line 0 Looks very hairy and there is agella at many locations I Ends Middle ALL OVER 0 Movement with peritrichous agella runs I 9 Bundled agella is moving in the CCW rotation to the right then the agella spread out all over around the cell tumbled agella and goes side to side to get to this point This middle stage ofmovement is done in a CW rotation It then goes back to being bundled agella rotating in a CCW manner moving to the left How Does a Motile Cell Determine Where to Go They compare the current environment with the environment they sensed moments before 0 If the environment is neededfavorable they will move towards it o If the environment is toxicunfavorable they will move away from o If there is no difference and the environment is indifferent the cell will have no net movement Types of Flagella Monotrichous o Vibrio Pseudomonas 0 One agella stemming from one end of the cell Lophotrichous o Rhodospirillium photometricum 0 Many agellum stemming from one end of the cell Ampitrichous o Spirillium 0 One agella stemming from each end of the cell Peritrichous o Proteus Salmonella Escherichia 0 Manny agellum surrounding the whole cell Note Flagella helps a cell move here and there with or without agella the cell needs to know where to go and how to get here and there Taxes Sensory Proteins Receptors Directed movements toward or away from a chemical or physical signal Sensory Proteinsreceptors in the cytoplasmic membrane detect the environment then interact with cytoplasmic proteins to affect motion Chemotaxis 0 Chemicals 0 Chemoreceptors sense chemical gradient Aerotaxis 0 Oxygen o Aertotactic receptors sense oxygen gradient 0 In 1881 TW Engelmann displayed bacterial cells moving towards algal cells giving off oxygen I Ex Of Aerotaxic algal cells releasing many amounts of oxygen many more cells around the algal cell than anywhere else 0 Aerobic Bacteria wants the oxygen being relesased by the algal cell Phototaxis 0 Light 0 Photoreceptors sense light gradient 0 Phototrophic bacterium thiospirillum jenense aggregates at wavelengths at which its pigment is absorbed I Absorb light at around 400 560 and 850 ATP can be made where light is absorbed I Photoreceptors phototaxic In a prokaryotl39c cell it will tumble and run to or from the attractant o If the net movement is away dispersed we no there is no attractant present 0 If the net movement is towards an attractant we know there is an attractant present Gas Vesicles Small gas filled structures made ofprotein that confer buoyancy on cells Seen in aquatic microorganisms like cyanobacteria Orient from either 0 Optimum light harvesting o In response to other environmental stimuli Note Can put itselfvertically in the water column depending on how much air is in the gas vesicles Magnetosomes Magnetic 0 Doesn t swim towards magnet on slide it is pulled there since it has magnetite within The intracellular particles are made of magnetite Fe304 Include aquatic microbes such as Magnetospirillum They orient cells in a particular direction Other Cell Structures Cell Inclusion Bodies Storage Depots o Glycogen I Sugar storage molecule I Feeds right offmetabolism o PolyBetaHydroxyalkanoate I The big white round holes I the cell I It is a lipid I Can be cut in half and used for energy source 0 Phosphate I Polyphosphate granules I Found in all cells I Found in DNA and RNA 0 The major molecule that nucleotides need Phosphate I Aids in DNARNA duplication replication and synthesis 0 Sulfur I Granules are stored in periplasm by gram negative bacteria they are black round structures in the cell 0 EX Purple Sulfur Bacteria I It is needed by all cells 0 Some cells need it for the start of the Electron Transport Chain System 0 ETS ATP produced as end produce Ferdinand Cohn 18281898 In 1876 he described the life cycle of Bacillus subtilis o Vegetative Cell Endospore Vegetative Cell Concluded that gram positive bacteria cells can make endospores Endospore Formation Sporulatl39on The formation of an endospore It is a survival mechanism used by some gram positive bacteria 0 Bacillus o Clostridium I Most are anaerobes o Thermoactinomyces Occurs when cell growth stops due to harsh conditions 0 Not all cells are alike so harsh conditions for each cell can vary I Temperature Salt levels pH levels Amount of pressure Radiation I Lack of oxygen Happens within and at the expense of a vegetative cell 0 When a cell forms endospore the spore will have everything it needs inside of it to make the cell The endospore is NOT a cell it is dormant inside the cell Endospore Dormant Used as a survival structure Contains 0 Several macromolecules I Carbohydrates Lipids Proteins 0 Calcium Dipicolinic acid 0 Small acid soluble proteins I Not present in vegetative cells only found in the endospore itself The endospore is very dehydrated o The cell that made the endospore is made up of 70 water yet endospore still dehydrated The cell itself is not what is harmful it is the endspore because it can travel 0 Spores germinate and thrive off different hosts Have intracellular locations 0 Terminal 9 at the ends I Clostridium Botulinum botulism 0 Note babies don t have immune system to deal with botulism o Subterminal within the end of cellnear end I Bacillus subtilis o Central within the middle I Bacillus anthracis anthraX Endospore has layers 0 Spore Coat CorteX Exosporium Core wall DNA I Surrounded by a lipid layer that is hydrophobic 0 When the cell sporulates it forms this white lipid layer Note Any white layer seen in a TEM image is a lipid 0000 0 Core 39 Spore coat V Cortex xosporium E core wall J DNA ankrllLT C Inmim Spore Released from a vegetative cell Can survive indefinite periods oftime Million of years potentially Note Nothing else other than fossils have the potential or can live that long spores are not technicallylivinglike fossils are though Resistant to heat chemicals radiation and dessication dying Clorox will not damage a sporeendospore since they are very resistant 0 You have to wipe it away this is how you get it out ofyour bath tub kitchen sink you squirtthen wipe cannotjust squirt The sporulating cell that is shown in the book is from a bees stomach o It is in amber and the bee was found in a lake fossilized 0 Shows that spores existed waybefore humans As long as the earth is here will have endospores since theyhave the ability to preserve themselves A spore cannot make another spore cannot grow or divide either At some point if conditions favorable the spore can germinate Germination SelfPreservation I Note Abacterial cell emerging from a spore is not reproduction with fungi it is though Germination When a spore transforms into a vegetative cell genetically identical to the vegetative cell that made the spore It is the reverse of sporulation Occurs only when conditions arebecome favorable o Occurs at expense ofthe spore The cell is IDENTICLE to the cell that made the spore inthe first place 0 Ex Going back to the bee found in lake in Minnesota it will be the exact same as that cell found 7000 years ago Can take several days to a week to occur But typically hours to days Note Vegetative Bacterial Cells do not survive in boiling water They will form when cooking butthe oiling water kills them they don39t hurt you BUT All sporesendspores CAN survive in boiling water The Viral Structure Virus Also known as virion orvirus particle Defined A nucleocapsid made of nucleic acid surrounded by a protective protein coat Nucleic AcidiViral Genom 0 Protective Protein CoatiCapsid C p id proteins arrange to give virion symmetry Helical Symmetry o RodShaped 0 Protein subunits twist up 0 lcosahedral Symmetry WW5 Roughly spherical o 20 equilateral triangles Will take over host cell in order to replicate No metabolism involved Two forms of existence 0 Extracellular I Outside of hostInert o In tracellular I Replicates inside of host by taking over metabolic machinery of the host itself Virion Small in size 0 2030 nm Small genome o 5kbZ30kb DNA or RNA Single or Double Stranded Linear or Circular Table 102 The Baltimore class icakiun system of viruses Examples Class Description olgsnoms and replication slrnegy Bacterial viruses Animal vimes l mmmemmden DNA genome Lambda Til Hcrpebiwum mix Wu H Singlesslralided DNA genom Mm ClIiLkan anemia virus m Daub gtsi39anded NA gen is lt 5mm v9 mi 1V Sing Ms v Singlr rslmudcd RNA gcrmml ml min nan I35 ViHI J VI 5 n i Ended RNA 9 mei nai rephcaies wrih Intermedime W Doublersvgnded DNA genomi mar rEplicaies mm Hepaims a virus RNA intermedlale CapsidEnveloped v Naked viruses 0 Lipid Bilayer o Derives from host cell Complex multiple parts assembled separately Vimids and Prions Viroids 9 enve oped outer membrane around capsid O A naked virus has no envelope surrounding 1t 0 Smallest known pathogens re 0 Structu Circular Single Stranded RNA Have NO protein 0 Cause plant diseases Prion 5 Ex Potato spindle tuber viroid o Proteinparticle o NO nucleic acid 0 Cause animal neurodegenerative diseases Creutzfeldt akob Disease C D o A rare dege nerative invariably fatal brain disorder Every one and one million persons affected yea rly worldwide US9 200 casesyear Seen later in life 0 O nset of 60 Ru ns rapid course 0 90 ofpatients die within the first year Occurs in humans Early stages failing memory behavioral changes lack ofcoordination and visual disturbances 0 As it progresses we see pronounced mental deterioration involuntary movements blindness weakness of extremities and coma may occur 0 Belongs to family of transmissible spongiform encephalopathies T5Es9 family of human and animal diseases 0 Spongiform refers to characteristics appearance of infected brains I Brain becomes filled with holes until it looks like a sponge o CID is most common known human TSE o Other TSEs I Kuru identified in people of an isolated tribe in Papua New Guinea now almost disappeared I FFI amp 655 9 extremely rare hereditary diseases found in just a few families around the world I Other TSEs are found in specific kinds of animals 0 ScrapIe affects sheep and goats o Mink encephalopathy o Feline encephalopathy Note TSEs families can also be found in yeast but has not been found in plants yet I Bovine Spongiform Encephalopathy BSE o Belongs to TSEs Family 0 Mad Cow Disease I Chronic Wasting Disease CWD o Occurs in elk deer etc Microbial Growth 1 Distinguish between macronutrients and micronutrients Macronutrients are nutrients that are required in large amounts Micronutreientsare nutrients required in trace amounts 2 Give at least 6 examples of macronutrients Carbon 0 A typicall cell is about 50 carbon 0 Carbon is needed to make organic compounds such as amino acids fatty acids organic acids sugars nitrogen base aromatic compounds and countless other things 0 Found in the environment as C02 and organic compounds 0 Autotrophs are able to build all of their structures from C02 0 Heterotrophs use organic compounds Nitrogen 0 Bacterial cells are about 12 nitrogen o Nitrogen is a key element in protein nitrogen acids and several other cell constituents o A bqu of the available nitrogen is in inorganic form either ammonia NIB nitrate N03 or N2 0 N2 can only satisfy the nitrogen needs of a few bacterianitrogen fixing bacteria Oxygen 0 Found in the environment as H20 and 02 o A requirement for growth and resperation Hydrogen Phosphorus 0 Required by the cell primarily for the synthesis of nucleic acids and phospholipids Sulfur o Is required because of its structural role in amino acids 0 Most sulfur originates from inorganic sorces in nature either sulfate 804239 or sulfide HS Potassium 0 Required by all organisms 0 Required for activity Magnesium 0 Function is to stabilize ribosomes membrane and nucleic acids and is also required for the activity of many cells Sodium o Is required by some but not all and is a reflection of the enviroment Calcium 0 Helps stabilize the cell wall and plays a key role in the heat stability of endospores Iron Microorganisims require various metals fir groth Iron plays a major role in cellular resperation Under anoxic conditions iron is typically found in the ferrous Fe2 form and soluble Under oxic conditions iron is found in the ferric Fe3 form and insoluble OOOO 3 Give at least 4 examples ofmicronutrients Boron Copper Cobalt Nickel Zinc Micronutrients typically play a role in components of enzymes and the cells catalysts 4 How do growth factors differ from macronutrients and micronutrients Growth factors are organic compounds They are only required in trace amounts and only by certain organisms Growth factors include vitamins amino acids purines and pyrimidines 5 Name the 5 groups describing an organism s relationship to oxygen Obligate Aerobes o Aerobic organisms that require oxygen for growth 0 Through cellular respiration these organisms use oxygen to oxidize substances such as sugars or fats In a test tube the position ofthe bacteria will all be as close to the surface as possible They want to get as close to the oxygen supply as possible Obligate Anaerobes 0 These are microorganisms that live and grow in the absence of molecular oxygen 0 In a test tube all the bacteria willall be at the bottom ofthe test tube This is because the bacteria moves as far away from the oxygen source as possible Facilitative Aerobes o Organisms usually bacteria that make ATP through aerobic respiration if oxygen is available but can switch to fermentation 0 Different from Obligate because the obligates die when there is or isn t oxygen 0 The test tube will show that there is more growth in the oxic zone but also growth throughout the entire tube This is because it does not die itjust switches to fermentation when oxygen is not present Microaerophiles o A microorganism that requires oxygen to survive but requires enviroments of low oxygen concentration 0 This means that the test tube will appear with most of the bacteria just below the oxic zone Aerotolerent Anaerobes o Organisms that are not affected by levels of oxygen 0 In the test tubes forthese types of organisms the bacteria will be spread evenly throughout the entire tube 0 6 What type of broth can be used to determine an organism s oxygen requirement What does this broth contain that makes it possible to determine an organism s oxygen requirement l G Thioglycolate is the broth that can be used to determine an organisms oxygen requirement It is a reducing agent A reducing agent may be added to a media where it reacts with oxygen and reduces it to H20 When thioglycolate reacts with oxygen throughout the tube oxygen can penetrate only near the top of the tube where the medium contacts the air Because ofthis reaction we can tell what level of oxygen is required for a certain organisms growth based on where it grows is the broth You inoculate the following organisms into tubes containing thioglycolate broth Where would you expect to see growth in the tube a C39 O O 39D Obligate aerobe 0 Only at the top Obligate anaerobe 0 Only nearthe bottom Facultative aerobe o Grows throughout the tube but best at the top Microaerophile o Grow near the top but not right at the top Aerotolerate anaerobe 0 Growth is not affected by the levels of oxygen What accommodations need to be made to grow an aerobe in the lab 0 In order for aerobes to grow in a lab the culture medium needs to be oxygenated 0 Like stated before aerobic organisms will die when there is no presence of oxygen 0 Allow exposure to air 0 Vigorous shaking o Bubbling sterile air into the tube 0 The bubbling and shaking increase the amount of oxygen and how far the oxygen goes with in the test tube to promote more growth 9 What accommodations need to be made to grow an anaerobe in the lab 0 In orderto grow anaerobes in the lab oxygen must be excluded 0 Bottles and tubes are completely filled with the culture media 0 Bottles are sealed tightly o A reducing agent is used Remember a reducing agent reacts with oxygen and reduces it to H20 therefor eliminating the oxygen content that might be in the test tube and ultimately promoting anaerobic growth 0 Use anoxic jars andor anoxic glove boxes 10 During what process are toxic oxygen molecules generated a One major form of toxic oxygen is singlet oxygen 0 Singlet oxygen is a higher energy form of oxygen in which outer shell electrons surrounding the nucleus become highly reactive and can carry out spontaneous and undesirable oxidations within the cell Singlet oxygen is produced both photochemically and biochemically the latter through the activity of various peroxidase enzymes 0 They are produced by peroxidase enzymes and others are produced as byproducts of respirationfrom PowerPoint o it is a fourelectron reduction of 02 to H20 by stepwise addition of electrons 11How does an organism protect themselves from toxic oxygen molecules 0 Super oxides and hydrogen peroxide are the most common toxic oxygen species so enzymes that destroy these compounds are widely distributed The enzyme catalase attacks hydrogen peroxide forming oxygen and water Another enzyme that destroys hydrogen peroxide is peroxidase o Differs from catalase because it requires a reductant usually NADH 0 Produce only water as a product as oppose to catalase that produces oxygen and water Superoxide is destroyed by the enzyme superoxide dismutase 0 An enzyme that generates hydrogen peroxide and oxygen from two molecules of superoxide o Superoxide dismutase and catalase work together to bring about the conversion of superoxide to oxygen and water Other means of superoxide disposal is present in certain obligater anaerobic archaea 0 when superoxide dismutase is absent a unique enzyme superoxide reductase is present and functions to remove superoxide 0 Unlike superoxide dismutase because it reduces superoxide to H202 without the production of 02 12What enzyme combinations would detoxify superoxide 0239 In what organisms might those combinations be found a The organisms that would detoxify super oxide and Superoxide dismutasecatalase and superoxide reductase o Superoxide dismutase o Reduces it down to oxygen and water 0 Are commonly found in aerobes and facultative aerobesorganisms o Superoxide reductase 0 reduce to H202withoutthe production of oxygen 0 found in obligater anaerobic organisms 13Where can an organism get nitrogen o Nitrogen is an important macronutrient because used to make nitrogen bases and amino acids 0 Nitrogen is found in the environment as NH3 NO3 and N2 0 Nitrogenfixing microorganismsuse atmospheric nitrogen N2 0 Nonnitrogen fixing microorganisms use the inorganic compounds ammonium NH3 and nitrate NO3 14 Name a molecule that requires phosphorus Where can a cell get phosphorus o Phosphorous occurs in the form of organic and inorganic molecules 0 P04339 is the usual form that phosphorus is found in the environment 0 Obtained from phosphate minerals and free phosphate PowerPoint 15 Name a molecule that requires sulfur Where can a cell get sulfur obtained from sulfates 804239 or sulfides 8239 16 Name a molecule that requires potassium Where can a cell get potassium 0 Potassium is required by all organisms 0 used to maintain solute concentration 0 obtained from free K or K salts 17 Name a molecule that requires magnesium Where can a cell get magnesium 0 Required by many enzymes 0 obtained from salts in various minerals o Mg2 is solutions or as various Mg salts same this as above different wording 18Name a molecule that requires iron What specialized molecule allows some organisms to sequester iron What are two examples of siderophores 0 Iron plays a major role is cellular respiration and needed for the electron transport chain proteins 0 Siderophores is a special iron binding agent that allows organism to sequester Iron 0 molecules bind iron and transport it through the cell 0 Two example of siderophores are 0 Enterobactins Sequesterthe limited iron supple in animalstissues to initiate infection 0 Aquachelin marine bacteria 19 Name a structure that requires calcium Where can a cell get calcium See Table 51 o Usual for in the environment Ca2 in solutions or as CaSO4 or as other otherCa salts 0 Calcium stabilizes the cell wall 0 Calcium is important in the development of endospores o Endospores haveare a calcium dipicolinic acid complex 20 Name a structure that requires sodium Where can a cell get sodiumSee Table 51 Can sodium motive force instead ofa proton motive force be used by some organisms to generate ATP 0 Usual for in the environment 0 Na in solution or as NaCl or other Na salts 0 Sodium is important to some but not all microorganisms and is typically a re ection of the habitaite o Seawater contains high levels of NA and marine microorganisms usually require sodium for groth o By contract fresh water sh are usually able to grow in the absence of sodium 0 Sodium ions bind to the outer surface of the Halobacterium cell wall to keep the cell wall together by shielding negative charges of nearby amino acids in cell wall glycoproteins If Na is in lacking the cell wall breaks apart and the cell lyses because the negatively charged amino acids repel one another 0 used by Napowered ATP synthase answer to question YES 21 De ne growth as it pertains to microorganisms o In microbiology growth is de ned as an increase in the number of cells 0 Knowledge of how microbial populations can rapidly expands is useful for designing methods to control microbial growth 0 Bacterial growth depends upon a large number of chemical reactions of a wide variety oftypes 22What is the most common process by which prokaryotes grow 0 The most common process by which prokaryotes grow is by binary ssion o In a growing culture a rod shaped bacteria will grow to approximately twice their original length and then form a partition and divide into two daughter cells 0 The partition that is formed is called a septum o The septum is the result from the inward growth of the cytoplasmic membrane and the cell wall from opposite directions which continues to grow until two daughter cells are pinched off 23What genomic event must take place before a cell divides 0 As stated above the genomic event that must occur beforethe cell divides is that the cytoplasmic membrane and the cell wall must start growing inward from opposite directions 0 This occurs until two daughter cells are pinched off they divide 24What proteins are required to move the copies of DNA to opposite sides ofthe dividing cell c A series of protein present in all prokaryotes called Fts proteins are essential for cell division 0 Fts stands for filamentous temperature sensitive which describes the properties ofthe cells that have mutations in the genes that encode for Fts proteins 0 Fts proteins interact to form a cell division apparatus called the divisome o FtsK proteins are DNA transloceses that move DNA during cell division 25 Following DNA replication what must form for cell division to continue 0 After DNA replication there must be a formation ofthe divisome in order for cell division to occur Divisomes and its proteins develop right in the middle of the cell because other wise it will be unevenly divided 26What proteins form the divisome Where are these proteins located How does there location correlate with where division will begin c There are three types of Fts proteins that work together to form the divisome 0 Min This protein ensures the divisome forms at the center of the cell Also the protein that makes up FtsZ The Min proteins present as a spiral structure that moves back and forth from pole to pole on the cytoplasmic membrane this is a concentration gradient and where the concentration is the highest at the center of the cell the FtsZ ring will form 0 FtsZ FtsZ molecules polymerases to form a ring that ring then attracts other divisome proteinssuch as FtsA o FtsA This protein is responsible for helping to hold the FtsZ ring the the cytoplasmic membrane Is a protein related to actin that and has an addition role in recruiting other divisome proteins 27 Does the divisome move 28 FtsZ protein has similar amino acid sequence to what eukaryotic cytoskeletal protein a The FtsZ protein is related to the tubulin the important cell division protein in eukaryotes o Tubulin form microtubules which are responsible for maintain cell structure and provide a platform for intracellular structure 29As the cell elongates new cell wall is laid down at certain sites determined by the location of what protein 0 MreB proteins are responsible for determining the location at which the new cell wall would be laied down forms sporal shaped bands just under the cytoplasmic membrane MreB cytoskeleton de nes cell shape by recruiting other proteins that orchestrate cell wall growth in a specific pattern It appears that MreB functions to localize synthesis of new peptidoglycan and other cell wall components to speci c locations along the cylinder of a rod shaped cell during growth 30As the cell elongates holes are made in the existing cell wall to make room for new cell wall material What enzyme makes these holes What happens if these holes are not quickly lled with new cell wall material lfa ce lacks Ftsl proteins what will happen once these holes are made Beginning at the FtsZ ring small openings in the cell wall are created by enzymes cell autolysins It is essential in peptidoglycan synthesis that new cell wall precursors be spliced into existing peptidoglycanin a coordinated mannerto prevent a breached a breach when the openings are not filled with the new cell material quick enough could cause autolysis which is spontaneous cell lysis Ftsl proteins also known as penicillin binding proteins are transpeptidation that insert new peptidoglycan into the openings made by the autolysin enzymes Without the Ftsl proteins the cell will breach and spontaneously lysis like stated before Transpeptidationforms the peptide crosslinks between muramic acid residues in adjacent glycan chains The opening only occurs where MinB different from the other Min contacts the cell wall 31 How does the new peptidoglycan precursor differ from existing cell wall peptidoglycan 32 How does the new peptidoglycan precursor get to the expanding cell wall 0 First there is the formation on the peptidoglycan precursors o Bactoprenol bonds to Nacetyl glucosamineNacetymuramic acid pentapeptide a Second there is a tranporst of the peptidoglycan precursors to expanding cell wall 0 Bactoprenoltransports the precoursor across the cytoplasmic membrane by rendering them sufficiently hydrophobic to pass through the interior of the cytoplasmic membrane 0 Lastly is the insertion of the peptidoglycan precursors into the existing wall 0 Bactoprenol interacts with an enzyme called glycolasesthat inserts cell wall precoursor into the growing point of the cell wall and catalyzes glycosideic bond formation Autolysin break gycoytic bonds in preexisting peptidoglycan while glycolase synthesizes them linking old peptidoglycan with the new 0 Then insertion done by transpeptidation using Ftsl 33What makes bactoprenol a good molecule to transport the new peptidoglycan precursor across the cytoplasmic membrane a The bactoprenol is a good molecule to transport the new peptidoglycan precursor across the cytoplasmic membrane because it is a hydrophobic lipid alcohol 0 Because ofthis it can render the precursor sufficiently hydrophobic so that is can pass through the cytoplasmic membrane 34 Fstl proteins catalyze what reaction 35What happensto the peptidoglycan precursor during transpeptidation 0 Initially there are two Dalanine residues at the end of the peptidoglycan precursor but one Dalanine molecule is removed during the transpeptidation reaction 36ln binary fission where is new well wa added relative to the divisome o In binary fission the new cell wall is added at the exact location the divisome directly in the middle of the cell c Budding is a result of unequal cell growth 37 Describe simple budding How is simple budding different than binary fission Give an example of a prokaryotic microorganism that divides by simple buddingGive an example of a eukaryotic microorganism that divides by simple budding 0 Illustration of simple budding Simple budding is different from binary fission because it forms a totally new daughter cell with the mother cell retaining it original identity The fundamental difference is not the formation of the buds but the formation of the new cell wall material from a single point polar growth rather than throughout the cell intercalary growth like binary fission Saccharomyces cervisiae is an organism that demonstrates simple budding 38 Describe budding by hyphae How is the new cell different than the parent cell What is a swarmer cell capable of doingGive an example ofa prokaryotic microorganism that divides by budding by hyphae 0 Illustration 0 Printed out slide 39 Describe cell division in stalked bacteriaHow is the new cell different than the parent cell Give an example of a prokaryotic microorganism that divides this way 0 Illustration 0 Printed out slide a One daughter cell is a swimmer and has one flagellum attached to one end and the mother cell is stalked 40Compare and contrast cell division in stalked bacteria and budding by hyphae 41 Describe the growth cycle Do the growth phases always occur in the order presented in figure 510610 in 12th edition 0 The growth cycle includes the lag phase the exponential phase the stationary phase and the death phase 0 Lag Phase is the transition period to allow synthesis of enzymes for binary fission When a microbial population is inoculated into a fresh medium growth usually begins only after a period oftime the lag phase Lag phase also occurs when the inoculum consist of cells that have been damaged not killed by treatment with heat radiation or toxic chemicals because of the time required for the cell to repair the damge o Exponential growth is the regular division of cells Each cell divides to form two more cells which go one to form two more cells and so on This can go on for a brief or extended period oftime depending on the available resources and other factors Stationary phaseoccurs when essential nutrients are depleted or there is a buildup of waste and the growth rate zero n stationary phase there is not net increase or decrease in the cell number and thus the growth rate ofthe population is zero Alothough the population may not grow many cell function may still occur including energy metabolism and biosynthetic processes In some cases there may be some cell division but no net increase in the cell number this is because some cells in the population grow while others die 0 This process is celled cryptic growth 0 Death phase is obviously when the cells can no longer function and die 0 0 Cell do not always follow this exact growth cycle different things can happen at different times and we can see this in the next few questions 42 If you use a loopful ofturbid culture of Ecoli in stationary phase to inoculate a new broth culture at what growth phase will the cells start 0 Because it is being but into a new broth it is most likely that the cell will go in to exponential growth But that is with assuming that the new broth has the essential nutrients that Ecoi requires for growth Ifthe new broth did not have the essential nutrients required for growth the cells would die as oppose to undergo exponential growth The answer to this question can very and is depended upon whether the essential nutrients are available or not 43 If you use a loopful ofturbid culture of Ecoli in exponential phase to inoculate a new broth culture at what growth phase will the cells start a If Ecoli was in exponential growth and inoculated into a new broth culture it is most likely that the cells will continue to undergo exponential growth without a lag phase 44 If you use a loopful ofturbid culture of Ecoli in lag phase to inoculate a new broth culture at what growth phase will the cells start Would it matter ifthe new broth culture was the same medium same composition or different medium 0 might be wrong The cells should start in the lag phase This is because since it is a new culture and it is already in lag phase the Ecoiprobably still need time to gain the essential nutrients required for biosynthesis When a microbial population is inoculated into a fresh medium growth usually begins only after a period of time the lag period 0 So the theEcoimay continue in lag period for a period of time or may go into exponential growth o It depends on the history ofthe inoculum and the growth conditions of the medium 0 Yes it would matter is the new broth culture was the same medium same composition or different 0 EXAMPLES o If an exponentially growing culture was inoculated in to the same medium under the same conditions ofgrowth there will be no lag and exponential growth begins immediately 0 However if the culture is taken from an old culture stationary phase and transferred in the same medium there is usually a lag phase even of the cells in the inoculum are alive This is because the cells are depleted of various essential constituents and tine is required for there biosynthesis 45 Plot on a graph what the growth cycle would look like during the entire process described here You grow an Ecoli culture in glucose broth Once it reaches stationary phase presumably due to consumption ofall the glucose you add additional glucose 46 During exponential growth cells predictably 0 During exponential growth cell doubles over time 0 Each cell divides to form two each ofthose then divide to form two more so on and so forth 47 Explain why generation time is often referred to as doubling time o A generation is when one cell divides into two 0 The time is takes to do this is known as the generation time o It is often referred to as doubling time because it is the time it takes for the cell to double 0 In the period is one generation all cellular constituents increase proportionally cells are thus said to be in balanced growth 48Graph the exponential growth examples on semilogarithmic graph paper both of which are posted on Moodle Be able to determine from the graphs the generation time how many cells after a period of time and at what time would there be a certain number of cells 49 Describe an everyday situation in which exponential growth would be relevant 0 During exponential growth the increase in the cell number is initially rather slow but increases at an even faster rate o In the later stages of growth this results in an explosive increase in cell number 0 For a nonsterile nutrient rich food product such as milk to stand under ideal bacterial growth conditions for a few hours during the early stages of exponential growth when the total cell numbers are relatively low is not detrimental 0 However standing forthe same length of time in the later stages of exponential growth when the cell numbers are much higher is disastrous 50 Know when to use NN02 Be able to calculate the nal cell number N if given the initial cell number and number of generations of growth Be able to calculate the initial cell number N0if given the final cell number and number of generations of growth 51 Know when to use gtln Be able to calculate the generation time 9 if given the time of exponential growth and number of generations Be able to calculate the amount of time of exponential growth t if given the generation time and number of generations Be able to calculate the number of generations n if given the time of exponential growth and generation time 52 Give an example of a batch culture 0 An example of a batch culture would be and enclosed vessel such as a tube or a flask A batch culture is a closedsystem microbial culture of a fixed volume A batch culture is continually being altered by metabolic activities ofgrowing organisms and is therefore a closed system In the early stages of exponential growth in batch cultures conditions may remain relatively constant but in later stages when the cell numbers become quite large the chemical and physical compositions in the culture medium changes drastically 53 How does a batch culture differ from a chemostat culture 0 In a batch culture the nutrients can affect both growth rate and growth yield 0 At very low concentrations of a given nutrient the growth rate is submaximal because the nutrients cannot be transported into the cell fast enough to satisfy the metabolic demand Atmoderate to high concentrations of a certain nutrient the growth rate plateaus but the nal cell yield may continue to increase in proportion to the concentration ofthe nutrients in the medium up to the some xed limit 0 In a chemostat broth both the growth rate and the growth yield population density of the culture can be controlled independently and simultaneously as opposed to being affected by nutrients and constantly changing with no control 0 The growth rate is controlled by the dilution rate 0 The growth yield is controlled by the limiting nutrients O 54What can be controlled in a chemostat that allows for a continuous culture to be maintained 0 The two factors that are important in such control are the dilution rate and the concentration of limiting nutrients o Dilution rate is the rate at which fresh medium is pumped in and spent medium is sent out The concentration ofa limiting nutrient are nutrients such as carbon or nitrogen that are present in the sterile medium and enter the chemostat vessel 0 55What are the two types of cell counts When would you use one type of cell count over the other a The two types of way you can count cells are by total cell count or viable count 0 Total cell count is the count of all cells dead or alive 0 this method would probably be most desirable when you are not concerned about the cell ability to divide and make offspring and you are interested in a quick and easy estimate 0 The most common total cell count method is the microscopic cell count A viable cell is the one that is able to divide and form offspring and in most cell counting situation these are the cells we are most interested in This method would probably be preferred when your concerned about the live cells We determine the number of cells in the sample capable of forming colonies in a suitable agar medium For this reason the viable count is also called a plate count 0 O O 56What instrument is used for a total cell count The total cell count can be determined by using a microscope to observe and enumerate the cells present in the culture or natural sample 0 Microscope counts canbe done on either samples dried on slides or on samples in liquid Dried samples can be stained to increase to contrast beteen the cells and their backgrounds With liquid samples specially designed counting chambers are used c A grid with squares of known areas 0 hemacytometer 57 Be able to determine how many cells are in a certain volume using a hemacytometer see weblink posted on Moodle 58Viable counts can be done in what 2 ways 0 Viable counts are done through a plate count 0 There are two ways to perform a plate count the spreadplate method of the pourplate method 0 The spread plate method uses a volume of approximately 01 mL or less of a diluted culture that is then spread over the surface of an agar plate volumes greater than that are avoided because the excess liquid goes not soak in and may cause the colonies to coalesce as they form making them difficult to count 0 In the pourplate method a known volume of a culture is pippetteed into the bottom of a sterile Petri dish Then melted agar medium is added and mixed well by gently swirling With this method the organisms to be counted must be able to with stand brief exposure to the high temperatures of the molten agar Another problem that may arise with the pourplate method is that any debris in the sample must the distinguished from the actual bacterial colonies for the plate will not be counted correctly erroneous 59What procedure usually precedes a plate count Why is this done 0 With both the spreadplate and the pourplate methods it is important that the number of colonies developing on the plate not be too many or too few 0 To obtain the appropriate colony number the sample to be counted must almost always be diluted 60What property ofthe culture is being measured by turbidity measurements 0 Turbidity measurement is actually the measurement of total cell mass 0 But because cell mass is proportional to the cell number we can use turbidity as a measurement of cell number in a growing culture 0 Therefore it measures both cell mass and cell munber 0 Looks cloudy because cells scatter light passing through the suspension 61 What instrument is used for a turbidity measurement 0 The instrument used for turbidity measurement is the spectrophotometer 0 An instrument that passes light through a cell suspension and detects the unscattered light that emerges o The spectrophotometer is measuring light not cell number and it should be understood that is may be hitting more than just cells The greater the turbidity the less light that get through the lower the reading on the spectrophotometer 62Turbidity is measure in units 0 In a spectrophotometer the unit of the turbidity measurement is optical density OD 63What exactly is the spectrophotometer measuring o It a measurement of the unfiltered light 64 Be able to interpret a graph of turbidimetric measurements as shown in figure 617b 65What does a standard curve represent 66 Describe the procedure for creating a standard curve a For unicellular organisms OD is proportional with in certain limits to the cell number 0 Turbidity reading can therefore be used as a substitute for total and viable counting methods Before this can be done a standard curve must be created The standard curve must relate a cell numbers dry weight or protein content to turbidity 67 Graph the standard curve examples on plain graph paper both of which are posted on Moodle From the graphs be able to determine the optical density ofa certain number of cells 68 Distinguish between species richness and abundance 0 Species richness is the number of different species present 0 Richness can be de ned in molecular terms by the diversity of phylopypes 0 Species abundance is the proportion of each species in the ecosystem 69 In what type ofenvironment would you expect to have specie richness 0 High species richness is common with most species present only at moderate abundance o The envirometn is one of many different types of nutrients and can therefore select for many different types of species 70 In what type ofenvironment would you expect to have specie abundance o In extreme environments species richness is often ver low and abundance of one or more species is very high 0 This is because the conditions in the environment exclude all but a handful of species and key nutrients are present at such high level that the highly adapted species can grow to high cell densities 71 In the real world how do microbes live as pure cultures 72What term refers to where a microorganism lives 0 Metabolically related microbial populations are called guilds 0 And a set of guilds form microbial communities 0 microbial communities interact with microoganisims and abiotic factors in the ecosystem in a way that defines the working ecosystem 73What type of microscopy does FISH use 0 The microscopy that FISH use is microautoradiography 74 Flourescent probes can differentially label organisms by recognizing specific sequences in their o FISH can be used to measure gene expression in organisms in a natural sample A method that amplifies the target is called in situ reverse transcription ISRT FISH This method uses a DNA probe to trap specific mRNA produced by cells in a natural sample Reverse transcription produces complementary DNA cDNA from mRNA The cDNA is then amplified by the polymerase chain reaction and detected by hybridization to the fluorescent probe Answer is DNA 75 List 3 reasons microbes sometimes grow differently in the lab than they do in their natural habitat 0 Estimates have shown that typical soil bacteria grow in nature at less than 1 of the maximal growth rate measured in the laboratory 0 These slow growth rate reflect 3 factors 0 1 Resources or growth conditions are suboptimal whereas in labs they are usually given the optimal amount to grow 0 2 The distribution ofthe nutrients throughout the habitat are not uniform whereas in the labs they are 0 3 In nature microorganisims grow in mixed population ratherthan pure cultures like they do in labs And organism that grow rapidly in a pure culture may grow very slow in a natural environment There is much more competition in a mixed population as oppose to a pure 76Where do biofilms form 0 As bacterial cells grow on surfaces they tend to form bio lms o Biofilms are assemblages of bacterial cells attached to a surface and enclosed in the adhesive matrix excreted by the cell 77Why do bio lms form What advantages does living in a biofilm offer a microorganism o Biofilms form because the trap nutrients for microbial growth and help prevent the detachment of cells on the surfaces present in owing systems c There are four reasons that underlie the formation of biofilms o Biofilms are means of self defense The resist physical forces that could sweep away unattached cells 0 Biofilm formation allows cells to remain in a favorable niche Biofilms attach to nutrient rich surfaces such as animal tissue or to surfaces in flowing systems such a rock in a stream These are fixed bacterial cell location where nutrients are more abundant or are constantly replenished 0 Form because they allow bacterial cells to live in close association with each other 0 Lastly biofilms seem to be the typical way bacterial cells grow in nature where nutrient concentrations are often one to two orders of magnitude lower than they are in laboratories 78 List the stages of bio lm formation What happens during each stage At what stage would the biofilm look like a mushroom 0 Formation of a bio lm begins with the attachment of freefloating microorganisms to a surface initial attachment The rst colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holdsthe biofilm together communicate Once colonization has begun the bio lm grows through a combination of cell division and recruitment The nal stage of bio lm formation is known as development and is the stage in which the bio lm is established and may only change in shape and size Development stage is the stage at which the bio lms look like mushrooms o The attachment of bacterial cells to the surface triggers biofilmspeci c gene expression 0 These are genes that are turns on and direct production of cell surface polysaccharides 0 These genes encode proteins that synthesize intracellular signaling molecules and initiate matrix formation Quorum sensing describes the ways in which bacteria determine how many of them there are in the vicinity o If enough are present a quorum they can get down to business 0 Acylatedhomoserine are the major intracellular signaling molecule 0 These molecules accumulate and signal other adjacent cells quorum sensing The quorum sensing signaling molecules used by Psuedomonasaeruginosa is the acylatedhomoserine These molecules then signal adjacent Paeruginosa cells and the population of the species enlarges and the biofilm develops Bonnie L Bassleris a researcher who has studied biofilms she has figured out some of the dialect the genetic and molecular mechanisms different species use She is best known for identifying a what might be a universal language all species share Something jokingly referred to as bacterial Esperanto The bacteria most talked about and that she most likely discovered this in is Vibrio fischeri which is the bacteria responsible forthe bioluminescence in the Hawaiian bobtail Squid 93Where are most terrestrial microbes found 0 The most extensive microbial growth takes place on the surface of soil particles usually with in the rhizosphere 94 In aquatic environments what types ofmicrobes are found the upper depths and at the surface 0 At the very top you care going to find oxygenic phototrophs o This is because this is where the most oxygen and the most light is found 95 In aquatic environments what types ofmicrobes are found the lower depths o The bottom of the ocean is an anoxic area 0 Because ofthisthe microbes that will have optimum growth at lower depths are anaerobic and fermentativemicroorganisms 96 Describe the events that take place in a river after the introduction of sewage or other highnitrogen or highphosphorus input 0 Look at graph slid 67 o The input of sewer water can lead to a marked oxygen shortage from bacterial respiration As the water moves away from that source of input organic matter is gradually consumed and the oxygen content returns to it previous level 0 You can see this trend by looking at the blue line on the graph When oxygen decrease the biochemical oxygen demand BOD increase as well as the growth ofanaerobic bacteria When the oxygen begins to rise again these anaerobic microbes release inorganic nutrients Inorganic nutrients such as PO4339 then increase the growth of algae and cyanobacteria and other aquatic plants This bloom of organic matter eventually leads to oxygen depletion which in turn leads to the death of the bloom When it dies it is mineralized by heterotrophic organisms 97What happens to the oxygen concentration afterthe introduction of sewage or other highnitrogen or highphosphorus input What microbes cause this What metabolic process is responsible When will the oxygen concentration return to previous levels What microbes are responsible for this What metabolic process is responsible 101 Where would you nd oxygenic phototrophs like cyanobacteria and algae within an aquatic environment 0 These would be found near the top or even on the surface a This is because these organisms require light and oxygen and the best source of these is at the top 102 Where would you nd anoxygenicphototrophs like the purple and green sulfur bacteria within an aquatic environment Why are they located here 103 Where in the water column do phototrophs chemoorganotrophs and chemolithotrophs each reside in a marine ie an ocean environment 104 What are terms used to describe an organism s requirement for elevated pressure 105 Describe the environmental conditions in the deep sea 0 Organisms that inhabit the deep sea face three environmental extremes 0 Low temperature 0 High pressure 0 Low nutrient level 106 What structures on the ocean oor provide warmth and inorganic nutrients to organisms that live down there 0 Thriving animal communities have been found clustered about thermal springs in deepsea waters throughout the world These undenNater hot springs are call hydrothermal vents diverse invertebrate communities develop near hydrothermal vents including tube worms large clams and muscles 0 also a suitable place for chemolithotrophic prokaryotes 107 Where does the carbon dioxide come from that autotrophs use in the deep sea 0 They are nourished through a symbiotic association with autotrophic bacteria 0 The bacteria actively live within the animal tissue and supply organic carbon to the animals in exchange for a safe residence and ready access to the electron donors needed for their energy metabolism 108 De ne mutualism Describe an example ofa mutualistic relationship in the deep sea 0 Mutualism is a symbiotic association in which both organisms bene t 0 An example is exactly like the one listed above with the bacteria and an animal such as a tube worm BIOL 2051 General Microbiology Laboratory Midterm Study Guide There will be four parts to the midterm Day 1 Section A 7 you will have to do a streak plate and perform a Gram stain You will have a total of 25 minutes to do both of these You must use your time wisely It is recommended that you do your streak plate while your smear for the Gram stain is air drying It is not recommended that you do one procedure first and then the other second because you may run out of time before getting done with the second procedure Day 2 Section B 7 questions about a set of materials tubes plates etc on the bench top in 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 during the exam Section D 7 short answer questions I Stains Stain Structure Primary stain Mordant Decolorizer Counterstain Result Organisms to Chemical stained remember Simple Cells Methylene None None None Cell Several blue morphology Safranin identi ed Crystal violet Gram Cell wall Crystal violet Iodine Ethanol Safranin G purple Staphylococcus structure G redpink epidermis peptidoglycan Spirosoma thickness lingual Endospore Endospores Malachite None Water Safranin Veg cells B megaterium vegetative cells green pinkred B sphaericus Endospore cultures on green slant Acidfast Cell wall Carbol Phenol and Acidalcohol Methylene Red acid fast Ec0li blue made in egg Mycolic Acid fuchsin red heat blue Blue not non albumin lipid Mycobacterium l smenmatis Capsule Background Congo Red None do Water Maneval s Background Azotobacter negative cell Capsule background not heat Stain cell red vinelandii stain polysaccharide Capsule polypeptide 7 colorless clear Cell redish Negative stain brown II Selective and differential media in the MPN agent component differential component agar 1 General tips Things to study Background material materials used procedures and expected results for each experiment 2 Quiz questions and questions at end of experiments in the lab manual 3 lVIicroscope parts and functions be able to focus on a smear 4 You will have to perform the streak plate technique properly You 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 of many of the materials used in lab can be seen in A Photographic Atlas for the Microbiology Laboratory by Leboffe and Pierce or online 2 Dilution problem 0 There WILL be at least one dilution problem on the midterm 0 You will have to be able to work out dilutions for tubes and cfuml of original culture Do examples provided on Moodle o No calculators will be allowed V Be sure you can spell the names correctly underline Genus and species separately and capitalize rst letter of Genus Go through experiments 120 looking for microorganisms used in the experiment or mentioned in the background information Write the full name of the organism in the first column and then in the second column write pertinent information such as characteristics lab results etc Form CENTRAL causes coccus NO M tuberculosis M leprae M marcescens to Coliform indicator organism for fecal contamination in US Green metallic sheen from colonies on EMB agar strong lactose fermenter Not salt tolerant aerogenes Coliform colonies on EMB lactose cerevisiae 39 extreme to many Pseudomonas Proteus Experiment 1 Culture Medium contains nutrients such as carbon and nitrogen which are required for cells to grow Complex Medium contains one or more chemically unde ned substances such as yeast and meat extract De ned Medium is one whose precise chemical composition is known Liguid Broth Solid uses agar extracted from Rhodophyta 7 after heating up to 100 C solidi es at 4042 C and won t melt again until 8090 C Plates in petri dish 7 used for isolation and growth of microorganisms Deeps in test tubes 7 used for anaerobic growth Slants in test tubes 7 used for maintenance of stock cultures of microorganisms Nutrient Agar pH 68 complex medium general purpose media for growth and culture of many microorganisms Tryptic Soy Agar pH 73 de ned medium Sabouraud Dextrose Agar ph 56 de ned medium 7 used for cultivation of yeast or fungi Sterilization removal or killing or all living organisms and their viruses from a growth medium 1 Autoclave moist heat sterilization of media glassware etc 7 121 C 15 psi pressure for 15 minutes 7 kills almost all bacteria and their endospores Dry Heat Sterilization kills by oxidation effect 7 aminghot air sterilization 7 useful for glassware loops Filtration for heat sensitive liquids or gases antibiotics beer 7 membrane made of cellulose acetate or cellulose with many tiny holes that physically trap the microorganisms but lets liquids gases pass through 4 Gaseous Chemosterilization cold sterilization 7 uses no heat 7 ethylene oxide toxic and explosive 7 heat sensitive materials like plastic petri dishes syringes leather wood paper metal and rubber products interplanatary spacecrafts Electromagnetic Radiation UV gamma Xrays microwaves and electrons 7 UV affects DNA by making thymine dimers and kills exposed organisms only good for exposed surface because of poor penetration capacity through solid opaque light absorbing surfaces 7 ionizing radiation causes ions and reactive molecules to fonn which degrades biopolymers such as DNA and proteins 7 used in food and medical industry 9 V39 How are the following sterilized Liquid media autoclave heat sensitive liquids ltration colal sterilization heat sensitive antibiotics ltration petri dishes colal sterilization bandages electromagnetic radiation Experiment 2 even though most microorganisms are not pathogenic they can cause diseases to sicldinjured people in hospitals 7 hand washing sterilization and cleaning is essential in preventing nosocomial hospital acquired infections Experiment 3 infectious dose number of microorganisms or viruses sufficient to cause an infection NormalResident Microbiota Flora lives and multiplies in or on the body 7 mostly associated with sebaciousapocrine gland where it is moist and sufficient nutrients are present skin is often dry and acidic 7 does not normally cause disease but it might 7 sometimes ora on the skin may cause disease if it reaches blood or tissue Transient Microbiota Flora may be present on body but not firmly entrenched 7 cannot multiply usually dies 7 skin pH is 46 acidic dry hand washing may wash off transient ora and expose normal ora 7 may wash some normal ora off too Experiment 4 Colony group of genetically identical bacteria arising from a single cell on an agar plate Colony Morphology characteristics of the colonies like shape margin type elevation pigmention 7 genetically different colonies can have the same colony morphology colony morphology depends on the medium it is growing on 7 size shape and even color can be affected by amount of nutrients in the medium Contaminant an unwanted organism accidentally introduced to the culture Colony Mogpholo gy 1 Whole colony shape round irregular rhizoid 2 Elevation convex umbonate at 3 Margin shape smooth lamentous lobate 4 Optical properties opaque translucent 5 Surface characteristics shiny dull 6 Color Experiment 5 Aseptic Technigue wipe bench top with vesphene and clean paper towel tie back long hair wash hands after lab keep only required materials on lab bence use aseptic technique sterilize inoculating loop pass glass tube through ame hold culture tube at an angle never place cap on bench top Experiment 6 Pure Culture contains only one kind of microorganism Streak Plate technique used for diluting bacteria on agar so as to isolate bacteria from a mixture Experiment 7 Prokaryotic cells 7 05 to 2 micrometers Oil immersion objective 7 1000x magnification Staining makes cells more visible by providing contrast most of the cell is water 7 rst step to making a smear determine cell morphology Heat Fixation kills the bacteria destroys autolytic enzymes and causes cells to adhere to the slide Experiment 8 l Cocci 7 39 39 J39 39 39 tetrad 2 Bacillus 7 diplobacilli divided cell did not separate streptobacilli chain of unseparated divided cells coccobacilli short oval cells 7 can be induced by less than optimal growth conditions or genetic makeup 7 staphylobacilli does not happen because bacilli do not divide along their short axis 3 Spiral 7 vibrio bent rods spirochete helical and exible like a corkscrew spirilla helical and rigid 4 Prosthecate cells 7 V make prosthecae cellular appendages that increase cell surface area and aid in transport of nutrients into the cell 5 Filamentous growth 7 actinomycete like I 7 produce lamentshyphae that makes a network mycelium and the hyphae later differentiate and fragment to produce spores Experiment 9 Total Magni cation magni cation of ocular lens 7 10x x objective lens 7 4x 10x 40x 100x depends on resolution Microscope used Compound consists of a multiple lenses 7 ocular and objective Binocular two eye pieces Resolving Power is the closest spacing between two objects at which the two objects can be identi ed as separate entities Immersion Oil 7 same refractive index as that of glass slide and lens 7 reduces the amount of light lost and increases resolution Experiment 11 GRAM POSITIVE violetblue GRAM NEGATIVE red Thick peptidoglycan layer 7 forms complex when mordant iodine is added to crystal violet stain Thin peptidoglycan layer Techoic acids present 7 alcohol and phosphate 7 gives antigenic properties No teichoic acids I39 I I No outer membrane 7 ides quotA l Outer 39 phospholipids Experiment 12 when water or essential Endospore 7 dormant highly durable and dehydrated survival structure gram positive rods 5 nutrients are depleted 7 no metabolic activities like in vegetative cell 7 cryptobiotic state Sporulation Forespore Replicated chromosomes and some cytoplasm is isolated by a spore septum ingrowth of the cytoplasmic membrane 7 later becomes a double layered plasma membrane around genetic material 2 Cortex Peptidoglycan laid down between the two plasma membranes 7thick layer 3 Spore coat Made by spore proteins 7 r quot for 39 to 39 39 39 7thick layer 4 Core contains cell wall cytoplasm cytoplasmic membrane and nucleoid contain DNA RNA ribosomes enzymes 7 dipicolinic acid dipicolinic acid and calcium complex heat resistance Germination 7 endospore returns to vegetative state and there39s outgrowth visible swelling due to water uptake and synthesis of DNA RNA proteins 7 cell immerges from spore coat resistant to heat boiling radiation dessication chemicals freezing Three common diseases caused by G endosporeforming rods 1 Botulism 7 Clastridium batulinum 2 Anthrax 7Bacillus anthracis 3 Tetanus 7 Clastridium tetani Experiment 13 7 mycolic acid cell wall complex branched chain hydroxy lipids Primary stain carbolfuschin red 7 mixture of the dyes basic fuschin and phenol phenol anal gentle heating drives the red fuschin in stain made in egg albumin 7 surface lipids causes cells to fonn aggregates hard to disperse in water Colony morphology compact wrinkled appearance 7 hydrophobic 7 impermeable to nutrients Experiment 14 Flagella 7 locomotion 7 Prokaryotes rotating motion Eukaryotes whipping motion Proka otic agella basal body or motor 7 connected to curved hook 7 connected to lament made of agellin proteins 7 driven by proton motive force when protons or Na ions come into the cell from periplasm through Mot protein and physically turn the moton Archaea agella mostly like prokaryote but has differences in width and how agella uses energy Types 1 Polar 7 monotricous l on one end lophotrichous a bunch on one end amphitrichous one or a few at both ends 2 Peritrichous several all over Motility Test Medium gt low agar concentration so motile bacteria can move around gt tetrazolium salt 7 electron acceptor 7 colorless and soluble when oxidized 7 red and insoluble when reduced MOTILE BACTERIA red spread around inoculation line NONMOTILE BACTERIA redness in just in the inoculation line Experiment 15 METHOD ADVANTAGES DISADVANTAGES Direct Microscope Count counting chamber Rapid no culturingincubation no selection against Counts alive and dead cells needs more skills need certain organisms hemacytometer Standard Plate Count viable count Measures only living cells Incubation selection against certain organisms counted as cfumL needs skill in dilution a Spread Plate for organisms that need oxygen Easy to do Needs skill in spreading same as above b Pour Plate No alcohol or ames involved Need melted agar trickier same as above Turbidimetric Assay Rapid no incubation Can t use for microorganisms that grow in clumps Experiment 16 Coliforms l aerobic or facultative anaerobic 2 rodshaped 3 gram negative 4 nonendospore forming 5 produce gas from lactose within 48 hours of incubation in 35 degrees C Potable water Most Probable Number Index 3 22 coliforms per 100 mL water Selective Media selects against certain types of organisms so only the organism of interest can grow in it Differential Medium gives visible indication of a physiological characteristic of a microorganism 1 Phenyl Ethyl Agar PEA selective medium 7 selects for G organisms 2 Manitol Salt Agar MSA differential and selective 7 selects for salt tolerant organisms and di erentiates manitol fermenters from non manitolfermenters 3 Eosin Methylene Blue agar EMB Lauryl Sulfate Lactose LSL or Lauryl Tryptose Broth selective and differential 7 selective for G7 bacteria and di erentiates lactose fermenters from non lactosefermenters Three steps to MPN Technique 1 Presumptive Test 7 count the number of tubes of each dilution in LSL broth that show gas production from lactose fermentation and consult table 2 Confirmed Test 7 take loopful of positive LSL broth and transfer to BGLB broth 7 positive is gas formation in Durham tubes 3 Completed Test 7 Do a streak plate on EMB plate with positive LSL or BGLB broth 7 lactose fermentation produces acids and pH drops to allow cells to uptake dyes 7 good fermenter black bad fermenters colorlesspink 7 ver good fermenter black with green metallic sheen on EMB plate 7 not very good though related to EColi 7 center of the colony takes up dye and outside is pinkish 7 sh eye colonies 7 not a lactose fermenter pinkish red colonies Experiment 17 Pasteur discovered that gentle heating can kill microorganisms 7 pasteurization heating rapid cooling Thermophiles love very high temperatures to grow in hot tubs hot springs hot geysers Mesophiles need medium temperatures comfortable to humans to grow in Psychrophiles love very low temperatures to grow in refrigerator artic regions Range of temperature temperatures that organisms can grow in Optimum Temperature temperature they grow in most rapidly Temperatures above optimum proteins enzymes become denatured thermal destruction of nucleic acids and cellular structures 7 irreversible 7 eventually causes death Temperatures below optimum rate of cellular enzyme activity diminishes lipids in cell membrane solidify and impair membrane functions 7 inhibit cell growth 7 likely not kill 17 A gt testing the capability of 395 L a o survive at various temperatures 7 not grow Deinococcus cannot really survive beyond 60 degrees C 7temperature sensitive 17 B gt test optimal pH and pH range for 3977 Optimum pH 7 pH at which organisms grow best 7 maximum rate of enzyme activity pH Range gt one unit above and below optimum at which organisms can still grow Out of pH range alterations to the 3D structures of proteins 7 extreme deviation can cause denaturation of proteins Acidophjle causes acne Sabouraud39s Agar pH 56 is selective for these ow best at s lives on skin pH 7 4 Neutrophiles 7 grow best at neutral pH 7 7 BasophilesAlkalinophiles gt prefer pH gt 7 7 quotquot he pH optimum of 9 can grow in pH 12 soil 17 C gt test bacteriocidal effect of UV 260 nm light is especially bad because it is absorbed by nucleic acids 7forms thymine dimers 7 result in mutation or cell death Lethal mutation when a mutation causes the cell to be unable to make necessary enzymes gt ability to repair damage done to DNA by UV light gt can survive UV by forming endospores 17 D gt test the tolerance of various organisms to different salt concentrations Osmosis ow of water from area of less solute to area of more solute through a semipermeable membrane to attain equilibrium Hypertonic more solute outside the cell than inside the cell 7 water ows out of the cell 7 plamolysis cell membrane pulls away from cell wall 7 bacteriostatic condition results Hypotonic more solute inside the cell than outside 7 water ows into the cell RBCs don t have cell wall and will burst 7 bacteria have cell walls that limit swelling when water goes in relatively unaffected Isotonic same concentration of solute inside and outside the cell 7 water ows equally in and out of the cell 7 equal osmotic pressure Halophiles 7 love salt 7 g Extreme Halophiles 7 grow in very high salt concentration 7 mostly Archaea species 7thrive in 153 Halotolerant 7 can grow in relatively high salt concentration but thrive best in low concentrations 7 grow best in less than 1 NaCl 39 grow in marine waters optimal salt concentration greater than 3 0 NaCl z grow in 75 NaCl but Experiment 18 Antimicrobial agents static agents inhibit growth only does not kill 7 fungistatic virustatic bacteriostatic 7 cidal agents kill cells 7fungicide virucide bacterocide Antiseptics 7 can be used on skin 7 ethanol iodine Disinfectants 7 use on inanimate objects 7 ethanol bleach Zone of inhibition clearing around antimicrobial disk where no growth has occured 7 bigger this is the more effective the antimicrobial agent is depends on concentration of bacteria type of medium effectivity of antimicrobial agent depends on contact time and concentration of agent Experiment 19 Minimum Inhibitory Concentration 7 amount of drug present at the outer edges of the zone of inhibition Antibiotic targets 1 ribosomes affect translation protein synthesis 2 cell wallcell membrane affect the stucture 3 DNA replication Mode of action the way in which the antibiotic kills or inhibits growth of the microorganism PENICILLIN prevents synthesis of peptidoglycan 7 weak cell wall cell undergoes lysis 7 G bacteria most affected Experiment 20 Capsule formed in excess of nutrients 7 polysaccharide or polypeptide water 7 prevent dessication and aid in sticking to surfaces reserve energy source 7 polysaccharide capsule 7 olypeptide capsule 7 roduce dextran and levan 7 stick to tooth enamel and cause tooth decay 7 capsule resists phagocytosis If capsule enhance the ability of a microorganism to cause disease it is called a Virulence factor V 1 1 J aureus Experiment 20 catalase l aerobic catalase positive Experiment 31 isolation amp ID staph normal ora yellow on MSA coagulase gram positive ferments mannitol pathogenic produces enterotoxin thick plasma appearance causes toxic shock causes plasma to clot Staphylococcus epidermidis Experiment 20 catalase l 39 quot aerobic catalase positive Experiment 31 isolation amp ID staph normal ora does not ferment mannitol few virulence factors runny plasma appearance white on MSA coa 11ase Experiment 33symbiosis facultative anaerobe removes oxygen so C I can survive Micrococcus I Experiment 20 catalase l 39 quot I aerobic catalase positive I Lactobacillus I Experiment 20 catalase l 39 quot I catalase negative I Experiment 28 yogurt produce lactic acid grow when pH drops below 6 i gram positive anaerobes Lactococcus lactis Experiment 23 fermentation of carbs I did not ferment milk pH 6 I Saccharomyces cerevisiae Ex eriment 23 fermentation of carbs roduce acid and as in both malt extract and a e 39uice Bacillus cereus Experiment 23 fermentation of carbs growth and acid production in glucose and sucrose S mutans I Experiment 20 catalase production I fermentative catalase negative I Experiment 27 isolantion amp ID strept produce alpha hemolysin green colonies aka viridians develop quot 39 39 39 S pyogenes Exp 20 catalase production fermentative catalase negative Exp 27 isolation amp ID strept strep throat produces a beta hemolysin scarlet fever colonies have a clearing produce streptokinase Enterococcus I Exp 20 catalase production I catalase negative Escherichia call 21 indole positive VP negative MR positive citrate negative Exp 26 nitrate reduction no gas production reduced to NO Exp 30 bacteriophage titration infected by phage lambda Bacillus subtillis Exp 21 amylase production I amylase positive I Exp 22 caseinase amp gelatinase I caseinase amp gelatinase positive Citrobacter 39eundii I Exp 23 fermentation of carbs I growth acid amp gas productions for glucose lactose amp sucrose I Exp 24 cysteine desulfhydrase I cysteine desulfydrase positive Enterobacter aerogenes Exp 25 IMViC may be found in feces MR negative found in environment VP positive found in decaying vegetation citrate positive indole negative Paracoccus denitri tcans Exp 26 nitrate reduction I produced gas reduced to N2 Clostridium sporogenes I Exp 33 symbiosis I obligate anaerobe ENZYME SUBSTRATE PRODUCT REAGENT RESULT MEDIA ORGANISM INDICATOR S epidermidis Streptococcus Catalase Hydrogen Water amp oxygen Bubbles of None perOXide bubbles 02 M icrococcus nr tnhnr illi ilt Oxygen Oxidase Tetramethylp Water OXidase Bluepurple TSA PEpL t39c phenyleneidamine reagent 39 Amylase Starch Oligosaccharides Grams iodine I Starch B subtilis of glucose hydrolysis agar E coli Oli osaccharides Yellow De radation DUbOS Cyto ha a Cellulase cellulose g 9 plate and p 9 of glucose pigmentation of paper broth hutchinsonii Caseinase Casein B subtilis Peptides hydrolysis Milk agar E 00 Gelatinase 1 N Gelatin peptides hydrochloric Gelatin B SUb HS hydrolysis Agar E coli acid Cysteine desulfhydrase H2Sferrous Ezlfaelfrl gt Peptone C freundii Cysteine sulfide Ferric citrate iron agar E coli sulfide Kovacs 1 E coli Tryptophanase Tryptophan lndole Red layer tryptone E aerogenes reagent broth Simmons E aerogenes C39trate39 Citrate source of Alkaline products BromOthymOI Blue C39trate permease blue agar slant Carbon E coli Alpha E coli reg rcat fse Nitrate Nitrite naphthylamine Red P acetate amp denitrificans sulfanilic acid Nitrite Nitrite Nitrous 0Xide or Bubble in Nitrate Paracoccus reductase Durham tube broth E coli coagUIase Coagulated S Fibrinogen Fibrin liquid 9 Plasma S epidermidis solid 4 39 Welcome to the Microbial World BIOL 2051 General Microbiology Dr Ginger Brininstool Of ce Phone 5784089 Of ce 602 Life Sciences Bldg Of ce hours 93011 MTh Email gbrininlsuedu 39 EIDLDGY OF Mtcaooamulsms H l a I hi Tl tli 39 39 T uHL quot39 l l Bookstores Bound w supplements 190 etext 126 Rent 93 Online Rent wwwcheggcom 87 Try old 5 discount code 00100089 Buy wwwamazoncom wwwmypearsonstorecom To purchase textbook new comes with supplements and full web access Bound 190 Etext 127 Moodle Syllabus Lecture material Lab Information and Quizzes Bring 1250 to lab Tuesday to buy kit with your lab partner kits cost 25 Bring disposable gloves or bring another 10 to buy a box of gloves in lab What is Microbiology The study of cells and organisms too small to be seen with naked eye Microbiologists study Bacteria Viruses Protozoans Fungi Algae Diatorns And more Diversity Fig 217 BACTERIA ARCHAEA EUKARYA Animals Slime Entamoebae molds Green nonsulfur Euryarchaeota bacteria M ethanosarcma Mitoch ndrion Met Extreme Crenarchaetoa bacterium halophiles Thermoproteus Proteobacteria Ciliates O 0 N SE tn Chloroplast Pyrodictium Thermoplasma C anobacteria Thermococcus Flavobacteria y Flagellates Mar ne Methanopyrus Trlchomonads Thermotoga Microsporidia Thermodesulfobactenum Di p lo mo n ad 5 Aquifex Giardia 3 Domains of Life Bacteria Archaea and Eukarya Microorganisms are Bacteriophage T4 Not cellular Bacillus Unicellular Anabaena Multicellular Form Groups Characteristics of cells Characteristics of cells f Cell 0 0 k ii Environment Spore p o a 0 l 7 1 3 i i Ancestral Q Slew I quot peCIes 00 ce 7 0 fquot N I fgtsies Figure 13 Microbial Communities Sewage sludge of Bacteria on Earth 25000000000000OOOOOOOOOOOOOOOOO Habitat Percent of total Marine subsurface 66 Terrestrial subsurface 26 Surface soil 48 Oceans 22 All other habitatsb 10 aData compiled by William Whitman University of Georgia USA refer to total numbers estimated to be about 25 X 1030 cells of Bacteria and Archaea This enormous number of cells contain collectively about 5 X 1017 grams of carbon bIncludes in order of decreasing numbers freshwater and salt lakes domesticated animals sea ice termites humans and domesticated birds Origin of Earth 46 bya Earth N2 C02 CH 4 atmosphere oxygenated How do microbes impact humans with regard to hS ONT Agriculture Energy Disease Food Agriculture Rumen 1 Grass b Cellulose leucose Microbial fermentation I Fatty acids CO2 CH4 Nutrition for animal Waste products Energy biofuels Methane Disease Cause amp Treatment Death rates for the leading causes of death in the United States Influenza and pneumonia Tuberculosis Gastroenteritis Heart disease Stroke Kidney disease Accidents Cancer Infant diseases Diphtheria 1900 100 200 Deaths per 100000 population Heart disease Cancer Stroke Pulmonary disease Accidents Diabetes Alzheimer s disease Influenza and pneumonia Kidney disease Septicemia Suicide Today I Infectious disease I Nonmicrobial disease 100 200 Deaths per 100000 population MICROBIOLOGY LAB FINAL Experiment 21 Metabolism is the sum of all reactions associated with the life ofa cell Enzymes a proteins biological reaction catalysts b without enzymes reactions would not occur fast enough for cells to live c spec c can only catalyze one reaction and react with one substrate Characterized by 1 Location Extracellular enzymes a act outside of the cell in which they are produced b break down large molecules into small ones for easy transportation in and out of cell membrane so it can be transported to where its needed Eg amylase enzyme that breaks down starch into glucose molecules Endoenzymes a used inside the cell b degrade molecules to release energy or use energy to build molecules 2 Time InducibleAdaptive enzymes a only produced when certain substrates are available b energy saving mechanism Constitutive enzymes a essential to the cell b produced at all times Macromolecules large made of several small subunits joined by dehydration reactions all reactions are reversible can be broken down into substituent components all the reactions are catalyzed by speci c enzymes Morphology vs Physiology Morphology to study the cell shape size arrangement of bacteria Physiology to study metabolism ofthe bacteria the kind of enzymes it produces and how it reacts biochemically tells you how an organism functions in the environment Catalase Test l useful for differentiation between genera 2 catalase breaks down HZO2 into water and 02 W this is what makes the bubbles 3 H202 byproduct of aerobic respiration toxic produced by all aerobic bacteria only i if fermentation or aerobic respiration occurs this is not produced Experiment 22 Oxidase Production 1 used to differentiate between genera like catalase test eg 39 2 Cytochrome oxidase enzyme catalyzes the oxidation of cytochrome a member ofthe electron transport chain and the subsequent reduction of 02 nal electron acceptor to H2O or H202 toxic catalase or peroxidase enzymes break it down 3 So organisms that use oxygen to produce energy obligate aerobes facultative aerobes microaerophiles will be oxidase positive since oxidase enzymes only work in the aerobic electron transport systems 4 tetramethylilrphenylenediamine dihydrochloride oxidase reagent fake substrate for cyt oxidase to act on in the presence of O2 put on the the colony directly positive bluepurple negative no color change Note Don t leave it on for too long or 02 from the air will react with it and make it purple and make you think the bacteria is Ox when it is really Ox Experiment 23 Signi cance of amylases component in laundry detergents used to produce glucose and syrup from starch remove coatings in the paper and textile industry coverting cellulose renewable energy resource to glucose glucose fermentation to ethanol and other chemicals 03M Types of Carbohydrates l Monosaccharides single sugars ve carbons ribose or six carbons glucose 2 Oligosaccharides composed of one of more monosaccharides 3 Polysaccharide polymer of glucose structural components reserve carbon and energy souce Cellulose glucose units joined by betal4glycosidic bonds upto 14000 glucose molecules in one cellulose bril major structural component of many plants produced by some bacteria A lot of bacteria and fungi in soil and rotting wood degradation of cellulose by cellulase aerobically or anaerobically Glycogen polymer of glucose reserve energy source for animal including human cells Dextran polymer of glucose Starch macromolecule reserve food source for some plants glucose units joined by alpha l4 glycosidic bonds Amylase extracellular enzyme can hydrolyze starch and break the glycolysidic bonds Starch Agar looks like nutrient agar starch is dissolved with agar sterilized starch reacts with iodine to give brownblue color Amylase produced gt after flooding with iodine there will be a zone of hydrolysis where no brownblue will be seen Amylase not produced gt no zone of hydrolysis Experiment 24 Proteins W macromolecules W composed of one or more polypeptides long chains of amino acids joined by peptide bonds Proteins can be W structural WW enzymes biological catalysts WW nutrientenergy souce Proteases are enzymes that hydrolyze proteins in shorter chains of amino acid called peptides Peptides are then degraded into individual amino acids for use by the cell for its metabolism Casein WWgt milk protein W gives milk its whiteness and opaqueness WWgt caseinase On milk agar plate if caseinase is produced there is a clear zone of hydrolysis Gelatin Wgt protein extracted from animal connective tissue and bone marrow WWgt gelatinase On Gelatin agar plate addition of HCl will precipate the gelatin in the agar and make it go white lf gelatinase is produced a clear zone of hydrolysis will be seen Why is gelatin not good as a solidifying agent in bacteriological media 1 it melts at 37 degrees C 2 Some microorganisms produced extracellular enzymes eg gelatinase that degrades gelatin Experiment 25 Carbon Source Energy Source Heterotroph W organic compound Phototroph W light Autotroph W atmospheric C02 Chemotroph W chemical Chemolithotroph W Chemoorganotroph W inorganic compound organic compound Fastidious organism has high nutritional requirements and will need special conditions to grow Glycolysis W process of converting glucose to pyruvic acid W common to fermentation and respiration 1 Terminal electron acceptor present gt respiration substrate and oxidative level phosphorylation produces water CO2 and ATP a Terminal electron acceptor is 02 gt aerobic respiration b Any other terminal electron acceptor gt anaerobic respiration 2 No terminal electron acceptor present Wgt fermentation only substraterlevel phosphorylation products can be acidic or neutral Lactic acid bacteria eg Lactobac us Streptococcus lactic acid from sugars gt used to make yoghurt and cheese from milk Yeast eg Saccharomyces cereVI sI ae produces CO2 and ethanol from sugar gt used to in bakery and brewery industries Phenol Red pH 7 quotgt orange pink neutral pH lt 69 gt bright yellow acidic pH gt 7 gt fuschia basic Durham Tubes inverted tubes meant to capture gas ifthere is any gas production if full with gas they oat on the broth Experiment 26 Amino acids 20 ofthem have an carboxyllic acid group and an amino group attached to the alpha carbon Cysteine and Methionine have a sulfhydryl group which plays a role in protein structure by making disul de linkages between amino acids These are used by some organisms as their electron source The cleavage of the sulfhydryl group is the first step of metabolic breakdown The reaction is catalyzed by cysteine desulfhydrase Cysteine quot cysteine desulfhydrase Wgt pyruvate ammonia H28 H28 ferric citrate gt ferrous sul de FeS W black ppt Medium PEP TONE IRON AGAR DEEP made of peptone which is made of animal proteins and is rich in cysteine and ferric citrate Experiment 27 See page 153 oflab manual Experiment 28 Anaerobic respiration uses anything BUT oxygen as terminal electron acceptor eg carbonate sulfate nitrate Some organisms use nitrate to process hydrogen produced in respiration Denitri cation reduction of nitrate iigt nitrite gt nitrous oxide gas gt dinitrogen gas Wgt ammonia by some prokaryotes Nitrogen Fixation using atmospheric nitrogen to make ammonia by some microorganisms Do tests for each product starting with ammonia and working backwards l Ammonia smell for it 2 N20 and NZ gas bubbles in Durham tubes 3 Nitrite red color after adding alphanaphthylamine acetate sulfanilic acid 4 Nitrate after testing for nitrite if there is no color change add Zn Zn turns nitrate to nitrite and gives red color since nitrite identi cation reagents are already in solution Medium nitrate broth with Durham tubes Experiment 29 Nitrogen is found in amino acids nucleic acids proteins and some phospholipids Most organisms need to ingest organic nitrogen Some prokaryotes can x atmospheric nitrogen to ammonia using two enzymes dinitrogenase and dinitrogenase reductase nitrogenase enzyme complex Dinitrogenase reductase can be inactivated by oxygen Protection mechanisms 1 Cyanobacteria compartmentalize nitrogenase into special cells called heterocysts whose sole purpose is to x nitrogen 2 Azotobacter uses a third protein to make a complex with the nitrogenase complex to conformationally protect it from oxygen inactivation and produces oxygeniretarding alginate capsule Symbiotic Nitrogen xing organisms Freeliving Nitrogen xing organisms Aerobic Anaerobic eg Rhlzoblum in root nodules oflegumes 8g Azowbactef eg COsmwufn Medium Nitrogenifree broth and plate used so that only organisms that can x atmospheric nitrogen can grow Mannitol is added as a carbon source because Azotobacter can easily use mannitol as a carbonisource and will ourish Experiment 30 Streptococci gram positive catalase negative cocci found in chains produce hemolysins virulence factors facultative anaerobes obligate anaerobes aerotolerant microacerophillic most human strains fastidious organisms need special controlled conditions to grow Diseases pneumonia strep throat meningitis impetigo scarlet fever Virulence factors hemolysins colonization factor Streptokinase invasive factor 1 Hemolysin proteins that cause damage to animal cells and cause them to lyse 2 Streptokinas dissolves brin clots that lets the organism invade tissue and spread infection Medium 5 blood agar place i 5 sterile sheep blood added to sterile tempered cooled to just above solidi cation medium Growth Conditions lt20 oxygen increased 5lO carbon dioxide environment incubate at 37 degrees C Hemolysis alphahemolysis betahemolysis gammahemolysis Incomplete lysis of RBCs Complete lysis of RBCs No lysis of RBCs Greenish around alpha Looks like a clearing around hemolytic bacteria betaihemolytic colony Green color produced from formation of methemoglobin from hemoglobin Called Viridans Streptococci Experiement 31 Lactic Acid Bacteria Gram positive Catalase negative some degrade peroxide using peroxidase Aerotolerant can grow in the presence of oxygen but do not use it for respiration Ferments sugars to produce lactic acid Homofermentatiye Heterofermentatiye Produces only lactic acid from sugar Produces lactic acid C02 and ethanol from sugar Succession one species has to establish itself rst before another species can grow 1 Streptoccus establishes itself and makes acids 2 Once the pH lt 6 LactobacIYHS can start to grow 3 LactobacIYHS ourishes and makes more acids and pH lt 5 4 Streptococcus dies Medium MRS DeMan Ragosa Sharpe Medium selective for LactobacIYUS because oflow pH Experiment 32 Microorganisms in nature occur in bio lms at waterrair or water solid surfaces Cell attach and secrete polysaccharides The polysaccharide matrix is sticky and more cells stick to it Cells communicate with each other using quorum sensing mediated by homoserine lactones when enough cells are present enough of the molecule is present and it induces them all to do something like secrete an enzyme or make virulence factors more ef cient that one cell secreting tiny amounts of stuff over a long time Bio lm infections are harder to treat immune cells and antibiotics cannot penetrate all the layers of the bio lm LIGHT MICROSCOPY Bright eld Dark eld Phasecontrast Light passes through directly Has a condenser that allows Direct rays light rays that from condenser to the eye light to only pass through the pass through the specimen specimen from the sides unaffected No intervening plate in Light that is not defracted does Diffracted rays light rays that condenser to de ect the light not enter the objective and are bent after passing through a contributes to the image you sample see Specimens need to be heat No staining no need to kill Uses condensers and objectives xed killed and strained for microorganisms that takes advantage of this viewing difference in refractive index and ampli es it Because most microorganisms lmages appear light on a dark lmage appears with signi cant are mostly water unstained background contrast to its surrounding specimens do not provide enough contrast viewing live motile organisms is hard Experiment 33 An infectious virus virion RNA or DNA genome has a protein coat called capsid around it i may have an envelope surrounding the capsid Very host speci c an animal virus can not only infect animals but only one type of animal Virus life cycle 1 Attachment to the host cell Injection release of viral nuclear genome into host cell Replication production of more viral components Assembly and packaging of viral components into new viruses Release of new virions 01 pr Viruses that affect bacteria phage or bacteriophage Lytic phases only lytic cycle and Temperate phases lytic and lysogenic cycle Plaque Assay Infected bacteria will lyse and cause clearings in the lawn viral plaques done by the lysereinfectionlyse nature ofviral infection new viruses get released from a lysed cell and infect adjacent cell counting the number of plaques and doing a dilution factor correction gives the concentration of phage in plaque forming units per mL p lmL pfumL is used because more than one virus can infect a cell and cause the same plaque to form Experiment 34 Staph00000115 gram positive catalase positive cocci found in clusters halotolerant part of the normal ora Medium MSA Mannitol Salt Agar selective and differential 75 salt concentration selective for halotolerant organisms and differential for mannitol fermentation Indicator phenol red pH 7 gt orange red pH lt 66 gt bright yellow pH gt 7 gt fuschia pink Plasma liquid portion of the blood containing brinogen To test between S aureus and S epidermidis check for mannitol fermentation and coagulase production Stapblococcus aureus Stapblococcus epIHermidis pathogenic nonrpathogenic Produces many virulence factors like beta Does not produce many virulence factors hemolysins and coagulase Responsible for diseases like staphlococcal food Does not cause many diseases poisoning gastroenteritis toxic shock syndrome Yellow pigment on MSA plate Colorless colonies of MSA plate Ferments mannitol yellow MSA Does not ferment mannitol red MSA Experiment 35 Symbiosis intimate relationship or association between two dissimilar organisms Five types Mutualism both organisms bene t from the association eg Lichens heterotrophic fungus and photosynthetic alga or cyanobacteria photosynthetic alga or cyanobacteria xes C02 and provides organic compounds to fungus and fungus protects it from high light intensity gives it water and minerals Rhizobium and Bradyrhfzobl um in legume root nodules nitrogen xing bacteria provide nitrogen for the plant and plant provide photosynthetic product organic molecules for the bacteria Commensalism one organism bene ts whereas the other is not affected eg Clostn dium Sporogenes is an obligate anaerobe that cannot survive in an oxic environment Staphylococcus epidermidis which is a facultative aerobe needs to rst grow and use up the oxygen before C Sporogenes can grow it survives until then by forming endospores Same thing between Bacteroia es obligate anaerobes in human gastrointestinal tract and Eschl39 chia c01 facultative aerobes Synergism both organisms bene t but the relationship is not necessary eg Enterococcus and LactobacIYHS can both grow separately in enriched media but needs the association in minimal media to grow Parasitism one organism bene ts while the other is harmed eg eas viruses obligate intracellular parasites Ede10wbe invading and multiplying within E c01 cell Antagonism one organism produces something to inhibit the growth ofthe other organism eg Pem c I um and Streptomyces produce antibiotics that prevent the growth of other organisms Lactobacillus and Streptococcus produce lactic acid which prevents the growth of other organisms too Experiment 36 39 gram negative aerobic rods live in root nodules of white clover Crop Rotation the main cash crop is substituted with a leguminous plant like beans alfafa peas plants that bear its seeds in a pod after a certain number of growing seasons to restore the nitrogen content ofthe soil alternative to adding artificial nitrogen fertilizers Stages of Rhizobfum root nodulation l Bacteria attaches the root hair oflegumes 2 A speci c polysaccharide on the outer later of bacterial cell wall associates with lectin a protein on the surface ofthe root hair 3 Bacteria enters the root hair cells and moves through them by forming an infection thread 4 The bacteria stimulates certain root cells to divide repeatedly and form a nodule 5 When the plant dies the nodules disintegrate and some ofthe dormant bacterial cells survive and reproduce in the soil and infect other plants Leghemoglobin ironicontaining protein with a heme group similar to hemoglobin in animals binds to oxygen in the nodules and regulates it so it is enough for the aerobic bacteria to grow but so high that it inactivates the nitrogeni xing nitrogenase enzyme gives nodules its red or pink color Experiment 37 Points of comparison between some important organisms Gram Rods Bacillus Sphaez icus vs Bacillus megaterium gt glucose and caseinase tests Baalus megaten39um Bacillus sphaericus Produces acid from glucose Does not produce acid from glucose Caseinase Caseinase Gram Cocci Staphylococcus vs Streptcoccus quotgt catalase test Staphylococcus Streptococcus Catalase Catalase Staphylococcus aureus vs Staphylococcus epidez midls Wgt mannitol fermentation coagulase test Staphylococcus aureus Staphylococcus epIHermidis Mannitol Mannitol Coagulase Coagulase Mlcrococcus luteus Wgt DOES NOT produce acids from glucose lactose or mannitol Gram Rods Appearance on EMB plate ability to ferment lactose Escherichia coli Enterobacter aerogenes Citrobacter eun d139139 Pseudomonas aeruginosa Green sheen on the colonies ton of acids produced from lactose fermentation Fislreye colonies dark only in the center a not as good at lactose fermentation Fishieye colonies dark only in the center i not as good at lactose fermentation No acids produced lactose not fermented Ehtefohacter aerogehes vs Citrohacter euhdii W Methyl Red and VoguesiProskaur tests lMViC Cysteine desulfhydrase production test for HES Enterobacter aerogenes Citrobacter eun d139139 MR Butanediol fermentation MR Mixediacid fermentation HES cannot use cysteine as electron source HES can use cysteine as electron source Proteus vulgans Wgt NO REACTION with lactose nonimotile Rhodopyta Escherichia coli Bacillus cereus Micrococcus luteus Streptomyces coelicolor Serratia marcescens Bacillus megaterium Deinococcus radiodurans Streptococcus mutans Staphylococcus epidermidis Spirosoma linguale Clostridium Bacillus Bacillus stearothermophilus Clostridium botulinum Bacillus megaterium Bm Bacillus sphaericus Mycobacterium in acid fast expr Mycobacterium turberculosis Mycobacterium leprae Entrobacter aerogens Ea Pseudomonas aeruginosa Proteus vulgaris Spirillum volutans Salmonella typhi MPN expr Vibrio cholera Shigella dysenteriae Lactobacillus genus Propionibacterium acnes Agrobacterium Saccharomyces cerevisiae Alcaligenes Names of to know tests to each from the others what is unique about each etc Details Name Staphylococcus aureus Exp 21 34 Gram cocci in clusters 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 a enterotoxin responsible for gastroenteritis l 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 epidermldls Exp 21 25 34 35 Gram staphylococci 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 Clostrldlum sporogenes Salt tolerant Glucose growth acid production no gas production Sucrose growth acid production no gas production Lactose growth acid production no gas production Mlcrococcus Catalase Exp 21 Use oxygen for respiration Lactobaclllus Gram Exp 21 31 Catalase Produce lactic acid in fermentation form sugars and is used commercially to produce yogurt and cottage cheese from mil 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 Lactococcus lactis Exp 25 Produce lactic acid Gram Homofermentative metabolism Important for manufacturing of dairy products Saccharornyces cerevlslae 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 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 hemolysin which 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 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 Cytophaga hutchinsonii Exp 23 Isolated originally from sugarcane piles Breaks down the cellulosecontaining sugarcane to use the simiple sugars as carbon and energy sources Degrades cellulose aerobically Citrobacter freundii 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 Enterobacter aerogenes Can be found in feces but may also be present in the environment and in Exp 27 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 Paracoccus den39tri cans 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 Azotobacter vineandii 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 Clostridium 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 Rhizobiurn trifoii Exp 36 Lives in symbiosis with white clover Media to know selective ingredients differential ingredients test it was used for reagents to add organisms to know significant ingredients what results look like m Details Mediu Trypticase Soy Agar TSA Exp 22 pH 73 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 Tryptophanase enzyme breaks down the tryptophan to make indole MRVP broth Methyl Red and Voges Proskauer 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 clearing v hemolysis no clearing no darkening MRS broth and agar Exp 31 Used to isolate Lactobacilus from 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 Enterobacter aerogenes fisheye phenyl ethyl alcohol agar Exp 37 17 Selective only for 6 organisms ENZYME SUBSTRATE PRODUCT REAGENT RESULT MEDIA ORGANISM INDICATOR Catalase Hydrogen peroxide Water H20 and Hydrogen peroxide Production of None Staphylococcus H202 molecular oxygen H202 bubbles 02 epidermidis oz bubbles Streptococcus mutans Oxidase Oxygen Water Oxidase reagent Turns purpleblue TSA used but Pseudomonas Ex tetramethylp where reagent is nothing speci c 39 Cytochrome phenylenediamine added needed oxidase EschIrIchIa colI if amylase is Amylase Starch Oligosaccharides use iodine to make Starch agar Bacillus of glucose a brown blue color yelloworange plate subtillus zone of hydrolysis is seen Escherichia colI Growth ofyellow Cellulase Cellulose Oligosaccharides None pigment on paper Dubos agar Cytophaga of glucose or paper platebroth hutchinsonii degradation A clear area is evident around Bacillus subtilis Caseinase Casein Peptides and under the Mi k agar plate None caseinase Ecoi producing colony Gelatinase Gelatin Peptides Hydrochloric acid Cleartransparent Gelatin agar Bacillus area plate subtillus E coli teine Cysteine amino Hydrogen sul de Ferric Citrate Black precipitate Deeps of Citrobacter desulmydrase acid w sulfhydryl H2S FeS eptone iron freundii group Ferrous sul de agar inoculated w needle E coli Tryptophanase Tryptophan lndole Kovac s Red layer on top Tryptone broth Escherichia coli reagent of broth Enterobacter aerogenes Citrate Citrate carbon Alkaline products Bromthymol blue Blue when pH Simmon s Enterobacter permease source buIlt Into agar up Citrate Agar aerogenes slantalkaline Slant indicator Nitrate Noa39 nitrate Nog39 nitrite umaphthylamine Dense deep Red Nitrate Broth Escherichia coli reductase acetate color sulfanilic acid Nitrite reductase NOQ39 Gas bubbles in Nitrate Broth 2 and N2 Nune Paracoccus nitrogen gas durham tube denitrificans Nitrogenase Dinitrogen N2 Ammonia NHa Ability to grow in Nfree Azotobacter Nune nitrogenfree brothagar vineandii brothagar S aureus Coagulase Fibrinogen Fibrin None Plasma plasma Solidi cation of S epidermidis p asma thickening Methyl Red Glucose Mixed acid Methyl Red Broth turns red Glucose MRVP Escherichia coli broth Enterobacter aerogenes Vogues Glucose Butanediol Alpha naphthol and Red not instant Glucose MRVP Enterobacter Proskaur acetoin broth aerogenes 40 Potassium Hydroxide falls from oxygen interface E coli
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