Micro 201 8/22-9/14
Micro 201 8/22-9/14 MICRB 201
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This 10 page Class Notes was uploaded by Julianna Sickafus on Thursday September 15, 2016. The Class Notes belongs to MICRB 201 at Pennsylvania State University taught by OLANREWAJU SODEINDE in Fall 2016. Since its upload, it has received 135 views. For similar materials see Introductory Microbiology in Microbiology at Pennsylvania State University.
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Date Created: 09/15/16
Microbiology 202 Notes: 8/22/16-9/14/16 Germ Theory of Disease (Koch’s Postulates 1) Microbe must be present in all cases of disease and absent from healthy organisms. 2) Pathogen must be isolated and grow in pure cultures. 3) Isolated organism must cause some disease in healthy host. 4) Same organism must be isolated again from newly diseased host. Growth of Bacteria on Solid Media -bacteria can grow in broths or on agar plates -liquid broths can contain many species -colonies grown on agar plates are from a single species; agar plates are better for isolating pure colonies Media Development -boiled potato (sterile) was used as a solid media -unreliable; not all cultures grow -an amylase enzyme is needed to break down starch in potato -adding gelatin to beef broth -this method was an improvement but: -gelatin melts at approximately 30 degrees Celsius -some bacteria contain protease enzymes which break down gelatin -finally added agar to liquid broth -agar is derived from seaweed Elements Found in Living Things -20 found -6 found in high amounts -C, H, O, P, S, N -some joined by covalent bonds: -H 2 CO 2 -broken by enzymes Formation of Proteins -primary: -ordering of amino acids in polypeptide chain -secondary: -beta pleated sheets or alpha helices -tertiary: -3D organization of individual secondary structures -primary, secondary, and tertiary structures are stabilized by weak non-covalent or covalent bonds -ie: disulfide bond -covalent -stabilizes some protein configurations -not to be confused with peptide bonds which hold polypeptides together which are covalent and hold amino acids together -quaternary: -combination of many tertiary structures -only possessed by proteins with 2 or more sub-units Resolving Distance d= 0.5/NA = wavelength NA= numerical aperature -smaller d value equals better resolution -to improve resolution: -decrease -increase NA -lower power lens= low NA 2 Types of Electron Microscopes -transmission -produces 2D image -good for showing internal structures -scanning -reveals more external structure produces 3D image Fixation and Staining of Specimens -fixation: -preserves cell structure, attaches specimen to slide -2 methods: -heat -quick, convenient, doesn’t preserve cell structure that well -chemical -more time consuming, preserves cell structure and shape -staining: -simple staining -use of one dye -differential staining -use of more than 1 dye -ie: gram staining Prokaryotic Cells -large size range 0.2-700 -2 major shapes -cocci -sphere shaped -can come in different orientations of spheres -staph: grape-like clusters -ie: staphylococcus aureus -diplo: pairs -ie: meningitis -strep: chains -ie: streptococci -bacilli -rod like -ie: salmonella -can also come in different orientations -Vibrio’s: comma-shaped -ie: cholera -bacillus can describe both cell shape and genus name other shapes: -spirochaetes -flexible, helical rods -ie: syphilis -most cocci are gram positive -with exception of Neisseria species -most bacilli are gram negative -with exception of Bacillus species Prokaryotic Cell Structure Protoplast -cytoplasm + plasma membrane Ribosome -made of 2 subunits -large subunit (50S) -small subunit (30S) -ribosomal particle is 70S Inclusion Bodies -storage granules for nutrients Nucleoid -genetic material, RNA, proteins Bacterial Cell Wall -do not exist in mycoplasmas or L-forms -mycoplasmas are genus of bacteria -many bacterial species have L-form Differences in Cell Walls of Gram Positives and Gram Negatives -peptidoglycan (PG) -single cross linked polymer consisting of: -a disaccharide (glycan) comprised of: -N-acetyl-glucosamine (NAG) -N-acetyl-muramic acid (NAM) -a short chain of amino acids -peptide of 4 alternating D and L amino acids connected to NAM -D-amino acids and DAP not found in proteins -linkage of subunits occurs between amino acid #3 and #4 of adjacent PG units -PG layer can be removed from cell by penicillin or lysozyme -in gram positives: -thick peptidoglycan layer rd -3 amino acid usually L-lysine -large quanitites of teichoic acid embedded in PG and extending into plasma membrane -aqueous space between PM and PG is called the periplasm -in gram negatives: -thin PG layer -outermembrane -endotoxin or lipopolysaccharide (LPS) on outer leaflet -lipoprotein on inner leaflet; anchored to PG -more permeable than PM Capsule -“slime layer” -external to cell wall -made of polysaccharides -virulence factor -resists phagocytosis by white blood cells -enhances adhesion to host tissues Pili or Fimbriae -short, hair like structures on cell surface -play role in attachment to host -sex pili -longer than regular pili -used in bacterial conjugation Flagella -for motility Bacterial Endospores -dormant structures formed as protection in response to adverse environmental conditions -only a few gram positives species can form them -ie: Bacillus, Clostridium -contain large amounts of dipicolonic acid (DPA) Eukaryotic Cell Structure Cytoskeleton -made up of actin (microfilaments), tubulin (microtubules), and intermediate filaments Endoplasmic Reticulum (ER) -network of tubules and flattened sacs -2 versions -rough: -contains ribosomes -involved in vascular transport and protein modification -smooth: -no ribosomes -involved in lipid metabolism and detoxification of chemicals -ie: pesticides, carcinogens Golgi Apparatus -flattened stack of sacs -receives material from ER -directs lipids/proteins to final destination -ie: secretory vesicle, plasma membrane, lysosomes Lysosome -intracellular digestion of: -macromolecules, harmful substances -ie: white blood cells in mammals via phagocytosis -contain enzymes called hydrolases -which function at low pH -lysosome must provide this environment using proton pumps to pump in H+ ions Ribosomes -active in protein synthesis -consist of 2 subunits -large subunit 60S -small subunit 40S -80S complete unit -each subunit composed of ribosomal RNAs (rRNA) and ribosomal proteins Nucleus -holds linear chromosomes -chromosomes packaged into chromatin by histones -nucleolus -site of rRNA synthesis and assembly of ribosomal subunits Mitochondria/Chloroplasts -responsible for cellular respiration (mitochondria) and photosynthesis (chloroplast) -contain own genomes and bacteria-like ribosomes Microbial Nutrition -nutrient -any compound required for biosynthesis and energy -major elements: -C, H, O, N, P, S -found in macromolecules -form structural and enzymatic components of cells -measured in g/L minor elements: -K, Ca, Mg, Fe, Na -required as enzyme cofactors or for activity of certain enzymes -measured in mg/L -major and minor elements referred to as macroelements collectively -microelements or trace elements: -Mn, Zn, Co -required as enzyme cofactors or for activity of certain enzymes -measured in g/L Classification of Microbes Based on Nutrient/Energy Sources Carbon: -autotrophs -use of CO 2 -ie: algae, photosynthetic bacteria -heterotrophs -use of pre-formed organic molecules made by other organisms Energy: -phototrophs -light -ie: algae, photosynthetic bacteria -chemotrophs -oxidation of organic/inorganic compounds Hydrogen/Electrons -lithotrophs -reduced inorganic molecules -ie: algae, photosynthetic bacteria -organotrophs -organic compounds -most microbes falls into one of two groups: -photo-lithotrophic autotrophs -chemo-oraganotrophic heterotrophs -prototroph -strain with “wild-type” nutritional requirements -auxotroph -mutant with nutritional requirements from environment Nutrient Uptake Diffusion (Passive and Facilitated) -high pressure to low pressure -with large concerntration gradient -diffusion rate decreases if substrate is not consumed -gases move across PM by passive diffusion -to increase diffusion rate, cells use membrane proteins (facilitated diffusion) -porins -help to transport compounds -substrate specific Active Transport -moving substances against concentration gradient -requires energy in the form of ATP or couple with ion movement -symports -energy released when a substrate moves down its concentration gradient -that energy is used to move another substrate against its concentration gradient -occurs in same direction as substrate moving down its gradient -ie: substrate in, substrate in -antiports -same concept as symports but substrate moving down gradient is going opposite direction and substrate moving up its gradient -ie: substrate in, substrate out -involves use of membrane proteins known as permeases Group Translocation -involves chemical modification (phosphorylation) of transported compound -happens while substrate is entering cell Culture Media Defined/Synthetic -known chemical composition -ie; M9 Complex -unknown chemical composition -ie: McConkeys Enriched -contains reagent for growth of picky microbes -ie; blood agar, charcoal agar Selective -permits growth of only certain microbes within a mixed population -ie: McConkeys contains bile salts -gram positives are sensitive -gram negatives are resistant Differential -distinguishes between different subsets of microbes -ie: McConkeys has pH indicator dye which can distinguish between lactose fermenting and non-lactose fermenting microbes Microbial Growth Growth -increase in cells’ components -usually followed by binary fission in bacteria -most eukaryotic cells divide by mitosis -in bacteria, growth in lab is measured as: -increase in cell population over time -growth curve -occurs in closed system -no additional nutrients -no removal of wastes -plotted as log10 -viable cell number vs. time -4 phases -lag phase -no immediate increase in cell number -synthesis of cellular components -variable in duration -occurs when cells are transferred: -old to new culture -different type of medium -exponential phase (log phase) -maximum growth rate -constant growth rate -cell number doubles at regular time intervals -phase of growth when cell population is most chemically and physically uniform -cells used in experiments are preferred in this phase -stationary phase -number of viable cells remains constant -death and growth rates about equal due to -depletion of nutrients -accumulation of wastes -death phase -decrease in viable cells -severe nutrient depletion and waste accumulation -logarithmic decline in viable cell number -cells don’t die all at once: -cell dies, other cells use its’ nutrients Indices of Growth Doubling/Generation Time -measured during log phase -time it takes for a 2-fold increase in cells number -E. coli doubling time = about 30 minutes -in 24 hours: 1x10 14 cells (1 gram) 28 -in 48 hours: 1x10 cells (7 tons) Growth Rate Constant -number of generations per unit of time Measurement of Microbial Growth Direct Counting -count cells in a chamber of known volume -ie: Petroff-Hauser Chamber -quick and simple -not a viable cell count unless a vital stain is used -hard to tell which cells are alive and which are not Electronic Counting -also not viable cell count -unless cells are used from exponential phase -ie: Coulter Counter
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