Microbiology: Week 2 Notes
Microbiology: Week 2 Notes 81382 - MICR 3050 - 001
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This 4 page Class Notes was uploaded by Amanda Biddlecome on Sunday September 4, 2016. The Class Notes belongs to 81382 - MICR 3050 - 001 at Clemson University taught by Dr. Rudolph in Fall 2016. Since its upload, it has received 5 views. For similar materials see General Microbiology in Biology at Clemson University.
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Date Created: 09/04/16
Microbiology 3050 Chapter 2: Study of Microbial Structure: Microscopy and Specimen Preparation Amanda Biddlecome August 29, 2016 1) The Light Microscopes -‐types in microbiology *bright-‐field, dark-‐field, phase-‐contrast, fluorescence, confocal ^bright and dark can be done with the same scope -‐compound *multiple lenses 2) Lenses and Bending Light -‐when light hits the lens, it bends (refracts) -‐refractive index *different for different mediums *measure of how much the velocity of light slows down (bends) as it enters the new medium *air=1; oil=1.25; water varies depending on what’s dissolved in it -‐direction and magnitude of the bend depends on the refractive index of both media 3) Lenses -‐collection of prisms -‐light bends toward the normal of the lens *the line perpendicular to the lens -‐focal point=where all rays meet behind the lens -‐focal length=distance between the lens and the focal point -‐strength of the lens: shorter focal length=greater magnitude 4) Bright-‐Field Microscope -‐condenser lens=focus light under the stage -‐image is dark against brighter background light *any light that can’t get through=darker object *denser objects=darker -‐objective lenses *parfocal=get in focus at lower power and should still be in focus as you move up in strength ^use coarse adjustments ONLY at 4 and 10 *parcentric=stays centered as you increase strength -‐total magnification=magnification of ocular lens (ours is 10X) X magnification of objective lens -‐upper limit of magnification for bright-‐field=1500X -‐upper limit of resolution for bright-‐field=0.2μm 5) Microscope Resolution -‐ability of the microscope to distinguish between two small objects that are close together -‐wavelength affects that distance (d) *shorter=greater resolution and smaller d *blue light=short wavelength -‐d=(0.5λ)/NA=(0.5λ)/(nsinθ) *NA=Numerical Aperture: how much light it captures *working distance=distance between specimen and lens ^longer objective=greater NA *n=refractive index ^oil gives better clarity, not better magnification 6) Dark-‐Field Microscope -‐reverse of bright-‐field microscope -‐bright image on a dark background -‐image is shown by the light reflected or refracted by the specimen -‐most bacteria don’t have pigments, so we have to stain them for a bright-‐ field, but that kills them -‐use dark-‐field to observe living microbes that aren’t stained *see motility -‐can see internal structures in eukaryotes -‐put dark field stop on a bright-‐field microscope to block light, except for the edges 7) Preparation and Staining of Specimens -‐we stain to be able to see them: contrast with the background -‐helps see morphological features *bacteria we look at overall shape -‐preserve the shape so it doesn’t change before our eyes *look normal and not distorted Chapter 7.5 1) Culture Media -‐they are nutrient preparations devised to support growth and reproduction of microorganisms *have macronutrients *have micronuetrients: inorganics, trace metals/minerals, Zn, Mg, Cu ^usually don’t have to worry about these because they are in water *growth factors=organics that they don’t make on their own: vitamins -‐can be liquid or solid *without agar=liquid broth *solid media are usually solidified with agar 2) Defined/Synthetic Media -‐all components and their concentrations are known *know EVERYTHING including formulas 3) Complex Media -‐very complex -‐some ingredients have unknown compositions and/or concentrations *ingredients could be different everytime -‐as soon as you add agar to something, it becomes complex, even if it started as defined 3) Complex Media Components -‐peptones *partial -‐extracts *aqueous extracts, usually beef or yeast -‐agar *sulfated polysaccharide used to solidify liquid media; most microorganisms can’t degrade it and it stays solid at 37 C o -‐ALL of these are complex 4) Functional Types of Media -‐general purpose media (supportive) *helps many microorganisms grow *trying to grow many different microorganisms -‐enriched media *try to grow many types of microorganisms *add something really complex to help the picky ones eat and grow ^blood *fortify it *chocolate agar=boiled blood -‐minimal media *contains minimal necessities for growth of wild type ^wild type=heartiest type of the strain *has inorganic salts, simple carbon sources, and water *looking to see if it’s a nutritional mutant ^know it is if it can’t grow on minimal media -‐selective media *takes enrichment to extreme *try to help things you want to see grow and kill other microorganism *MacConkey agar: bile salts and crystal violet: kills gram-‐positive and selects for gram-‐negative -‐differential media *distinguish between different groups of microorganisms by biological characteristics ^blood agars: hemolytic versus nonhemolytic bacteria (alpha, beta, and gamma) *MacConkey agar: lactose fermenters make lots of acids and turn colonies pink/medium pink and lactose nonfermenters 5) Isolation of Pure Cultures -‐pure culture=population of cells coming from a single cell *all are the same strain *pick up one colony and it should be pure *individual cells need to be well-‐separated to tell if they’re pure *use streak plate, spread plate, and pour plate 6) Streak Plate -‐use inoculating loop -‐each cell can reproduce to form a separate colony 7) Aseptic Transfer -‐hold loop down -‐flame enough of the loop to kill all bacteria -‐flame mouth of the tube right after you open it, never put the lid down on the bench, and flame it again right before you close it 8) Spread Plate and Pour Plate -‐spread plate *dilute out the culture, put it on agar surface, spread it out with a hockey stick -‐pour plate *mix diluted sample with liquid agar and pour it into the plate -‐both give isolated colonies but you can also count isolated colonies *enumerate bacteria in the sample *count 30-‐300 colonies because less than 30 isn’t an accurate representation and more than 300 is impossible to count -‐1:10 dilution *1mL of sample into 9mL of water *dividing original concentration by 10 each time -‐1:100 dilution *1mL of sample into 99mL of sample -‐to get original concentration from dilution: multiply counted colonies by reciprocal of dilution
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