11723185
Description
Flashcards by Candice Young, updated more than 1 year ago
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Created by Candice Young
over 6 years ago
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| Question | Answer |
| significance of small size of bacteria | cell volume small --> surface/volume ratio large --> transport of metabolites (nutrients in, waste products out) rapid --> LESS need for intracellular compartments |
| Bright field microscopy | magnification infinite, RESOLUTION limited by wavelength of light used to capture image + light capturing ability of lens limit usually ~0.2 μm or 200 nm objects scatter light more than surrounding material and look dark against light background |
| total magnifying power | the product of the objective (10-100x) and ocular (10x) magnifications |
| resolution | the smallest distance at which two points can be detected as separate objects --> a function of the wavelength of light used to create the image and the light-gathering ability of the objective (AKA its numerical aperture) R=λ/2NA "higher resolution" = R or NA SMALLER |
| How can we create contrast (in GENERAL) in bright-field microscopy? | Try staining the sample: spread culture over slide, dry in air, pass slide through flame, flood with stain, and examine dyes are cationic; bind to nucleic acids, acidic polysaccharides or cell surfaces use to see if there's any cells at all; DONT use if you want to keep cells alive!! |
| gram stain | differential stain that can be used to distinguish between G+ and G- flood heat fixed-smear with crystal violet, cells turn purple --> add iodine (makes CV harder to remove), all cells still purple --> decolorize with alcohol (dissolves OM of G-, dehydrates PG of G+), G+ purple, G- colorless --> counterstain with safranin, G+ purple, G- pink |
| disadvantages of gram staining | usually kills the cells, can't observe living specimen --> lose details about growth and motility |
| How can we create contrast optically? | use Phase contrast or differential inference contrast (DIC) microscopy --> observe LIVE cells that have a higher refractive index than surrounding medium requires special equipment: polarized light, specialized lens |
| Transmission Electron Microscopy (TEM) | λ [nm] = h/mv ≈ 1.23/V^(1/2) [Volts] typically: λ = .0055 nm has a very small NA b/c they only gather refracted electrons over very small angle θ BUT decrease in λ compensates --> resolution in TEM is much better than in light microscopy (0.2 nm, 1000x better!) |
| Visualization: Immunofluorescence Microscopy | antibody that recognizes specific protein attached to a fluor --> fixed, permeablized cells treated with the antibody and observed --> light of a specific λ excites fluor --> light emitted by the fluor detected |
| Pros/Cons of Immunofluorescence | Pros: can locate individual proteins, can use WT cells (as long as you have an antibody to the target protein), uses a light microscope (easier to get) Cons: cells are fixed, can get a nonspecific signal everywhere, fixation can destroy protein or structure recognized by the antibody |
| Visualization: Protein fusions to Green Fluorescent Protein (GFP) | 3 amino acid side chains react to form fluor that absorbs BLUE light, emits GREEN light GFP = protein, not a chemical fluor using recombinant DNA tech: connect the gene for target protein to the gene for GFP --> express GFP fusion protein in bacteria --> observe location using fluorescence microscopy |
| Pros/Cons of GFP visualization | Pros: don’t need antibody (takes months to produce and then might not work), can see GFP fusions inside LIVING CELLS --> watch protein movements over time Cons: have to genetically manipulate cells, having GFP connected to protein can interfere w/ normal function/location, GFP signal weaker than non-protein fluors, hard to see if there aren’t many copies in cell |
| Steps AFTER GFP visualization | always double check results with immunofluorescence/DIC/phase & see if GFP fusion protein can substitute functionally for the wild-type protein |
| How do we know an IF or GFP experiment is showing us the REAL location of the protein? | *see if both IF and a GFP fusion give you the SAME result about a protein’s location* AS WELL AS IF: compare fluorescence signal in WT to mutants lacking the target protein GFP: determine GFP fusion protein is functional by making sure it works in case of deletion of the target protein |
| Immunofluorescence vs GFP | IF: must fix the cells, need antibody that recognizes target protein BUT don’t need to genetically manipulate bacteria GFP: don’t need an antibody, can use living bacteria BUT do need to express fusion protein in bacteria and ensure it's functioning like WT protein |
| Direct Microscopic Count | count number of cells under a square grid in a counting chamber (Ex: depth of liquid in counting chamber is 0.02 mm --> volume under 25-square grid = 0.02 mm^3 = 0.02 μl = 2 x 10^-5 ml # of cells in 25-square grid = # in 2 x 10^-5 ml --> # per 1 ml can be calculated) |
| Viable cell count (including disadvantages) | Counts # of cells in sample capable of growing up into colony on solid medium; ONLY living cells are counted Cons: counting cells from environmental sample --> medium only suitable for some species, could take days/weeks for growth |
| Turbidity | cultures appear turbid b/c light passing through sample is scattered by bacteria: turbidity of a culture/its optical density proportional to # of cells present (exact relationship is diff for each species due to cell size/shape) --> generate standard curve to relate OD to cell # counted by an independent method |
| Pros/Cons of OD count | Pros: rapid, no sample destroyed Cons: does not distinguish between live & dead, nor between different kinds of cells |
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