Laboratory Methods
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| General principles of microscopy | wavelength of radiation
magnification
resolution
contrast
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| magnification | an object is magnified in size so that it becomes visible to the observer
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| resolving power (resolution) | the ability to see fine details
to resolve 2 separate objects as distinct structures
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| Total magnification of the final image | a product of the separate magnifying powers of the two lenses
-- power of objective x power of ocular= total magnification
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| Contrast | -differences in intensity between two objects or between an object and a background
-importance in determining resolution
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| What increases contrast? | staining
use of light that is in phase
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| Compound microscopes | - series of lenses for magnification
-light passes through specimen into objective lens
-oil immersion lens increase resolution
-have 1/2 ocular lens
-most have condenser lens
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| Total magnifaction= | magnification of objective lens x magnification of ocular lens
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| Bright-field | most widely used, specimen is darker than surrounding field
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| Dark-field | brightly illuminated specimens surrounded by dark field
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| Phase-control | transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures, very detailed viewing
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| Flourescent microscope | -direct UV light source at specimen
-specimen radiates energy back as a longer visible wavelength
-UV light increases resolution and contrast
-some cells are naturally fluorescent; others are stained with fluorescent dyes
-used to identify pathogens a
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| Confocal microscope | -use fluorescent dyes
-use uv lasers to illuminate fluorescent chemicals in a single plane
-computer constructs 3D image from digitalized image
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| Electron microscope | -greater resolving power and magnification
-magnifies 10000x to 100000x
-detailed images of bacteria, viruses, internal cellular structures, molecules, and large atoms
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| 2 types of electron microscopes | transmission and scanning
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| Staining | -increases contrast and resolution by coloring specimens with stains and dyes
-smears of microorganisms (thin film) made prior to staining
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| Examples of stains | Gram, acid-fast, endospore, capsule, flagellar
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| Culture | visible (macroscopic) growth of organisms in or on a medium
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| Colony | macroscopic cluster of cells appearing on a solid surface; each arising from a single cell
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| Pure culture | contains a single specimen
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| Mixed culture | contains more than one specimen
-when you know that you will have more than one species
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| Contaminated culture | contains "unwanted" species
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| Media can be classified according to 3 properties | physical state
chemical composition
functional type
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| Physical state | liquid, semisolid, and solid
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| Chemical composition | synthetic (chemically defined) and nonsynthetic (complex)
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| Functional type | general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration
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| Liquid | broth; does not solidify
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| Semisolid | viscous consistency; contains some solidifying agent
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| Solid | firm surface for colony formation
-contains solidifying agent
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| Agar | -solidifying agent
-a complex polysaccharide isolated from red algae
-solid at room temp, liquefies at 100C, does not resolidify until it cools to 42C
-provides framework to hold moisture and nutrients
-not digestible for most microbes
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| Fastidious | bacteria that require growth factors and complex nutrients
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| Inoculation | introducing microorganisms into culture medium
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| Incubation | providing a controlled environment for culturing (grows best at 37C)
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| Identification of microbes | -biochemical assays
-immune assays- can distinguish diff strands
-nucleic acid fingerprints
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