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Microbiology Ch. 4

Laboratory Methods

General principles of microscopy wavelength of radiation magnification resolution contrast
magnification an object is magnified in size so that it becomes visible to the observer
resolving power (resolution) the ability to see fine details to resolve 2 separate objects as distinct structures
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
Contrast -differences in intensity between two objects or between an object and a background -importance in determining resolution
What increases contrast? staining use of light that is in phase
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
Total magnifaction= magnification of objective lens x magnification of ocular lens
Bright-field most widely used, specimen is darker than surrounding field
Dark-field brightly illuminated specimens surrounded by dark field
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
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
Confocal microscope -use fluorescent dyes -use uv lasers to illuminate fluorescent chemicals in a single plane -computer constructs 3D image from digitalized image
Electron microscope -greater resolving power and magnification -magnifies 10000x to 100000x -detailed images of bacteria, viruses, internal cellular structures, molecules, and large atoms
2 types of electron microscopes transmission and scanning
Staining -increases contrast and resolution by coloring specimens with stains and dyes -smears of microorganisms (thin film) made prior to staining
Examples of stains Gram, acid-fast, endospore, capsule, flagellar
Culture visible (macroscopic) growth of organisms in or on a medium
Colony macroscopic cluster of cells appearing on a solid surface; each arising from a single cell
Pure culture contains a single specimen
Mixed culture contains more than one specimen -when you know that you will have more than one species
Contaminated culture contains "unwanted" species
Media can be classified according to 3 properties physical state chemical composition functional type
Physical state liquid, semisolid, and solid
Chemical composition synthetic (chemically defined) and nonsynthetic (complex)
Functional type general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration
Liquid broth; does not solidify
Semisolid viscous consistency; contains some solidifying agent
Solid firm surface for colony formation -contains solidifying agent
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
Fastidious bacteria that require growth factors and complex nutrients
Inoculation introducing microorganisms into culture medium
Incubation providing a controlled environment for culturing (grows best at 37C)
Identification of microbes -biochemical assays -immune assays- can distinguish diff strands -nucleic acid fingerprints
Created by: KatelynnJoy