Stokes Microbiology Ch 3

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p87 in pdf / p54 in book

micrometer (µm), nanometer (nm)

measurements to measure microorganisms and their structural components:
_______ = 0.000001 m = 10⁻⁶ m
_______ = 0.000000001 m = 10⁻⁹ m
(additionally, picometer = 0.000000000001 m = 10⁻¹² m)

simple, compound

______ microscopes only have one lens (used by van leeuwenhoek, making him the first person to see bacteria);
______ microscopes have multiple lenses

light microscopy

the use of any kind of microscope that uses visible light to observe specimens

compound light microscope (LM)

an instrument that has a series of lenses and uses visible light as its source of illumination; light goes from the *illuminator* (source) to the *condenser* (directs through specimen) to the *objective lenses* to the *ocular lens* (eye piece)

total magnification

______ = objective lens magnification (power) x ocular lens magnification (usually 10x);

resolution (resolving power)

the ability to distinguish fine detail/structure with a magnifying instrument; the ability of the lenses to distinguish two points a specified distance apart (ex/ if 0.4 nm, can distinguish between two points ≥ 0.4 nm); generally, the shorter the wavelength of light used, the greater this is

refractive index

a measure of the light-bending ability of a medium; the relative velocity with which light passes through a substance;
changed by staining so that specimens are contrasted sharply with their medium under a compound LM

immersion oil

oil that is placed between the glass slide and the certain objective lens, so that light rays do not get lost after it passes through the specimen; high magnification with good resolution is achieved

brightfield illumination

when dark objects are visible against a bright background; most commonly used in laboratories; can be used with live, unstained, preserved, stain specimens; inexpensive, easy; can see internal structures

darkfield microscope

a microscope that has a device to scatter light from the illuminator so that the specimen appears white against a black background; does not distort living cells; shows outline, not internal details

phase-contrast microscope

a compound light microscope that allows [more precise] examination of structures inside [living] cells through the use of a special condenser; not necessary to fix/stain (which distorts/kills);two lights: from source and what's reflected/diffracted from a particular structure

DIC (Differential interference contrast) microscope

an instrument that provides a three-dimensional, magnified image; similar to phase-contrast (refractive indexes) but uses two beams split by prisms so has higher resolution; shows bright colors and is 3-D

fluorescence microscope

a microscope that uses an ultraviolet light source to illuminate specimens that will *fluoresce* (absorb short wavelengths of light [UV] and give off light at a longer wavelength [visible]); cells may be stained with fluorochromes (dyes); used in diagnosing infections (ex/ FA technique)

fluorescent-antibody (FA) technique (immunofluorescence)

a diagnostic tool using *antibodies* (natural defense molecules that react to foreign substances called *antigens*) labeled with fluorochromes and viewed through a fluorescence microscope

confocal microscope

a light microscope that uses fluorescent stains and laser to make two- and three-dimensional images; similar to fluorescence but only illuminates one plane at a time with short-wavelength (blue) light, resulting in improved resolutions; can scan various depths of specimen up to 100 µm deep

TPM (two-photon microscope)

~ a light microscope that uses fluorescent stains and long wavelength (red) light; allows imaging of living cells in tissues up to 1 mm deep (compared to confocal's 100 µm); less damaging than confocal and can track activity in cells in real time

SAM (scanning acoustic microscope)

~ a microscope that uses high-frequency ultrasound waves to penetrate surfaces; interprets the action of a sound wave sent through a specimen (what is reflected back from an interface within the material); resolution about 1 µm; used to study living cells attached to another surface

electron microscope

a microscope that uses electrons instead of light to produce an image (shorter wavelengths give greater resolution); uses electromagnetic lens (not glass); examines objects smaller than about 0.2 µm; always black and white but can be colored artificially for details

transmission electron microscope (TEM)

electron microscope that provides high magnifications (10,000-100,000x) of thin sections of specimen; final image is a "micrograph"; resolution: 2.5 nm; metals "stain" to absorb more electrons for more contrast; can see internal structures; preparation to prevent electron scattering kills specimen and can distort ("artifacts" may appear)

scanning electron microscope (SEM)

an electron microscope that provides 3-D views of the specimen magnified 1000x-10,000x; "micrograph" final image; useful for surface structures; resolution 10 nm; preparation to prevent electron scattering kills specimen and can distort (adds "artifacts")

scanned-probe microscopy

~ microscopic technique used to obtain images of molecular shapes (see their surface), to characterize chemical properties, and to determine temperature variations within a specimen, all without modification or damaging radiation
ex/ STM (scanning tunneling microscopy) and AFM (atomic force microscopy)

staining

coloring the sample of microbes with a dye to view through a microscope or emphasize certain structures (before so, the microorganisms must be *fixed* [attached], which kills them but distorts only minimally)

smear

a thin film of material containing microorganisms, spread over the surface of a slide (then fixed [by passing over a flame facing up, or covered with methyl alcohol for 1 minute] and stained which is washed off)

basic dye, acidic dye

____ ___: salt (chromophore) in which the color is in the positive ion (cation), used for bacterial stains (bacteria surfaces are slightly negative = attract); ____ ___: chromophore in which the color is in the negative ion (anion), used for *negative staining* (colorless bacteria repel, stains background instead)

simple stain

an aqueous or alcohol solution of a single basic dye; to highlight the entire microbe so that shapes and basic structures are visible

mordant

chemical added to a staining solution to make it stain more intensely/to coat a structure/specimen to enlarge it; forms large crystals with the dye that are too large to escape the cell wall

differential stains

a stain that distinguishes objects into distinctive groups on the basis of reactions to the staining procedure;
ex/ Gram stain, acid-fast stain

*crystal violet* (purple) (+), *safranin* (basic red) (-)

*Gram stain* (differential stain, classifies bacteria into two groups, gram-positive/gram-negative; dvlped by Hans Christian Gram) procedure:
1. *primary stain* to all cells: ____ ____ 2. iodine (mordant) 3. *decolorizing agent* (alcohol) removes color from gram-neg 4. *counterstained* with ____, then blotted

positive, negative

in Gram stains, bacteria that retain purple color after being decolorized = gram-_____;
bacteria that lose purple color after being decolorized (then get colored red/pink) = gram-_____

negative, positive

gram-______ bacteria: contain a layer of lipopolysaccharide within their cell wall, thin layer of peptidoglycan, more resistant to antibiotics, easier to destroy with alcohol; gram-______ bacteria: have a thicker peptidoglycan cell wall, teichoic and lipoteichoic acids, CV-I complex (purple) not washed out, tend to be killed by penicillin, lysozyme, detergents

acid-fast stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol; only binds to bacteria that have waxy material (lipids) in their cell walls (*Mycobacterium* tuberculosis/leprosy and *Nocardia*)

*carbolfuchsin* (red) (acid-fast), *methylene* (blue) (non-acid-fast/negative)

acid-fast staining procedure: 1. ____ to fixed smear, heated, cooled, washed 2. acid-alcohol (decolorizer) removes red stain from bacteria that are not acid-fast; 3. counterstains with ____ non-acid-fast cells (carbolfuchsin is more soluble in the acid-fast cell wall lipids, non-acid-fast lack lipids)

acid-fast, non-acid-fast

______ retain red color because carbolfuchsin is more soluble in their cell wall lipids than in acid-alcohol; ______ lose red color (then colored blue) because their cell walls lack lipid components

special stains

stains used to color and isolate/distinguish specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capules

capsule stain

stain that demonstrates presence of a microbe's gelatinous covering, can determine the organism's *virulence* (degree to which a pathogen can cause disease); done by *negative staining*, then staining the bacteria cells; structure does not accept most dyes, so "halos" appear

endospore

special resistant, dormant structure within a cell that protects it from adverse environmental conditions; ordinary stains/dyes do not penetrate wall; highly refractive, can be detected under LM unstained, but cannot be differentiated from inclusions of stored material without special stain

endospore staining

Schaeffer-Fulton ______ _______ procedure: 1. primary stain: *malachite green*, heated to steaming 2. decolorized with water for 30 seconds, other parts washed colorless 3. counterstained with safranin

flagella staining

a tedious and delicate special staining procedure using mordant and the stain carbolfuchsin to build up the diameters of extremely small structures of locomotion so that it can be seen with a LM

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