Microbial Laboratory techniques
Terms
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- Microscopy
- total magnification f an object is calculated by multiplying the magnification of the objective lens byh the magnification of the ocular lens.
- compound microscope
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uses visible light.
Maximum resolution, or resolving power (the abilityh to distingulish between two points) of a compound light microscope is 0.2um, max magnification is 20003l. - staining
- Specimens are stained to increase thew difference between the refractive indexes of the specimen and the medium.,
- Immersion oil
- used with the oil immersion lens to reduce light loss between the slide and the lens.
- Brightfield illumination
- used for stained smears.
- Modified compound microscopes
- Unstained cells are more productively observed using darkfield,phase-contrast, or DIC microscopy.
- Darkfield Microscopy
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Shows a light silhouette of an organism against a dark background.
It is most useful for detecting the presence of extremely small organisms. - Phase contrast microscopy
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A phase-contrasts microscope brings direct and reflected or diffractedc light rays together (in phase) to form an image of the specimen on the ocular lents.
It allows the detailed observations of living cells. - Fluorescence Microscopy
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Specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near ultraviolet) light source
dThe microorganisms appear as brfight objectws against a dark background.d
Fluorescence microscopy is used primarily in a diagostic procedure called fluorescent-antibody (FA) technique, or immunofluorescence. - Confocal Microscopy
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In confocal microscopy, a speciamen in stained with a fluorescent dye and illuminated one plane at a time.
Using a computer to process the images, two dimensional and three dimensional images of cells can be produced. - electron Microscopy
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A bean of electrons, instead of light, is used with an electron microscope.
Electromagnets instead of glass lenses, control focus, illumination and magnification
Thin sections of organisms can be seen in an electron micrograph produced using a transmission electron microscope (TEM) Magnification: 10,000-10,0003.
Resolving power: 20nm. - Scanning tunneling and Atomic Force microscopy
- Scanning tunneling microscopy(STM) and atomic force microscopy (AFM) produce three dimensional images of the surace of a molecule.
- Preparing Smears for Staining
- Staining means coloring a microorganism with a dye to make some structures more visible.
- Fixing
- Uses heat or alcohol to kill and attach microorganisms to a slide.
- colored negative
- colored negative ion of an acidic dye will stain the background of a bacterial smear; a negative stain is produced.
- Simple Stains
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simple stain is an aqueous or alcohol solution of a single basic dye.
Used to make cellular shapes and arrangements visible.
A mordant may be used to improve bonding between the stain and the specimen. - Diffenential Stains
- Differential stains such as gram stain and acid fast stain, differentiate bacteria according to their reactions to the stains.
- Gram stain procedure
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`Uses a purple stain (crystal violet) iodine as a mordant, an alcohol decolorizer and a red counterstain.
Gram positive bacteria retain the purple stain after the decolorization steop; gram-negative bacteria do not and thus appear pink from the counterstain. - Acid fast microbes
- such as members of the genera Myco-bacterium and Norcardia, retain carbolfuchsin after acid alcohol decolorizationa and appear blue.
- Special Stains
- Then endospore staina and flagella stain are special stains that color and isolate only certain parts of bacteria.
- Negative Staining
- is used to make microbial capsules visible.
- Stain procedures
- Wet mount staining unnecessary organisms are very large.
- Hanging Drop
- place a drop of culture on coverslip (glass cover for a slide and then inverting it over a hallowed out slide.
- heat fixing
- kills organism then adheres to slide.
- Pure Cultures or clones of bacteria
- The isolation method most commonly used to get pure cultures is the streak plate method.