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Ch. 3 Microscope

UCC Microbiology 108

What is focal point? The lenses focus light rays at a specific place.
What is focal length? The distance between center of lens and focal point. The shorter the focal length, the more magnification.
What is refraction? The bending or changing in the angle of the light rays as it passes through a medium such as a lens.
What is refractive index? A measure of how greatly a substance slows the velocity of light. Or a measure of the light-bending ability of a medium. Immersion oil has same index as glass so light won't be bend any further. Air and water have different index(bends light).
Objective lens Magnification: Scanning=4x, low power= 10x, high power=40-45x, oil immersion= 90-100x
What is the illuminator of a compound light microscope? Light source
What is the condenser of a compound light microscope? Has lenses that direct rays through the specimen
What are the objective lens of a compound light microscope? Lenses closes to the specimen and used to magnify the images.
What are the ocular lens/eyepiece of a compound light microscope? Magnifies the image and also used to view the image. Magnification power is 10x.
What is a light microscope? Any kind of microscope that uses visible light to observe specimen. They are compound microscopes ( uses 2 or more lenses). Microscopes used by Leewenhoek was a simple one.
What is a bright-field microscope? Produces dark image against a brighter background(field). Has several objective lens but these are PARFOCAL(remain in focus when lens are switched)
What is total magnification? Product of the magnifications of ocular lens and the objective lens
What is dark-field microscope? Used to study living microorganisms. Specimen appears light against black background. Very thin spirochete such as TREPONEMA PALLIDUM(causes syphilis) is examined.
What is electron microscope? Used to examine objects smaller than 0.2 micrometer like viruses and internal cellular structures. A beam of electrons is used instead of light. Better resolution(clarity) because of shorter wavelength of electrons.
What is the transmission electron microscope (TEM)? ELECTRONS scatter when they PASS THROUGH ultra-thin sections of a SPECIMEN. Resolution about 2.5nm. Magnifies 10,000-100,000x. Internal structures seen. Images can be viewed thru eyepiece.
What is the scanning electron microscope (SEM)? Uses electrons reflected from the SURFACE of a specimen to create image. Produces 3D image of the specimen's surface features. Resolution about 20nm. Objects magnified 1,000 to 10,000x. See external structures. Images viewed on viewing screen
What is a smear? A dried preparation of bacterial cells on a glass slide. Good smear: microbes= evenly spread on surface of slide, not washed away during staining, forms are not distorted.
What is heat fixation? Passing air-dried smear several times over the flame having smear side up OR by using blow dryer from the underside of the slide(smear side up). COAGULATE bacterial PROTEINS so bacteria stick to the slide surface. KILLS BACTERIA.
Chemistry of a stain Stain is organic compound. 3 parts of a stain: BENEZENE ( organic color solvent) CHROMOPHORE (chemical that is color) AUXOCHROME (ionizes the chromogen so it can bind with the cells, fibers, or tissues)
What is chromogen? Benezene+ chromophore. It is a colored compound not a stain.
Why do we do staining? To study the microbial structures and to group the microbes in a specific group for diagnosis. It creates a contrast between the bacteria and the background.
What is a basic dye? It contains colored postive ions. Ex=crystal violet, methylene blue, malachite green, safranin
What is acidic dye? It contains colored negative ions. Ex=Eosin, acid fuchsin, nigrosin
What is negative staining? Bacteria are slightly negatively charged so acidic dyes are repelled by most of the bacteria. A dye stains the background. ( Heat fixation is not needed). It is used to observe the overall shape, size, capsule ( if present)
What is simple staining procedure? Uses aqueous or alcohol solution of a single basic dye. Highlights the entire microorganism to STUDY THE CELL SHAPE, SIZE, ARRANGEMENT. Sometimes uses a mordant (helps bind dye more strongly)
What is differential staining procedure? Uses at least 3 chemical reagents applied to a heat fixed smear. 1.) PRIMARY STAIN 2.) DECOLORIZING AGENT(may or may not remove primary stain from entire cell or certain cell structures) 3.)COUNTERSTAIN (contrasts color of primary stain)
What is gram staining procedure? It classifies bacteria into positive and negative groups. It has 4 steps.1. ) Primary stain crystal violet 2.) Wash off primary stain and apply iodine 3.) Alcohol/ alcohol acetone is used as de-colorizing agent 4.) Wash off alcohol, stain slide Safranin
What is gram stain positive bacteria? Thicker layer of peptidoglycan in the cell wall. Will remain purple.
What is gram stain negative bacteria? Thinner layer of peptidoglycan. Has a layer of lipopolysaccharide layer. Remains colorless unless stained with counter stain Safranin which makes it a pink color.
What is acid-fast staining? Distinguish MYCOBACTERIUM species and some species of NOCARDIA. Binds strongly to waxy material in the cell wall. 1) Carbol fuchsin (red dye) is applied 2) Cool and wash with water,then wash with alcohol( decolorizer) 3.) Counter stain methylene blue.
How can you tell if a bacteria is non-acid fast? Non acid fast bacteria loses primary stain (stays blue). Acid fast bacteria retain the dye (red) because it is more soluble in the cell wall lipids than in the acid alcohol.
What is Endospore staining? SCHAEFFER- FULTON endospore stain. Heat fixed smear is prepared. Primary stain Malachite Green. Counter stain is Safranin. Spores appear green and cells pink.
What is capsule staining? Capsule determines a microbe's virulence. INDIA INK OR NIGROSIN is used to stain the background dark. SAFRANIN is used to stain the cells. Capsules do not accept most dyes like Safranin. They look like halos (unstained area) around stained cells
What is flagella staining? Flagella are very thin. A mordant is used to build up diameter of flagellum and when stained with CARBOLFUCHSIN it becomes visible under light microscope. Number and location of flagella is used in diagnosis.
In electron microscopy, the specimen is placed on? A copper mesh grid
Distortion of cell size and shape are minimized in? Negative staining
Bright field illumination is produced by? Condenser
Most ocular lenses magnify specimens by a factor of? 10
The ability of lenses to distinguish fine detail and structure is called? Resolution
Which microscope would you choose to view the cellular contents of a microbe in its natural state? Phase-contrast microscope
Which microscope uses an ultraviolet light source? Fluorescence microscope.
Which microscope achieves highest magnification and greatest resolution? Electron microscope.
Created by: smwondr



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