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Microbiology - Week 2

by: Mallorie Jones

Microbiology - Week 2 Bio 183

Mallorie Jones


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This chapter covered a lot about microscopes and how staining a specimen works and why we do it. Hope it helps!
Brian N. Albrecht
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This 4 page Class Notes was uploaded by Mallorie Jones on Wednesday September 7, 2016. The Class Notes belongs to Bio 183 at Northwest Iowa Community College taught by Brian N. Albrecht in Fall 2016. Since its upload, it has received 3 views.


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Date Created: 09/07/16
Chapter 3 ­ Observing Microorganisms Units of Measure ● Micrometer ( m) = .000001 m (10 m)  μ → 1  μ m = 10  m = 10  mm → 1000 nm = 1  μ m ● Nanometer (nm) = .000000001 m (10  m)­9             → 1 nm = 10  m = 10  mm → .001  μ m = 1 nm A) Light Microscopy ● Refers to the use of  any kind of microscope that  uses visible light to observe specimens ● Types: ○ Compound light → we’ll use this in class ○ Darkfield ○ Phase­contrast  ○ Differential interference ○ Fluorescence ○ Confocal 1) Compound Light Microscopy ○ Has a series of lenses use visible light as its source of illumination  → image is magnified again by the ocular lense ○ Total magnification ­ how many times you magnify the image ■ obj. lense mag X ocular lense mag ○ Resolution ­  the ability of the lenses  to distinguish fine detail and  structure ■ Example: a microscope has r.p. Of .4 nm, it can  distinguish 2 points that are .4 nm apart → Shorter wavelengths of light provide greater resolution ○ Refractive index ­  a measure of the light­bending ability  of a medium → light may bend in air so much that it misses the small high­mag lens ○ Brightfield illumination ­ produced through condenser by focusing the  light → Immersion oil keeps light from bending Parts of the microscope: B)  Electron Microscopy ●  A beam of   electron   is used instead of  light ○ Use for examination of objects smaller than .2  m μ ■ Viruses, internal cell structures ● 2 types: 1. Transmission → 2D ○ Ultra thin section of the specimen 2. Scanning → 3D → The shorter wavelength of electrons give greater resolution  → 100,000x’s small than visible light Preparation of Specimens for Light Microscopy A) Preparing Smears for Staining *Most microbes are colorless ● Staining ­ coloring the microorganisms with a dye that emphasizes certain structures ○ Stains consist of + and ­ ions ● Fixed ­ when the specimen is attached to the microscope slide and to kill microbes by  heat or chemicals ● Smear ­ thin film of material containing microorganisms on a slide  → must be allowed to air dry ● Basic dyes ­ color is in the cation (+) ● Acidic dyes ­ color is in the smion (­) ● Negative staining ­ preparing colorless bacteria against a colored background ○ Useful in determining morphology ○ Less likely to distort the shape since heat fixation is not used B)  Simple Stains ● *a single basic dye ● *makes cellular shapes and structures visible ● Mordant ­ additive that intensifies the stain → Methylene blue, cabofuncusim, safranin, crystal violet C)  Differential Stains ● *react differently with different kinds of bacteria ○ Can be used to distinguish different bacteria 1) Gram Stain ○ *developed in 1884 by Danish bacteriologist, Hans Christian Gram ○ Classifies bacteria into 2 large groups: i) Gram ­ positive ii) Gram ­ negative ○ Procedure: i) Primary stain ­ purple stain → crystal violet ii) Dye is washed off and smear is covered with iodine → mordant ○ Gram­positive ­ bacteria that retain purple ○ Gram­negative ­ bacteria that lose the purple color after decolorization i) That’s why they’re stained with safranin­red  ii) Counterstains ­ stains that have contrasting color → safranin 2) Acid­Fast Stain ○ Binds strongly to bacteria that have a waxy material in their cell wall ○ used to ID all bacteria in gums such as Mycobacterium and Nocardia             → acid-fast vs. not acid fast → primary stain - cabolfucusim → counterstain - methylene blue D) Special Stains ● *used to color parts of microorganisms ○ Flagella, endospores, capsules 1) Negative Staining for Capsules ○ Capsules ­ a gelatinous covering → can’t hold stain ○ Virulence ­ the degree to which a pathogen can cause disease → Negative stain ­ india ink or nigrosin 2) Endospore Staining ○ Endospore ­ a special resistant, dormant structure formed within a cell  that protects a bacterium from adverse environmental conditions. → Primary stain ­ Malachite green, usually with heat → Counterstain - safranin → Schaffer - Fulton method 3) Flagella Staining ○ Flagella ­ structures of locomotion too small to be seen with a light  microscope without staining → Mordent on flagella → Carbolfuchsin simple stain


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