Micro Ch 4 Microscopy
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| Describe the image produced by a convex lense | Inverted, reversed, enlarged
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| Usefulness of microscope depends on ability to clearly resolve two objects close together | Resolution
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| Minimum distance between two objects where those objects still appear distinct | Resolving power
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| Determines how much detail can be seen | Resloving power
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| Two things that determine resolution | Quality of lense and illuminating light's wavelength
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| Maximum power of most bright field microscopes | 0.2 micrometers or 1x10(^-6)m, you can see bacteria but not viruses
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| Ability to see differences between two objects or an object and its background | Contrast
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| Increase contrast by... | staining
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| Light microscope with a single magnifying lense similar to a magnifying glass, like Leeuwenhoek's | Bright field microscopes
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| How to determine toal magnification of a bright field microscope | Objective lense x ocular lense
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| What is the purpose of a condenser lense, present on most bright field scopes? | Focuses illumination on specimen and does not affect magnification
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| Why is oil used on higher magnificaiton? | it reduces light refraction because it has a very similar refractive index to glass
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| Best for observing pale objects, the only light seen is that scattered by the specimen | Dark field microscopy
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| Which light microscope is like someone standing in the dark while people shine flashlights on him? | Dark field
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| What type of organisms are used in phase microscopes? | Living microbes that would be damaged by attaching them to slides or staning
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| How is contrast created in a phase microscope? | Light waves are put out of phase, which creates shadow-like
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| A phase microscope is a type of light microscope, name the two types of phase microscopes | Phase-contrast and differential interference contrast (Nomarski)
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| This type of microscopy uses UV light which specimen radiates back as visible wavelengths | Fluorescent microscopes
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| Fluorescent microscopes are used in immunoflorescence to... | identify pathogens and make proteins visible
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| If a specimen is not naturally fluroescent, what must be done? | Attach fluorescent die to antibodies, which attach to specimen
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| Light microscope using fluorescent dies, UV lasers and producing images in planes | Confocal microscope
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| How close can light microscopes resovle structures? | 200nm
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| Microscope that allows greater resolving power and magnification up to 100,000 giving detailed view of bacteria, viruses, even large atoms | Electron Microscope
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| Two types of electron microscope | Transmission (TEM) and Scanning (SEM)
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| Microscope that magnifies over 100,000,000 times | Probe microscopy
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| Two types of probe microscope | Scanning tunneling, and atomic force
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| What needs to be done to a specimen before dying it? | Spread, Dry completely, Heat fix
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| If you don't dry your sample completely before heat fixing it, what happens to your bacteria? | It explodes
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| What are stains made of? | organic salts
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| Basic dyes carry a _________ charge | positive (Bases absorb H+)
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| Positively charged basic dyes bond to negative charged structures like... | The Cell
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| Acidic dyes carry a _________ charge | negative (acids release H+)
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| Negatively charged acidic dyes are repelled by the cell and commonly stain... | The background (negative staining)
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| From this we can tell that the cell has an overall _______ charge | negative
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| Commonly used basic dyes | Methylene blue, Crystal violet, Safranin, Malachite green
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| Simple stains will allow for increased contrast between a cell and the background, what is the drawback? | There is no differentiation because they are all the same color
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| Two most common types of differential stain | Gram stain, Acid-fast stain
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| What do differential stains use to differentiate? | reagents
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| Most widely used procedure for staining bacteria developed over a century ago | Gram Stain
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| Gram staining separates bacteria into two major groups. What are they? | G+ is purple. G- is pink.
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| In Gram staining, what is the primary stain? | Crystal violet. This stains all cells violet.
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| In Gram staining, what is the second step? | Mordant, which makes stain stick to peptidoglycan better. In this case it's Gram's iodine.
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| In Gram staining, what is the third step? | Decolorizing agent like alcohol removes dye from some cells
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| Which cells will be decolorized by the alcohol? | Gram negative cells will quickly decolorize, Gram positive cells can eventually be decolorized
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| In Gram staining, what do the cells look like after the third step? | Gram negative are clear, Gram positive are clear
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| In Gram staining, what is the final step? | Counterstain/Secondary stain/recoloring with Safranin
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| What type of organism is Acid-fast staining used on? | Resistant to convential staining, like mycobacterium, which has mycolic acid, a waxy protectant
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| How does acid-fast stain get into cell? | Heat or concentrated dye
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| In Acid-Fast staining, what is the first step? | Primary dye Carbol fuschin
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| What color are the cells after the first step of acid-fast staining? | All are fuschia
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| In Acid-Fast staining, what is the second step? | Decolorize with an acid alcohol
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| What color are the cells after the second step of acid-fast staining? | The acid fast cells stay fuscia (dye that's hard to get in is hard to get out), the rest are colorless
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| In Acid-Fast staining, what is the final step? | Counterstain with methylene blue
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| What color are the cells after the final step of acid-fast staining? | Acid fast still only have the fuscia. Non acid fast are blue.
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| An example of a negative that only stains the background and causes a "halo" of no dye? | Capsule stain
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| Example of using heat to facilitate staining of resistant microbe structures like those formed by Bacillus and Clostridium | Capsule stain
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| What dye is typically used for endospore staining? | Malachite green
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| Flagella Stain increases the diameter of flagella and is made more visible by using a... | mordant
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| What is a micrometer? | 1/1000th of a milimeter
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| What is a nanometer? | 1/1000 of a micrometer
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| Why does a lens refract light? | it is obtically dense compared to the surrounding medium, it moves more slowly through the lens
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| In what way do light rays move as they pass through the focal point? | They spread apart, creating an enlarged inverted image
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| Type of magnification combining many lenses to obtain a large, blury, faint image | empty magnification
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| What are the two properties of a clear image? | resolution and contrast
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| The ability to distinguish objects that are close together | resolution
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| Part of the microscope which remagnifies the image formed by the objective lense | Ocular lense
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| Part of the microscope which transmits the image from the objective lens to the ocular lens using prisms | Body
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| Part of the microscope which holds the body onto the base | Arm
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| Part of the microscope which contains primary magnification lenses | Objective lenses
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| Part of the microscope which holds the microscope slide in position | Stage
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| Part of the microscope which focuses light through the specimen | Condenser
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| Part of the microscope which controls the amount of light entering the condenser | Diaphragm
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| Part of the microscope which contains the light source | Illuminator
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| Part of the microscope which moves the stage up and down to focus the image | Coarse and fine focusing knobs
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| Resolution distance is dependent on these two principles | Wavelength of electromagnetic radiation x0.61 / numerical aperture of lens
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| Term refering to differences in intensity between two objects, or between an object and its background | Contrast
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| Two ways to increase contrast between microbes and their background | Staining and in phase light
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| What do we call microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a specimen? | confocal microscopes
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| What is the difference between a simple and a compound microscope? | Simple have one lens (like Leeuwenhoek's), Compound have more than one (like Galileo's)
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| Part of the microscope which holds 3 or 4 objective lenses | Revolving nosepiece
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| Why does immersion oil increase resolution? | It has a similar refractive index to glass
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| Name for the distance between the lens and the specimen | working distance
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| How is total magnification of a microscope determined? | multiply objective by ocular
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| What is a photograph through a microscope called? | Micrograph
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| What type of objects do we observe through dark field microscopes? | pale
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| What type of microscope is used to examine living specimens that would be damaged or altered by atatching them to slides or staining them | phase microscopes
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| What's the difference between in-phase and out-of-phase | In means crest and trough align, out mean they are not. If wavelengths are out of phase enough, you can see a difference
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| What's the difference between phase-contrast and differential interference contrast microscopes? (both are phase microscopes) | Phase is simple, letting you see fine structures like cilia and flagella. Differential (or Nomarski) is more advanced, using prisms to creat a 3-D appearance with unnatural colors
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| Molecules that absorb energy from invisible radiation and radiate it back as longer, visible wavelengths | fluorescent
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| Why must air be evacuated from the column of an electron microscope? | Air would absorb electrons so there would be no radiation to produce an image
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| How does a Scanning Electron Microscope work? | Specimen is covered in platinum or gold, then the electrons go back and forth across its surface knocking electrons off the surface which ultimately produce an image
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| What are the 2 probe microscopes? | Scanning tunneling and atomic force
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| 2 ways to fix a smear to a slide | Heat fixation or chemical fixation. Both cause desiccation.
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| Name of the colored portion of a dye | chromophore
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| Give components of the dye methylene blue chloride | Cationic chromophore, methylene blue, chloride anion. The methylene blue is postiviely charged
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| Anionic chromophores are also called... | acidic dyes (anions = negative charge because they gave all their H+ away, making them acidic)
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| Basic dyes work best under (acidic/basic) conditions | Basic. But they stain acidic structures
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| Why does Sudan Black stain adhere to membranes? | it is lipid soluble and accumulates in phospholipid bilayers
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| In Gram Staining, what color will the G+ and G- microbes be after the first, basic dye Crystal violet? | both Purple
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| In Gram Staining, what color will the G+ and G- microbes be after the mordant, iodine is applied? | Both purple
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| In Gram Staining, what color will the G+ and G- microbes be after the decolorizing agent, ethanol/acetone? | +purple -colorless
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| In Gram Staining, what color will the G+ and G- microbes be after the counterstain, safranin is applied? | +purple -pink
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| What genera are typically identified using an acid-fast stain? | Mycobacterium and Nocardia
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| In acid-fast staining, what color with the acid-fast cells and the not-acid-fast cells be after the primary stain, carbolfuchsin? | All red
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| In acid-fast staining, what color with the acid-fast cells and the not-acid-fast cells be after decolorizing with hydrochloric acid/alcohol? | Acid fast cells retain red color. Those that aren't lose their color, as does the background
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| Explain counterstaining in acid fast staining. | Use methylene blue, which stains only the bleached non-acid-fast cells. The acid fast cells remain red.
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| Which genera of bacteria produce endospores? | Bacillus and Clostridium
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| What dye colors the endospore? | Malachite green dye turns endospores green and vegetative cells remain red.
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| Histological stain Gomoroi methenamine silver (GMS) is commonly used to screen for the presence of... | Fungi and carbohydrates
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| Histological stain Hematoxylin and eosin (HE) is used to | Delineate features of histological specimens such as presence of cancer cells.
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| What two dyes are used in a negative (capsule) stain? | Negative, acidic dye repulsed from negative cell surface staining background, and basic counterstain
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| Explain flagellar stain | Pararosaniline, carbolfuchsin, and mordants such as tannic acid and potassium alum. This increases diameter and contrast.
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| What are electron microscope "stains" | Heavy metals like lead, osmium, tungsten, uranium. These absorb electrons to increase density.
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| What are the 3 constituents of a taxa? | Classification (based on similarities), Nomenclature (rules of naming), and identification (science of determining classification and nomenclature)
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| Scientist who first published taxonomy system | Carolus Linneaus, in Species Plantarum
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| Species definition: | group of organisms that interbreed to produce viable offspring. Microbial: strains arising from a single cell
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| Name the taxonomic groupings in order from largest to smallest | Domain Kingdom Phyla Order Class Family Genus Species
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| Binomial nomenclature is comprised of a genus name (always a noun) and a... | Specific epithet (species name, which is generally an adjective)
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| Name of the goal of modern taxonomy which groups organisms in a manner reflecting their evolution from common ancestors | phylogenetic heirarchy
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| Three domains proposed by Carl Woese | Eukarya, Bacteria, Archaea
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| Books containing information used for the identification of microbes | Bergey's Manual
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| term meaning shape of colony or microbe | morphology
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| term meaning that the microbe triggers an immune response in the host that results in the production of antibodies. | Antigenic
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| Substance that binds to the antigen that triggered its production | Antiserum
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| Procedure where antiserum is mixed with potentially pathogenic target cells. If antigenic cells are present, clumping occurs | Agglutination test
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| What is a phage? | Bacteriophage; a virus that infects and usually destroys bacterial cells
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| In phage typing, what are the clear areas on the bacterial lawn called? | plaques
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| mutually exclusive paired statements that lead either to the identity of an organism or to another paired statement | Dichromous keys
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