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Nissing Micro Ch4
Micro Ch 4 Microscopy
| Question | Answer |
|---|---|
| Describe the image produced by a convex lense | Inverted, reversed, enlarged |
| Usefulness of microscope depends on ability to clearly resolve two objects close together | Resolution |
| Minimum distance between two objects where those objects still appear distinct | Resolving power |
| Determines how much detail can be seen | Resloving power |
| Two things that determine resolution | Quality of lense and illuminating light's wavelength |
| Maximum power of most bright field microscopes | 0.2 micrometers or 1x10(^-6)m, you can see bacteria but not viruses |
| Ability to see differences between two objects or an object and its background | Contrast |
| Increase contrast by... | staining |
| Light microscope with a single magnifying lense similar to a magnifying glass, like Leeuwenhoek's | Bright field microscopes |
| How to determine toal magnification of a bright field microscope | Objective lense x ocular lense |
| What is the purpose of a condenser lense, present on most bright field scopes? | Focuses illumination on specimen and does not affect magnification |
| Why is oil used on higher magnificaiton? | it reduces light refraction because it has a very similar refractive index to glass |
| Best for observing pale objects, the only light seen is that scattered by the specimen | Dark field microscopy |
| Which light microscope is like someone standing in the dark while people shine flashlights on him? | Dark field |
| What type of organisms are used in phase microscopes? | Living microbes that would be damaged by attaching them to slides or staning |
| How is contrast created in a phase microscope? | Light waves are put out of phase, which creates shadow-like |
| A phase microscope is a type of light microscope, name the two types of phase microscopes | Phase-contrast and differential interference contrast (Nomarski) |
| This type of microscopy uses UV light which specimen radiates back as visible wavelengths | Fluorescent microscopes |
| Fluorescent microscopes are used in immunoflorescence to... | identify pathogens and make proteins visible |
| If a specimen is not naturally fluroescent, what must be done? | Attach fluorescent die to antibodies, which attach to specimen |
| Light microscope using fluorescent dies, UV lasers and producing images in planes | Confocal microscope |
| How close can light microscopes resovle structures? | 200nm |
| Microscope that allows greater resolving power and magnification up to 100,000 giving detailed view of bacteria, viruses, even large atoms | Electron Microscope |
| Two types of electron microscope | Transmission (TEM) and Scanning (SEM) |
| Microscope that magnifies over 100,000,000 times | Probe microscopy |
| Two types of probe microscope | Scanning tunneling, and atomic force |
| What needs to be done to a specimen before dying it? | Spread, Dry completely, Heat fix |
| If you don't dry your sample completely before heat fixing it, what happens to your bacteria? | It explodes |
| What are stains made of? | organic salts |
| Basic dyes carry a _________ charge | positive (Bases absorb H+) |
| Positively charged basic dyes bond to negative charged structures like... | The Cell |
| Acidic dyes carry a _________ charge | negative (acids release H+) |
| Negatively charged acidic dyes are repelled by the cell and commonly stain... | The background (negative staining) |
| From this we can tell that the cell has an overall _______ charge | negative |
| Commonly used basic dyes | Methylene blue, Crystal violet, Safranin, Malachite green |
| 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 |
| Two most common types of differential stain | Gram stain, Acid-fast stain |
| What do differential stains use to differentiate? | reagents |
| Most widely used procedure for staining bacteria developed over a century ago | Gram Stain |
| Gram staining separates bacteria into two major groups. What are they? | G+ is purple. G- is pink. |
| In Gram staining, what is the primary stain? | Crystal violet. This stains all cells violet. |
| In Gram staining, what is the second step? | Mordant, which makes stain stick to peptidoglycan better. In this case it's Gram's iodine. |
| In Gram staining, what is the third step? | Decolorizing agent like alcohol removes dye from some cells |
| Which cells will be decolorized by the alcohol? | Gram negative cells will quickly decolorize, Gram positive cells can eventually be decolorized |
| In Gram staining, what do the cells look like after the third step? | Gram negative are clear, Gram positive are clear |
| In Gram staining, what is the final step? | Counterstain/Secondary stain/recoloring with Safranin |
| What type of organism is Acid-fast staining used on? | Resistant to convential staining, like mycobacterium, which has mycolic acid, a waxy protectant |
| How does acid-fast stain get into cell? | Heat or concentrated dye |
| In Acid-Fast staining, what is the first step? | Primary dye Carbol fuschin |
| What color are the cells after the first step of acid-fast staining? | All are fuschia |
| In Acid-Fast staining, what is the second step? | Decolorize with an acid alcohol |
| 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 |
| In Acid-Fast staining, what is the final step? | Counterstain with methylene blue |
| 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. |
| An example of a negative that only stains the background and causes a "halo" of no dye? | Capsule stain |
| Example of using heat to facilitate staining of resistant microbe structures like those formed by Bacillus and Clostridium | Capsule stain |
| What dye is typically used for endospore staining? | Malachite green |
| Flagella Stain increases the diameter of flagella and is made more visible by using a... | mordant |
| What is a micrometer? | 1/1000th of a milimeter |
| What is a nanometer? | 1/1000 of a micrometer |
| Why does a lens refract light? | it is obtically dense compared to the surrounding medium, it moves more slowly through the lens |
| In what way do light rays move as they pass through the focal point? | They spread apart, creating an enlarged inverted image |
| Type of magnification combining many lenses to obtain a large, blury, faint image | empty magnification |
| What are the two properties of a clear image? | resolution and contrast |
| The ability to distinguish objects that are close together | resolution |
| Part of the microscope which remagnifies the image formed by the objective lense | Ocular lense |
| Part of the microscope which transmits the image from the objective lens to the ocular lens using prisms | Body |
| Part of the microscope which holds the body onto the base | Arm |
| Part of the microscope which contains primary magnification lenses | Objective lenses |
| Part of the microscope which holds the microscope slide in position | Stage |
| Part of the microscope which focuses light through the specimen | Condenser |
| Part of the microscope which controls the amount of light entering the condenser | Diaphragm |
| Part of the microscope which contains the light source | Illuminator |
| Part of the microscope which moves the stage up and down to focus the image | Coarse and fine focusing knobs |
| Resolution distance is dependent on these two principles | Wavelength of electromagnetic radiation x0.61 / numerical aperture of lens |
| Term refering to differences in intensity between two objects, or between an object and its background | Contrast |
| Two ways to increase contrast between microbes and their background | Staining and in phase light |
| What do we call microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a specimen? | confocal microscopes |
| 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) |
| Part of the microscope which holds 3 or 4 objective lenses | Revolving nosepiece |
| Why does immersion oil increase resolution? | It has a similar refractive index to glass |
| Name for the distance between the lens and the specimen | working distance |
| How is total magnification of a microscope determined? | multiply objective by ocular |
| What is a photograph through a microscope called? | Micrograph |
| What type of objects do we observe through dark field microscopes? | pale |
| 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 |
| 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 |
| 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 |
| Molecules that absorb energy from invisible radiation and radiate it back as longer, visible wavelengths | fluorescent |
| 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 |
| 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 |
| What are the 2 probe microscopes? | Scanning tunneling and atomic force |
| 2 ways to fix a smear to a slide | Heat fixation or chemical fixation. Both cause desiccation. |
| Name of the colored portion of a dye | chromophore |
| Give components of the dye methylene blue chloride | Cationic chromophore, methylene blue, chloride anion. The methylene blue is postiviely charged |
| Anionic chromophores are also called... | acidic dyes (anions = negative charge because they gave all their H+ away, making them acidic) |
| Basic dyes work best under (acidic/basic) conditions | Basic. But they stain acidic structures |
| Why does Sudan Black stain adhere to membranes? | it is lipid soluble and accumulates in phospholipid bilayers |
| In Gram Staining, what color will the G+ and G- microbes be after the first, basic dye Crystal violet? | both Purple |
| In Gram Staining, what color will the G+ and G- microbes be after the mordant, iodine is applied? | Both purple |
| In Gram Staining, what color will the G+ and G- microbes be after the decolorizing agent, ethanol/acetone? | +purple -colorless |
| In Gram Staining, what color will the G+ and G- microbes be after the counterstain, safranin is applied? | +purple -pink |
| What genera are typically identified using an acid-fast stain? | Mycobacterium and Nocardia |
| 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 |
| 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 |
| Explain counterstaining in acid fast staining. | Use methylene blue, which stains only the bleached non-acid-fast cells. The acid fast cells remain red. |
| Which genera of bacteria produce endospores? | Bacillus and Clostridium |
| What dye colors the endospore? | Malachite green dye turns endospores green and vegetative cells remain red. |
| Histological stain Gomoroi methenamine silver (GMS) is commonly used to screen for the presence of... | Fungi and carbohydrates |
| Histological stain Hematoxylin and eosin (HE) is used to | Delineate features of histological specimens such as presence of cancer cells. |
| What two dyes are used in a negative (capsule) stain? | Negative, acidic dye repulsed from negative cell surface staining background, and basic counterstain |
| Explain flagellar stain | Pararosaniline, carbolfuchsin, and mordants such as tannic acid and potassium alum. This increases diameter and contrast. |
| What are electron microscope "stains" | Heavy metals like lead, osmium, tungsten, uranium. These absorb electrons to increase density. |
| What are the 3 constituents of a taxa? | Classification (based on similarities), Nomenclature (rules of naming), and identification (science of determining classification and nomenclature) |
| Scientist who first published taxonomy system | Carolus Linneaus, in Species Plantarum |
| Species definition: | group of organisms that interbreed to produce viable offspring. Microbial: strains arising from a single cell |
| Name the taxonomic groupings in order from largest to smallest | Domain Kingdom Phyla Order Class Family Genus Species |
| Binomial nomenclature is comprised of a genus name (always a noun) and a... | Specific epithet (species name, which is generally an adjective) |
| Name of the goal of modern taxonomy which groups organisms in a manner reflecting their evolution from common ancestors | phylogenetic heirarchy |
| Three domains proposed by Carl Woese | Eukarya, Bacteria, Archaea |
| Books containing information used for the identification of microbes | Bergey's Manual |
| term meaning shape of colony or microbe | morphology |
| term meaning that the microbe triggers an immune response in the host that results in the production of antibodies. | Antigenic |
| Substance that binds to the antigen that triggered its production | Antiserum |
| Procedure where antiserum is mixed with potentially pathogenic target cells. If antigenic cells are present, clumping occurs | Agglutination test |
| What is a phage? | Bacteriophage; a virus that infects and usually destroys bacterial cells |
| In phage typing, what are the clear areas on the bacterial lawn called? | plaques |
| mutually exclusive paired statements that lead either to the identity of an organism or to another paired statement | Dichromous keys |
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