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Nissing Micro Ch4

Micro Ch 4 Microscopy

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
Created by: jenissing