Chapter 4 Flashcards
(46 cards)
1
Q
What are Units of Measurement?
A
-the metric system: decimal system in which each unit is one-tenth the size of the next largest unit.
• The basic unit of length in the metric system is the meter
• One-thousandth of a meter is a millimeter (mm)
• about the thickness of a dime
• One-thousandth of a millimeter is a micrometer (um)
• which is small enough to be useful in measuring the size of cells.
• One-thousandth of a micrometer is a nanometer (nm)
• which is one-billionth of a meter and is used to measure the smallest cellular organelles and viruses.
2
Q
what is Microscopy? what are the 4 principles?
A
Microscopy refers to the use of light or electrons to magnify objects.
• 4 principles: wavelength, magnification, resolution, and contrast
3
Q
what is wavelength?
A
- Various forms of radiation differ in wavelength
- The distance between two corresponding parts of a wave.
- The human eye distinguishes different wavelengths of light as different colors.
- Moving electrons also act as waves, with wavelengths dependent on the voltage of an electron beam.
- Electron wavelengths are much smaller than those of visible light, and thus their use results in enhanced microscopy.
- the smaller the wavelength, the better the resolution
4
Q
What is magnification?
A
- the apparent increase in size of an object and is indicated by a number followed by an “X”, which is read “times.”
- Magnification results when a beam of radiation refracts (bends) as it passes through a lens.
- Because of the curvature of the lens, light rays pass through a focal point then spread apart to produce an enlarged and inverted image
- Empty magnification: when magnification of an image to a point where it is too faint or too blurry to be useful, occurs when magnification has exceeded the resolving capability (resolution) of the microscope
5
Q
What is resolution?
A
• the ability to distinguish between objects that are close together.
• In less technical terms, it is the degree of clarity
• Expressed as the distance between 2 objects
-Modern microscopes can distinguish objects as close as 0.2 µm
• 2 things that limit resolution
• wavelength of light used (inverse relationship to resolution)
• numerical aperture of lens (ability to gather light)
6
Q
What is the limiting factor in obtaining good resolution?
A
wavelength of light
7
Q
What else, besides wavelength, impacts resolution?
A
- The numerical aperature of the lens also impacts resolution
- Numerical aperture is a measure of the ability of lens to collect light from the specimen.
- Lenses with low numerical aperture collect less light than those with higher numerical aperture.
- Higher numerical aperture objective yields best resolution.
8
Q
What is contrast?
A
• Contrast
• Differences in intensity between two objects, or between an object and background
• Since most microbes are colorless, staining increases contrast
• Polarized light may also be used to enhance contrast
o phase contrast microscope
9
Q
What are bright-field microscopes?
A
- Bright-field microscopes
- The most common microscopes are bright-field microscopes, in which the background (or field) is illuminated and the specimen is colored (stained) to improve contrast
• There are two basic types:
o Simple microscopes contain a single magnifying lens and are similar to a magnifying glass.
• Leeuwenhoek’s microscope had a single lens
o Compound microscopes uses 2 lenses for magnification.
• This is what we use in class
10
Q
What are the 4 objective lenses on compound light microscopes? (bright-field)
A
- ocular lens: the eyepiece; typically 10x
- objective lens: usually 3-4 on each microscope
- Scanning (4x)
- Low power (10x)
- High Power (40x)
- Oil Immersion (100x)
11
Q
what is total magnification
A
Total Magnification: ocular x objective
• Thus, the maximum magnification our microscopes in lab can achieve is 1000x (10 x 100)
• The limit of useful magnification for a light microscope is 2000x, after which empty magnification results (little to no useful resolution)
12
Q
what is the relationship between light refraction and oil immersion?
A
Light refraction and Oil Immersion
• Light travels through different substances at different speeds.
• Generally speaking, the denser the substance, the slower light moves.
• This causes light to bend
13
Q
what is refractive index?
A
Refractive index measures the light bending ability of a specimen
• things that do not bend light are invisible
• Due to the refraction of light, the 100x objective lens often does not capture enough light to view the specimen with proper resolution (or view it at all)
14
Q
How do you increase resolution with oil immersion?
A
- The oil-immersion lens (100x) is exceedingly narrow, and most light misses it.
- Therefore, the object is seen poorly and without resolution.
- To increase the resolution with the oil-immersion lens, a drop of immersion oil is placed between the lens and the glass slide
- Immersion oil has the same light-bending ability (refraction index) as the glass slide, so it keeps light in a straight line as it passes through the glass slide to the oil and on to the glass of the objective lens.
- With the increased amount of light entering the objective (increased numerical aperature), the resolution of the object increases, and one can observe objects as small as bacteria.
15
Q
What are dark-field microscopes?
A
- Dark-field microscopes
- utilizes a dark-field stop in the condenser that prevents light from directly entering the objective lens.
- Instead, light passes onto the slide at an oblique angle.
- Only light rays scattered by the specimen enter the objective lens and are seen, so the specimen appears light against a dark background.
16
Q
In dark field microscopes, what does the specimen appear like?
A
Only light rays scattered by the specimen enter the objective lens and are seen, so the specimen appears light against a dark background.
17
Q
What is the advantage of dark field?
A
- Advantage of dark field:
- Increases contrast, allowing observation of more details
- Best for observing unstained or pale objects, unseen by using bright-field
18
Q
what is a phase-contrast microscope? What are the disadvantages and advantages?
A
- Phase microscopes use a phase plate to retard light rays passing through the specimen so that they are ½ wavelength out of phase with neighboring light waves
- Brings out differences in refractive indices, producing sharp contrast
- Advantages:
- produces sharply defined images in which fine structures (including within cells) can be seen
- Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining
- Disadvantage:
- false halos around image; makes it hard to measure size
19
Q
what are fluorescent microscopes?
A
Fluorescent microscopes
• Direct UV light source at specimen to fluorescent specimen
• Specimen radiates energy back as a visible wavelength
• UV light increases resolution and contrast
• UV light has shorter wavelengths than visible light
• Because fluorescing structures are visible against a black background
• Used in immunofluorescence
• Antibodies with fluorescent dyes attached bind specially to antigens
• Used to identify pathogens and to make visible a variety of proteins (such as antibodies)
20
Q
what are confocal microscopes?
A
- Confocal microscopes
- use fluorescent dyes in conjunction with UV lasers to illuminate the fluorescent chemicals
- the lasers can target only one thin plane of a specimen at a time
- several images are taken and digitized, and then computers construct three-dimensional images of the entire specimen.
21
Q
what is electron microscopy?
A
- Electron Microscopy
- Light microscopes cannot resolve structures closer than 200 nm (.2 microns) due to the limitation of wavelength of visible light
- Electron microscopes provide both greater resolving power and magnification
- Electrons travels in much shorter wavelengths than visible light
- Can also magnify objects 10,000X to 100,000X
- Allows for detailed view of bacteria, viruses, etc.
22
Q
what is a transmission electron microscope (TEM)
A
- Transmission Electron Microscope (TEM)
- Generates a beam of electrons that passes through a thinly sliced, dehydrated specimen, through magnetic fields that manipulate and focus the beam, and then onto a fluorescent screen that changes the electrons’ energy into visible light.
- TEM produces a flattened extremely high resolution picture of the specimen
- Can resolve objects up to 2.5 nm apart
- Allows us to see internal structures of the cell
23
Q
what are scanning electron microscopes (SEM)?
A
- Scanning Electron Microscope (SEM)
- the surface of the specimen is first coated with a metal such as platinum or gold.
- The SEM then focuses the beam of electrons back and forth across the surface of the coated specimen, scanning it rather than penetrating it.
- Electrons scattered off the surface of the specimen pass through a detector and a photomultiplier, producing a signal displayed on a monitor.
- SEM produces a high resolution 3D image of the specimen
- resolves images about 20 nm apart; not quite as high resolution as TEM
24
Q
what is a probe microscope?
A
- Probe
- Probe microscopes use miniscule electronic probes to magnify specimens more than 100,000,000X.
- There are two types: Scanning tunneling microscopes and Atomic force microscopes
- Both pass a pointed metallic on the surface of a specimen and measure the amount of electron flow. They can reveal details on a specimen surface at the atomic level.
25
what is a smear? What are the steps?
* Smear: thin film of specimen applied to a slide
* Melts polysaccharides (sugars) so that they stick to the slide; kills specimen
* Steps:
* Add drop of water to slide
* Smear applied to slide
* Air dried (or heated lightly)
* Specimen fixed to slide (attached)
* Usually heat-fixed: kills and attaches specimen to slide
* Heat fix by passing thru flame 1-2 times
* Stain smear so you can view it
26
what do you do after the bacteria is fixed to the slide? Why do we do this thing?
* After the bacteria is fixed to the slide, we can add stains
* Stain: coloring a specimen with dye
* Why we stain:
* provides contrast between organisms and background (incr resolution)
* allows us to see morphology (shape) and arrangement (grouping)
* Allows us to view structures of organisms (nucleus in Euks)
* some staining techniques help identify organisms further (differential stains)
27
what are the types of staining?
basic dyes, acidic dyes, simple stains, differential stains (gram and acid-fast stains), Endospore stain
28
What are basic dyes?
* Basic dyes: the colored ion (chromophore) is positively charged
* opposites attract!
* the positively charged chromophore sticks to the negatively charged bacteria giving it color
* this is called “direct staining”
29
what are acidic dyes?
* Acidic dyes: the chromophore is negatively charged
* Like charges repel
* the dye colors the background, not the specimen
* this is called “negative (indirect) staining”
* Allows us to view capsules (outside of cell), giving us an idea of the true size
30
what are simple stains?
* Simple stains
* Composed of single basic dye
* Crystal violet, safranin, methylene blue
* Used to determine size, shape, and arrangement of cells
31
what are differential stains, and what are the 3 different kinds?
* Differential Stains: used to differentiate bacteria into groups; useful step in identification of bacteria
* 1) Gram stain
* Most common technique in Microbiology
* Breaks bacteria in 2 groups: Gram (+) and Gram (-)
* Capitalizes on differences in cell wall thickness
* 2) Acid-fast stain
* Used to identify Mycobacterium, which has a waxy cell wall and doesn’t readily take up dye
* Members of this genus include the pathogens that cause tuberculosis and leprosy
* 3) Endospore stain
* Allows us to view endospores, which are produced by 2 genera: Clostridium and Bacillus, both of which include pathogenic species
* Malachite green heated to promote penetration of stain into endospores
* Counterstained with safranin
32
what are the 4 steps in gram staining?
* Step 1: primary stain
* Crystal violet (purple)
* Wait about 20 seconds
* Rinse with distilled water
* Outcome: all cells (Gram + and - are stained purple)
* Step 2: Gram stain
* Creates an CV-Iodine complex (which is larger than crystal violet alone) within the bacteria; this makes it harder for the stain to leave the cell during the alcohol wash
* Leave on for 20 seconds
* Rinse with distilled water
* Outcome: all cells still purple
* Step 3: Decolorizer
* Alcohol is used as the decolorizer
* Removes primary stain from gram (-) cells
* Leave alcohol on for 20 seconds
* Rinse with distilled water
* Outcome: Gram + remain purple, - are colorless
* This step is the MOST IMPORTANT step of the gram stain
* The timing is critical
* Step 4: Counterstain
* Safranin, which is pink, is used as the secondary stain (counterstain)
* Leave on for 20-30 seconds
* Rinse with distilled water
* Dry slide
* View under microscope
* Outcome: Gram + still purple, Gram - pink
33
What is Mordant?
Mordant - intensifies the stain (makes it STICK) and coats a structure to make it thicker and easier to see after it is stained
• e.g., Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained
• e.g., we use a mordant during gram staining so that the crystal violet stain stays inside the bacteria
• Iodine is a common mordant
34
what is the key to gram staining?
* The key to the gram stain is the alcohol wash
* Timing of this step is absolutely critical
* The alcohol dehydrates the cell wall of Gram (+), prevent the CV-I complex from leaving the cell
* The alcohol burns holes in the cell of the Gram (-), allow the CV-I to escape
* Because the cell wall of Gram (-) is much thinner
* If you leave the alcohol on too long, Gram (+) cells lose the CV-I complex, and will appear pink
* If you do not leave the alcohol on long enough, Gram (-) cells will retain the CV-I, and look purple
35
how to scientists sort organisms? Who came up with this?
•Scientists sort organisms on the basis of mutual similarities into non-overlapping groups called taxa
o Taxonomy : science of classification, nomenclature (rules of naming), and identification
• Linnaeus and Taxonomic Categories
o Carolus Linnaeus invented a system of taxonomy, grouping similar interbreeding organisms into groups
o He gave each species a descriptive name consisting of a genus name and specific epithet
o This practice of naming organisms with two names is called nomenclature
• Today, taxonomists place less emphasis on comparisons of organisms’ physical and chemical traits and greater emphasis on comparisons of their genetic material
36
what did carl woese do?
Carl Woese compared nucleotide sequences of rRNA subunits; proposed three domains as determined by ribosomal nucleotide sequences
• Eukarya, Bacteria, and Archaea
• Cells in the three domains differ by other characteristics
• Including cell membrane lipids
• Types of tRNA
• Sensitivity to antibiotics
37
What is Bergey's manual of systematic Bacteriology?
* Bergey’s Manual of Systematic Bacteriology
* 5 volumes (when finished)
* Based on evolutionary relatedness
* rRNA sequencing
* all organisms have ribosomes!
* Bergey’s Manual of Determinative Bacteriology
* Provides identification schemes based on criteria such as morphological features, biochemical tests, etc.
38
What are dichotomous keys?
Dichotomous keys
• Series of paired statements where only one of two “either/or” choices applies to any particular organism
• Key directs user to another pair of statements, or provides name of organism
39
What 5 procedures are used to identify and classify organisms?
```
Taxonomists use one or more of five procedures to identify and classify microorganisms:
• Physical characteristics
• Biochemical tests
• serological tests
• phage typing
• nucleic acid test
```
40
What 5 physical characteristics can be used to identify microorganisms?
```
For bacteria:
• Colony appearance
• Morphology and arrangement of cells
• Gram stain results
• Acid fast results (identify Mycobacterium)
• Flagella (location, number, etc.)
```
41
what biochemical tests are used to identify microbes?
Microbiologists also use biochemical tests
• Distinguishes microbes by their ability to utilize or produce certain chemicals.
• This includes
• the used of selective and/or differential media
• Fermentation tubes
• Rapid Identification methods
42
What are serological tests
Serological Tests: The study of antigen-antibody reactions in laboratory settings
• Many microorganisms trigger an immune response that results in antibody production
• In an agglutination test, antiserum is mixed with a sample that may be antigenic: clumping of antigen with antibodies (agglutination) indicates the presence of the target cells.
43
What are bacteriophages?
Bacteriophages are viruses that infect and usually destroy bacterial cells.
• Whenever a specific phage is able to infect and kill bacteria, the resulting lack of bacterial growth produces within the bacterial lawn a clear area called a plaque.
• Phage typing may thus reveal that one bacterial strain is susceptible to a particular phage, whereas another is not, and allow scientists to distinguish between them.
• Scientists also analyze a specimen’s nucleic acids
• The DNA and RNA inside a cell can be used to identify species and strains of a species
• Includes rRNA sequencing (ribotyping)
• Used in Bergey’s Systematic Manual
• All organisms have ribosomes AND they tend to be less degenerative (fewer mutations) than DNA
• G+C %: percentage of guanines and cytosines in DNA
44
What is the G+ C ratio?
The G + C ratio is the percentage of all base pairs in a genome that are guanine-cytosine base pairs.
• Taxonomists use this ratio in classifying microbes.
• Low G + C gram positive
• High G + C gram negative
• Bacteria with G + C ratios below 50% are considered “low G + C bacteria”; the remainder are considered “high G + C bacteria.”
45
what are the LOW G+C Gram (+) examples?
Low G + C Gram (+) examples
* Clostridia are rod-shaped, obligate anaerobes, many form endosporesIncludes causative agents of gangrene, tetanus, botulism, and diarrhea.
* Mycoplasmas lack cell walls; frequently associated with pneumonia and urinary tract infections.
* Bacillus forms endospores and is common in soils. Some bacilli are beneficial in agriculture, but others cause anthrax and food poisoning.
* Listeria can contaminate meat and milk products and causes bacteremia and meningitis.
* Lactobacillus are rods normally found in the mouth, gastrointestinal tract, and vagina, where they provide beneficial microbial antagonism through lactic acid fermentation. They are also used in the production of yogurt, buttermilk, and pickles.
* Streptococcus causes strep throat and other diseases
* Staphylococcus is found growing harmlessly on human skin, although some strains can invade the body and cause pneumonia, toxic shock syndrome, and a variety of other serious infections.
46
What are the HIGH G+C gram (+) examples?
High G + C Gram Positive examples
* Corynebacterium are pleomorphic aerobes and facultative anaerobes that reproduce by snapping division. They are characterized by their stores of phosphate within inclusions called metachromatic granules.
* Mycobacterium includes species that cause tuberculosis and leprosy; grow slowly and have unique cell walls containing waxy mycolic acids.
* Actinomycetes resemble fungi in that they produce spores and form filaments; includes Actinomyces, which is normally found in human mouths and Streptomyces, which is used to produce important antibiotics.
