Lecture Exam 1 Flashcards
1
Q
What is cytology?
A
Study of cell structure
2
Q
What is cell physiology?
A
Study of cell function
3
Q
Why is the study of cells important?
A
Levels of biological organization
Cell structure and disease
4
Q
Why is it important to study cell structure?
A
Biologists must understand cell structure to understand how cells function like cell membrane structure/support and diseases
5
Q
What is the cell membrane?
A
Membrane made of phospholipid bilayer (polar and non-polar regions) that contains proteins
not all molecules can move freely across membrane
6
Q
What disease was mentioned in the importance of studying cell structure?
A
Listeria monocytogenes (a bacterial infection of eukaryotic cells)
7
Q
What are microscopes?
A
The main tool used to study cell structure
8
Q
What is focus?
A
The ability to see an object clearly
9
Q
What is magnification?
A
The degree to which an object is enlarged
4x, 10x, 40x, 100x
10
Q
What is resolution?
A
The ability to see two objects as distinct
11
Q
What is contrast?
A
The ability to see an object against a background
12
Q
How is contrast created?
A
Via staining or type of microscope (phase contrast) that creates contrast
13
Q
How are microscopes classified?
A
Based upon the wavelengths of light used to magnify an object and their resolution
14
Q
What are the 3 elements of all microscopes?
A
Source of illumination (light source or beam of electron)
Specimen
System of lenses
15
Q
What does the system of lenses do in a microscope?
A
Focuses the illumination source on the specimen and forms the image
16
Q
What is a compound microscope?
A
Uses 2 sets of lenses to generate the image
17
Q
What are the two sets of lenses called in a compound scope?
A
Objective and ocular (in eyepiece)
18
Q
What is total magnification?
A
Objective times ocular magnification
19
Q
What are the 3 general types of scopes?
A
Light
Electron
Scanned probe microscope (atomic force)
20
Q
List the types of light microscopes?
A
Bright field, dark field, phase contrast, fluorescence, confocal
21
Q
What are bright field scopes?
A
Image is obtained by the transmission of light through objects
22
Q
What are dark field scopes?
A
Generally used to view living specimens, light is directed from the side or the back
Only scattered light enters microscope lenses (lowlight source)
a condenser and/or a stop is present below the stage causing the light rays to hit the specimen at different angles
Specimens appear as a bright object against a dark background
23
Q
what is a phase contrast scope used for?
A
widely used to visualize living cells
24
Q
how does a phase contrast scope work?
A
uses a change in the phase of light as it passes through the specimen in order to generate contrast
beam of light made up of many individual rays of light
as ray passes from the light source through the specimen, their velocity may be affected by density of the specimen
25
in phase contrast microscopy, the waves in phase appear as what? out of phase?
in: bright area
out: dim area
26
how does the setting of phase contrast microscopy work?
setting of phase plate is controlled by the phase ring
phase ring setting must match the setting of the objective lens (ex: 10x objective, PH1)
27
what is fluorescence microscopy used for?
to detect structures, molecules, or proteins within a cell (types of stains)
28
How does fluorescence microscopy work?
fluorescent molecules absorb light at one wavelength and emit light at another, longer wavelength
in fluorescence microscopy, a cell is stained with a dye, and the dye is illuminated with filtered light (barrier filter #1:excitation filter) at the absorbing wavelength
the light emitted from the light source is directed towards the cell by a beam splitting mirror (dichroic mirror)
the light emitted from the dye is viewed through a filter (barrier filter #2) that allows only the emitted wavelength to be seen
29
give an example of a fluorescent stain
DAPI (4'6-diamidino-2-phenylindole)
nucleic acid stain that stains double stranded DNA
excitation maximum for DAPI bound to ds DNA is 358 nm
the emission maximum is 461 nm
30
what other fluorescence stains were discussed?
acridine orange
nucleic acid stain that stains ds DNA green and ss DNA orange
31
how does the confocal microscope work?
the confocal microscope is able to filter the out of focus light from above and below the point of focus in the object using a confocal pinhole
light from above and below the plane of focus of the object is eliminated from the final image
32
describe electron microscopy
uses a beam of electrons (heated tungsten filament) instead of a light beam
uses electromagnets as lenses to direct the electron beam
contrast is created by the scattering of the electron beam
33
why use an electron microscope?
revolving power of electron microscope is far greater than other microscopes due to the short wavelengths of electrons (as compared to wavelengths of light)
thin sections (resolution)
34
what are the two types of electron microscope?
transmission (TEM)
scanning (SEM)
35
describe the transmission electron microscope
forms an image from electrons that are transmitted through the specimen being examined
generates a flat 2-D image
used to view internal cell structures
36
what are general stains?
used to stain a population of molecules or structures inside a cell
37
what are specific stains?
used to stain specific molecules in a cell (usually proteins)
stain is covalently bound to an antibody that is specific for the protein being stained
38
what are the disadvantages of TEM?
sections must be thin (no 3D aspect)
specimens must be fixed, dehydrated and view under a high vacuum to prevent scattering of electrons
treatment kills the specimens
treatments can also cause shrinkage and distortion
39
how are sections created for microscopy?
microtome
cryosectioning
40
what is a microtome and how is it used for sectioning?
instrument that cuts specimen into thin sliced to be put on slides
specimen must first be dehydrated--water replaced with paraffin wax, then wax block is sliced
41
why is paraffin wax used with a microtome?
hardens the specimen for slicing
42
describe scanning electron microscopy
produces an image from electrons that are deflected from a specimen's outer surface (overcomes problem of sectioning with TEM)
beam of electrons scans the surface creating a 3D image
useful in studying the surface structures of intact cells and viruses
43
what is freeze fracture?
a type of electron microscopy technique used to view the interior of cell membranes
tissue block is frozen-- frozen block is cracked to create a cross section
cracked cross section is coated with a heavy metal stain (scatters electrons)
44
what are scanned probe microscopes?
use various kinds of probes to examine the surface of a specimen using electrical current
electrical current does not modify or damage the specimen
45
what are atomic force microscopes?
uses a metal and diamond probe
the probe is forced down onto the specimen and scans across the surface
no special sample preparation
provides 3D images of samples at high resolution
46
what is the purpose of using stains in microscopy?
stains can be used to create contrast and therefore label cell structures/molecules
47
what must be done prior to staining?
tissues must be sectioned (microtome or cryosectioning)
cells must be permeabilized and fixed
48
why must cells be permeabilized prior to staining?
permeabilizing solutions make holes in the cell membrane that allows cells to take up stains
49
what is cell permeabilization?
treatment of cells usually with a mild detergent such as NP-40 or Triton X
50
what is cell fixation?
allows for preservation of cell structure with only minimal distortion
often kills the cells
51
how are cells fixed?
heat fixation
treatment with cell fixative (forms chemical bonds between proteins)
52
describe the process of heat fixing
simple method: air drying and heat fixing
organisms are heat fixed by passing an air-dried smear of the cells through the flame of a bunsen burner
heat coagulates the cellular proteins causing the cells to stick to the slide (don't overheat)
53
give examples of cell fixatives
formaldehyde (paraformaldehyde, paraformalin)
gluteraldehyde
alcohol (ethanol or ethanol/methanol mix
actual stain (alcohol or methanol based)
54
what is in vitro staining?
staining of non-living fixed cells or tissues
55
what is in vivo staining?
staining of living tissues using vital stains which are introduced into the cells/tissues while the cells are still living
56
what is an intra vital stain?
solution or dye is injected into the body or living tissue
57
what is a supra vital stain?
staining of living cells outside the body
58
give examples of in vivo staining
GFP (green fluorescent protein) [not a general stain]
nile blue (nuclear stain)
59
describe the use of intra vital stains in ophthalmology
sodium fluorescein (water soluble and non-toxic)
can be used to determine the shape of the eye for fitting of contact lenses; studying dry eye; diagnosis of corneal abrasions (corneal staining)
easily flushes out of eye
60
describe GFP (green fluorescent protein)
originally isolated from jellyfish
traditionally used to study protein expression in living cells
can also be used to track cell movement or development
can also be used to label cellular proteins/structures/organelles
61
what is a mordant?
salts of aluminum, iodine or iron
helps some types of cells take up stains
mix dye with the mordant to form an insoluble colored precipitate
excess dye is washed away and the mordant remains behind
62
give an example of the use of a mordant in staining
gram staining of bacteria
add crystal violet follow by iodine (iodine acts as a mordant)
63
give examples of organic dye stains and what they stain
fuchsin- mitochondria
sudan black- fat granules
coomassie blue- protein stain
hematoxylin/eosin- nucleus/cytoplasm stain
64
what is a counterstain?
stains that have a contrasting color to the primary stain
65
give 2 examples of a counterstain
gram stain: safranin (stains gram neg bacteria pink) acts as a counterstain because gram pos bacteria retain the original purple stain
hemotoxylin/eosin: eosin acts as a counterstain
66
what are the two types of fluorescent stains?
general stains
specific markers
67
describe general fluorescent stains
stain a entire group of molecules/structures in a cell
example: acridine orange (nucleic acid stain)
68
describe specific marker fluorescent stains
will stain a specific structure or molecule
carried into cells by antibodies (dye is covalently linked to the antibody structure)
69
how are antibodies named?
animal used to produce the antibody is listed first
animal that produced the antigen protein is listed second
70
give an example of antibody naming
inject a goat actin protein into a mouse for production of an actin antibody
name: mouse anti goat actin antibody
71
give examples of fluorescent dyes:
fluorescein
rhodamine
DAPI
acridine orange
ethidium bromide
72
describe fluorescein
fluoresces green when excited with blue light
not a general stain: carried into cells by an antibody
73
describe rhodamine
fluoresces red when excited with green-yellow light
not a general stain: carried into cells by an antibody
74
describe DAPI
DNA stain (nuclear stain) fluoresces blue when excited by UV light
75
describe acridine orange
Nucleic acid stain
fluoresces green or red/orange when excited with light 500 nm in wavelength
76
describe ethidium bromide
DNA stain that stains cells in the final stages of apoptosis
fluoresces when excited by UV light
77
what is immunofluorescence staining?
specific staining that uses fluorescently labeled antibodies
78
what are the two types of immunofluorescence staining
direct and indirect
79
what is direct immunofluorescence staining
uses a single antibody carrying a fluorescent label
80
what is indirect immunofluorescence staining
uses two antibodies (primary and secondary)
81
what is a primary antibody?
specific for the cell molecule or structure to be stained
82
what is a secondary antibody?
specific for the primary antibody and carries the fluorescent label
83
what types of staining are used for electron microscopy?
negative stain
immunogold labeling
84
describe negative staining
use of heavy metals
some biological molecules (C, H, O, N) have low atomic numbers and cannot scatter electrons well
heavy metal stains are electron dense and can be used to scatter electrons
the heavy metal can be linked to an antibody specific for a protein or the specimen can be suspended directly in an electron dense stain
antibody attaches to the protein and the protein can be visualized in the cell
85
when is negative staining used?
used on small intact specimens (viruses) (no sectioning is needed but larger specimens can be sectioned)
used in SEM to visualize the shape and surface appearance of small specimens
86
describe immunogold labeling
used to localize specific macromolecules (proteins)
uses two antibodies: primary and secondary
primary antibody: specific for the macromolecule
secondary antibody: specific for the primary antibody labeled with gold particle (electron dense)
gold particles visualized as dark spots
87
what is an organelle?
membrane enclosed compartment found in cell interior
88
what is the lumen of an organelle?
the interior of the organelle
89
what are the characteristics of organelles
each organelle contains its own set of specialized molecules/enzymes
proteins are synthesized in the cytoplasm (or other places) and these proteins carry a signal sequence that directs it to a specific cellular compartment or organelle
90
what are signal sequences?
sequences that direct proteins to cellular destinations
91
where are proteins synthesized?
cytoplasm
ER membranes
mitochondria/chloroplasts
92
what are the two major steps in the isolation of organelles?
homogenization and fractionation
93
what is homogenization?
disrupts cells so that intact organelles can be isolated
94
what is fractionation?
isolates organelles (usually by centrifugation)
95
what are acid stains and give an example
color comes from presence of negative ions
eosin: cytoplasmic stain, stains pink
96
what are basic stains and give an example
color comes from positive ions in sample
hematoxylin: nuclear stain, stains purple
97
what are neutral stains?
color comes from presence of both positive and negative ions
98
describe the process of antibody production
1. inject protein (for antibody production) into an animal, animal produces antibodies against protein (immune response)
2. sacrifice animal and collect spleen cells
3. hybridoma cells create immortal cell line
4. purify antibodies from immortal cells, lyse cells nd collect cell lysate, apply cell lysate to affinity column
5. pack affinity column with beads bound to protein used to produce antibody
6. apply cell lysate to column, only antibody will bind to beads in column
99
where are antibodies produced?
the spleen
100
what is an immortal cell line?
cells continuously growing, dividing, and producing antibodies
101
what is a hybridoma cell?
spleen cell +tumor cell
102
what makes up homogenization buffer?
detergents (NP-40 or Triton X), proteases (collagenase or trypsin) and chelating agents (EDTA)
103
how are tissues homogenized?
addition of homogenization buffer
mechanical action
104
what forms of mechanical action are used in homogenization?
homogenizer, blender, sonicator
105
what does a sonicator do?
electrical pulses converted to vibrations to disrupt cells
106
what do chelating agents do?
break adhesions between cells in tissue layer-- role of cadherins
107
wat is the ECM made of and what part of the homogenization buffer breaks it down?
carbohydrates and proteins
broken down by proteases (break linkages between ECM and cell layer)
108
what is the role of cell adhesion molecules (CAMs)?
keep cells together
example: cadherins are calcium dependent cell adhesion molecules
109
what breaks down cell adhesion molecules?
EDTA binds calcium and disrupts adhesion
110
what is the role of tight junctions?
link cells tightly together in a tissue layer, mechanical force important to break these
111
what is the role of integrins?
family of proteins that link cells to the ECM
112
what are the two types of centrifugation used for fractionation?
differential velocity centrifugation
density gradient centrifugation
113
describe differential velocity centrifugation
separates organelles based on size and density
vary time and speed to isolate organelles
generally uses a fixed angle rotor
the homogenate is repeatedly centrifuged at varying force
114
what is a fixed angle rotor?
holds the centrifuge tubes at one angle during centrifuge process
115
describe density gradient centrifugation
used to further purify organelles after differential velocity centrifugation
uses sucrose, cesium chloride or Percoll gradients (very viscous solutions)
organelle is isolated when the density of the surrounding solution matches the density of the organelle
uses a swinging bucket rotor
116
Why do we isolate organelles into cell free systems?
Use isolated organelles to determine the specific details of cell processes
117
What is a cell free system?
Function of organelles without the whole cell
Experiments with cell processes by adding or taking away a component
118
What is the structure of the nucleus?
Surrounded by a double membrane (nuclear envelope)
Membrane contains pores that allow for transport of molecules into and out of the nucleus
119
What types of molecules are transported into the nucleus?
Enzymes involved in DNA replication and transcription
Histone proteins
120
121
What types of molecules are transported out of the nucleus?
mRNA
ribosomes
122
What is the function of the nucleolus?
Produces ribosomes needed for protein synthesis and translation
123
What is the function of the nucleus?
Contains genetic material (DNA)
Organization (chromatin, chromosomes)
Site of DNA and RNA synthesis
124
Describe chromatin
DNA and bound histones, tightly packed
Tight because - DNA and + histones
Occurs during interphase (G1,G2,S)
125
Describe chromosomes
Condensation (unpacking) of chromatin
M phase of cell cycle
126
Describe the general structure of the endoplasmic reticulum
Organized into branching, tubules and flattened sacs that extend through the cytoplasm, tubules interconnect so the membrane informs a continuous sheet
127
What is a membrane that encloses a single internal space called?
Lumen
128
What is the difference between the rough endoplasmic reticulum and the smooth endoplasmic reticulum?
The rough endoplasmic reticulum has ribosomes associated with the ER membrane. The smooth ER does not.
129
What is the function of the rough ER?
The rough ER functions in protein, synthesis, and protein folding
130
What types of proteins does the rough ER function in?
Transmembrane proteins and water soluble proteins
131
What are transmembrane proteins?
Proteins that span the width of the membrane (plasma membrane, or organelle membrane)
132
What are examples of transmembrane proteins?
Integrins, channel proteins, etc.
133
What are water soluble proteins?
Proteins that are fully translocated across the endoplasmic reticulum membrane and released into the ER lumen OR proteins destined for secretion or the lumen of an organelle
134
135
Give examples of water soluble proteins
Resident ER proteins, hormones
136
What are the functions of the smooth ER?
Lipid synthesis: phospholipids, fatty acids, steroids
Carbohydrate metabolism
Detoxification of the cell
Calcium storage
137
What type of cells have a large smooth ER?
Liver cells
138
Cystic fibrosis is caused by a mutation in gene encoding regions of what?
Chloride ion channels
139
What is CFTR?
Transmembrane glycoprotein (protein with sugar side chains)
140
Describe how the chloride ion channels work in a person without cystic fibrosis
Chloride ions are secreted from cells lining the airways of the lungs
Chloride ions and sodium ions secreted from the cell
Water follows the chloride and sodium ions and results in the hydration of the mucus lining the air passages
141
Describe the chloride ion channels in a person with CF
Chloride ion is not transported
Sodium and water don’t exit the cell
Lack of hydration of mucus lining the air passages favors the growth of bacteria
142
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