Bio 2415- Module 2
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Bio 2415- Module 2
methods cell biology
Methods in Cell biology
what are the factors that determine science? (what is science?) (6)
focuses on the natural world
aims to explain the natural world
uses testable ideas
relies on evidence
involves the scientific community
leads to ongoing research
benefits from scientific behavior
what are the 6 qualities that allow science to be used as a tool for exploration?
adaptable, not rigid
only falsifies, does not prove
cumulative, builds upon previous results
self-critical, because science is social activity
devoid of moral content
is limited in its application
describe the 2 main approaches to scientific inquiry.
: scientists use some aspect of the world and use i
to draw general conclusions
: scientists use "scientific method" and
to explain nature
an initial observation leads to the discovery of a certain pattern
allows a tentative prediction to be made which leads to a general theory about how things work
general principles from a large number of specific observations
what is an example of observational science and inductive reasoning?
the cell theory
after 2 centuries of scientists discovering cells in every biological specimen they observed, they could conclude "all organisms are made of cells"
what are the problems with observational approach?
cannot establish cause and effect
(can form correlation)
groups are not random
refers to something known as the
flows in the opposite direction from inductive reasoning
from general to specific
what are the 7 steps in the scientific method?
methods and predications
what are the 3 important properties to a good hypothesis?
statement consistent with most of the data, may take the form of a model (an explanation that appears to account for the data); must be testable
a hypothesis that has been extensively tested by many investigators, using different approaches, widely accepted
a theory that has been tested and confirmed over a long period of time with virtually no doubt of it's validity
what are the 3 main points to remember during the experimenting stage of the scientific method?
tests should be
measurements that are
quantitative are better than those that are qualitative
experiments designed to test hypothesis must be "controlled" experiments
describe the 2 types of variable.
: values that can be changed in a given model or equation
: values that result from the independent variable
3 reasons controlled groups are important.
1. clarify experimental results by
providing a standard for comparison
2. without control, it's no possible to say if experimental outcome is
due to variable or some other variable
3. used to
minimize the unintended influence of other variables
on the same system
in an experiment that correlates with the variable and
can lead to erroneous "false positive" conclusions
type of cofounding variable
is some aspect of the study process itself that produces results independent of what we are testing
a mathematical body of science pertaining to the collection, analysis, interpretation, or explanation, and presentation of data
number of the same samples in the experiment
greater the N-value, greater the value of interpretations of scientific method
important because it tells you if you're consistent
have to have at least 3 repetitions in bio to perform statistics
what are the 5 sections in a research paper?
materials and methods
discussion and conclusion
peer review and it's important
is the evaluation of work b one or more people of similar competence to the producers of the work
important because it's a form of self-regulation by qualified members of a profession within the relevant field
3 main approaches of experimenting in cell biology
: using purified chemicals and cellular components or live cells
cells in petri dish of multicellular organism
: using live cells (from unicellular organism such as yeasts) organisms
: using computer analysis of large amounts of data
what are the 3 common laboratory models using in cell biology?
: easy to grow in agar medium
S. cerevisiae (eukaryotic unicellular-yeast)
: easy to grow in simple growth medium
A. thaliana (photosynthetic eukaryotic cell)
: clonal growth
who was Robert Hooke?
observed compartments in cork, under a microscope, and
first named cells
(basic unit of bio)
used dead cells with 30X enlargement
who was Anton van Leeuwenhoek?
few years later
produced better lenses
in microscope that magnified up to 300X
first to observe living cells
including single celled organism in pond water and sperm
Who as Robert Brown?
coined the term nucleus
discovered in leaves of orchids
who was Felice Fontana?
glimpsed the nucleus in epidermal cell so snakes (animal material)
described the nucleolus
what were the 2 factors that restricted progress in early cell biology?
(ability to see fine detail)
focus was observation
, with little emphasis on explanation
define chromatic aberration and how to fix it
type of distortion in which there is a failure of a lens to focus all colors to the same convergence point
blue and red most impacted
when moving through lens; they separate from each other and it depicts a blurred image
double lens system used to correct
chromatic aberration (with concave and convex lens)
what methods were used to identify intracellular structures?
oil immersion and better staining and cutting procedures led to the identification of intracellular structures
oil immersion reduces bending & velocity of light
what are the 4 requirements for microscopy?
source of illumination
system of lenses
a method of interpretation
what is the difference between compound light microscope and electron microscope in terms of lens source of illumination and visualization?
compound light microscope
: lens is glass
directly visualized though eyepiece, or focused on a detector like photographic film or digital camera
light is source of illumination
: lens is electromagnets
focused on a florescent screen or film or digitally imaged with a detector
source of illumination is beam of electrons from a heated tungsten filament
how does light help us visualize an image under a microscope?
photons and electrons behave as waves
when wave form is disrupted, object becomes visible under microscope
the ability for an object to disturb the wave form is dependent on the size of the object and the wavelength within the waveform
how close can two points be in an image before they are no longer seen as two separate points?
what are the 3 things resolution is related to?
1. numerical aperture of the object lens:
higher the numerical aperture, the better the resolution (the amount of light you can gather and focus on to the specimen)
2. The wavelength of light passing through the lens
: the shorter the wavelength, the better the resolution
3. refractory index
: a measure of the change in velocity of light as it passes through one medium to another (oil slows velocity of light)
limit of resolution
how far apart adjacent objects must be in order to be distinguished as separate entities
smaller the limit of resolution, the greater the resolving power of a microscope
distinguish the difference between magnification and resolution.
: expanding the image to get the details; is a characteristic of resolution
how did microtome improve resolution?
allowed preparation of very thin slices of samples which improves the limit of resolution
what kind of images do light microscopes create? what can be a problem?
bright field image
most cells and cells structure are transparent and colourless
how is bright field microscopy improved?
differential interference contrast
microscopy exploit differences in the phase of light passing through a structure with a refractive index different than the surrounding medium
uses a phase plate to allow rays of light to differ in wavelength
(picks up contrast on membrane)uses a prism to split the light to different wavelengths
what is one way of enhancing contrast? problem with this?
developed by German industrial chemists in second half of 19th century
key role in staining and identifying subcellular structures
have to kill specimen
Hematoxylin- blue nuclei
Eosin- red to pink proteins
Periodic Acid Schiff Stain
stains glycoproteins, glycogen, and mucin purple
hematoxylin (nuclei) + chromotrope (muscle red) + aniline blue (collagen)
ex. staining blood vessels
stains DNA and RNA blue
don't have to kill specimen to use
can wash away dye with water
detects fluorescent dyes, or labels, to show locations of substances in the cell
can view living cells or subcellular phenomena
excitation filter only allows one wavelength of light from the mercury arc lamp to pass through and that light is the one that will be picked up by specimen
(excites the specimen)
what are the 4 types of ways to make cells fluorescent?
: natural emission of light by biological structures when they have absorbed light and used to distinguish light originating from artificially added fluorescent markers
: use of glass micropipette to inject fluorescence at microscopic level (whole cell fluorescent)
: using antibodies that are directly labeled with a fluorescence dye
: direct manipulation of an organism's genome using biotechnology
uses a laser beam to illuminate a single plane of a fluorescently labeled specimen and in this way improves resolution
Computer-Enhanced Digital Microscopy
you can use a computer to subtract the noise (background junk in your image) to get a more focused picture
digital reconstruction of 2D images
stack the image together using a computer
Time lapse Microscopy
4D imaging where the 4th dimension is time
Fluorescence recovery after photobleaching (FRAP)
measure the ability of a molecule to move around over time
you can determine how fluid the cells are by seeing how long it takes the fluorescence cells to recover after bleaching
Describe the 2 types of electron microscopy.
Transmission Electron Microscopy (TEM):
electrons are transmitted though the specimen; 2D imaging
Scanning Electron Microscopy (SEM):
the surface of a specimen is scanned, by detecting electrons deflected from the outer surface; 3D imaging
TEM: Negative stain
the mount is suspended in an electron dense stain and then the specimen is dripped on top with water so to be able
to visualize the light specimen against a darkly stained background
darker image shows in electron dense
heavy metal vapor is sprayed at an angle across the specimen causing the accumulation of the metal on one side and a shadow region without metal on the other side
3D feel in a 2D image
TEM: Freeze Fracture (7 steps)
1.cells are frozen in liquid nitrogen
2. block of frozen cells is fractures
3. surface ice is removed by a vacuum
4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica
5. surface is shadowed with a platinum vapor
6. organic material is digested away by acid, leaving a replica
7. carbon-metal replica is put on a grid and examined by a transmission electron microscope
tissue contains more than just cells. what other materials might you find in tissue?
how do you isolate cells from acellular material? (separating tissue materials)
you need to chemically disrupt the extracellular matrix that holds the cells together
treat with proteolytic enzymes (trypsin and collagenase) to digest proteins in the extracellular matrix and with agents (EDTA) that bind Ca2+ on which cell-to-cell adhesion depends
tissue can then be teased apart into single living cells by gentle agitation
what are 3 methods of separating different cell types?
insert cells in tube, shake tube and dense cells fall to the bottom, less dense float to the top
Antibody, bead, or medium based centrifugation:
fill tube with antibodies and the antigens will get pulled to the bottom
Fluorescence activate cell sorting (FACS):
take antibody and flag it with flurochrome and pass it through a machine and one by one it deflects them into diff tubes
Tissue culture dishes and medium
culture dishes must be coated with ECM components
medium components vary depending on cell type
what is the function of growth factors in tissue culture?
growth factors are added to ensure that cells can undergo cell division
plant cell culture (media and how cells proliferate)
plant cell culture
requires growth regulators
many types of plant cells proliferate in culture,
producing a mass of undifferentiated cells
what is the striking feature that differentiates plant cell culture and animal cell culture?
totipotency: ability of a single cell to divide and produce all of the differentiated cells in the organism
cell culture assays
experiments with cells in culture
ex. migration assay
how are organelles and macromolecules separated using ultracentrifugation?
disrupt the cell membrane to form a homogenate
separate the components of the homogenate by centrifugation at high speeds
repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components
smaller the subcellular component, the greater the centrifugal force required to sediment it