1. (5 points) Briefly explain the idea of differential centrifugation . What is the point of the multiple rounds of centrifugation?
The idea of differential centrifugation is isolate different components of the cell. For example, the nuclei, the cell wall, mitochondria, or other organelles in the cell. The use of increasing force and time will sediment organelles based upon their size, with the largest organelle that will sediment first. The point of multiple rounds of centrifugation is to be able to separate the different organelles in the cell based on their size and density. Since some organelles might have similar densities, so they might sediment together. A method of purifying the sample that might contain organelles with similar densities is a density gradient sedimentation, which uses different solutions of different densities to obtain purer fractions. In this experiment, differential centrifugation will be used to isolate chloroplasts from spinach and the nuclei and mitochondria from cauliflower.
2. (5 points)RCF (Relative Centrifugal Force) can be calculated using the following equation:
RCF (in g units) = 28.38 (R) (N/1000)2 R = radius of rotor of centrifuge
N = revolutions per minute (rpm)
How would you achieve the same g force with two rotors, one with an R that is longer than the other?
To achieve the same g force with two rotors, the revolutions per minute (N) would decrease for a rotor of a longer radius (R) and vice versa. A shorter radius (R) would yield an increase in the revolutions per minute (N). So a change in revolutions per minute (N) due to a change in the radius of rotor to the outer margin of the centrifuge tube (R) can yield the same g force depending on which variable is changed.
3. (5 points) Fill in the appropriate letter in the matching questions below::
The following equation is used to determine resolution: d = 0.61/(n sin ). Please match the following terms with their definitions.
d ____c_____ a. the numerical aperture
d b. 1/2 the cone of light entering the objective lens.
n ____e_____ c. resolution
____b_____ d. the wavelength of light used
n sin ____a_____ e. the refractive index of the medium between the
specimen and the objective lens
4. (5 points) Why is an ocular micrometer used and how is it calibrated?
An ocular micrometer (stage micrometer) is used to calibrate the microscope for the different lenses used since the microscopic slide already has markings on it with known dimensions, either a line or a grid. To calibrate the ocular micrometer, the grid is aligned with the micrometer and then the relationship between the eye piece and the distances on the micrometer is determined.
5. (5 points) A step by step flow sheet that indicates what you will do during the lab.
• Collect cauliflower sample (chill and place in buffer with some sand and grind)
• Filter
• Centrifuge filtrate (make sure they are balanced)
• Pour postnuclear supernatant into clean tube and centrifuge
• Pour away postmitochondrial supernantant
• Scrape mitochondrial pellet from centrifuge tube
• Transfer mitochondrial suspension to test tube
• Collect fresh spinach leaves and perform similar procedure as above with the mitochondrial preparation.
• Calibrate lenses on microscope to examine samples.
• Stain mitochondria and determine sizes of at least five mitochondria
• Look at prepared bacteria slide and determine sizes.
• View nuclei and determine sizes.
• View chloroplasts and determine sizes.
• Observe effect of urea on chloroplasts.
• Observe effect of non-ionic detergent on chloroplasts.
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