DATA

Discussion

In this experiment cell fractionation- the process of isolating various components of the cell via centrifugation was performed on spinach leaves in order to isolate and observe chloroplast. Upon analyzing samples from each fractionation step the following was determined: The initial homogenate contained little debris but what debris that was found consisted of clumps of plant tissue that exhibited signs mutilation. This most likely occurred from the mechanical methods used to break down cellular tissue. Determination of chloroplast was uncertain. Supernatant #1 contained lots of free floating small debris throughout the media, most likely the result of pieces of cell wall and cell membrane disrupted during the initial mechanical degradation. A moderate number of chloroplast were observed free floating in the media. It was not entirely clear if other organelles (possibly mitochondria) were floating along with the debris. In Pellet #1 lots of large stationary debris was observed along with possible cell structures containing chloroplasts. The debris may be the result of cellular membranes and proteins isolated during the centrifugation. In Supernatant #2 very little debris as well as very few free floating chloroplast were seen. A very few plant cells could be identified with chloroplast inside however this may have likely resulted from contamination with the pellet. Comparisons with other groups data for this fraction will aid in determining just how "clean" this layer should be.

Pellet #2 by far yielded the cleanest layer of chloroplast with little to no other debris. The reason for this must lie in the observation that most all other larger cellular components were isolated in the previous fractions, leaving this particular organelle. A sample for this last fraction was used along with the methyl green reagent in order to observe nuclei. After about 2 minutes some of the chloroplast nuclei could be observed although the staining was only slightly darker than the chloroplast and appeared to stain an area much smaller than what appeared to be the observed boundaries of the nucleus. Reasons for this may be that the reagent was only staining for a particular component within the nuclei or that the stain may not be as effective as predicted in staining nuclei. Using a fresh sample of the reagent as well as a different reagent may aid in determining this. During analysis by spectrophotometry the sample showed a relatively high absorbency for the dark blues and the yellowish-oranges, while showing relatively low absorbencies for green, orange, and red. These observations are consistent with the fact that chlorophyll absorbs most wavelengths of light except for green which it reflects back. Based on the absorbency at 652nm a calculation of the amount of chlorophyll was made.