Lab Report 1
by Guiherme and Siyang
The objective of the first week is to use microscope as a tool to measure biological
samples and get a feeling of the size. First we take images of scale bars of known
dimension under microscope. Then we take images of different samples under different
objective magnification. For 100x, oil immersion technique has to be used. When it
comes to data analysis, we first use ImageJ to convert pixels to microns so that we can
label all the images with scaling bars.
We observed the fascinating single cell organism stentor. We measured the cilia length
and the number of cilia per cilia group. Both non-fluorecent and GFP-fluorescent
Escherichia coli (usually abbreviated to E. coli) were observed. The phase contrast
images have better representation of fine structures than ordinary bright field images.
For images of non-fluorescent E. Coli cells taken at 40x objective, we did a particle
analysis in ImageJ and get a cell count and average cell diameter. The diameter value
we obtained is 0.7μm, which is smaller than expected. We believe it is due to the fact
that the cells are not in the same focusing plane and the threshold operation generates
particles of smaller diameters. Change to some special coating glass slides might help
the cells stay in the same focusing plane. We studied photobleaching effect of GFP
protein by taking a series of images every ten seconds under fluorescent microscope
illumination. The images are analyzed in photoshop and we fit the data with
exponential decay and resolved a time constant of 85 seconds. The number seems to be
at the large end. But it tells us that if we only illuminate the sample a couple of seconds
when taking pictures, we can probably ignore the photobleaching effect with the current
GFP stain. We need to be cautious that GFP has a lot of mutants and their properties are
different. Also the photobleaching is dependent on the illumination intensity, so it will
be better if we measure the light intensity during the testing. We also observed slides of
fibroblasts stained with FITC for actin and Texas Red for mitochondria. We use imageJ
to assign pseudo colors for different channels and obtained composite images. Both cell
size and cell nucleus size are measured. The largest organism we observed is
Drosophila melanogaster under stereo microscope. The two mutants we have is
apterous which does not have wings developed and scarlet which lacks brown pigment in
eyes.
The second week of the lab we are supposed observe and measure the growth of yeast
Saccharomyces cerevisae. One way is to make special agar slides and drop some yeast
cells. Then every twenty minutes take an image. With the images stacked, we
successfully observed the growth of yeast population. It will be even better if we
shorten the time interval for pictures. We also set up a growth experiment in media and
measure OD to monitor growth. But the first time we tried, we have to grow the yeast at
room temperature. We did not observe any significant growth in the lab period of time.
From the plates we have, we do know the cells are fine. When we tried the second time
with another group, we got contaminations and the data is not reliable.