Lab Report : Introduction to Light Microscopy

Hello everyone! :)

As I told you before, in this year we made nine different experiments in our biology lab classes. And I'll share the important parts of my lab reports regularly with you through this blog. As a result, you can see what we did and learned in this year in BIO 106. In addition, I think, these reports can give you some advice about how biological lab reports should be written, etc. I hope, they will be useful for you :) 

Here is the first experiment of this semester: 

INTRODUCTION TO LIGHT MICROSCOPY

THE AIM OF THE EXPERIMENT:

The aims of observations in this experiment were to specify different eukaryotic cell structures of animal and plant cells, to determine their morphological properties which can be observed under a light microscope, to estimate the sizes of animal and plant cells and, to determine and compare the structural and basic differences between Gram (+) and Gram (-) bacteria.

INTRODUCTION:

Until the invention of the first microscope in the seventeenth century, scientists were not be able to see and observe the cells and other tiny living and non-living things which couldn’t be seen with naked eyes. Light microscopes were the first microscopes which is used by scientists to see the complex structure that underlies all living things. After that, scientists developed different technologies such as Fluorescence Microscopes, Confocal Microscopes, Transmission and Scanning Electron Microscopy to observe specific structures of different cells.

The Magnification of a microscope is the measurement of enlarging of a sample’s image under a microscope and total magnification of a compound light microscope which includes two different lenses: objective lens and ocular lens, can be calculated by multiplying both two lenses’ magnification degrees. Resolution is described as the ability of a microscope to distinguish the details of the samples and the power of resolution (resolving power) of a microscope is determined by the numerical aperture of its objective. The difference in the intensity of light, between the image of a sample and its background relative to the intensity of the overall background is defined as the “Contrast” of a microscope and when a specimen is transparent or lack of color, the contrast of this specimen is needed to improve by using dyes, etc. In addition, the immersion oil is a synthetic oil which is used by scientists to increase the resolving power of a microscope and to arrange the brightness of the image of the specimen which is observed through the microscope.

Under a light microscope, some structural differences between animal, bacterial and plant cells can be observed. None of animal cells has a cell wall, however the plant cells and most of the bacterial cells have their own special type of cell walls. In addition, plant cells have an ordinary structure together and their cells’ shapes are mostly like a quadrilateral, however bacterial and animal cells doesn’t have a specific shape and they don’t build an ordinary view together. On the other hand, the chloroplast excited by the light can only be seen in plant cells.

By using Gram Staining Method, Gram(+) and Gram(-) bacteria can be distinguished with crystal violet dye. Gram(+) bacteria retain the dye and can be seen purple, Gram(-) bacteria cannot retain the dye inside because of its second outer membrane and appear pink.

METHODS:

Preparing and Cleaning The Microscope:

·         To be able to use 100X lens with immersion ail properly, the 100X lens was cleaned by using Isopropanol and a tissue paper at the beginning and at the end of the experiment.

·         The microscope was prepared according to the directions of the Lab Assistant and focused for both eyes of performers for each observation individually.

Observation of The Printed Letter “e”:

·         A printed black “e” letter was put on a microscope slide.

·         Two or three drops of water were dripped on the letter properly by using a Pasteur pipette.

·         A coverslip was placed on the letter and water on the microscope slide.

·         To prevent and reduce the bubble formation between the coverslip and the paper, the back side of a Pasteur pipette was pressed to the coverslip gently.

·         The microscope slide was placed on the microscope stage.

·         The letter “e” was centered and the microscope was focused for both eyes.

·         For observing the letter “e”, three different objective lenses were used. Firstly, 4X lens, and then 10X and 40X lenses were used.

·         For each step, the observations under different lenses were made and drawn to the lab notebook.

·         The pictures of the letter under different lenses were taken.

The Observation of Buccal Smear:

·         By using a Pasteur pipette, one or two water droplets were placed on a slide

·         A toothpick was used to get epithelial cell samples from the mouth of the performer’s partner (E.D) by scrapping the toothpick inside of his cheek.

·         The toothpick with cell samples was stirred into the water droplets on the slide.

·         A coverslip was placed on the sample.

·         One or two droplets of a dilute methylene blue solution were added to one edge of the coverslip on the sample.

·         The dye was drawn under the coverslip by using a tissue paper.

·         The dyed specimen was observed under three different objective lenses. Firstly, 10X lens was used. After that, 40X and 100X lenses were used.

·         When 100X lens was used, one or two droplets of immersion oil were dripped on the coverslip.

·         The observations under different lenses were made and drawn and their pictures were taken.

Using The Hemocytometer:

·         By using another toothpick, a new epithelial cell sample was gotten from the performer’s mouth (K.E.Ç).

·         One or two water droplets were placed on a glass slide with a hemocytometer.

·         The toothpick with new sample was stirred into the water droplets on the slide.

·         A coverslip was placed on the sample.

·         Under the coverslip, one droplet of methylene blue was added to the sample.

·         The glass slide was placed on the microscope stage.

·         The samples were observed under 10X and 40X magnifications.

·         By using the squares and lines on hemocytometer under 40X lens, the diameter of the field of view and the diameter of one cell were calculated.

·         The calculations and the observations were written and drawn.

The Observation of Elodea Cells:

·         The samples from Elodea leaves were taken from the Lab Assistant.

·         The Elodea sample was placed on a slide and one or two droplets of water were placed on the sample.

·         A coverslip was placed on the sample.

·         The sample was observed under 10X, 40X and 100X magnifications.

·         During the observations under 40X magnification, the horizontal and lateral sizes of the Elodea cells were calculated, according to the calculations in epithelial cell samples under 40X.

·         For the observation under 100X magnification, one droplet of immersion oil was placed on the coverslip.

·         The observations for each step and the calculations under 40X were written, drawn and their pictures were taken.

The Observations of Gram(+) and Gram(-) Bacteria:

·         Two different bacteria samples were taken from the Lab Assistant on prepared microscope slides.

·         The one labeled as “only E.coli” was observed under 4X, 10X, 40X and 100X magnifications.

·         For the observation under 100X, one droplet of immersion oil was placed on the coverslip of the sample.

·         The observations for each magnification were written and drawn.

·         The other slide with the label “E. coli + B.subtilis” was observed under 4X, 10X, 40X and 100X magnifications.

·         Immersion oil was used for the observation under 100X magnification.

·         All observations under each magnification were written and drawn.

DISCUSSION:

During the observations of this experiment, some specific differences between animal, bacterial and plant cells could be observed, as expected at the beginning of the experiment. In addition to that, estimating the sizes of animal and plant cells, determining the morphological properties of these cells and making a structural comparison between Gram(+) and Gram(-) bacteria were the other expectations of this experiment.

For these purposes, firstly a printed letter “e” was observed under 4X, 10X and 40X magnifications. As can be observed in Figure 1 and Figure 2, the images of the letter “e” were inverted and reversed. The causes of this altered view of the letter are the focal length of the objective lens and the lens’ curvature. The focal length of the objective lens of a microscope is very short and after the light passes through the printed “e”, the light also passes the objective lens of the microscope and the focal point of the objective lens. As a result of these steps, the images are inverted and reversed.

As expected at the beginning of the experiment, under different magnifications, the images were not in the same size. As long as the power of magnification of the lenses increased, the size of the images which were observed also increased. However, the directions of the movements of the images were the same for each power of lenses and they were to the opposite direction of the sample’s movement, because the objective lens of the microscope inverts the image of the sample.

As it can be observed, by using methylene blue to stain the buccal smear, the nucleus and the organelles of the epithelial cells which contains nucleic acids such as ribosomes and mitochondria could be observed. The methylene blue dye interacts and dyes the components of cells which contains nucleic acids darker than the other parts of the cell. The mitochondria have their own DNA and RNA molecules inside. In addition to that, ribosomes consist of rRNAs and they also contain nucleic acids to stain with methylene blue. On the other hand, on the rough Endoplasmic Reticulum in the cells there can be observed ribosomes, too and the Endoplasmic Reticulum is placed around the nucleus in the cells. As it can be seen in Figure 6, as a result of ER ribosomes’ existence, the density of the ribosomes around the nucleus are higher than other parts of the cell. If the buccal smear samples wouldn’t be dyed or stained with methylene blue, these observations couldn’t be made under a light microscope, because of the transparent existence of the epithelial cells. 

In the figure, it can be observed that because of their tetragonal shaped cell walls, plant cells have an ordinary structure in their tissues. In addition to that, there is no empty place between the cells of this sample and, the thickness of the cell walls are not the same for each cell in the sample, because of the difference between their lifetimes. On the other hand, there can be also seen the transportation channel which consists of Xylem and Phloem cells of the Elodea Leaf sample, in the Figure.

In the figure, the plant cells can be seen in more detail and the chloroplasts of the cells can also be seen during their movement because of the light excitation. However the nucleus of these plant cells couldn’t be seen under the 100X magnification.

The bacterial cells which were observed in the Gram Staining Experiment were in different colors. As it can be observed in the figure, the E.coli Bacteria have a line shaped structure and their color was pink. On the other hand, as it can be seen in Figure 14, the B.subtilis Bacteria have a circular cell structure and their color was violet. Because the Gram (+) B. subtilis Bacteria can retain the violet dye inside. However, the Gram(-) Bacteria E.coli cannot and appear in pink.

In the end of the experiment, it can be observed that animal cells, plant cells and bacterial cells have some differences in their structural basis such as having a cell wall for bacterial an plant cells, having an ordinary structure in their tissues for plant cells and having chloroplasts inside the their cells for the plant cells. In addition to that, the sizes of the cell samples were also different from each other. The buccal cell samples are bigger than the plant cells and the bacterial cells are the least. Because of that, the details of the observations of the inside cell structures were not the same for each cell type, too.