Purpose: To experiment with the use of a compound light microscope, to learn the use of video and still picture capturing hardware and software, to experiment with cell staining and indicators, to observe the structures of various plant and animal cells, as well as pond water and newspaper fiber, and to observe the effect of heat (cooking) on the cells.

Observations:

The letter "e"-40x+

This shows a letter "e" cut out of a newspaper. One can observe the fibers of paper and ink in the picture. The small bubbles of air visible were trapped when the coverslip was put over the slide

Pond water sample-400x+

One can observe the algae in the water, along with a small, probably unicellular protist or amoeba moving across the screen when the picture was captured.

Cheek cells, unstained-100x+

This picture shows several epithelial tissue cells from a human cheek. The cell walls and nuclei are clearly visible. The cells are suspended in a fluid matrix of saliva.

Cheek cells, stained with methylene blue-400x+

This picture, taken at a higher magnification, shows the same cells stained with methylene blue solution to show clearly the nuclei and cell walls. The cell in the middle which appears to have two nuclei is most likely two transparent cells stacked on top of each other.

Cheek cells, stained with Biuret Reagent

The test was negative--the reagent remained a light blue, indicating the lack of proteins in epithelial cheek cells.

Potato, uncooked, unstained-400x+

This picture shows several raw potato cells. They are of a rigid polygonal structure, and are packed tightly together. The cellulose walls are clearly visible.

Potato, uncooked, stained with iodine-100x+


100x+


400x+

These pictures show potato cells packed together, stained with iodine. Their intense blackness, a very positive indicator of the iodine solution, reveals the presence of starch in the cells. The black dots are leucoplasts, granules of coiled starch within the cells.

Potato, uncooked, stained with Benedict’s Reagent

The test was negative—the reagent remained a light blue, indicating the lack of simple sugars in the cells.

Potato, cooked, unstained-400x+


100x+

These two pictures show potato cells after boiling. The changes seemed to be that the cells grew larger and separated somewhat from their neighbors, enabling a clearer picture (as cell were not piled on top of one another). These cells are transparent and suspended in a fluid matrix of water, probably from cooking. The chain-like structures visible at 100x+ may be chromosomes coiled up from the heat.

Potato, uncooked, unstained—Scanning Electron micrograph, unknown magnification

Courtesy http://www.ifrn.bbsrc.ac.uk/fb/tex/2_5.html

This picture shows the surface of an uncooked potato; the polygonal cellulose cell walls have been cut cleanly by the knife, and leucoplasts are clearly visible within the cells. The cells are about 100 microns across, with 1-micron thick cell walls.

Potato, cooked, unstained—Scanning Electron micrograph, unknown magnification

Courtesy http://www.ifrn.bbsrc.ac.uk/fb/tex/2_6.html

This picture shows the surface of an cooked potato; the polygonal cellulose cell walls have remained intact, in contrast to the uncooked cells, cut cleanly by the knife. The cells are about 100 microns across, with 1-micron thick cell walls.

Potato, cooked, stained with iodine-100x+


400x+

These two pictures show cooked potato cells after being stained with iodine. They retain the properties of cooked cells as described above, but leucoplasts been turned black, indicating that the starch had not been destroyed by cooking. However, the leucoplasts are significantly enlarged, a phenomenon that we discuss in our conclusions.

Potato, cooked, stained with Benedict’s Reagent

The test was negative—the reagent remained a light blue, indicating the lack of simple sugars in the cells.

Egg White, stained with Biuret Reagent

The test was positive—the reagent turned purple, indicating the presence of proteins in the albumin.

Egg White, stained with Benedict’s Reagent

The test was negative—the reagent remained a light blue, indicating the lack of simple sugars in the albumin.

Egg White, tested for lipids

The test was positive—the paper became transparent, indicating the presence of lipids in the albumin.

Egg White, cooked

The albumin protein became denatured; it became thicker, and white.

Egg Yolk, stained with Biuret Reagent

The test was positive—the reagent turned purple, indicating the presence of proteins in the egg yolk.

Egg Yolk, stained with Benedict’s Reagent

The test was negative—the reagent remained a light blue, indicating the lack of simple sugars in the cells.

Egg Yolk, tested for lipids

The test was positive—the paper became transparent, indicating the presence of lipids in the yolk.

Onion interior, uncooked, unstained-40x+


100x+


400x+

These three pictures show the brick-like array of rigidly structured onion cells in a single layer of interior onion tissue. At the highest level of magnification, cell junctions are clearly visible. The polysaccharide "glue" between cells is also visible.

Onion interior, uncooked, stained with methylene blue—100x+

40x+

400x+

These three pictures show, at differing magnifications, the scale of an onion’s cellular structure—at 40x, many polygonal cells packed together closely in a rigid structure, at 100x, showing several tightly jammed cells, and at 400x, showing the separation between two adjacent cells. The round, dark spots in the 100x+ and 40x+ magnifications are bubbles of air trapped when the coverslip was put over the slide.

Onion interior, cooked, unstained—100x+

This picture shows several cooked onion cells. The nuclei are nicely visible. The cells appear larger than their uncooked counterparts, a phenomenon probably due to water intake during boiling.

Onion interior, cooked, stained with iodine—40x+


100x+


400x+

These three pictures show cooked onion cells stained with iodine. The cells have barely changed color, indicating a lack of significant levels of starch, or, at least at much lower levels than in a potato. The highest magnification shows a clearly visible cell junction with a nucleus appearing to the right of the cell walls.

Onion interior, cooked, stained with Benedict’s Reagent

The test was negative—the reagent remained a light blue, indicating a lack of simple sugars in the cells.

Onion skin, unstained—400x+

40x+

These pictures show the closely-packed structure of a section of onion skin—made of rigid cellulose. The cells, at 40x, are visible as a very dense array of brick-like structures. At 400x, we get a much closer view of many overlapping cells in a small space, giving a sense of how tightly packed the cells are. The circular spots visible, however, are not cells; they are air bubbles.

Onion skin, stained with iodine—40x+

This picture beautifully depicts the "brick wall" of onion epithelial (single squamous) tissue cells in a section of onion skin. The edges of the cells are stained mildly black by the iodine solution, indicating the presence of some starch in the cells.

Apple cells, uncooked—40x+

100x+

This picture shows apple cells, tightly packed in a rigid cellular structure, at two different magnifications—at 100x, several cells are clearly visible, while at 40x, a greater array of cells is visible.

Cooked apple, 100x+

This picture shows apple cells after boiling. They appear to have lysed due to excess water intake during boiling.

Banana cells, 100x+

This picture shows several banana cells. They appear very unique, with pom-pom like projections from the cells. These may be cilia, cytoskeletal fibers, or structural fibers to hold the cells together.

Banana cells, stained with Biuret Reagent, 400x

This picture shows several banana cell walls. Their lack of color change after infusion with the reagent indicates the lack of many proteins.

Banana cells, stained with methylene blue, 100x+

This picture shows banana cells as described above, but with enhanced detail due to the staining. We can see that the cells are more or less transparent, but overlapping in places. They are packed closely together.

Cucumber cells-400x+

This picture shows several bands of pinwheel-like structures in a cucumber cell. They could be some kind of structural tissue, or they could be large organelles-we don’t know!

Apple peel—400x+

This picture shows an array of rigid, tightly arranged, red-pigmented cells from the peel of an apple.

Cauliflower--100x+

This picture shows an array of small, tightly packed cauliflower cells. The cells, interestingly, are less polygonal than many other plant cells. Perhaps this is because the cells were taken from the floret, not the ground tissue, of the plant.

Celery with chloroplasts, 400x+

This picture shows two celery cells with a cell wall between them. The cell on the right contains a clearly visible nucleus, and both cells contain what we believe may be chloroplasts, the small green dots.

Peach skin, 100x+

This picture shows cells from the skin of a peach. They appear similar in size, structure, and pigmentation to the apple skin cells, but their coloring is more blotchy rather than uniform—perhaps this accounts for the orangish, rather than red, color of a peach—the red pigment is not uniformly present.

 

 

Conclusions:

From the completion of this lab, we learned many things about plant and animal cell structure, chemical staining and video microscopy techniques, and the impact of cooking (boiling) upon plant cells.

Over a three-day period, we spent about three hours constructing and viewing wet-mount slides of various fruit and vegetable skin and ground tissue, cheek cells, a piece of newspaper with the letter "e", and a drop of pondwater. We also cooked pieces of fruit and vegetables, stained them (as well as drops of egg white and yolk) with reagents, and tested some for lipids. Reagents we used included Biuret Reagent (testing for proteins), Benedict’s Reagent (testing for mono- and disaccharides with free aldehyde groups—i.e., glucose and maltose, but not fructose or sucrose), and Iodine Reagent (testing for starch), as well as a methylene blue solution that improved contrast and clarity of our micrographs. Our cooking procedure, while makeshift, was sufficient to cause softening and structural changes in the items we cooked. It consisted of boiling specimens in a test tube with water inside it, placed in a water-filled beaker on a hot plate for an indeterminate amount of time. Staining was done by placing a piece of paper towel on one edge of a coverslip and several drops of reagent on the other so that the reagent would be drawn across the slide by the cohesion of the water moving up the paper towel. Additionally, we tested for lipids in egg yolk and white by placing some liquid on a piece of brown paper, allowing the samples to dry, and holding them up to a light source to observe their transparency (if light was transmitted, it was an indicator of lipids, because the hydrophobic lipids would not evaporate from the paper and would keep it moist, filling in pockets of air and transmitting light.) Because of our lack of extensive, thorough, and accurate testing for macromolecules, we were able to make few conclusions about the results of our testing, beyond the obvious--i.e., potatoes contain starch; egg white contains proteins. However, we did observe several things. First, the tests we performed for starch exposed significant quantities only in potatoes. This in itself is a valid observation; however, we believe it has wider ramifications. Obviously, all plant cells contain leucoplasts—they are required to store energy, a necessity of life. The same holds true for proteins—we know that all cells have at least some proteins, in their plasma membranes. Why, then, did tests we performed for these two substances, as well as simple sugars, turn up many negative results? The answer, we believe, lies both in the sensitivity of the reagents to varying levels of these molecules and our highly unskilled observation of the indicator results. This meant that, without a skilled eye, we could not discern small levels of indicating color change in the reagents; thus, as a practical matter, they were limited in their indicating effectiveness to significant or unusually high levels of these molecules.

Despite the shortcomings of our methods, we are now able to make several conclusions from the analysis of our observations. First, we were able to observe distinguishing characteristics between plant and animal cells. In each of the fruit and vegetable specimens we observed, the cells had a rigid polygonal structure and were packed closely together. This phenomenon is a clear result of necessity—that is, for plants without dense structural support (such as a skeleton) to maintain their structural integrity, they must have some kind of uniform matrix of support—the cellulose in their cell walls. But even this is not enough if the cells are not kept together—plant cells must be packed tightly together in order to form rigid intercellular junctions (polysaccharide "glue" and plasmodesmata) to facilitate structural support, communication, and transfer of nutrients, including water. Additionally, the banana cells had pom-pom-like projections from each cell, which we thought might serve as some kind of extracellular fibers, or perhaps as microtubule components of the cell cytoskeletons. The cheek cells, on the other hand, were good representatives of the animal cell family—they were not packed tightly together (this is unnecessary in an animal body, with widespread connective tissues and fibrous extracellular matrices to hold other tissues in place), and were not rigid or polygonal. A summarizing principle of these observations is that the structure of cells is dependent largely on the environment in which they interact with other cells. Plant cell structure is described above, a necessity of the nature of plant tissue. Animal cell structure, too, is a direct result of their environment. Unicellular organisms, including the amoeba we believe we observed in the pond water, have another unique property—like Hydra, their cells must be in constant contact with their environment—in this case, the cell lives in an environment in which it has no need for intercellular junctions; it is fully self-sufficient, and is accordingly structured, with its properties of cytoskeletar movement. Observations of more biologically significant distinctions between animal and plant cells were impossible for us to make, a limitation primarily of equipment—the light microscopes we were using were not nearly powerful enough to reveal organelles smaller than the nucleus (i.e., we could not observe confidently the presence or absence of chloroplasts), or to expose more significant structural differences (absence of rigid cell walls in animal cells, for instance). The third category of matter we observed was the letter "e" from the newspaper—it was abiotic, and therefore not composed of a cellular structure. Instead, its structural support (communications or nutrient transport were, obviously, unnecessary in a non-living body) was provided by intertwining fibers of paper. This, interestingly, provides a unique link between the newspaper and any of our plant specimens—even though the newspaper was non-living, it still relied on the structural cellular foundation of the plant cells to maintain its own structure, as wood cellulose fibers are the main component of paper.

Additionally, we made several observations about the correlation of structure and function in plant cells. Plants are made up of five major types of cells: parenchyma cells (most abundant, performing many functions), collenchyma cells (providing support and elongation for growing plants), sclerenchyma cells (forming structural and protective hard tissue, including fibers and sclereids), water-conducting cells, and food-conducting cells. Each has a specific structure based directly on its function in the plant—for instance, sclerenchyma cells have very thick cell walls for support. Several types of complex plant tissues are comprised of these cells—epidermis, vascular tissue, and ground tissue (making up the bulk of a plant, similar to muscle cells in the human body). In most of our specimens, we observed ground tissue samples, composed primarily of parenchyma cells. However, the onion skin we observed may have been composed of sclerenchyma cells. One observation we made which would support this is that the cells in an onion’s skin are much more rigidly arranged than interior cells; additionally, there were more layers of them in a similar sample thickness, indicating perhaps that they were very layered in order to bear pressure. The structure of cells in a specimen also, we found, influenced the way it appeared and felt to us, on a practical level. For instance, the very rigid cells in a potato made the ground tissue of that potato feel much firmer to the touch than, for instance, the tissue of an apple, with its less rigid cells.

Next, we were able to make several observations about the effect of cooking on plant cells. Note that we are excluding from this the effect of cooking on egg albumin, as the changes that occurred in that strictly protein-based substance were the result of protein denaturation, not cellular change. Generally, when we cooked specimens, we observed that the cells grew less polygonal, larger, and separated from each other. The overall specimen became slightly larger and softer. We believe that these results are all based on either the increased velocity of heated water molecules in the test tube, the hydrolysis and chemical degradation of the middle lamella pectic polysaccharides that provide structural support for intercellular junctions, the denaturation of the fibrous protein intermediate filaments supporting the cell, or any combination of those factors. The pectic hydrolysis would have led to the intercellular separation we observed, as a result of loss of structurally reinforcing rigid intercellular junctions. The enlargement of the cells, we believe was a result both of the increased plasma membrane exposure of the separated cells and of the increased velocity of water molecules in the test tube, perhaps heightening the water diffusion into the cells by forcing water molecules through plasma membranes, cannon-style. The cells would lose their rigid polygonal structure as a result of both loss of structural intercellular support, denaturation of the intermediate filaments, and cell expansion, which would cause the cell walls to balloon outward to form a circle, the most efficient storage space based on perimeter. This phenomenon was exaggerated in the apple cells, which appeared to actually have lysed from excess water intake. This combined circular structure and expansion would have reciprocally led to further cell separation, as circles, especially large circles, do not fit into a space as efficiently as polygons, because they must have additional space between cells to compensate for their lack of angular projections to fill space. This phenomenon would lead to overall expansion of the specimen to accommodate the more spaciously distributed cells, a common observance. Because of the lack of connection of the cells after cooking, they could also be easily displaced, which lead to their softening and susceptibility to crumble at the touch to a greater extent than uncooked cells. Interestingly, this phenomenon also causes changes in our taste perception of cooked and uncooked cells. As shown in the electron micrographs, cell walls in uncooked cells split cleanly when cut or torn, opening the cells. In cooked specimens, however, the cells are not interconnected and are able to move, which leads to the maintenance of the integrity of their walls, as cells can move away from a knife rather than be cut (this is similar to attempting to cut through balloons with a saw, either when they are tied together and held in place (uncooked), or are loose (cooked)). Thus, when we eat uncooked plants, we receive much more of the taste and nutrients inside the cell than we do with cooked plants, whose cell walls are not broken.

Additionally, we performed a test for starch in potato cells before and after boiling. Expecting no change in the inner composition of the cell, we were somewhat surprised by what we found—the leucoplasts were still present, but seemed to have swelled a great amount. Additional research still provided no definite answers as to the cause of this phenomenon. Leucoplasts are composed of two different types of starch—linear amylose, and irregular, branched amylopectin. When heated, they swell little, until they reach 140 degrees Fahrenheit, at which point their intermolecular hydrogen bonds break, allowing water to invade the previously crystalline and impermeable granule. The amylose in the leucoplast spills out, forming hydrogen bonds with water molecules and thereby gelatinizing the water. The amylose-water bonds, however, are weaker than amylose-amylose bonds; thus, the starch gel tends to shrink together to form a denser gelatinous structure. This phenomenon is greatest in leucoplasts with a high amylose-amylopectin ratio; conversely, in leucoplasts with a low ratio, the gel is more dispersed and less viscous. We cannot be certain, in this case, whether the leucoplasts in our cells burst and gelled due to this phenomenon (as would seem logical) or simply became enlarged (which would indicate that while the water in the beaker was boiling, the water in our test tube may not yet have reached 212 degrees Fahrenheit.)

Error Analysis:

There was much potential for error in this study, largely as a result of the freelance laboratory environment, which generally precluded careful planning and standardization of results shared between colleagues and periods. Human errors could have been and most likely were made in slide preparation, staining, microscope technique, observations, and cooking. Especially destructive to the integrity of the results may have been cooking and staining techniques. Cooking procedures were never standardized or properly timed, so boiling times ranged from a few minutes to twenty. There was no protocol for water temperature; it may have been too hot, or, as our results describe above may indicate, not nearly hot enough. Also, boiling specimens in water may well have leached matter out of the specimen—this could have been avoided by cooking in an oven or other method not requiring immersion in water. Staining was done by a multitude of different methods. For instance, I spoke with several people about staining of egg yolk for proteins. One put a drop of liquid on a slide and a drop of reagent on top of it. One put egg yolk in a test tube, along with the reagent. One then cooked that mixture. Not only were procedures not standardized, but amounts of specimen and reagent or water involved in both the cooking and staining aspects were not specified or standardized. Observation of stained specimens was also highly erratic and unreliable. This study could be greatly improved by pre-planning of procedural protocol and standardized recording of observations.

Sources:

Campbell, Neil A., Lawrence G. Mitchell, and Jane B. Reece. Biology: Concepts and Connections. 3rd ed. San Francisco, CA: Benjamin/Cummings-Addison Wesley Longman, Inc., 1997.

http://www.ifrn.bbsrc.ac.uk/fb/tex/

All micrographs ©1999 The Bio-Web Group

Pictures courtesy Daniel Winik, Cynthia Collier, Ben Weinberg, Devin Symons, Mark Parker, Mira Guo, and any others whom I may not have recognized!