Exercise 1. the Discovery of Cells

Reprinted from Gallik S., Cell Biology OLM Page | 1

Exercise 1. The Discovery of Cells

A. Introduction

Ancient Greek philosophers famously espoused the idea that life generated spontaneously. Aristotle recognized that some animals come from parent animals of the same kind, but thought that others appear spontaneously from a nonliving substance he called pneuma and from four terrestrial elements, earth, air, fire and water. Aristotle condensed these ideas into a theory that we now call the Theory of Spontaneous Generation. The theory basically states that living organisms form spontaneously, from nonliving matter. Cork (Microscopic magnification 400X) It wasn't until the 1800s that scientists made the important discoveries that unequivovally dispelled the theory of spontaneous generation. One of these developments was the formulation of the Cell Theory. The Cell Theory, one of the fundamental unifying principles of biology, establishes the cell as the basic unit of life. As is summerized in the timeline below, the theory grew out of hundreds of years of scientific research and technical developments, beginning with the development of glass lenses and the microscope and continuing with the discovery of the cell. In this exercise, we trace some of the major events in the history of the development of the microscope, discovery of the cell and the formulation of the cell theory. In lab, we microscopically examine some of the same specimen early biologists examined when they first observed cells and finally came to the conclusion that all living things are made of cells. B. Invention of the Microscope

The invention of the glass lens set the stage for the development of the microscope. The earliest glass lenses date back to approximately 700 BC, but it is widely reported that the glass magnifying lens was invented by Roger Bacon in the mid 13th Century. Bacon, an English Franciscan monk, working from the studies of early Arab scientists (circa 1000), investigated the optical properties of glass magnifying

Copyright © 2011, 2012, 2013 by Stephen Gallik, Ph. D. Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM Page | 2

lenses, which was reported in his major treatis, Opus Majus, written at the request of Pope Clement IV and presented to the Pope in 1267.

Glass lenses were first used to correct human eyesight. There is a debate among historians as to when eyeglasses were invented and by whom. Some claim they were invented in Italy about 1286. However others claim they were invented earlier, in India.

The first microscopes were developed about 300 years later, the same time the first telescopes were developed, between 1590 and 1610. The first working telescopes were built by Dutch lensmakers at the beginning of the 17th century (1608). Credit for the invention of the telescope is given to three individuals: Hans Lippershey, Zacharias Janssen and Jacob Metius.

Zaccharias Janssen is also associated with the invention of the simple and compound light microscopes. Confusion surrounds the role of Zaccharias in the microscope's invention. It is generally reported, and seems highly likely, that Hans Janssen, Zaccharias' father, either helped his son build the first microscope or built the first microscope himself in 1595.

Janssen's microscope was a hand-held microscope, The Janssen Microscope consisting of two sliding tubes, each inserted into the end Image Source: Molecular Expressions of a middle stationary tube. The eyepiece lens, which was inserted at one end of one of the sliding tubes, was a bi-convex lens. The objective lens, inserted at the far end of the other sliding tube, was a plano-convex lens. One could focus on the specimen by simply sliding either of the sliding tubes in and out. The micrsocope was capable of magnifications ranging from 3X to 10X.

Galileo improved upon the designs of early telescopes and microscopes. He built his first telescope in 1609, a year after the Dutch, and later built improved models. The Galilean telescope characteristically had one concave and one convex lens mounted in a rigid tube. They were the first telescopes to achieve magnifications beyond 10X, with some achieving magnifications of nearly 30X. Using these telescopes, Galileo discovered the moons of Jupiter and many other significant astronomical phenomena.

Soon after his work on the telescope, Galileo used the instrument at close range to magnify small objects, and by 1624 he was routinely using his compound microscope. Like the Galilean telescope, the Galilean compound microscope had one concave and one convex lens. Galileo's illustrations of Copyright © 2011, 2012, 2013 by Stephen Gallik, Ph. D. Licensed under a Creative Commons AttributionGalileo's-NonCommercial Microscope-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under thisImage copyright Source: and license History are those of sourced the to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Microscope.org Reprinted from Gallik S., Cell Biology OLM Page | 3

microscopic views of insects made using one of his microscopes were published in 1625 and appear to be the first clear documentation of the use of a compound microscope. But it wasn't for another 40 years that the first cells were observed, by Robert Hooke and Antoni van Leeuwenhoek.

C. Robert Hooke and Antoni van Leeuwenhoek

Robert Hooke (1635-1703) was an English chemist, physicist, architect, and surveyor. He designed microscopes, he didn't build them. His designs improved upon microscope mechanics and illumination, which improved resolution and increased the magnification to approximately 50X. These improvements enabled Hooke to microscopically view greater detail of objects smaller than those reported by Galileo.

The Hooke microscope consisted of three glass lenses, an eyepiece lens, a bi-convex objective lens and an intermediately-placed tube lens, inserted at various places Robert Hooke's Microscope. along a rigid tube assembly. The tube assembly was Image Source: Molecular Expressions mounted to a base through a moveable joint that permitted adjustment of the incline of the microscope tube. The microscope also used a special illumination system that concentrated light on the specimen, making the final image brighter.

Hooke used his compound microscope to microscopically study a variety of organisms and biological structures, including thin slices of cork, the outer-most layer of the bark of trees. In 1665, he reported some of his observations in his famous treatise entitled Micrographia, which included his now-famous drawing of his microscopic view of cork. In describing what he observed, Hooke coined the term "cell", in reference to the microscopic compartmental structure he observed. Today, Robert Hooke is credited with the first microscopic view of the cell and is, along with Antoni van Leeuwenhoek, credited with the discovery of the cell.

Antoni van Leeuwenhoek (1632-1723), a contemporary of Robert Hooke, was a Dutch draper, tradesman, and amateur scientist. He was also a masterful lensmaker. He taught himself how to grind and polish glass lenses, and he developed a level of lensmaking skill unmatched in his Robert Hooke's drawing of the day. He produced lenses with a magnification greater microscopic image of cork. Micrographia, 1665. Copyright © 2011, 2012, 2013 by Stephen Gallik, Ph. D. Licensed under a Creative Commons AttributionImage -Source:NonCommercial Wikipedia-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM Page | 4

than 250X, a power that far exceeded the lenses used by Galileo and Hooke.

Leeuwenhoek used his lenses to make relatively high quality simple microscopes. His hand-held microscopes were approximately two inches long and contained one of his small, high-quality bi-convex lenses sandwiched between two brass plates. With the specimen mounted on a pin and the entire instrument held up to the light and close to the eye, Leeuwenhoek microscopically viewed biological specimen at a resolution and magnification not attained by any other scientist of his day. He observed and, in 1674, gave the first relatively detailed description of red blood cells, was the first to describe colonial alga Spirogyra and was the first to describe the small unicellular organisms found in pond water, "animalcules" as he called them. In addition, he was the first to observe and describe bacteria and is considered by many to be the "Father of Microbiology".

Leeuwenhoek reported his observations to the Leeuwenhoek's Microscope. Royal Society. In 1678 the Royal Society asked Image Source: Molecular Expressions Robert Hooke to confirm Leeuwenhoek's findings. Hooke not only confirmed Leeuwenhoek's findings, but noted that Leeuwenhoek's simple microscope gave clearer images than Hooke's compound microscope. Today, Leeuwenhoek is credited with the first microscopic view of living cells and is, along with Robert Hooke, credited with the discovery of the cell. D. Schleiden, Schwann, Virchow and the Cell Theory

While Hooke and Leeuwenhoek were the first to observe cells and are rightfully credited with the discovery of cells, neither one of them understood the role cells played in the organization of living systems or the universality of cells among living organisms. It wasn't until the 1800s, roughly 200 years after the discovery of cells, that the work was done that firmly established the Cell Theory and convincingly dispelled the Theory of Spontaneous Generation.

The notion that the cell is the basic Matthias Schleiden & Theodor Schwann unit of life, which is the core tenet of

the Cell Theory, was first declared by Henri Dutrochet in 1824. Dutrochet was an accomplished French biologist credited with, among other things, early research into the development of embryos and determining the basis for osmosis. Following up on the work of Ludolph Treviranus and Johann Moldenhawer, who put forth the notion that cells were separable into individual units, Dutrochet proposed the idea that "The cell is the fundamental element of organization". But it wasn’t until the work of Matthias Scleiden and Theodore Schwann was published, about 15 years later, that a concise cell theory was formulated.

Matthias Jakob Schleiden (1804 – 1881) was a German botanist. He spent a significant part of his career studying plant structure under the microscope. In his monograph Beiträge zur Phytogenesis (Contributions of Phytogenesis), which was published in 1838, he described his observations that different parts of the plant are made of cells, and he concluded that all plant tissues are made of cells and that the embryonic plant grows from a single cell.

Theodor Schwann (1810 – 1882) was a German physiologist and a close associate of Schleiden. He studied the microscopic structure of various animal tissues and collaborated with Schleiden on studies of the microscopic structure of plants and animals. Among his many accomplishments is his discovery of the cells that form sheaths around neurons in the peripheral nervous system, now known as Schwann cells. In 1839, a year after Schleiden reported his studies on plants, Schwann published his manuscript entitled Microscopic Investigations on the Accordance in the Structure and Growth of Plants and Animals, in which he formally concludes that "All living things are composed of cells and cell products".

The Work of Robert Remak and Rudolf Virchow

Robert Remak (1815-1865) was a Polish physician, biologist and contemporary of Schleiden and Schwann. His work in embryology resulted in his determination that adult animal tissues come from three embryonic germ layers, the ectoderm, mesoderm, and endoderm. Remak's experimental observations also showed, for the very first time, that cells come from pre-existing cells through a cell divsion process.

Robert Remak & Rudolf Virchow Rudolf Virchow (1821 - 1902) was a German physician, pathologist and contemporary of Remak. His microscopic study of diseases earned him the moniker "Father of Modern Pathology". Virchow popularized the maxim Omnis cellula e cellula ("Every cell originates from another existing cell like it."). His genuine experimental contribution to our knowledge about the origin of cells

has been questioned. It has been authoritatively claimed that Virchow plagarized Remak's work. Moreover, the maxim Omnis cellula e cellula has its origins with two other scientists, Francesco Redi and François-Vincent Raspail. Francesco Redi carried out the landmark experiments that showed, for the first time, that fly larvae (maggots) only develop on decaying meat when living flies had access to the meat, leading Redi to conclude that life only comes from pre-existing life. He coined the phrase Omne vivum ex vivo ("Every living thing comes from life"). François-Vincent Raspail extended Redi's conclusion to the realm of cells and coined the phrase Omnis cellula e cellula. Thus, Virchow's genuine contributions to the notion that cells come from other cells should probably be limited to a recognition that he popularized Raspail's maxim.

Despite the controversy surrounding Virchow's work on the origin of cells, the scientific community credits Theodor Schwann, Matthias Jakob Schleiden, and Rudolf Virchow with the formulation of the Cell Theory. The theory has three basic tenets:

 All living things are made of cells.  The cell is the basic unit of all living things.  Cells only come from pre-existing cells.

The Cell Theory is now considered one of the great unifying principles of biology. It establishes the cell as the basic structural and physiological unit of life and formally establishes the principle that life is not generated spontaneously but comes directly from pre-existing life. Thus, the formulation of the Cell Theory during the 1850s, based on work that began with the development of the microscope in the 1600s, finally and unequivocally dispelled the notion that life originated spontaneously from non-living materials. E. This Week’s Laboratory Experiment

Introduction

This week's experiment is a celebration of the scientific work that went into the discovery of cells and the formulation of the Cell Theory. In lab, we will perform a simple observational study of the basic microscopic structure of some of the tissues examined by Robert Hooke and Antoni van Leeuwenhoek, specifically cork, carrot, green algae and pond water. Our observational study is designed to answer the basic that we are asking: What is the basic microscopic structure or composition of cork, carrot, green algae and pond water? The specific objective of the scientific work to be performed is to record and describe the basic microscopic structure of cork, carrot, green algae and pond water, for the simple purpose of developing an appreciation for the scientific work that lead to the discovery of cells.

Experimental Design

As an observational study, this experiment is designed to determine the microscopic structure of various tissues in their native condition, with no application of an experimental treatment.

Each pair of students will microscopically examine 3 independent samples of each of four specific specimen: cork, carrot, green algae and pond water. Students will record their observations in one of two ways, either by drawing simple sketches of the observations or by photographing the microscope image through the eyepiece.

Materials

Equipment & Supplies  Compound microscope  Hand microtome  Glass microscope slides  Coverslips  Forceps  Optional: students should bring their compact digital cameras or cell phone cameras if they want to take photographs of the microscope images through the microscope's eyepiece.

Biological Specimen  Cork  Carrot  Pond Water  Green Algae

Experimental Protocol

A. Microscopic Study of Cork

Cork was one of the many materials Robert Hooke studied with his microscope. It was through his description of his microscopic observations of cork, published in 1665 in his monograph Micrographia, that he coined the term “cell” in reference to the microscopic compartments that make up cork tissue.

Hooke's Protocol:

Hooke's report of his study of cork is found in the section of Micrographia entitled Observ. XVIII. Of the Schematisme or Texture of Cork, and of the Cells and Pores of some other such frothy Bodies. In describing his basic methodology, he wrote:

I took a good clear piece of Cork, and with a Pen-knife sharpen'd as keen as a Razor, I cut a piece of it off, and thereby left the surface of it exceeding smooth, then examining it very diligently with a Microscope, me thought I could perceive it to appear a little porous; but I could not so plainly distinguish them, . . .

. . . , I with the same sharp Penknife, cut off from the former smooth surface an exceeding thin piece of it, and placing it on a black object Plate, because it was it self a white body, and casting the light on it with a deep plano-convex Glass, . . .

Cell Biology OLM Protocol:

Your protocol is similar to Hooke's.

1. Randomly retrieve 3 corks from the bowl.

2. Using a fresh single-edge razor blade, very carefully slice a very thin section from one of the three corks with a sharp single-edge razor blade. You might need to make several attempts before you get a satisfactory section. The, using a pair of forceps, carefully place the section on a clean microscope slide (Do not add water).

3. Place the slide on the stage of your microscope, turn on the illuminating lamp and focus on the specimen with the 4X objective lens. Once the specimen is in focus, rotate through the objectives (4X, 10X and 40X) and study the microscopic structure of the specimen at each magnification. As you study your specimen, determine whether the specimen is thin enough to get a clear view of the microscopic structure of the specimen. If it isn't, try to make thinner sections until you get a satisfactory section.

4. Prepare two additional slides of cork from samples taken from the other two corks in a fashion similar to that above (for a total of three slides) and inspect the sections as you did before. As you study your specimen, determine whether the microscopic structure seen in each slide is consistent with that seen in the other slides.

5. If the structure is consistent from slide to slide, choose one of the slides as a representation of your thin sections of cork, place it on the microscope and view the specimen at the magnification that appeals to you. Record the image by either drawing a simple sketch of the observations or by photographing the microscope image through the eyepiece.

B. Microscopic Study of Carrot

Cork was not the only plant structure studied by Hooke. He also studied a variety of other plants, including carrots. After describing his microscopic observations of cork in Observation XVIII of Micrographia, Cooke wrote:

Nor is this kind of Texture peculiar to Cork only; for upon examination with my Microscope, I have found that the pith of an Elder, or almost any other Tree, the inner pulp or pith of the Cany hollow stalks of several other Vegetables: as of Fennel, Carrets, Daucus, Bur-docks, Teasels, Fearn, some kinds of Reeds, &c. have much such a kind of Schematisme, as I have lately shewn that of Cork, save only that here the pores are rang'd the long-ways, or the same ways with the length of the Cane, whereas in Cork they are transverse.

Cell Biology OLM Protocol:

1. Randomly retrieve 3 carrot pieces from the bowl.

2. As was done with cork, carefully slice a very thin section of carrot from one of the three carrot pieces with a sharp single-edge razor blade. Then, using a pair of forceps, carefully place the section on a clean microscope slide (Do not add water).

4. Prepare two additional slides of carrot from samples taken from the other two carrot pieces in a fashion similar to that above (for a total of three slides) and inspect the sections as you did before. As you study your specimen, determine whether the microscopic structure seen in each slide is consistent with that seen in the other slides.

5. If the structure is consistent from slide to slide, choose one of the slides as a representation of your thin sections of carrot, place it on the microscope and view the specimen at the magnification that appeals to you. Record the image by either drawing a simple sketch of the observations or by photographing the microscope image through the eyepiece.

C. Microscopic Study of Pond Water

Antoni van Leeuwenhoek is credited with the first published observations of living cells. One of his very first discoveries was that of the organism that we now know as the green alga Spirogyra. He discovered it in a vial of pond scum taken from a small lake near Delft, Holland. In a letter to the Royal Society dated 1674, Leeuwenhoek writes:

About two hours distant from this Town there lies an inland lake, called the Berkelse Mere, whose bottom in many places is very marshy, or boggy. Its water is in winter very clear, but at the beginning or in the middle of summer it becomes whitish, and there are then little green clouds floating through it; which, according to the saying of the country folk dwelling thereabout, is caused by the dew, which happens to fall at that time, and which they call honey-dew. This water is abounding in fish, which is very good and savory. Passing just lately over this lake, at a time when the wind blew pretty hard, and seeing the water as above described, I took up a little of it in a glass phial; and examining this water next day, I found floating therein divers earthy particles, . . .

In 1702, Leeuwenhoek reported, for the first time, observations of a wide variety of living "animalcules", as they were called at the time, in pond water. Based on his descriptions and drawings, we now know he observed ciliated protozoa Vorticella and Stentor, the colonial protozoon Volvox, diatoms and rotifers.

Cell Biology OLM Protocol:

You are going to prepare three independent samples of pond water and from each of those samples identify as many different single celled organisms as possible.

1. Using a Pasteur pipette, take a random sample of pond water and prepare a wet mount.

2. Place the slide on the stage of your microscope, turn on the illuminating lamp and focus on the specimen with the 4X objective lens. Once the specimen is in focus, search the slide for the single celled "animalcules". Once located, find the objective that gives you the best view of these organisms. Then, using the identification guide provided, identify those organisms you can find. Record your observations with drawings or photographs.

3. Repeat steps 1 and 2 two additional times so that you have identified organisms in each of three samples of pond water.

D. Homework Assignment

Your instructor may give you a homework assignment. Make sure you understand the assignment before you leave lab today.

E. Clean Up

Before you leave you must clean up your place so it looks the way it did when you walked into lab today.

F. Bibliography & Further Reading

1. Bryson, Bill. A Short History of Nearly Everything. Broadway Books, New York, 2003. 2. Dutrochet, Henri "Recherches anatomiques et physiologiques sur la structure intime des animaux et des vegetaux, et sur leur motilite, par M.H. Dutrochet, avec deux planches.” 1824 3. Ford, B.J. The Leeuwenhoek Legacy. Biopress, Bristol, and Farrand Press, London. 1991. http://www.sciences.demon.co.uk/whistmic.htm 4. History of the Microscope. http://www.history-of-the-microscope.org/. 5. Hooke, Robert. Micrographia. Royal Society, London. 1665. Reprinted by The Project Gutenberg, E-Book #15491. http://www.gutenberg.org/files/15491/15491-h/15491-h.htm. Release date: March 29, 2005 6. Hume. The Microscope. University of Dayton, Dayton, Ohio. http://campus.udayton.edu/~hume/Microscope/microscope.htm 7. Inwood, Stephen. The man who knew too much: the strange and inventive life of Robert Hooke, 1635–1703. London, 2003. 8. Karling JS. "Schleiden's Contribution to the Cell Theory". The Amer. Naturalist 73, 517–537, 1939 9. Mallery, Charles. The Cell Theory. Department of Biology, University of Miami. Last updated Feb, 2008. http://fig.cox.miami.edu/~cmallery/150/unity/cell.text.htm#text1 10. Museo Galileo, Institute and Museum of the History of Science. Galileo's Microscope. Florence, Italy. http://brunelleschi.imss.fi.it/esplora/microscopio/dswmedia/storia/estoria1.html 11. Pedrotti, Peter W. Thonis Philipszoon, Antoni van Leeuwenhoek. http://www.vanleeuwenhoek.com/. 2011 12. Porter JR. Antony van Leeuwenhoek: tercentenary of his discovery of bacteria. Bacteriol Rev 40 (2): 260–9, 1976. 13. Schleiden, Matthias Jakob "Contributions to Phytogenesis." 1839. 14. Schwann, Theodor and Schleyden, M. J. Microscopical researches into the accordance in the structure and growth of animals and plants. London: Printed for the Sydenham Society. 1847. http://vlp.mpiwg-berlin.mpg.de/library/data/lit28715/index_html?pn=1&ws=1.5. 15. Silver, GA. Virchow, the heroic model in medicine: health policy by accolade. American Journal of Public Health 77 (1): 82–8, 1987. 16. Turner, W. The Cell Theory Past and Present. J Anat Physiol 24 (Pt 2): 253–87, 1980. 17. Waggoner, Ben. Robert Hooke. http://www.ucmp.berkeley.edu/history/hooke.html. Jan, 2001. 18. Waggoner, Ben. Antony van Leeuwenhoek. http://www.ucmp.berkeley.edu/history/leeuwenhoek.html. 19. Wikipedia. Cell Theory. http://en.wikipedia.org/wiki/Cell_theory 20. Wikipedia. Robert Hooke. http://en.wikipedia.org/wiki/Robert_Hooke 21. Wikipedia. Zacharias Janssen. http://en.wikipedia.org/wiki/Sacharias_Jansen 22. Wikipedia. Henri Dutrochet. http://en.wikipedia.org/wiki/Henri_Dutrochet 23. Wikipedia. Theodor Schwann. http://en.wikipedia.org/wiki/Theodor_Schwann 24. Wikipedia. Matthias Jakob Schleiden http://en.wikipedia.org/wiki/Matthias_Jakob_Schleiden 25. Wikipdia. Rudolf Virchow http://en.wikipedia.org/wiki/Rudolf_Virchow 26. Wilkins, John S. Spontaneous Generation and the Origin of Life. http://www.talkorigins.org/faqs/abioprob/spontaneous-generation.html Posted April, 2004.