Cells, Microscopes, and Domains of Life

Cells, Microscopes, and Domains of Life

Name:______Due Date: ______Section: ____

Partners: ______

Cells, Microscopes, and Domains of Life

Introduction

In 1665, the English scientist Robert Hooke used a primitive microscope that magnified objects 30 times (30X) to observe a thin slice of cork (bark from an oak tree). There he saw “a great many little Boxes” which he called “cells”, because they reminded him of the tiny rooms, or cells, occupied by monks. Today, powerful electron microscopes which magnify objects up to 500,000 times reveal the complex internal structure of cells. In the several centuries between Hooke’s original discovery and today’s scientific frontiers, research by generations of scientists has formulated and supported the basis of modern cell theory:

(1) Every living organism is made up of one or more cells.

(2) The smallest living organisms are single cells, and cells are the functional units of multicellular organisms.

(3) All cells arise from preexisting cells.

There are two fundamentally different types of cells: prokaryotic and eukaryotic. “Karyotic” refers to the nucleus of a cell, a membrane-enclosed sac which contains the genetic material. “Pro” means “before” in Greek, and prokaryotic (“before a nucleus”) cells do not have an organized nucleus surrounded by a membrane. “Eu” means “true” in Greek, and eukaryotic (“true nucleus”) cells do have an organized nucleus enclosed by a membrane.

All cells, whether prokaryotic or eukaryotic, have at least three components: (1) a cell membrane which surrounds the cell and regulates the flow of materials between the cell and its environment, (2) genetic material, and (3) cytoplasm, which consists of all the material inside the cell membrane except the nucleus. In addition to the presence or absence of a nucleus, there are additional important differences between prokaryotic and eukaryotic cells. Prokaryotic cells are very small, with a relatively simple internal structure. Although the functions carried out by a prokaryotic cell may be quite complex, these functions are not associated with discrete, membrane-bound structures, or organelles, inside the cell. In contrast, eukaryotic cells contain a variety of membrane-enclosed organelles that lend structural and functional organization to the cell. Eukaryotic cells are also larger than prokaryotic cells.

For many years, biologists classified the astonishing diversity of life on Earth (at present, around 1.4 million species have been named) into five kingdoms: Monera, Protista, Fungi, Plantae, and Animalia. As indicated in your text, taxonomy at the kingdom level of classification is presently in a state of flux. Criteria for classification have historically been based on similarity in physical form. However, modern technologies, including analysis of the genetic material, are allowing biologists to revise and refine traditional classification schemes, with the ultimate goal that classification should reflect the evolutionary relationships of organisms. The 5-kingdom system of classification must be viewed as a dynamic theory which, like all good scientific hypotheses, must be amended as new information comes to light.

Recent studies have determined that the “kingdom” Monera actually consists of two radically different kinds of organisms, the bacteria and the archaebacteria. (“Archae-“ is a prefix indicating antiquity, as in the word “archaeology”.) Their evolutionary divergence from each other is so ancient, setting the two groups on such uniquely different pathways, that it demands a re-working of our highest levels of taxonomic classification. Although not all biologists are in agreement, an alternative to the 5-kingdom system that has increasingly gained acceptance is one that introduces a higher level of classification than the “kingdom”, a level called the “domain”. This is the alternative system adopted by your textbook, and which will therefore be adopted in this laboratory course. According to this system, all living organisms are classified into three domains: the Bacteria, Archaea, and Eukarya. All organisms in the domains Bacteria and Archaea are prokaryotes, and all organisms in the domain Eukarya are eukaryotes.

Although this system solves certain problems, in that it recognizes the ancient and fundamental differences between Bacteria and Archaea, it leaves others unsolved, notably the lines along which to designate kingdom status. Your text treats this dilemma by retaining four kingdoms in the domain Eukarya: kingdoms Protista, Plantae, Fungi, and Animalia. The kingdom Protista consists of generally single-celled eukaryotic organisms. All of the organisms in the kingdoms Plantae, Fungi, and Animalia are also eukaryotic, but most of them are multicellular. These can be further classified on the basis of their way of acquiring nutrients. Members of the kingdom Plantae photosynthesize, that is, they combine carbon dioxide and water in the presence of light to create the sugar glucose. Members of the kingdom Fungi secrete enzymes outside their bodies and then absorb the externally digested nutrients. Members of the kingdom Animalia ingest their food and then digest it.

Your text remains non-committal with regards to the kingdom status of organisms in the domains Bacteria and Archaea. In keeping with your text, we will avoid referring to prokaryotes as members of any “kingdom” and only refer to their “domain”. Please keep in mind, however, that your Teaching Assistants were brought up with the 5-kingdom system, that it is hard to break old habits of thought, and they may occasionally “slip” in their classification references to prokaryotes! Although this may seem like a tedious academic discussion, remember that classification reflects our most fundamental ideas concerning the origin and diversification of life on Earth over 4 billion years of evolution.

In today’s lab, you will use a compound light microscope to observe representatives of two of the three domains (Bacteria and Eukarya) and, within the domain Eukarya, members of each of its four kingdoms. Neither as primitive as Robert Hooke’s instrument, nor as sophisticated as an electron microscope (nor as expensive!), light microscopes remain the mainstay of modern biological research and education.

The Compound Light Microscope(see adjoining figure)

The compound light microscope is a precision instrument, and should be handled with great care. All of the instructions given in this laboratory are designed to help you get the most from your use of the microscope and to ensure that it is well maintained for other students. To help you learn how to use the microscope, we will show you a short video (How to use a Microscope) before you begin the lab exercises.

Working with a partner, obtain a prepared “letter e” slide. Find the stage on the microscope you will share. Its purposes are (1) to support the slide, and (2) to allow the slide to be moved beneath the objective lens so you can examine different parts of the specimen on the slide. Open the spring-loaded finger of the specimen holder with one hand, and insert the “letter e” slide into the holder with the other hand. Release the finger gently after the slide is placed inside the holder. Using the stage control (the

vertical dial below and to the right of the stage), observe how the slide can be moved right & left, forward & backward, so as to position different parts of the specimen above the hole in the stage.

This hole allows light emitted from the illuminator at the bottom of the scope to pass through the specimen you are viewing. The light is turned on and off by a switch, and its intensity is controlled by a rheostat (voltage control dial). For the Olympus CH2 model microscope, the switch is at the front left corner and the rheostat on the right-hand side; for the Olympus Ch30 model, the switch and rheostat are on the right arm of the scope. Make sure the rheostat dial is set no higher than “3”, and then turn on the illuminator switch. Turning on the illuminator at higher intensities quickly burns out the microscope bulb or fuse.

This tool is called a compound microscope because the image is magnified by a set of two lenses: one in the eyepiece (the ocular lens) and one which is mounted on a revolving nosepiece, the objective lens. Note that there are three different objective lenses mounted on the revolving nosepiece. The shortest lens (color-coded red) magnifies the specimen four times (4X), and the next longest lens (color-coded yellow) magnifies it ten times (10X). These are both considered low power objectives. The longest lens (color-coded blue) is that of the high power objective, which magnifies the specimen forty times (40X). You should always begin your observation of a specimen on a low power objective, 4X or 10X. Make sure now that the 4X objective is in place and, if it is not, turn the nosepiece until it “clicks” into place.

Locate the coarse and the fine adjustment knobs on the side of the scope. The coarse adjustment knob is the larger of the two knobs, and the fine adjustment is the smaller. Now look through the eyepieces, which contain the ocular lenses. Turn up the rheostat just enough so that you can comfortably see the image. Use the coarse adjustment knob to bring the specimen (the letter “e”) into view. This action changes the distance between the objective lens and the stage. Now turn the fine adjustment knob to bring the specimen into the sharpest view you can. When using the fine adjustment, it is best to choose some sharp edge in the specimen to focus on.

The distance between the two oculars of the eyepiece can be adjusted to match the distance between your eyes. Move the two oculars in and out until you can comfortably see a single image. Note that there is a numerical scale between the two oculars. Write down the number on the scale at which you comfortably see a single image. By returning to this inter-ocular distance after another student has used the microscope, you can customize its setup for yourself. The left ocular also contains a diopter adjustment ring, to accommodate any difference in acuity between your right and left eyes. To customize this feature, first bring the specimen into focus with both eyes open. Next, close your left eye and use the fine focus adjustment to bring the specimen into best focus for your right eye. Now, open your left eye and close your right eye. Rotate the diopter ring on the left ocular to bring the specimen into its sharpest focus for your left eye. Finally, with both eyes open again, you will have the best focus possible for your eyes.

Q. What do you notice about the appearance of the letter “e” when viewed through the microscope?

The magnification of the ocular lenses is 10X. Thus, the ocular lenses make the specimen appear ten times larger than it actually is. Since both the ocular and objective lenses magnify the image, the totalmagnification is computedby multiplying the magnification of the ocular lens (10X) by the magnification of the objective lens. In this case, with the lowest power objective (4X) in place, the total magnification is 10X x 4X, or 40X. The letter “e” appears 40 times larger than when viewed with the unaided eye. (Refer also to the poster “Microscope Magnification” in the lab; a small copy is included with this laboratory).

Q. What is the total magnification of a specimen when the high power objective (40X) is being used?

Now, turn the knurled ring on the revolving nosepiece to change from the 4X objective to the 10X objective. As long as the specimen was in good focus at the lower magnification, it should only need minor adjustment with the fine adjustment knob to bring it into sharp focus at a higher power objective. You may need to increase the light intensity somewhat at this higher magnification. Again, use a sharp edge of the specimen when making fine focusing adjustments. As you turn the focal adjustment knobs, you may reach a stopping point. Do not force the knob beyond this point. Doing so may strip the threads of the focal adjustment and damage the microscope. Instead, inform your Teaching Assistant of the problem.

Finally, after the specimen is in sharp focus with the 10X objective, switch to the highest power objective (40X) by rotating the revolving nosepiece until the objective clicks into place. Increase the light intensity slightly. Notice that the distance between the objective lens and the slide is very small at this point. You should never use the coarse adjustment on high power! Even a small turn of the coarse adjustment knob may cause the objective lens to crash into the slide, irrevocably damaging both. Instead, useonly the fine adjustment knob to bring the specimen into sharp focus.

There is one other lens system on the compound microscope of which you should be aware. This is the condensor lens, which is situated between the illuminator and the stage. As its name suggests, its function is to “condense”, or concentrate, the light from the illuminator onto the specimen through the hole in the stage. It does not magnify or otherwise change the apparent size of the specimen. Notice the condensor height adjustment knob on the left-hand side of the condensor lens, which moves the condensor lens towards or away from the stage. While looking through the eyepiece, use the knob to move the condensor lens up and down, and observe how the light intensity changes. For the purposes of this course, you should keep the condensor lens as far towards the stage as it will go (i.e., maximum brightness).

You should also know about another way the light intensity can be adjusted. Locate the aperture iris diaphragm lever, which controls the amount of light coming through the condensor lens. While looking through the eyepiece, move the lever back and forth and observe how the light intensity changes. A common problem among beginning students of the microscope is to use far more light than is necessary to view the specimen. This not only decreases the life of the illuminating bulb but, more importantly, may give you a nasty headache from eye strain by the end of the lab session. Look at the number on your rheostat dial. With the 40X objective in place and the iris diaphragm opened for maximum brightness, if the number is higher than 6 (for the Ch2 model; 4 for the CH30 model), your illuminator is too bright and you should turn down the rheostat! In general, you should keep the iris diaphragm all the way open and use only as much light from the illuminator as necessary to clearly view the specimen.

Return the “letter e” slide to its box when you have completed the above exercises.

Microscopic Measurement

To find the length of something like this piece of paper, you need a ruler marked in specific units. To measure the size of a cell under the microscope, you also need a unit of measure or some frame of reference. The circle of light you see when looking through the ocular, called the field of view, can be used as a frame of reference if you know its diameter. You should have noticed that, as you switched from lower to higher powers (4X to 10X to 40X), you saw less and less of the letter “e”. As the magnification increases, the diameter of the field of view becomes smaller.

The following table shows the diameter of the field of view with the three different objectives:

Table 1. Diameter of field of view with three different objective lenses

Objective Lens / 4X / 10X / 40X
Diameter of field of view (millimeters, mm) / 4.5 / 1.8 / 0.45
Diameter of field of view (micrometers, m )

Before the use of the microscope as a biological tool, scientists had no need of units smaller than a millimeter. For microscopic measuring, the millimeter itself is divided into 1000 parts. One of the parts is called a micrometer, abbreviated m.

A micrometer is one thousandth of a millimeter. How many micrometers are there in 4 mm? Since there are 1000 m in 1 mm, there are 4000 min 4 mm.

Q. How many m in 56 mm?

Q. How many m in 2.5 mm?

You should also be able to change micrometers to millimeters. To do this, you divide the number of micrometers by 1000. How many millimeters are there in 8000 m? 8000 m divided by 1000 = 8 mm.

Q. How many mm in 1800 m?

Q. How many mm in 425 m?

Complete Table 1, converting the diameters of the field of view from millimeters to micrometers. You will use this information to determine the size of the cells you will be observing. How? Let’s say you are looking at a leaf specimen using the 4X objective. To determine the size of the individual cells in the leaf, first estimate how many cells would fit end to end across the field of view. Then divide this number into the diameter of the field of view (in m). This is the average size of the leaf cells you are observing.