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LABORATORY 2 Introduction to Microscopic Anatomy Objectives: 1. Identify the major components of a binocular compound microscope and identify their functions. 2. Properly use a microscope to focus on specimens using each of the objective lenses. 3. Define the following terms: total magnification, resolution, microscopic field, working distance, parfocal and contrast. 4. Differentiate between the following specimen preparation methods used to prepare tissues for microscopy: whole mount, smear, squash, cross section, longitudinal section 5. Describe the three major parts of a typical mammalian cell. 6. Identify the ectoderm, endoderm and mesoderm of a late gastrula and identify the tissues to which they give rise in the fully developed organism.

Introduction The microscope is a biologist’s most powerful observational tool. A light microscope functions by enlarging the image of an object illuminated by sunlight or an electric light source. Enlargement of the image is achieved when glass lenses bend the light rays that pass through the image. The rays that carry the image are focused at a position on the opposite slide of the lens, generating beyond this focal point an enlarged inverted image of the object. Microscopes used in modern biology labs are compound microscopes, and the light passes through two lenses arranged in series. This provides greater magnification than the use of a single lens.


The quality of a microscopic image is determined by magnification, resolution and contrast. Proper adjustment of the microscope will maximize these qualities and help to reduce eye strain. This lab exercise will focus on proper use of the binocular compound light microscope and its application to the study of human cells. The diversity of cell structure and the specialization of cell function will be stressed. Review the information below before you begin. I.

Care and Storage: Every part of a good quality microscope is incredibly expensive, so be very careful when handling it.

1. Use two hands to carry the microscope. Place one hand under the base and use the other to hold the arm. Always keep the microscope in an upright position. 2. Always place the microscope on a flat surface and make sure that it is not too close to the edge of the lab bench. 3. Clean every lens before and after use. Use lens paper only, and if necessary, lens cleaner. Never use any other materials to clean the lenses. 4. Always turn off the light when not actually using the microscope.


5. Always unplug the electrical cord by pulling on the plug, not the cord. 6. Never remove parts from the microscope. Notify the lab instructor if there is a problem – don’t try to fix it yourself. 7. When you are finished with the microscope, return it to its numbered location in the microscope cabinet. 8. Microscopes should be stored with the lowest powered objective lens in position and the lens should be positioned as close to the stage as possible (use the coarse adjustment knob). Remember to remove any slides from the stage and return it to its proper location. 9. Focus smoothly; don't try to speed through the focusing process or force anything 10. Always begin focusing using either the scanning or the low power 11. (10X) objective. Because the microscopes are parfocal, an image brought into focus at low power will be quickly and easily focused at high power. 12. Only use oil when using the 100X (oil immersion) lens; always use oil when using the 100 X lens. Always clean the slide and the oil immersion lens after use using the lens cleaner solution and lens paper.



Parts of the Microscope

Ocular lens:

also called an eyepiece. The microscopes in our lab have two eyepieces at the superior end of the body tube (are binocular). The ocular lenses on your microscope have a magnification power of 10X. One of the ocular lenses has a pointer in it that may be positioned by rotating that ocular lens

Body tube:

Holds the ocular lenses in place

Revolving nosepiece:

A circular mechanism located at the bottom of the body tube and attached to three or four objective lenses. It can be rotated to position the desired lens in the light path

Objective lens:

One of either three or four magnifying lenses attached to the revolving nosepiece. Each objective lens is marked with its magnification factor and is marked with unique colored rings for quick identification. Microscopes may have any or all of the following objective lenses: scanning lens:


either 4X or 5X; use this for initial location of a specimen

10 X (low power) lens:

may also be used to locate a specimen; this objective lens may be suitable for viewing some specimens that do not need greater magnification

40 X (high power) also called a high, dry objective; lens: used for specimens that require a greater degree of magnification 100X (oil immersion) lens:


used for viewing extremely small objects, such as blood cells or bacterial cells; this lens must be immersed in oil when in use

Lower powered lenses are short in length and higher powered lenses are longer.

Mechanical stage: Flat, horizontal platform below the objective lenses that the slide rests on. The stage has an opening so the light can pass through the specimen from below. This stage is equipped with a mechanical device that can be used to move the slide backwards and forward and side to side Substage light:

A high intensity light source. Light rays from this source pass through the specimen, through the objective lens and the ocular lenses to the eye


A small lens located beneath the stage that concentrates the light onto the specimen. The condenser can be raised and lowered. For most work, the condenser should be positioned as close to the stage as possible (raised position)

Iris diaphragm:

Regulates the amount of light that passes through the specimen. The diaphragm is adjusted by rotating a lever attached to the side of the device

Course Used to bring the specimen into focus by adjustment knob: increasing or decreasing the distance between the objective lens and the specimen


Fine Like for course adjustment knob, but is used Adjustment knob: for finer focusing after course focusing knob has been used


Focusing Procedure 1.

Turn on the light source.


Secure the slide in place on the stage using the brackets. Use the mechanical stage device to position the specimen as best as you can over the light beam passing through the slide. (Note: make sure that the slide is clean and avoid getting fingerprints on the cover slip).


Place the lowest powered objective (4X or 10X) into position using the revolving nosepiece. Bring the objective and stage as close together as possible using the coarse adjustment knob.


Look through the ocular and adjust the light intensity using the iris diaphragm. With a binocular microscope you need to adjust the eyepiece separation just like you do a pair of binoculars. Make sure that the substage condenser is in its highest position. Use the coarse adjustment knob to slowly move the slide and the objective lens apart. When the image becomes clear, switch to the fine adjustment know to make the image sharper. One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope. If you wish to view the specimen using a higher magnification (either 10X or 40X), rotate the next higher magnification lens into position without touching either the coarse or fine adjustment knobs. These microscopes are parfocal, that is, once a specimen is in focus at low magnification, it remains focused when higher objectives are placed into position. Some minor adjustment with the fine adjustment knob may be needed. Never adjust the focus using the coarse adjustment knob when using the 40X or 100X lens. Readjust the diaphragm to increase the light intensity if necessary.







You may wish to use the 100X (oil immersion) lens for very small objects. If so, first focus on the object using the 40X lens, as described above. Then, follow these steps: a. Center the object that you are interested in magnifying in the center of the field. b. Using the revolving nosepiece, swing the 40X objective lens out of the way, about halfway to the next position. c. Carefully place a small drop of immersion oil directly on the slide over the center of the region of interest. d. Rotate the oil immersion objective into position e. Using the fine focus and looking through the ocular lens, focus on the specimen. It will probably be necessary to increase the light intensity using the iris diaphragm. f. Make sure that you clean the lens and the slide when you are finished.

Note: Never use an oil immersion lens without the oil. Never get oil on any other lens. Clean up all oil when finished.


Useful Terms Contrast:

The difference in intensity between the specimen and the surrounding background. It is most conveniently altered by using the iris diaphragm located below the substage condenser.

Field of vision:

the surface area which can be seen when looking through the light microscope. The area decreases with increasing power of magnification.


The ability to distinguish between closely positioned objects. The human eye can resolve objects that are about 100 m apart. Under ideal conditions, the compound light microscope has a resolving power of 0.2 m. Without good resolution, magnified objects may appear blurry and indistinct. Resolution is specified as “r”, the minimum distance at which the objects can be perceived as distinct from each other. Thus, smaller the “r”, the better the resolution


Total magnification:

. Working distance:

The perceived increase in the size of an object when viewed using a microscope. TM is equal to the magnifying power of the ocular lens multiplied by the magnifying power of the objective lens being used; thus for a 10X ocular lens and a high dry objective, the total magnification is 10X x 40X, or 400X . The distance between the bottom of the objective lens and the top of the cover glass on the slide. The higher the magnification the smaller the working distance. The working distance between the highest powered objective and the slide can be less than a mm, so care must be taken to keep an objective lens from hitting the slide.

Materials 1. Each student should have a compound microscope. 2. Each pair of students should have: Lens Paper Immersion Oil Lens Cleaner Box of prepared slides Colored pencils Activity 1: 1.

Using A Compound Microscope

Use a prepared slide of the letter “e” for this section. a. Remove a prepared slide of the “letter e” from the box and look at the letter under the cover slip with your unassisted eye (the slide label should be on the left). Sketch the letter e in the space found in Question #1 of the Lab Activities Worksheet. b. Secure the slide on the stage of your microscope and bring the letter e into focus using the lowest powered objective (follow the directions below). Sketch the letter e as it appears in the field in the space found in Question #1 of the Lab Activities Worksheet. c. Move the slide slowly away from you on the stage using the mechanical stage while continuing to view it through the ocular lenses. Note the direction the image moves in Question #1 of the Lab Activities Worksheet.


d. Move the slide slowly to the right using the mechanical stage while continuing to view it through the ocular lenses. Note the direction the image moves in Question #1 of the Lab Activities Worksheet. e. Increase the magnification using the low powered objective. and refocus. Use the iris diaphragm to increase the amount of light passing though the specimen. When you are satisfied with the image, increase the magnification to the high dry objective and refocus. Answer the remaining portions of Question #1 of the Lab Activities Worksheet. Activity 2:


Slide Preparation: Sectioning and Mounting Specimens Textbook: Photographic Atlas:

page 1032 page 153 (Fig. 16.10); page 154 (Figure 16.13)

Review the material below before you begin this activity: 1.


Go to the demonstration area and view the slides that demonstrate various types of slide preparations: Lymph: wm Renal (kidney) tubule: ls Renal (kidney) tubule: cs (ts) Sperm: sm Chromosomes: sq Make drawings of each slide in Question #2 of the Lab Activities Sheet.

Background Information Even if you know what type of specimen you would like to examine, there are a variety of ways to prepare the specimen for the finished microscope slide. Here are some of the preparations used for human cells and tissues: Wholemount:

An entire structure, uncut, is embedded in mounting resin directly on the slide and covered with a glass coverslip.

Smear or Drop:

The specimen(s) are in suspension then dried directly onto the glass slide where they are fixed, stained, and mounted in resin under a coverslip. This preparation is usually used for blood cells.


The cell specimen is broken using pressure – usually used to release chromosomes from nuclei, then processed as a smear is.



A thin piece of specimen is shaved from the whole specimen to permit light to reveal greater structural detail. Sections are usually between 10 and 100 microns thick, which is usually thicker than one cell diameter. Therefore, several layers of cells may be present in the section. Some sections, called thin sections, are on the order of 1 micron thick, which is usually less than one cell diameter. Therefore, subcellular structures are more easily discerned than in thicker sections. Sections can be either longitudinal sections which are made lengthwise, parallel to the long axis of the structure, or transverse (cross sections) which are made perpendicular to the long axis.

Look at the label of a prepared slide to determine how the specimen has been prepared. "wm" means whole mount (the complete structure); "cs" is a cross section, such as a thin transverse section; "ls" is a longitudinal section, a section cut lengthwise; "sq" is a squash preparation; and, "sm" is a smear, such as a blood smear. Activity 3:

Generalized mammalian cell

Read the material below before you begin this activity: The basic structural unit of the body is the cell. Cells must maintain boundaries, take in nutrients, dispose of wastes, reproduce, grow, move, and undergo metabolism. While cells of the human body are varied in their morphology they all have three major parts: the plasma membrane, cytoplasm, and nucleus. Resources:


Pages 62-63 page 120 (Fig. 4.3) Page 11 (Fig. 2.1) Page 21 (Fig. 3.3)

Photographic Atlas:

1. Focus on a single cell on the microscope slide. Draw one cell from the specimen and label the plasma membrane, cytoplasm, and nucleus. Activity 4:

Embryonic Germ Layers

Read the material below before you begin this exercise. 1.

Use the models and/or prepared slide in the lab to identify the primary germ layers of a gastrula.


Make a labeled drawing in Question #4 of the Lab Worksheet.


A stem cell is a cell that has the ability to self replicate (divide) for indefinite periods, perhaps throughout the entire life of an organism. In addition to replicating, stem cells may also differentiate into any of the mature cells that make up each of the tissues of the organism. These mature cells have characteristics sizes, shapes and composition, along with specialized functions. A fertilized egg is often referred to as a totipotent stem cell, since it has the capacity to divide and produce cells that may differentiate into any of the cells of the mature organism. After an egg is fertilized it begins a series of rapid mitotic divisions, forming a ball of cells (day 3) called a morula. The cells of the morula continue to divide, and reorganization of the structure occurs, leading to the formation of a blastocyst (day 4) and then a layered structure called a gastrula (day 16). The cells of the embryonic germ layers of the gastrula are called pluripotent stem cells because they can each give rise to a more limited variety of cell types. The differentiation of stem cells is regulated by intrinsic signals and by the external microenvironment.


Fertilized Egg


. .

Ectoderm brain spinal cord all other neurons sensory receptors adrenal medulla skin (epidermis) pituitary gland connective tissue of head

Mesoderm muscles blood bone sex organs adrenal cortex lymphatic tissue urogenital system heart and blood vessels most connective tissues


Endoderm lining of gut lining of lungs lining of the bladder liver pancreas larynx, trachea, lung thymus gland thyroid, parathyroid gland urinary bladder, vagina, urethra

Checklist: A. Microscopy Location and functions of: ____Ocular lens ____Body tube ____Revolving nosepiece ____Mechanical stage ____substage light ____Condenser ____Iris diaphragm ____Course adjustment knob ____Fine adjustment knob

Terminology ____Contrast ____Field of vision ____Resolution ____Total magnification ____Working distance

B. Preparing specimens ____Types of preparations (wm, sm, sq, etc.) C. Cells ____Simple squamous cell D. Embryonic germ layers ____Ectoderm





Lab 2 worksheet

Name: ______________ Score: ______________

1. Microscopic Techniques: a.

Draw the letter “e” as it appears under each magnification and note the total magnification for each image:

Unaided Eye

Scanning Objective

Low Power Objective High, Dry Objective

TM =

TM = _____________

TM = ______________


TM = _____________


As you move the slide toward you while looking at the letter through the ocular lenses, in what direction does the image move? ________________________________


As you move the slide to the left while looking at the letter through the ocular lenses, in what direction does the image move? ________________________________


As you increase the magnification using higher powered objectives, how does each of the following change? Circle your answer:


Size of the image:

Decrease/Increase/No Change

Size the field

Decrease/Increase/No Change

Working distance

Decrease/Increase/No Change

Is it better to increase or decrease the light intensity as you change to a higher magnification? __________________________


2. Specimen Preparation Make sketches of the specimens at the demonstration area:

Lymph (wm) TM:

Renal (kidney) tubule (ls)




Sperm (sm) TM:

Renal (kidney) tubule (cs) TM:


Chromosomes (sq)





3. Generalized cell Prepare a labeled sketch of the following slide as seen under the microscope:

Simple Squamous Epithelium Total magnification: _________ Label: nucleus, cytoplasm, and plasma membrane

Simple Squamous Epithelium (cs) Total magnification: _________ Label: nucleus, cytoplasm, and plasma membrane


4. Development a. Make a sketch of a gastrula and label the endoderm, ectoderm and mesoderm.

Gastrula Total magnification: _________ Label: ectoderm, mesoderm, and endoderm

b. Indicate from which embryonic layer the following tissues/organs develop: Brain and spinal cord ________________________________________________________ Muscle _______________________________________________________ Liver __________________________________________________________


Post lab worksheet lab 2 4. Complete, or respond to, the following questions: a.

The _____ is a lens under the stage that concentrates the light.:


The area of a slide seen when looking through __________________ a microscope is the:


If the stage has a device that moves the slide from side to side and up or down, the stage is called a(n) _______________ stage.


The ability to distinguish between closely positioned objects is called:


If, after focusing using a low power objective __________________ lens only the fine adjustment needs to be used to focus under a higher power, the microscope is said to be:


If a microscope has a 10X ocular lens and the objective lens in use is the 100X, what is the total magnification achieved?



The distance from the bottom of the objective Lens to the specimen is called the:



The set of lenses closest to the viewer’s eyes __________________ are:


What device allows the user to alter the amount of light that passes through a specimen from a light source?


You are looking into the microscope and are surprised to see that only half of the field is illuminated. What is the probable cause? __________________________ You are viewing a specimen using a microscope and you attempt to change the position of the specimen in the field using the mechanical stage. You are surprised when the slide does not move. What is the probable cause? _____________________________________________________







The letter “J” appears in this orientation under the cover slip of a microscope slide. How will this letter appear when viewed using a microscope? (Draw it in the space): __________________________

5. Indicate which type of preparation (whole mount, smear, longitudinal section, cross section or squash) you would use in the following situations: To do a blood cell count


To view the lumen (opening) in the trachea


To view an entire, intact human embryo


To view the internal tissues of a tooth ________________ from the superior portion to the inferior portion To release pathogens from infected cells in preparation for microscopy


6. Using your textbook, determine the function(s) of the following: Plasma Membrane:____________________________________________________ ______________________________________________________________ Cytoplasm:_____________________________________________________

Nucleus: _____________________________________________________ ______________________________________________________________


7. Indicate whether the tissue/cell below is derived from ectoderm (EC), mesoderm (ME), or endoderm (EN): _____ blood

_____ lymphatic tissue

_____ skin (epidermis)

_____ bones

_____ brain and spinal cord

_____ adrenal medulla

_____ heart

_____ liver

_____ lining of the respiratory tract

_____ thyroid gland



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