Biology 143
Lab 2
2-1
LAB 2: INTRODUCTION TO MICROSCOPY, CELL STRUCTURE
AND TISSUES
The purpose of this laboratory is to learn the correct care and use of the microscope. This lab
also provides an introduction to epithelial tissues and reviews the proper procedure for making a
temporary wet mount of a specimen.
Prelab
•
•
•
•
•
•
•
Review microscope use (p 7)
Complete introduction to cell structure self review (p 8)
Complete table of organelle functions (p 9)
Label animal cell diagram (p 10)
Complete p 12
Fill in the ‘representative locations’ for the tissues (p 13-15)
Complete p 16-17
I. INTRODUCTION TO MICROSCOPY
Introduction
The microscope provides a means of viewing specimens that are not visible with the naked eye
and allows small details of visible specimens to be examined. This instrument is expensive and
proper care is critical to ensure good results and to prevent damage. In order to understand the
instructions for use, you must first become familiar with the components of the microscope and
their functions. Once you are familiar with the components, you may proceed to set up critical
(Kohler) illumination if time allows. Following the steps for critical Illumination (p 6) will ensure
that the proper amount and intensity of light is focused on the specimen to allow you to see
minute details. Failure to follow the instructions can cause distortion of the images and interfere
with specimen identification.
Objectives
1.
To identify the component parts of the microscope
2.
To learn the functions of the component parts of the microscope
3.
To describe and demonstrate the proper techniques for care of the microscope
4.
To demonstrate the proper technique of illuminating and focusing the microscope using
Kohler illumination
5.
To define terms relevant to microscopy
Camosun College 2008
Biology 143
Lab 2
2-2
PART A. IDENTIFICATION OF COMPONENT PARTS OF THE MICROSCOPE AND THEIR
FUNCTIONS
With the assistance of your instructor, locate the microscope parts identified below on your
microscope, and label them on the diagram provided.
Oculars, Head, Head clamp screw, Diopter rings, Revolving nosepiece, Objectives, Stage,
Condenser, Iris diaphragm ring (or lever), Mechanical stage with slide holder, Stage
travel knobs, Condenser adjustment knob (not shown in diagram), Coarse focus knob,
Fine focus knob, Power switch, Rheostat
NOTE: there are several different models of microscope in the lab; some of the labeled parts
may not be in exactly the same location.
Camosun College 2008
Biology 143
2.
Lab 2
2-3
Become familiar with the functions of the component parts
1. Oculars (eyepieces)
Function: To magnify the specimen. Magnification is usually 10X and is engraved
on the top of the objectives. The oculars are actually simple magnifying glasses and
are useful if you wish to examine the lenses for dirt or oil.
2. Diopter rings
Function: To correct for differences between the viewer's eyes. Your microscope
may have one or two diopter rings, depending on the model.
3. Revolving nosepiece
Function: To house the objectives and allow the various objectives to be moved into
or away from the viewing position
4. Objectives
Function: To increase magnification and resolution. The objectives are engraved
with several numbers.
These numbers give information about the quality,
requirements and capabilities of the objectives. Magnification of the four objectives
on the Kyowa Medilux-12 are 4X (scanning), 10X (low), 40X (high dry) and 100X
(oil immersion). The 100X objective is only used when immersion oil is placed
between the slide and objective. This objective will not be used this term unless
instructor demonstrates and/or adds the oil.
5. Stage
Function: Where slide is placed (moved up and down by focus knobs)
6. Mechanical stage with slide holder
Function: To hold the specimens in place for viewing and allow movement of the
slides in two directions to enable the viewer to examine the entire slide. The
mechanical stage has readings which permit relocation of specific sites.
7. Condenser clamp screw
Function: To hold condenser in place.
8. Condenser
Function: To focus the light from the illuminator on the object being viewed. N.B.
Incorrect adjustment of the condenser is the most common error in microscopy. The
condenser must be adjusted as described in the following section. The condenser is
always at or near the stage. Generally the condenser is not moved to reduce light.
Moving the condenser out of its correct position causes distortion of the image.
9. Iris diaphragm ring (or lever)
Function: Controls the amount of light from the light source.
Camosun College 2008
Biology 143
Lab 2
10. Head clamp screw
Function: To hold head in place and to allow rotation of the head. Care
should be taken to ensure that the head clamp screw is secure.
11. Stage travel knobs
Function: To move the slide over the stage surface
12. Head
Function: To house the two oculars
13. Condenser adjustment knob
Function: To move the condenser up and down for critical illumination.
14. Coarse focus knob
Function: To focus specimen with scanning objective.
15. Fine focus knob
Function: To fine focus on the specimen.
16. Power switch for light
Function: To provide light for viewing specimens.
17. Interpupillary distance scale
Function: To give setting for distance between the viewer's eyes.
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2-4
Biology 143
PART B.
Lab 2
2-5
CARE AND USE OF THE MICROSCOPE
1. Students are responsible for the care and maintenance of the microscopes for the
semester.
2. The microscopes should always be stored in the microscope cabinet with the 4X objective in
place and the cord neatly and securely wrapped around the microscope.
3. Take the microscope assigned to your station from the cupboard. Carry with one hand on
the arm or limb and the other underneath the base. Ensure that the microscope is held in its
normal position with the base horizontal to the floor. If microscope is tilted, there is a danger
of the oculars or condenser falling out.
4. Check your microscope at the beginning of each laboratory period and report any problem
or malfunction to your instructor so that you will not be held responsible.
5. Check that all objectives are screwed in firmly and that the head clamp screw is tight.
6. Lower stage as far as possible, using the coarse adjustment to separate the objective and
stage as widely as possible.
7. Wipe all lenses of the oculars, objectives, condenser,
kimwipes before use.
and the field diaphragm with
8. Obtain a stained, prepared slide, turn the light on, center the specimen over the light and
using the 4X (scanning) objective, raise the stage as high as it goes. Now lower the stage
to focus with the course focus knob. N.B. The coarse focus knob is only used with the
scanning (lowest power lens)
9. Using the handling strips, adjust the interpupillary distance so that it is comfortable for you
and you see only a single circle of light (viewing field). Note the reading on the scale.
My reading on the interpupillary distance scale is _____________
10. Rotate the diopter ring on the right eyepiece tube to the same reading as noted on the
interpupillary scale. Switch to the 10X objective. Keep your left eye closed, look through
the right ocular and adjust with the fine adjustment until the best image is obtained. Then
close the right eye, look through the left ocular and adjust if necessary. Use ONLY the left
diopter. Do NOT use either focusing knob during this adjustment.
11. To focus with the 10X objective, rotate the low power objective into place, and focus with the
fine focus only. These microscopes are parfocal and remain basically in focus when you
change from one objective lens to another.
12. Repeat the last step with the 40X lens to focus at high power. NOTE: the working distance
is very small (the lens almost touches the slide); NEVER turn the coarse focus knob with
any lens except the scanning objective...you can crush slides and damage lenses.
13. When you are finished using the microscope, rotate the scanning lens back into place, then
lower the stage fully to remove your slide. Return the microscope to the cupboard.
Camosun College 2008
Biology 143
CRITICAL
Lab 2
2-6
ILLUMINATION (optional)
1. With the specimen in focus, move the condenser up toward the top of its range.
2. Open the substage condenser diaphragm.
3. Close the iris diaphragm.
4. Move the condenser up or down until the iris diaphragm is in sharp focus, i.e., the edges are
clear and are surrounded by a bluish light.
N.B. If the cone of light is not centered, ask the instructor for assistance.
5. Open the iris diaphragm until it clears the periphery of the field. Turn down the light if it is
too bright.
N.B. Once you have set the condenser, do NOT move it out of this position. The condenser
should not be lowered to improve illumination, instead adjust the condenser diaphragm
6. Adjust the condenser diaphragm until the image is optimal.
7. Switch to high dry (40X) power.
8. Adjust the condenser diaphragm until the light is optimal for you.
9. Turn off the light when you have completed the observations. It is not necessary to turn the
light off between slides but if the microscope is not being used for part of the lab, the light
should be turned off.
10. Clean all lenses, rotate the 4X objective into place, wrap cord firmly and neatly around the
microscope and return to the correct location in the microscope cupboard.
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Biology 143
Lab 2
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RELATED TERMINOLOGY:
1.
Magnification - the enlargement of a specimen
2.
Primary magnification - magnification of one of the components of the microscope,
e.g., the primary magnification of oculars is 10X, objectives are 4X, 10X, 40X or 100X
3.
Total magnification - the combined magnification of the oculars and the objective being
used, e.g., the total magnification when using the 10X objective is 100X
4.
Resolution - the ability to separate fine lines or structures so that they can be seen
clearly as separate entities; the ability to see fine differences in detail. With poor
resolution, two lines or points that are very close together could or would appear as one
line or point.
5.
Depth of focus (field) - the vertical range of the specimen that appears to be in sharp
focus. As the magnification increases, the depth of field decreases.
6.
Working distance - the space between the lowest part of the objective and the
specimen when the specimen is in focus.
7.
Viewing field – the actual area that you see when looking through the microscope. The
field of view decreases as the objective magnification increases.
9.
Parfocal - the characteristic of a microscope that allows you to maintain focus as you
change from one objective to another once the specimen has been focused with one of
the objectives. Only small adjustments with the fine focus should be required.
Complete the table below for your microscope:
Objective lens
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Objective
Ocular
Total
magnification magnification magnification
Biology 143
Lab 2
2-8
II. INTRODUCTION TO CELL STRUCTURE (self review)
A.
THE EUKARYOTIC - CELL
1.
Briefly - distinguish between
Eukaryotic Cell
________________________________________________________________
________________________________________________________________
Prokaryotic Cell
________________________________________________________________
________________________________________________________________
2.
Complete the following flow chart showing the levels of organization in living
organisms:
___________________
(eg.
)
(eg.
)
(eg.
)
(eg.
)
(eg.
)
(eg.
)
↓
___________________
↓
cells
↓
____________________
↓
____________________
↓
____________________
Camosun College 2008
Biology 143
3.
Lab 2
2-9
Using your text as a reference, complete the table below by summarizing the function of
each of the organelles. Locate the following structures and organelles on the animal
cell model and in the electron photomicrographs, then label the diagram of the cell
which follows.
Table 1. Organelles and their functions
ORGANELLE
plasma membrane
nucleus with its chromatin
nucleolus
nuclear envelope
nuclear pores
cytosol
ribosomes
smooth endoplasmic reticulum
rough endoplasmic reticulum
golgi complex
lysosomes
mitochondria
centrioles
vesicles or vacuoles
cilia or flagella
cytoskeleton
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FUNCTION
Biology 143
THE ANIMAL CELL
Camosun College 2008
Lab 2
2-10
Biology 143
Lab 2
4.
Examine a living eukaryotic cell:
2-11
•
Using a toothpick, gently scrape the inside of your cheek and spread the
material on a slide. Add a drop of methylene blue stain to the smear, and
cover the preparation with a coverslip.
•
Examine the preparation with your compound microscope and locate the
flat, squamous epithelial cells that line your oral cavity.
•
Bring the cells into focus with the high power objective (400X) and sketch
a few cells below (to scale, as you see them in the viewing field)
What organelles are visible? _________________________________________
Why are most of the "organelles" you just studied not visible?
___________
Fig 1. Sketch of squamous epithelial cells from lining of mouth
Magnification power =
Camosun College 2008
Biology 143
Lab 2
2-12
PART III: INTRODUCTION TO TISSUES
In this lab you will be examining various epithelial tissues. You should be able to identify the
cells and tissues studied, know the functions of the tissues, and know where in the body each
tissue type is found. As the tissues are reviewed in the lab, you should identify each tissue
image (at the back of the lab), paste the image in the appropriate space and label any
structures / cells indicated.
TISSUES
1.
Define a "tissue"
2.
Name the 4 major tissue types
a)
b)
c)
d)
EPITHELIAL TISSUES AND GLANDS
Epithelial tissues
•
cover body surfaces and line body cavities, organs and vessel lumens.
•
are characterized by a basement membrane, on which the cell layer(s) sit.
•
form both exocrine and endocrine glands
You will examine slides of various epithelial tissue types. You should learn to locate and
recognize the various types and become familiar with their functions and locations in the body.
Describe 4 functions of epithelial tissue:
1.
______________________________________________________________________
2.
______________________________________________________________________
3.
______________________________________________________________________
4.
______________________________________________________________________
Camosun College 2008
Biology 143
Lab 2
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Note the flow diagram below indicating the major types of epithelial tissues in terms of cell
shape, (squamous, cuboidal, columnar); surface specialization, (plain, ciliated, microvillous):
and the number of layers, (simple, pseudostratified, stratified, transitional).
Apical
specializations
Differentiation
Layering
Nonkeratinized
stratified
squamous
Simple
squamous
Keratinized
stratified
squamous
Microvilli
Newly
mitosed cell
Simple
cuboidal
Cilia
Transitional
Stratified cuboidal
Microvilli
Simple
columnar
Stratified
columnar
Cilia
Pseudostratified
ciliated columnar
SIMPLE EPITHELIAL TISSUES
A. Simple squamous
FUNCTION: Rapid diffusion of materials through the tissue
DESIRED FEATURES: Shortest possible diffusion distance, i.e. thinnest possible tissue
REPRESENTATIVE LOCATIONS IN THE BODY: _______________________________________
SLIDES: Mammalian lung, section (label squamous cell, alveolar sac)
Camosun College 2008
Biology 143
Lab 2
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B. Simple cuboidal
FUNCTION: Form walls of small-diameter tubules, some secretion and absorption; general
metabolism
DESIRED FEATURES: Thicker cells than squamous, but not too thick for small tubules
REPRESENTATIVE LOCATIONS IN THE BODY: _______________________________________
SLIDES: Mammalian kidney, section. (Note the presence of microvilli in some, but not all of
the tubules near the periphery. Those that have them are responsible for about 80% of the
kidney's impressive reabsorption capacity) Label cuboidal cell, basement membrane.
C. Simple columnar
FUNCTION: Absorption of nutrients
DESIRED FEATURES: Relatively large cells to house the necessary metabolic machinery and
many microvilli
REPRESENTATIVE LOCATIONS IN THE BODY: _______________________________________
SLIDE: Duodenum, section (label columnar cell nucleus, basement membrane, connective
tissue)
Camosun College 2008
Biology 143
Lab 2
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STRATIFIED AND PSEUDOSTRATIFIED EPITHELIAL TISSUES
A. Stratified squamous
FUNCTION: Protection against abrasion and tearing; can be stretched
DESIRED FEATURES: Layers of cells on the surface that are expendable and that can be
replaced continually from below
REPRESENTATIVE LOCATIONS IN THE BODY: ______________________________________
SLIDES: Esophagus, section (label squamous cells, basement membrane, connective tissue)
B. Pseudostratified ciliated columnar
FUNCTION: Trapping of airborne particles that could otherwise become lodged in the lungs
DESIRED FEATURES: Production of mucus in which to trap particles and presence of cilia to
move the mucus and trapped particles to where they can be disposed of
REPRESENTATIVE LOCATIONS IN THE BODY: _______________________________________
SLIDE: Trachea, section (label basement membrane, connective tissue, cilia, goblet cell).
NOTE: locate a goblet cell; goblet cells are unicellular exocrine glands which secrete mucus
NOTE: Glands are typically formed from epithelial tissue.
Camosun College 2008
Biology 143
Lab 2
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GLANDULAR EPITHELIUM
Epithelial tissue has also become specialized for secretion. Modified epithelial cells form the
functional basis of all the glands of the body including both exocrine and endocrine glands.
Examine the text and answer the following:
A.
B.
DISTINGUISH BETWEEN:
a)
Endocrine gland ___________________________________________
b)
Exocrine gland _____________________________________________
STRUCTURALLY: exocrine glands may be . . .
a)
Unicellular:
Locate the unicellular exocrine gland, the goblet cell, in the pseudostratified,
ciliated, columnar epithelium.
Goblet cells secrete a polysaccharide called mucin.
When mixed with water, what does this secretion become? _________________
What are the functions of this substance?
________________________________________________________________
Where else in the body would you
expect to find goblet cells? __________________________________________
b) Multicellular:
If multicellular, duct structure may be
i) Simple
_____
ii) Compound
_____
Both simple and compound multicellular glands may have secretory units described as
i) Tubular
ii) Alveolar (acinar)
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______
Biology 143
Lab 2
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C. FUNCTIONAL CLASSIFICATION OF EXOCRINE GLANDS on the basis of how they work.
Define each of the following terms - with examples.
a)
Merocrine
b)
Holocrine
c)
Apocrine
EXOCRINE
GLANDS
Unicellular
Multicellular
Simple
Tubular
D.
Compound
Acinar
Tubular
Tubuloacinar
Acinar
ENDOCRINE GLANDS (Structure and Function will be studied in association with the
endocrine system).
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Biology 143
EPITHELIAL TISSUES
Camosun College 2008
Lab 2
Biology 143
Camosun College 2008
Lab 2
1