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Histology
Lab, Biology 302 Spring 2003 Dr. Ed Devlin Webpage for Course: http://people.hsc.edu/faculty-staff/edwardd/edsweb01/histology.htm Lab
Topic
Section 1. Introduction to histological techniques I, II, III, IV
2.
Blood and Connective Tissue Proper
V, VI 3.
Epithelium
VII
4.
Muscle and nervous tissue
VIII, IX
5.
Lab Exam I
--------
6.
Cardiovascular system and Integument
X, XI
7.
Cartilage, bone,
XII
8.
Lymphoid and respiratory system
XIII, XIV
9 .
Digestive system
XV
10.
Lab Exam II
--------
11.
Glands of the digestive system
XVI
12.
Urinary and endocrine system
XVII, XVIII
13.
Reproductive system
XIX, XX
14.
Lab Final III - Turn in slide projects
--------- CHECKING
SLIDE SETS After
your slide boxes have been issued, check each slide listed on the list
provided.
1. Note that your slide box is numbered and that each slide in the set
lists both this box number and the number of that particular slide. Only the
slides with your box number belong in your set.
2. All slide sets have blank spaces for future slides.
3. If a slide in your set is missing, place an X next to its number on
the master slide list. If you have a slide in your set that is from another
box, see your lab instructor.
4. If any slides in your set are damaged, note this on your master
slide list next to the slide number.
5. Hand in the completed list at the end of the lab. THE
STUDY OF HISTOLOGICAL SLIDES The
tissues you will be observing were prepared by a variety of different methods.
Your impression of how you perceive the tissue will be influenced by the
specific technique used in the preparation of the slide or the electron
micrograph. Each of the slides or micrographs you will be examining are
unique. Force
yourself to integrate information from discrete observations and develop
generalizations about the cells and intercellular substances under
consideration. This should involve both analysis (the separation of
constituent parts) and synthesis (combination of the constituent parts
into a whole). A concept is formed by a process of generalization. The
development of concepts in histology or science in general includes both
inductive and deductive reasoning.
Inferences involve:
induction
particular details
-----------------> generalizations,
"facts"
<----------------
concepts, ideas
deduction When
you examine a new slide, study it at low, medium and high magnifications.
Attempt to classify it into one of the major categories of tissue.
Refine your classification as far as possible. Formulate hypotheses about the
tissue and test them. Does your educated guess stand up to critical analysis?
A listing of the slides in your set are on the next page, check to make sure
all the slides in the set are present. 2.
Phagocytosis liver sec. 3.
Animal Mitosis Fish Blastula sec. 4.
Glycogen Liver sec. Best's Carmine 5.
Areolar tissue spread film 6.
Reticular Tissue sec. 7.
Ligamentum nuchae
c.s. & l.s. 8.
Elastic cartilage epiglottis sec. 9.
White fibro--cartilage sec. 10.
Membranous bone fetal skull sec. 11.
Compact bone decalcified c.s. 12.
Muscle composite sec. 13.
Cardiac muscle c.s. & l.s. 14.
Muscle-tendon connection sec. 15.
Nissl bodies neurocytes
sec. 16.
Motor end organs w.m. 17.
Spinal ganglion sec. 18.
Cochlea guinea pig l.s. 19.
Optic nerve mammal c.s. 20.
Artery & vein c.s. 21.
Artery, vein & nerve elastic tissue c.s. 22.
Entire heart l.s. 23.
Heart rat c.s. 24.
Lymph vessel valve w.m. 25.
Bone marrow red section 26.
Hypophysis sag. sec. 27.
Thyroid & Parathyroid sec. 28.
Enamel formation pig sec. 29.
Developing tooth l.s. 30.
Vallate papillae v.s. 31.
Esophagus composite sec. 32.
Esophagus and stomach l.s. 33.
Stomach & duodenum l.s. 34.
Duodenum c.s. 35.
Jejunum c.s. 36.
Ileum 37.
Recto-anal junction l.s. 38.
Liver sec. 39.
Liver pig sec. 40.
Bile duct mammal c.s. 41.
Pancreas sec. 42.
Epiglottis l.s. 43.
Trachea & esophagus c.s. 44.
Lung & bronchioli sec. 45.
Lung elastic fibers sec. 46.
Mammary gland active sec. 47.
Kidney entire l.s. 48.
Urethra female c.s. 49.
Urinary bladder collapsed c.s. 50.
Vas deferens c.s. 51.
Seminal vesicle sec. 52.
Penis mammal c.s. 53.
Ovary mature follicle cat sec. 54.
Ovary corpus luteum of pregnancy sec. 55.
Umbilical cord c.s. 56.
Bone dry ground human c.s. 57.
Joint human fetus l.s. 58.
Skeletal muscle human c.s. & l.s. 59.
Medullated nerve human osmic
acid 60.
Sympathetic ganglion human sec. 61.
Aorta elastic tissue human c.s. 62.
Vena cava human c.s. 63.
Human blood smear Wright 64.
Palatine tonsil human sec. 65.
Adrenal gland human sec. 66.
Pineal body human sec. 67.
Thymus human infant sec. 68.
Parotid gland human sec. 69.
Appendix human sec. 70.
71.
Human skin white v.s. 72.
Human skin Negro v.s. 73.
Human scalp white v.s. 74.
Skin cornified human sec. 75.
Mammary gland inactive human sec. 76.
Kidney human sec. 77.
Ureter human c.s. 78.
Urinary bladder human c.s. 79.
Testis human sec. 80.
Epididymis human sec. 81.
Sperm smear human 82.
Fallopian tube ampulla human c.s. 83.
Uterus human fall. phase sec. 84.
Uterus human progravid phase sec. 85.
Cervix uteri human c.s. 86.
Vagina human l.s. 87.
Placenta human sec. 88.
Barr's bodies human female 89.
Meissner's corpuscle's primate sec. 90.
Cerebellum primate sec. 91.
Lymph node primate sec. 92.
Spleen primate sec. 93.
Lip primate sec. 94.
Cardiac stomach primate sec. 95.
Fundic stomach primate sec. 96.
Pyloric stomach primate sec. 97.
Submaxillary gland primate sec. 98.
Sublingual gland primate sec. 99.
Eyelid primate sec. 100.
Prostate gland primate sec. ASK
QUESTIONS 1.
Is
the image the best that can be obtained with the equipment available? 2.
How
do the cellular and extracellular elements fit into the tissue? 3.
What
would these elements look like at higher levels of resolution (EM)? 4.
What
would this structure look like in another plane of section (longitudinal,
frontal, transverse, oblique)? 5.
What
features are elucidated by the specific techniques used in preparing this
slide? 6.
What
features are obscured as a result of the technique used? 7.
Is
there a close relationship between the fixed tissue and the living tissue? 8.
What
has been added or removed to the slide or micrograph as a result of the
technique of preparation? MAKE
DRAWINGS You
will be required to study the tissues under consideration carefully and make
drawings of the tissues. Drawing the tissues forces you to see detail and get
a better understanding of its structure. REVIEW FOR EXAMINATIONS 1.
Review your concept of each tissue type or organ. 2.
Examine unlabeled slides and micrographs (or slides with the label covered). 3.
Examine with the naked eye first or at lowest power. 4.
Test the other students in your class and allow them to test you. I. INSTRUCTIONS FOR
USING THE COMPOUND MICROSCOPE 1.
Be able to identify all of the parts of the microscope including:
A. Ocular lens or eyepiece
B. Objective lens
C. Stage
D. Condenser and condenser diaphragm*
E. Condenser focus knob*
F. Course and fine focus
G. Iris diaphragm Histological
and cytological details will be seen clearly when your microscope is set up
according to the following outline. Microscopes
of several designs may be provided. Learn
to use each of them correctly. 1.
Identify all Parts of Microscope with your Instructor 2.
Turn On Illuminator Turn
on the light and set the intensity control to a comfortable level.
The higher levels of intensity will be needed only for oil immersion.
On some instruments there are red numbers. These are rarely needed.
When they are used extensively the life of the bulb is shortened Levels
of intensity should always be adjusted with the light intensity control or
with a filter. Do not get in the habit of adjusting the intensity with the substage
diaphragm (vide infra). 3.
Check for Ground Glass in Front of Light Source On
some Nikon microscopes (early models) there should be a circular piece of
ground glass on top of the illuminator or attached to the underside of the substage
condenser. On other instruments
the top element of the field lens (illuminator) will have a ground surface.
Some models (with a field diaphragm) have a ground glass surface built
into the base. All three types of
microscopes may be encountered in the lab.
The purpose of the ground glass is to provide an even illumination with
coiled filaments used in the bulbs of all our instruments.
If a ground glass surface is not present, consult with your instructor. 4.
Make Preliminary Examination of a Histological Specimen with the 10X Lens;
Focus the Image of Specimen Place
a slide containing a stained section on the stage of the microscope and
examine it using the 10X objective. Focus
with the coarse adjustment, then focus with the fine adjustment. 5.
Adjust Substage Condenser Adjust
the substage condenser lens so that is top element
nearly touches the underside of the specimen slide.
It should always be used in a high position. On microscopes with a
field diaphragm (an adjustable aperture in the front or top of the
illuminator), the substage condenser lens should
be adjusted (upward or downward) until a sharp image of the field diaphragm is
visible in the plane of the specimen. In
order to accomplish this it may be necessary to reduce the diameter of the
field diaphragm in order to see its edges clearly when you examine a specimen
as in step 4. After
the field diaphragm is in focus, with a given objective lens (10X, 40X, 100X),
open the diaphragm until the light just fills the field of view as seen
through the eyepieces. If there is no field diaphragm on your microscope set
the substage condenser as high as it will go
without actually touching the slide. The
purpose of the field diaphragm is to reduce glare.
It is most useful for oil immersion work but it is not essential.
If you do not have one, don't worry about it.
(It is possible to make one by punching a round hole in a card and
centering it over the illuminator. You
may wish to try this as an experiment. A
hole can be made with a cork borer.) 6.
Adjust Eyepieces After
you have a sharp image of the specimen with the right eye, adjust the left
binocular tube independently so that the image appears sharp with both eyes.
Note that you can also adjust the interocular
distance for comfortable vision. 7.
Adjust Substage (Condenser) Diaphragm for Each
Objective Remove
one of the eyepieces and peer down the tube.
The aperture you see is at the back focal plane of the objective lens.
The degree to which this aperture is filled with light will depend on
the opening of the substage (condenser) diaphragm.
Set
the substage diaphragm correctly for the 10X
objective by opening or closing it until it just matches the diameter of the
opening at the rear of the objective lens (back focal plane).. Switch to the
40X objective. You will notice that the substage
diaphragm will now need to be opened wider to fill the back focal plane of the
40X objective.
Beyond
this point details are lost because of glare.
You must compromise. The
most common error is to close the substage
diaphragm down too far. Play
around with this adjustment until you can begin to appreciate the influence it
has on what you see. Remember this
point! Never use the substage
diaphragm to control the intensity of light.
The lamp power supply should be used to regulate the intensity of the
light. 8.
Use Oil Immersion with 100X Objective (optional) To
use the oil immersion objective, place a small drop of Type "A" (low
viscosity) immersion oil on the specimen slide after first finding the area
you want to examine under the 10X objective.
Swing the oil immersion objective into position without allowing the
40X objective to pass over the oil. Swing
it the other way! If
this precaution is not followed you will get oil on the high dry lens (not
intended for oil immersion) and it is troublesome to remove.
Dry lenses are useless if they have even a trace of oil on their front
elements. If oil gets on any of
the dry lenses, call your lab instructor.
He will remove it with lacquer thinner. For
critical work with the oil immersion objective Type "B" immersion
oil (higher viscosity than "A") should be used between the slide and
the top element of the condenser. We
will demonstrate this procedure. It
is necessary if you want to achieve the highest possible resolution of
specimens that are thin and well preserved (Epon
section). It is a somewhat messy
procedure; the oil must be carefully removed when you are through. Remove the
oil from slides when you are through. Toluene
is the best solvent for this purpose. 9.
Be Careful With The Instrument Avoid mechanical damage to the microscope. Objective lenses are especially fragile. Avoid any situation that would allow an objective lens to hit a slide or the mechanical stage. If you have problems (mechanical or optical) consult your instructor. Please do not try to make repairs yourself. *
may not be available on your microscope 1.
Andrew,
W. and C. P. Hickman. Histology of the Vertebrates.
C. V. Mosby Co.
1974. 2.
Bergman,
R. A. & A. K. Afifi. Atlas of Microscopic
Anatomy. W. B. Saunders Co.
1974. 3.
*Bloom,
W. and D. W. Fawcett. A Textbook of Histology.
W. B. Saunders Co. 1975. 4.
Copenhaver,
W. M., D. E. Kelley and R. L. Wood. Bailey's Textbook of Histology.
Williams and
Wilkins Co. 1978. 5.
DiFiore,
M. S. H. Atlas of Human Histology.
Lea & Febiger.
1974. 6.
Freeman,
W. H. and B. Bracegirdle. An Atlas of Histology.
Dover Publications Inc. 1964. 7.
Gardner,
D. and T. C. Dodds. Human Histology.
Churchill Livingston. 1976. 8.
Ham,
A. W. Histology. J. P. Lippincott
Co. 1974. 9.
Hammersen,
F. Sobotta/Hammersen Histology.
A color Atlas of Cytology, Histology and
Microscopic Anatomy. Urban
and Schwarzenberg.
1980. 10.
Reith,
E. J. and M. H. Ross. Atlas of Descriptive Histology.
3rd edition. Harper &
Row. 1977. 11.
*Rhodin,
J. A. G. Histology, A Text and Atlas. 12.
13.
K.
R. and M. A. Bonneville. Fine Structure of Cells & Tissues.
Lea & Febiger.
1974. 14.
Warwick,
R. and P. L. Williams. Grey's Anatomy. 35th
edition. W. B. Saunders Co.
1973. 15.
Weiss,
L. and R. O. Green. Histology. McGraw-Hill
Book Co. 1977. 16. *Wheater, P. R., H. G. Burkitt & V. G. Daniels. Functional Histology. Churchill Livingston. 1979. II.
EXPERIMENTS WITH THE MICROSCOPE The
following factors are important in how the image of microscopic specimens will
appear:
1. Refractive index of the mounting medium
air = 1.00
water = 1.33
R = 0.61
immersion
oil = 1.52
N
A Directions _____________________________________
_____________________________________
water
air
oil Set the condenser diaphragm to the proper setting as described earlier and examine the pieces of lens paper with the 10X and 40X objectives. Make sketches of typical paper fibers as they appear in air, water and oil. Give careful attention to detail and any perceived differences in contrast. You
will notice that the refractive index of the medium around the fibers has a
marked influence on their appearance. Which medium do you feel provides the
most faithful image? Examine the papers again with the diaphragm adjusted
to a smaller size (stopped down). Try to determine what influence the
diaphragm has on the details of the fibers appearance.
III.
HISTOLOGICAL TECHNIQUE - TISSUE
PREPARATION One
part of your laboratory grade will be determined by your ability to prepare
stained slides of mammalian tissue. This work will be done independently with
the help of the instructor. This section will give you a basic understanding
of the theory and process of histological tissue preparation. Feel free to ask
for assistance or clarification during the tissue work-up. A.
Fixation A
common fixative for routine histology is Neutral Buffered Formalin. We will
use this fixative in our preparations. It preserves the basic tissue types
(epithelium, connective, muscle and nervous) so they can be easily recognized.
One flaw is that it does not stabilize lipids. Organelles such as
mitochondria, Golgi bodies and endoplasmic
reticulum contain a high percentage of lipid and are rendered unrecognizable. Bouin's
fluid coagulates proteins. Delicate filaments and microtubules are totally
disorganized. Although the nucleus and chromosomes are
generally rendered easily stainable. In
our procedure small pieces of tissue (5-10 mm3) are usually fixed
for overnight at room temperature. Larger pieces may not be properly fixed at
the center. Following the fixation the specimens must be washed to remove the
fixative this is usually done with water or ethanol. We will use 70% ethanol.
The washing will be accomplished by placing the specimen into two changes of
70% ethanol. Your specimen can be kept for several months in ethanol, although
the sooner it is embedded the better. You will prepare 3 samples of your
tissue. B.
Dehydration There
are trade-offs in dehydration as in all the other steps. We want the tissue to
remain in each of the concentrations of ethanol long enough for the water to
be removed, but exposure of the tissue to 95% or 100% ethanol for too long a
period of time hardens to tissue excessively and sectioning is difficult. Also
care must be taken not to let the tissue dry out while moving it from one
solution to the next. C.
Clearing The
dehydrating agent is replaced with a substance called an antemedium.
This is a general name for a substance that will mix with both the dehydrating
agent and the embedding medium. Toluene or xylene
are commonly used antemedia. A
property of these materials is that they have a high refractive index and tend
to clear the tissue. They are therefore often called clearing agents. If you
have properly dehydrated your tissue sample, when placed in the clearing agent
it should appear clear and translucent. If it appears milky it has not been
dehydrated properly. There
are problems with clearing agents. They are highly volatile and if the tissue
is exposed to the air the clearing agents will evaporate and leave air pockets
in the tissue. Another concern that is shared with ethanol is these compounds
are highly flammable and appropriate care must be taken with their use.
D.
Infiltration and Embedding Cleared
specimens are placed in a solution of molten paraffin in an oven set at about
60o C. The antemedium is gradually
removed by dilution in several changes of paraffin. The paraffin infiltrates
the tissue and more or less takes the place of water in the living tissue.
This procedure causes the tissue to shrink down to as much as 40-50% of its
original volume. Maintaining the tissue in the oven may also cause the tissues
to become hard, especially is the temperature is too high, and as we will see
hard blocks are hard to section. The
tissues are finally transferred to a small container of pure molten paraffin
and oriented in the direction they will be sectioned. The paraffin containing
the specimen is rapidly cooled and solidified by placing it in ice water. The
protocol we will use is as follows (3 blocks): 1. Neutral Buffered formalin
2 hours to overnight 2. 50% ETOH
2 hours 3. 75% ETOH
2 hours 4. 95% ETOH
2 hours 5. 100% ETOH
1 hour 6. 100% ETOH
1 hour 7. 100% Organic Solvent
1 hour 8. 100% Organic Solvent
1 hour 9. 100% Molten Paraffin*
2 hours 10. 100% Molten Paraffin*
3 hours to overnight 11. Embed and orient tissue in block 12.
Place in ice water to solidify
* The Best Kind of Paraffin is: Baxter Scientific Products Ameraffin
Tissue Embedding Medium Cat.
# M7346-1A E.
Sectioning The
tissue sample now surrounded by hard paraffin is trimmed to a trapezoid shape
and placed in the chuck of a microtome. A microtome is a instrument for
holding and advancing the specimen while moving it past the cutting edge of a
sharp knife. Paraffin specimens are usually cut at thicknesses of 5-10
microns. Sections are cut then removed from the blade with a small paint brush
and placed on a slide covered with a thin layer of albumin and water (4 drops
of water). The
albumin acts as an adhesive and the water allows the section to expand before
it attaches itself to the slide. The slides are then placed on slide warming
trays to help flatten the sections out (1 hour+). Sectioning is the most
difficult part of the tissue preparation process and you should read chapters
4 and 5 in Humason (1979) before signing up to do
your sectioning. Your initial sectioning attempts should be done in the
presence of the instructor or lab assistant. Everyone encounters difficulties
in the process of sectioning, here are some suggested remedies (Modified from
Richards, 1949).
1.
Ribbons are crooked
1.1
Wedge-shaped sections caused by poor trimming; sides of paraffin block
are not parallel or not parallel to edge of knife.
1.2
Part of knife edge may be dull; try another part of it.
1.3
Uneven hardness of the paraffin; one side may be softer than the other,
or contain areas of crystallization; re-embed.
2.
Sections fail to form ribbons
(usually due to hardness of paraffin)
2.1
Use softer paraffin (lower melting point).
2.2
Blow on knife to warm it or dip it in warm water.
2.3
Cut thinner sections.
2.4
Place table lamp near knife and block to warm them both.
2.5
Resharpen knife.
2.6
Lessen tilt of knife and clean edge.
2.7
Dip block in softer paraffin and retrim so
a layer of this paraffin surrounds original block.
3.
Sections are wrinkled or
compressed
3.1
Resharpen knife; a dull knife compresses
badly.
3.2
Paraffin too soft; re-embed in harder paraffin.
3.3
Cool block and knife.
3.4
Increase tilt of knife.
3.5
Clean edge of knife with finger or xylene;
remove any paraffin collected there.
3.6
Tissue is not completely infiltrated (Poor infiltration is usually
caused by traces of water or alcohol). Correct by first removing paraffin that
is present. Soak in xylene
for 2 or 3 hours (or more); change twice, then place in absolute alcohol for 1
or more hours. This should remove
all traces of water. Clear again
in xylene (check against milky appearance); reinfiltrate
and embed.
3.7
Soak tissue block in water. When
soaking in water is recommended, the cut face of the tissue is exposed to tap
water for 30 to 60 minutes. This
treatment is generally satisfactory; however, some technicians advocate the
addition of glycerin (1 part to 9 parts water) or fabric softeners, same
ratio, or several drops of liquid dish detergent per 100 ml of water, or 60%
alcohol instead of water (Lendrum 1944).
These fluids work in through the cut tissue surface and soften tough
parts. (Exception:
Do not soak nervous system tissues at any time and lymph nodes and fatty
tissue only briefly. Paraplast
will not absorb water.)
4.
Ribbons are split or scratched
longitudinally
4.1
Nick in knife; move to another part of edge or resharpen
knife.
4.2
Knife dirty or gritty along edge.
4.3
Dirt or hard particles in tissue or in paraffin; crystals from fixing
solution not adequately removed; filter stock paraffin or decalcify tissue.
4.4
Decrease tilt of knife.
4.5
Tissue too hard; soak in water or RDO decalcifier
briefly (1 to 5 minutes).
5.
Tissue crumbles or falls out of
paraffin
5.1
Poor infiltration; reinfiltrate and
re-embed.
5.2
Not completely dehydrated.
5.3
Not completely de-alcoholized.
5.4
Too long in paraffin bath or too hot while there; soak in water.
5.5
Clearing fluid made tissue too brittle; soak in water.
6.
Sections cling to block instead
of knife
6.1
Knife dull or dirty.
6.2
Increase tilt of knife.
6.3
Paraffin too soft or room too warm; try harder paraffin or cool block.
6.4
Infiltrating paraffin too hot, or too long exposure to solutions that
harden; soak in water.
7.
Tissue makes scratching noise
while sectioning
7.1
Tissue too hard; paraffin too hot or exposed too long to solutions that
harden; soak in water.
7.2
8.
Knife rings as it passes over
tissue
8.1
Knife tilted too much or too little.
8.2
Tissue too hard; soak in water.
8.3
Knife blade too thin; try a heavier one.
9.
Sections curl, fly about, or
stick to things, owing to static electricity from friction during cutting,
especially in weather of low humidity.
9.1
Increase humidity in room by boiling water in open pan.
9.2
Ground microtome to water pipe.
9.3
Postpone sectioning until weather is more humid; early morning
sectioning often is best.
9.4
See p. 60 for suggestions concerning clothing, furniture, and static
eliminator.
10.
Sections are skipped or vary in
thickness
10.1
Microtome in need of adjustment or new parts.
10.2
Tighten all parts, including knife holder and object holder clamp.
Always!
10.3
Knife tilt too great or too little. F.
Staining and Mounting Hematoxylin stains the
nuclear materials and the eosin stains the cytoplasmic elements of the cells.
During this portion of the process you should wear old clothes or lab coats.
You should also understand the whole procedure before you get start. There are
a number of different protocols for staining the one we will use is as
follows:
1.
Place slide in solvent for 10 minutes (removes paraffin)
2.
Place slide in a second change of solvent for 10 minutes
3.
Place in 100% ETOH for 3 minutes with agitation (hydration)
4.
Place in second 100% ETOH - up and down motion 10X (hydration)
5.
Place in 95% ETOH for 3 minutes, agitate gently (hydration)
6.
Place in 80% ETOH for 3 minutes, agitate gently (hydration)
7.
Place in distilled water for 3-5 minutes, agitate gently
8.
Place in Harris Hematoxylin stain for 3-5
minutes
9.
Rinse in lightly running water
10.
Examine wet slide under microscope, if too proceed to step 11, if too
light return to step 6, if just right proceed to step 12
11.
Remove excess stain with acid-alcohol (35% ethanol with 0.1 m HCl) 12. Neutralize in alkaline alcohol (35% ethanol with pinch of NaCO3)
13.
Place in 70% ETOH for 1 minute (dehydration)
14.
Place in eosin in 70% ETOH for 2-5 minutes (counterstain)
15.
Place briefly in 70% ETOH to rinse off excess eosin
16.
Place in 95% ETOH for one minute (dehydration)
17.
Place in a second change of 95% ethanol for 1 minute (dehydration)
18.
Place in 100% ethanol for 1 minute (dehydration)
19.
Place in 100% solvent for 2-10 minutes (clearing)
20.
Add small drop of mounting media and a cover slip
21.
Allow to dry before examining References 1.
Galigher,
A. E. and E. N. Kozolff. 1971. Essentials of
practical microtechnique. Lea
and Febiger, 2.
Humason,
G. 1972. Animal Tissue Techniques. Fourth Ed. W. H.
Freeman & Co. 3.
Sheehan,
D. C. and B. B. Hrapchak. 1980. Theory and
Practice of histotechnology. The C. V. Mosby
Co. , 4.
Clark,
G. 1981. Staining Procedures. Fourth Edition. Williams & Wilkins. IV.
PARTS OF A TYPICAL CELL A
good example of a typical cell is a lymphocyte, one of the white blood cells.
Lymphocytes can be seen in slides of blood smears (slide 25). Find this slide
in your set and examine under low power. Identify the numerous erythrocytes
(red blood cells) which appear pink and are non-nucleated in mammals.
Generally one or two lymphocytes appear in each low power view. Lymphocytes
can be identified by their large nucleus with masses of chromatin material.
The cytoplasm is a clear region that is so small compared with the nucleus
that it often appears as a small rim at on side of the cell. Examine the
lymphocytes at all magnifications. Draw the cell as it appears under high
power. This
is a good opportunity to be able to examine a typical cell at higher
magnification and resolution using electron micrographs (photographs of cells
taken on a transmission electron microscope). Micrographs such as these are
used extensively today and you will be seeing a lot of them both in your text
and in the lab. Within a short time you should be able to recognize the
greater resolution that characterizes an electron micrograph from a light
micrograph (photograph from a light microscope). Examine the electron
micrographs in chapter 1 of the text and in addition to the structures listed
above, make drawings and be able to identify the following structures:
1. Ribosomes
(15 nm)
2. Endoplasmic reticulum (both smooth and rough)
3. Golgi apparatus
4. Mitochondria (0.5 um)
5. Lysosomes
6. Microvilli
7. Filaments
8. Inclusions
9. Cell membrane and cell division
10. Nucleus and nuclear membrane |