CELL BIOLOGY
EXPERIMENT
Department of Cell Biology & Genetics
Fujian Medical University
2010.3
CONTENTS
1. Introduction of Light Microscope .......................................................................
and Observation of Cell Morphological Structure ................................................. 3
2. Cell Chemistry .................................................................................................... 5
3. Cell Disruption and Subcellular Fractionation ............................................... 8
4. Animal Cell Fusion Induced by Polyethylene Glycol (PEG) ............................
and Cell Division ...................................................................................................... 10
5. The Making and Observation Of Cytoskeleton Specimens ..............................
and Ultra-structure of Animal Cell Organelles .................................................... 15
6. Preparation and Observation of the Mouse Chromosome ...............................
And Observation of the Human Chromosome ..................................................... 24
1. Introduction of Light Microscope
and Observation of Cell Morphological Structure
Instructor: Dr. Changwen ZHOU, Dr. Xiaoqing YOU
Objectives
At the end of this experiment, you will:
1. Acquaint with the structure of the light microscope and learn how to operate it properly.
2. Grasp the morphological character and the structure of cell under the light microscope.
Materials
1.
2.
Light microscope, Cedar oil, Lens paper, Xylene;
Prepared slide of Letter, Fiberglass, Hair, Onion root tips, Mouse intestinal epithelial cells,
Rabbit spinal cord cell, Human smooth muscle cells, Frog blood cells, Mouse liver cells,
Human oral epithelial cells, Human fibroblasts, Equine ascarid uterus.
Experimental Procedure
A. Structure and Operating of Light Microscope
1. Carry the microscope with two hands. Keep one hand underneath the microscope and the other
on the arm.
2. Learn the parts of your microscope. Never touch any lens with your fingers. If a lens needs
cleaning, use lens tissue, a lens cloth or a lens pen and be gentle.
3. Rotate the coarse-focusing knob forward to lower the stage down. Adjust the objectives on the
nosepiece of the microscope until the 4×or 10×lens (shortest objective) clicks into working
place. ALWAYS START WITH THE LOW POWER OBJECTIVE!
4. Insert the plug into the socket, press the button to turn on the light, push the knob back and forth
to make the light intensity proper for you. Use only as much light as you need (Not too dim, and
not too bright ).
5. First watch the slide directly in order to know the position of the specimen, and then put the
slide (e.g.Prepared slide of Letter) on the stage and hold it firmly with the clip, and move the
specimen to the center of the light pore.
6. Rotate the coarse-focusing knob backward to raise the stage until you cannot move it any more.
Rotate the knob forward slightly（just a bit）, then you can observe the image through the ocular
lenses. Rotate the fine-focusing knob to make the image clear.
7. If you want to see some part more clearly, move the part to the center of the field of vision.
Rotate the nosepiece to 40×lens (high power magnification). Rotate fine-focusing knob to make
the image clear. NEVER USE coarse-focusing knob for 40×lens !
8.
If you want to make some part magnified further and see it more clearly after observing it under
40×lens, you can move 40×lens away from light pore. (Don’t move the stage and the slide any
more!) Then add a drop of cedar oil (Its function is to increase the refraction of light.) onto the
specimen, and rotate the nosepiece to 100×lens(oil lens). Make sure that there is only oil
(without any air bubbles) between the lens and the specimen. Rotate fine-focusing knob slowly
to make the image clear. NEVER USE coarse-focusing knob FOR 100×lens !
9. When you finish observing, you have to use a piece of lens paper with xylene to clean oil lens
and the specimen.
10. After observing, rotate the nosepiece to make all the objective lenses away from light pore.
Lower the stage down, and remove the slide. Decrease the light intensity to “0”, and turn off the
light. Romove the plug from the socket.
Cautions
1. Always carry the microscope with two hands.
2. Use only lens paper to clean all lenses.
3.
Never allow the lens to touch the cover slip or the slide, or wet stains. Corrosive chemicals may
destroy the lens.
4. Move the slide in the opposite direction you want your specimen to go.
5. If your microscope doesn’t work very well, please notify your instructor at once. Never try to
repair it by youself.
B.
a.
1.
2.
3.
4.
5.
6.
b.
1.
2.
3.
4.
Observation of Cell Morphological Structure with Light Microscope
Different Cells
Prepared slide of Onion Root Tips
Prepared slide of Mouse intestinal epithelial cells
Prepared slide of Rabbit spinal cord cell
Prepared slide of Human smooth muscle cells
Prepared slide of Frog blood cells
Prepared slide of Mouse liver cells
Different Organelles
Golgi Complex: Prepared slide of Rabbit spinal cord cell
Mitochondria: Prepared slide of Human oral epithelial cells
Cytoskeleton: Prepared slide of Human fibroblasts
Centrosome: Prepared slide of Equine ascarid uterus
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2. Cell Chemistry
Instructor: Ms. Li LI , Mr. Mingquan WANG
Objectives
At the end of this experiment, you will:
1. Know the distribution of the chemical composition of cells, such as carbohydrate, nucleic acid,
protein, and enzyme.
2. Familiarize with the principles and methods of the cytochemical reactions ,which reveal the
chemical composition in situ.
Materials
1.
2.
3.
4.
Microscope, Slide, Cover slip, Alcohol burner, Lighter, Lens paper, Staining dish, Paper towel,
Knife, Pipette, Beaker, Forceps
Xylene, Cedar oil, iodine solution, 95% Alcohol, Methyl Green-Pyronine Solution, 2%H 2 O2 ,
benzidine, 1% Safranin Solution, 0.5% CuSO 4
Toad blood, Potato, Mouse blood
Prepared slide of acidic proteins
Prepared slide of basic proteins
Principles
Cytochemical reactions are based on the original structure of cells. Chemical reagents will react
specificly with some chemical compositions in cells, and followed by the deposition of colored
precipitates in situ. According to the colored precipitates you can know intacellular localization of
biomacromolecules such as carbohydrates, proteins, nucleic acid, and enzyme as well.
Experimental Procedure
C. Observation Of Starch(Starch-iodine reaction)
a. Principles:
Starch is produced by all green plants as an energy store. It consists of two types of molecules:
the linear and helical amylose and the branched amylopectin .Amylose in starch is responsible for
the formation of a deep blue color in the presence of iodine.
b. Procedure:
1. Chip the potato carefully to get a thin slice as thin as possible, spread it on a slide.
2. Add a drop of iodine solution and put a cover slip onto it, then observe.
c. Result:
Every cell crowded with elliptical granules of various sizes, which is in a beautiful blue or purple
black colour.
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D. Observation Of Nucleic Acids in cells(Methyl Green - Pyronine staining)
a. Principles:
The two types of nucleic acids are deoxyribonucleic acid (DNA), found in the nucleus, and
ribonucleic acid (RNA), found in the nucleolus and cytoplasm. Methyl green - Pyronine is a alkaline
mixed dyes ,which is the basis of selective staining of nucleic acids. Methyl green stain selectively
highly binds to polymerized DNA, thus staining the nuclei green. Pyronine stain preferentially binds
to low polymers of nucleic acid,such as RNA and any significant extent, depolymerized DNA.
b. Procedure:
1. Making the smear: Place a small drop of toad blood near one end of a slide, bring the edge of
another slide in contact with the drop and allow the drop to bank evenly behind the spreader. The
angle between the two slides has to be 30-40 degrees. Now, push to the left in a smooth, quick
motion. The smear should cover about half the slide. Allow the smear to air dry.
2. Fixing:Dip the slide in 95% alcohol for 5~10 minutes, then dry it at room temperature.
3. Staining: Place the slide on a staining rack, add several drops of methyl green - pyronine on it
until the smear is fully covered 5~10 minutes later, rinse it thoroughly in running tap water, until
purple disappears. Suck redundant liquid with paper towel carefully or let it air dry. Then observe
under light microscope.
c. Result:
Under low-power magnifination, the nuclei are green, which indicates the distribution of DNA,
whereas the cytoplasm and the nucleoli are red, which indicates the distribution of RNA.
E. Observation Of Acidic protein and basic protein in cells
a. Principles:
Proteins are polymers made of amino acid monomers. Different protein contain different acids
and bases because of their unique sequence of constituent amino acids. At physiologic pH, if the
protein is negatively charged, it’s acidic protein. On the contrary, it’s basic protein. After you
remove DNA and RNA prior to fast green staining with trichloroacetic acid (TCA), the acidic
protein and the basic protein can be shown respectively by fast green staining solution at different
pH. Those responding to fast green at pH 8.0~8.5 are basic proteins. The positive reaction at pH
2.0~2.5 is attributed to acidic proteins.
b. Results:
1. Prepared slide of acidic proteins:It was stained by acid fast green, showing the location of
acidic proteins. You’ll see cytoplasm and nucleoli are green while the nuclei are mostly colorless in
cells.
2. Prepared slide of basic proteins:The smear stained by basic fast green will show the location
of basic proteins.In the field of vision, you’ll see the nuclei are green while the cytoplasm do not
stain in cells.
F. Observation Of Enzyme in cells
a. Principles:
The catalase enzyme plays an important function in living organisms ,which degrades hydrogen
6
peroxide into water and oxygen gas because hydrogen peroxide is very toxic to living cells. When
this reaction occurs, the oxygen gas is released as bubbles. In low concentrations of hydrogen
peroxide, the catalase enzyme also exhibits peroxidase activity. A benzidine test could be employed
to detect the presence of of of catalase as it can oxidize benzidine into blue or brown product.
b. Procedure:
1. Showing the catalase enzyme of potato cell
(1) Chip to get a thin slice of potato, spread it on a slide.
(2) Place a few drops of hydrogen peroxide on the potato specimen ,wait a minute.
(3) Result:you’ll find it bubble profusely.
2. Showing the catalase enzyme of mouse blood cell
(1) Get a drop of mouse blood and make the smear, air dry it.
(2) Dip it in 0.5%CuSO 4 for 30s-1 minute.
(3) Dip it in benzidine for 5 minutes, rinse it in running tap water.
(4) Dip it in Safranin Solution 1% for 1 minute, rinse it in running tap water, let it air dry.
(5) Result: There are benzidine blue or benzidine blown granules in some big and irregularly
shaped cells, which are macrophages that contain lots of catalases.
Exercises
Draw a cell from prepared slide of acidic proteins to show the distribution of acidic proteins
7
3. Cell Disruption and Subcellular Fractionation
Instructor: Dr. Hekun LIU, Mr. Zhaohua LIN
Objectives
At the end of this experiment, you will:
1. Be familiar with the experimental principle of the fractionation of cellular components.
2. Be able to identify the basic method of the fractionation of nuclear and mitochondrial.
Materials
1.
2.
Low-speed centrifuge, High-speed centrifuge, Glass homogenizer, Absorbent paper, Coverslip,
Slides
0.9% NaCl solution, 0.25M sucrose-3 mM CaCl 2 solution, 1% toluidine blue, 0.02% Janus
Green B
Principles
The proportion of cells with different structures and sizes are not the same, the venue at the same
centrifugal settling velocity is not the same, according to this principle, used a different speed
centrifugation, cells were isolated within the classification of various components.
Separation of organelles of the most commonly used method is an organization made of
homogenate, suspended in a uniform media using differential centrifugation were separated by the
process including the organization of cell homogenate fractionation and analysis of three-step, this
approach has become a study sub-cellular components of the chemical composition, physical and
chemical characteristics and function of the principal means.
Homogenization：low -temperature conditions will be organized on the homogenizer by adding
isotonic homogenate media (0.25M sucrose-3 mM CaCl 2 ) to make it a variety of broken cell
organelles and It includes objects homogenate.
Fractionation from the low-to high-speed centrifugal gradual subsidence. First, so that larger
particles with low precipitation, and then a higher speed, will be floating in the supernatant of the
particles could settle down, so that a variety of cell structures, such as the nucleus, mitochondria, etc.
can be separated. As the samples of various sizes and densities of different particles in the
centrifugal start evenly distributed throughout the centrifuge tube, so every stage centrifugal
sedimentation to be the first time most certainly not a pure particle, subject to repeated suspension
and centrifugation to be purified.
Analysis of isolated components classification, available chemical and biochemical methods of
cell shape and function of identification.
8
Experimental Procedure
A. separation and extraction of the nucleus
1. After the mice was sacrificed by cervical dislocation, promptly cut open the abdomen, remove
the liver into the beaker that containing 0.9% NaCl, cut into small pieces (remove the
connective tissue) as soon as possible, repeated washing, try to remove blood, suck the liquid on
the surface with the filter papers.
2. Place liver tissue which wet weight is about 1g in a small petri dish, take 8ml pre-cooling 0.25M
sucrose-3mM CaCl 2 with a cylinder volume, firstly add a small amount of the solution in the
petri dish to make the live tissue into shredded, and then add all the solution in.
3. Pour the shredded liver tissue homogenate into the tube, so that the bottom homogenizer
immersed in containers filled with ice, the left hand holding the right hand perpendicular to the
homogenized ramming rod inserted tube, the upper and lower rotating grinding 3 ~ 5 times, with
3-layer gauze filters homogenate in the centrifuge tube, and then prepare a smear
make
①,
marks, and dry naturally.
4. Will be equipped with the filtrate, after balancing the centrifuge tube placed in ordinary
centrifuges to 2500rpm, centrifuge for 15 minutes; (1), then centrifuged slowly, moved into
high-speed centrifuge tube, stored in a beaker of ice until the separation of mitochondria; (2)
prepare smear ②, and dry naturally; (3) the remaining sediment to the next step.
5. With 6ml 0.25M sucrose-3 mM CaCl 2 , suspended sediments in order to 2500 rpm supernatant
centrifuged for 15 minutes, the residual liquid through a straw dogs new tricks into the
suspension, drop a drop on clean glass slide, prepare smear
dry naturally.
③,
6. All smears ①, ②, ③ stained with l% toluidine blue stain, cover with coverslip and observe
under light microscope.
B. High-speed centrifugal separation and extraction of mitochondrial
1. The high-speed centrifuge tube containing supernatant from a beaker with ice taken out of trim,
with its 17000rpm centrifuge for 20 minutes, discard supernatant, specimens from sediments.
2. Adding 0.25 M sucrose-3 mM CaCl 2 solution lml, wind and percussion into a suspension with
the straw in order to 17000 rpm centrifuge for 20 minutes, the supernatant to another test tube
suction, the specimens from the sediments by adding 0.1ml 0.25 M sucrose-3 mM CaCl 2
solution, blending into the suspension (available toothpick).
3. Supernatant and sediment suspension, respectively, drip drop on a clean glass slide
(respectively marked
④,⑤ sm ea
20 minutes covered with lid piece dyeing.
4. Results：Oil microscopy, granular mitochondria are stained blue-green Janus Green B.
9
4. Animal Cell Fusion Induced by Polyethylene Glycol (PEG)
and Cell Division
4.1
Animal Cell Fusion Induced by Polyethylene Glycol (PEG)
Instructor: Dr. Yingchun LIU , Ms. Jun SONG
Objectives
At the end of this experiment, you will:
1. Grasp the concept of cell fusion, and basic principles and method.
2. The preliminary master PEG-induced cell fusion.
Materials
50% w/v PEG （M W =1000, pH8.0
Solution
（H B SS,GKN; 0.2% methylene blue
pH8.0）,
Principles
Cell fusion, which under natural conditions or artificial means (biological, physical, chemical)
enables two or more cells in the process of melding to form a cell. Artificially induced cell fusion, in
the sixties of last century as a new technology . The induced cells could be of the same or different
types. The newly created hybrid cell by fusing two cells of different types will have properties of
both the parent cells. A heterokaryon may reproduce itself for at least several generations. Cell
fusion provides a method for assigning specific genes to specific chromosomes, so it is widely used
in cell biology and medical research. Cell fusion could be induced by many kinds of materials. There
are many methods such as biology methods( e.g. sendai virus), chemical methods (e.g. PEG) and
physical methods (e.g. electrical pulses). Currently the most widely used is PEG, because it is easy to
get, simple to operate, and its fusions efficiency is stable. PEG is a highly hydrated polymer, there
are a range of different molecular weight polymers. PEG-promoting financial mechanism has not
been entirely clear, it may cause the membrane phospholipid acyl bond and polar groups to
structural rearrangement.
Procedure
A.
1.
2.
B.
1.
2.
3.
Stock Solution Preparation of Chicken RBC (red blood cells):
Add chicken blood in a tube containing heparin four times the volume of 0.85% NaCl solution.
Place the stock solution of chicken RBC in the refrigerator for use within a week.
Fusion Procedure
Take 1 ml stock solution of chicken RBC in 10 ml refrigerate tube.
Add and mix 4 ml 0.85% NaCl with the pipette
Spin the cells down. (5 min, 1200 rpm)
10
）, w arm ed
4. Carefully discard the supernatant. Stir the pellet by gently tapping the bottom of the tube.
5. Add and mix 5 ml 0.85% NaCl. Spin the cells down (5 min, 1200 rpm) again. (To achieve the
removal of cell surface adhesion material purpose. )
6. Carefully discard the supernatant. Resuspend the cells in 10 ml GKN.
7. Take 1 ml chicken RBC in 10 ml refrigerate tube. Add and mix 4 ml HBSS.
8. Spin the cells down. (5 min, 1000 rpm)
9. Carefully discard the supernatant. Tap the pellet to resuspend.
10. Place the tube in a 39°C water bath and kept there during the fusion.
11. Add 0.5 ml 50% PEG 1000 pre-warmed to 39°C to the pellet using a 1 ml pipette, over a period
of 1 min, continually stirring the cells with the pipette. (Add slowly, drop by drop)
12. Continue stirring the cells in 50% PEG 1000 for further 1-2 min.
13. Incubate for 15 min at 39°C.
14. Add and mix gently 5 ml HBSS. Incubate for 5 min at 39°C.(To terminate reaction)
15. Spin the cells down. (5 min, 1000 rpm)
16. Discard the supernatant. Add and mix gently 5 ml HBSS. Spin the cells down again.
17. Discard most of the supernatant. Resuspend the pellet with the pipette.
18. Take some cell suspension on the glass slide by adding methylene blue staining 3 minutes.
19. Covered with coverslip, observed under the microscope.
Results
Observing cell fusion under the microscope, you could see the different states of cell fusion: two
cell membranes contact each other, causing adhesion; A fusion pore forms at the junction of the
membrane and spreads outward to form a cytoplasmic channel; The two nuclei of the adjacent cells
share a common cytoplasm after fusion. Finally, you should find all kinds of fusion cells containing
two or more nuclei.
Note: distinction between overlapping cells and fusion cells under the microscope.
Exercise
Draw a typical fusion cell.
Solutions
1.
2.
Hanks' Balanced Salt Solution （H B SS）: 137.93 mM NaCl, 5.33 mM KCl, 4.17 mM
NaHCO 3 , 1.26 mM CaCl 2 , 0.493 mM MgCl 2 , 0.407 mM MgSO 4 , 0.441 mM KH 2 PO 4 , 0.338
mM Na 2 HPO 4 , 5.56 mM D-Glucose
GKN
：0.
1g G lddH
ucose,
3 , 100ml
2 O 0.04g K C l,0.80g N aC l,0.03
11
4.2
Cell Division
Objectives
At the end of this experiment, you will:
1. Be familiar with the stages of both mitosis and meiosis
2. Be able to identify the stages of mitosis in both animal and plant cells via the microscope
Materials
1.
2.
3.
Microscope slide, Cover slip, Lens paper
Xylene, Cedar oil
Prepared slide of onion root tip
Prepared slide of horse roundworm (Parascaris equorum) uterus
Prepared slide of locust testes
Theory
Mitosis, the process where a single cell divides resulting in generally two identical cells, each
containing the same number of chromosomes and genetic content as that of the original cell. Mitosis
consists of four stages: Prophase, Metaphase, Anaphase, and Telophase, and very specific cellular
events occur during each stage to insure that the cell divides properly.
Meiosis is the process by which the gametes (sperm and eggs) are produced. Meiosis consists of
two nuclear divisions, meiosis I and II. Meiosis results in a sum total of four cells from each parent
cell. Each new cell contains half the number of diploid chromosomes.
Experimental Procedure
C. Mitosis
a. Observing mitosis of plant cells- the slide of onion root tip
1. Put an onion root tip slide on the microscope stage. Under low power magnification (10×),
observe the area near the root cap, because this area is the growing area, in which all cells are
cubic and in different phases of mitosis.
2. Locate and observe cells in each phase of mitosis under high power magnification (40×). It will
be necessary to move the slide to find each of the four phases.
(1) Interphase----Before mitosis, cells are in interphase. In the center of cytoplasm, there is a
round nucleus, while chromatins are diffused in the nucleus, and the nuclear membrane and
the nucleolus can be seen clearly.
(2) Prophase----Chromatins undergo condensation gradually and then turn into chromosomes.
At this point, each chromosome contains two DNA molecules organized as sister
chromatids held together by a centromere. At the end of prophase, the nuclear membrane
dissolves and the nucleolus disappears. Another characteristic which is not visible on the
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following slides are the centrioles (only in animal cells), which migrate to opposite poles
and will become the core of the microtubule organizing center (MTOC).
(3) Metaphase----The simplest phase of mitosis to identify is characterized by the “lining up”
of the chromosomes along the metaphase or equatorial plate. Spindle fibers may or may not
be visible connecting the centromeres of the sister chromatids to opposite ends of the cell.
(4) Anaphase----The centromeres split and all sister chromatids separate. Pulled by spindle
fibers, sister chromatids move to the opposite poles respectively.
(5) Telophase----The chromosomes become decondensed gradually, and finally turn into
chromatins. The nuclear membrane and the nucleolus appear once again, forming two new
nuclei. Spindle fibers disappear. Cytokinesis, the process of dividing the cytoplasm, began
in anaphase and is now leaving some definitive characteristics during telophase. In plant
cell, the appearance of the cell plate (a precursor to the cell wall) is a characteristic of
telophase.
b. Observing mitosis in animal cells- the slide of horse roundworm horse roundworm uterus
Put a slide of horse roundworm uterus on the microscope stage. Find a cross section using the
scanning objective, switch to the 10X objective, focus, and switch to the 40X objective. You will see
many elliptical zygote cells in different phases. Each zygote cell is surrounded by an ovum shell.
There is an ovum lumen between zygote cell membrane and ovum shell. Find the five stages of the
cell division stage as above.
Although the behavior of the chromosomes is the same as in plant cells, several distinctive animal
structures should be visible:
 Centrioles----small dots at the poles around which the microtubules of the spindle seem to
organize.
 Asters----a star-like array of microtubules surrounding each centriole pair.
 Cleavage Furrow----A constriction near the center of the dividing cell. In animal cells, the
appearance of the cleavage furrow is a good indication that the cell has entered telophase. The
cleavage furrow is an invagination of the cell membrane at the point at which the cell will be
split at.
D. Meiosis
Meiosis is divided into two steps: Meiosis I and Meiosis II. Each step of meiosis is divided into
the appropriate Prophase, Metaphase, Anaphase, & Telophase stages with the number I or II
following it to identify meiosis I or meiosis II. Let us look at the stages of meiosis in order by
observing the slide of locust testes.
Meiosis I
Prophase I: Similar to prophase of mitosis with one important difference:
Crossing Over: Pairs of homologous chromosomes synapse together to form tetrads and
exchange genetic information (DNA). Crossing over creates new, recombinant chromosomes.
Prophase I encompasses five stages.
(1) Leptotene----During this stage, individual chromosomes begin to condense into long
13
strands within the nucleus. However the two sister chromatids are still so tightly bound that
they are indistinguishable from one another.
(2) Zygotene---- At this stage, the synapsis (pairing/coming together) of homologous
chromosomes takes place.
(3) Achytene----This stage contains the following chromosomal crossover. Non-sister
chromatids of homologous chromosomes randomly exchange segments of genetic
information over regions of homology.
(4) Diplotene----Homologous chromosomes separate from one another a little. However, the
homologous chromosomes of each bivalent remain tightly bound at chiasmata, the regions
where crossing-over occurred. The chiasmata remain on the chromosomes until they are
severed in Anaphase I.
(5) Diakinesis----This is the first point in meiosis where the four parts of the tetrads are actually
visible. Sites of crossing over entangle together, effectively overlapping, making chiasmata
clearly visible. Other than this observation, the rest of the stage closely resembles
prometaphase of mitosis; the nucleoli disappear, the nuclear membrane disintegrates into
vesicles, and the meiotic spindle begins to form.
Metaphase I: Brief stage in which tetrads line up in the equatorial plane of the cell.
Anaphase I: Homologous chromosomes separate and migrate to opposite ends of cell. Each
chromosome still contains a pair of sister chromatids.
Telophase I: Two groups of chromosomes come together at opposite poles. Each group becomes
surrounded by a new nuclear membrane. Chromosomes uncoil, nucleoli reappear. Cleavage of the
cytoplasm occurs in as mitosis.
Interphase: may be very brief or absent between meiosis I and meiosis II.
Meiosis II: Meiosis II is the second part of the meiotic process. Much of the process is similar to
mitosis. The end result is production of four haploid cells. From the two haploid cells produced in
meiosis I. The four main steps of Meiosis II are: Prophase II, Metaphase II, Anaphase II, and
Telophase II.
Prophase II: DNA condenses into chromosomes. No crossing over occurs.
Metaphase II: Individual chromosomes line up in the equatorial plane of the cell.
Anaphase II: Chromatids separate and begin to migrate to opposite poles of the cell.
Telophase II and Cytokinesis: A full set of chromosomes reaches each pole of the cell. Four
different gametes are produced.
Exercise:
Examine prepared microscope slides of both animal cells and plant cells. Using your microscope,
scan the slides to find a cell in interphase and each one of the four stages of mitosis. Draw a
schematic representation of your observations for plant cells at each stage in the paper, labeling all
structures.
14
5. The Preparation and Observation Of Cytoskeleton Specimens
and Ultra-structure of Animal Cell Organelles
Instructor: Mr. Zhiguang ZHANG, Mr. Ling CHEN
5.1
The Making and Observation Of Cytoskeleton Specimens
Objectives
At the end of this experiment, you will:
1. Master the method of preparing cytoskeleton specimens.
2. Master the method of staining cytoskeleton with Coomassie blue.
3. Understand the basic shape and structure of cytoskeleton.
Materials
1.
Light microscope, Incubator, Small beaker, Pipette Forceps, Scissors, Reagent bottles, Slides,
Cover slips, Paper towel, Lens paper
2. 6mmol/L PBS (PH6.5), 2%TritonX-100, 3%Glutaraldehyde, 1%TritonX-100, M-buffer,
solution(10×original solution), 0.2% Coomassie blue
3. Onion bulb
Principle
Cytoskeleton is a complicated netlike structure composed of many protein fibers. It consists of
three distinct structures: microtubules (MT), microfilaments (MF) and intermediate filaments (IF).
They have great effects on the maintenance of cellular shape, and on the growth, movement, division
and differentiation of cells, as well as on cellular substance transportation, energy transformation,
information transmission and gene expression.
In the experiment, we use inner epidermis cell of onion scale leaf to show cytoskeleton under
2% TritonX-100, in order to extract most proteins and all fat, but save the proteins of cytoskeleton.
Secondly, stain the specimen with Coomassie blue. Finally, observe under microscope.
Experimental Procedure
1.
Cut an onion bulb into pieces, and take a middle piece of scale leaf and cut it in the inner side to
obtain a piece of inner epidermis (about 5mm×5mm), then rend the thin layer with forceps. Dip
it in the 6mmol/L PBS (pH6.5) in a small beaker for 5~10 min.
2. Suck the PBS with a pipette and discard it. Add 2 ml of 2%Triton X-100 in the beaker and
incubator at 37℃ for 20~30 minutes.(This step is to extratct proteins in the cell except for
cytoskeleton proteins.)
3. Suck 2%TritonX-100 with a pipette completely and discard it . Add M-buffer solution to wash
15
the specimen three times. 5minutes each time. (The function of this treatment is to keep
cytoskeleton stable.)
4. Suck M-buffer solution and discard it. Add 3% Glutaraldehyde to fix the specimen for 10 ~
20minutes.
5. Suck the fixative and wash the specimen with PBS three times, 15minutes each time.
6. Suck the PBS and add 5 drop of 0.2% Coomassie blue to stain for 15 minutes.
7. Suck the staining solution and wash the specimen with distilled water for 2~3times.
8. Spread the specimen on the slide and cover the slip.
9. Observe: Under light microscope, you can see the outlines of epidermis cells. In each cell, there
is a netlike structure composed of many blue fibers in different thickness. These fibers are
bunches of microfilaments. You can also see that there are some radial fibers around the nucleus
in some cells.
Observe the cells stained well under high power magnification. If you adjust fine focusing knob,
you’ll find the three-dimension structure of cytoskeleton.
5.2. Ultra-structure of Animal Cell Organelles
Objectives
At the end of this experiment, you will:
1. Master the ultra-sturcture of cell organelles
2. Observe the electron micrograph of cell organelles
Materials
the electron micrograph of cell organelles (album)
Contents
To master the ultra-sturcture of cell organelles through observing the electron micrograph of cell
organelles.
1. Cell Membrane
Cell membrane, or plasma membrane, are continuous, unbroken sheets and encloses every cell
defining the cell’s extent and maintaining the essential differences between its contents and the
environment. This membrane is a highly selective.
Electron micrograph showing the three-layered (trilaminar) structure of the plasma membrane of
an erythrocyte after staining the tissue with the heavy metal osmium. Osmium binds preferentially to
the polar head rows denote the lipid bilayer, producing the trilaminar pattern. The arrows denote the
inner and outer edges of the membranes (Figure 1).
2. Mitochondria
Mitochondria are surrounded by a double-membrane system, consisting of inner and outer
mitochondrial membranes. The inner membrane forms numerous folds (cristate).The membranes of
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the mitochondrion divide the organelle into two aqueous compartments, one within the interior of
the mitochondrion, called the matrix, and a second between the outer and inner membrane, called
the intermembrane space. Transmission electron micrograph of a thin section through a
mitochondrion revealed the internal of the organelle, particularly the membranous cristate of the
inner membrane (Figure 2). The matrix has a gel-like consistency owing to the presence of a high
concentration of water-soluble proteins.
Figure 1. An electron micrograph
of a human red blood cell membrane
Figure 2. Ultra-structure of Mitochondria
3. Endoplasmic Reticulum (ER)
The endoplasmic reticulum (ER) is divided into two subcompartments, the rough endoplasmic
reticulum (rER) and the smooth endoplasmic reticulum (sER). Both types of ER comprise a system
of membranes that enclose a space, or lumen.
The rough ER has ribosomes bound to its cytosolic surface, whereas the smooth ER lacks
associated ribosomes. The rER is typically composed of a network of flattened sacs(cisternae), as
shown in Figure 3A. The rER is continuous with the outer membrane of the nuclear envelope, which
also bears ribosomes on its cytosolic surface. In contrast, the membranous elements of the sER are
typically tubular and form an interconnecting system of pipelines curving through the cytoplasm, as
shown in Figure 3B.
4. Ribosome
Ribosomes are complexes of ribonucleic acid (RNA) and protein; they form the catalytic site for
the translation of messenger ribonucleic acid (mRNA) into a peptide chain. In the cytoplasm, the
subunits of the ribosome behave as independent particulars until they participate in the translation of
mRNA, at which time the 40S and 60S associate with mRNA to give rise to the functional 80S
ribosemes.
When a messenger RNA in the process of being translated is examined in the electron microscope,
a number of ribosomes are invariably seen to be attached along the length of the mRNA thread. This
complex of ribosomes and mRNA is called a polyribosome, or polysome. The ribosomes are aligned
in loops and spirals, indicating their attachment to mRNA molecules to form polysome(Figure 3C).
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5. Golgi Apparatus
The Golgi apparatus is usually located near the cell nucleus. It consists of a collection of flattened,
membrane-bounded sacs(cisternae, singular cisterna), which are piled like stacks of plates. Each
Golgi stack has two distinct faces: an entry, or cis, face and an exit, or trans, face. The bulk of the
Golgi complex consists of a series of large, flattened cisternae, which are divided into several
functionally distinct compartments arranged along an axis from the cis face closest to the ER to
medial cisternae and to the trans face at the opposite end of the stack.(Figure 3D ).
A
B
C
D
Figure 3 The electron micrograph of four cell organelles. A. rough endoplasmic
reticulum, rER. B. smooth endoplasmic reticulum, sER. C. ribosome D. Golgi
complex, Golgi
6. Lysosomes
Lysosomes are membrane-enclosed organelles that contain an array of digestive enzymes capable
of breaking down all types of biological polymers-proteins, nucleic acids, carbohydrates, and lipids.
Lysosomes function as the digestive system of the cell, serving both to degrade material taken up
from outside the cell and to digest obsolete components of the cell itself.
In their simplest form, lysosomes are visualized as dense, uniform-staining spherical vacuoles,
called primary lysosomes, which are just recently isolated from Golgi apparatus, and contain no
materials. While, when materials have been taken up for digestion, the primary lysosomes turn to
secondary lysosomes, which can display considerable variation in size and shape according to the
differences of the materials that have been taken up.(Figure 4) Lysosomes thus represent
morphologically diverse organelles defined by the common function of degrading intracellular
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material.
A
B
C
D
Figure 4 Electron micrograph of lysosome
(A) Primary lysosome, which is visualized as dense, uniform-staining spherical
vacuoles.
(B,C,D) Secondary lysosome, usually has taken up some materials, displays
variation in size and shape according to the differences of the materials.
7. Cytoskeleton
The cytoskeleton is defined as a network of protein filaments extending throughout the cytoplasm
of all eukaryotic cells. The cytoskeleton provides a structural framework for the cell, serving as a
scaffold that determines cell shape, the positions of organelles, and the general organization of the
cytoplasm. In most cells, the cytoskeleton is composed of three principal types of protein filaments:
microtubules, microfilaments, and intermediate filaments, which are held together and linked to
subcellular organelles and the plasma membrane by a variety of accessory proteins (Figure 5).
(1) Microtubules
The microtubules are composed of a single type of globular protein called tubulin. The building
blocks of microtubules are tubulin dimers which consist of two closely related tubulin: α-and
/β-tubulin. Dimers of α-and /β-tubulin polymerize to form microtubules, which are approximately
20~25 nm in diameter and composed of 13 protofilaments assembled around a hollow core. The
microtubules can function as: 1.singlet, which disperse in the cytoplasm; 2. doublet, which can be
found in the flagellum and cilium; 3. triplet, which can be found in the centriole and basal body.
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(2) Microfilaments
The actin, which is a major cytoskeletal protein, polymerizes to form the microfilaments. The
microfilaments display a thin, flexible fibers approximately 5~7 nm in diameter and up to several
micrometers in length. Microfilaments (MF) are particularly abundant beneath the plasma
membrane where they form a network that provides mechanical support, determines cell shape, and
allows movement of the cell surface, thereby enabling cells to migrate, engulf particles, and divide.
Figure 5 Electron micrograph of cytoskeleton
In most cells, the cytoskeleton consist of microtubules (MT), microfilaments (MF), and
intermediate filaments (IF), which are held together and function as a scaffold that
determines cell shape, the positions of organelles, and the general organization of the
cytoplasm.
(3) Intermediate filaments
Intermediate filaments (IF) have diameters between 8 and 11 nm, which is intermediate between
the diameters of the two other principal elements of the cytoskeleton. Whereas microfilaments and
microtubules are polymers of single types of proteins (actin and tubulin, respectively), intermediate
filaments are composed of a variety of proteins that are expressed in different types of cells. In
contrast to microfilaments and microtubules, the intermediate filaments are not directly involved in
cell movements. Instead, they appear to play basically a structural role by providing mechanical
strength to cells and tissues, especially in epithelia and myocyte.
Figure 6 Structure of the axoneme of cilia and flagella
(A) Electron micrograph of a cross-section of the axoneme of a rat sperm fla-gellum.
(B) Schematic cross-section of an axoneme. The nine outer doublets consist of one
complete A tubule and one incomplete B tubule. The outer doublets are joined to
each other by nexin links and to the central pair of microtubules by radial spokes.
Each outer microtubule doublet is associated with inner and outer dynein arms.
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8. Cilia and flagella
Cilia and flagella are microtubule-based projections of the plasma membrane that are responsible
for movement of a variety of eukaryotic cells. The fundamental structure of both cilia and flagella is
the axoneme, which is composed of microtubules and their associated proteins. The microtubules
are arranged in a characteristic "9 + 2" pattern in which a central pair of microtubules is surrounded
by nine outer microtubule doublets. (Figure 6)
9. Centriole
Most microtubules extend outward
from the centrosome, which contain a
pair
of
centrioles,
oriented
perpendicular to each other and
surrounded
by
amorphous
pericentriolar material. The centrioles
are cylindrical structures based on nine
triplets of microtubules, similar to the
basal bodies of cilia and flagella, while
there is no central pair of microtubules
in centrioles. (Figure 7)
10. The structure of nucleus
Under light microscope, the
structure of nucleus from eukaryotic
cell morphologically consists of the
nuclear envelope, the chromatin, and
Figure 7 Structure of the centriole
A:
Schematic
cross-section of centriole. The nine outer
the nucleolus.
triplets consist of A, B and C tubule; the centrosome
(1) Nuclear Envelope
contain a pair of centrioles, oriented perpendicular to
each other and surrounded by amorphous pericentriolar
The critical function of the nuclear
material.
membranes is to act as a barrier that
B,C: Electron micrograph of a cross-section of the
centriole.
separates the contents of the nucleus
from the cytoplasm. The nuclear envelope has a complex structure consisting of two nuclear
membranes, an underlying nuclear lamina, and nuclear pore complexes (Figure 8A). The nucleus is
surrounded by a system of two concentric membranes, called the inner and outer nuclear membranes.
The outer nuclear membrane is continuous with the endoplasmic reticulum, so the space between the
inner and outer nuclear membranes is directly connected with the lumen of the endoplasmic
reticulum. In addition, the outer nuclear membrane is functionally similar to the membranes of the
endoplasmic reticulum. In contrast, the inner nuclear membrane carries proteins that are specific to
the nucleus, such as the nuclear lamina. The nuclear pore complexes are the only channels through
which small polar molecules, ions, and macromolecules (proteins and RNAs) can travel between the
nucleus and the cytoplasm (Figure 8B). The nuclear pore complex is an extremely large structure
with a diameter of about 120 nm and an estimated molecular mass of approximately 125 million
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daltons—about 30 times the size of a ribosome.
A
B
Figure 8 Electron micrograph of the nuclear envelope and nuclear pores
A: Electron micrograph of a cross-section of nuclear envelope, the inner and outer
nuclear membranes are joined at nuclear pore complexes (arrows). The inner
nuclear membrane is lined by the nuclear lamina, which serves as an attachment site
for chromatin.
B: Electron micrograph showing nuclear pores, Many nuclear pores (arrows) are
visible in this freeze-fracture preparation of the nuclear envelope.
(2) Chromatin
The chromatin, which appears to be uniformly
distributed, becomes highly condensed during
mitosis to form the compact metaphase
chromosomes that are distributed to daughter
nuclei. During interphase, some of the chromatin
remains highly condensed, that is the portion of
heterochromatin,
which
is
condensed,
dark-staining, transcriptionally inactive, and
usually distributed beneath the nuclear envelope.
Except for the heterochromatin, the remainder of
Figure 9 Chromatin in interphase nuclei
the chromatin is the euchromatin, which is The euchromatin is distributed throughout
decondensed, light-staining transcriptionally the nucleus. The heterochromatin is usually
distributed beneath the nuclear envelope
active, and distributed throughout the nucleus indicated.
(Figure 9).
(3) Nucleolus
The most prominent nuclear body is the nucleolus, which is the site of rRNA transcription and
processing as well as aspects of ribosome assembly. Morphologically, nucleoli consist of three
distinguishable regions: the fibrillar center, dense fibrillar component, and granular component.
These different regions are thought to represent the sites of progressive stages of rRNA transcription,
processing, and ribosome assembly. The fibrillar center, also called nucleolar organizing regions
(NOR), is the light-staining central region of nucleolus. The NOR contains a cluster of tandemly
repeated, transcriptionally active rRNA genes, surrounded by the growing RNA chains; The dense
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fibrillar component is the dark-staining
region, which locates in the periphery of
the fibrillar center, contain the synthesized
RNA, which is undergo processing; The
granular component, that surrounds the
dense fibrillar component is the processing
ribosomes (Figure 10).
Figure 10 Structure of the nucleolus
An electron micrograph illustrating the fibrillar
center (FC), dense fibrillar component (DFC), and
granular component (G) of a nucleolus.
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6. Preparation and Observation of the Mouse Chromosome
And Observation of the Human Chromosome
Instructor: Dr. Qilian ZOU,Ms. Min WANG
6.1
Preparation and Observation of the Mouse Chromosome
Objectives
At the end of this experiment, you will:
1. Be familiar with the method of preparing chromosome specimens from animal marrow cells.
2. Be able to identify dividing phases and count chromosomes under light microscope.
Materials
1.
2.
3.
Microsope, Incubator, Refrigerator, Centrifuge, Balance, Dissecting pan, Scissors, Forceps,
Gauze, Injector, Pipette, Centrifugal tube, Test-tube surpport, Measuring cylinder, Alcohol
burner, Marker pen, slide glass, drier
Hypotonic solution(0.075 mol/L KCl), Fixative solution (methyl alcohol：glacial acetic acid
=3：1, v/v), Giemsa staining solution
Immature KUNMING mouse
Principle
Chromosomes can only be observed in dividing cells. Especially , in the metaphase, their
morphology is typical for observation and their number is easy to count. Metaphase chromosomes
are obtained from either the diving tissues or suspensions of mitosis cells by special technique.
Marrow cells are abundant and at a high level of dividing activity. And they are easy to obtained
without in vitro culture or sterile operation. Here we treat KUNMING mouse by colchicine, which
deters the dividing cells at metaphase. Then we prepare chromosome specimens from mouse
marrow cells by a series of relevant steps.
Experimental Procedure
1.
Treat Mouse by Colchicine
Treat KUNMING mouse by intraperitoneal injection of colchicine(2 ug/g weight) 3~4 hours
before experiment. Colchicine prohibits formation of mitotic spindle, leading to the determent
of mitosis at metaphase.
2. Separation of Thighbone
Kill the mice by cervical dislocation(Method: Press mice neck with forceps or fingers,
meanwhile pull its tail with your other hand.) Place mice in the dissecting pan, remove the skin
and muscle of two legs with scissors, expose the thighbones and their joints. Cut the thighbones
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3.
4.
5.
6.
7.
8.
9.
at the joints(Caution: Do not break up the thighbones, make sure that the whole thighbones are
intact.) Use a piece of gause to remove the muscle and tendon thoroughly.
Collect Marrow Cells
Cut two ends of the thighbone to expose the marrow cavity. Fill the injector with hypotonic
solution (0.075 mol/L KCl). Then insert the syrine needle into the marrow cavity from the
upper end of thighbone. Inject the hypotonic solution into the marrow cavity to wash the
marrow cells out. Repeat this step several times until the thighbone turn white. Collect marrow
cells into a 10ml centrifugal tube. Wash both legs of a mice into the same tube. Add the
hypotonic solution to about 10ml/tube.
Hypotonic Expansion
Mark your tube with marker pen. Place the tube in the incubator at 37℃ for about 20 minutes.
Marrow cells will expanse after hypotonic treatment, which leads to dispersion of
chromosomes at metaphase.
Fixation and Centrifugation
Add 5 drops of fixative solution (methyl alcohol :glacial acetic acid =3:1, preparing whenever
necessary) and mix gently by the pipette. Balance and centrifuge at 2,000 rpm for 5 minutes.
Discard the supernatant. Add 5ml fixative solution gently and disperse the cell precipitation
gently by the pipette(Caution: This step must be carried out gently, or the marrow cells will be
broken up and the chromosomes will be lost). Make the fixative step last for 10 minutes.
Balance and centrifuge at 2,000 rpm for 5 minutes again.
Prapare the Marrow Cells Suspension
Discard the supernatant. Add 8~10 drops of fixative solution and disperse the cell precipitation
by the pipette to prepare the cell suspension.
Prepare Chromosome Specimens
Suck the cell suspension into the pipette, then drip 2~3 drops onto the slide( precool by the
refrigerator, use it as soon as possible ) at the height of 25~30cm. Move the slide over the fire
back and forth until the glass become dry.
Staining
Stain the specimens with Giemsa staining solution for 7~10 minutes, then wash the redundant
staining solution away. Dry the specimens by drier.
Observation
Observe the prepared specimens under low power magnification. You will find many rounded
nuclei at interphase, which are stained into reddish purple and different from size. Look for the
chromosomes dispersing among the interphase nuclei. Choose a better field of vision, in which
the chromosomes disperse properly and have distinct morphology. Then move them into the
field center and change to high power magnification for detailed observation. The mouse
chromosomes are like letter “U” in shape and all of them are telocentric chromosomes. Choose
1~2 dividing cells to count the number of mouse chromosomes. A mouse diploid cell contains
20 pairs of chromosomes( 2n=40 ), of which 19 pairs are autosomes and the other pair are sex
chromosomes called XX in female or XY in male.
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6.2
Observation of the Human Chromosome
Principle
Human genome includes 23 pairs of chromosomes, of which 22 pairs are autosomes numbered
from 1 to 22, while the other pair are sex chromosomes called XX in female or XY in male.
Experimental Procedure
Take a prepared chromosome specimen from human peripheral lymphocytes and observe firstly
under low-power magnificantion. Find a good metaphase, in which chromosomes disperse properly
and have distinct morphology. Then move them into the field center and change to high power
magnification for detailed observation. You can also change to oil lens to observe the basic
conformation of chromosomes and count the number of them.
E. The basic conformation of chromsomes
A chromosome includes two sister chromatids with a centromere joining them together. A
centromere divides a chromatid into two parts. The shorter part is called short arm (p), while the
longer part is called long arm (q). According to the position of centromeres, human chromosomes
are classified into three types:
1. Metacentric chromosomes (mc) ---- Their centromeres are between 1/2 and 5/8 along the
longitudinal axis of chromosomes.
2. Submetacentric chromosomes (smc) ---- Their centromeres are between 5/8 and 7/8 along the
longitudinal axis of chromosomes.
3. Acrocentric chromosomes (ac) ---- Their centromeres are between 7/8 and the end along the
longitudinal axis of chromosomes.
F. Counting chromosomes
1. Find a diploid cell in meterphase, of which chromosomes disperse very well without any
overlapping.
2. Divide all chromosomes into 3~4 parts according to their natural distribution.
3. Count the number in each part and add them up, then you will get the total number of
chromosomes.
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