Download Histological Sample Preparation (EXERCISE)

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Techniques: Histological Sample Prep
Background
Living cells go through a sequence of events called the cell cycle during
which DNA is
duplicated and the cell divides. This helps organisms to develop, grow,
replace and repair body
tissues. The cell cycle is divided into interphase, mitosis and cytokinesis.
A cell spends most of its time in interphase, during which it prepares for
cell division.
Interphase can be divided into three phases: G1, S and G2. The G1, the first
gap phase, is where
the cell starts making new proteins and organelles. During S phase, the cell
duplicates its DNA.
The G2, the second gap phase, is where the cell goes through its final growth
prior to the mitotic
phase, including the duplication of centriole pairs.
Mitosis is a series of events that result in the division of the nucleus to
form two genetically
identical daughter nuclei with the same number of chromosomes as the parent
cell. Although
mitosis is a continuous process, (yet for the sake of understanding) it has
been divided into four
phases: prophase, metaphase, anaphase and telophase. Some textbooks may
classify late
prophase as an additional phase called pro-metaphase. During prophase,
chromosomes begin to
condense, the nuclear envelope and the nucleolus disappear, duplicated
centriole pairs move
towards the opposite poles of the cell, microtubules organize to form a
mitotic spindle and
chromosomes made of two chromatids attach to the spindle fibers. In
metaphase, chromosomes
are fully condensed and aligned at the equator of the cell called the
metaphase plate. During
anaphase, the cell elongates and each chromosome splits at the centromere to
separate the
identical chromatids. The two daughter chromosomes are pulled towards the
opposite poles by
the kinetochore microtubules. In telophase, the two sets of chromosomes reach
the opposite
poles and begin to de-condense. The mitotic spindle disappears and the
nuclear envelope and
nucleolus re-appear resulting in the formation of two genetically identical
nuclei.
Cytokinesis is the division of the cytoplasm that often begins during late
anaphase or early
telophase and separates the nuclei to form two daughter cells. In animal
cells, cytokinesis is
marked by the formation of a cleavage furrow where a ring of actin fibers
forms around the cell
equator and contracts to pinch the cell into two parts. In plant cells, small
vesicles containing
cell wall materials collect between the two newly formed nuclei. These
vesicles fuse to form a
cell plate, which grows outwards to complete the formation of two daughter
cells.
In order to study cell division, it is essential to examine tissues that are
growing and where cells
are actively dividing. In animals, mitotic cell division can be easily seen
in the whitefish
blastula, a stage of embryo development. In plants, special growth areas
called meristems are
found in the root and shoot tips. These cells are actively dividing to cause
growth of the roots
and shoots. In this exercise, you will use slides to understand mitosis and
cytokinesis.
Purpose
This laboratory activity introduces the preparation of simple stained slides
and their nuclear
observation with other prepared slides, and the identification of interphase
and different stages of
mitosis in animal and plant cells.
Materials per team
Compound Microscope and Immersion Oil
Very Small Clean Beakers (50 mL)
Carnoy Fixative Solution
70% Ethanol Preserved Onion Root Tips
Hydrochloric Acid Solution (18%)
Microscope Slides and Coverslips
Scalpel and Forceps
Paper Towels and Lens Paper
Prepared slides of:
- Allium (Onion) Root Tip
-Whitefish Blastula
Toluidine Blue Solution
Procedures
A. Preparing Mitotic Squashes
1. Obtain two small beakers. Label one “HCl” and pour enough 18% hydrochloric
acid
solution into it to cover the bottom. Label the other beaker “Carnoy” and
pour enough
carnoy fixative solution into it to cover the bottom.
2. Use forceps to transfer an onion root tip into the HCl. Incubate for 4
minutes.
3. Use forceps to transfer the root into the carnoy fixative. Incubate for 4
minutes.
4. Place the root on a slide.
5. With a razor blade or other sharp instrument (forceps), cut off 1 to 2 mm
from the root tip
and save it on the slide (discard the remainder of the root).
6. Cover the root tip with a few drops of toluidine blue for 2 minutes. After
2 minutes, blot
away the stain; be careful not to touch anyting with your fingers!
7. When the stain has been blotted away, cover the root tips with 1 to 2
drops of water.
Gently lower a coverslip over the root tip. Cover the slide with a paper
towel or other
absorbent paper, and with your thumb, firmly press on the coverslip; do not
twist the
coverslip. This pressure will spread the cells into a single layer.
8. Observe your preparation under low power of the microscope. If the cells
are not
sufficiently separated to permit viewing of each cell, squash the preparation
again.
9. Using high-dry or oil-immersion magnification, identify the different
stages of mitosis.
B. Prepared slides
Whitefish blastula:
1. Obtain a slide of whitefish blastula and clean it with a paper towel.
2. Hold the slide against the light to locate the stained sections of the
embryo.
3. Focus the slide using the 10X objective lens of the compound microscope.
Note that in each section of the embryo, there are cells at different stages
of division.
4. Switch to the high-dry (40X) objective and identify cells that are at
specific stages of the
cell cycle: interphase, prophase, metaphase, anaphase and telophase. Give it
a shot at
100X (oil-immersion) if you can’t see well enough.
5. Find a cell showing cytokinesis. Note the cleavage furrow, a constriction
forming
between two daughter nuclei.
6. Draw one representative cell of each stage and complete the worksheet (A
and B).
Allium (onion) root tip:
1. Obtain a slide of Allium (onion) root tip and clean the slide with a paper
towel.
2. Hold the slide against the light to locate the stained sections of the
root tip.
3. Focus the slide using the 10X objective lens of the compound microscope.
Note that in each section of the root tip, there are cells at different
stages of division.
4. Switch to the high-dry (40X) objective and identify cells that are at
specific stages of the
cell cycle: interphase, prophase, metaphase, anaphase and telophase. Give it
a shot at
100X (oil-immersion) if you can’t see well enough.
5. Find a cell showing cytokinesis. Note the cleavage furrow, a constriction
forming
between two daughter nuclei.
6. Draw one representative cell of each stage and complete the worksheet (A
and B).
C. Microscope Clean-up
1. When you are done with the microscope, remove the slide. Pre-prepared
slides are
saved; student-prepared slides and coverslips are discarded in the Biohazard
box.
2. Clean the oil immersion lens and the slide after you are done by placing a
drop of 70%
ethanol (or xylene) on a piece of lens paper and gently wiping the lens until
there is no
more oil residue is left on the lens. Do not use any other type of paper to
clean the lens –
paper towels will scratch the sensitive lenses. The lens must be cleaned of
oil,
otherwise the lens will become damaged.
3. Loosely wrap the electric cord around the base of the microscope before
returning it to
the cabinet.
Whitefish embryo
Allium (onion) root tip
Interphase
Prophase
Metaphase
Anaphase
Telophase & Cytokinesis
WORKSHEET
Histological Sample Prep
A. Interphase and Mitosis
1. Draw a cell that represents interphase in whitefish blastula and onion
root tip.
Whitefish blastula
Allium (onion) root tip
Key characteristics for identifying interphase:
_______________________________________________________________________
2. Draw a cell that represents prophase in whitefish blastula and onion root
tip.
Whitefish blastula
Allium (onion) root tip
Key characteristics for identifying prophase:
_______________________________________________________________________
3. Draw a cell that represents metaphase in whitefish blastula and onion root
tip.
Whitefish blastula
Allium (onion) root tip
Key characteristics for identifying metaphase:
_______________________________________________________________________
4. Draw a cell that represents anaphase in whitefish blastula and onion root
tip.
Whitefish blastula
Allium (onion) root tip
Key characteristics for identifying anaphase:
_______________________________________________________________________
5. Draw a cell that represents telophase in whitefish blastula and onion root
tip.
Whitefish blastula
Allium (onion) root tip
Key characteristics for identifying telophase:
_______________________________________________________________________
6. During which phases of mitosis are chromosomes composed of a single
chromatid?
______________________ _______________________
7. Answer the following questions about the mitotic spindle:
a. What is a mitotic spindle composed of?
______________________________________
b. What is its function?
_____________________________________________________
8. The two sister chromatids of a chromosome have ________________
(identical/different)
DNA molecules.
9. If a cell has 4 chromosomes during metaphase of mitosis, how many
chromosomes will it
have during anaphase? _________________
10. During which phase of the cell cycle are chromosomes maximally condensed?
__________
11. During which phase of the cell cycle are chromosomes least condensed?
_______________
12. During prophase, each chromosome is made up of _____________ chromatids
that are held
together by a ____________________.
13. What is the difference between prophase and prometaphase?
________________________
_________________________________________________________________________
B. Cytokinesis
1. Draw a cell that represents cytokinesis in whitefish blastula and label
with the following
terms: cell membrane, nuclei, chromatin, cytoplasm and cleavage furrow.
Key characteristics of animal cell cytokinesis:
_________________________________________
_________________________________________
2. Draw a cell that represents cytokinesis in onion root tip and label with
the following terms:
cell wall, nuclei, chromatin, cytoplasm and cell plate.
Key characteristics of plant cell cytokinesis:
_________________________________________
_________________________________________
3. If a cell undergoes mitosis but does not complete cytokinesis, it would
become a cell with
_________ nuclei.
4. During cytokinesis in plants, the cell plate is formed by fusion of
vesicles.
a. Which organelle makes the vesicles?
________________________________________
b. What is contained in these vesicles?
________________________________________
5. Colchicine is a chemical that inhibits spindle formation. Explain what
will happen if a
dividing animal cell is treated with colchicine?
__________________________________
________________________________________________________________________
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Histological Sample Preparation
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