Laboratory one

Mitosis on onion root tip

Objectives:

Students are able to:

 Recognize and distinguish the various stages of mitosis

 Understand the changes in DNA content and chromosome number as cells progress through the cell-
cycle and mitosis.

Except for the gametes, the ordinary method of cell division is mitosis, a morphologically visible
process. The hereditary characteristics are carried by the DNA-protein complexes which are
found in equal amounts in nearly all nuclei of the body.

Mitosis is preceded by an exact duplication of the DNA and associated proteins in the nucleus.
This occurs during interphase—the period between actual mitotic divisions. After this doubling,
the chromosomes are organized, complete division and are then equally distributed to the two
daughter cells during mitosis.

All somatic cells pass through the mitotic and interphase periods at one time or another. The
duration of the mitotic period is usually about 0.5 to 2 hours, while the intermitotic period can
vary from a few hours to many years.

In humans and other mammals, the cells are relatively small in size and the number of
chromosomes great, thus making accurate observations rather difficult. For this reason, cell
division will be first studied in a plant (onion root tip) which has larger cells with fewer
chromosomes.

Interphase (not a stage of mitosis): The chromosomes cannot be distinguished and appear as
scattered granules connected by a network of pale-staining strands within a distinct nuclear
membrane. A nucleolus is usually present.

In prophase: Morphologically distinct chromatin threads appear and these shorten and thicken,
forming distinct chromosomes. The two pairs of centrioles begin to separate; (onion root cells

1
lack centrioles). Around the centrioles fine fibrils appear forming aster rays. The whole structure
is called an amphiaster and some of the aster rays elongate to form the spindle fibers. The
nucleolus and the nuclear membrane begin to disappear.

Metaphase: The chromosomes become arranged at the equatorial plate which is midway
between the two pairs of centrioles. The spindle is now fully formed. It does not stain
(achromatic) and consists of fine microtubules, some of which are attached to the chromosomes.
During this period the chromosomes begin to show indications of splitting.

At anaphase: Each chromosome pair completes its splitting, and the two daughter chromosomes
move toward opposite poles. Spindle microtubules may be seen between the retreating
chromosomes.

Telophase: The chromosomes have reached the spindle poles and appear as a dense, basophilic
mass within which individual chromosomes cannot be defined. The nuclear membrane
reappears, and the outlines of the chromosomes disappear leaving scattered chromatin granules
connected by a pale-staining network. Nucleoli reappear and seem to be associated with a
particular chromosome. Cytokinesis, the division of the cytoplasm, usually occurs during the
telophase, but the synchrony between nuclear and cytoplasmic telophase, but the synchrony
between nuclear and cytoplasmic events is not constant; cytokinesis may begin as early as late
anaphase or be delayed beyond the nuclear reconstruction of telophase.

Materials:
■Test tube ■scissors
■fixative solution (9 part 45% acetic acid ■razor blade
and 1part 1N HCL) ■metal spatula
■watch glass ■aceto-orcein stain
■onion ■microscope slides
■beaker or small cup (150mL, ■cover slips.
approximately)

2
Procedure
 Cut off four root tips, each approximately 1 cm long;
 Fill test tube 3cm full of fixative.
 Place four root tips into the test tube of fixative and incubate at 50 degrees Celsius for six
minutes.
 Then dump heated fixative and tips into watch glass.
Staining the Cells:

 Take root tips, one at a time, out of watch glass

 Place each on in the middle of a microscope slide.
 Cut all excess from the root tips except for 2mm at the very tip of the root (end that was
not cut from the plant).
 Place two drops of aceto-orcein stain on top of the 2mm root tip.
 Let stain soak into root tip for two minutes.
 Squash the root tip, on each slide, pressing straight down so as not to overlap the cells.
 Place two more drops of stain upon the root tip and wait for another two minutes.
 Then place cover slip flat upon the root tip, making certain not to move the cover slip
horizontally.
 Press the cover slip gently with a pencil eraser, again only straight down without moving
the cover slip.
 Soak up extra stain from slide around cover slip with a paper towel without moving cover
slip.
 Observe and record steps of cell division under microscope (at 400x)

3
 Laboratory two
Meiosis
Objectives
Students are able to:
 Describe major events occur during meiosis
 Understand the advantages of meiosis
 Differentiate meiosis from mitosis
Meiosis is division for sexual reproduction and results in gametes, either egg or sperm in
mammals. Meiosis is also called reduction division since the number of chromosomes is
reduced by half. .
Meiosis is a unique biological event that not only maintains the chromosome number constant
for a species of plants or animals, but provides a means of genetic variability because of
"crossing over", an event which allows the exchange of genetic material. In animal meiosis,
immature germ cells undergo a "reduction" from the diploid number of chromosomes and
become mature haploid gametes. Mature gametes are sperm and eggs. In plants, meiosis may
result in the formation of spores.
Meiosis involves two cellular divisions instead of one, resulting in the formation of four
gametes, each with half the original chromosome number. The two cellular divisions are
designated as Meiosis I and Meiosis II. Major events are as follows:

A. Meiosis I
 Chromosomes appear as double structures consisting of two chromatids.
 Chromosomes carrying similar (but not identical) traits pair up. This pairing is called
synapsis and may result in an exchange of genetic material between chromosomes called
crossing over. Thus, tetrads of chromatids are formed.
 Tetrads gather on the equatorial plane.
 Whole chromosomes (consisting of two chromatids each) move to the opposite poles. At
this point, homologous (like) chromosomes are essentially separated into two different new
cells. The chromosome number has been halved.

4
Question: Which chromosomes go to: which pole is entirely by chance, what is the significance
of this?
B. Meiosis II
 Chromatids, still joined, move to the equatorial plane.
 Chromatids separate and move to opposite poles.
 We now have four (4) cells each with half the normal number of chromosomes.

Question: What does homologous mean? What are homologous chromosomes?

Question: Where in the human body would you expect meiosis to occur?
Question: How many chromosomes are in a human somatic cell? How many chromosomes
are in a sperm or egg?
Question: Are the end results of meiosis always gametes?
Explain.
Question: Disregarding crossing over, how many different gametes are possible from these six
different chromosomes?
Notes

 Requires patient students that have used compound microscopes before.

 Preparation time needed - The plant buds and animal testes need to be collected in the
late spring and early summer, when meiosis is most to be occurring. The buds and testes
can be stored in fixative for at least a year.
 All the material from the plants and animals needs to put in fixative (3:1 fixative
consisting of 3 parts 100% ethanol and 1 part glacial acetic acid) for several days to
bleach out any pigment present.
 If petroleum jelly is put around the edges of the cover slips, the slides can be kept for
several days.
 Hazards / Precautions - The stain used can stain skin and clothes. The fixative solution is
flammable. Avoid contact with and breathing of any of the chemicals used to kill the
insects.

Materials & Equipment Needs

5
Materials needed for lab:

 Buds from a variety of pollen producing flowering plants (such as beans, spiderworts and
lilies)
 Microscope slides and overspills
 A probe and scalpel or razor blade
 Forceps
 A compound microscope capable of at least 400X magnification
 A Bunsen burner or hot plate
 Acetocarmine stain. Most biological supply companies carry prepared acetocarmine that
is ready to use.
 3:1 fixative consisting of 3 parts 100% ethanol and 1 part glacial acetic acid. (600 ml
ethanol and 200 ml glacial acetic acid). 100% methanol can be used in place of the
ethanol.
 Testes from grasshoppers, crickets and other insects

Collection of plant buds for the meiosis lab:

It is a good idea to gather flower buds when they are available, especially in the spring. It is
important that the buds are very young, preferably before the production of pollen. Any species
of pollen producing flower will do. Lilies, spiderworts and beans work well because they are
easy to find and have large chromosomes that stain well. Otherwise, greenhouses and florist
shops are good sources of flower buds throughout the year. Try to pick young inflorescences
with series of intact flower buds. To preserve the buds, first carefully remove the buds from the
plants. Put the buds in containers that can be stoppered and stored easily. Add enough (3:1
ethanol/acetic acid) fixative to each container to insure that the buds stay covered. This lab can
be done at any time of the year if the materials are collected and preserved beforehand. If you
are using freshly picked buds, you need to store them in the fixative solution for at least several
days before the lab. The fixative will bleach out pigments such as chlorophyll in the bud that can
interfere with the staining process. They can be stored for over a year.

Collection of animals’ testes for the meiosis lab:

6
Collect the insects you plan on using in the late spring and early summer when the male insects
are producing sperm. Grasshoppers are common insects that are easy to catch, have testes that
are easy to locate and breed in the late spring while school is still in progress. House crickets
may be a good source of specimens, too. Consult a zoology or entomology text so you can
distinguish between male and female insects. Put the insects in a killing jar and add a chemical
such as dichlorobenzene or ethyl acetate to kill the insect. Finger nail polish remover or moth
balls will work too. Once the male insects are dead, remove the testes from the insects and store
them in the fixative solution (3:1 ethanol / acetic acid). Directions for excising and preserving
the testes are included in most invertebrate and micro techniques lab books. I have included a
frontal section diagram of the posterior of a male grasshopper in case; an appropriate text cannot
be located. The shape of the internal organs will vary between species of grasshoppers. They
can be stored for over a year in fixative.

Procedures for Preparation of Plant and Animal Meiosis Slides:

For Plants:
1. Remove the bud from the fixative and place it in an open flat container (petri plates work
well).

2. Use an eye dropper to add some of the fixative to the bud so the bud doesn't dry out. The
container should be open so that you can use pointed forceps to dissect out the anthers. Use a
dissecting microscope if you need to. Good lighting and manual dexterity are helpful. Check a
diagram in any good botany book for the location and shape of the anthers in a typical plant bud.

3. Once the anthers have been isolated, transfer them to a slide and cover the anthers with
acetocarmine stain.

4. Use a probe or razor blade to mash up the anthers in the acetocarmine stain. Use a fine tipped
forceps to remove debris such as anther walls. Make sure that you have removed the outside of
the anthers. Let the remainder of the anthers set in the stain for several minutes.

5. Add more acetocarmine stain to the slide if dries out.

7
6. Cover the anther remainders on the slide with a cover slip.

7. Check the heating directions and squash technique that is used for both plant and animal
slides below.

For Animals:

1. Remove the testis from the fixative and place it in slide.

2. Use forceps or a razor blade to smash and smear the contents of the testis over the central part
of the slide.

3. Remove any large pieces of debris with fine pointed forceps.

4. Use an eye dropper to cover the material with acetocarmine stain.

5. Let the material set in the stain for at least several minutes.

6. Add more acetocarmine stain to the slide if dries out.

7. Cover the testis material on the slide with a cover slip.

Heating to set the stain and the squash technique:

The next step is heating the slide gently. If a Bunsen burner or alcohol lamp is used, hold the
end of the slide and move it back and forth through the flame. You have to be careful not to heat
the slide so the stain boils or burn your fingers. Gentle heating will improve the staining process
considerably. Take your time. If you heat the slide on a hot plate, it has to be set at 80 degrees
C. Allow the slide to set on the hot plate for 3 - 5 minutes. Make sure the stain on the slide does
not boil. If the material starts to dry out add more stain to the edge of the coverslip. Capillary
action will pull it in. After heating the slide, let it cool for a few seconds. Place the slide
between layers of paper towels or napkins. Press straight down firmly. You need to be careful
not to break the slide. This squashing process flattens the cell's nuclei and spreads out the
chromosomes

8
Examination of the Slides:

The slides are now ready to be examined using a compound microscope. With hard work, you
might find meiotic cells. Remember that you may have to look through quite a few slides to
locate cells in different stages of meiosis. Having diagrams or photos of meiotic cells as
reference points is quite useful in the process of searching the slides. Remember, that you may
search quite a few slides before you get lucky. If you find a slide you want to keep for several
days, adding petroleum jelly to the edge of the cover slip will prevent it from drying out.

9
 Laboratory three

Probability and the Chi-Square Test

Objectives

Students are able to:

 Describe chi-square-test
 Apply chi-square test in genetics
 Test hypothesis and goodness of fit in mono and di hydride crosses

Types of Data:

There are basically two types of random variables and they yield two types of data: numerical
and categorical. A chi square (X2) statistic is used to investigate whether distributions of
categorical variables differ from one another. Basically categorical variable yield data in the
categories and numerical variables yield data in numerical form.

The Table-1 below may help you see the differences between these two variables.

Possible
Data Type Question Type
Responses
Categorical What is your Gender? Male or Female
Discrete- How many cars do you
Numerical 2 or 3
own?
Numerical Continuous - How tall are you? 72 inches

Notice that discrete data arise from a counting process, while continuous data arise from a
measuring process.

10
The Chi Square statistic compares the tallies or counts of categorical responses between two (or
more) independent groups. (Note: Chi square tests can only be used on actual numbers and not
on percentages, proportions, means, etc.)

Chi Square Goodness of Fit (One Sample Test)

This test allows us to compare a collection of categorical data with some theoretical expected
distribution. This test is often used in genetics to compare the results of a cross with the
theoretical distribution based on genetic theory.

Materials:

 Coin
 Colored beads
 White paper
 Pencil

Procedures -1

 Toss a coin several times

 Record the output for each toss
 Count each head and tail and sum up
 Calculate the chi-square value of the toss
 Justify the hypothesis that the head and tail are independent or the event is free
from bias (fair).

Procedure-2

 Collect the beads with same color

 Make two different groups of beads (same size, but different color) in two containers.
 Label their color and size for specific genetic trait
 Draw up (pick) each beads from each container
 Pair and put on a table in a line for each consecutive event
 Count the paired beads which represents two trait of the offspring formed

11
  Find and count the number of phenotypic classes
 Calculate the chi-square value for each phenotypic classes
 Justify the trial is acceptable or not

Suppose you preformed a simple monohybrid crosses between two individuals that were
heterozygous for the trait of interest.

Aa x Aa

Table- 2. Shows the results of monohybrid cross between two heterozygous for the “A” and “a”
gene.

A a Totals
A 10 42 52
a 33 15 48
Totals 43 57 100

The phenotypic ratio 85 of the A type and 15 of the a-type (homozygous recessive). In a
monohybrid cross between two heterozygotes, however, we would have predicted a 3:1 ratio of
phenotypes. In other words, we would have expected to get 75 A-type and 25 a-type. Are or
results different?

Calculate the chi square statistic X2 by completing the following steps:

1. For each observed number in the table subtract the corresponding expected number (O —
E).
2. Square the difference [(O —E) 2].
3. Divide the squares obtained for each cell in the table by the expected number for that cell
[(O - E) 2 / E].

12
 4. Sum all the values for (O - E) 2 / E. This is the chi square statistic.

For our example, the calculation would be:

Observed Expected (O (O — E)2 (O — E)2/ E

—
E)

A-type 85 75 10 100 1.33

a-type 15 25 10 100 4.0

Total 100 100 5.33

x2 = 5.33

We now have our chi square statistic (x2 = 5.33), our predetermined alpha level of significance
(0.05), and our degrees of freedom (df =1).

Entering the Chi square distribution table with 1 degree of freedom and reading along the row we
find our value of x2 5.33) lies between 3.841 and 5.412.

The corresponding probability is 0.05<P<0.02.

This is smaller than the conventionally accepted significance level of 0.05 or 5%, so the null
hypothesis that the two distributions are the same is rejected.

In other words, when the computed x2 statistic exceeds the critical value in the table for a 0.05
probability level, then we can reject the null hypothesis of equal distributions.

Since our x2 statistic (5.33) exceeded the critical value for 0.05 probability level (3.841) we can
reject the null hypothesis that the observed values of our cross are the same as the theoretical
distribution of a 3:1 ratio.

13
Table-3. Chi Square distribution table.

Probability level (α)

Df 0.5 0.10 0.05 0.02 0.01 0.001

1 0.455 2.706 3.841 5.412 6.635 10.827

2 1.386 4.605 5.991 7.824 9.210 13.815

3 2.366 6.251 7.815 9.837 11.345 16.268

4 3.357 7.779 9.488 11.668 13.277 18.465

5 4.351 9.236 11.070 13.388 15.086 20.517

Chi Square Test of Independence

For a contingency table that has r rows and c columns, the chi square test can be thought of as a
test of independence.

In a test of independence the null and alternative hypotheses are:

HO: The two categorical variables are independent.

HA: The two categorical variables are related.

We can use the equation Chi Square = the sum of all the (FO - FE) 2 / FE or

Here FO denotes the frequency of the observed data and FE is the frequency of the expected
values.

14
The general table would look something like the one below:

An important question to answer in any genetic experiment is how we can decide if our data fits
any of the Mendelian ratios.

A statistical test that can test out ratios is the Chi-Square or Goodness of Fit test.

Laboratory Four

15
 Karyotyping
Objectives
Students are able to:
 Define karyotyping
 Explain the advantages of karyotyping
 Exercise karyotyping

Introduction
Karyotyping is the process by which geneticists take pictures of the chromosomes while the cell
is undergoing mitosis. The picture is then enlarged. The picture of the chromosomes is then cut
up so that each chromosome is removed. The chromosomes are matched up and attached to a
paper according to size. The chromosomes pairs are numbered from largest to smallest. The
somatic chromosomes match up firstly. Then the sex chromosomes are paired, lastly.
Centromere positions: Each chromosome has two arms, labeled p (the shorter of the two) and q
(the longer). The p arm is named for "petit" meaning 'small'; the q arm is named q simply
because it follows p in the alphabet.
Metacentric: A chromosome is metacentric if its two arms are roughly equal in length.
Acrocentric: If the p (short) arm is so short that it is hard to observe, but still present, then the
chromosome is acrocentric
Telocentric: A telocentric chromosome's centromere is located at the terminal end of the
chromosome.
Holocentric: With Holocentric chromosomes, the entire length of the chromosome acts as the
centromere.
Materials:
 Scissor or razorblade
 Glue
 White paper
 Chromosomes
 Pencil
 Board

16
Procedure:

 Obtain a set of chromosomes

 Select chromosomes which have same size and centromere position
 Cut out the single chromosomes on board using razorblade or scissor turn by turn
 Match the chromosomes with their homologous mate using glue.
 Number the largest pair and then to the smallest pair until sex chromosomes

INFORMAL LAB REPORT

1. Explain what the word cytogenetic means.

2. Briefly discuss the chromosome abnormality in the karyotype. Be sure to include
the type of abnormality, the total number of chromosomes in the karyotype, the
specific chromosome involved, the gender of karyotype, and the reason for the
gender.
3. Using your knowledge of meiosis discuss how the chromosome abnormality
could have occurred.
4. After comparing disorders, which of the chromosome disorders seems to be the
most damaging? Least? Explain giving an example. Why do you think that there
was only one monosomy example?
5. Karyotyping can be used as a prenatal screening tool. Is a baby guaranteed from
genetic disorders if the chromosome configuration appears normal? Explain.

Lab # 5

17
 Title Blood Typing

Objective

 To determine blood group

Introduction:- Blood is a specialized bodily fluid in animals that delivers necessary substances
such as nutrients and oxygen to the cells and transports metabolic waste products away from
those same cells.In vertebrates, it is composed of blood cells suspended in a liquid called blood
plasma. Plasma, which constitutes 55% of blood fluid, is mostly water (92% by volume), and
contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the
main medium for excretory product transportation), platelet and blood cells themselves.

Almost always, an individual has the same blood group for life, but very rarely an individual's
blood type changes through addition or suppression of an antigen in infection, malignancy, or
autoimmune disease. Another more common cause in blood type change is a bone marrow
transplant. Bone-marrow transplants are performed for many leukemias and lymphomas, among
other diseases. If a person receives bone marrow from someone who is a different ABO type
(e.g., a type A patient receives a type O bone marrow), the patient's blood type will eventually
convert to the donor's type.

A blood type (also called a blood group) is a classification of blood based on the presence or
absence of inherited antigenic substances on the surface of red blood cells (RBCs). These
antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood
group system. Some of these antigens are also present on the surface of other types of cells of
various tissues. Several of these red blood cell surface antigens can stem from one allele (or very
closely linked genes) and collectively form a blood group system. Blood types are inherited and
represent contributions from both parents. A total of 30 human blood group systems are now
recognized by the International Society of Blood Transfusion (ISBT).

Many pregnant women carry a fetus with a blood type different from their own, and the mother
can form antibodies against fetal RBCs. Sometimes these maternal antibodies are IgG, a small
immunoglobulin, which can cross the placenta and cause hemolysis of fetal RBCs, which in turn

18
can lead to hemolytic disease of the newborn, an illness of low fetal blood counts that ranges
from mild to severe.

Based on the presence or absence of antigen and antibodies, human blood is classified into 4
groups A, B, AB, and O.
 The A group contains antigen A and antibody B.
 The B group contains antigen B and antibody A.
 The AB group contains both antigens A and B and no antibodies.
 The O group contains no antigen and both antibodies A and B.
 The ABO blood group is inherited by a set of multiple alleles.
Presence of a particular factor is denoted by Rh factor discovered by Weiner from the rabbits
immunized with the blood of the Macaca rhesus monkey

Materials
 Blood sample
 Applicator sticks
 Antisera A, B, and Rh
 Glass slide
 Cotton Spirit and disposable needles
Procedure
1. Clean a glass slide thoroughly.
2. Place a drop of antiserum A on the left side, antiserum B on the right side, and antiserum D at
the center of the slide.
3. Clean the tip of the index finger with cotton soaked in spirit.
4. Prick the fingertip with the help of a sharp, sterilized disposable needle.
5. Place 3 drops of blood near the antisera.
6. Mix the blood and antiserum using application sticks.
7. Observe your experimental result.

19