BIO 141 (GENETICS)

LABORATORY EXERCISE I
The Physical Basis of Heredity

PART III. Mitosis in Allium cepa L.

INTRODUCTION

Genetics is the study of heredity and variation in organism (Laude et al., 1997). Heredity is
the sum of all biological processes by which biochemical, physical and behavioral traits are
transmitted from parents to offspring (Hartwell et al., 2000). The concept of heredity
encompasses the diversity of individuals within species. The answer lies in our genes that
serve as functional unit of heritable material within living cells. In cells, genetic traits that are
transmitted and form variations are carried out.

MATERIALS AND METHODS

A. Growing onion root tips

1. Pour clean water into the clean (and clear) glass/plastic jars - about three-
quarters full.
2. Carefully place the onion bulbs in the glass/plastic jars so that only the lower
surface of the onion comes in contact with the water.
3. In a case where the onions are too small, they can be supported using
toothpick/splints.
4. Let the onions rest on water for 3-4 days. Only the lower surface (base from which
roots emerge) should be in contact with water (Figure 1). Grow the roots until they
are about 2‐3 cm long

Figure 1. Growing onion root set-up

 https://www.microscopemaster.com/onion-root-tip-mitosis.html#gallery[pagegallery]/1/

B. Harvesting and fixing of root tips

Early morning is the best time to harvest onion root tips as the root tips
actively undergo cell division in the morning.
1. Cut the 1.5‐2 cm end off the root.
2. Place immediately into acetic ethanol fixative.
3. Fix for 2-24 hr before staining.
4. Store at 4°C for future use.

C. Staining and squash preparation

1. Get one onion root fixed with acetic ethanol.
2. Place the fixed root on a slide.
3. Cut the terminal 1mm of the root to remove the root cap. Discard the root cap.
Actively dividing cells will be found in the meristematic region of the root, which
follows the root cap (Figure 1).

Figure 1. The mitotic region of the root

4. Cut a very thin section of the meristematic region. Discard the rest of the root.
5. Add one drop of acetocarmine on the thin root section. Let stand for 1min.
6. Cover the stained root section with a coverslip.
7. Heat the slide gently by passing it over the flame of an alcohol lamp to
facilitate staining of the chromosomes.
8. Squash the slide with your thumb using a firm and even pressure. (Avoid
squashing with such force that the cover slip breaks or slides)
9. Observe it under a compound microscope in 10x objective. Scan and narrow
down to a region containing dividing cells and switch to 40x for a better view.
Preparation of acetic ethanol fixative
Mix 3 parts of absolute ethanol (ethyl alcohol) with 1 part glacial acetic acid
e.g. for 40mL fixative: 30mL absolute ethanol + 10mL glacial acetic acid

Preparation of 1N HCl solution

Take 8.3 ml of concentrated (37%) HCl and 91.7 ml of dH2O to make 100 ml 1N HCl.
Preparation of acetocarmine stain
1. Heat a solution of 45% acetic acid (45ml glacial acetic acid/55ml of distilled
water) to boiling.
2. Add 0.5g of Carmine and continue heating for 15-20 minutes while stirring.
3. Cool resulting solution.
4. Filter to remove any precipitate.
 BIO 141 (GENETICS)
LABORATORY EXERCISE I
The Physical Basis of Heredity

PART III. Mitosis in Allium cepa L.

WORKSHEET

Name: Simbulan, Lia Franchesca C. Date:

Course/Section: BSBIO-2B Group No.: 7

I. Place a photo of your onion set-up here.

Onion set-up at Day 0

Onion set-up with growing roots (Day 4 onwards)

II. For each of the stages of mitosis identified below, draw one onion root tip cell you
have observed under the microscope.

Prophase Metaphase

Anaphase Telophase
STUDY QUESTIONS. Answer the following questions. Refer to the given rubrics.

1. What is the significance of mitosis?

Since it influences an organism's growth and development, the mitotic

process is important because, long as it progresses normally, an organism's
growth and development would be normal. Therefore, the sequence of the
mitotic cycle should provide indications to issues related to aberrant growth
and development.

2. What is the purpose of the following chemicals used in this experiment?

a. Acetic ethanol
Treatment with acid and heat is used to break up the cellulose cell wall
allowing stain to permeate the tissue and makes it easier to squash the tissue
on a microscope slide.

b. HCl solution
The HCl helps break apart the tissue so the cells can be seen better

c. Acetocarmine stain
This stain is used to study chromosomes as it stains them in a
deep red tint without staining the cytoplasm.

Rubrics:
5 points – detailed response 3 points – adequate response 1 point – inadequate
response
REFERENCES

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272485/#:~:text=The%20decrease
%20in%20the%20mitotic,the%20total%20number%20of%20chromosomes.