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Bio 1140 Lab 4

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Bio 1140 Lab 4

Uploaded by

carmen.alsh01
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© © All Rights Reserved
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Bio 1140 Lab 4 – Mitosis

Preparation of a root tip squash.

General Instructions:
Use the plastic pipette to transfer all liquids.
Put the transfer pipette on double layer of brown paper when you're not
using it (not directly on bench).
Use only enough staining solution to cover the root tips (do not fill the
microtube).
Use a lot of water (fill the 3/4 of the tube) for the washes.
Discard all liquids in waste beaker located on each bench.
Keep the stained roots in water so that they won’t dry.
Keep your fingers away from Feulgen stain especially if bright pink is not
your favourite colour.
You must wear safety goggles during all steps of the preparation of root tip
squash.

Protocol:

1. Write your name or station number on the microtube containing the broad
bean root tips in Carnoy Lebrun fixative.

2. Using a plastic pipette, remove the fixative and wash the root tips once in
95% alcohol. Then, remove the alcohol.

3. Add hot 1N HCl then place microtube in tube rack in water bath at 60°C for
10 min. Timing is important.

4. Quickly discard the HCl (using the plastic pipette) and gently
add cold distilled water to stop the hydrolysis.

5. Discard water and add Feulgen stain (1-2 drops, just enough to submerge
the sample).
Leave roots in stain for about 30 minutes, until the tips are deep red. If not
deep red, leave them in longer (up to 1 hour). Note that the deep colour is in
the root tip, where the cells are small and actively dividing. The zone of
elongation has much less colour.

6. Discard the staining solution in the chemical waste bucket.

7. Rinse roots three times with room temperature water (fill 3/4 of the tube
each time). Keep roots in water after rinsing them.
8. Place one stained root tip on microscope slide and using a razor blade, cut
off the last 2 mm of the deeply stained tip and discard the rest. Then, add
one drop of acetic acid on the root tip.

9. Put on a coverslip and squash by pressing with the eraser end of a pencil.
Squash until the circle of tissue is about 1 cm across. Be careful not to break
the coverslip.

10. Show squash slide to your demonstrator for technical evaluation.

11. Using the squashes you have prepared, find and take a picture of all cell
cycle stages you see.

Instructions and Submission of the Mitosis Report

Print

Mitosis Lab Tasks

1- Make observations of cell cycle phases on 2 species (count at least 150


cells per species)

2- Submit your observations (number of cells in each stage of cell cycle)


on Brightspace at the end of your lab session.

3- The day after your lab, download the combined data for your lab
section.

4- Calculate the mean (arithmetic mean) of cells in each stage and the
standard error for the class data

4- Use your data and the class data for your graph in the report.

Mitosis report Content (3 Pages):

1- Title Page: see example in appendix.

2- A two-panels graph that shows the percentage of cells in each phase of


the cell cycle, for both organisms (Onion and Broad Brean).

Upper Panel: Combined data from your section: % average + standard


error: posted on Brightspace. Download your class data from
Brightspace to get data for the graph.
Lower Panel: Your own measurements (% of cells) for both species. Use
your own Data to calculate the percentage of cells in each stage:

See example of graph in the Prelab presentation, read the appendix about
Graphing Data and Watch the Video showing how to make the 2-panel
graph in Excel. You TA will also demonstrate how to do the graph.

3- Conclusion Page : Compare the results on graph: Onion vs Broad bean


and Class data vs your own observations.
Use clear language and examples with values taken from the graph to
illustrate your conclusions.
Page 3 Length (total): 1/3-page max. (about 20 lines at 1.5-line space). It Can
be shorter.

Submit Report on Brightspace before

Format: 1 PDF file with 3 pages. Name of File:


first_last_ID_section_mitosis.pdf

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