1. BIO1140 Lab 4: Mitosis

2. Lab Objectives Visualize DNA following staining procedure
Identify and compare stages of the cell cycle using plant and animal tissue
Determine relative time cells spend in each stage of the cell cycle

3. Phases of mitosis Recorded as Prophase
No chromosome visible nucleolus present
chromosomes visible
Nucleolus disappeared
chromosomes visible
nuclear membrane disintegrated
Recorded as Prophase
Copyright © 2008 Pearson Cummings. All rights reserved

4. Phases of mitosis Chromosomes align
on the equator plate
sister chromatids separate and start migrating toward pole
Chromosomes unfold and new nuclear membrane forms around new nuclei
cytoplasm starts to divide
daughter cells split and enter interphase
Copyright © 2008 Pearson Cummings. All rights reserved

5. Mitosis Lab activities
Staining and squash of broad bean (Vicia faba) root tip.
Identification and count of mitotic stages on various samples:
Whitefish (Coregonus clupeaformis) blastula
onion (Allium cepa) root tip
broad brean (Vicia faba) root squash
Collect class data

6. Vicia faba (broad bean) root squash
Material Provided:
Root tips fixed in Carnoy-lebrun fixative (stops cell processes and preserves tissues).
Step by step procedure described in lab manual:
Step 1: Label microtube with your name before starting staining procedure.
Step 2: Remove Fixative using plastic pipette
Dispose of fixative in chemical waste bucket
Use toothpick to hold in your sample if necessary
DO NOT DISPOSE OF ANY LIQUID IN THE DRAIN USE THE LIQUID WASTE BEAKERS

7. Vicia faba (broad bean) root squash
Step 3: Washes
Rinse tips once in 95% alcohol then discard alcohol using plastic pipette
Step 4: Hydrolysis
Add 1N hot HCl to fixed roots.
Incubate at 60°C for 10 minutes precisely.
Discard HCl then GENTLY add ice-cold distilled H20 (stops hydrolysis)

8. Vicia faba root squash Step 5: Staining Discard water
Add Feulgen stain (be extremely careful it stains virtually anything instantly)
Stain for minutes or until root tip is red (observe first prepared slide in the meantime: either onion root or whitefish embryo)
Remove staining solution using plastic pipette and rinse with water 2-3 times (caution with rinsing water  it still can stain)
Keep stained roots in water so that they won’t dry out

9. Vicia faba root squash Step 6: Preparing the root tip squash
Add 1 drop of acetic acid to microscope slide
Add 1 root tip
Using a razor blade, cut off last 2 mm (deeply stained) discard the rest
Add coverslip

10. Vicia faba root squash Step 7: Squash
Apply direct downward pressure – No twisting, banging, striking etc!!!
Press hard
Check under Microscope then press again if needed.

11. Evaluation of squash Show your preparation to your TA
Present one picture of your squash to TA for evaluation (light, colour balance, focus…..)
Choose your best shot!

12. Counting cell stages
Under the 40X objective, count the # of cells in each stage of the cell cycle – count at least 50 cells (take a snapshot at 40x then count on screen).
Repeat count twice (or until you counted at least 150 cells).
Record results in lab manual table and then in lab website (Lab4 page).
Start with one prepared slide, then your squash, then the second prepared slide

13. Onion (Allium cepa) root tip (40 min.)
faculty.clintoncc.suny.edu/ faculty/Michael.Gr...

14. Whitefish (Coregonus clupeaformis) blastula (30 minutes)
CAUTION – SLIDES ARE THICK – USE FINE FOCUS
biopsy/images/mitosis.jpg

15. Lab 4 evaluation Prelab quiz
Technical skills count for 20% of this lab. You will be evaluated on:
Cleanliness
Microscope skills
Ability to produce a good quality squash (= not too thick, cells not damaged…)
Quality of picture presented to Tas
Lab report
Remember:
Liquids in Liquid Waste Beakers
Stained slides in broken glass containers
Protect your eyes: Always wear goggles!!

16. Lab 4 Report Read instructions on lab web site Report: 3 Pages
Title Page
A graph (two panels) presenting the % of cells in each of the cell cycle phases, for all 3 different organisms.
Upper Panel: Combined data from the class (average + standard error: posted on lab website)
Lower Panel: Your own measurements (calculate the % of cells in each stage from your data)

17. Don’t forget to add a caption below the graph
Multi panel graph instructions:
Stack two graphs one on top of the other. Follow the same rules as for a one-panel graph except:
Two panels share a common Y axis
X axis on both panels with tick marks
X-axis label is only present on lower panel
Same scale on Y axis
Symbol key
Upper panel: class data
(average PLUS standard error)
series 1
series 2
series 3 1 1 2 3
Percentage of cells
Y axis 2 4
Lower Panel group data
THIS IS NOT A
PERFECT GRAPH
JUST AN EXAMPLE
OF 2-PANEL GRAPH LAYOUT
Don’t forget the caption
X Axis: Stages of the cell cycle 3
Don’t forget to add a caption below the graph

18. Lab4 report Page 3: Conclusion about graphed data
Brief comparison of data: between organisms and group vs. class.
Answer the question: based on your observations during the lab, determine the relative length of each phase of the cell cycle.
Page 3: max. 2/3 page long (1.5 space, 12 points font = 15 lines max).

19. Lab4 report (cont’d)
Combined DATA is available on the Lab4 page of the lab website
DUE DATE: ONE WEEK FROM TODAY
All lab4 samples are exam material (= you must be able to recognize a mitosis phase on a picture)