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BIO1140 Lab 4: Mitosis
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
Phases of mitosis
Copyright © 2008 Pearson Cummings. All rights reserved
chromosomes visibleNucleolus disappeared
No chromosome visiblenucleolus present
chromosomes visiblenuclear membrane disintegrated
Recorded as Prophase
Phases of mitosis
Copyright © 2008 Pearson Cummings. All rights reserved
Chromosomes alignon the equator plate
sister chromatids separate and start migrating toward pole
Chromosomes unfold and new nuclear membrane forms around new nucleicytoplasm starts to divide
daughter cells split and enter interphase
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
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
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)
Vicia faba root squash
• Step 5: Staining• Discard water• Add Feulgen stain (be extremely careful it stains virtually
anything instantly)• Stain for 30-40 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
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
www.npc.edu/Bio105/media/m1_l6-05.gif
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.
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!
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
faculty.clintoncc.suny.edu/ faculty/Michael.Gr...
Onion (Allium cepa) root tip (40 min.)
www.karlloren.com/ biopsy/images/mitosis.jpg
CAUTION – SLIDES ARE THICK – USE FINE FOCUS
Whitefish (Coregonus clupeaformis) blastula (30 minutes)
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!!
Lab 4 Report
• Read instructions on lab web site• Report: 3 Pages
1. Title Page2. 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)
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
Upper panel: class data(average PLUS standard error)
Lower Panel group data
2
3
1 2 3 4 5
THIS IS NOT APERFECT GRAPHJUST AN EXAMPLEOF 2-PANEL GRAPH LAYOUTDon’t forget the caption
Symbol key
1
X Axis: Stages of the cell cycle
1
2
3
Y a
xis
Per
cent
age
of c
ells
series 1series 2series 3
4
Don’t forget to add a caption below the graph
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).
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)