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)