BIO1140 Lab 4: Mitosis

Objectives of the lab 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 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

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

Mitosis Lab activities Staining and preparation of 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

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

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 put 1 root tip on slide (don’t let it dry) Using a razor blade, cut off last 2 mm (deeply stained) discard the rest Add 1 drop of acetic acid to microscope slide Add coverslip

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. www.npc.edu/Bio105/media/m1_l6-05.gif

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

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

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

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 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)

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 1 2 3 4 5 X Axis: Stages of the cell cycle 3 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)