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Mitosis: Onion Root Smash! Sure, you know your birthday. You've huffed and puffed harder every year to blow out the candles on your special day. But do you know your physical age? Seventeen, you say? Look at your arm; nothing you see there is 17 years old. Those skin cells are only 2 to 4 weeks old. Had a paper cut lately? The blood that oozed out of that wound was probably no more than 4 months old. The cells that line your stomach bail out only 5 days after that greasy cheeseburger combo meal you devoured. Your liver cells are a little hardier, they hang around for a year and a half or so. Only a few types of cells in your body are lifetime buddies. In fact, the billion-plus cells in your body regenerate, on average, every 7 years. Where do all those cells come from? Does a single organ produce all those new cells, or are you born with a supply of them? Does your brain direct cell production? When your body sleeps, are your cells inactive? No single organ produces all your cells, you're not born with ready-made replacement cells, and your brain doesn't direct all of the cell production going on 24/7 in your body. Cells are created 1 at a time as each cell divides. Mitosis, meiosis, cell division, and genetic inheritance are complex concepts. So it’s helpful to be able to better visualize them when we are able to. In this lab you will be using the root tips of onions to investigate mitosis. Why onion root tip cells? Because the root tip is a fast growth area of the onion plant, cells are rapidly dividing, Also, the cells are large, so they are relatively easy to see, and the 16 chromosomes stain easily. Mitosis in Allium root tip cells is not synchronized. You can see various phases of mitosis and many intermediate configurations. Part 1: Visualizing Mitosis Hypothesis: Based on the information you have about the cell cycle and mitosis, predict where a cell would spend most of its time, justify your prediction. 2 cell DK Ly Speeds ke. oP e tips in Setectias, ting 2 hoe ohle setts tect db ttde often Procedure *Note: Root tip smashing is not an easy bed 5 Figure 2a. Onion root tip anatomy. Only the cells at technique to master. Don’t get discouraged if you the very tip of the root (Zone of Cell Division) are don’t visualize mitosis occurring. We have backup undergoing mitosis. These are visually distinct ina pre-mounted slides if we need to use them. fresh root tip, appearing more round or square than the elongated cells in the Zone of Elongation above it. 1. Cut three roots from an actively growing plant using a scalpel. Caution. The scalpel is extremely sharp. 2. Trim the tip of each root to 1 cm; use only the tapered end of the root up. 3. Use forceps to place 2-3 root tips (use only the 1-cm tips) ona glass microscope slide. 4. Place 2-3 drops of 1 M hydrochloric acid to cover the root tips on the microscope slide. 5. Light a match and heat the BOTTOM of the slide for approximately 5 seconds. 2 6. Allow the root tips to soak in the acid for 5 minutes. Scanned with CamScanner | 7. After 5 minutes, use a paper towel and carefully blot away excess hydrochloric acid from the slide. Cautior Avoid contact of the acid with skin. 8. Place 2-3 drops of deionized water on the root tips. Use a paper towel to blot away excess water. 9. Repeat steps 7 and 8. q 10. Add 1 drop of methylene blue stain to the root tip. Note: Methylene blue stain is a permanent stain. q 11. Allow the root tips to soak in the stain for 3 minutes. Use a paper towel to blot away excess methylene blue stain. 12. Add 1 drop of deionized water to the root tips. 13. Use forceps to move one root tip to a clean microscope slide. 14. Place a cover slip on the root tissue. Using the eraser end of a pencil, gently apply pressure on the cover slip to squash the root tissue. Apply an even downward pressure on the root tips and cover slip but not so hard as to break the cover slip. Do not twist or grind the coverslip. 15. Using low magnification on the microscope, focus on the root cells. Switch to medium power or high power as necessary to easily visualize the inside of the onion root cells. 16. Study all of the squashed tissue to locate cells in each stage of the cell cycle. Note: All stages of mitosis may not be present within a single field of view. 17. Repeat steps 14-17 using the remaining two root tips. Analysis 1. Find a cell in each phase of mitosis. Draw and label (including phase name) what you see. Remember the rules for making scientific drawings! | ives mus be Earellet Mucleoles 1) Nedleus Nucleas Lhrercsenes)—— threrserenes Nucleokess Dasha gargs lenly tote Naclews 2. Estimate the time spent in each phase: a. Count 150 cells in the field of view. For each cell, note the stage of the cell cycle/mitosis that you see. You may have to use multiple fields of view. Be sure you do not duplicate fields of view in your count. Determine the percentage of cells in each stage. Do this for 3 different root tips, then take the average. d. Using the average, estimate the time spent during each stage of the cell cycle if a normal cell cycle takes 13 hours to divide. Here is an example. is) If ten cells out of 150 were found to be in prophase, then percentage of cells is 10/150 x 100 = 6.7% This shows that any one of the hypothetical cells spends 6.7% of the time in prophase or... 0.067 x 13 hours (780 minutes) = 067 x 780 = 52 minutes spent in prophase. Scanned with | CamScanner’: 3. a. Click on Stem Cell Quick Reference. Complete the table below. Embryonic Stem Cells Possibilities of stem cells/therapies Considerations in treatment/research Somatic (adult) Stem Cells type |a the kody lould produce any cell leulé be rejected due te beiny foreign ond tne feeds Sheald be eveated te nef rely on embrye destyuctter Induced Pluripotent Stem Cells fer patients. lealé help fre duce Uecd Trarsflants Free clners tould be rejected L aequiving them trem the subject 1s both fithy £ afticul? Could allow fey ethial easily harvestable reploce Sofely must be entered due to gene meditication i r heats for eebryente (il bet § ¢ tater treatment Therapeutic Cloning lould Fepslt any 2resn Ts tmereditty expensive pI ny. 
