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Fill the other 3 about 2/3 full with 0. 2% Enhance solution. Set these aside for later. 2. Pour about 100 ml of 0. 2% Enhance solution into a flask with a side arm. 3. Get three or four spinach leaves and cut 40-50 disks with a cork borer. Do not include the large veins in the disks that you make. Stack the leaves so you can cut through them all and cut 3 or 4 disks at once. Use a glass rod or a straw to push the disks out of the cork borer into the flask containing Enhance solution. Take the disks in the flask to your instructor, who will use a vacuum pump to draw the air out of the disks.

As the flask is evacuated, you will see the water bubble and you may see bubbles coming out of the edges of the disks. Evacuate for about 30 seconds, then stop to see if the disks sink to the bottom of the flask. If they do not sink, continue to apply the vacuum. It is not necessary to sink all of the disks. The tissue will be damaged if it is over- aspirated. 5 Take the flask of disks back to your bench and pour its contents into large dish. Discard any disks that are still floating (in the wastebasket, not the sink).

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With forceps, gently transfer 10-15 disks to each of the 4 Petri dishes. Spread the disks apart so that they are not resting on each other and replace their lids. Label them with your name and place as follows: 1 – on the plant stand as close as possible to the bright light (bright light – low CO) 2 – next to 1 on the plant stand (bright light – high CO) 3 – on your lab table under normal room light (low light – high CO) 4 – inside a beanie or drawer in complete darkness (no light – high CO) Results: The Petri dishes placed in the bright light both had 100% of the disks floating.

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