1. Organelles Ordered from Ward Science Organelles Lab Activity #36V3505 Presented by Deb Semmler St. Joseph’s High School dsemmler@saintjoehigh.com

2. The kit The kit provides an introduction to subcellular components (organelles) and internal cell membranes suitable for life science or biology courses. It provides materials for 15 groups.

3. Contents of the kit: Microscope slides (72) Cover slips (100) Forceps (6) Razor blades (15) Plastic cups (15) Janus Green B Stain ( 30 ml) Dialysis Tubing (4 m) Hydrogen peroxide solution (240 ml) Twist ties (60) Teacher and student manuals

4. Additional required material Fresh celery (4-5 stalks) Potato Microscopes funnels

5. Overview of the labs in kit to observe organelles in plant cells to simulate the function of an organelle and organelle membrane with the help of Janus Green B stain, to observe an organelle (mitochondrion) in action

6. Advanced teacher preparation For the first activity, fresh celery stalks will be used. These should be pre-cut into 4-5 cm. length pieces. For the second activity, the students need a source of fresh potato. The teacher should prepare a potato ‘slushie’ by putting small chunks of potato into a food processor with a small amount of crushed ice. Make sure mixture is fairly liquid (add more water, if necessary). 4-5 small potatoes is more than enough for a class of 30. Store in the refrigerator until needed. The dialysis tubing needs to be cut into 18- 20 cm. segments. The last activity also requires 4-5 cm. length pieces of fresh celery.

7. First Activity Uses a microscope slide of living plant tissue for a general observation of organelles within cells. The preparation of the sample for viewing requires some patience. The sample should be as thin as possible.

8. How to cut the celery Take the small piece and place it on the slide with a drop of water. Add a coverslip and observe under high power.

9. What will you see The nuclei of the cells, as well as the chloroplasts, which are a bright green color, and several clear, elliptical bodies, the mitochondria, should be clearly visible under high power. The rest are very difficult to see. Students are instructed to draw and label what they see.

10. Second activity This activity sets up a model to simulate organelle activity. The dialysis tubing, which allows small molecules to pass through, will simulate an organelle membrane. The blended potato provides a source of enzymes such as would be present within an organelle.

11. Enzymes First enzyme: catalase- catalyzes the following reaction: 2 H 2 O 2 → O 2 ↑ + H 2 O The hydrogen peroxide simulates the raw materials which an organism would acquire. The enzyme, catalase, within the semi-permeable membrane of dialysis tubing, processes this raw material, and the final products are water and oxygen. The water can pass through the tubing, but not the oxygen. It builds up in the tubing causing it to swell.

12. Secondary reaction The second enzyme in the potato is catacholase, which, in the presence of oxygen, catalyzes the formation of the pigment melanin, which has a characteristic brown or pinkish brown color. As the first reaction releases oxygen, the second enzyme begins to work, turning the potato extract within the sack a pinkish brown color within a few minutes. This secondary reaction represents a side reaction within an organelle, producing a by-product that might be detrimental to the organelle.

13. Second activity set-up Pour some tap water into the plastic cup. Soak the dialysis tubing in the water for about two minutes. After it has soaked, wrap a twist tie around the tubing about 1” from the end and twist it tightly. Open the other end of the tubing by gently rolling the tubing between your fingers. Don’t’ puncture the tube- handle carefully. Place a funnel in the open end and add about 3-4” of potato mixture. Gently squeeze it up into the tube until its about 1” from the end, getting out as much air as possible. Carefully add a twist tie to the opposite end. The tube should be rather limp.

14. continued Empty the plastic cup and pour about 1/3” of hydrogen peroxide into the bottom. Add enough tap water to fill the cup to a little over ½ full. Describe the water/hydrogen peroxide mixture. Place the potato tube into the cup of hydrogen peroxide and water and observe for 5-10 minutes. Record your observations.

15. Disposal After about 10 minutes, the reaction should have slowed down. If the membrane has become very full with oxygen, be careful undoing the twist tie (it’s best to open it over a sink). Contents can go down the drain. Tubing should be collected for disposal.

16. Comments Students should check tubing after 5 minutes, to make sure tubing isn’t too expanded as it can pop. The tubing can be reused if it doesn’t have any holes in it. It should be washed gently in warm water and soaked in warm water for a while.

17. Activity discussion Semi-permeable tubing will allow one-way flow of materials, simulating both the intake of raw materials and the retention of enzyme by an organelle. The enzyme in the ‘organelle’ processes the raw material and produces a definite product. Although the oxygen is retained, it will eventually be released by diffusion, as are the products of real organelles. Side reactions will occur in organelles, just as the side reaction with melanin shows. The production of melanin simulates the accumulation of waste products and demonstrates one reason why real organelles have a fixed lifetime and must eventually be replaced.

18. Third Activity Students will observe the mitochondria at work, since it is possible to satin this organelle in such a way as to demonstrate chemical activity. The stain used is Janus Green B which is a greenish blue under normal conditions. Removal of a pair of hydrogen atoms causes loss of color. During the normal activity of mitochondria, an enzyme- dehydrogenase- catalyzes the removal of hydrogen atoms from substrate molecules. By staining the celery molecules with Janus Green B, it is possible to watch this enzymatic reaction.

19. Procedure Obtain another piece of celery and prepare a slide like the one in activity one. Observe it under the microscope under high power. While it is still under the scope, get some Janus Green B stain and a paper towel. Place a drop or two of stain next to the coverslip and place the paper towel on the other side of the cover slip. Observe the cell being stained as the paper towel draws the stain under the coverslip. The stain will be drawn into the mitochondria and turn them greenish blue. If the dye looses hydrogen, however, the dye loses color, showing what mitochondria does to food (sugar) molecules that enter the organelle. Observe the slide for several minutes and note any changes over time.

20. Lab Evaluation First activity- – Using the celery as a source for viewing organelles isn’t necessarily the best. Although you can see the nucleus and can somewhat see the chloroplasts, dyed onion cells show nuclei better and fresh elodea is a better source for viewing chloroplasts. The mitochondria was barely visible if you knew what you were looking for. I would probably still use onion and elodea for identification, but it’s nice to know there are other things that can be used.

21. Second Activity: dialysis tubing This worked great. Not only did the dialysis tubing expand when oxygen was generated, but it also changed color showing the secondary reaction. – Draw-backs- it was a bit tough to get the ‘slushie’ into the tubing but by cutting off the end of a disposable plastic pipet to make a larger intake, the material was easier to handle by piping it into the tubing. – Note: If the potato ‘slushie’ could be made right before the class, it would be more visual. After about an hour, the potato turns brown and there isn’t as much of a color change.

22. Third Activity: staining with Janus Green B This activity had mixed results. The Janus Green did indeed stain the mitochondria green, but it also stained many of the other organelles green as well so it was hard to point out which was the mitochondria at first. However, after a few minutes, the mitochondria did change back to clear while the others remained green, so that helped.

23. Final Comments: I did like the dialysis experiment a lot, so the kit was worth purchasing to get that information. Other than that there was nothing special about the kit with the exception of the Janus Green B stain that you can pick up at most chemical supply companies. I would, however, still use onion tissue and elodea tissue when trying to show actual organelles.