AP Bio Final Project- Dissection Labs

Dissection Lab

1.  Starfish

    Pre-Dissection:

 

    Pedicellariae: keep the body surface clear of algae, encrusting organisms, and other debris

    Ambulacral Spines: used to aid arm in movement

    Ambulacral Groove: aid arm in movement

    Disk: protects the stomach and other internal organs

    Mouth: used for capturing and eating food

    Spines: protect the starfish from predators

    Anus: expels fecal matter 

    Arm: used to move

    Madroporite :used to filter water into the water vascular system

    

    Post Dissection:

    Incision Guide

1. Lay the starfish with its aboral side up.

2. Cut off the tip of a ray and then remove the flap of skin to observe the two long digestive glands called the     pyloric caeca.

3. Cut a circular flap of skin from the central disc to observe the stomach.

4. Remove the pyloric caeca from the dissected ray to observe the gonads.

5. Cut off the tip of a ray to observe the parts of the tube feet.

6. Locate the bulb-like top of a tube foot called the ampulla. This sac works like the top of an eyedropper to     create suction. The bottom of the tube foot is a sucker. 

7. Embedded in the soft body wall are skeletal plates called ossicles.

8. Running down the center of each arm is a lateral canal to which tube feet are attached. 

9. In the central disc the five lateral canals connect to a circular canal called the ring canal. 

10. A short, canal called the stone canal leads from the ring canal to the madreporite where water enters.

    Dissection Video
http://youtu.be/fBDWEHzKHS8

2. Clam

    Pre-Dissection:

Growth Rings- show how old the clam is
Exhalent Siphon- allows water flow for locomotion, feeding, respiration, and reproduction
Umbo- hinge that allows for the opening and closing of the clam

   Post Dissection: 

    Incision Guide

1. Locate the umbo, the bump at the anterior end of the valve. This is the oldest part of the clam shell. Find       the hinge ligament which hinges the valves together and observe the growth rings.

2. Turn the calm with its dorsal side down and insert a screwdriver between the ventral edges of the valves.

3. Turn the screwdriver so that the valves are about a centimeter apart. Leave the tip of the screwdriver              between the valves and place the clam in the pan with the left valve up.

4. Locate the adductor muscles. With your blade pointing toward the dorsal edge, slide your scalpel                 between the upper valve & the top tissue layer. Cut down through the anterior adductor muscle, cutting as     close to the shell as possible.

5. Repeat step 4 in cutting the posterior adductor muscle.

6. Bend the left valve back so it lies flat in the tray.

7. Examine the inner dorsal edges of both valves near the umbo and locate the toothlike projections. Close       the valves & notice how the toothlike projections interlock.

8. Locate the muscle "scars" on the inner surface of the left valve. The adductor muscles were attached here       to hold the clam closed.

9. Identify the mantle, the tissue that lines both valves & covers the soft body of the clam. Find the mantle         cavity, the space inside the mantle.

10. Locate two openings on the posterior end of the clam. The more ventral opening is the incurrent siphon         that carries water into the clam and the more dorsal opening is the excurrent siphon where wastes &             water leave.

11. With scissors, carefully cut away the half of the mantle that lined the left valve. After removing this part            of the mantle, you can see the gills, respiratory structures.

12. Observe the muscular foot of the clam, which is ventral to the gills. Note the hatchet shape of the foot            used to burrow into mud or sand.

13. Locate the palps, flaplike structures that surround & guide food into the clam's mouth. The palps are             anterior to the gills & ventral to the anterior adductor muscle. Beneath the palps, find the mouth.

14. With scissors, cut off the ventral portion of the foot. Use the scalpel to carefully cut the muscle at the top       of the foot into right and left halves.

15. Carefully peel away the muscle layer to view the internal organs.

16. Locate the spongy, yellowish reproductive organs.

17. Ventral to the umbo, find the digestive gland, a greenish structure that surrounds the stomach.

18. Locate the long, coiled intestine extending from the stomach.

19. Follow the intestine through the calm. Find the area near the dorsal surface  that the intestine passes               through called the pericardial area. Find the clam's heart in this area.

20. Continue following the intestine toward the posterior end of the clam. Find the anus just behind the                 posterior adductor muscle.

21. Use your probe to trace the path of food & wastes from the incurrent siphon through the clam to the             excurrent siphon.

    Dissection Video

http://youtu.be/kN-yzS47HFs

3. Grasshopper

    Pre-Dissection:

    Post Dissection:

    Incision Guide

1.Using forceps, remove each of the appendages from the head.

2. Examine the following appendages on the thorax.

3. Using forceps, remove one of the walking legs and identify these parts --- the coxa connects the femur          (the thickest part of the leg) to the grasshopper's body; a slender, spiny tibia connects the femur to the           tarsal segments (lowest part of the leg).

4. Remove a jumping leg.

5. Raise both pairs of wings and locate the first abdominal segment.

6. Locate the tympanic membrane or eardrum on the first abdominal segment.

7. Using a magnifying glass, locate the spiracles or tiny pores for respiration on each side of the abdominal         segments.

8. Determine if your grasshopper is a male or female by looking at the end of the abdomen. Females have a       tapered abdomen that ends in a pointed egg laying tube called the ovipositor. Male have a more rounded       abdomen that turns upward.

    Dissection Video
http://youtu.be/3sCpXPAYYxE

4.Crayfish

    Pre-Dissection:

   Post Dissection:

    Incision Guide

1. Place a crayfish on its side in a dissection tray. Use the diagram below to locate the cephalothorax and the    abdomen. The carapace, a shield of chitin, covers the dorsal surface of the cephalothorax. On the                  carapace, observe an indentation, the cervical groove, that extends across the midregion and separates the    head and thoracic regions. On the thoracic region, locate the prominent suture or indentation on the                cephalothorax that defines a central area separate from the sides. Note the individual segments of the            abdomen.

2. Turn the crayfish with its DORSAL side upward, and locate the rostrum, which is the pointed extension of    the carapace at the head of the animal shown in the diagram above. Beneath the rostrum locate the two          eyes. Notice that each eye is at the end of a stalk.

3. Locate the five pairs of appendages on the head region. First locate the antennules in the most anterior           segment. Behind them observe the much longer pair of antennae.

4. Locate the mouth. Then observe the mandibles, or true jaws, behind the antennae. Now locate the two         pairs of maxillae, which are the last appendages in the cephalic region.

5. On the thoracic portion of the cephalothorax, observe the three pointed maxillipeds.

6. Next observe the largest prominent pair of appendages, the chelipeds, or claws. Behind the chelipeds           locate the four pairs of walking legs, one pair on each segment.

7. Now use the walking legs to determine the sex of your specimen. Locate the base  segment of each pair      of walking legs. The base segment is where the leg attaches to the body. Use a magnifying glass to study        the inside surface of the base segment of the third pair of walking legs. If you observe a crescent-shaped       slit, you have located a genital pore of a female. In a male, the sperm duct openings are on the base                segment of the fourth pair of walking legs. Use a magnifying glass to observe the opening of a  genital pore.

8. On the abdomen, observe the six distinct segments. On each of the first five segments, observe a pair of         swimmerets.

9. On the last abdominal segment, observe a pair of pointed appendages modified into a pair of uropods. In       the middle of the uropods, locate the triangular-shaped telson.

10. Now turn the crayfish ventral side up. Observe the location of each pair of appendages from the ventral         side.

11. Remove all jointed appendages of the crayfish.

12. Using one hand to hold the crayfish dorsal side up in the dissecting tray, use scissors to carefully cut              through the back of the carapace. Cut along the indentations that separate the thoracic portion of the              carapace into three regions. Start the cut at the posterior edges of the carapace, and extend it along both        sides in the cephalic region.

13. Use forceps to carefully lift away the carapace. Be careful not to pull the carapace away too quickly.             Such action would disturb or tear the underlying structures.

14. Place the specimen on its side, with the head facing left. Using scissors, start cutting at the base of cut             line. Extend the cut line forward toward the rostrum (at the top of the head).

15. Use forceps to carefully lift away the remaining parts of the carapace, exposing the underlying gills and           other organs.

16. Locate the maxillae that pass the pieces of food into the mouth. The food travels down the short                  esophagus into the stomach. Locate the digestive gland, which produces digestive substances and from         which the absorption of nutrients occurs. Undigested material passes into the intestine. Observe that the          intestine is attached to the lobed stomach. The undigested material is eliminated from the anus.

    Dissection Video
http://youtu.be/idGeyRXd0OU

pGLO Lab

PURPOSE 

   The purpose of this lab was to help us understand genetic transformation because moving genes from one organism to another through help of a plasmid. We did genetic transformation in order to determine the rate of a plasmid carrying a gene that transforms into E. Coli. 

INTRODUCTION

Genes are changed by genetic transformation because a certain gene is placed into an organism. Antibiotic ampicillin treats certain bacterial, but pGLO is resistant to it. When sugar arabinose is in the cell it allows GFP to be activated in transformed cells. We hypothesized that the bacteria will gloom the tray with the ampicillin and they will not glow on the one without arabinose. Arabinose will turn on the glowing expression. 

METHOD

1) First things first, we grabbed two micro test tubes, labeled one +pGLO and the other -pGLO, and then four LB nutrient agar plates and labeled them as shown below.

2) Using a sterile transfer pipet, we dropped 250µL of transformation fluid (CaCl2) into each tube and then put them on ice.

3) Using a sterile loop, we took one colony of E-coli from a starter plate and put it into the +pGLO tube. We then did the same for -pGLO but using a new loop.

4) Using yet another new loop, we transfered pGLO plasmid DNA into the +pGLO tube.

5)The two tubes were then kept on ice for ten minutes, followed by a 50sec heat shock delivered in a water bath set to 42 degrees Celsius, followed by yet another ice incubating period for two minutes.

Ice incubation

Heat shock

6) Finally, we added 250µL of broth to each test tube and then using a pipet we pipetted 100µL of the test tubes’ contents onto the appropriate nutrient agar plates.

 

All packed up until the next day!

DATA

Aaaaaand our experiment failed. After being exposed to ampicillin, all of our E-coli died. Luckily for us, we were able to get our hands on the another groups last two agar plates to show what the results should have been. 

DISCUSSION

   The results of the two control plates were as expected. The untransformed E. coli prospered in the agar without ampicillin, covering the surface of the plate almost entirely, and was eradicated from the agar with ampicillin. Both of the experimental plates contained ampicillin, which was meant to select against untransformed E. coli and leave a few colonies of E. coli that had been transformed by pGLO, while the fourth and final plate also included arabinose to make the transformed E. coli a fluorescent green under UV light. The expected result would have been several isolated colonies of nonfluorescent transformed E. coli on the first experimental plate and a similar number of colonies of fluorescent, ampicillin-resistant E. coli on the second, as observed in the agar plates from the other group's lab. The other group was found to have 30 colonies of transformed fluorescent E. coli, which was calculated to be a transformation efficiency of 191.25 transformants per microgram of pGLO DNA spread on the plate. However, as all E. coli was eradicated from both plates in this experiment, it can be concluded that the transformation failed. The process produced zero colonies and a transformation efficiency of zero. The gene for ampicillin resistance from the pGLO plasmid was not incorporated into the E. coli's DNA, so the E. coli on the agar plates containing ampicillin was killed.

     Errors may have occurred at many steps along the way. Both control plates resulted as expected, so the error was probably not in the E. coli or the agar plates. Also, several other groups used the same pGLO and had successful transformations, so the chances of having faulty pGLO are also low. Thus the most likely option is that the E. coli underwent too much heat shock, allowing its proteins to denature irrevocably, or too little heat shock, preventing the pGLO genes from bypassing the cell membranes. The problem may also have occurred immediately following the heat shock if the tubes had not been in close enough contact if the ice to allow the cell membranes to reform. As other groups were able to transform bacteria with a higher efficiency using the same materials and procedure, to improve this experiment would be a simple matter of following the procedure and the timing of the steps more precisely.

CONCLUSION

   The experiment was ultimately unsuccessful in transforming E. coli into a fluorescent, ampicillin-resistant bacteria. The transformation efficiency was zero.