Wednesday, May 28, 2014

Dissection Guides

   
Starfish
The external anatomy: 
The first thing we did was determine which side was the top and which was the bottom. The side that was more rounded and had the madreporite close to the middle was the top, and the side with an indent for the mouth was the bottom. Then, we found the axis of symmetry of the starfish. All of these features are labelled below. Other features on the exterior of the starfish are also labelled. The spines can be seen on both the top and bottom, and are used for protection. Our starfish had five arms, each with an ambulacral groove containing tube feet. These allow the starfish to move. Then, there is the central plate. This is at the center of the top side, and contains the madreporite. This is a bony spot that is also used for protection.

The internal anatomy: 
On the inside, the features that line each arm include the digestive tracks, the ambulacral ridge, and ampullae. The digestive tracts allow the starfish to process nutrients that are received from the stomach. The stomach is in the middle of the starfish, below the skin and bony madreporite. The ambulacral ridges, which link to the ring canal, transport water throughout the body of the starfish. There are also gonads at the top of the ampullae, which line the ambulacral ridges. These are like the sex organs of the starfish. 

Incision guide for dissection: 
The first step to dissect a starfish is to determine the line of symmetry. Then, cut off the end of one of the arms. Now, you can see the skin and the end of a digestive tract. Make a rectangle-shaped cutout that only pierces the skin, and you can see the top of the digestive tract. If you pin back the skin, you can see the top of the digestive tract. Upon removing the digestive tracts, you can see the ambulacral groove lined by ampullae. If you follow the groove towards the center, you can see the small gonads. The next step is to cut around the madreporite to create a circular flap of skin. When this is removed, you can see the stomach. The mouth is under the stomach.

Dissection Video:
http://m.youtube.com/watch?v=VBNOCCL4l5s

Clam
The external anatomy:
The first thing we had to do was to find the anterior and posterior sides of the clam. Next, we located the umbo, as well as the dorsal, lateral and ventral surfaces. The lines on the outside of the shell are called growth rings, and they tell us how long the clam has been alive. 

The internal anatomy:
Once the clam is opened, we can find the anterior and posterior adductor muscles. These muscles are responsible for opening and closing the clam, much like the jaw muscles of humans. Next, we find the gill. This is what the clam uses to breathe. Next to the gill is the foot, which is used to burrow into mud or sand. Under the gill and the foot we find the yellowish, spongy, reproductive organs. 

Incission guide for dissection:
The first step of dissecting a clam is to determine the anterior, posterior, ventral, and dorsal sides of the clam. Place the clam on its dorsal side and insert a screwdriver into the clam. Carefully work the screwdriver back and forth to losen the jaws. Pry the clam open and look at the internal organs. Locate the anterior and posterior adductor muscles and the gill and the foot and reproductive organs. 








Dissection Video:
http://m.youtube.com/watch?v=qfMVg3Zrrr8


Frog

The External Anatomy:
To decide whether the frog we had was a male or female we looked at the frogs hands to see if it had enlarged thumb pads or not. Our frog did, meaning that it was a male. The frogs external features on its head are its external nares, which the frog uses when it is immersed in water. Frogs normally just breathe out of their mouth, but the nares allow it to swim with their external breathing points only outside of the water. The mouth is what the frog uses for most of its breathing and eating. The tympani are the frogs structure for hearing. The eyes of a frog do not move like human eyes, their eyes bulge out from their head so much so they can see in different directions at once. The nictitating membranes are basically a third eyelid for the frog that is used for extra protection when the frog is outside of the water.
The Internal Anatomy:
Frogs have two types of teeth: vomerine and maxillary. The vomerine teeth are mostly vestigial in frogs. Their function is to hold and capture prey. The maxillary teeth are small and sharp and located near the upper jaw. Their functiom is to chew and crush prey. There maxillary teeth are not really used that often because in most cases frogs swallow their prey hole and don't chew them. Internal nares are a frogs nostrils that are used when their mouths are closed to breathe. Their tong mostly aids in grabbing their prey. The Eustachian tubes balance pressure in the frogs inner ear while it is swimming. The glottis is the tube leading to the frogs lungs and the esophagus is the tube leading to the stomach. The pharynx  is what food, liquid, and air passes through. We were not able to get a good picture of the mouth. But we were able to get one picture with the tongue and the maxillary teeth. 
The organs of the digestive system of the frog are the cloaca which is found between the hind legs of the frog and sperm, urine, and feces exit out of and then the esophagus, stomach, small intestine, large intestine, liver, gallbladder, and pancreas. The heart is located above all of these organs. 

Incision Guide for Dissection:
The incision we made for the frog was at the opening of the cloaca, between its hind legs. Then we cut all the way up to the frogs mouth on the underside and then at each leg we made a cut as well so we could pull back the skin to pin it down and get a better view. We used scissors instead of a scalpel so we wouldn't pierce any of the internal organs. We cut through the muscles and breastbone to be able to look at the internal organs. 
  

      Dissection Video:

http://m.youtube.com/watch?v=8KH6e26sPhI

Perch 
The external anatomy:
The three main sections of the fish are the head, trunk, and tail. The pectoral, dorsal, pelvic, anal, and caudal fins cans all be located on the exterior. The lateral line can also be located on the side of the perch. Next to the eyes the gill chamber can be found, covered by the bony operculum.  The nostrils are in front of the eyes. If you open the mouth, the teeth of the perch can be seen. The fish itself is covered in scales; the scales, if seen under a microscope, have lines that indicate age. 

The internal anatomy:
The cream covered liver is at the front of the body cavity. The gall bladder is between the lobes of the liver. Under the gall bladder and liver, the asophagus can be seen attaching to the stomach. At the posterior end of the stomach are the coiled intestines. The spleen is the small reddish-brown organ near the stomach. Below the operculum are the bony gill rakers. In front of the liver and below the gill rakers is the heart. The heart has two chambers: the atrium and the ventricle. Below the lateral line is the swim bladder, which gives the fish buoyancy. Below the swim bladder are the gonads, and testes/ovaries. The kidneys are the two long, dark organs near the posterior end of the perch. These filter its blood. 

Dissection guide:
Take pins to secure the fish. Use scissors to cut off the operculum, so that the gill rakers can be seen. One can be removed to observe. Now, make a rectangle cut under the lateral line. It should extend close to the head of the fish, as this is where most of the perch's organs are. Cut through both the skin and muscle, removing scales if need be. Removing the flap of skin makes these organs visible. 




Dissection Video: 


http://m.youtube.com/watch?v=tGSz1UTeQ90

Thursday, March 6, 2014

PGLO Lab

Purpose
Thre purpose of this lab was to find the transformation efficiency of the plasmid in E. Coli in different mediums. The independent variables were the presence of plasmid and the type of medium. The different mediums were +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. The dependent variable was the transformation efficiency. 

Introduction
Transformation efficiency is the total number of cells growing on the agar plate divided by the amount of DNA spread on the agar plate, and it shows approximately how many cells take on the the DNA of the plasmid. Plasmid was the DNA that was inserted into the E. Coli, and it was called pGLO. Heat shock is the procedure used to form holes in the plasma membrane of the E. Coli. 

Methods: First off, we labeled two micro test tubes with +pGLO and -pGLO. Then with a new pipet, we put 250 micro-liters of the transformation solutions which is CaCL 2 into both of the test tubes. Then after adding the transformation solution we had to put the tubes in a cup of ice.  We had to then go over to the starter plate and get a single colony of bacteria and put it into the +pGLO tube.  We had to spin the loop with the bacteria on it in the tube to get it off and make sure all of it stayed in the tube. Then, we repeated the same thing for -pGLO. Only for the +pGLO tube we put pGLO plasmid DNA into it. Then, we put the tubes back in the foam rack and put it in an incubator for 10 minutes. While we were waiting we labeled four LB agar jars:  +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB. we put the tubes into a heart shock for 50 seconds, which was set at 42 degrees Celsius, then we had to put them on ice for 2 minutes. We took the tubes off the ice and with a sterile pipet, added 250 micro-liters  LB nutrient broth into both the the tubes and let them sit at room temperature for 10 minutes. Once again with a new pipet, we put 100 micro-liters  transformation and control suspensions onto the agar plates. Using a new sterile pipet for each plate, we spread the suspensions evenly around the surface. Then we stacked our plates and put them in a 37 degree celcius incubator overnight.  

Data
We took our data from the pGLO positive LB/Amp/Ara plate in order to determine the Transformation efficiency. This would tell us how many cells out of our total maintained the plasmid.
Using all of this data, we determined that there are 2.17*10^3 transformants per microgram. This means that approximately 2170 cells in each microgram take on the plasmid and its traits. The ones that take on the plasmid will have the ampicillin resistance and glow in the dark trait. 

Discussion
This lab was only possible because of the heat shock. When a heat shock occurs, it allows cellular membranes to pull apart just enough to let in a plasmid. This is how the pGLO plasmid entered into the E. Coli cells, allowing the traits to be adapted. When pGLO was not present, however, growth depended on the presence of ampicillin. With ampicillin in the growth medium, there was no growth of E. Coli cells. Without ampicillin, there is one large colony of E. Coli growing unimpeded.  But when pGLO is incorporated into the cell, E. Coli gains the trait of ampicillin resistance. The plasmid also allows the bacteria to glow in the dark, but only when supplied with sugar. This is why one plate, the one without sugar, has normal E. Coli colonies while the other, with sugar, was able to glow. The last two plates, with both pGLO and ampicillin, have sparse colonies compared to the unimpeded growth of the first. This is because the plasmid is not incorporated into every cell, causing not every cell to become ampicillin resistant. The transformation efficiency is calculated in order to tell us the number of transformants per microgram of DNA. Our transformation efficiency was in the range we expected, with 2.17*10^3 being a sensible number of transformants in each microgram of pGLO.


Conclusion: The conclusion we got was exactly what we were expecting it to be. The +pGLO LB/amp/ara plate had dots of bacteria basically covering the whole surface. Once we shined the ultraviolet light on it, it became green, so we knew we did it right. With all the combinations of substances in this one, is what made it glow. The +pGLO LB/amp had dots of bacteria as well, but didnt glow like the other one. The -pGLO LB/amp had no growth on it at all. And -pGLO LB was almost covered completely with bacteria.