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2. What are the two things you need to look at immediately after starting a gel running (or soon thereafter)?
3. Give the gel running settings (agarose concentration, voltage, time, etc.) that you would choose in order to observe good resolution between a 300 nt and a 400 nt band.

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Related questions

Please answer questions at the end.

In this lab, you will add different volumes of “cell-free supernatant” (CFS) to fresh cultures of V. harveyi and observe bioluminescence. The CFS is prepared by culturing V. harveyi in liquid medium overnight, then separating the cells from the supernatant by centrifugation.

MATERIALS • overnight culture of V. harveyi in AB medium • fresh AB medium • 1 microcentrifuge tube • 3 glass culture tubes

PROCEDURE

1. This experiment will be performed with your lab group. Label the microcentrifuge tube and 3 glass cultures tubes with your group number. Also, label the 3 glass culture tubes as follows: “Media only,” “Cells+fresh media,” and “Cells+CFS.”

2. Using aseptic technique, pipet different volumes of fresh AB medium into the glass tubes as follows: 1 mL in the “Media only” tube, 800 ul in the “Cells+fresh media” tube, and 500 ul in the “Cells+CFS” tube.

3. Pipet 500 ul of the overnight culture into a microcentrifuge tube and centrifuge at maximum speed (>13,000 x g) for 5 min to pellet the cells.

4. After centrifugation is complete, transfer 300 ul of the cell free supernatant (CFS) from the microfuge tube to the glass tube labeled “Cells+CFS”. Be careful to avoid the cell pellet.

5. Remove the remaining supernatant from the microfuge tube using a micropipette and discard as culture waste.

6. Add 500 ul of fresh AB medium to the cell pellet in the microfuge tube and pipet up and down to resuspend until no clumps are visible.

7. Add 200 ul of the cell suspension to the “Cells+fresh media” tube and 200 ul of the cell suspension to the “Cells+CFS” tube. Note the time.

8. Place the 2 glass tubes containing cells in the 30 degree incubator/shaker.

9. After 1 hour and 2 hours, observe your tubes next to the “Media only” tube in a dark room. You will need to let your eyes adjust for at least 30 seconds (count to 30 slowly in the dark) before you can evaluate whether the cultures are luminescent or not. Also, make sure you try swirling the tubes in the darkroom and observe the tubes for a minute after you have stopped swirling. After the observation at the 1 hour time point, put your tubes back in the incubator shaker until the 2 hour time point.

10. Note your observations. At the end of the lab, place your tubes in the tube waste.

Questions

1. What were the expected results? Explain the rationale of the experiment. If you didn’t obtain the expected results, hypothesize why not and describe how the experiment could be improved.

2. What are differences between luminescence and fluorescence?

3. Why do V. harveyi cultures glow more brightly when swirled vigorously? Be specific.

4. Imagine you have a mutant V. harveyi strain that can’t synthesize autoinducer and another mutant V. harveyi strain that makes a defective luciferase. If you streaked both strains near (but not touching) each other on the same plate, would you expect to see light produced? If so, by which strain, where, and why?

5. You have two identical tubes of wild-type V. harveyi (with the same cell concentration in each tube) in fresh media. To one tube, you add CFS from a light-producing V. fischeri culture, and to the other tube you add an equal volume of fresh media. If both tubes of V. harveyi have the same light production, what can you conclude? Explain.

6. Many biomedical research labs study quorum sensing in V. fischeri or V. harveyi. How is studying light production in marine bacteria relevant to human health? List at least 3 different examples.

7. Why do you think it is important for bacteria to be able to control the expression of some genes based on the number of individuals present?

Background

Almost all animal and plant cells require oxygen and give off carbon dioxide. This is called gas exchange and it occurs by passive diffusion. Some animals do all or most of their gas exchange across their skin (for example amphibians) but they have very low metabolisms and use very little oxygen and get rid of very little carbon dioxide.

On the other hand humans (and other mammals) have very high metabolic requirements and we can’t rely on gas exchange over the limited 1.5 to 2.0 square meters of our skin to get enough oxygen in and carbon dioxide out fast enough. Our gas exchange takes place across a very thin membrane in the Alveoli of the lung and the gas exchange surface in humans is estimated to be between 50 and 100 square meters, or between 25 and 67 times our skin surface area.

Our lungs provide a huge surface for gas exchange but they are a dead-end system. Once the oxygen in the lungs is used up it needs to be replenished and carbon dioxide builds up in the air spaces in the lungs and needs to get out. This is where the diaphragm and a few of the skeletal muscles become important. In its relaxed state the diaphragm is at its longest and the space in the thoracic cavity (and the lungs) is small. When the diaphragm contracts it shortens, which increases the volume of the thoracic cavity (and the lungs). The increase in size means the pressure is lower in the lungs than outside so air comes into the lungs (inhalation). As the diaphragm relaxes, the lungs decrease in size enough that the pressure inside the lungs is higher than the external air pressure so air comes out (exhalation).

Introduction

In this lab you will be building a model of a lung and experimenting with different ways to breath.

Supplies:


In the Lab Kit

One round 9" balloon (one lung, we don’t need two for this)
medium-sized plastic bag large enough to fit over bottom end of bottle (diaphragm)
medium-size, thin rubber band

You supply

plastic 2-liter soda bottle with bottom cut off (thoracic cavity)

Tape (optional)

Directions:

1. Insert the balloon through the open bottom of a modified 2-liter soda bottle and up over the neck of the bottle. Pull the end of the balloon over the edges of the neck of the bottle. The balloon is held open by the neck of the bottle.

2. Place the plastic bag over the bottom (open) end of the 2-liter bottle and use the rubber band to hold it in place. This will serve as the diaphragm. To inflate the lung, blow into the balloon just before you fasten the rubber band. You can also wrap tape over the edges of the plastic bag to minimize air leaks

3. Grasp the bottom of the plastic bag and pull it down and push it up. Watch as the "lungs" (the balloon) expand and contract as you do this. The balloon has to change size because the air space between the balloon and bottle must remain constant. (Well, it would if the seal around the plastic bag and bottle were perfect).

You may block the "trachea" (the neck of the bottle) so that the air supply is cut off and watch as nothing happens when the "diaphragm" is pushed in and out.

This model isn’t perfect. None of the joints are air tight as they would be in a real thoracic cavity. Pull the “diaphragm down and watch the lungs expand . If you hold it in place, the lungs will collapse again over time. This is because of leaks and is not how real lungs work. Pushing the diaphragm in and pulling it out over and over does give you an idea of how lungs work. As the diaphragm tightens, contracts (pull down on the plastic bag) the lungs fill with air. As the diaphragm relaxes (push up on the plastic bag) air is expelled from the lungs .

There are several different ways we breath, i.e. expand our thoracic cavity so that our lungs can expand. You have just made a demonstration model of the diaphragm at work. You can also use your rib cage to expand and contract so that you can inhale and exhale. There are muscles between and around your ribcage which you can use to change the size of your thoracic cavity.

This is approximately what your model of a lung will look like.

Answer the questions on the Report Page

Report Page Name

1. Put a hand on your lower ribcage and try moving your ribs out and in. Does air also move in and out?

2. Someone with an upper back or neck injury may not have the options of using diaphragm or ribs to breath. They can learn to use the clavicle and the muscles attached to expand their thoracic cavity a small amount and breathe independently for short periods of time. Put your hand on you clavicle and try to raise and lower just that bone without using your diaphragm or your ribs. It isn’t easy but you should be able to get a little air into and out of your lungs this way. How effective do you think this way of breathing is?

3. When you take a really deep breath you use all these muscle groups to help. Take a really deep breath and notice that your ribs expand, your lower abdomen comes out (a sign that the diaphragm has contracted) and your shoulders lift a little (the upper chest and clavicle are working). Now take a relaxed breath. Only your abdomen should go in and out. The movement of your abdomen is a result of the contracting diaphragm pushing on the contents of your “gut” which forces your abdomen to push out.

Do you breath in or out when your diaphragm contracts?\

4. If a hole were poked in your thoracic wall (the wall of the pop bottle – the hole should be at least the diameter of a pencil)

a. what would happen when you tried to breath? (This is a pneumothorax).

b. Is the space between the balloon and plastic bottle still required to remain constant?

5. Would it be possible to live with a paralyzed diaphragm? Explain your answer.

It’s Friday morning and Sal Volpe is sitting in Dr. Lorraine’s exam room, dozing after another night of disrupted sleep. When the doctor knocks and walks in, she finds the 66-year-old man looking exhausted and uncomfortable. Sal gets to the reason for his visit immediately: He’s been suffering from “stomach aches” (dyspepsia) that wake him at night and nag him in between meals during the day. He describes his pain as gnawing, burning (maybe a 4 out of 10 on a pain scale) and points to the epigastric region of his abdomen. When he eats, he tells Dr. Lorraine, the pain goes away, but then he feels bloated and a little nauseated. The pain usually returns 2–4 hours later, depending on what he eats. Sal explains that he has had some pain relief from the over-the-counter drug Pepcid® (famotadine).

Dr. Lorraine proceeds with the history and physical exam. She discovers that Sal has a family history for gastrointestinal cancer and has unintentionally lost 10 pounds since his checkup a year ago. His epigastric area is modestly tender to palpation. She suspects a peptic ulcer (gastric or duodenal), but the weight loss and family history make it prudent to eliminate the diagnosis of stomach (gastric) cancer. “Mr. Volpe, I think you may have a stomach or intestinal ulcer,” Dr. Lorraine says. “I suggest we perform an endoscopy to have a look. This involves passing a small tube with a small camera through your mouth and into your stomach. We can look at the wall of your stomach and small intestine, check for an ulcer, and remove a very small piece of tissue to test for infection. We call this a biopsy. We’ll also test the biopsy for cancer because of your family history. But, I really think we’re dealing with an ulcer here and not cancer.”

Later that month, the endoscopy is performed and it confirms Dr. Lorraine’s suspicions. Sal has a duodenal ulcer and infection with the bacterium Helicobacter pylori (H. pylori). This is not surprising since H. pylori is the cause of most peptic ulcer disease, particularly in the duodenum. Treatment involves complete eradication of the H. pylori with two different antibiotics, and a drug that decreases gastric acid secretion, a so-called proton pump inhibitor (PPI). Dr. Lorraine explains to Sal, “Mr. Volpe, you do not have stomach cancer, but you do have a duodenal ulcer caused by the H. pylori bacteria I was telling you about. Too much acid and inflammation from this infection is causing your pain. The good news is we can probably cure your ulcer by killing the bacteria, but you will have to take three different medications twice a day for 14 days. I’ll see you again in 3 weeks; we can do a simple breath test to determine if the H. pylori has been successfully eliminated.”

Short Answer Questions:

The structures in the epigastric region share a common nerve supply. Can you name the specific cranial nerve that serves this region and the part of the nervous system to which it belongs?

In order to understand the disease in Mr. Volpe’s alimentary canal, one must know the layers that make up its walls. Design a chart that identifies the four basic layers of the alimentary canal, the tissues that make up each layer, and the general function of each layer.

Dr. Lorraine suspects a peptic ulcer. This is an inflammatory lesion in the stomach or duodenal mucosa, which may extend through all layers of the alimentary canal wall. Describe the basic histological (tissue) structure of the mucosa layer in the alimentary canal. Identify the unique features of the mucosa in the stomach and in the duodenum, and explain how this uniqueness determines the function of the stomach and the duodenum.

Mr. Volpe asks, “What do the bacteria have to do with the ulcer?” Dr. Lorraine tells him that the H. pylori increases stomach acid secretion and, at the same time, breaks down the lining of your stomach and duodenum. What is the source and normal function of acid in the stomach and what regulates its production

Why is Mr. Volpe’s dyspepsia relieved by food, and aggravated 2–4 hours after a meal?

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