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DROSOPHILA GENETICS LAB I PURPOSE: To become familiar with the stages of the life cycle, the sex differences and mutations of Drosophila. To perfect techniques for anesthetizing, identifying, and counting Drosophila. BACKGROUND MATERIAL: LIFE CYCLE There are four distinct stages in the life cycle of the fruit fly: egg, larva, pupa, and adult. At 25 C a fresh culture of Drosophila will produce new adults in 9-10 days. Five days in the egg and larval stages and four days in the pupal stage. The adult flies may live for several weeks. Drosophila cultures should not be exposed to high temperatures (e.g.. above 30 C) which result in sterilization or death of the flies no to low temperatures (e.g. below 10 C) which result in a prolonged life cycle (perhaps 57 days) and reduced viability. The adult Drosophila female starts to deposit eggs on the second day after emergence. Embryonic development of the egg takes about one day at 25 C. The larva is white, segmented and worm like. The larval stage is a feeding stage and consists of three subdivisions called instars. The first and second instars stages end in molting which allows the larva to grow. The third instar ends with pupation. Prior to pupation, the animal stops feeding and crawls to some relatively dry surface and the cuticle hardens and darkens to form the puparium. Metamorphosis occurs in the puparium and takes about four days. The pupa begins to darken just prior to the emergence of the adult fly. Most flies eclose (emerge) from the pupa in the early morning hours. CULTURE MEDIUM We will use instant Drosophila Medium. It is essential that you use clean vials and plugs as mites and fungus infections can wipe out your Drosophila and lead to agony as you have to start your whole experiment over. C are at the beginning will save you grief in the long run. Mix equal amount of medium and tap water in the bottom of a vial. Try to keep the sides relatively clean. you do not need more than an inch of medium in the bottom. Too much can be as bad as not enough. Sprinkle a few grains of dry yeast on the medium. After about 1 minute flies can be introduced into the vial. Always place anesthetized flies on the side of the vial and leave the vial on its side until they awaken. Anesthetized flies can become stuck in the medium and die. Be sure to label the vial with the type of cross, the date, and your name. ANESTHETIZING FLIES We will use carbon dioxide and ice to anesthetize our flies. This is the safest, cleanest, and least obnoxious way of anesthetizing the flies. We will have several carbon dioxide generators. These are side arm flasks with rubber tubing attached to the side arm. The other end of the tube will have a Pasteur pipette attached. Drop 2 Alka Seltzers into about 2 inches of fresh water in the bottom of the flask. Replace the cork. Place the vial of flies on its side and insert the Pasteur pipette between the vial and the plug. Watch until all flies have stopped moving. Remove the plug and empty the flies on a piece of filter paper in a plastic petri dish. Place the dish on a container of ice that you can get from the freezer. Keep the lid on and the flies on ice while you are observing them. You may remove the top for separating and moving but it is a good idea to keep the lid on most of the time while you are just observing. If you are placing the flies back into a culture vial be sure you keep the vial on its side until the flies awake. If you will not need the flies for further use place them in the morgue. Be sure you are through with them before you send them to meet their maker. You do not have the ability to resurrect fruit flies! Please return all ice containers to freezer when not in use. SEXING FLIES It is essential that you learn to distinguish the males from the females. With practice this becomes relatively easy and you will probably be able to sex the flies without magnification. At first it will be best to use the dissecting scopes. There are several ways of distinguishing the males from the females. I think the most reliable way is to look for the sex comb on the front legs of the male. They do not appear on the front legs of the female. This characteristic is present even in the pupa. In older flies the posterior part of the abdomen is quite dark in males and considerably lighter in females. The tip of the abdomen in more rounded in males than in females. In general the male is smaller than the female. Use a camel hair brush for moving flies around. It is easy to damage the flies if you use pencils or other objects. PROCEDURE You and your partner will be given a vial of flies. Label this with your name and the date. Guard it with your life! Anesthetize the flies in your culture and observe. Practice sexing the flies. When you are familiar with your flies look at the other types of flies in the lab. You will be expected to recognize all the different mutants we have. Make a new vial of culture medium and return your flies to the new medium. Be sure to label the new culture. This will serve as a back up in case anything happens to your original culture. Bring a shoe box and create a lovely home for your new fly culture. Clean up and prepare for a quiz over the material in this lab. Record the making of your new culture in you log book. Each person must keep his own log book. Write in this book only what you do and observe. Log book checks will not be announced and are vital to your grade. Keep you book up to date and bring it to class every day. If you do not have it when I check you will receive a 0. No exceptions! Be sure to date each entry and to record each observation and every cross you make. Be sure to put your flies away and to keep your supplies with your flies. If you leave your supplies out you will have to buy them back and if you lose your flies WOE BE UNTO YOU! Please be careful.
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