How to Dissect Drosophila melanogaster

Why fruit flies? The use of Drosophila melanogaster, or the fruit fly, as a model system for researching human biology has several benefits:

Fruit flies conform to the three main biological research techniques: genetics, biochemistry, and molecular biology. A large percentage of the fruit fly genome and proteome is conserved in humans. The cellular and molecular biology of the fruit fly is well established.

Why dissection? Dissection is essential to studying biological processes in living organisms. It provides researchers with a direct approach to viewing the molecular composition and cellular characterization of fly tissue. In an educational setting, dissection often refers to a complete investigation of the innards composing an individual body. You may recall the dissection of a frog or fetal pig from high school. In such a situation, the goal is to find and observe various organs systems. In a research laboratory, dissection usually involves the harvesting of a single organ for further experimentation. Ten or more fruit flies must be dissected to prepare for an experiment. This instruction set details the steps to dissecting larval salivary glands. Note to Readers: Dissection is a skill that requires much cultivation. Practice dissection one to two hours a day over the course of a week. This should enable you to gain a level of proficiency appropriate for experimentation, i.e. a minimal dissection time and maximal quality of the dissected organs. Also, there are a few short but important procedural steps that must be performed the day before you dissect. These are listed in the section titled Preparation The instructions that follow are designed for readers with some laboratory knowledge, but no experience in dissection is necessary. Specifically, you should have some experience with pipetting less than 1 mL of solution, and know how to focus a microscope.

Safety Hazards and Equipment Usage

Research laboratories are filled with dangerous tools and chemicals. In addition to any hazards your supervisors have already reviewed with you, there are a few guidelines specific to safety and proper equipment management in salivary gland dissection:

Always wear wrist protection when you are dissecting.

Dissection is characterized by repetitive pinching motions and specific wrist posture. These factors can lead to chronic hand and wrist pain, and, over time, carpal tunnel

syndrome. In order to prevent these conditions, wrist guards should be worn on both wrists whenever you are dissecting. Even if you will only be dissecting for a short period of time, it is important to become accustomed to wearing wrist protection.

Be cautious when using No. 5 forceps.

No. 5 forceps are extremely pliable, and their tips will bend and/or break easily. Dissecting with twisted or uneven forceps is very difficult, so you will want your forceps in the best possible condition. Be careful never to drop a pair of forceps, and refrain from placing the forcep tips on hard surfaces unless it is during dissection.

Materials
The following list of materials is based on the assumption that dissection is being performed a Drosophila laboratory setting. Materials such as a 4C refrigerator or an ice machine, which are always available in such a laboratory, are not listed.

Vialed larvae 15-mL conical tube 10 mL phosphate-buffered saline (PBS) Ice bucket Depression wells 200-1000 L pipetman 20-200 L pipetman 200 L Pipet tips Dissecting microscope Left and right wrist guards 2 pairs of No. 5 forceps Small Petri dish Laboratory wipes

Drosophila Background Information

Larval Anatomy
The anatomy of the larvae is divided into three parts: anterior, midsection, and posterior. The anterior is distinguished by the presence of a black u-shaped organ, called the mouth hook. The mouth hook is visible by the naked eye. The posterior has no prominent distinguishing characteristics, and is most easily identified as the end lacking the mouth hook. The midsection, as its name implies, is located in between the posterior and the anterior of the larvae.

Life Cycle
There are four major stages in the life cycle of the fruit fly: egg, larva, pupae, and adult. For our purposes, the only relevant stage is that of the larva. The larval stage is further categorized three sub-stages: first instar larva, second instar larva, and third instar larva. The early third instar larvae will yield the best salivary glands. Therefore, it is important to be able to differentiate between second and third instar larvae. Grab a fly vial, and try to identify the second and third instar as follows: Second instar larvae: Look closely at the food in the vial. Try to locate darting black specks along the fly food-glass interface. These are the mouth hooks of second instar larvae. The defining characteristic of second instar larvae is that they have not emerged from the fly food. Third instar larvae: Look closely at the walls of the vial. Try to locate off-white larvae crawling along the inside of the glass. Any larva that has emerged from the fly food is in its third instar stage. However, inactive larvae should not be selected for dissection, because they undergoing transformation from a larva to a pupae. and Early third instar larvae are on the glass that is closest to the fly food

Preparation
The day before dissection . . .
1. Check that there are second instar larvae in several vials. Note: Many of the second instar larvae will become early third instar larvae overnight, ensuring that you will have larvae in the proper stage on the day of dissection. 2. Fill the 15 mL conical tube with 10 mL of PBS and place it in a 4C refrigerator.

The day of dissection . . .

1. Load the ice bucket about 80% full with ice. Note: Often, several labs share a communal ice machine. Ask any member of your lab (or a nearby lab) where one is located. 2. Create a rectangular indentation in the ice large enough to fit the depression wells. 3. Place the depression wells in the indentation, and pack the ice tightly with your hands. 4. Insert the conical tube of PBS into the ice bucket so about 75% of its length is surrounded by ice. 5. Using a pipetman, place 500 L of cold PBS into one of the depression wells. 6. Using a pipetman, place a 100 L drop of cold PBS onto a Petri dish.

7. Locate an early third instar larva in a vial, and remove the stopper from that vial. 8. Hold the forceps in your right hand as you would a pair of tweezers. 9. Firmly grasp the midsection of the larva between the forcep tips, and remove the larva from the vial. 10. Insert the forceps (still holding the larva) into the PBS droplet on the dissection stage, and slowly release your grip on the forceps. Note: This action should deposit the larva into the droplet. 11. Repeat steps 7 to 10 until there are ten to twenty larvae contained in the drop of PBS.

Dissection
1. Put on left and right wrist guards 2. Using the pipetman, place a 100 L droplet of cold PBS onto the center of the microscope dissection stage. Hereafter, this will the referred to as the dissection droplet. 3. Place another 100 L droplet of cold PBS several inches to the left of the dissection droplet. Hereafter, this will be referred to as the waste droplet. 4. Choose a larva from the Petri dish to dissect. 5. Hold the forceps in your right hand as you would a pair of tweezers. 6. Firmly grasp the midsection of the larva between the forcep tips, and remove the larva from the Petri dish. 7. Insert the forceps (still holding the larva) into the dissection droplet, and gently release your grip on the forceps. Note: This action should deposit the larva into the droplet. 8. Still holding one pair of forceps in your right hand, pick up the other pair of forceps with your left hand and hold them (once again) as you would a pair of tweezers. Hereafter, the forceps in your right hand will be referred to as the right forceps. Likewise, the forceps in your left hand will be referred to as the left forceps. 9. Focus the microscope on the larva. Note: Steps 11 to 17 are to be performed while looking through the microscope. 10. Try to orient the larva horizontally, with the posterior end facing left and the anterior end facing right. Note: Larvae are buoyant and may be moving during dissection, so this step may take several minutes. You will probably need to use both forceps to pin the larva to the surface of the dissection stage.

Before moving onto the next step, check that the larva is probing with its mouth hook; this is a reassurance that the larva is alive. 11. Using the left forceps, grasp the midsection of the larva between the forcep tips. Note: Only grasp as tightly as is required to stop the larva from swimming away. If you grasp the larva too tightly, it will start to squirm, which will make the next step more difficult. 12. Using the right forceps, firmly grasp the mouth hook of the larvae between the forcep tips. 13. Pull the left and right forceps apart firmly and slowly until the tissue held by your right forceps is completely detached from the tissue held by your left forceps.
Gut

Note: You may have to pull the forceps five or six inches apart before you get complete detachment of the tissue; it is alright to move outside the dissection droplet and off the dissection stage, if necessary. As you pull, try to locate the salivary glands among the exposed organs. Initially, the glands will often appear stretched and attached to the gut. The glands may eventually be released from the gut while you are pulling, in which case they will assume their normal shape. After detachment, the right forceps should contain the mouth hook connected to two salivary glands, the end of the gut, and excess tissue, as the picture to the left illustrates. The left forceps should contain a mostly intact larva body.

Salivary glands

Source: http://biology.clc.uc.edu/ fankhauser/Labs/Genetics/Drosophila_chromosomes/

Drosophila_Chromosomes.htm

14. Insert the left forceps (still holding the tissue) into the waste droplet, and gently release your grip on the forceps. Note: This action should deposit the tissue into the waste droplet. If the waste droplet is out of the range of the microscope, you should stop looking 15. Detach the gut from the salivary glands. Minimize direct contact with the salivary glands as much as possible. 16. Carefully put the left forceps down beside you.

17. Firmly grasp the mouth hook of the larvae between the right forcep tips, and remove the mouth hook (along with the attached salivary glands and excess tissue) from the dissection stage. 18. Insert the right forceps (still holding the mouth hook, salivary glands, and excess tissue) into the pool of PBS in a depression well, and gently release your grip on the forceps Note: This action should deposit the mouth hook (along with its attachments) into the depression well. 19. Clean the remaining larval tissue and PBS droplets off the dissection stage with a laboratory wipe. 20. Repeat steps 2 to 19 until you have dissected all the larvae in your Petri dish.

Troubleshooting
The most common problem in dissection occurs when you are trying to locate your organ of interest in the dissected material. Details in the notes and picture of step 13 are included to help you identify the salivary gland. However, the results depicted and described are the products of very specific technique. If you are not getting these results, this is likely a consequence of one of two factors: The location at which you grasp the larvae in steps 11 and 12.

The force with which you pull apart your forceps in step 13.

Try varying these factors slightly to see if you can get the expected results. If you are still getting undesirable results, a common oversight in salivary gland dissection is that the forceps must be pulled apart IN BOTH DIRECTIONS, at the same time, during step 13. Sometimes, it is impulsive to pull on only one of your forceps and keep the other stationary. This action will cause the larva to rip apart randomly. If this occurs, you may have to dig through a lot of tissue to find the salivary glands, probably damaging them in the process.