LAB instructions

1. Read the paragraphs about making a pick above. 2. Decide whether you want a flattened pick or loop pick. 3. The instructor will complete steps 4 and 5, but if you decide you want to modify your pick, I’m including those instructions so you can. 4. To construct a pick, first cut ~an inch of wire off of the roll with wire clips. (**NOTE: Platinum and Platinum-Iridium wire is EXPENSIVE, so try not to waste it! If you break your pick, try to find the wire from the pick and use this to create a new pick.) 5. If you want a loop pick, use needle-nose pliers to curve the end of your platinum-iridium wire into a blunt loop. You can fine-tune your loop with forceps and your fingers. If you want a flattened pick, use the flat edge at the back of the pliers, or a hammer, to flatten the very tip of a Pt wire. Once it is flattened, sand the tip to remove jagged edges. 6. AFTER YOU HAVE THE WIRE IN THE SHAPE YOU WANT, attach it to a glass Pasteur pipette. We will demonstrate this and be on hand to help, but here is an outline of the procedure: - Turn on and ignite your Bunsen burner using the flint. - Insert the end of the wire that is not curved/flattened into the end of a Pasteur pipette. - Hold the end of the glass pipette with your hand and the wire with forceps/pliers. Hold the platinum wire/Pasteur pipette over a Bunsen burner until the glass begins to melt. Once the wire is adhered to the glass, you can let go of the wire and hold your hand so the pick is almost straight upside down. Turn in the flame until the glass has melted Developmental Genetics Lab Spring2025 7 around the wire and the wire is held firmly in place. Allow to cool. You have created your pick! 7. I like to label my pick at the glass end with color tape. You can write your initials on the tape. 8. At the end of class, store your pick in the rack for your section in the slot designated for your bench. 9. If (when) your pick breaks during the semester, you now know how to make a replacement! KEEP THE WIRE and throw away the glass. Please ask for help if you need assistance. B. Setting up your microscope 1. We will use ‘trans’-illumination rather than ‘epi’-illumination to view C. elegans. We have 3 sets of microscopes. The new Olympus microscopes have a built-in illuminator. The other 2 types of dissecting microscopes have detached light sources that you need to align. One microscope is at each student bench. We encourage partners to take turns trying the different microscopes. 2. To set up the tan microscopes with external light sources, place one of the arms from the illuminator into the back of the dissecting microscope. DO NOT JAM IT IN; YOU WANT TO HAVE THE ENTIRE FIELD EVENLY ILLUMINATED CREATING AS LARGE A CIRCLE OF LIGHT AS POSSIBLE, WHICH REQUIRES THAT THE LIGHT SOURCE NOT BE TOO CLOSE. [You can also experiment with epiillumination by having both gooseneck illuminators shine down directly on the stage. In this case, you will want to swap out the round glass circle on the stage for a black circle to minimize glare and maximize contrast.] 3. To set up the black Bausch and Laumb microscopes with external light sources, place one of the arms from an illuminator into the back of the dissecting microscope. It is fine to place the illuminator into the hole as far as it goes. 4. Place your plate of worms on the stage of the dissecting microscope. You have received a plate of N2’s; N2 is the standard wild-type strain for C elegans researchers. 5. The cover of the worm dish should be facing up so that it can be removed easily and so that you don’t need to look through agar to observe worms. In general (assuming the optics are ok), keep the lid on the plate until you need to remove it for transferring worms. When you do transfer worms, you can “clamshell” the plate to avoid contamination. 6. Adjust your microscope to the lowest magnification so you have the largest field of view. 7. Focus your microscope until you can clearly see all the worms. Adjust the illumination knob until the worms are visible, but not shiny. 8. Experiment with the tilt of the mirror to see differing contrasts. Different contrasts are useful to observe different features. In worms older than L4, the gut is dark and the gonad arms are lighter. Developmental Genetics Lab Spring2025 8 C. Observing C. elegans 1. Read the setting up your microscope instructions. All the way through. 2. Select worms of various sizes and zoom in to view more details of individual worms. You may need to adjust the microscope focus as you zoom in and out, but you generally do not need to adjust the illumination. 3. Identify which end is the head and which is the tail. The head is more blunt-ended and contains the lighter pharynx. The tail is long and tapered. 4. Intestinal cells are darker than surrounding tissue, so you can see where the pharynx ends and the gut starts. Do you see the black dot going up and down at the posterior of the pharynx? That dot is the grinder (or “E. coli mulcher”) opening and closing. Can you see the pharynx pumping? At what frequency? 5. One way to get C. elegans to move is to gently drop the plate onto the stage from about a half-centimeter height or to lift the lid a few millimeters and let it drop back down. The resulting vibration activates their touch-sensitive neurons causing them to move. Feel free to gently poke and prod them with your pick. Do all parts of the body move in the same way, or do some parts appear to have more freedom to move in more directions