Supplement 3 Student Instructions for Lab 1
Total Page：16
File Type：pdf, Size：1020Kb
Supplement 3 Student Instructions for Lab 1 Exercise 1: The purpose of this exercise is multi-fold. At the conclusion you will have: • gained familiarity manipulating C. elegans strains (to become a master worm-picker!) • learned how to identify the different life history stages of C. elegans hermaphrodites and males • learn how to mount worms on a microscope slide and acquire a digital DIC image using a compound microscope There are 6 teams in your section made up of X individuals / team. Each team will be responsible for correctly identifying, isolating and capturing images of a specific life history stage. Team 1: L1 ⚥ Team 2: L2 ⚥ Team 3: L3 ⚥ Team 4: L4 ⚥ Team 5: L4 ♂ Team 6: Adult ⚥ & embryos Instructions: Step 1: Pick 5 examples of your target to a new OP50 plate. See page 3 for instructions. Step 2: Take a camera phone picture of your target on the plate at maximum zoom Step 3: Make a slide, mount 3-5 examples and capture a transmitted light (DIC/Phase/Bright field) image of your target (depending on microscopes/objectives available use 10x, 20x and/or 40x). See page 5 for detailed instructions. Step 4: Save/Export the files as a .jpg or .tif labelled as: Section#Team#_Phenotype_Stage_Magnification (e.g., 1.2_WT_adult_10X.jpg) Step 5: Upload your images to the correct folder of shared class data Step 6: Create a single file with the different C. elegans life history stages following the instructions provided on page 2 and the grading rubric. 1 Supplement 3 Student Instructions for Lab 1 For your lab report: Figure 1. Title. (A) Descriptive explanation (B) Descriptive explanation Figure 2. Title. (A) Embryo (B) L1 (C) L2 (D) L3 (E) L4 (F) Male (G) Adult hermaphrodite The figure legend should include what magnification the compound microscope images were acquired with. You must include your images and can use images from other groups as long as you acknowledge them in the figure legend. 2 Supplement 3 Student Instructions for Lab 1 Exercise 2: The purpose of this exercise is to obtain a synchronized population of animals to use for future experiments. Each group will be provided with a stack of 6 plates. Each plate will have many gravid animals and should be near starvation which you can determine by the low amount of OP50 on the plate. Half of the groups will have DQM583 and the other half will have DQM594. Instructions: 1. Follow the protocol to synchronize worms on page 5. 2. Make sure to use the dissecting microscope to ensure that you have a sufficient number of embryos when you finish the steps. Additional Information and Protocols: General worm maintenance: C. elegans are best maintained by feeding monoxenically in the laboratory using E. coli strain OP50 as a food source on Nematode Growth Medium (NGM) agar which has been poured into petri dishes. Your TAs and instructors have prepared the first batch of plates for you, but for experiments later in the semester, we will ask you to seed your own plates with E. coli OP50. This is done using standard sterile techniques by pipetting (using a sterile 5ml/10ml/25ml serological pipet) a small volume (a couple of drops or a single drop for mating plates) of OP50 on NGM plates. Allow the bacterial lawn to dry overnight. 1. Picking up worms. Flame the platinum wire of your pick until it glows orange and let it cool briefly in the air. Put a glob of bacteria on the end of the wire (you may find it helpful to take it from a worm-free plate, e.g., the one to which you will be transferring worms). Touch the glob to a worm, using the sticky bacteria to pick up the worm. (Don't "scoop" the worm; just touch the bacteria on the flattened underside of the pick to it.) Then set the worm down on a new plate in the thick part of the E. coli lawn. Be careful not to gouge either plate, as this will encourage the worms to burrow into the agar, where they become inaccessible. Start by transferring one worm at a time; eventually it is possible to carry 10 or more worms on one pick-full. Sometimes worms don't like to come off a pick easily; in this case, soften the glue glob by moving the loaded pick back and forth gently in the E. coli of the plate to which you are transferring. TO DO: Learn how to identify: (1) eggs and embryos, (2) L1 larvae, (3) L2 larvae, (4) L3 larvae, (4) L4 larvae, (5) adults, (6) males. Practice transferring worms from plate to plate with a pick. Eggs are oval and easy to identify. Adults are the largest worms on the plates and carry eggs and embryos visible in the middle of the ventral side (worms crawl on their side, not their ventral surface). L1 larvae are the smallest things crawling on your plates. They are sufficiently small to fit inside an eggshell, which they recently did. At their youngest, they have no dark coloring, unlike other larvae and adults. L4 hermaphrodites are best identified by their invaginated developing vulvae that look like pale half-moons on the ventral side in the middle of the worm. There is a black dot in the half moon in L4 larvae. 3 Supplement 3 Student Instructions for Lab 1 2 layers of tape molten agar agar pad 4th slide guide2. Mounting slides live larvae for microscopy. You will have the chance Figureto view 2. the Set-up development for preparing of larval agar stage pads worms and embryos using the compound microscope scope. The following outlines the general steps. The techniques are discussed in more detail below. To prepare a slide you need to: 1. identify the desired stage of embryo, zygote, or worm 2. transfer it to an agar pad on a slide in a small volume (~1.2 µl of M9) 3. add a cover slip Instructions: Prepare an agar pad by placing a drop of molten 5% Noble agar on a plain microscope slide. This slide should be placed between two guide slides whose surfaces have been raised slightly with two layers of tape so that the agar pad has the proper thickness (shown above). Work quickly so the agar does not solidify before being flattened. Perpendicular to these slides, place a fourth slide over the agar drop so that a pad is formed. Do this by placing one side of the fourth slide at the edge of the molten agar, avoiding trapping air bubbles in the agar. Press the top slide down against the supporting slides to flatten the agar. Wait a couple of minutes for the agar to solidify. Once the desired specimens are ready to mount, lift the two slides contacting the agar away from the guide slides. Carefully tease the two apart by a back and forth horizontal motion that will leave the agar pad on one slide only. Do not rip the pad as you tease the slides apart. Try to work steadily now so that the agar pad does not dry. Mounting larvae (L1-L4) or adult worms. To transfer larvae or adult worms to a slide you first add a drop (~1-2 µl) of M9 on top of your agar pad. Collect worms as outlined above with your platinum wire, and then gently move the glob of bacteria containing your worms side-to-side in the drop of M9. This should free most of the worms from the bacteria. Using your fingers, gently cover the worms with an 18 x 18 mm coverslip. 4 Supplement 3 Student Instructions for Lab 1 3. Obtaining synchronized larvae Worms can be synchronized in development for experiments through alkaline hypochlorite treatment more commonly called bleaching. The protocol is as follows: 1. Ideally, you will want to bleach 6 plates filled with gravid adults. Add 1 mL of distilled water to each plate. Swirl the plates and/or use your pipette to remove worms from the bacteria. Tilt the plates and transfer the water and worms to a labeled 15 mL conical tube. Repeat with all of the plates. 2. Add water such that the final volume in the conical tube is 8 mL. 3. Add 600 µL of 5M base (NaOH or KOH) to the tube. 4. Add 1200 µL (600 µL x 2) of bleach to the tube. 5. Incubate the tube on the rocker for 5 minutes. After 5 minutes, inspect the tube by holding it up towards the light or by viewing it under a dissecting scope. You should wait until no (or very few) adult bodies are left. Note that this is very time sensitive -- bleaching for too short a time will result in undissolved adults, whereas bleaching for too long will dissolve the eggs. 6. Spin down the tube in the conical tube centrifuge at level 5 for 2 minutes. Use a balance (with about ~10 mL of fluid) if necessary. Dump the supernatant down the sink (do this in one motion - repetitive pouring can disrupt the pellet). 7. Fill the tube with water (up to the 15 mL mark). Start by adding a little bit, flicking the tube to resuspend the pellet, before filling to the top. Spin down the tube at level 5 for 2 minutes. Remember to change your balance, if necessary. Dump the supernatant. 8. Repeat the water wash. 9. Perform two washes with M9. 10. After the final wash, resuspend with ~2-3 mL of M9 buffer and incubate on the rocker at 20 or RT.