Embryo Dissection and Micromanipulation ToolsHazel L. Sive, Robert M. Grainger, and Richard M. Harland Adapted from \"Equipment for Embryo Experiments,\" Chapter 4, in Early Development of Xenopus laevis by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000. INTRODUCTION This article describes the creation and maintenance of tools for use in dissection and micromanipulation of embryos. All tools must be kept clean and rinsed with 70% ethanol to keep them sterile. RELATED INFORMATION For excellent advice on dissection and micromanipulation tools, see Hamburger (1960). Forceps Stainless-steel forceps, such as the Dumont 5 or 5A (Fine Science Tools), are adequate for membrane removal and most micromanipulations. They do not rust easily, but they cannot be sharpened to as fine a point as carbon-steel forceps. To sharpen the forcep points, use a fine stone or fine emery paper, observing the process under a dissecting microscope. Use only slight pressure, and keep the points together so that the sharpened points meet cleanly at their tips. Slight bends can be introduced or corrected by pressing the tips against the microscope stage. Label the forceps for different uses: good forceps should be reserved for fine work, and old battered forceps (which can be bent and filed back to a blunt end) should be designated for all noncritical purposes. Needle-nose pliers or coarse forceps can be used to make adjustments to the angles of fine forceps, but any excessive bend will break the tips. Hair Loops Loops of human hair mounted in beeswax at the tip of a cut-off Pasteur pipette are used for steadying and moving embryos around during micromanipulations. It is useful to have loops in a variety of sizes. Small loops are resilient and useful for fine dissections, whereas larger loops are good for sorting and pushing embryos around. Hair loops can be prepared easily in the laboratory as follows: 1. Heat a long-stem Pasteur pipette below the shoulders in a small Bunsen burner flame, and pull to ~15 cm. 2. Make a second and finer pull to ~25 cm on the same pipette. 3. To create an opening big enough to thread the hair easily, score the end of the pipette with a diamond pencil, and break the end. It may be necessary to use a dissecting microscope and forceps to view and hold the hair. 4. (Optional) Carefully flame-polish the severed glass tip to help prevent the hair from being damaged. 5. Select a human hair longer than the pipette, and thread it through the smaller opening of the pipette until the end emerges from the larger opening. Then push the free end of the hair (at the smaller opening of the pipette) far enough into the small opening so that it does not pop out. Tighten the loop by pulling on the end of the hair from the larger opening (see Fig. 1). View larger version (4K):[in this window][in a new window] Figure 1. Hair loop.6. Scrape a little beeswax into the end of another Pasteur pipette, and warm it over a flame until molten. 7. Apply the waxed tip to the small opening of the hair loop. Wax is drawn by capillary action into the tip. 8. Make final adjustments to the size of the loop by pulling on the hair, and then remelt the wax with a warmed pipette tip. Once set, the wax holds the loop in place. A hair loop can also be set in beeswax by dipping it into molten beeswax (melted on a hotplate). After removing the loop from the wax, blot excess wax from the loop by touching the loop to a piece of tissue (e.g., Kimwipe) that has been preheated on a warm hotplate (or on a hot piece of metal heated using a Bunsen burner). Eyebrow Knives Many embryos are sufficiently soft to be cut with human eyebrow hairs, which offer a combination of resilience and sharpness that is difficult to match. Eyebrow knives are made in the same way as hair loops, but more easily. Eyebrow (or eyelash) hairs vary enormously from person to person in their quality as cutting tools. Try different people for sources of hair. The ideal hair is strong but not brittle. Simply insert the root of the hair far enough into the drawn-out pipette so that it leaves the desired amount of curvature/resilience (2-5 mm). Set in place with beeswax as described above. Eyebrow knives are not used to saw down into the embryo as are conventional knives; instead, they are pushed or threaded through the tissue and flicked upward through it. They can also be used to trim isolated tissue that has been removed from the embryo. Trimming is done by pressing the eyebrow knife against the solid base of the dissecting dish. Fine Tungsten Needles Tungsten needles and glass knives are useful for manipulating and dissecting embryos of more advanced developmental stages when the embryonic tissues become tougher and somewhat resistant to eyebrow knives. Tungsten needles with 1-mm tips can be purchased from Fine Science Tools as an alternative to eyebrow knives. In contrast to eyebrow knives, tungsten needles can become permanently bent. To avoid this, dissect the embryos on an agarose bed. Tungsten needles can be made quite easily. Mount short pieces of fine tungsten wire (California Fine Wire, size 0.001) in pipette tips that have been prepared as described above for hair loops. Cut the wire to a length of 10 mm, insert it into the drawn-out tip, and then carefully fuse the tip to the wire by gently heating it in the edge of a flame. Overheating makes the wire brittle. Alternatively, tungsten wire holders can be purchased from Fine Science Tools or Carolina Biological. Glass Needles Glass needles are useful for dissecting older embryos. They are made very simply from microinjection needles or hand-pulled capillaries. The needles are mounted in holders used for tungsten needles (available from Fine Science Tools or Carolina Biological). The fine-needle tips should be broken off to provide a tool sturdy enough for dissection. Because this method leaves the tips open, the needles are difficult to sterilize and thus should be discarded after use. REFERENCE Hamburger V. 1960. A manual of experimental embryology. University of Chicago Press, Illinois.