Cells should be taken directly from the YES plate or, for liquid cultures, after harvesting by centrifugation. Several crosses can be set up on the same 9-cm diameter plate.
Do not exceed this level or mate on plates containing all amino acids because the nitrogen from the amino acids will be catabolized, which reduces the efficiency of G 1 arrest and, therefore, mating efficiency. Allow the cell suspensions to dry completely. Monitor the formation of asci by removing some cells and examining them under a light microscope.
Some mutants are completely sterile and cannot be studied by routine crosses. However, it is possible to force such crosses by digesting the cell wall to form protoplasts that can then fuse to form a zygote Nadin-Davis and Nasim Transfer cells to mL screw-cap tubes and harvest cells by centrifugation for 3 min at g. Wash cells by resuspension in 10 mL of 0. Discard supernatant and resuspend cells in 5 mL of 0. Discard the supernatant, and resuspend protoplasts in 2 mL of 1.
If the two strains are ready at slightly different times, the first strain can be left in sorbitol at room temperature. Combine the two protoplast samples and mix by gentle pipetting up and down. Harvest the protoplasts by centrifugation for 3 min at g. Add to the protoplast mixture. Do not use any physical means of spreading beads or glass rod as this will burst the protoplasts. Problem: Step 4 : Some mutants mate inefficiently using the standard protocol. Efficiency of mating is often improved on one or more of these media.
It may also be helpful to use a higher ratio of mutant to wild-type cells in the cross e. A further way to increase efficiency can be to reduce the number of auxotrophic markers in the strains. We acknowledge Agnes Grallert for providing the protoplast fusion protocol. From the Fission Yeast collection, edited by Iain M. A Positioning top panel and gluing bottom panel the eyebrow hair to the tooth pick.
The thickened black line indicates the shaft of the hair; the gray area indicates the location of the glue. B Animals should be touched by stroking the hair across the body at the positions of the arrows. The six touch receptor neurons are indicated.
Wild-type animals will move adults usually reverse direction in response to their plate being tapped Chalfie and Sulston, This stimulus often happens when plates are placed on the stage of a dissecting microscope. Touch-insensitive animals do not respond to this tapping. Although not as accurate a measure of touch sensitivity as touching with an eyebrow hair, this is a rapid assay that has been used to screen for touch-insensitive mutants Chalfie and Au, Plates with F2 progeny of mutagenized parents were dropped from about cm above the stage of the microscope and then examined for animals that did not move.
Candidate touch mutants were then tested with the eyebrow hair and worm pick to determine if they were touch insensitive.
Cathy Rankin and coworkers designed an electric tapper to deliver a taps at defined intervals to plates for their studies examining the touch circuitry and habituation of the touch response Rankin, ; Wicks and Rankin, The above assays treat touch sensitivity as an all-or-none phenomenon. Often, however, animals give a partial response to the touch stimulus. In addition, even touch-insensitive animals sometimes respond to the first touch. Two general methods provide a more quantitative measure of the touch response: counting the responses to multiple touches and touching with defined stimuli.
The first instance of using multiple touches with an eyebrow hair to measure the degree of insensitivity to touch was by Hobert et al. In this assay animals are touched ten times alternating head and tail touches and a score is giving for the number of positive responses. At least thirty animals are examined and mean percentage score is obtained. Since individual touches are not always the same, the values obtained in this assay are not truly quantitative. Nonetheless, by using multiple animals relatively subtle difference in touch sensitivity can be revealed e.
In addition, by separately scoring head and tail responses differences between the two can also be uncovered Zhang et al. In using this method one should be careful to distinguish between animals that habituate more rapidly than wild type from those that respond less frequently to the touch stimulus. The animals with the former defect should show a response pattern in which the animals respond less frequently to successive touches; animals with the latter defect should show no pattern to the failures.
The experimenter should also determine whether the animals are responding equally in terms of number of responses to touches in the head or tail. They consist of a series of flexible fibers that are touched end-on to skin and bend once a specific force has been applied. The longer and thinner the fiber, the less force is required to bend it. These calibrated fibers are then used to determine the forces needed to provoke a touch sensation when placed on the skin. We have adapted this method to C.
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Typically, we mount the capillary on a manual micromanipulator with the fiber perpendicular to the agar surface, position a worm underneath the fiber, and make contact by moving the z-axis of the micromanipulator. Animals are touched in the same locations as described above for testing body touch with eyebrow hairs, and the response is noted.
Extracted from the literature. Comments from researchers who use this assay would be appreciated. Harsh touch to the body is measured by prodding animals with a platinum wire in the midsection of the body Chalfie and Sulston, ; Way and Chalfie, Nonmoving gravid adults are prodded at or just posterior to the vulva. Animals respond by initiating locomotion, usually by backing up.
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Animals to be assayed should be grown in the continuous presence of food. Animals who are starved and animals that have passed through the dauer stage often fail to respond to harsh touch regardless of the functional status of the PVD sensory neurons. Harsh touch is assessed in animals in which the function of the ALM and PLM neurons have been perturbed as these neurons mediate response to both gentle and harsh touch. Although the animals are reported to avoid the edge of an agar chunk, little is known about this behavior.
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Croll and Matthews, An overtly similar behavior called nictation is reported to play a role in the dispersal of parasitic nematodes. An assay for this behavior has not been developed. The nose of C. This rhythmic nose motion is called foraging in this context. The rate of basal foraging is dependent on the RMD motorneurons and on glr-1 function Hart et al. Animals respond to touch to the side of the nose by rapidly moving their nose away from the stimulus; this is called head withdrawal.
Response is again mediated by the RMD motorneurons and requires glr-1 function. Hart et al. Animals respond by rapidly moving the nose away from the hair. Significant practice is required learn where to lay down the hair and to learn to discriminate between the rapid withdrawal motion versus normal foraging motion. Foraging and head withdrawal assays should be undertaken by observers blind as to genotype or treatment. Thin bacterial lawns for these assays resemble those used in nose touch assays. Allow the liquid to soak into the plate.
To prevent bacterial growth and too much thickening of the bacterial lawn, the plate should be used within a few hours or should be sealed with Parafilm and stored at 4 degrees for weeks. Allow stored plates to return to room temperature before use. The tap-withdrawal protocol measures the responses of a worm to a single tap or a series of successive taps trains of taps , given to the side of a 4 cm Petri plate filled with 10 ml of NGM agar. This technique produces a quantifiable measure of the magnitude of this reversal response.
For this assay worms are placed on the center of a Petri plate and are videotaped through the lens of a dissecting microscope. The mechanical tapper is arranged to hit the center of one side of the plate. The tapper is composed of an electromagnetic relay run by a stimulator. Rankin et al. These experiments are usually run with the lids off of the plates while taps are being delivered.