The overall goal of this procedure is to introduce and grow sea elegance in CENIC liquid media for numerous downstream applications. This is accomplished by first bleaching grave worms from starved agar plates. The second step of the procedure is to transfer the released eggs to M-C-E-H-R with antibiotics and allow them to develop to the grave stage, which may take seven to 10 days.
The third step is to bleach the graves and allow them to develop a second time to the grave stage without additional antibiotics. The final step is to synchronize the Axe worms and utilize them for nutritional studies examining worm growth and development. Ultimately, results show that wild type C elegance grow well in Axe Liquid media and M-C-E-H-R media can be utilized to examine the nutritional needs of the worms without bacterial byproducts.
The main advantage of this technique over existing methods like growing worms on agar plates or in liquid media with bacteria, is that the effects of nutritional elements or toxins can be tested without confounding bacterial byproducts. Generally, individuals new to this method will struggle because of media preparation and the initial adaptation of the worms to the EMIC liquid media. This procedure describes the preparation of M-C-E-H-R-A liquid media that allows C elegance to grow without a bacterial food source.
Using stringently sterile technique under a hood mix the components of the M-C-E-H-R in the following order. Begin with choline, diac acid citrate, and follow with the vitamin and growth factor. Mix the myo acetol, the human chloride, and last add DI water Filter the M-C-E-H-R mixture through a 0.22 micron filter with suction.
Next in the following order, add the nucleic acid, mix the mineral mix, lact, albumin, hydrolysate, essential amino acids, non-essential amino acids, potassium phosphate, deg, glucose heap, sodium salt, and DI water. Now filter the mixture again with suction. After the filtering, add cholesterol to the media and then check the pH of an aliquot of media.
The pH should be between six and 6.5. If the solution was made correctly, add a 20%volume of the pasteurized milk to the M-C-E-H-R media using scrupulous sterile technique. Store the media at four degrees Celsius.
Have ready 10 60 millimeter NGM plates full of GR worms that have consumed most of the available OP 50 E coli. Rinse these worms into a 50 milliliter conical tube. Using five milliliters of M nine buffer per plate, allow the worms to settle and then carefully remove the supernatant.
Repeat this, rinse two more times By adding M nine buffer, allowing the worms to settle and removing the supernatant, then add six volumes of 0.1 normal sodium chloride to the worm aggregate now compared to the worm suspension, prepare a mixture of one volume of five normal sodium hydroxide and two volumes of fresh bleach. Solution Bleach the worms by adding this mixture to the suspension. Next vortex, the mix until the grave worms are dissolved and only eggs remain in the suspension.
This takes between five and 10 minutes. Use phase contrast microscopy with a 10 x objective to monitor this reaction in the tube. Once the eggs are isolated, pellet them at 800 G for 45 seconds and at four degrees Celsius, aspirate the supernatant and rinse the pellet twice in 10 milliliters of sterile water in the hood, repeat the centrifugation and aspiration for these rinses.
Next, resuspend the egg pellet with M-C-E-H-R media and transfer the released eggs to a tissue culture flask. Add 100 micrograms per milliliter of tetracycline to the eggs in the flask. This step should be carried out in a laminar flow hood.
Using stringently sterile techniques, transfer the liquid cultures to a 20 degrees Celsius incubator and culture them on an orbital platform. Shaker at 70 RPM monitor the worms development daily. After seven to 10 days, the worm should develop to the Ravi stage, transfer the worms to a conical tube and pellet them at 800 G for five minutes at four degrees Celsius in a swinging bucket rotor.
After aspirating the supernatant resuspend the worm pellet in one volume of 0.1 molar sodium chloride and allow the worms to settle on ice for five minutes. Now, repeat the aforementioned bleaching procedure and culture the eggs in the cenic liquid media. As before, It's essential that sterile techniques are utilized when culturing the worms ally.
Avoiding the use of antibiotics ensures that the sea elegance cultures are indeed free of contaminating bacteria. When the culturing process is repeated past, the second generation emit the tetracycline from the media. At this point, synchronized cultures can be prepared for synchronization.
Add 10 milliliters of M nine buffer to eggs released following bleaching of GR worms, and allow them to hatch overnight at 20 degrees Celsius on an orbital platform. Shaker the next day, pellet the larvae at 800 G for five minutes and resuspend the L one larvae. In 10 milliliters of M-C-E-H-R, transfer the suspension to a 25 square centimeter flask and grow them up to their maximum density.
Checking the growth of the worms every few days is necessary to ensure that the worms do not become too crowded and deplete the available nutrients in the media. To store an axen worm culture, resus suspend a pellet of worms in 0.5 milliliters of S buffer in storage vials, add one volume of S buffer with 30%glycerol. Then transfer the vials to negative 80 degrees Celsius, followed by long-term storage in liquid nitrogen to thaw the worms incubate the vial at 37 degrees Celsius until almost all the ice is melted, which takes about two minutes.
Then under sterile conditions, transfer them back to M-C-E-H-R media. One advantage of using M-C-E-H-R was seen in studies that examine the exact nutrient requirement on worm development. Worms were grown in M-C-E-H-R supplemented with increasing amounts of heme to determine low optimal and toxic levels of heme.
In addition, an inversely heme responsive reporter strain was utilized to indirectly assess the heme status of the worms in a smaller range of concentrations. These worms grown at four micromolar heme showed high fluorescence indicating that the worms were in a low heme environment. When the heme concentration was increased to eight micromolar, the GFP expression declined.
When the concentration of heme was increased to 10 micromolar, the worms showed very little GFP expression and at 20 micromolar of heme, they showed no GFP expression. While attempting this procedure, it's important to remember to use scrupulously sterile techniques to ensure that the cultures are emic and to prevent bacterial contamination. After watching this video, you should be able to make M-C-E-H-R establish and maintain a xenex sea elegance cultures that can be utilized for a wide number of applications, which require a large number of sea elegance.
