This protocol presents an aseptic technique for use with C.Elegans that eliminates the need for an open flame. The main advantages of this technique are that it reduces safety hazards by eliminating an open flame and can be used in laboratory spaces without gas lines and in laminar flow hoods. Moving instruments in and out of the sterilization area without touching the sides can be difficult, but adding a guide and stabilizing your hand makes the movement easier.
Begin by attaching a loop holder accessory guide to the micro-incinerator by clipping it onto the outer barrel. Plug in the micro-incinerator to a standard 120-or 230-volt electrical outlet as appropriate. Turn the micro-incinerator to the High setting and allow it to warm up for 10 to 20 minutes to reach an optimum temperature between 800 to 825 degrees Celsius.
Insert the instrument into the cylindrical sterilization area without touching the sides by sliding it along the guide. Hold the instrument in the sterilization area for five to seven seconds. Remove the instrument without touching the sides by sliding backward along the guide.
For picks, allow the instrument to cool for three to five seconds before touching a worm to avoid burning it. After picking worms, reinsert the pick in the chamber for five to seven seconds to incinerate any worms on the pick. Connect a Bunsen burner to the gas line using rubber tubing.
Make sure to secure the tubing tightly and position the burner away from overhead objects. Turn on the gas by turning the knob on the gas line. Ignite the burner using a striker or lighter.
Adjust the flame using the gas knob and the air intake until a blue cone is visible. Hold the pick, spatula, or scalpel in the flame until it glows red. For picks, allow the instrument to cool for three to five seconds before touching a worm to avoid burning it.
After picking worms, reinsert the pick in the flame to incinerate any worms on the pick. Culture OP50 E.coli bacteria in Luria broth overnight on a 37 degree Celsius shaker. After overnight culture, dilute the culture in sterile water at a ratio of 1:100.
Dip a worm pick sterilized using a Bunsen burner or a micro-incinerator in the bacterial solution, re-sterilize, and swirl it in 100 microliters of sterile water. For positive control, dip the pick in bacterial solution, swirl in sterile water, and for the negative control, sterilize the pick without dipping into bacterial solution and then swirl into the sterile water. Plate the 100 microliters of water on a 10 centimeter Luria broth agar Petri dish, spread the water using a sterile cell spreader, and incubate at room temperature for 24 hours with the lid on.
Count the colonies manually after the incubation period. Relative contamination rate was measured by counting the number of colonies per sample. In negative control plates, zero colonies were found.
A mean count of 4.7 colonies with a standard error of 0.3 colonies was obtained when the Bunsen burner was used to sterilize picks. A mean count of 3.3 colonies with a standard error of 1.2 colonies was observed in the micro-incinerator condition. However, a mean count of 298.3 colonies was obtained in the positive control condition.
Thus, the micro-incinerator achieved sterility results equally effective to the Bunsen burner. Following this procedure, any methods that involve picking or chunking of C.Elegans can be performed. Thus, it is applicable to a wide variety of research questions.
