The overall goal of this procedure is to manufacture a three dimensional cultivation system for nematodes that may better simulate natural conditions. This method can answer key questions in the field of C elegans development, physiology and behavior. We first had the idea for this method when we were studying natural nematode behavior in rotting fruit which is a 3D environment.
This technique is advantage compared to standard two dimensional cultivation techniques because nematodes reside in 3D habitats in nature. Though this method has been developed C elegans cultivation, it can also be applied to other nematodes. Demonstrating this procedure will be Tong Young Lee, a graduate student in my laboratory.
To begin this procedure, Otto Clave the prepared solutions at 121 degrees Celsius for 15 minutes. Next, prepare 50 milliliters of the sterile cholesterol solution at 5 milligrams per milliliter. In a 50 milliliter conical tube, add 0.25 grams of cholesterol and 50 milliliters of 99.99%ethanol and mix well.
Next, sterilize the cholesterol solution using a 50 milliliter syringe with a 0.45 vented syringe filter and remove any undeveloped cholesterol. Then, inoculate 10 milliliters of lysogeny broth with bacteria. Culture the bacterial inoculation at 37 degrees Celsius in a shaking incubator overnight.
In preparation for a serial dilution, allocate nine milliliters of the Sodium Chloride solution into seven sterile 15 milliliter conical tubes. To make a tent of the seventh fold dilution of Opi 50 Strain E Coli. Afterward, pop out one milliliter of the bacterial culture or diluted bacterial culture to a new tube with nine milliliters of Sodium Chloride solution.
And vortex the mixture well. After preparing the bacterial culture, add 0.6 grams of Sodium Chloride, one gram of granulated auger and 0.5 grams of peptone in a 500 milliliter flask. Place a magnetic stir bar into the flask.
Then add 195 milliliters of distilled water and cover the flask with aluminum foil. Next, Otto Clave the sample at 121 degrees Celsius for 15 minutes. Subsequently, place the hot Otto Claved flask into a stir plate, stir at a moderate speed for at least two hours until it reaches 40 degrees Celsius.
Alternatively, the flask can be cooled to 40 degrees Celsius by placing it in a 40 degrees Celsius water bath for 15 minutes before transferring it on a stir plate. Be sure to cool sufficiently as continuing from here. A high temperature may lead to clouding of the finished auger but temperature that is too low may result in premature hardening of the auger.
When the temperature of auger media reaches 40 degrees Celsius, add Calcium Chloride, Cholesterol Solution, Magnesium Sulfate and Potassium Phosphate Buffer and continue to stir the solution. For NGT-3D lifespan assay, add 80 microliters of 150 millimeter FUDR to the media to result in a final concentration of 120 micromolar. Then, remove six milliliters of auger media into a sterile conical tube and keep it warm in an incubator.
This media will be used for the bacteria free top layer. Next add six milliliters of the diluted bacteria culture into the flask directly on a sterilized bench. For NGT-3D, pour 6.5 milliliters of media into the eight milliliter clear plastic test tubes to form a bacteria auger layer.
And carefully dispense 200 micro liters of the bacteria free media on top of the semi-hard 3D media to make a thin bacteria free layer on top. Then close the cover of the chamber tightly. For NGB-3D, pour 65 milliliters of media into a 25 centimeter squared clear plastic cell culture bottle.
Then position the chamber vertically at room temperature for one week to allow bacterial colonies to grow to considerable size of at least one millimeter diameter. In this procedure, pick a L four stage worm and transfer it to a bacteria free NGM plate. Then allow the worm to freely move around for a few minutes in order to remove bacteria from it's body.
And repeat the procedure two more times. Afterward, carefully place the clean worm on the surface of the 3D media. The worm should've actually entered the auger into the 3D auger matrix.
Then close the cover loosely to incubate for 96 hours at 20 degrees Celsius. After four days, close the lid tightly and place the NGT-3D culture chamber into a water bath at 88 degrees Celsius to melt the auger. Using a glass pipette, transfer the melted media onto a nine centimeter plastic petri dish.
Afterward, using a transmission stereo dissecting microscope, count the number of worms at the L3, L4 and adult stages. As shown here, the construction of NGT-3D is a simple and straightforward protocol that results in an auger filled test tube with small bacterial colonies spaced throughout the auger. To confirm whether C elegans can reduce and grow normally in NGT-3D, fertility and larva development are compared in 3D with the standard 2D NGM plates.
In the Relative brood size assay, adult C elegans hermaphrodites in NGT-3D reproduced just as well as hermaphrodites in the standard 2D MGM plates when bacterial colonies are plentiful with at least 60 colonies. Furthermore, larva development in NGT-3D also proceeds normally when there are plenty of bacterial colonies with the majority of worms in the adult state after 96 hours. However, if bacterial colonies are sparse in the 3D matrix, both relative brood size and larva development are negatively impacted.
Here is a survival curve of the wild type C elegans in NGT-3D or NGM plates. Once mastered, this technique can be done in three hours if performed properly. Following this procedure, other methods like life span assays and behavior analysis can be performed in order to answer questions about aging and behavior in 3D.
After its development, this technique can pave the way for researchers in the fields of genetics to explore questions about ecology and evolution in C elegans. After watching this video, you should have a good understanding of how to cultivate nematodes in a 3D laboratory environment.
