BISC 219/2009: Mod 1 Media and Tools
Tools and Techniques
Sterility: Aseptic technique should be practiced at all times while conducting nematode genetic analysis. Check for contaminants (mold, yeast, non-feeder strain bacteria) on agar culture medium plates and discard any that show signs of contamination. If a contaminant arises after worms are transferred , remove 10-20 worms to fresh medium containing new E. coli "food", let the worms crawl for 1/2 hour, then transfer a few of the worms to second fresh plate.
NGM-Lite Worm Medium:Eric Lambie originally described NGM-lite culture medium in Worm Breeder's Gazette 13(5):11 (February 1, 1995). This medium contains a simple but complete nutrient rich environment for culturing C. elegans.
2 g of NaCl
4 g of Bactotryptone
3 g of KH2PO4
0.5 g of K2HPO4
20 g of agar
1 mL of 5 mg/mL cholesterol (dissolved in 100% ethanol)
Dissolve all ingredients in 1 liter of distilled water and autoclave.
Escherichia coli: Strain OP50 or a streptomycin-resistant derivative of E. coli OP50 is used as nematode food. OP50 is a uracil auxotroph, meaning that this strain of bacteria can not synthesize the essential nutrient uracil; therefore, growth of this strain is not as robust as in wild type E. coli, even when the culture medium provides sufficient uracil. Because bacterial growth is limited by the auxotrophy, the "lawn" of bacteria does not grow so thick as to obscure observation of nematodes. NGM-lite agar medium poured into sterile plastic petri plates are seeded with a liquid culture of OP50 E. coli bacteria by applying a drop or two on the middle of the plate. C. elegans worms will generally confine themselves to the bacterial lawn; therefore, leaving the edges of the plate unseeded with bacterial "food" allows us to observe fairly easily all the animals.
Several hundred adult worms may be grown on a single 60 mm x 15 mm NGM-lite plate without exhausting the bacterial supply. However, each animal produces close to 300 offspring, meaning that the capacity of a plate can be exhausted within one to two days if too many worms are transferred. When the bacterial food is exhausted, the adults die of starvation and the population becomes primarily geriatric adults and young larvae, neither of which are useful for setting up further experiments or crosses. Plates inoculated with 5 or fewer worms can support one generation of growth and last up to a week in good condition if the temperature is controlled carefully.
Methods for Handling Worms
- Wire Tool (“worm pick”): One end of a fine platinum wire is embedded in a Pasteur pipette and the other end is flattened to make a tiny shovel. The tool is sterilized by briefly passing it through a flame. Older larvae and adults can be lifted off a plate by scooping under them and lifting up. In this way, individual worms can be "captured" and transferred. With practice more than one worm may be picked up at a time. This process can be facilitated by first touching your worm picker to the bacterial lawn to acquire some “bacterial glue”. The transfer must be accomplished as quickly as possible in order to avoid killing the worms by dessication on the tool. It is important to avoid gouging the plate, or animals will burrow into the agar despite the fact that all food is on the surface. After a worm is transferred, the tool is sterilized by flaming again. Care should be taken not to cross-contaminate strains of worms by carrying over unwanted worms, especially small larvae and eggs. In some cases (such as stock transfer) a mixture of worms is not problematic; however, when setting up a genetic cross it is essential that only late larvae and adult males are transferred. This is difficult at first but you will become an expert worm picker as the semester progresses.
- Agar Chunk: When you don't care exactly how many worms are transferred (especially on old plates when most of the worms may have burrowed into the agar), simply cut out a 1 cm square "chunk" of agar using a sterile scalpel and transfer it to a fresh plate. The scalpel should be dipped in alcohol and flamed before and after each use.
- Transfer by Liquid: If a small amount of liquid is gently put on a plate, adults and larvae tend to float off in the liquid while eggs tend to stick to the surface of the plate, creating a useful way of fractionating worms. If the liquid is transferred to a conical centrifuge tube, the adults will settle to the bottom in a few minutes, allowing adults to be purified from young larvae. The adult worms may be collected off the bottom of the centrifuge tube in a small volume of liquid taken and then transferred to new NGM-lite agar, where the liquid will be absorbed, leaving adult worms on the surface of the new culture.
Genetic Manipulations
Matings:
To start a mating, place 4 to 5 young adult (or L4) males on a small plate with 2 to 3 young adult (or L4) hermaphrodites. We will use plates seeded with a very small drop of OP50 bacteria for our crosses. Using only a small central area of the plate helps our worms find each other quickly and mate. The self-cross progeny produced by the hermaphrodite must be distinguished from outcross progeny. Generally, the hermaphrodite is homozygous for a visible marker and the progeny from self-crossing will show the same phenotype as the hermaphrodite, while progeny from a cross with a wild type male (outcross) will be heterozygous for the maternal marker and thus appear wild type if the mutation is recessive. When no other way is available to distinguish selfcross from outcross progeny, only males are scored. It is the outcross progeny of a mating that are of interest; essentially, all male progeny are crossprogeny. For this reason it is critical than the male transfer not be contaminated with any eggs or hermaphrodites, as they will be confused with crossprogeny.
When the male progeny from a cross are scored, remember that the parental males remain on the plate (usually 3 or 4 animals). These males will be the oldest and largest males and should not be scored among progeny phenotypes. Generally, the parental males are no longer fertile by the time the cross is scored.
In cases where male progeny are to be used for a subsequent cross, care must be taken to use the young adult males and to avoid the parental males, since only the young males will carry the marker of interest.
In cases where outcrossed hermaphrodite progeny are required, only L4 hermaphrodites are picked since adult hermaphrodite progeny will not be virgin: adults will have mated with sibling males.
Scoring Phenotypic Ratios
Since worms crawl constantly around the plate, the only reliable method of counting animals is to remove them from the plate as they are scored. Individuals can be picked up with the wire pick and incinerated in the flame. Many investigators prefer to count one phenotypic class at a time. Unless you have a very good memory it is probably best to jot down sub-totals several times while scoring a plate. In general, populations are scored after four days of incubation at 20°C. At this time virtually all F1 progeny are adults, while small larvae on the plate represent second generation progeny and are not scored.
C. elegans General Information
Lab 1: Welcome to C. elegans and Mutant Hunt
Lab 2: Linkage Test and Backcross
Lab 3: Linkage Test, Backcross and Mapping
Lab 4: Mapping Part 2
Lab 5: Score
