1Department of Cancer and Human Molecular Genetics, Bellvitge Institute for Biomedical Research, 2C. elegans Core Facility, Bellvitge Institute for Biomedical Research
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Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cerón, J. Basic Caenorhabditis elegans Methods: Synchronization and Observation. J. Vis. Exp. (64), e4019, doi:10.3791/4019 (2012).
Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10.
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others.
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.
1. Protocol A: Culturing Worms for Bleaching 11
Large populations of C. elegans can be obtained by culturing them either in liquid media or on solid media in plates. They are usually grown on solid NGM (Nematode Growth Media) and fed with E. coli bacteria, which is added to the plates either alive or dead (killed by UV12, by heat13 or by cold14). The most common procedure uses live OP50 E. coli, which is defective in the synthesis of uracil and cannot overgrow into a thick layer that would obscure the worms.
2. Protocol B: Treatment with Alkaline Hypochlorite Solution ("Bleaching")11
The bleaching technique is used for synchronizing C. elegans cultures at the first larval stage (L1). The principle of the method lies in the fact that worms are sensitive to bleach while the egg shell protects embryos from it. After treatment with alkaline hypochlorite solution, embryos are incubated in liquid media without food, which allows hatching but prevents further development.
3. Protocol C: Worm Plating
4. Protocol D: C. elegans Observation
D.1 Nomarski observation
Differential interference contrast microscopy is an optical microscopy illumination technique used to enhance the contrast in unstained transparent samples. The word Nomarski refers to the prism used, named after his inventor. By observing animals alive we are able to examine the physiology of the animal with the only alterations derived from immobilization. In addition, as no fixative is added, fluorescent markers can be observed in vivo. This fact and the possibility of fusing fluorescent markers to a gene of interest make it feasible to follow processes in which the protein of study may be involved. By using the technique described in this protocol, not only live worms can be observed, but they can also be recovered and plated again.
Agar pad preparation (just before use):
Mounting live animals
D.2 Ethanol fixation and DAPI staining
The protocol described here represents a fast way of dyeing worms with DAPI, however because of the dissecation of the worm some structures may present some alteration. There are several other methods to fix worms previous to DAPI staining such as fixation with Carnoy's solution or formaldehyde that preserve better the integrity of the worm 17.
Ethanol fixation (modified from 18)
4',6-diamidino-2-phenylindole (DAPI) staining
Nematode Growth Medium (NGM)
1.7% (w/v) Agar
50 mM NaCl
0.25% (w/v) Peptone
1 mM CaCl2
5 μg/ml Cholesterol
25 mM KPO4
1 mM MgSO4
42 mM Na2HPO4
86 mM NaCl
1 mM MgSO4
Bleaching solutions tested
|recipe #1||recipe #2||recipe #3||recipe #4||recipe #5|
|sodium hydroxide (ml)||1.25 (1M)||0.5 (5M)||2.5 (1M)||2.5 (2M)||2.5 (1M)|
|sodium hypochlorite ~ 4% (ml)||1||1||1||2||1|
Table I. Different bleaching solution recipes tested for this article. Recipes #3 and #4 are 2x, and should be added to the same volume of M9. Recipes for #1, #2 and #5 have been previously reported 2, 11, 19. Final concentrations: #1 NaOH 0.25M, NaOCl ~0.8%, #2 NaOH 0.5M, NaOCl ~0.8%, #3 NaOH 0.625M, NaOCl ~1%, #4 NaOH 1 M, NaOCl ~1.6%, #5 NaOH 0.5M, NaOCl ~0.8%.
5. Representative results
Figure 1. Comparison of five different bleaching solutions at two different incubation times. N2 worms washed twice with M9 were split into five 15 ml conical tubes containing each bleaching solution. Tubes were shaken vigorously and 1 ml transferred to a new tube with M9 to stop the reaction after the time specified. After bleaching procedure worms were incubated with 1 ml of M9 at 20 °C for 24 hours. In each case, lower picture was taken just after bleaching, upper picture 24 hours later.
Figure 2. Temperature of bleaching solution affects the effectiveness of the treatment. Equal volumes of N2 worms were bleached with the same bleaching solution either previously chilled on ice for 20 minutes or kept at 25 °C for the same time. The two columns on the left show pictures just after bleaching. After treatment worms were incubated in 15 ml conical tubes with 1 ml of M9 at 20 °C for 24 hours. Columns on the right display pictures 24 hours later.
Figure 3. The ratio worm pellet:time of alkaline hypochlorite incubation affects the effectiveness of the treatment. 50, 100, 250 and 500 μl of worm pellet were incubated with 2 ml of bleaching solution #3 for 3, 6 and 9 minutes. Hatched L1, dead embryos and remains of adult fragments were quantified after incubation at 20 °C for 24 hours in 15 ml conical tubes with 1 ml of M9 buffer. Approximately three confluent 55 mm plates with adult worms are needed to get a 100 μl worm pellet.
Figure 4. Proper aeration is required for hatching and survival of C. elegans embryos. A 100 μl pellet of N2 worms were bleached for 6 minutes and incubated in 15 ml conical tubes with 1, 5 or 10 ml, as specified, of M9 at 20 °C for 24 hours. The upper part of the figure displays pictures of the cultures after 24 hours, where arrows indicate eggs that did not hatch. At the bottom, there is a graph depicting the amount of larvae (light grey) and dead embryos (dark grey) 48 hours after bleaching at the stated conditions.
Figure 5. Life cycle of C. elegans. a . Approximate length of the worms at different stages. Hours required to reach each stage depending on the temperature (modified from 20). b. Nomarski (up) and DAPI (down) pictures of different worms at the indicated developmental stages. Most significant features in each phase are magnified. L1: arrow indicates the precursors of the somatic gonad and the germ line. Early L4: black arrow (Nomarski) indicates the developing vulva; white arrows (DAPI) indicate the two gonadal arms. Mid-late L4: arrow indicates the developing vulva at the so-called Christmas tree stage. Young Adult: black arrow indicates an embryo inside the uterus, arrowhead points to the spermatheca, white arrow indicates an oocyte. Gravid adult: arrowhead (DAPI) points out fertilized embryos. Arrow in DAPI image indicates spermatheca.
Figure 6. Vulva morphology at L3, L4 and Adult stages. At the L3 stage only a small lumen where the vulva is formed can be observed. At L4, this lumen expands forming the so-called "Christmas tree". In the adult the vulva is already closed. Yellow lines indicate the location of the vulva at these three stages.
Figure 7. DAPI staining at L3, early L4, late L4 and Adult stages. At L3, germ line is elongated. At L4, gonad arms present U-shape morphology. At late-L4 stage sperm can be observed in the distal part of the gonad. Young Adults present oocytes. The Adult germ line presents oocytes and embryos. Yellow lines delimitate germ lines at the different stated stages.
Figure 8. C. elegans development at 15 and 25 °C. N2 worms were bleached, incubated overnight in M9 and agitation at 15 °C, transferred to plates and grown the indicated times at the stated temperatures.
Several bleaching solutions have been described. We tried five different recipes (Table I) and, in our hands, they did not show significant differences in the synchronization of worm populations (Fig. 1). However, our experiments did show that parameters such as temperature (Fig. 2), the ratio bleaching solution:volume of worms (Fig. 3) and the volume of M9 with which the embryos are incubated for hatching (Fig. 4) do affect the survival of the worms, being related to proper aeration of the culture. In our shaking conditions, while in a tube of 15 ml a volume of 1 ml allows survival of all worms, a volume of 5 ml is already too much to allow proper egg hatching and comparable to the maximum volume of 15 ml (not shown).
C. elegans Development
During its development, C. elegans goes through four larval stages (Fig. 5) prior to the adult stage. The germ line is a good indicator of the developmental stage of C. elegans. The easiest feature of C. elegans development that can be observed under Nomarski optics is the development of the vulva, which starts to form at early L4 stage. At first, only a small lumen is observed, which later expands to the so called "Christmas tree" shape, by mid-late L4. Finally, by the end of L4 the vulva closes (Fig. 6). On the other hand, DAPI staining allows the observation of the development of the gonad. From the four cells in L1 to the dividing cells and elongating gonad in L2 and L3. At L3 the distal tip cells can be observed, starting to migrate dorsally. Meiosis also starts by the end of L3. At L4 distal tip cells reach their definitive position and germ cells differentiate to sperm. By the end of L4 sperm production ends and oocyte production starts. In adult worms embryos can be observed inside the uterus (Fig. 7).
Development and Temperature
C. elegans develops at a different rate depending on the temperature: while it takes about 90 hours from the moment the egg is laid until the new worm starts to lay its own eggs at 15 °C, 45 hours are enough when grown at 25 °C (Fig. 6). The study of the differential developing rate at diverse temperatures leads to relative flexibility in setting up conditions and performing experiments. Additionally, it offers the possibility not only to monitor the effects of a particular treatment or alteration (for example temperature sensitive alleles), but also to establish the best conditions in which carrying out a particular experiment.
We have nothing to disclose.
Authors would like to acknowledge MICINN (PTA program supporting Montserrat Porta de la Riva), AGAUR (Phd Fellowship to Laura Fontrodona), Instituto de Salud Carlos III (Miguel Servet program supporting Julián Cerón), and Marie Curie IRG, ISCIII and IDIBELL for financing the lab.