The WBG

An online publication service of WormBook

A rapid nematode preparation for microscopy

Shamble Saha1, Vivek Gowda1, Chelsea Trengrove1 and Benjamin Wolozin1
1The Laboratory of Neurodegenerative Research, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston MA

Correspondence to: Shamol Saha (saha@bu.edu)

The use of 2% agarose pads is the conventional current method of choice for nematode microscopy.  Although reliable, agarose pads are cumbersome to prepare and require the use of an immobilizing drug to keep nematodes motionless during the microscopy. Furthermore, a fast solidifying agarose drop on a glass slide requires some extra caution to make sure that the pad height remains minimum for microscopy with 40x or 100x lenses. We have developed a simpler and faster method for mounting nematodes for microscopy that does not require anesthetic drug. We report that a blend of equal percentage (20:20) of PEG 20000 and glycerol in 1x PBS provides enough viscosity to immobilize nematodes without distorting the cuticle/body shape. The treatment does not induce any observable osmotic shock when used on adult nematodes.

To perform the procedure, nematodes cleansed of bacteria are picked and then released into a PEG/glycerol droplet (6.5 μl) on a glass slide.  A cover slip is then gently placed on the droplet containing the nematode. Due to the small volume of PEG/glycerol droplet, nematodes need to be transferred to PEG/glycerol droplet, and rapidly overlaid with a cover slip before mounting medium (PEG/glycerol) dries. This quick step instantly immobilizes the nematode and leaves them well suited for microcopy, including acquiring images for extended periods of time. We find the method is particularly useful for quick screening of GFP expressing lines, which frequently requires analysis of many nematodes, and use of high-resolution, compound microscopy for detecting low levels of GFP expression (such as due to certain weak promoters).

We have also experimented with other mixtures of PEG and glycerol.  For instance, a mixture of 30% PEG 8000 in 25% glycerol also renders the nematodes amenable for microcopy, however increasing PEG concentrations above 30% renders nematodes unstable and unsuitable for microscopy.  Larval nematodes appear to be slightly more sensitive to the procedure.  We noticed slight cuticle distortion in younger nematodes (L1-L3) when PEG/Glycerol (20:20) solution was used for microscopy.  A further dilution of PEG might be required to overcome the distortion and 1 μl of levamisole (20mM stock) can be used to insure that the nematodes are motionless.

Figures



Figure 1: Images showing C. elegans dopaminergic neurons and their projections in a transgenic line carrying a Dat1:eGFP construct. DIC pictures of the related areas are shown in the left panel.
Published by
Categories: Volume 18, Number 3, WBG submissions

The wet plate protocol: an efficient way to obtain dauer larvae

Andreas M. Weller
1Department of Evolutionary Biology, MPI for Developmental Biology, Tübingen, Germany

Correspondence to: Andreas M. Weller (andreas.weller@tuebingen.mpg.de)

Developmentally arrested dauer stages in nematodes for laboratory assays have so far been obtained either by simply starving culture plates or by liquid culture in large Erlenmeyer flasks. Harvesting dauers from starved agar plates is straightforward but the yield is usually very low. Liquid cultures on the other hand produce high yields, but their maintenance is labor-intensive. We have combined the simple setup of starved plates with the increased yield of liquid cultures in the “wet plate protocol”:

  1. Wash 3 well-populated culture plates of worms into a centrifuge tube using sterile dH2O. Pellet down and remove supernatant.
  2. Add 20ml of fresh E. coli OP50 grown in L-Broth medium.
  3. To start the wet plates, use 9 freshly poured 10cm NGM plates. The plates should not have been dried after pouring.
  4. Seed each plate with 2ml of worm/OP50 mixture.
  5. Place the plates in a Tupperware® box. Add moistened tissue to keep the interior of the boxes humid and incubate at 25° C.
  6. Check the plates every second day to ensure conditions are still humid.
  7. Depending on species, the plates are ready to harvest with lots of dauer larvae present after about 10 days.

It is important to maintain a layer of liquid on the plates at all times. If the worms are able to crawl instead of swim, the plates are already too dry. Just like liquid cultures in an Erlenmeyer flask, liquid plates may become contaminated. It is therefore important to prepare the plates to be as sterile as possible (i.e. under a flowhood). If problems persist, the culture may either be started with freshly bleached eggs instead of a mixed culture. Additionally, antibiotics and fungicides may be added to the worm/OP50 mixture.

For large-scale experiments that require huge numbers of dauers (e.g. microarrays), the use of liquid cultures is preferable. For smaller experiments however, the wet plate protocol has proven to be simpler, quicker and less prone to contamination in many assays in our lab.

Published by
Categories: Volume 18, Number 3, WBG submissions

glp-1(e2141) sequence correction

Diana Dalfó1, James R. Priess2, Ralf Schnabel3 and E. Jane Albert Hubbard1
1New York University School of Medicine, New York NY, 2Fred Hutchinson Cancer Research Center, Seattle WA , 3Technische Universitaet Braunschweig, Braunschweig, Germany

Correspondence to: E. Jane Albert Hubbard (jane.hubbard@med.nyu.edu)

The Notch-like receptor GLP-1 is defined by several temperature sensitive and non-conditional alleles. The glp-1 alleles e2141 and e2144 were isolated by Ralf and Heinke Schnabel in a temperature-dependent embryonic lethal screen at the MRC, Cambridge. Our results suggest that there was a mix-up in the distribution or propagation of the e2141 allele, such that the “e2141” allele that the Priess lab has maintained and subsequently distributed appears to be identical to e2144. The Priess lab apologizes for any inconvenience associated with previous distribution of “e2141“. In short, contrary to what was previously thought and published (Kodoyianni et al., 1992 and also in WormBase), e2141 and e2144 do not bear the same sequence change. e2141 actually carries two mutations in exon 8: c2920t and a3610g.

We urge the community to sequence their “e2141“-bearing lab strains prior to future publication or dissemination. We note that in the prior literature, strains designated as “e2141” may have been carrying the e2144 (c2785t) mutation. In light of our sequencing results, the literature indicates that the e2144 (c2785t mutation variously attributed to “e2141” or “e2144“) results in a much stronger embryonic phenotype than e2141 (c2920t a3610g mutations). See, for example, Mello et al., 1994, and Hutter and Schnabel, 1994. The e2141 allele is in the CGC collection as strain CB4037, and the e2144 allele is now deposited at the CGC as strain GE68.

Figures



Figure 1:

References

Hutter H and Schnabel R. (1994). glp-1 and inductions establishing embryonic axes in C. elegans. Development 120, 2051-64. PubMed

Kodoyianni V, Maine EM, and Kimble J. (1992). Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans. Mol. Biol. Cell 3, 1199-213. PubMed

Mello CC, Draper BW, and Priess JR. (1994). The maternal genes apx-1 and glp-1 and establishment of dorsal-ventral polarity in the early C. elegans embryo. Cell 77, 95-106. PubMed

Published by
Categories: Volume 18, Number 3, WBG submissions

Modifying the gene model of rrf-3

Ayelet T. Lamm1, Jonathan I. Gent2 and Andrew Z. Fire1,2
1Department of Pathology, Stanford University School of Medicine, Stanford CA, 2Department of Genetics, Stanford University School of Medicine, Stanford CA

Correspondence to: Ayelet T. Lamm (ayeletla@stanford.edu)

RRF-3 is one of the four putative RNA-directed RNA polymerases (RdRPs) in C. elegans and plays a major role in endogenous synthesis of siRNAs. By deep sequencing of mRNA tags (mRNA-seq) from N2 worms and from worms with mutational backgrounds, at different stages, we found that the annotated seventh exon of rrf-3 contains an intron. The position of the intron is chrII: 8165255-8165300 (WS190 version of the C. elegans genome), and it is 45 bp long (Figure 1). The 5′ end of the intron starts with GTATATTCTA and the 3′ ends with AAATATTCAG. The intron does not change the reading frame and shortens the protein by 15 amino acids.

Figures



Figure 1: (A) PCR and RT-PCR on a region in rrf-3 ’s seventh exon spanning the newly defined intron. (B) View of the rrf-3 gene by UCSC genome browser with the WS190 version of the C. elegans genome. The primers that were used for (A) are shown in red. Sanger sequencing of the RT-PCR is shown in black and validates the intron. The purple arrow points to the newly identified intron.
Published by
Categories: Volume 18, Number 3, WBG submissions

JFlux – a platform for flow cytometry with focus on the COPAS biosorter

Johan Henriksson1 and Thomas R. Bürglin1
1Karolinska Institutet, Department of Biosciences and Nutrition, Huddinge, Sweden

Correspondence to: Johan Henriksson (mahogny@areta.org)

We recently acquired a COPAS worm sorter and found that the analysis software included could not do what we needed. For this reason, we began developing a framework tailored to analyze the data. So far we have developed the functionality to produce chronograms (Dupuy et al., 2007) and to compare subregions statistically. The scatter plots produced are easy to navigate and can be overlaid to compare different runs.

The software is open source, enabling anyone to extend or modify it to fit their purpose. It is split into two parts: a library that can be used within your own code (e.g. for high-throughput analysis) and a graphical user interface for manual visualization and analysis of the data. All code is written in Java.

The code is already available at jflux.sourceforge.net, but the software is still in early development. We hope that other COPAS users are interested in contributing to this project. Having an extensible platform will allow us to incorporate new analysis methods to make the most out of the data.

Figures



Figure 1: A chronogram showing how the expression changes during development. Each horizontal line represents a histogram at one time point, with time of flight (hence length of the worm) increasing downwards.


Figure 2: One profile along the worm of expression (green) together with the extinction (red).


Figure 3: Two different runs (red and blue) overlaid in a scatter plot highlights the differences

References

Dupuy D, Bertin N, Hidalgo CA, Venkatesan K, Tu D, Lee D, Rosenberg J, Svrzikapa N, Blanc A, Carnec A, et al. (2007). Genome-scale analysis of in vivo spatiotemporal promoter activity in Caenorhabditis elegans. Nature Biotechnology 25, 663-668. PubMed

Published by
Categories: Volume 18, Number 3, WBG submissions
%d bloggers like this:
%d bloggers like this:
%d bloggers like this:
%d bloggers like this:
%d bloggers like this: