Hitchcock DR et al. (1998) Arch Environ Contam Toxicol "Determining toxicity trends in the ozonation of synthetic dye wastewaters using the nematode Caenorhabditis elegans."

Law SE, Hitchcock DR, Williams PL, Wu JH

[Arch Environ Contam Toxicol, 1998]

The nematode Caenorhabditis elegans was used in 72-h toxicity tests to evaluate the influence of ozonation on the toxicity of three synthetic azo dye wastewaters (two reactive dyes and one acid-based dye). The two reactive dye wastewaters contained high concentrations of NaCl (89-112 g/L) in addition to potentially toxic dye components. To determine the contribution of NaCl to toxicity, simulated dye wastewater samples with and without NaCl were tested. Samples were collected at various times during ozonation (t = 0, 8, 32, 64 min); nematodes were exposed to the samples for 72 h. The influence of ozonation on toxicity varied between dye wastewater types. For the acid-based dye wastewater, toxicity increased as duration of ozonation increased. For the reactive dyes without NaCl, toxicity did not appear to be influenced by ozonation. For the reactive dyes with NaCl, mortality was 100% with or without ozonation. Range-finding experiments with NaCl in water and NaCl in dye wastewaters suggested an additive toxic interaction between NaCl and the dyes in wastewater to the nematodes. The duration of ozonation for acid-based dyes and the relatively high NaCl concentrations for the reactive dyes appear to influence effluent toxicity in the ozonated dye wastewaters.

Tong YG et al. (2010) J Neurosci Methods "Conditions for dye-filling of sensory neurons in Caenorhabditis elegans."

Burglin TR, Tong YG

[J Neurosci Methods, 2010]

Dye-filling is a common method used to stain Caenorhabditis elegans sensory neurons in vivo. While the amphids and phasmids are easy to stain, a subset of sensory neurons, the IL2 neurons, are difficult to stain reproducibly. Here we examined the conditions under which the IL2 neurons take up the lipophilic fluorescent dye DiI. We find that IL2 dye-filling depends on salt concentration, but not osmolarity. Low salt prior and during incubation is important for efficient dye uptake. Additional parameters that affect dye-filling are the speed of shaking during incubation and the addition of detergents. Our modified dye-filling procedure provides a reliable method to distinguish mutant alleles that stain amphids and phasmids, IL2 neurons, or both. An additional benefit is that it can also stain the excretory duct. The method allows genetic screens to be performed to identify mutants that selectively affect only one of the sensory structures or the excretory duct.

Jardim NS et al. (2024) J Vis Exp "Using Low-cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans."

Silva AC, Avila DS, Jardim NS

[J Vis Exp, 2024]

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

Mota AAR et al. (2018) ACS Omega "From Live Cells to Caenorhabditis elegans: Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative."

Neto BAD, da Silva WA, de Souza Cintra GA, Mota AAR, de Oliveira HCB, Assumpcao JAF, Freitas-Junior LH, Correa JR, de Andrade LP

[ACS Omega, 2018]

The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD-coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named <b>BTD-Lip</b>). The use of <b>BTD-Lip</b> for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live <i>Caenorhabditis elegans</i> (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.

Amin S et al. (2020) Front Microbiol "Degradation and Toxicity Analysis of a Reactive Textile Diazo Dye-Direct Red 81 by Newly Isolated Bacillus sp. DMS2."

Madamwar D, Amin S, Desai C, Divecha J, Rastogi RP, Chaubey MG, Jain K

[Front Microbiol, 2020]

An efficient diazo dye degrading bacterial strain, <i>Bacillus</i> sp. DMS2 was isolated from a long-term textile dye polluted environment. The strain was assessed for its innate ability to completely degrade and detoxify Direct Red 81 (DR81) textile dye under microaerophilic conditions. The degradation ability of strain showed significant results on optimizing the nutritional and environmental parameters. Based on statistical models, maximum efficiency of decolorization achieved within 24 h for 100 mg/l of dye supplemented with glucose (0.02%), MgSO₄ (0.002%) and urea (0.5%) at 30C and pH (7.0). Moreover, a significant catabolic induction of a laccase and azoreductase suggested its vital role in degrading DR81 into three distinct metabolites (intermediates) as by-products. Further, toxicity analysis of intermediates were performed using seeds of common edible plants, aquatic plant (phytotoxicity) and the nematode model (animal toxicity), which confirmed the non-toxic nature of intermediates. Thus, the inclusive study of DMS2 showed promising efficiency in bioremediation approach for treating industrial effluents.

Shakir MA et al. (1993) Neuroreport "A role of ADF chemosensory neurones in dauer formation behaviour in C. elegans."

Siddiqui SS, Shakir MA, Miwa J

[Neuroreport, 1993]

IN Caenorhabditis elegans, mutants in osmotic avoidance behaviour (osm), which fail to avoid high concentrations of salts and sugars, have been previously identified. These osm mutants are also defective in dauer larva formation, and fail to take up fluorescein dye in six pairs of amphid neurones (ADF, ADL, ASH, ASI, ASJ, and ASK) and two pairs of phasmid neurones. Analysis of the FITC dye uptake by osm-3 mutants show that seven of the eight osm-3 alleles can take up FITC dyes in one pair of amphid neurones, ADF. Comparison of dauer larva formation behaviour in different osm-3 alleles shows a direct correlation between improved behaviour and FITC dye uptake. Therefore, these allelic strains are useful in defining the role of ADF neurones in dauer larva formation.

Mendes S et al. (2011) Bioresour Technol "Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters."

Mendes S, Martins LO, Viegas CA, Leitao JH, Ramos CG, Farinha A

[Bioresour Technol, 2011]

The azoreductase PpAzoR from Pseudomonas putida shows a broader specificity for decolourization of azo dyes than CotA-laccase from Bacillus subtilis. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We show that addition of CotA-laccase to PpAzoR reaction mixtures lead to a significant drop in the final toxicity. A sequential enzymatic process was validated through the use of 18 representative azo dyes and three model wastewaters that mimic real dye-containing effluents. A heterologous Escherichia coli strain was successfully constructed co-expressing the genes coding for both PpAzoR and CotA. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourization levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.

Bossinger O et al. (1992) Dev Biol "Cell-cell communication in the embryo of Caenorhabditis elegans."

Schierenberg E, Bossinger O

[Dev Biol, 1992]

We have investigated the pattern of cell-cell communication in embryos of the free-living soil nematode Caenorhabditis elegans. For this, we have established a method for microinjection of tracer dyes into individual blastomeres. After iontophoresis of fluorescent dyes of different molecular weights (Lucifer yellow, LY, M(r) 457; rhodamine-labeled dextran, RD, M(r) 4000), we can visualize intercellular communication pathways. The dye-spread of LY, indicating communication via gap junctions, becomes first visible in the late 2-cell stage. From the 4-cell stage onward all cells appear to be well coupled by communication channels, which allow the free diffusion of LY. In contrast, RD remains restricted to the injected cell and its descendants. After the primordial germcell P4 has been generated in the 24-cell stage, dye-spread of LY into this cell and its somatic sister D is delayed. However, the restricted dye-coupling of D is only temporary. After a brief period it joins the somatic compartment. With the beginning of the morphogenesis phase the two existing germline cells (the daughters of P4) are completely uncoupled from the soma, while the latter still forms a single dye-coupling compartment. Only during the second half of embryogenesis different separate somatic communication compartments are established. We followed the pattern of intercellular communication in the alimentary tract and found a progressive restriction into smaller dye-coupling units. Our data are compared to those found in other systems and discussed with respect to cellular determination and differentiation.

Phiri AM et al. (2017) Parasitology "A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases."

Behnke JM, de Pomerai DI, Buttle DJ, Phiri AM

[Parasitology, 2017]

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Guo YM et al. (2008) Nat Methods "Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP."

Zheng G, Lin R, Li WH, Guo YM, Chen S, Shetty P

[Nat Methods, 2008]

To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan-local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.