Household compounds to reveal the invisible

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Introduction:

Common chemicals and compounds can be used to good effect for visualizing things that are otherwise hard to see. Whether using art supplies, household cleaning agents, or other things, one can gain a lot of information with simple techniques. These approaches have been used in numerous scientific publications for different purposes, and are still useful today. This article will be a place to share these.


Sumi ink and India ink

Dendraster_SumiInk.jpg


Fig. 1: Sumi ink reveals the jelly coat around sand dollar embryos. Unhatched Dendraster excentricus embryos were placed in sumi ink diluted about 1:5 with seawater, and imaged using a compound microscope. Without sumi ink (left) and with sumi ink (right).

Sumi and India inks are suspensions of fine soot particles. Because the particles are so dark, they show up well in the light microscope. The ink has multiple uses: visualizing the structure of highly transparent gels1, such as the jelly coats around invertebrate eggs (Fig. 1)[1], visualizing flow in the microscope [2], and tracking cells during tissue growth and development [3], among others.

For example, to visualize the jelly coat around sea urchin or echinoderm eggs one can put the eggs or embryos into a suspension of sumi ink in seawater. Shroeder (1980) used this method to visualize the sea urchin "jelly canal" and investigate how it was related to the site where the sperm fuses with the egg, and the future axis of the embryo (to see the jelly canal, the embryos had to be put in the ink suspension immediately at spawning, before the jelly swells [1]). Shroeder ground a sumi ink stick to make the sumi ink suspension, but one can also use pre-made sumi ink: just dilute it in seawater, mix with embryos on a slide, and observe in a microscope.

One can also use India or sumi ink to visualize the structure of the extraordinarily intricate mucus house of larvaceans. Larvaceans are small, planktonic invertebrates, closely related to vertebrates. They inflate a complex set of filters made out of mucus, and pump water through this "house" to catch small particles of food. Finding larvaceans can be challenging, but if one can find them and keep them alive long enough to make their house, all one has to do is add some dilute sumi ink to their seawater. The filters will appear dark.

Food coloring.

One can visualize transport with food coloring or other inks and dyes quite well. For example, one can cut a flower stem and stick it into food coloring, which will travel up the xylem, staining the network of vessels in the petals {photo needed}.

Detergent and bleach

Sometimes the easiest way to see something is to get rid of everything else. One can often get rid of cells using detergent to break cell membranes, leaving the extracellular matrix after washing (Fig. 2){citation needed; would be nice to have a better example}. One can also see fine skeletal structures well by bleaching away organic matter (Fig. 3).

DendrasterPlusDetergent.jpg


Fig. 2: Detergent reveals the extracellular matrix around sand dollar embryos. At fertilization, echinoderm embryos produce a layer of clear extracellular matrix (the hyaline layer) that surrounds the cells of the developing embryos and helps hold them together. In some species this layer is several micrometers thick, but in the sand dollar Dendraster excentricus it is quite thin and hard to see. On the left is a living 1-cell stage sand dollar embryo. From outside in, one can see a sphere of red pigment granules embedded in the jelly coat (see Fig. 1), the fertilization envelope (elevated at fertilzation), and the zygote itself. The hyaline layer is quite thin and difficult to see. Placing an embryo into dilute detergent (Triton-X 100) destroys the cell membrane, revealing the hyaline layer (right). The method is very crude, and disrupts a lot of things, does show the bag of material that surrounded the cell.

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Fig. 3: Bleach reveals how individuals within a colonial animal share nutrients and neural signals. Bryozoans are a type of filter-feeding animal that grows by budding off new individuals which remain connected to each other. The individuals share neural signals (even between genetically distinct colonies[5]!) and nutrients. In one major group of bryozoans, the individuals are enclosed in skeletal boxes lined with calcium carbonate. To grow, nutrients have to pass through the skeletal walls through pores. Shown here is one wall that connected a parent individual (of Membranipora membranacea) to the individual that budded from it, and the pores that connected them. The pores were revealed by bleaching the colony in household bleach for a few minutes, then rinsing in distilled water, to destroy the tissues. The wall (approximately 0.1 mm wide) was broken off from the rest of the colony and imaged with a microscope.

Glycerin and clearing

One of the biggest factors that makes biological samples opaque is differences in refractive index between the denser material and water. To make those samples transparent enough to see inside the sample, one can equalize the refractive index differences by substituting water with glycerin (or glycerin mixed with water). {photos, citation, and methods needed}

Literature Cited

1. Schroeder TE. 1980. The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos. Exp Cell Res.128(2):490-4. DOI:http://dx.doi.org/10.1016/0014-4827(80)90088-9. http://www.sciencedirect.com/science/article/pii/0014482780900889
2. von Dassow M, Davidson LA. 2009. Natural variation in embryo mechanics: gastrulation in Xenopus laevis is highly robust to variation in tissue stiffness. Dev Dyn.238(1):2-18. PMCID:PMC2733347. DOI:10.1002/dvdy.21809. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19097119
3. Campbell RD. 1973. Vital Marking of Single Cells in Developing Tissues: India Ink Injection to Trace Tissue Movements in Hydra. J Cell Sci.13(3):651-61. http://jcs.biologists.org/content/13/3/651.abstract
4. McLane Louis T, Chang P, Granqvist A, Boehm H, Kramer A, Scrimgeour J, Curtis Jennifer E. 2013. Spatial Organization and Mechanical Properties of the Pericellular Matrix on Chondrocytes. Biophysical Journal.104(5):986-96. DOI:http://dx.doi.org/10.1016/j.bpj.2013.01.028. http://www.sciencedirect.com/science/article/pii/S0006349513001331
5. Shapiro, D., 1996. Size-dependent neural integration between genetically different colonies of a marine bryozoan. J Exp Biol 199, 1229-1239.

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