Electromagnets and Worms: Methodology

In a conference in D.C. this week my mentor, Dr. Deheyn, is presenting some of the work I've done in creating an experiment that will test the effects of a magnetic field on the bioluminescent mucus of the tube worm Chaetopterus variopedatus. Since the mucus is Ferrous and a reaction involving Fe⁺² and H₂O₂ (peroxide) is necessary for the organism to luminesce, there is a possibility that magnetism might increase the luminosity (brightness) of the mucus by somehow stimulating the ferrous reaction within the mucus. Previous tests were conducted with rare earth magnets (see this blog for the previous test procedures), but after I obtained erratic results I decided to construct an electromagnet (click here for construction process), which would produce a uniform electromagnetic field that could be regulated.

PROTOCOLS

An electromagnet was created by coiling 30-Gauge wrapped wire around a plastic tube multiple times (more coils = stronger magnetism) designed to hold a 1.5 ml tube. This electromagnet was connected to a Laboratory DC Power Supply and supplied with 17.35 Volts of coarse current. For the experiments, two 1.5 ml tubes containing 750 µl of Artificial Sea Water and 750 µl of Raw C. variopedatus mucus apiece were prepared.

One C. variopedatus worm was extracted from its tube and brought into the lab, where it was subsequently decapitated and soaked in 1000 µl of Potassium Chloride for 7 minutes and 30 seconds. Then the extracted mucus/KCl solution was diluted with 750 µl ASW. 100 µl samples were taken from each tube and analyzed for Kinetics on a Luminometer.

TEST 1

   The samples from Test 1 were analyzed in 5 minute intervals for 50 minutes and showed no significant trends over the 10 analyzed samples.

TEST 2

Test 2 was conducted with the same protocols. The altered variable was the interval at which samples were taken. 100 µl samples were pipetted in 10 minute intervals and analyzed rather than 5 minute intervals. The data showed no significant trends in Test 2 either, however.

TEST 3

The very nature of this bioluminescent mucus (of any mucus, in fact) is that it is very viscous and adhesive. Therefore, one reason why the results were so erratic could have been due to the fact that the samples were from the same batch, but there was no guarantee that the samples had contained similar proportions of mucus to ASW. Testing one sample of mucus and then testing a totally different sample to observe the luminescent decay would not necessarily demonstrate the decay of the first sample. So for Test 3, no samples were taken at intervals.

Instead, two Luminometer analysis tubes were filled with750 µl of the raw mucus/KCl mixture apiece. One was placed in the control container and the other in the electromagnet. The whole 750 µl solution was placed into the luminometer at 10 minute intervals. The solution remained undisturbed by pipets, as no samples were extracted. The ability to place the whole tube inside the luminometer allowed for a consistent luminous decay to appear on the graph since it was the same (not different) sample being tested each time. In addition, the accuracy of the reading on the mucus within the magnetic field was equivalent to the accuracy of an extracted solution. In other words, if there were any bad effects from taking the sample away from the magnetic field, that discrepancy was consistent throughout all tests as far as data collection goes, however, if the mucus required constantsubjection then the 10 seconds for which it was outside of the field might have significantly affected the experiment. 

The 3^rd column shows how much the luminosity has decreased during the ten minute intervals. Surprisingly enough, the Total Decay, which was the difference between the luminosity after 70 minutes and the initial luminosity, was greater in the mucus in the electromagnet than in the control. The mucus in the electromagnet initially started out much higher, but subsequently dropped to hover above the luminosity of the control. Although in the end it was still brighter, it decayed much more. If anything, it stimulated higher initial luminosity which possibly led to more decay afterwards. Nonetheless, the Control had a quicker rate of decay than the Electromagnet.

So far, the best method seems to call for 750 µl of raw mucus/KCl solution in luminometer tubes, analyzed for luminosity every five minutes and testing the effects of adding metal or any other modifications in the apparatus. The length of the test would have to be determined by the point at which the luminescent activity is no longer significant. Test 3 was stopped before the luminescent activity dropped to that level.

The chart below displays the differences in average luminosity throughout the test and the rate of decay in order to determine which one decayed more and which one decayed faster.

Since the Control decayed at a faster rate than the mucus subjected to an Electromagnetic field, I'm going to continue using the protocols of Test 3 for more experiments to make sure that this trend is not anomalous. 

Electromagnets and Worms: Construction

I have been working on constructing a simple electromagnetic apparatus to test the effect of a magnetic field on the bioluminescent mucus of the tube worm Chaetopterus variopedatus. Since the mucus is Ferrous and a reaction involving Fe⁺² and H₂O₂ (peroxide) is necessary for the organism to luminesce, there is a possibility that magnetism might increase the luminosity (brightness) of the mucus by somehow stimulating the ferrous reaction within the mucus. Previously, I used rare earth magnets to stimulate the mucus (see this blog for the previous test procedures), but after erratic results and no significant data, I decided to construct an electromagnet so that the magnetic field would be uniform throughout the sample and regulatable. 

Below are some photos that show the construction process:

I took a conical plastic test tube that wasn't too wide or thin, but just right to wrap wire around and fit Eppendorf tubes in.

I cut the test tube at the top, but only cut part off the cone bottom so that the diameter was small enough to allow me to rest the lip of a 1.5 ml tube on the rim.

I placed a spool of 30 gauge wrapping wire around a screwdriver suspended by a column chromatography clamp so that I could easily spin the wire into my own electromagnetic coil (bottom left with the blue cap).

This is what the coil looked like halfway through. It was already thick, but the more coils in an electromagnetic there are, the stronger the magnetic field will be.

I had designed the holding tube around which the wire was coiled so that a 1.5 ml tube could rest inside the lip. 

Here, the electromagnet is finished and thick with coils; a luminometer testing tube rests inside.

The electromagnet is connected to the positive and ground connections on a Laboratory DC Power Supply. The current and voltage are turned up to full power and 17.36 Volts are being sent through to the electromagnet. The DC power is constant and the electromagnet has a uniform field inside of the tube, creating dependable constants for the experiment.

ICP Analysis Update

We have results from the Inductively Coupled Plasma Mass Spectronomy (ICP-MS), but have not yet processed the data. (Click here for a short explanation of what the ICP-MS is). Two things that seem promising, though, are the facts that the Calcium Carbonate was so saturated that the machine couldn't obtain an accurate measurement and aluminum was present in the ossicles. The first thing is unusual due to the fact that the sample size consisted of milligrams (not even grams) of ossicles which, like all bones, contain lots of calcium carbonate, but usually not in such high saturation. Also, the fact that aluminum is embedded in the tiny bones is strange in light of recent findings that aluminum is a neurotoxin that, though abundant in nature, is not an essential element and poses a threat to organisms. However, in an analysis of iridescent fish scales it was found that there were high concentrations of aluminum that might play a part in the scales' light refraction. Since we already found out that the ossicles are specialized for fluorescent light transmission by transmitting green visible light wavelengths, it is possible that aluminum also enhances the ossicles' light enhancing properties.

Since we were unable to get a calcium reading with our previous samples, I'll be sorting and prepping new samples (about 1/10 of the amount we tested last time) for another ICP analysis. Hopefully there won't be a saturation of calcium in these readings since the proportion of Calcium Carbonate is what we compare all the other measurements to in order to create ratios of metals to calcium. (I.e. there are 10 parts Calcium and 1 part Magnesium.)  Once that's done we'll have to make sense of all the measurements and numbers and proportions.

Prepping for the Plasma Machine (ICP)

Hours upon hours of ossicle sorting have almost paid off—we're finally going to analyze the samples using the ICP-MS.


I'll explain in a moment, but first: some ossicle samples from the brittle star O. californica were analyzed using hypo-spectronomy and found to transmit light between wavelengths of 530 and 590 nanometers—the spectrum of visible green light—which happens to be the color of brittle star bioluminescence. Therefore, it is very likely that the tiny bones in the arms of certain brittle stars are optimized to transmit and ideally amplify the light from their luminescent reactions. How amazing is that?! These are specialized bones in a specific luminescent system that aid in defense or mating or whatever else the brittle stars use luminesce for. Well, anyways, I was excited.

Okay. Back to the issue of ICP analysis.

ICP-MS stands for Inductively Coupled Plasma Mass Spectronomy. As another intern explained to me, it is the process of heating up argon gas into the fourth state of matter, that is, plasma. Once there is plasma in the chamber of the machine, samples (which need to be in liquid form) are sent into the chamber in a kind of mist after being nebulized. The drops vaporize near the plasma and solids break down into individual atoms after being vaporized and certain chemical elements are ionized. These ions are sorted in a special device for mass spectronomy, which can determine the presence of elements of atomic mass 7 to 250 and their proportions in the sample. In the case of the brittle star ossicles, we want to find out how much of what elements are in the bones of luminescent brittle star species compared to proportions of the same elements in non-luminescent species in order to determine if those elements (mainly metals) play any role in specialized light transmission and refraction.
During this and last week, I continued to sort more of the A. pujetana ossicles since I had less of that sample compared to my O. californica and A. squamata samples. I sorted as much as I could on Thursday and then weighed all the samples. All the weighing and digesting, etc. had to be done in order so that it would be easier to organize the samples once we got the results back from the ICP. The order I used was: Blank_1, Blank_2, Blank_3, OCL, OCT, OCV, ASL, AST, ASV, APL, APT, APV. The first two initials stand for the species name (i.e. O. californica) and the last letter stands for what kind of ossicle it is, that is, Lateral Shield, Top/Bottom Shield, or Vertebrae. 
After the ossicles were weighed, I added approximately 150 microliters of 70% Nitric Acid (or about two large drops from a disposable glass pipette) to each tube in order to dissolve the samples into a liquid. A glass pipette was used instead of the more accurate mechanical pipette because Nitric Acid is so strong it or its fumes could potentially harm the sensitive measuring device in a normal pipette and make it unusable or in need or serious repair. Once the acid was added, the ossicles sat for 12 or more hours to insure optimum digestion.

The container and tube were tared, then the sample was added. The weight (usually in milligrams) was recorded in a text file on the computer that the scale was hooked up to. [Note: in the picture it shows a tube with a cap on; in the actual weighing the cap was never put on in the scale due to the fact that if more grams are tared the less accurate the measurements of the samples will be.]

"JUSTRITE: Acids and Corrosives Storage Cabinet"

Not More than 70% Nitric Acid. It's extremely bad for your health (note the skull and crossbones), severely reactive, and extremely dangerous on contact, but worry not! it's not flammable. You need to wear goggles & shield, lab coat & apron, and proper gloves when using this acid under a vent hood.

The amazing vent hood, which, in the clean room, is always blowing on high, keeping this part of the lab at high negative pressure and sucking out any harmful or plain stinky acid or chemical smells.

 In the pictures below, acid is added to the sample of O. californica vertebral ossicles and bubbling occurs due to the reaction between the calcium carbonate in the tiny bones and the Nitric Acid, which facilitates the creation and release of Carbon dioxide gas.

The tubes, once the sample and acid are weighed, are put in a tube rack under the fume hood so that the acid can fully dissolve the ossicles into a fluid liquid to be diluted with MilliQ water after full digestion. The resultant liquid will be nebulized into the ICP-MS for analysis.

I recorded the weights on the computer and in my notebook. Notice how minuscule the sample sizes are in terms of weight (in grams).

I have yet to get the results from the ICP, but am nonetheless optimistic that they will be quite interesting and certainly worth a follow-up post!

In Search of Beetles

Today my mentor, Jenny (another intern who attends UCSD) and I went to the Natural History Museum to search their entomological archives. Basically, they have a very large collection of dead bugs. Technically, their collection consists of over 900,000 insect and arachnid specimens, which is relatively small compared to some other collections (of course, the Smithsonian has 35 million specimens). In this vast archive of thousands of organisms we were to search for iridescent or fluorescent organisms. This required searching through cabinets full of glass display cases that contained tens of dead insects pinned to foam mats.

Why?

My mentor had an upcoming funding presentation and when it comes to research science, you need to have some great PR once in a while to keep the public informed and supportive, and your funding steady. We have been working on iridescent fish scales and butterflies (the standard specimen when it comes to structural light coloration studies), but we hadn't yet looked at other land organisms. However, if we found some intriguing insects or what have you that were found in the San Diego area, it would be a good continuation of our structural coloration research and possibly serve as a contrast between marine and terrestrial iridescence.

When we arrived at the Natural History museum and taken behind-the-scenes to the entomological section, we had no idea what we were looking for. Granted, we knew what the glimmer and shine of iridescence looked like, but we were to look through hundreds, if not thousands of insects. At least the entomologists had an idea for where we should start.

The room was full of those large rolling shelving units like they have in the archives of big libraries. The metal cabinets lined the walls and only opened for one small passageway at a time before they closed their dull teeth behind you with the slow creaking sound of the old wheels. These were definitely archives--stored away behind dull gray doors that often stubbornly refused to open, unwilling to reveal the amazing specimens that were trapped in this fluorescent-lit prison, a zoo and a cemetery alike.

Inside the gray doors were wooden cases with glass on top to display the specimens, every one of which was pinned to the foam beneath. Many of them had labels that revealed (in what has to be some of the smallest legible writing in the world) the date of collection, genus/species, location of collection, and other notes. All of this information was crammed perfectly onto small tags pinned underneath the bugs.

The curator suggested we look at insects of the order Coleoptera, commonly known as beetles. They make up a lot of the collection at the Natural History Museum besides butterflies and Coleoptera actually has more organisms than any other order. So in a way, it was good to start there because there was so much to choose from, but that also meant that between the 3 of us, there were thousands and tens of thousands of beetles waiting to be seen. In the picture above there are about 45 cabinets. In each cabinet, there were anywhere from 6 to 12 display cases full of tens of beetles. And we looked through all the cabinets shown in that picture and more!

Some beetles looked plain and unassuming, but had great color contrast, which is important in the natural world. Mostly, we were looking for some beautiful examples of iridescence that was preferably not green iridescence, which is really common. We wanted more reds or purples, which are harder colors to produce and much rarer in nature.

Although these beetles don't seem to be anything special, they are an example of nature's less showy wonders.
Their coloration (which is structural [all beetle coloration is structural]) consists of a white, which deflects all wavelengths of light, and a brown, which absorbs many wavelengths of light. This contrast is probably used for camouflage or to confuse predators, but to produce two very contrasting colors simultaneously is an amazing development.

Some very cool beetles. There's a very iridescent purple-blue-teal-green beetle in the middle and a scarab that looks like it truly was preserved in gold.

These otherwise boring light brown beetles iridesce green as you can see on many of their backs. There are a few that haven't been hit by the light in the right way or at the right angle, but many iridesce a nice, light green.

From Arizona, these green beetles had amazing silver stripes down their backs. The light refracting off of them is the iridescence of the silver lines.

Striking reddish purple coloring with green trimming defines these beetles.

The common green iridescence with red highlights and ...posteriors.

An assortment of bees, in the upper left there are some smaller bees that the picture below depicts (in poor lighting).
These bees had iridescent "fuzz" on them and usually the smaller ones tended to be more colorful.
Also, in the picture above, a normal common bee that we usually see would be a size between those small bees and those giant bees.

It might be hard to see in the poor lighting of the photo, but these small bees have some strong iridescent green colors. Surprisingly enough, the none iridescent portions of some of them turned out to be fluorescent, which is certainly intriguing!

The entomological curator (left) removes an insect that my mentor (right) is renting from the Natural History Museum.
We are allowed to borrow the specimens for a year and if we need them even longer we can just contact them and extend our lease.

In the end, we ended up borrowing 13 insects for further examination in the lab. One of the most amazing things was that we borrowed a beetle that was collected in 1832! We were allowed to lease a 180 year old insect and take it back to the lab with us. Amazing, huh?

Although looking through all those beetles took us the better part of a day, ever the gluttons for punishment in the name of science, it was decided that we would probably go back in the near future to look for good damselfly or dragonfly specimens. Our only concern was being a burden to the ever helpful curator, who had the daunting task of rearranging the collection according to the ever-changing taxonomy standards that seem to be constantly re-classifying insects. It's no small task to reorganize a small entomological collection (only 900,000 bugs, remember?) The best of luck to him and to us as we delve deeper into the world of terrestrial organisms, which us marine biologists know surprisingly little about...

A Day of Photo Microscopy

Two other interns in my lab, David and Caitlin are photographically analyzing fish scales to see if their coloration and iridescence is due to structure or pigmentation. In order to do this, they take photos of raw fish scales under white, green, cyan, and blue light. The whole scale, the outside and inside edges, and the center are all photographed. Different exposure settings (as determined by Auto-Exposure) are used for white light and green light; but the exposure under green, cyan, and blue light remains the same. Once pictures of the raw scales are complete, scales soaked in an enzyme that digests fish flesh and any pigmentation are photographed using the same protocols. If there is still coloration or iridescence on the digested scales (which end up being very translucent), then that might signify structural coloration or iridescence in the scales of that particular fish. There have been no conclusive results as of yet, however, we are still in the process of photographing numerous samples from quite a few different colorful fish species.

The scales below were pulled from an orange and black Clarion Angelfish that probably looked like the picture below when alive:

These are all pictures of the black scales. Most of the scale looks translucent. This is because most of the coloration is at the very end of the scale (far left in picture). When the magnification is increased, there are black dots that become more distinguishable. Is it these small dots alone that give the fish its black color? Is the coloration due to pigment or the structure of the scale? These are questions we seek to answer. But first, at least a couple of black scales had to be photographed.

Note: If you click on the picture, it will open up in its actual size, allowing you to see the high resolution and detail that the microscope camera is capable of.

Black Scale One Face Up (Some Overlap)

Full Scale View:

Detail of Tips, note the black dots especially in the second picture

Bottom edge that is usually under other scales on the fish

A small overlapping scale with lots of visible black dots

(from what Caitlin told me, an overlapping scale isn't as common as you'd think)

Middle of the Scale

Higher Magnification Center of Scale

Black Scale One Underside

Well, if you made it to the end (or just skipped on down here), then I'd first like to congradulate you. When a phase of this project is nearing conclusion, tens and tens more photos will have to be looked through, compared, analyzed, etc. to give an accurate photographic analysis of whether or not fish scale iridescence and coloration is due to pigmentation or structure. It is interesting to note that this is only one set of photos for only one scale. There are always more than 2 sets of photos per scale color according to location on the fish, which means there are anywhere from 6 to 10 photo-sets per fish, and this was Fish #7. Not to mention that I'm not counting the photo sets of digested scales, which these photos will be juxtaposed with.
In addition, this is one of several photo sets I took of the Clarion Angelfish scales.