In this laboratory¹ you will apply the techniques of dilution and measurement of concentrations to investigate a physiological parameter: the hemolymph (or blood) volume of an insect. A parameter is a quantifiable variable of a system you wish to study. Determining values for a parameter such as hemolymph volume allows you to measure the effect of a treatment in an experiment, or to calculate other things. For example, you can determine the absolute amount of a metabolite such as an amino acid if you know both its concentration and the volume of the hemolymph containing it. We have chosen the cockroach because it is readily available and it is large (for an insect!).

Your objectives are:

1. To determine cockroach hemolymph volume by injecting a known amount of Amaranth Red dye, and measuring its dilution by the hemolymph.
2. To think about and correct for possible errors in the method.

Outline:

1. Derive a standard curve for dye concentration by a method simulating the dilution by the hemolymph.
2. Inject a cockroach with a known amount of Amaranth Red.
3. Extract hemolymph samples at given times
4. Plot your data and calculate hemolymph volume.

My friend Steve and I were partners for the cockroach experiment. Steve was the data recorder, I was the grunt who did the actual dirty work. I told you he was smart.

The experiment consisted of us diluting cockroach blood in situ with Amaranth Red dye. Some reports have indicated a degree of carcinogenicity² with this dye, and the lab manual that we were working from told us to be careful with it because it was carcinogenic. As part of my grunt responsibilities, I had to draw the extracted blood/dye mixture up into a pipette (a long measuring tube).

Drawing liquid into a pipette is done a number of ways. These ways usually involve a mechanical suction device that you attach to the top end of the pipette. The least desirable, and most dangerous method is to use your mouth as the mechanical suction device. You suck on the end of the pipette while the tip is immersed in the fluid that needs to be drawn.

Because we had spent so much time arguing deciding which one of us should do the grunt work, all of the mechanical suckers ended up being taken by the other groups in the class. It fell to me to pipette this mixture of cockroach blood and Amaranth Red by mouth.

Life in the fast lane has destroyed many of my useful brain cells over the years — especially the ones responsible for memory. At this point in the story, however, my memory becomes even hazier. That is simply because of emotional anguish and mental trauma.

I remember looking at the bottom of the test tube that contained an extremely red mixture. It wasn’t a lot. This is to be expected. Cockroaches are not huge hemolymph reservoirs since they are so small. The liquid was red and for some reason had a bubbly kind of froth on top.

I can’t remember why we couldn’t just stick the test tube into the spectrometer to measure the optical density then. There’s obviously a good reason and if I followed my footnote number 1 below I’d probably figure it out, but that’s a digression from my story.

I remember Steve looking at me with an expression of, “So what are you waiting for? Start sucking.”

So I did.

The pipette that I was using had a relatively thin lumen and required quite a bit of suction to get it started. Once started though, capillary action helped out a bit.

I drew the blood mixture up into the pipette and was quite pleased with the way it was pulling in. I was so pleased, in fact, that I decided to try to empty the test tube if possible.

Sucking away at the end of the pipette, the test tube slowly emptied — and then I got to the froth that had been sitting on the top of the blood.

Drinking milkshakes has never been the same since that day.

I remember the gurgling within the test tube as the froth sucked up. That’s the same sound that a nice chocolate milkshake makes when you get to the bottom of it. Unfortunately the way the air bubbles in the milkshake’s straw break the suction and cause the drink to deliciously sputter into your mouth is the same way the bloody froth broke the suction in the pipette and caused the bright red cockroach hemolymph to sputter into my mouth.

Steve’s eyes slammed open. I think he might have even started running around in circles with his hands clutching out for something to help me with. But what?

He remembers seeing my surprised expression, mouth half open, and red stuff pouring out of the corners of my mouth. It was like I had just feasted on a vampiric meal, only to be surprised that it wasn’t actually blood. In my case, however, it was actual blood — insect blood.

I ran over to the nearest sink and spat out the potentially carcinogenic stuff all the while trying to keep things quiet so that the other people in the class wouldn’t notice what a couple of idiots we were. A ton of copious rinsing was performed afterwards.

I don’t remember if we were able to salvage enough hemolymph to continue with the experiment. As usual, I probably got close to a failing grade on that experiment.

So what did the blood taste like?

To be honest I don’t remember. When I think of the taste, all I remember is the colour red, and the smell of the chemicals around us in the lab. I’m sure the mixture was tasteless.

The point of all of this is that my friend Steve has remained a good friend even though somehow I ended up getting the wrong end of the stick in our joint ventures. That wasn’t the only time we worked together and I got screwed. I remember at least one other experiment: The physics one where we were doing something with a big glass sphere containing Mercury.

What a pretty thing Mercury is at room temperature flowing around in an amorphic homogenous mass. So precious. This neurotoxic metal that gets absorbed through skin is not so pretty, however, when the glass container it is in breaks and it leaks out in the blink of an eye onto the hand of the grunt holding the container.

Yes. that would have to be me.

For some people, death flirted with them in university because of their social activities, but not for me. Death flirted with me during the process of learning.

I’m lucky I’m still alive after those hazy university days.

Footnotes:

1. http://www.ableweb.org/volumes/vol-15/8-smith/8-smith.htm [↩]
2. http://msds.chem.ox.ac.uk/AM/amaranth.html [↩]