by Richard L. Howey, Wyoming, US
Killing delicate and contractile organisms presents a set of difficult and frustrating problems for the amateur scientist. Many elaborate, and sometimes tedious, methods have been developed over the past 150 years. Some of them involve the use of substances which today are unobtainable legally by amateurs and legally obtainable by professional researchers only after coping with copious bureaucratic procedures and forms. In the 19th Century, medical schools often had great difficulty obtaining cadavers for dissection and, as a result, medical science frequently ended up subsidizing grave robbers, thus creating a cottage industry.
Narcotics, such as cocaine hydrochloride, were often used as a means of anesthetizing small invertebrates before killing and preserving. Ironically, today, any street-wise college or high school student can obtain it with little difficulty and, if he or she is careful, with only minimal risk from the authorities. Physiological risks are quite another matter. What the authorities should do is donate confiscated drugs to medical research institutions, hospitals, Dr. Kevorkian, and universities. After all, what are a few more addicted doctors and drugged-up, spaced-out professors?
Chloral hydrate was often used as an anesthetic for small invertebrates and also as an ingredient for Hoyer's medium, which is an excellent mountant for nematodes and other small creatures. Today an amateur can't obtain it and professionals have to go through all the paperwork nonsense. So what is the amateur to do?
Fortunately, there are a few simple techniques which can be quite effective for aquatic invertebrates and I will mention a few convenient ones before I go to the topic of natural asphyxia.
1) Some marine invertebrates can be effectively relaxed and killed by transferring them to fresh water. In general, it is best to do this by means of a gradual infusion of freshwater into a small amount of salt water in which the specimens have been allowed to relax. With this method and the others discussed below, timing is crucial. When the specimen no longer responds to prodding or other significant tactile stimuli, then it may be ready for fixation and preservation. I say "may", because some organisms are so contrary as to not respond to any tactile stimuli, but will contract violently the moment a fixative is added.
With those that are less perverse, one has to time the addition of a fixative, so that contraction does not occur, but maceration of the tissues has not yet begun. This has to be determined by trial and error.
2) A very useful field technique for many marine invertebrates and some freshwater ones is the use of Epsom salts (magnesium sulfate) and the good news is that it's cheap and easily obtained at a local store. Furthermore, plain old Epsom salts seems to work as well, if not better, than more expensive laboratory or research grades of magnesium sulfate. It is easy to use, safe, convenient to carry into the field and inexpensive—an unbeatable combination.
The crystals can be added directly to the water or you can make up a solution (about 33%) and add it gradually. Again, timing is crucial; you have to observe the progressive effect on your specimens. For some very small specimens, it may take only a matter of minutes; for larger ones, sometimes hours or even overnight treatment is required.
3) For some micro-invertebrates, such as, rotifers, suddenly flooding them with boiling water can produce a number of nicely extended specimens.
4) A cell biologist I know, recommends a simple mixture of 1 part undiluted formaldehyde to 1 part absolute alcohol. This must be used with care as it is both toxic and flammable. Trial and error is the only way of determining whether or not it will be effective for the organisms you are working with.
5) Ordinary soda water, such as that which you can by in any market, can also be useful with some organisms.
6) A few researchers have begun to investigate the effectiveness of microwaves as a means of killing micro-organisms prior to fixation. This is an area where the amateur can readily carry out experiments and make interesting contributions. This should be done only on micro-organisms and only with the proper glass or plastic containers.
7) At last we get to natural asphyxia. The key factor here is learning to communicate with the particular organism in question so that you can convince it to hold its breath until it passes into unconsciousness. (Sorry, I just couldn't resist.)
Most aquatic organisms require oxygen and a prolonged absence often induces a stupor which causes the organism to relax. Place your specimens in a jar or vial in a small amount of water. Take a suitable quantity of filtered pond water or artesian water and boil it vigorously to drive off the dissolved oxygen. Avoid tap water or distilled water. Tap water often contains excessive amounts of salts as well as chlorine and distilled water is virtually free of all salts, including those which many organisms require, and it can produce negative effects, such as contraction, of the organisms. After boiling the water, let it cool to room temperature and then fill the jars or vials containing the specimens right to the very brim. This is important, because when you insert the stopper or put on the lid, you don't want any airspace between the lid or stopper and the water. If there is such an airspace, the water will begin to absorb oxygen and you may end up defeating your purpose.
It is best to use containers that you can put on the stage of your stereo microscope and check from time to time. Again, the timing is critical and can be determined only by trial and error, so it's a good idea to keep careful records for future use. Too little time and the organisms will contract when a fixative is added; too much time before adding the fixative and the organisms will begin to deteriorate.
With the new computer technologies available for building elaborate databases, I sometimes think that we, as amateurs, are neglecting an important opportunity, especially with regard to creating a reference base for all kinds of techniques that apply to specific organisms. Why should all the trial and error work go to waste, so that person after person ends up repeating essentially the same trials? Records of such experiments could provide valuable guidelines and techniques for other amateurs. What about the professionals? Well, let them pay us for it and help support our researches.
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