Happy new year! How appropriate that today begins a new step in the preparation of a whole insect mounted with pressure. The previous step was maceration and some specimens will always macerate faster or slower than others. Seeing as I must return to work tomorrow I decided to move things along today, even though one specimen appeared unready.
After the (Chemical) Fire
No there hasn’t been an accident, but after just over fifty hours of macerating the ants may appear as though there has been. When they were initially placed in the macerating solution each ant was quite buoyant, floating on the surface of the liquid. Withing twenty-four hours small bubbles were in evidence on the surface of their abdomens and they were noticeably less so. In the early evening of yesterday one of the ants was observed to be exhibiting neutral buoyancy. This morning it was resting on the bottom of the macerating jar with its abdomen appearing markedly lighter in color than the other. Enough time was felt to have passed for the next stage of slide preparation to begin on the sunken ant at least. Operations were carried out on each in any case.
Until today every activity performed on the specimens has consisted of nothing more than placing them in some container of liquid and waiting. At first today will prove no different. The ants will be transfered to an empty container and washed of the macerating solution. After this, any remaining traces of the macerating solution will be neutralized chemically. Finally the specimens will be pressed, dehydrated, and hardened.
A Wash and a Brush Up
Remove the macerated specimens to a small container. I find that a low profile beaker is best but any small container with a spout will prove convenient. Once the specimens are in the empty container pour over them a quantity of distilled water and leave them for not less than fifteen minutes. After fifteen minutes pour off and replenish the water. This activity may be carried out more or less extensively depending on the preparers confidence in ridding the specimens absolutely of the macerating solution.
Some mounters (among them R. M. Allen in his manual The Microscope) recommend performing the process of washing over the course of several hours. It is also possible to hasten the procedure by washing in the more cursory way described above provided an acid is used to ensure the absence of any Sodium Hydroxide after washing. I have read in one account or an other that after removal from the macerating solution the specimens may be washed and placed into a vessel of concentrated acetic acid and left indefinitely. I have never done so personally, I may post about the effect if I ever do.
After the second wash the ants were removed to a Syracuse glass of distilled water. Once there they were lightly spread with a camels hair brush to remove them from the posture they had taken after killing, five drops of glacial acetic acid was introduced to the Syracuse glass. For particularly bulbous insects the abdomen may be pressed gently with a camels hair brush while in the Syracuse glass. This will discharge the liquefied organs and help to make the following step easier. For the small ants I used this was not warranted.
Layout and Pressure
Put out two clean glass slips for each specimen, these will be used to apply the pressure needed to flatten the subject for mounting later. With four slips on the table, one need not use expensive slips of first rate quality provided only that they are clean, remove one ant to the center of one slide and using a corner of blotter paper draw off as much liquid from around the specimen as is possible without drying it entirely. Removing the liquid is not essential, but will help to ease the process of arranging the specimen.
With a clean camels hair brush and fine pair of forceps (or fine, blunted, dissecting needle) arrange the ant as desired on the slip. Care should be taken to extend the antennae, mouth parts, legs, or any portion which is of interest. One should act quickly so that the specimen is never permitted to dry out completely. Once satisfied with the arrangement lower a second slip onto the first so that the head contacts the lowered slip first and the edges of each line up. Carefully apply pressure while maintaining the alignment, failure to do so (lateral movement of either slip) may tear specimen.
Once the slips are as close to each other as careful pressure with the hand permits the slips must be bound together. An assistant could tie a short length of strong thread around either end of the slips, or any number of a variety of spring clips may be applied. Clothes pins may be used but they are quite bulky. The traditional binder clip is superior but one must search out a supply that is composed entirely of plain metal.
Hard and Dry
As soon as the slips are bound together they are placed into a vessel of alcohol sufficiently large and full that the entire slip is submerged. The alcohol should be not less than 90% pure, but denatured alcohol is perfectly acceptable. Two things will be accomplished by the alcohol in this step; the specimen will be dehydrated, and its tissues will be hardened in its flattened condition.
Once in the alcohol the specimens must be left for a period of time that is largely dependent on their size. Allen describes the required time only as “short” and I will not hazard a guess as to the unit of time that best describes. I can say that the slips may be left in the alcohol until it is convenient to carry on. I have left harvester ants pressed as described above in alcohol for as short as three hours and as long as twenty-five without noticing any difference.
Next time, things finally become clear!
How do you clean your cover slips and slides. I can never get mine clean enough.
Cleaning is always a bit of personal preference. I advocate a simple soap and recourse to a weak acid or alkali for stubborn dirt. Once my slip or slide is clean I’ll dry it with an old linen handkerchief and then polish it between two blocks covered with chamois leather. For particular preparations with more stringent requirements I’ll clean a number as above and store them in alcohol ahead of time. Giving each an additional polish with a worn silk handkerchief as it is removed from the alcohol before use.
Ahh! I shall try chamois leather blocks. That’s a good solution. It’s always the drying stage that messes me up. I use lint free microfibre cloths but even they are too fluffy for drying. I’ve broken a few coverslips trying to dry them while holding them by the edges.
I generally wash in soapy water and rinse in deionised because the water here is very hard and inevitably leaves a spot of limescale somewhere.
I have tried alcohol but I haven’t tried weak acids or alkalis. I’m always terribly impatient with slides and coverslips.
Oooh! I have just had an idea! Contact lens solution! That should have lots of proteases and yummy enzymes in it. I also happen to have a few unused bottles upstairs from when I stupidly decided to try contact lenses. They’re horrible.
I’ll confess to impatience as well, that’s in truth the reason for the expedited cleaning method I use as described above. Yeah, that’s the cut corners method, for some really good advice on initial cleaning, and cleaning for re-use the best procedure I’ve ever come across is described in sections 168-171 of Kingsbury and Johannsen’s book Histological Technique. Here’s a link below. Does wordpress allow links in comments? Suppose I’ll find out, rather inept at all of the finer points of blogging I’m afraid…
If your having trouble with contamination from your wash water then that contact lens solution may prove just the thing. I hope to hear of your fantastic success. Coincidently, I agree they are a horrid contrivance, responsible for my only bout with conjunctivitis.