Truly Traditional Mounts I

The holidays is nothing if not cause to remember traditions. -K

In microscopy perhaps no slide is quite as traditional as the dry cell mount, a sealed air filled compartment protecting a dry object. Dry mounts took all sorts of forms from glass covered wooden slides, to full portions of insect, to diatoms and podura scales. However, of all the objects mounted dry perhaps none was quite so enjoyable (and now some common) as the mount of “Forams.”

IMG_1522_scalecrop

Forgive the catalog numbers.

 

Forams, or more accurately foraminifera, were among the most popular of the small fossil specimens to be mounted. In the present day one may be forgiven for questioning the designation as in the past it was somewhat of a common practice to label any small fossil thusly. The ease of acquiring them was no doubt a reason for their ubiquity. If one happens to live in an area that was formerly a body of water simply digging down a few feet in the garden (more about this come spring) will often reveal a layer of sediment full of a quantity of microfossils. Such were commonly mounted as an enjoyable object for examination with low power.

The traditional dry cell mount of forams is nothing more than a glass slip on which is mounted a specimen over an opaque surface. Around this is placed a cell, either a ready made one of aluminum (or these days of glass), or one built up of layers of shellac. The whole is covered by a circular cover glass which is sealed or cemented to the cell. The specimen may be then used as any other object for the microscope, secure from contamination and viewed as intended below a cover of recommended thickness.

To produce a traditional dry mount a few items will be required. Chief among them are a slide ringing table (for more on that see my earlier post on ringing a slide), a supply of round cover glasses, and a cell. For most practitioners the simplest means of producing a cell is by building one up from layers of shellac. One may use store bought, ready-made shellac of course, or the dry material dissolved in ethyl alcohol. If one is already possessed of an arbor press and a variety of circular punches one may form cells from aluminum with little trouble. Whether using shellac or aluminum one should fabricate cells so that they are only just thicker than the specimen vertically, and slightly larger than the cover glass diametrically.

For opaque mounted objects (such as forams) one will need a means of preventing light from penetrating the bottom of the slide. That need may be met easily by any matte finish black paint, though some practitioners will no doubt prefer to use black paper glued to the slip. One may try either and decide which is more to their liking, or trust to the greater convenience of either depending on the available equipment. With the contemporary popularity of scrap-booking circular paper punches may be had inexpensively and the chief argument against paper (the difficulty of true circles, discarded). Paper (of sufficient quality) is of uniform appearance and opacity throughout and lends itself to any number of adhesives. Paint is self adhesive in a way even modern gummed papers will never be and permits one to ensure perfectly central placement (as it is applied on the turntable), but may tend to be applied less then uniformly.

With a cell formed around the opaque disc one then affixes the perfectly dehydrated specimen so that it will not move. Historically, mounters used a variety of gum or albumen glues. Some in the present day may elect to use any of the plethora of artificial adhesives currently available, from rubber cement to cyanoacrylate the choices seem limitless. I confess to having always found tragacanth gum as the most effective and long lasting option. One need only add too a small quantity of the powdered resin (as it is commonly available) distilled water, which will then provide an effective and long-lasting adhesive that tends to prevent the condensation of moisture (by absorption of the same) in imperfect cells.

Do come by for the next (practical) post if you have any interest and in the mean time consider the application of the same methods for transmitted light mounts and other (think liquid without pressure) preparations as they will be elucidated some later time.

Ringing a Slide: Practice

I accept full responsibility for the following as the majority is not attributable to any particular authority but only my own experiences. -K

In practice ringing a slide (this deals only with circular covers, when treating with rectilinear covers I will refer to it as sealing) is a simple task and once performed is easily repeated indefinitely, provided a few points are always observed. The keys to successful ringing are entirely mechanical and essentially every other aspect can be overlooked provided the mechanics are given priority. For example the sealing cement is less important than it’s viscosity and the position of the ring much more crucial than its appearance.

The easiest cement is often the one on hand, but for beginners or those wishing only to get a feel for the practice I can not more highly recommend gold sizing. For those unfamiliar, gold sizing is a gilders sizing cement for the application of (primarily) gold leaf. Any of the larger art supply houses can provide a suitable gold sizing and it may generally be found among the model paints. Available in a number of grades, look for one which is described on the label as “fast drying” or “suitable for exterior varnish.” It will likely be the least expensive of the sorts on offer. Do not purchase gold size which is described as “for picture varnish.” Higher grades of gold sizing dry incredibly slow, some behaving more like an oil paint in that they never set up completely which is wonderful protecting art which needs to breath, but not for sealing a coverslip.

Acquire also a fine camels hair brush, any will do provided it may be pointed (either purchase a pointed brush or plan on trimming it) and has natural bristles. Gold sizing may be cleaned from the brush with xylene (xylol in Germany and many continental European countries) and many synthetic bristles will dissolve partially (or completely) in xylene and similar solvents (benzene &ct.).

One should already have a ringing table of some sort. If not, Brunnel in the UK and BioQuip in the US appear to be the only contemporary sources. If one would purchase a second hand turntable or construct one for themselves it must be outfitted so that it turns perfectly without wobble, and freely completes many revolutions at a fair speed without slowing. There should be a simple means of holding the slide quite firmly and some convenient and sturdy rest for the wrist and forearm. I prefer a rest which supports the hand above the surface of the turntable so that the wrist may be held straight while holding the brush vertically over the slide.

Prior to beginning one should clean the slide thoroughly; first scraping any exuded mountant with the dull side of a heated scalpel and following by a lens paper wet with a small amount of an appropriate solvent. When positioning the slide to be sealed upon the turntable one must be sure to center not the specimen, but the coverslip. With turntables that are marked by concentric rings (as most are) it is simple to get the position if the coverslip is near to the size of a marking. In any case once believed to be centered set the turntable spinning and look down from above to determine that it is indeed centered. A ring which is not concentric with the edges of the cover will serve but is less secure and less effective.

Effective position seen at right.

Effective position seen at right.

As seen in the right half of the above image holding the wrist straight helps to maintain a steady hand and places the point of motion at the more sturdy joint of the shoulder rather than at the wrist. It’s rather similar to the form one uses when writing in the Palmer method, for any old enough to remember being instructed in that technique.

Why though should it matter if the hand is kept steady? The seal is applied in an instant by just touching the tip of the loaded brush to the slide, and as the turntable should be rotating a fair clip it only requires a second or two for the ring to be built up by many revolutions. In that short time there is little opportunity for motion to be sure, but the place at which the ring is applied is paramount and ensuring a minimum of motion keeps the ring from being located improperly.

The author is of the oppinion that an improperly positioned ring is worse than no ring at all. Being improperly positioned a ring fails to adequately seal the slide and presents significant chance for damage when it must be removed either to remount the spoiled specimen or to properly ring the slide in the future. For preservation, the ring should be applied so that it’s thickest portion is adjacent to the corner created by the bottom of the coverslip and the top of the slip. Covers which have been first properly sealed may then be added to with decorative and functional pigments which extend farther onto the other surfaces of the coverslip or slip. Concentric rings of contrasting colors may be laid down with ease that present an eye-catching and very finished appearance, as long as their is an underlying ring located correctly such additional layers can not but help, however superfluous they may be.

Note the outline at the top right

Note the outline at the top right

Taking a sidelong glance at a cutaway provides an illustration of properly positioned rings. On the top are rings which are excellent; just covering a portion of the coverslip and slide and presenting a significant barrier to oxygen infiltration or volatile compound escape. The example at the upper-left is representative of the sort of seal one is likely to find on antiquarian slides and is achieved by successive layers of cement or the use of a highly viscous cement. Asphalt varnishes or gum/rubber bearing shellacs will produce a ring of this character in a single application. The image at the upper-right is what one can expect of a properly located ring of gold size.

Below are rings which although correctly located will provide almost no protection. On the left one can see the product a ring which suffered for either being applied with too much pressure on the brush (just the faintest touch is needed) or with a cement of to low a viscosity, the thin cement spreading out because it was not substantial enough to support itself in the place of the force created by the spinning turntable. At the right one sees the cut-away of a ring that was applied without flowing freely from the brush. If the cement will not flow properly onto the slide one might need to first wet the brush in a small amount of the solvent to encourage a healthy flow. Another cause of rings of this sort is an unclean slide, which can result in rings that flow excessively or are seemingly repelled by the surface of the slide (depending on the nature of the contamination).

At some later time I will try to post some of the very serviceable sealing cements I have employed over the years and speak to the virtues and vices of each. For the time being I recommend those who desire an excellent all-around sealing cement stick with gold size as it is perfectly serviceable for most needs and of high quality. I must say that many cements available, at first seemingly excellent are much less impervious to the compounds apt to find their way onto a slide as it is used. Simply being available from a reputable supply house is not a proof of serviceability and one should never rely on an untested cement for a valuable preparation.

Rapid Process Mounting

Sorry for the lack of updates, the summer is always a less leisurely time than one expects. -K

When last macerated and pressed mounts were mentioned the method employed required a long weekend at a minimum, but the entire process can be done in a day. Some people will appreciate progressing from specimen collection to finished slide so rapidly and some are sure to prefer the day by day process of a longer method. For the summer months when both specimen availability and social obligations are at a peak, the following entries from my notes may prove welcome. Embellishments not appearing in my notes but provided here for clarity appear in parenthesis:

8 Jun. ’14 10:00am Captured 5 spiders from under the addition. Collected them into stoppered test tubes finding it easier than Mason jars (a flat of small jelly jars often makes up my collecting kit when working in the garden). Three of the spiders are larger and of orange hue which leads me to identify them as males of the common house spider. Two despite being smaller I take as females of the same species. (The description in the Audubon field guide describing males as orange and smaller than females, while Comstock elaborates at the striking variety of forms exhibited by the species and its tendency for differing specimens to be taken for differing species by novices. The habitat and cohabitating species contribute to my identification.)

Killed them by introducing Ether soaked cotton swabs to the test tubes. One stopper was ejected by the expanding gas and I feel a better poison will need to be collected (for use with this method I imagine chloroform will prove superior and more desirable than for instance, ethyl acetate, as speed is a factor).

11:18am Have processed the three males through to the pressing jar. Each was boiled in 3ml of 10% NaOH until quite transparent. (The test tubes into which the spiders were initially collected were used and heated over an alcohol burner. When doing so the test tube should be slightly tilted to face away from the preparer and towards bare floor or table to minimize danger in the case of bumping. Agitating the test tube slightly and holding over rather than in the flame will also help to prevent bumping.) The solution was then drawn off and aprox(imately) twice the volume of distilled water added to the test tube. Into this was placed several drops of glacial acetic acid until diffusion currents were no longer observed. The solution was then poured off and replaced w(i)t(h) aprox(imately) 2ml of distilled water.with a rubber stopper inserted the specimen was swirled in the tube until near the mouth and then rolled to be on the side away from the water adherent to the side of the tube. With the stopper removed the tube was manipulated so as to wash the specimen into a Syracuse glass.

(Here in my notes is a sketch depicting the motions required to carry the spider from the test tube with such a small amount of water. It is of course simpler to simply employ a larger volume of water but then it requires a larger Syracuse and in the end more potential for spillage. The ease of adding a sketch to handwritten notes is reason enough for me to never adopt an electronic medium for note taking.)

The females were placed into a Syracuse in the vacuum desiccator together with a jar of Drierite to await processing after the males are completed. (I try to work with specimens of a single type only at one time for the sake of simplicity. It’s far easier than one might expect to mix things up when working with only five specimens.)

(After a lite lunch with my wife, which gave the alcohol sufficient time to harden and dehydrate the specimens, I returned.)

1:50pm Removed pressed males and transfered them through to clove oil one by one. Find the first macerated to be a bit to opaque and hope the clearer will improve its appearance. All removed intact and came away from pressing slips without incident. (I use slips of the cheapest sort for pressing and some are never quite clean enough-or smooth enough-to release the specimens without damage. The cheap slips is a compromise I make for budgetary reasons as the spring clips which I use to hold the pressing slips together can be quite hard on the slips.)

2:45pm Mounted the males on plain beveled edge slips from Ted Pella in balsam under 22×35 Gold Seal covers. (I quite like the plain economy slips from Ted Pella, the beveled edges decrease immensely the tendency of chips to form when used on spring loaded mechanical stages.) One specimen became far off center and the cover was lifted and the specimen repositioned under a new glass, a leg was separated in the process but was left in approximately proper posit(ion). (Misplacement of specimens can be upsetting but unless it approaches the edge of the cover should be overlooked as the risk of damage is quite large once the balsam has begun to penetrate the specimen. I properly should have placed the whole into xylene to dissolve the balsam and then remounted as normal but with several specimens on hand I become less careful than I should be.)

3:12pm Slides placed in attic to cure. (If more rapid curing is required a cool oven or hot plate on very low setting can set the mountant nicely in a few hours. I prefer the heat of the attic of my home during summer as the hot [32-35C] dry [below 5% RH] attic cures slowly enough to permit even the largest and deepest air bubbles time to escape.)

 

Slightly More (Complicated) Hair

I’m constantly amazed at the ease with which one can obtain certain chemicals; in the old days you had to know the right people, now you just have to know how to search the web. Of course in the very old days your local pharmacist would just give you some… -K

The slide I’ll describe today is very much the same as the previous, differing only in the treatment one uses prior to mounting. While very fine results may be had by the process described previously, a little more effort can deliver a superior preparation. This method is not the acme of hair mounts either. There is seemingly always a little bit of improvement that one can make when it comes to mounting. Once one begins to gain a degree of comfort and familiarity with the process, ideas for improvement abound. The successful student is one who does not see the procedures of others as a rule-book but as an inspiration.

Hair is among the materials that could be mounted directly into resin after collection. In the previous entry that is very nearly what was done. A brief rinse in alcohol was performed primarily to ease manipulation, rather than to provide for dehydration as one may have reasonably guessed. Fortunately, the composition of hair is such that even freshly collected hair is essentially “dry” to begin with. Natural oils are the primary moisture in any hair and this oil will have some effect on the refraction of the mounting medium after the slide has cured. To provide a better level of resolution of the margins of a strand of hair one must remove the oil from the hair so that the refractive index of the mountant is uniform.

Most every microscopist will posses a number of solvents that may be used with success to clean a hair of oils prior to mounting. One solvent in particular enjoyed a profound popularity, for this purpose and others fifty years ago: ether. Sulfuric ether (more apt to be found today as ethyl or diethyl ether) is a highly volatile inflammable solvent. It is heavier than air and has a rather distinct odor that is markedly less beckoning than chloroform, neither of which one should inhale, and both of which have their uses in microscopic mounting.

The distillation of sulfuric ether is not complicated and is one of the more common bits of home chemistry achievement one sees on various web sites. Do not attempt the distillation of sulfuric ether at home without the proper training. Far too many people have far too high an opinion of their abilities and will run into all manner of problems. I have seen videos of ether being distilled on kitchen hot-plates that make me marvel at the fortune of others in not catching fire. Ether is available from many reputable chemical supply houses at a very economical cost. If one is unable to locate ether, various similar solvents may be used. Xylene and toluene are effective substitutes if local laws or personal caution dictates.

To remove totally the natural oils from a sample of hair one should make a solution of one part sulfuric ether to one part anhydrous alcohol. This is one of the situations where I do not recommend the use of denatured alcohol as an alternative to anhydrous alcohol. Some formulations of denatured alcohol may provide suitable results, but as the amount of solution required is very small, anhydrous ethanol can be used without it being a great waste. Alternatively, one may use anhydrous isopropanol or even anhydrous methanol, though isopropanol is likely to be the most readily available and least expensive. Be sure to mix the solution in an amber glass vial as ether has the potential to form hazardous peroxides in the presence of light.

With the ether-alcohol solution prepared, take in a forceps the hair to be mounted and carefully agitate it in the vial for from forty to sixty seconds. Afterwards deposit the hair very near the center of a clean slip and mount it as described in the previous post. As mentioned previously this treatment will remove the natural oils from the hair and allow for the mountant to form a material of uniform refractive index around the hair. This will permit the scale pattern, and interior structure of the hair to be observed somewhat better than that of a hair which is mounted directly, or through alcohol and a clearing agent alone. It it will not provide as clear an image of the scale pattern as a scale cast, but that is another sort of slide entirely.

Hair

As a bald gentleman I find it distasteful to spend any time thinking of a better title. Fortunately, the gravitas that comes of being a bald chap with eyeglasses and a deep voice nearly makes up for the lack of coiffe. -K

Hair (note in the following the word hair is meant to indicate animal hair) is to be found very nearly everywhere. Entering any building which humans occupy even on an intermittent basis one is sure to find a strand here or there. If one examines a shirt or jacket, hair will be found, this time of year one might well be wearing a shirt knit from the hair of sheep. Most people don’t give the hair much thought beyond grooming their own, but the microscope owner might do well to consider it for a time.

Hair is among the easier things to examine in temporary or permanent mounts. Far from the smooth thread that it appears to the naked eye, under the lens of a microscope it can be surprisingly diverse, and I’m not writing of curl, coarseness, or color. Hair is the subject of a good deal of forensic investigation and a the techniques surrounding it are myriad. Specialized means of taking longitudinal and cross sections have been developed, differing mountants advocated for different types of hair, various adhesives recommended for scale impressions et cetera. I imagine most schools still introduce students to the microscope with the printed letter “e” slide, and leave out hair.

Two slides will be produced, one using more advanced methodology, and the other more expedient. The results will be largely similar and will demonstrate that fine permanent mounts of hair may be produced without overmuch difficulty by those of any skill level. Forgoing more complex methods only regular segments of hair will be mounted, no sectioning is required other than to cut to length the hair so that it will fit beneath the coverglass.

For the first, simpler, mount one will require a fine forceps, a cleaned coverslip and coverglass, the preferred resinous mountant, alcohol of the highest concentration available (for Euparal and similar mountants) or an essential oil or xylene (for Balsam or Damar mountants) and a fine shears. If using alcohol or a clearing agent a watch glass or similar vessel will be needed. One will also require hair, human hair can be employed, but will often prove less interesting than that of other mammals. Because hair is dead, composed primarily of keratinous protein, and actually holds moisture very poorly, the use of alcohol or any clearing agent may be considered optional. They will be used in the following example only because it makes the manipulation of the hair to be mounted less delicate.

In the slide I will make the hairs used will be human, and mouse. The hairs will appear very different under the lens and to better illustrate that difference on each slide will be placed a human hair alongside a mouse hair. If one has a pet mouse, or an acquaintance who does, then the mouse hairs may be collected without too much difficulty from within the nest the next time the pets enclosure is cleaned. Human hair may be of course obtained by plucking a strand from any willing head.

Cut the hairs to be mounted so that they are in length less than half of the diameter of the coverglass used. Depending on the color of the hairs collected it will be helpful to work over a sheet of black or white paper. Place the cut hair from one subject into a watch glass of alcohol (if using Euparal) or Xylene (if using balsam) for a moment and then transfer to the cleaned glass slip. Place the hair very near to the center of the slip and release it from the forceps while it still remains moist, the liquid will help to cause the hair to cling to the slip and make positioning it more simple. Do likewise with the hair from the second subject, placing it very near to that of the first on the slip.

Place only the smallest drop of mountant in the center of a cleaned coverglass. Allow the mountant to spread out until it almost reaches the edges of the cogverglass and lower it onto the slip. If the mountant is not allowed to spread out before it is placed on the slip, the hairs will be displaced outward as the coverglass settles. Using hairs that have been cut to less than half the diameter of the coverglass used will ensure that even if they are displaced they will not approach the edge of the coverglass and spoil the mount.

If only hair from one specimen is to be mounted, placing it in the exact center of the slip and lowering the coverglass so that the mountant presses it to the slip will help also help to prevent its being displaced as the coverglass settles. Alternatively one may place two or more hairs so that they intersect in the center of the slip and thereby create a mount that allows for one to practice identifying the relative depth of specimens on a slide.

General Program for Preparation of a Chitinous Specimen with Pressure

In the previous series I stretched a rather simple mounting technique out to a few thousand words. I did my best to make things clear and explain the why and how of things. It’s easy to understand that way, but it’s easier to follow along with brevity. Here’s the same information all in one page and about 500 words. Easy to print out and refer to as required.

1) Remove killed and fixed specimen from storage, clean superficially with a camel hair brush, and bring into distilled water if necessary (removing fixatives such as alcohol or formalin). Dry specimens do not need to be brought into distilled water.
2) Place superficially cleaned specimens into a 10-15% solution of caustic potash (Potassium Hydroxide) or caustic soda (Sodium Hydroxide) for macerating. Ensure sufficient of the macerating solution is present to remain at 10% concentration when the fluid diffused from the specimen(s) are added to it.
3) Retain specimen in solution for 12-72 hours, or until visual signs of internal organ decomposition. Alternatively, heat the specimen in solution for up to 30 minutes to accelerate the process, do not allow the vessel to “boil dry.”
4) Remove specimen from macerating solution and wash in several changes of distilled water.
5) Add 5 or more drops of acetic acid to specimen in distilled water, both to ensure complete removal of macerating solution and to further soften chitinous tissue. If required specimens may be stored in strong acetic acid until ready to continue.
6) Apply pressure to bulbous portions of the specimen with the butt of a camel hair brush or needle holder, working from the head and expressing liquefied internal organs through the anus. For small ants and thin bodied specimens this treatment is not required.
7) Lay specimen out in position desired for mounting on slip not suited for general mounting (slips with chips or imperfections may be used). Arrange all appendages as desired before continuing.
8) Place a second slip over the first applying pressure first at the head of the specimen, hold slips together closely and prevent slipping of one across the other.
9) Use clips or other convenient apparatus to bind slips tightly together.
10) Place bound slips into vessel of anhydrous alcohol (95% denatured if none better is available) and leave for not less than 1 hour to dehydrate and harden. Specimens may be left in alcohol until convenient to proceed.
11) Take bound slips from alcohol and hold close while removing clips holding them. Separate slips flooding with alcohol so that the specimen does not adhere.
12) Wash specimen into watch glass with alcohol and use a camel hair brush to remove any internal debris that adhere to the specimen.
13) Transfer specimen to clearer required by the final mounting medium to be used. Leave for as long as the particular clearer demands. 1-24 hours is the usual time.
14) Take specimen from clearer and allow excess to run off before placing onto clean slip for mounting.
15) Apply mountant to one side of a cleaned cover glass of appropriate size for the specimen and lower directly onto specimen.
16) Add mountant at edge of cover glass if insufficient mountant was used, clean exuded mountant from slide in the event of excess.
17) Affix temporary label to slide and put up for mountant to cure as required.

Clearing and Mounting

The next bit is rather simple compared to everything previous but does require a bit more dexterity. It is important to remember that not all the specimens that have reached this step will come out unscathed. In fact, some specimens might not be suitable because they have been damaged in those earlier steps! Ever heard some tough walking cliché say “pain is weakness leaving the body”? Well friends, failure is just experience entering the body. Believe me, if I can do this, so can you.

At this point one should have a container of alcohol (denatured is fine provided it’s 95% or better) with specimens inside held together between two slips. During their time in the alcohol the water in the specimens has been dehydrated out by the alcohol. After having been used a number of times (more or less depending on the initial quantity of the alcohol and size and number of the specimens) the small amount of water present in the specimens will be enough to dilute the alcohol noticeably and it should be retained for other uses that require less complete dehydration.

One might be aware that besides dehydration alcohol will act as a hardener on many organic substances, chitin among them. Ardent hikers should know the old trick of preventing blisters by hardening the souls of ones feet with rubbing alcohol before a big hike. The hardening effect of alcohol will ensure that our specimens remain in shape for mounting, but it will also make them quite brittle. Avoid the temptation to rush and be careful as possible in the upcoming steps.

With a large watch glass or Syracuse glass on the table, and a wash bottle or pipette of alcohol at hand, remove one set of slips from the container of alcohol. Slowly remove the clips holding the slips together and separate them a small amount by sliding one or the other slip away. Resist the urge to lift the slips apart as an antenna or leg might more easily break off with that motion. Once the slips are slid apart a small amount it should become apparent that the specimen is more affixed to one slip than the other. Work with the slip to which the specimen is most attached at the bottom so that the specimen is facing upwards and keep the specimen wet with alcohol while gradually sliding the other slip away.

Once the top slip has been removed use a stream of alcohol from a wash bottle or pipette to rinse the specimen into the watch glass. With the specimen in the watch glass give it a quick look with a hand lens or dissecting microscope to see if any debris is clinging to it. Use a camel hair brush, fine needle, or eyelash to remove any debris from the specimen. If only a few specimens are being processed they may all be washed into the same watch glass before proceeding, or one may wish to finish each specimen before beginning the next. The choice largely depends on how much space is available in which to work, but I prefer to move each specimen though the process one at a time.

Image

An ant washed into a Syracuse glass for cleaning

If the specimen has been macerated too long, not long enough, or has been otherwise damaged it should be obvious at this point. Whether or not to continue with a damaged or poorly treated specimen is up to the microscopist. An ant that has broken off legs or antennae, or one that is falling apart from too long a maceration can still make a serviceable mount. If the abdomen has been disintegrated (as sometimes happens when macerated too long) consider mounting only the head, mouth parts, antennae, or legs. If the specimen is clearly too thick or still retains its internal organs one can wash it in water and return it to the macerating solution, or start anew with a fresh specimen. Whatever the case, keep written notes on the quality of the specimen had at this point, as it relates to the operations performed on it. It’s one thing to read someones account saying a day or two of macerating is sufficient and something else entirely to know from experience that 48 hours of macerating is too much for one specimen and not enough for an other.

Once satisfied that the specimen is free of debris it must be cleared. Clearing is a simple operation in which the fluid of the specimen is replaced with one which is miscible in the mounting medium to be used. If working with Euparal for instance the clearer used would be Euparal essence or even simple the purest most anhydrous alcohol available. With Canada Balsam (sometimes called xylol Balsam, neutral Balsam, fir Balsam or simply Balsam) one has their choice of clearers, the one selected being largely a matter or preference the procedure is the same whichever is used. Three of the most popular and readily available clearing agents for Balsam are turpentine, xylene, and Clove oil. Of the three Clove oil is the safest to work with (though it is also more expensive and harder to find than either turpentine or xylene) and it is worth noting it has the least offensive scent.

With a fair amount of clearer in a small jar close at hand, remove the specimen from the watch glass using a section lifter, dry camel hair brush, fine forceps or whatever is convenient. Once lifted from the watch glass allow as much alcohol as possible to flow from the specimen without its drying out completely. A small corner of filter paper can be touched to large specimens if necessary. Place the specimen into the jar of clearer. The amount of time required for the clearing agent to penetrate the specimen is dependent on its size and just how well macerated it is. I find that 24 hours is nearly always enough time but have used as little as one or two hours in the past.

Image

Ant in clearer with section lifter and centering card

After 24 hours the clearer should have penetrated the specimen entirely and replaced the alcohol that was used to dehydrate the specimen. If xylene was used as the clearer and it was observed to become milky when the specimen was placed into it then specimen was not fully dehydrated and should be placed into pure alcohol for an hour or so then put into a new dish of clearer. With a fresh and scrupulously clean slide laid out on a centering card the specimen should be removed from the clearer and laid out as desired. It will not be quite so brittle as it was after being taken from alcohol but one must still be exceedingly careful with the delicate appendages.

With the specimen centered on the slide take a small corner of filter paper and leach away any excess clearer clinging to the slide without removing that which wets the specimen. At this stage I find that Clove oil is not only the best smelling clearer but the most forgiving. It will not evaporate nearly as fast as turpentine or xylene and provides some valuable extra time with which to ensure that the specimen is positioned properly. Unfortunately, this also means that specimens produced with Clove oil as the clearer will also require a longer curing period.

Cover slip placement is often treated as a matter of preference but I vigorously oppose that notion. For particular styles of mounting, particular methods of cover slip placement are undoubtedly superior. The best method is however that with which the mounter is able to achieve the best results. In the case of pressed and macerated insect mounts it is easiest to place a drop of mountant on the cover slip and then lower it directly onto the specimen. This method does the most to prevent displacement of the specimen and is the method by which the amount of mountant used is best gauged, an invaluable asses to the novice. One large drop or two small drops from a slender glass rod is about right for most specimens the size of a harvester ant.

Once the cover slip is set one may chose to apply a small amount of pressure with the eraser end of a pencil or a camel hair brush but its not often necessary. Any excess mountant exuded may me cleaned up with bit of filter paper, but take care not to disturb the cover slip. The slide may now be placed in a cool oven or incubator (if one is available) for a few hours to cure. The time required for curing depends on the mountant and clearer used as well as the temperature. If time is of the essence the slide may be warmed over a spirit lamp or Bunsen burner to hasten things along, but superior results are often achieved by simply placing the slide in a case of some sort to keep the dust off and putting it on a shelf or a hot summer time attic for a few weeks. At room temperature a natural Balsam mount with Clove oil clearer can take as long as two months to cure.

Don’t be discouraged by the waiting time, the longer the period which is available for curing the more opportunity there is for any bubbles in the mountant to work their way out. In some up coming posts I’ll write about putting together a simple slide dryer and ringing a mount of this sort, but for now here’s an ant thats been produced over the course of these blog entries:

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Note the air bubble to the right of the lower antenna, and how unconcerned I am about it

New Year, Next Step

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.

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Todays supplies

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.

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A specimen pressed between slips for placement in alcohol

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!

Maceration

Be careful and nobody gets hurt. It’s great advice but seriously if one does not feel confident in performing the following operation safely then by all means do not build your confidence with this exercise! Chemical burns are not the sort of thing one experiences lightly. However, common sense and some basic protective equipment are all that’s necessary, more about that below.

Supplies for preparing the macerating solution.

Supplies for preparing the macerating solution, etc.

What, Why, and How

Continuing on with the previous slide prep, at this point a quantity of specimens should have been aquired. It’s unimportant if they are in alcohol as the preceding post describes or dry. It’s assumed that some manner of ant (order Hymenoptera) or spider (order Araneae) has been collected and it will be required. In order to prepare a pressed insect (even if spiders are no insect I trust the simplicity of writing as such will be forgiven) one must first do something about all the bits and pieces inside it’s chitinous skeleton. Secondly, the chitin will need to be acted upon to soften it so that it may be made to fit under a cover-glass, thirdly it will need to be rendered a bit more transparent so that the microscope may be used as with a transparent object.

The process by which these three operations occur is called maceration. During maceration soft tissues (proteins, fats, and nearly everything else organic) undergo a process of chemical decomposition. The organs will be liquefied and the chitin itself softened and somewhat bleached. If one observes a specimen of Hymenoptera post maceration it will be seen that the chitinous plates of its abdomen will be somewhat separated at their joints. The specimen as a whole will exhibit a noticeably flabby appearance and be quite pliable. This is a good thing, don’t worry.

Maceration can be performed by any number of operations but the simplest is by the use of a strong base in 10% solution with distilled water. Of the bases Potassium Hydroxide (caustic potash, potash lye, KOH etc.) and Sodium Hydroxide (caustic soda, lye, NaOH etc.) are the most frequently employed. Each has it’s particular merits but Sodium Hydroxide is somewhat more forgiving in its action on the specimens and is generally more widely available. For our purposes the differences between the two are inconsequential so one is free to employ whichever is available.

One can order the chemical from a reputable supplier online without any great difficulty or expense. It may also be found at a well stocked hardware or grocery, often in the plumbing isle. Each is likely to be available as a liquid of varying concentrations, or a solid in flakes or pellets. The pure, pelleted solid is perhaps the best choice and easiest to work with.

Safety; or, Please Ignore Tyler Durden

Does anyone remember the movie Fight Club? Perhaps the scene where Brad Pitt burns the back of Edward Norton’s hand comes to mind? They were making soap and Sodium Hydroxide just happens to be used in the soap making process. Pitt burned the back of Norton’s hand with lye to make some point about the value of being a masochistic luddite or some such and then proceeded to dump a quantity or vinegar on the site of the burn as treatment once he’d made his point. This is really a terrible idea.

There are two ways a strong base can cause a burn, chemically and physically. Neither is pleasant. The chemical burn is caused by the strong base breaking the bonds of ones very flesh, in essence macerating skin just as we will be macerating the specimens to be mounted. The physical burn is caused by heat. When a strong base is added to a solvent like water, or say the moisture in the cells of human skin, it will separate into its ions (a chemical reaction that causes a chemical burn, but) this is an exothermic reaction that can give off quite a bit of heat. The more totally it separates and the faster it happens the more heat is produced.

Vinegar is a weak acid, lye is a strong base, and water is ideally neutral. If one puts an equal amount of lye into 100 ml of water and 100 ml of vinegar, the lye will separate more completely and much faster in the vinegar. It will give off more heat and not provide the instant relief the film might lead one to expect. Proper first aid for an accidental application of lye in solution (or dry) is water, lots and lots of water, for 15 minutes. Don’t apply vinegar, and don’t take advice on chemistry from movies or blogs on the internet. If there is an accident and some water is applied till it stops hurting, but for less than 15 minutes (until all of the base is neutralized) the chemical reaction will continue and the burn will become worse. Look up the safe handling requirements of Sodium Hydroxide (and any chemical) before handling it if there are any concerns. Wear gloves and goggles and work in a room with immediate access to plenty of running water. Clean up any spills immediately using plenty of water and wash everything used to handle the Sodium Hydroxide throughly.

A final note, the Sodium Hydroxide (or Potassium Hydroxide) is going to come packaged in an air tight container. The chemical inside will be as free from moisture as the packager could make it. When exposed to the air it will absorb some moisture from the air. Seal it tightly to keep it from forming a solid block during storage. When handled it may stick unpleasantly to whatever it contacts either as a result of its extreme dryness or its affinity for moisture. Be careful that no little bits go unnoticed sticking to the underside of this or that. Always add the base to the water, and always add it a little bit at a time. 

Weights and Measures

Mixing up a 10% solution of Sodium Hydroxide is a simple matter. Add one part of Sodium Hydroxide to ten parts of distilled water in a suitable container. What makes a container suitable? Particularly high concentrations of Sodium Hydroxide can etch glass over time. Sodium Hydroxide will also react with some metals such as aluminum. So for a suitable container a tightly sealed plastic jar may serve. However, as 10% is not a particularly high concentration, and many jars used for canning employ stainless steel lids, a wide mouth pint Mason jar or similar is recommended. Clearly label the jar ahead of time so it isn’t forgotten later.

When comparing one part of a dry compound with ten parts of  liquid a common unit of measure will be necessary, in this case mass (though weight will be used if one feels like being technically accurate). Distilled water (in microscopy one should nearly always employ distilled water) has a practical mass of one gram per centimeter cubed. One cubic centimeter just happens to be equivalent to one milliliter. So if one intends to make up approximately 100 ml of 10% Sodium Hydroxide solution, one will need 100 grams of distilled water and 10 grams of Sodium Hydroxide.

Chemistry professors might cringe at the preceding but exact concentration is not really important, it just needs to be in the neighborhood of 10%. Remember, Sodium Hydroxide is a bit more forgiving than Potassium Hydroxide so a concentration varying as much as 5% in either direction is no great failure. Supposing however, a significant textbook of chemistry resides on a nearby book self… one might consult it to find that a 10% concentration of Sodium Hydroxide in water has a density of 1.10890 kg/L at room temperature (1.1089 g/mL). This would mean one should add 11.089 grams of Sodium Hydroxide to 100 ml of distilled water for a 10% solution.

That same textbook will likely describe a number of formula for the calculation of pH. With a digital pH meter or some indicating paper one might measure the pH of the solution produced and work a few equations to determine the final concentration after the solution is mixed. But Molarity and pH calculations are only fun for a special few and all this chemistry is just taking away from time at the optical bench!

In any case measure 100 ml of distilled water into the jar using your graduated cylinder or whatever measure is handy. Then weigh out the proper amount of Sodium Hydroxide on a balance or scale and carefully add it a small amount at a time to the water. If a sensitive enough scale or balance isn’t handy then one should note that two teaspoons of pure pelleted NaOH weighs in at 12.6 g. Add it slowly a little at a time to the water to give the heat of the reaction time to dissipate. It will dissolve slowly and for a time the water will appear quite cloudy. Do not put the lid in place until the solution becomes clear (pressure may build up and burst the container!) which may be some minutes. Cleaning the utensils used to measure is a good activity while one waits for the solution to clear. Do not be alarmed if the jar becomes warm to the touch (it will no matter how slowly the Sodium Hydroxide is added) it will not pose a hazard if due care and time is taken.

Finally

At this point one should be in possession of a 10% solution of a strong base in water, and a quantity of insects in alcohol. Carefully remove the specimens to a dish of water. A long set of forceps wielded carefully is ideal for the operation. I find a set of stainless steel thumb dressing forceps invaluable.  Pour off and replenish this water once or twice to remove the alcohol. Then carefully introduce the specimens to the macerating jar. If working with dried specimens rinse them in distilled water to remove any debris before adding them to the macerating jar. A wash bottle of distilled water and a Syracuse glass makes the operation the work of a moment.

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Washing the specimens prior to maceration

The time required for maceration is widely variable and dependent on the size and toughness of the specimens. As a general guide to begin with; a period as short as 24 hours will prove sufficient for small, lightly colored ants and spiders. As long as 48 or 72 hours is often required for larger ants (like the black carpenter ants one finds on decks and trees). Trial and error provides a better eye for success than any text so do not be afraid to experiment with different periods of maceration.

When in a hurry the prepared macerating solution can be made more potent or heat my be applied to speed its action on the specimens. Several handbooks recommend boiling the specimen in the solution for a few minutes. Doing so is markedly more hazardous and the savings in time is not worth the added risk. Boiling will not generally produce superior results. If a specimen is possessed of a particularly large abdomen it may be necessary to puncture its underside with a dissecting needle prior to placing it in the macerating jar to ensure the action of the solution on the internal organs.

The next step will be posted after my specimens are prepared for it!

Tomb of the Cluster Flies

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When one buys a big old house it generally comes with a big old attic, and in my part of the world that means scores of dead flies as well. However unsavory it may be to a new housekeeper it is a boon to the microscopist. Yesterday I put together a quick little centering card and today I put it to use. Just as the general plan of that card was taken from the pages of Gage’s The Microscope, so to are our current efforts. A page or so beyond his illustration of the centering card is the innocuous little paragraph shown here:

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The entire operation sounds simple enough but let us first assemble the required supplies. One will of course need a number of dead flies, or rather their wings but generally the fly remains with the wings. A similar number of cleaned slips, and their attendant cover-slips should also be readied with labels attached. Labels could be neglected until the slides are finished, but I find it convenient to attach blank labels to my slides before mounting so that I might mark penciled notes upon them if the need arises. The centering card which was made previously should be brought out, or some other means of positioning the specimens in the center of the slip. A fine camel hair brush, a pair of forceps or two, and a pin in a holder will prove invaluable, but could be dispensed with by the specially careful and dexterous. Finally a resinous mountant is required. I will be using natural balsam today but Euparal or one of the synthetics could be used in its place.

I collected nearly a dozen dead flies this morning from the floor of my attic and a quick look at my Audubon field guide showed them to be some species of Pollenia, very likely P. rudis. Identification is somewhat secondary to the goal here, though It is nice to be able to properly label the prepared slides and enter the scientific name into ones catalog. If an identification can be made wonderful, but we are really out for mounting practice today. My specimens gathered I retired to my work area. It is often a great help to work over a surface on which the specimen stands out in sharp contrast, so I laid a sheet of plain white copy-paper on my table and deposited the flies in one corner.

The specimens being exceedingly dry at first rendered dissection quite tedious. Each attempt at grasping the body of a fly with forceps resulted in a powdered portion of Pollenia. I tried holding the fly to the table with a camel hair brush and carefully pulling the wings off by grasping them at their base with my forceps and had some success. In the end I found grasping the abdomen lightly with one pair of forceps and pulling the wings off with a second pair of forceps by grasping them their root the most expeditious method. I took care to keep aware of which side of each wing was dorsal and completed each slide before beginning an other dissection.

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With a set of wings removed I placed a clean slip upon my centering card and transfered the wings to it on the tip of a camel hair brush. Once there it was easier to position the wings using a needle as the wings showed a tendency to adhere naturally to the brush. I positioned the wings around the center of the slide as recommended by Gage with one dorsal side up and the other ventral.

Next I placed a large drop of balsam in the center of a cover-slip and allowed it to spread out a bit of its own accord. I found the amount of balsam excessive on my first attempt and in the remaining slides I was careful to apply less and spread it out on the cover-slip with the glass applicator rod.

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Gage recommends placing the cover-slip by grasping it with forceps. Some people find this a simple operation but I often end up grasping it rather too firmly and damaging it. When placing a cover-slip I find it simpler to grasp it by the edges between the thumb and fore-finger, lowering it vertically so that the suspended mountant touches the specimen first. Upon releasing the cover-slip a small amount of even pressure is all that is then needed to complete its application. This method helps to prevent specimens which are not affixed to the slip from moving from their intended position.

In the space of forty minutes or so I was able to produce ten slides and each one showed at least one air bubble. I placed each slide in a covered tray and carried them to the attic. Gage wrote that “if the slide is put in a warm place these will soon disappear” and I expect it will reach eighty degrees Fahrenheit in the attic some time this afternoon. One can only hope that the bubbles are expelled.