Saving Slides With Mountant Issues: III

The Process Thus Far

A quantity of well stained serial sections was obtained for nearly nothing. The slides so far have suffered greatly, some catastrophe reducing their original mountant to a crackling obscure matrix secured under a cover glass yet utterly unsuited for microscopy. Softened by a period in turpentine the fouled mountant was dissolved enough for the original cover to be stripped from the preparation. Thus exposed, the remaining mountant was washed from the sections with further turpentine. Now an essentially new specimen has only to be mounted.

Considerations

Serial sections are laid out according to various conventions and generally in a way so as to fit beneath covers of any of a half dozen readily available sizes. When the original covers were removed they might have easily been retained and reused after a thorough cleaning. In this instance the cover slips were simply placed into a jar with all manner of other cover slips which are being retained until such time as the author forgets about them or gets around to cleaning them. If the sizes were uncommon or otherwise not available it would have been a different manner. Any cover glass removed that was observed to be in any way damaged during removal should of course not be used for mounting again.

When selecting new cover slips one should consider size above all else; size matters. Size of course referring not only to the dimensional area of the cover slip but to the thickness (or gauge number) as well. One should select a cover slip so as to protect as much of the specimen as possible without covering an excessive amount of empty space and having a thickness corresponding to that for which the objectives likely to be used are corrected. Nearly every slip required a #2 cover slip measuring 24mm by 50mm.

Working with large cover slips can be a great challenge. The large size renders them more easily broken in measuring, cleaning, and general handling. One need only break a couple to get the necessary feel for it. Fragility aside, if they are not often used one will have a considerably harder time at applying a sufficient but not excessive amount of mountant than one will have when using a more familiar size. If too much mountant is applied one will find that air bubbles will more easily get in. In part this may be overcome by using a more viscus mountant and applying it over a thin film of solvent on the surface of the specimen.

One must also select a mountant which is suited to the specimen. Stained sections prepared in a laboratory setting often make use of a mountant which is convenient rather than one which is permanent. Convenience may mean the mountant is rapidly cured or particularly well suited for showing the structures looked for. Before selecting a mountant ensure that the solvent used in removing the previous mountant is miscible in that which one would choose. If it is not one will need to get the slips into that solvent by degrees to avoid damage to the specimen and ensure proper penetration. Turpentine being miscible in balsam meant that this tried and true mountant was a logical choice.

Mounting

The process for mounting the reclaimed slip anew is precisely that used for any mount. Ensuring that the specimen does not dry out, but is not flooded with excess clearer, place it on a low temperature warming table, or draw it over (but not through) a burner to warm it. Place then upon the slip a quantity of mountant and permit it to rest somewhat, allowing the mountant time to spread out but always ensuring that the specimen is not permitted to dry out. After a minute or so use a cover glass forceps to place a well cleaned cover glass upon the surface of the mountant. If just the right amount of mountant was used one may count on the cover glass alone for weight and may set the slide aside or into a cool oven to cure.

In general practice one may be sure that too much mountant was applied. This eventuality requires that steps be taken to ensure that the cover glass lays securely in place and is uniformly flat. If steps are not taken one may find that one side of the cover is significantly higher than the other. Place the slide into a cool oven or upon a warming table and permit it to stand for an hour. After this time one may be reasonably sure that the mountant is marginally more dense but still quite fluid. Place upon the center of the cover a small weight corresponding to the shape of the cover and return it to the warming table or oven. After a quarter hour check upon the slide to ensure that the cover slip has not displaced. If it has far too much mountant was used, or the oven (or warming table) is not level, and one will need to slowly but smoothly reposition the cover.

When the slide has cured it is a simple matter to clean any exuded mountant.

Results

In part I of this series we saw a representative example of one of the damaged slides, so badly degraded as to be entirely useless for microscopy. Permit then a look at it now, and yes it is indeed the same slide; note the engraved number.

This slide might look familiar.

This slide might look familiar.

A quick look with the microscope shows that in spite of the severity of the damage shown in the old mountant, the specimen came out unscathed. Any small particles of the old mountant which were not removed will be immediately apparent but one might always repeat the process if one was not scrupulous in their cleaning.

At low power (10x Plan-Achro objective) with the BalPlan.

At low power (10x Plan-Achro objective) with the BalPlan.

High-dry (43x Plan-Achro objective) with the BalPlan.

High-dry (43x Plan-Achro objective) with the BalPlan.

Notes:

∗Correction collars and draw tubes not withstanding most manufactures correct for a particular cover glass thickness across their entire line, in perpetuity. Bausch & Lomb has corrected their objectives for .18mm (#2 gauge) from the first and (considering that a variation of as little as .03mm in thickness will require an adjustment of the draw tube of 30mm) one would do well to gauge their cover slips before use.

†It is my considered opinion that in no case is it better to use too little mountant, err on the side of caution but do not drive to excess.

Once again an excessively long post, sorry for that. Next time I’ll try something shorter, I swear! -K

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Saving Slides With Mountant Issues: II

Correcting the Damage

After determining that the slide is a good candidate for rehabilitation one must remove the the degraded mountant and the cover glass. Doing so is not a difficult or tedious task but it will require a degree of patience.

Removing the Cover

The first step is to determine a solvent that will dissolve the mountant without unduly harming the specimen. Working with the knowledge that most mountants which discolor to yellow in areas where it has been exposed to the air contain a natural resin, one finds a starting point to identify a suitable solvent. In many cases a comparatively gentle solvent such as the pure gum spirit of turpentine will prove satisfactory. Whatever the mountant and solvent, placing a drop of solvent on an area where the mountant extends beyond the cover slip is the simplest means of determining its suitability. After a few minutes the heretofore brittle mountant will take on a sticky, thick syrup consistency if the solvent is able to dissolve the mountant.

For some synthetic mountants one may find alcohol works well, however, the frequent use of alcohol as a destain in the initial slide preparation may lead to the loss of vibrancy in the specimen. Other options of more rapid action (but more pronounced hazard) may be benzine, xylene, dichloroethane, or chloroform. On the opposite end of the spectrum in cases where more gentle action or greater safety is desired one may employ various essential oils such as the classic, clove oil.

Once a solvent is identified one need only place the damaged slide into a suitable vessel filled with an ample quantity of solvent. If many slides are to be rehabilitated one may choose to employ a large staining vessel and rack. If only a few must be processed a small Coplin staining jar or in a pinch a slide mailer may be used. One should keep in mind that as the solvent acts upon the mountant its strength will be depleted and the time required will be greater.

From left, vertical Coplin, plastic slide mailer, horizontal Coplin.

From left, vertical Coplin, plastic slide mailer, horizontal Coplin.

Above may be seen a few suitable vessels. The plastic slide mailer is useful only for the less volatile solvents and is recommended for no more than one or two slides. The vertical Coplin jar has a volume more than double that of the slide mailer and is well suited for from two to four slides. The horizontal Coplin jar has slots for ten slides but the most that can be recommended is eight. One may of course load each vessel to capacity if one is willing to permit the dilution of the solvent to such an extent that the process will take a week or more.

After a period of from 24 to 72 hours one may expect the mountant to be largely dissolved. At this time one may remove the cover glasses. Occasionally the condition of the mountant upon removal from the solvent will be such that the cover will have come off without the need for additional effort. If a cover glass should remain in place one will need to raise it by carefully inserting a needle or blade under one edge. Once accomplished, surface tension should be overcome and the cover easily removed. If the cover is seen to flex significantly one must forgo removal for the time being. A cover glass support (or more economically a portion of a broken cover glass) should be inserted just enough to maintain a slight lift on one side of the cover glass and the whole then returned to to the solvent.

Provided the slides were well prepared initially and the solvent selected was not undually harsh there should be no risk of sections coming away from the slide or adhering to the cover.

Removing the Mountant

With the cover removed one should take care not to permit the surface of the sections to dry. There is liable to remain a significant quantity of partially dissolved mountant on the slip. To remove this, one will require additional solvent. Resist the urge to employ the same solvent that was used previously; it has been rendered too weak and will now contain all manner or particulate contaminants. Depending on the time one has available one may proceed with one of two methods described below.

The fast method requires a slide clamp, a circle of filter paper, a funnel, a ring stand, a beaker, and a pipette. Pour a quantity of solvent into the beaker and placing the filter paper in the funnel position it in the ring stand over the beaker. Grasp the slide with the clamp and holding it in one hand over the mouth of the funnel use the pipette to send a gentle stream of solvent over the slip so that any particulate matter that washes off is caught on the filter paper. Refill the pipette from the beaker and repeat until the slip appears free of undissolved mountant. This method is particularly well suited for use with more profoundly damaged slides or particularly delicate specimens as it affords a degree of fine control and permits the technician to immediately notice any detriment to the specimen.

The fast method. At the bottom of the frame may be seen a watch glass holding a quantity of removed cover glasses.

The fast method. At the bottom of the frame may be seen a watch glass holding a quantity of removed cover glasses.

The slow method is simply a continuation of the previous process. One should use a new vessel and again additional solvent. If one is forced to use the same vessel one must be sure it has been scrupulously cleaned. Take care that all slips are arranged so that any particulate mater dislodged from one slip will not fall upon another. In practice this means that wherever possible slips should be positioned back to back. After a period of three to twelve hours all the solvent should be removed. Rinse the slip then with a pipette of fresh solvent to ensure no particulate contaminants are present on the surface. This method should not be used with powerful or excessively volatile solvents. It is also unsuitable for deeply or contrast stained specimens.

The next post will treat with re-remounting the slip. If you’re not used to working with large cover slips you’re in for a treat! -K

Notes:

∗For some subjects it was accepted practice to adhere sections to the cover glass rather than the slide. This is exceedingly uncommon in preparations younger than 80 years or so. Thicker sections are sometimes attached to the cover but such preparations were likely made by students in an effort to salvage an otherwise unusable section, so be cautious with slides that have the signature or a novice. There are methods for handling such slides but that would make a long post longer.

†Of course one could go right ahead and completely destain the specimen. Followed to the extreme this method would provide one with what is in essence a newly prepared slip ready for staining. Again though, I am trying (and failing) to keep this short.

Saving Slides With Mountant Issues: I

Obtaining Damaged Slides

Very often the amateur may come upon the opportunity to obtain significant quantities of prepared slides of a type that makes use of methods or materials that are not generally within the scope of ones own activities. Such slides may provide valuable objects for study. Regrettably, one may find that many slides prepared in professional circumstances only find their way onto the market at excessive cost or when damage sustained in one way or another has rendered them unsuitable for study.

Recently a quantity of slides were obtained by the author that consisted of serial sections from Leptonycteris sp. The exceptionally thin and expertly stained sections were mounted in series under rectangular 18x40mm or 24x50mm coverslips. All of the slides showed degradation of the mountant to a greater or lesser degree characteristic of repeated stress from heating and cooling.

Due to the visible damage 75 individual slides were had for the incredible price of ten United States dollars. One would be hard pressed to obtain a similar number of blank slips or coverslips for the same price.

Assessing the Damage

Visibly degraded mountant, black background.

Initial observation showed that the slip was in fine condition without chips or cracks which would greatly complicate the task of rehabilitation. Despite the significant degradation of the mountant the cover slip remained firmly in place, a good sign that the sections beneath would be intact. Running a fingernail over the cover is often all that is required to determine if the coverglass itself is similarly undamaged.

Visual inspection showed the mountant to be significantly degraded on most of the slides. Pronounced yellowing at the edges gave way to white patterns where mechanical damage had caused the mountant to crack to an extent that it was no longer in optical contact with the coverslip. In some instances the mountant had become brittle to the point that the coverslip had come off entirely. The areas occupied by the specimen were invariably damaged even in places where the surrounding mountant remained sound, as may be seen at the extreme right of the image at left.

Under the microscope one could observe the extensive cracking of the mountant shown below. Focusing on the specimen with a 16mm objective was nearly impossible as the depth of focus afforded by the objective ensured that the cracked mountant would intrude into the image plane. At higher levels of magnification one could optically section the image enough that the cracks were not immediately evident, but resolution was impaired to the point of near total occlusion of any detail.

Photomicrograph showing degraded mountant.

Photomicrograph showing degraded mountant.

The Classic Blood Smear

Blood smears can be fun, but be safe and sterile! -K

Assemble everything required whether only the most basic slide is being made or a more complex preparation. It’s always a good idea to begin with the required materials and a clean work area.

  1. Use a sterile lancet to procure two drops of blood from ones own, or a volunteers, (alcohol swabbed) finger tip. Discard the first drop and place the second on a very clean slip.
  2. With a second slip pull a smear on the first slip.
  3. Dry and fix the smear by ones preferred method. It’s simplest to grip the slide firmly and wave it about until dry.
  4. Stain the smear with ones preferred stain. For Wright’s stain: a. Drop on stain solution to cover the smear and leave for minimum of two minutes. b. Drop on an equal volume of distilled water and leave in place until a greenish scum forms on the surface (1-4 additional minutes). c. Rinse with a few more drops of distilled water. d. Dry in air, do not blot.
  5. If desired mount under a coverslip with neutral balsam or green euparal.
  6. Clean, label, and store slide.

Now for a few notes:

If you have trouble getting a suitable amount of blood try using a larger gauge lancet. A common 33 gauge lancet is very narrow and might not be successful for some people, but a 10 gauge (most common size for Unistik spring loaded lancets) might be too painful for others. A 28 gauge is apt to be more universally acceptable.

When pulling the smear one may find that by holding the second slip at a shallower or steeper angle the thickness of the smear can be controlled in a limited way. The speed at which the smear is made also has some effect on thickness. Try to maintain a consistent speed every time but don’t be afraid to experiment to find the angle that is most successful at ones own speed.

Do not try to fix with any substance (except for some methods of vapor fixation) before the smear has been dried, it will come off, the smear will be ruined. If one is working with a large number of smear it may be better to dry and fix in an oven.

Stains are available in many forms. Even something as common Wright’s stain may be found in a one step buffered solution, as a more traditional un-buffered preparation, or a powder. The directions above will give acceptable results with a buffered or un-buffered solution. When provided, follow the manufacturers directions for the stain used. Additionally, remember that all stains have a shelf life. Most solutions of Wright’s will only work their best after seasoning for a few weeks, and lose their potency after eighteen months.

A stain like Wright’s will bleach out somewhat in an acid mountant. It is therefore advisable to use an ostensibly neutral mountant like euparal or green euparal (which retains stain brilliance better than regular euparal). Alternatively one might keep a few pieces of marble in their balsam bottle to cut down on its acidity.

Depending on the size of the smear one may find a certain need for longer than average coverslips. 22x50mm and 22x35mm covers are widely available but one may also use a smaller cover that only covers a portion of the smear. In any case one should take the time to clean the area not covered after the mountant had cured.

There’s a great deal more to the microscopy of blood and for many people a simple smear can get dull quickly. However, it’s still an important skill and covers many of the various aspects of mounting in a way that can be moved through rapidly in just a few minutes. It’s useful also as a demonstration of the importance of specimen preparation as even an unstained smear will show more than a simple drop of blood under a coverslip. -K

More Blood!

Not done yet, this is just how to make the smear. -K

There are more than a few methods for making smears in general, and a couple for making blood smears specifically. By far the most generally useful method, and the easiest, is what I refer to as pulling a smear. In pulling a smear one may produce a large, thin, and even smear that is rather more uniform and consistent than that which might be produced by other methods. It’s very easy for beginners to read the method and then  perform the action incorrectly, so please observe the pictures and practice with a bit of fountain pen ink or food coloring before getting set to make the smear with blood.

Practice the method with ink before working with blood.

Practice the method with ink before working with blood.

In A we see a drop of blood (in this case ink for practice) placed near one end of a slip. In B a second slip has been placed on the first at an acute angle. The second slip is then slowly pushed back until it comes into contact with the blood. Once contact is made the blood will spread out along the line the edge of the second slip. Maintain firm and even contact with the slips. The blood should remain primarily on the side next to the drop; within the acute angle. As in C, the second slip is then drawn in a smooth even motion along the first so that the blood is pulled along leaving behind an suitable smear. In D care was made to produce some of the more common troubles. Midway along the smear a lighter line is observed where the slip was stopped as it was drawn along. Further along we observe two blank areas where the slip was allowed to lose contact at the lower edge.

In the above image the camera is positioned at six o’clock, and I am seated at three o’clock. Most depictions of the process provide the impression that smears are best drawn along a horizontal from left to right (or right to left). It is easiest to ensure a smooth motion (for those who are right handed) to pull the smear from from eleven o’clock towards four o’clock.  When pulling along a perfect horizontal it is more difficult to keep constant contact and pressure along the entire length of the smear. It is in some respects a matter of preference but above all do not make the common mistake of drawing over the drop or of positioning the second slip so that the drop of blood spreads out in the obtuse angle side of the arrangement.

Apart from everything else, it’s very important to form the smear on only the cleanest of slips. Oil, dust or other imperfections can prevent the smear from adhering properly once made, or from being made effectively in the first place. Always take a moment to exhale on even a clean slip prior to beginning. If the vapor of ones breath forms an even cloud upon the slip, and it dissipates rapidly it is likely to provide good results.

With the smear made it must be fixed. One may fix via heat or chemical methods but for blood smears it is often better to simply wave the slide about rapidly (with a firm grip of course) until the smear is dry. Microbiologists are apt to follow Ehrlich and place the slide on a hotplate or in a cool oven for hours on end, but such efforts are not generally practical when only a few slides are being produced. If one desires to employ chemical fixation, the film must first be adhered to the slip and the primary method for that is… waving the slide about rapidly! Because the cells in the blood smear will retain the shape when dried, it is best to forgo complex fixation and stick with that tried and true method, regardless of how indecorous it seems.

After fixation the smear may be examined immediately, with or without further treatment (even with oil immersion). Or it may be stained to improve its appearance. One my use a popular traditional stain such as Wright’s or whatever is on hand. In the next post we’ll cover the use of Wright’s, but one may wish to try the stains they are familiar with. Methylene blue is a widely available stain that will make leukocytes much more noticeable.

Blood And…

Please refer to first image in the previous post for clarity. -K

Where can blood smears be acquired?

The first two slides were purchased as part of larger collections from a student cleaning house in the first instance, and a collector in the second. If one wishes similar specimens know that they are generally not on the expensive side (as far as prepared slides go) and available wherever second-hand goods of a curious sort may be had. The second two slides and others of similar sort are available singly, or as components of general sets from dealers in educational materials. Without being too indirect let me just comment that Carolina is an excellent source if one elects to purchase commercially prepared slides of considerable quality.

As an obvious alternative, one may produce blood smears of their own without excessive trouble or exotic materials. A few slips, a stain or two and a pin are the bare minimum of supplies required. Unfortunately, things get tricky here. Blood smears are usually produced for a particular diagnostic purpose, and although they may be safely made by an amateur, it’s a medical procedure and can present some hazards. One may hardly rush about pricking friends and family with a pin for the sake a few drops of blood and a bit of curiosity. Blood-borne pathogens are very real and potentially dangerous, so that pin mentioned above is right out.

What is needed to make one?

Toss out that pin and replace it with a sterile lancet. Pharmacies provide an extensive selection of disposable lancets and reusable lancing devices for diabetics. One can purchase a box of one hundred single use sterile lancets for a few dollars and a compatible variable depth lancing pen for a few more. Spring-loaded disposable lancets are an option as well but might not be as readily available.

Any two slips of the traditional one by three inch size may be used. It is often best to select high quality slips of one millimeter thickness however, cleanliness is the most important factor for success with smears; fine smears may be made on the cheapest economy slips, provided they are clean. It’s worth noting that although the smear may be made upon any slip it is most easily produced when using a slip with plain cut edges, beveled or ground edged slips may be used but are apt to result in difficulty.

Cover slips are not absolutely necessary, but if desired one should use the largest possible and ensure all are of a thickness for which ones objectives are corrected. It may occasionally be best to form the smear directly on the cover slip and in that case one should avoid circular covers.

If employing a cover slip one will need a mountant. Any of the standard permanent mounting mediums will do. For most users this will mean Euparal or Balsam. When using balsam it should be as neutral in pH as possible as acidity will tend to bleach the stains more or less rapidly. Histomount, Mount-Quick, SHUR/mount, Meltmount, and any other of the modern permanent mountants may be used with only slight modification to the smear preparation as required.

Stains are a must when it comes to blood smears. Wright’s stain is perhaps the most popular, followed by hematoxylin and eosin (H&E). Depending on the supply of stain one already has on hand it is possible to prepare a suitably stained preparation with any of numerous combinations. Eosin and methylene blue may be used individually to produce an effect identical to that of Wright’s stain (which is of course a combination of eosin and methylene blue in methyl-alcohol). Giemsa, on its own or in conjunction with Wright’s is a popular option as well. It is always possible to try whatever is on hand provided one is familiar with its action.

What else is needed?

Nothing, nothing else is absolutely needed. At it’s most basic one needs only two slips, a drop of blood, a bit of stain, and access to running water. The next step beyond requires only the addition of a cover slip and mountant. Different sources might lead one to believe that a host of other supplies are required. Access to a Bunsen burner or hot plate is frequently required in historic methods, notably the Ehrlich method. A dozen Coplin jars and different solutions for each might be called for in others. One might be directed to add normal saline or buffer solutions to the smear while staining. Some works see fit to recommend methyl-alcohol as a fixative or drying bath.

All the extras are really just that, extra. While specialized methods are recommended for particular needs, all the extras are truly superfluous for smears intended only for general examination. If one happens to have other materials on hand and wishes to employ them effectively the points at which they come into play will be mentioned when the process is explained in the next posting. It is no great loss if only the basics are employed.

The bare minimum for a blood smear.

The bare minimum for a blood smear.

Next time: making the smear!

Blood

I’ve been putting it off but… let’s have a look at blood smears. -K

What are Blood Smears?

Blood smears are the sort of slide most people imagine when asked to think of a specimen for microscopy. They are thin, generally well-stained, films of blood secured to slips for observation under high power. Such slides also happen to be among the most commonly produced slides on earth. Medical practitioners all over the world, be they in state of the art laboratories or primitive field collection sites produce countless blood smears annually while working to diagnose and treat profoundly diverse diseases. To drive home their abundance; I’ve nearly one thousand smears provided by a student who produced them all while working on a single research project. There was a time when blood smears were routinely used in biology classrooms to introduce microscopy and cellular anatomy to students from grade school through to university, but concerns for safety left blood smears by the wayside and educators began to focus on onion skins and relied on ready made smears.

A variety of blood smears.

A variety of blood smears.

At left one may observe a variety of blood smears. The top is a double smear of blood from a laboratory mouse that was made as part of a study relating to malaria vaccination. Bellow that is a human blood smear that was used as a representative example of sever chronic anemia for diagnostic comparison as part of hospitals hematology lab collection. The final two slides are representative of modern, mass-produced slides of the sort one might find in a young students biology classroom or a popular science store. Differences among the slides are immediately obvious. One might consider that all the slides are utilitarian; constructed for particular purposes to which they are well suited.

The first two present an observable area of considerable size, such that a range of structures may be found in quantity. Large smears allow one to consider the percentages of the various types of leukocytes, for example. The size of the smear likewise ensures that some portion of it is apt to be of optimum thickness. Additionally neither of the first two slides is furnished with a cover slip. While this makes the smears rather more vulnerable, it also permits the use of very high power, high numerical aperture immersion objectives with exceptionally limited working distance.

The second two slides feature comparatively limited area for observation. What material is available for observation is insufficient to provide much for study but is instead exhibits a representative specimen, uniform, and secure beneath a cover slip. Making use of such a limited portion of the slip permits the slides to be used on microscopes lacking mechanical stages and relying instead on simple stage clips for securing the slide. Stage clips would severely damage the smears on either of the first two slides. Struck by the curious rectilinear outline of the smear in the second two slides, one might not be surprised to know their smears are actually cut from a sheet and then mounted as a transparent object. Mechanically neither of the bottom two slides is suitable for oil immersion work. However, they are widely available, inexpensive, and sturdy enough for prolonged use in a classroom setting.

If your kisses can't hold the man you love... mononucleosis will!

If your kisses can’t hold the man you love… mononucleosis will!

More on blood smears next time! I have got to stop being so long-winded… -K

Truly Traditional Mounts III

An ounce preparation is fine but a pound will go farther, so here’s hoping no one skipped the previous post. -K

Preparation

The following is only slightly different depending on the specimen/specimens to be mounted. In the case one may wish to mount objects beyond those described, the following may be helpful. Consider that what may be mounted in a dry cell is extensive but, in general all may be divided into three categories.

  1. Those which are adhered to the cover glass: diatoms and other very small delicate objects.
  2. Those which are adhered to the slip: small objects that may be damaged by moving about within the cell.
  3. Those which are loose within the cell: soil, sand, and portions of feather are the usual.

In this case, objects of the second variety are being utilized; specifically microfossils, but any other items failing into the same category may be used. Due to wide availability and comparatively large size, microfossils, micrometeorites, and other similar objects are an excellent first choice for one not yet acquainted with the technique. Whatever is selected, first see that it is prepared for mounting by ensuring it is totally dry and devoid of contaminating debris.

Mounting

Bausch and Lomb Dissecting microscope W

Bausch and Lomb Dissecting microscope W

A petri dish, watch or Syracuse glass is useful for laying out objects. Select a quantity of objects which are of a size to fit beneath the cell vertically and lay them out (on a handy surface) in a pleasing or utilitarian way horizontally. Place the slip (with cell) near to the surface on which the specimens have been lain out; some will find a dissecting microscope or loupe useful if inherent vision merits. An old style dissecting microscope may be found suitable, as one may easily move the lens aside when it is not required.

Depending on the adhesive employed… Gum tragacanth is the finest choice so far as the author is concerned. It is inexpensive, sufficiently able to adhere most objects, of a quality to persist over time (as evidenced by historical use), and is hydrophilic enough that it will absorb moisture to an extent that condensation will be prevented from forming within the cell. …one may be required to act differently.

Five small conispiral gastropod shells

Five small conispiral gastropod shells

Using a fine camels hair brush one must apply adhesive either to the surface on which the specimen will be mounted or onto the underside of the specimen itself. For some adhesives a needle in a holder or hemostat will serve. Some adhesives are more forgiving than others as regards positioning, but at all costs one must avoid allowing any adhesive adhere to an observable surface. One should be aware that some adhesives dry opaque or with a gloss finish that will show conspicuously in the finished mount. For the smallest objects one may paint the entire surface with gum and allow it dry. When an object is placed one need only direct a breath of moist air onto the whole to render the gum adhesive.

Geometric arrangements are pleasing but utilitarian layouts have some advocates as well. One may wish to layout specimens so that they are within the field of view of a particular objective (I should post about that sometime) and such an arrangement may be made to permit isolated observation of an individual specimen.

This arangement offers differing perspectives of similar objects

This arrangement offers differing perspectives of similar objects

Post Placement

When the specimens have been placed one need only permit sufficient time for the moisture (that may or may not be) inherent in the adhesive to dissipate. In some areas (geographically speaking) and circumstances this may only entail permitting the slip to stand out in a dust free area for some minutes or hours. It is noteworthy that (in many locations) humidity will be lower in the winter and contribute to lower than general humidity (at ambient temperature) within the cell, which is desirable and will tend to prevent condensation on the underside of the cover glass.

Covering

Prior to sealing a cell mount one must be confident that the humidity of the specimen is lower than the humidity of any environment in which it is apt to find itself, and that the positioning is as perfect as it might be. Many adhesives will require several hours before the required conditions are met. However, once such is the case the cell should be sealed. Examine the slip under low power to ensure no dust has landed on the objects and all is as it should be.

Sealing

Sealing is distinct from covering in the sense only that a cell which is covered might still be exposed to the environment at large (yes such a thing exists). To ensure that the cell is sealed one must use a cover of a size for the cell. The underside of the cover should rest upon the top of the cell, and the edge of the cover should not extend beyond the outer surface of the cell. Place the cover across the cell first to verify the size. In the below image the figure on the left is correct. The above on the right shows a cover glass which is too small, below is one which is too large.

Post-it notes are excellent!

Post-it notes are excellent!

Some texts recommend heating the slip so that the shellac cell is rendered soft and then applying gentle pressure to the cover to form a seal. This method has the advantage of eliminating imperfect seals which result from imperfections in the top surface of the cell. Regrettably, this method may also tend to deform the cell if the slip is heated too much or for too long. One may achieve as effective a seal by painting a ring of fresh shellac on the cell before placing the cover glass. Once the cover glass is in place set a ring of shellac around the margins of the cover to ensure that the seal is hermetic. A few layers of shellac are recommended so that the cover is well secured mechanically.

At this stage the slide is complete. Once it has dried many persons elect to give the cell a coat of decorative paint. I find the appearance of plain (dark) shellac acceptable.

Note the errant adhesive on #130, that is why I do not reccomend rubber cement.

Note the errant adhesive on #130, that is why I do not recommend rubber cement.

 

 

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.

The Microscopists Obeisance

The foulest sin in microscopy is to rack down with the coarse focus while ones eye waits at the ocular. -K

Always treat ones instrument with respect and consider the object upon the stage as well. Fine slides and delicate microscopes have come down through the years in such condition and numbers that even a casual enthusiast may have a first rate instrument and broad collection of slides at quite a reasonable price. It’s a terrible thing for such wonderful resources to become lost due to lack of observation of the microscopists obeisance.

When first placing a slide upon the stage orient a likely area beneath the objective and bow. There, with ones head low in deference to the care shown by others whose similar consideration permitted these items to persist long enough to become ones own, look askance towards the stage and rack the focus to set the objective within its focal distance. Then raise ones head and gaze into the eyepiece, hand upon the fine focus; never to touch the coarse unless one again bows low, for science and the preservation of history for generations to come.

Too many a wondrous specimen and unique objective have perished at the hand of the eager searcher, irreverent hand always upon the coarse focus and eye forever glued to the ocular. Why, just take a look at this poor eye:

Image

The things this slide has suffered, dear me.

A section, showing a retinoblastoma, spoiled by one to proud to take care. Balsam brittle and yellow it will still endure for ages but its perfection is spoiled by a crack (and missing portion) spanning the full length of the 30mm x 35mm cover. At least I can believe that the fool who damaged it learned his error-even at the cost of this slide-for the rest of the series I posses is thankfully free from evidence of such abuse.

So bow, low, and often! Save yourself and the slides in your care from damage such as this, take pride and make the microscopists obeisance!