Equipping A Laboratory
by Richard L. Howey, Wyoming, US
I'm assuming that you already have a microscope or even better two; a good compound microscope and a stereo dissection microscope. Frankly, I consider that the bare minimum. They don't have to be elaborate or expensive, but a good quality instrument of each sort is really indispensable for any serious amateur microscopist. What we don't tend to think so much about is all the miscellanea which go along with doing microscopy.
Let's start with a relatively modest instancedissection. Suppose you want to dissect a starfish; what tools will you need? Most biological supply houses sell dissecting kits which are largely useless, unless you are building miniature ships made out of matchsticks. Generally they contain a pair or two of scissors barely suitable for cutting paper; a pair or two of forceps that work so long as you don't have to pick up anything very small; a couple of dissecting needles useable to tear the hell out of a frog that's been sitting in formaldehyde for 10 years, but useless if you are trying to dissect an insect larva; a probe which is handy for cleaning your fingernails, but why else it's included is a mystery; an eye dropper, whose presence is also mysterious, and finally, the one thing that may truly be of some use, a scalpel, but even here it is often one with a large curved blade that is not of much help to the microscopist, unless he or she is cutting sizeable pieces of material for sectioning. There are student dissecting kits (a very misleading descriptionin most countries, dissecting students is illegal), basic dissecting kits, instructor's dissecting kits, advanced dissecting kits, and micro-dissecting kits and these last two can cost over $100 each. Forget it! You might want to buy one of the empty vinyl folding cases that hold dissecting tools and then put your own selection in it for use on field trips.
Otherwise buy or make the individual tools that you will really be using. Make a scalpel?!? Well, there are some amateurs who take great pleasure in making their own tools and some have produced very effective micro-scalpels by cutting and mounting small sections of double-edged razor blades in wooden handles. This is the sort of work that needs to be done with considerable caution, otherwise you may end up with lots of samples of slides of your own blood. For most of us, it is much more reasonable to buy a scalpel handle or two and some disposable blades. You can buy a number 3 stainless steel scalpel handle for $1.00 and it will accommodate #10, 11, 12, and 15 style blades; a #4 handle, for the same price, will take #20, 21, 22, 23, and 24 style blades and you can buy the blades for 30 cents each. This is a real bargain and it allows you to select from 9 different styles of blades to acquire just what fits your needs.
Steel scalpel handles and some blade styles
I used to buy some very nice little micro-scalpels, 3 different varieties, and I still use them and have learned how to sharpen them, but for micro-dissection, nothing beats a disposable surgical scalpel with a #11 blade. For those of you who are Internet auction addicts, you can sometimes find terrific deals, IF you know in advance the regular prices on the items you bid on. For example, recently I bid on 500 disposable scalpels with #11 blades and got them for about $45 with shipping. I recently saw the same scalpels in a catalog for $1.99 each! With shipping that would be over $1,000 for the same quantity. What am I going to do with 500 scalpels? Well, of course, I'll use some, but my proximate friends will also benefit when I sell them to them for only $1.00. Just kidding. For my friends, only 89 cents.
You might also wish to consider investing in a disposable blade remover or two at $3.95 each. These are a wise safety move if you are using the stainless steel scalpel handles with the replaceable blades. You simply insert the blade into the small box and push the lever to remove the blade and store it safely. Remember, these are surgical steel blades and are extremely sharp.
I'm rather fussy about scissors, but I also don't believe in spending $700-$800 for special micro-scissors made for eye surgery (nor will I ever be able to afford such an extravagance.) What supply houses often sell under the general description of dissecting scissors may not be very useful for the microscopist. I tend to buy three kinds of scissors.
1) Iris fine point scissors, which one can get with either straight or curved tips at $1.10 each or $1.00 each if you buy ten. These are stainless steel and made in Pakistan. They are not what one would call "first quality", but they are really quite good and, at this price, one can use a small diamond hone to touch them up and keep them the way you want them.
2) Micro-dissecting scissorsagain from Pakistan, but of good quality and the blades are only 11 mm. long with a separation at the tips of just 5 mm. The handles form an elongate bow which when squeezed close the blades for cutting. This provides very nice control and they do work quite nicely and cost only $8.50.
3) Iridectomy scissorsThese have a long stainless steel handle which has one of the blades at its end and then the other blade comes in at an angle with a short lever, so that when you push the short blade it connects with the other blade to cut. These are also made in Pakistan, work well, and cost between $20 and $25. The big supply houses used to supply an American made version, but they cost over $100 each! and so they have now gone to the product from Pakistan.
What about dissecting needles? Buy some. They are cheap ($1.50 for 10, straight or curved) and they are handy. However, for serious micro-dissection, it is best to make your own. I use insect pins and some very small dowels for handles. I bought the smallest clamp-on vice (sorry, I meanvise) I could findthe jaws are only about 3" acrossand I cut a piece of dowel to the length I want for a handle and clamp it in firmly. Then I take an ordinary dissecting needle and push it gently into the dowelgently, because if you push too hard, it will split the dowelyou need to make a hole just deep enough to comfortably seat the needle before you glue it. Oh, I forgot to tell youI take one of those ordinary generic dissecting needles and I rotate it on a hone until it has an extremely sharp point. this is the reason for the vise. If you try to hold the doweling in your fingers you will once again have an ample supply of material for making blood slides.
The insect pins are steel and have a small knob as a head. Determine the length you desire and then using a pair of wire cutters, snip off the section with the head. It is best not to carry out this procedure in the bathroom where eventually these little flying steel fragments will embed themselves in your feet after showering. Put a drop of glue in the hole of the dowel and using some sturdy blunt forceps or a small pair of needle-nose pliers, place the needle firmly into the handle and hold it for a few moments to let the glue set. After the glue has firmly set (usually 12 to 24 hours), carefully remove any excess glue with a scalpel. It is a good idea to make a dozen or so at a sitting, then you will have them whenever you need them. These needles are very handy not only for dissection, but also for moving small objects on a slide when you are making dry mounts. They take a bit of getting used to as they are very springy and just when you are trying to nudge a foram into a drop of glue, the needle may send the specimen flying off the slide.
The other type of dissecting needle I make is even smaller and finer. For these I use a kind of insect pin called "minuten Naedeln" or "minute needles". These are the type entomologists use to mount gnats and other very small insects, although now they tend more toward gluing the specimen on a small triangle of paper and then using a larger sized pin through the paper for mounting in a specimen case. What this means is that minuten Naedeln are harder to obtain and somewhat more expensive, but for $15-$20, you get a small paper packet with 500 needlesa lifetime supply. Share the cost with some fellow enthusiasts and divide up the packet. Bewarethese tiny needles are so thin and so extremely sharp that they can imbed themselves in your fingers with such facility that you hardly notice. The procedure for mounting them in handles is the same as for the larger needles; it just requires a bit more care.
Next, let's consider forceps, of which there is a wide variety. You can buy "student forceps", which usually means that they are cheap and rather poorly made; "cover glass forceps" which I have always found rather awkward, "cartilage forceps"now, how many of us are going to be working with cartilage?what this describes is a fairly sturdy forceps with serrated tips which provide good grip and that can be quite useful for certain types of specimen material; and there are fine point forceps, curved forceps, micro-dissecting forceps, watchmaker's forceps, hemostats, with scissor-like handles with tips ranging from "mosquito" hemostats to sizes more appropriately designated as "tongs', and finally, plastic forceps. I know that you suspect I'll probably say something sarcastic about plastic forceps, but you're wrong. Plastic forceps are ideal for removing specimens from liquids, such as, formaldehyde and alcohol.
Back to Pakistan. I buy what one company describes in its catalog as "micro-fine forceps". There are very interesting as the quality can vary from pair to pair, so I usually buy 10 at a time for the very reasonable price of $13.00. Even though they are "micro-fine", the tips are usually not fine enough nor sharp enough for my purposes, such as handling minute forams. I examine each pair under my stereo dissecting microscope and select the best ones (the others, I use for general purpose work), then I sharpen the two tines using a pocket diamond hone. This is probably not something that you can get away with as an engagement present for your fiance, since these little gems cost less than $10. I strongly recommend the model the fishermen use with a grove down the middle which is used to sharpen hooks. This is a real advantage when you are shaping the forceps tines. I run each tine along the groove 10 times, then I examine, then usually another 10 times; occasionally, a third 10 times. When I'm satisfied, I very gently rub the inside of each tine to remove the dross from the grinding of the outside. With a bit of practice and patience, you can produce exceptionally fine forceps. I have one pair of Swiss watchmaker's forceps which cost $27.50. These handmade ones are every bit as good and you don't need to be afraid to give them hard use, because if they sustain a bit of damage, you can always touch them up or make another pair at a small fraction of the cost of Swiss forceps.
You are also going to need some pipets and I generally use two different kinds on a regular basics and infrequently, a third type.
1) Disposable Glass Pasteur pipetsFor me, these are indispensable. You can buy them in boxes of 250 in two different sizes. The 5¾" ones can be purchased for $4.95 and the 9" ones for $6.20 in flint glass. If you want borosilicate glass, they're somewhat more5¾" for $8.25 and 9" for $10.50. When simply examining samples, one can use a single pipet again and again, but if you are looking at special cultures or using them for chemicals, then they are cheap enough to use once and discard.
From these Pasteur pipets, you can make your own micro-pipets. You will need an alcohol lamp. You can obtain a heavy base glass one for $4.50 and 40 cents for 12 replacement wicks or an aluminum one for $8.50 and $2.50 for 12 replacement wicks or a wickless burner of $9.85. These use 95% denatured ethyl alcohol. [CAUTION: This is highly flammable and toxic. It cannot be denatured and can be fatal if taken internally. Avoid inhaling the fumes and if you spill it on the skin, wash immediately with soap and water.] If you are sensible, the risks are minimal. Don't use it around flammable materials or around appliances which might generate sparks. Also, don't try to use other types of alcoholsI have, and I ended up with soot covered micro-pipets.
When I want to make a batch of micro-pipets, I clear off an area of my lab table and set out a metal tray. If this is your first attempt, try using the 9" pipets; they have a much longer tip and you're less likely to burn your fingersdo be attentive, as molten glass can inflict some very nasty burns. I don't want to make this sound like you are walking through a chemical factory with a vial of nitroglycerine, but with the use of common sense and a bit of practice, you will soon become adept.
Place the center of the tip in the hottest part of the flame and slowly rotate the pipet. You will quickly learn to sense when the glass is soft enough to pull. Make the pull firmly, smoothly and quickly, but not too quickly, as you remove it out of the flame. Do not pull the pieces completely apart; leave a narrow, fine tube of glass connecting the tip you are pulling on and the rest of the pipet. (When this does happen and the tips seal over, set these pieces aside and use them for micro tools.) Let the glass cool for a few seconds and then bend it until the thin tube of glass snaps. This is best done over the metal tray to try to catch any minute glass fragments. Then place both the pipet and the broken tip on the tray to cool. If you have carried the procedure out properly, you will have a micro-pipet and you may discover later that the tip is too fine for a particular task, in which case, you simply use a pair of your micro-forceps to break the tip at the place you want. I usually make about 50 micro-pipets at a time and when they are thoroughly cooled, place them in a heavy cardboard box on a shelf next to my microscope table for easy access. You will also want to purchase a packet or two of small soft latex bulbs for the Pasteur pipets.
2) Another type of pipet which is quite handy is the transfer/dropper pipet which is polyethylene, is a single piece and includes the bulb, and they come in 14 different sizes and styles ranging in price from $13.50 per 500 for most types to $13.50 per 100 for an extra long 23 ml. pipet. Naturally, my Scottish ancestry leads me to go for the $13.50 per 500 styles which are, in fact, the ones I like best anyway (Sure, sure, I can hear you saying. One of my graduate students said to me a couple of years ago: "If someone had those old paper pill boxes on sale, you'd buy them. Well, they were only $2.00 per 1,000, so what's $8.00? I have to admit that I am extremely susceptible to bargains, which is why I shall probably have to keep teaching long after retirement age.) In any case, this type of pipet is very useful for a one time use with strong chemicals or stains. When I use one for such a purpose, I toss it in a cheap, plastic, sealable sandwich bag before discarding it, to protect me from possible fumes and the disposal people from coming in contact with any toxic residues.
3) Occasionally you many want to have a pipet that allows you to dispense very precise amounts of fluid. A very reasonable solution is to buy a few disposable serological pipets. These are college educated pipetsthey've graduated. For $2.90 you can get 10 one milliliter pipets with 0.01 ml. graduations and for $4.90, you can get 10 ten milliliter pipets with 0.1 ml. graduations. These can be especially helpful when making up solutions of vital stains where dilutions of from 1 to 10,000 up to 1 to 500,000 are required. They are also useful if you are making up solutions of various salts as media for the culturing of aquatic micro-organisms.
If you are working with larger organismssomething larger than a rotifer, but smaller than a rhinocerosthen you may want to add some heavier instruments, such as, bone cutters, a bone saw, large tissue forceps, and a dissecting pan to two along with a packet of dissection pins. These pans have a thick layer of wax in them and as you proceed with your examination of the specimen, you can use dissecting pins to position skin, muscles, etc. so that you have a clearer view of the structure and organization of the organism.
At the beginning of this essay, I mentioned a starfish. If your local beach doesn't have anyunfortunately, my nearest local beach is 1,100 miles awaywell, then biological supply houses will be glad to provide thesepreserved, of course. If you want to buy live ones, the air freight costs are phenomenal. You can get a 3"-4" preserved starfish for $1.20 or if you feel like splurging, you shell out $1.90 and get a 6"-7" one.
A starfish is an absolute bundle of mysteries and wonders and I'm going to whet your appetite by mentioning a few of them and letting you have the fun of figuring out the patterns and details. Starfish are not, of course, fish; no more than sea cucumbers are cucumbers of any sort. Some zoologists have tried to insist that people call them sea stars, rather than starfish, but people, being stubborn, still call them starfish and probably always will. Starfish belong to the phylum Echinodermata or "spiny skinned" creatures derived from two Greek words, one meaning "skin" and the other meaning "hedgehog," a description particularly apt for sea urchins.
To begin to understand a starfish, you have to observe it with exceptional care and patience. This is not something you can learn in an hour or a few weeks; it takes constant practice. The genius of Darwin, in large part, resided in his exceptional ability to observe, describe, and contemplate what he had seen without imposing preconceived models of interpretation. Pick up the starfish, if you're not allergic to formaldehyde and/or alcohol, and one of the first things you will notice is the spiny skin. In some species, there are literally spines; in others, knobs, plates, and bumps; in Dermasterias, the so-called "leather star", the texture of the surface is indeed leathery and the skeletal plates are imbedded just under the surface; in some other genera, the surface texture is rough and scaly, but with no distinct spinesinstead the small plates form an intricate, lace-like set of patterns across the surface.
On the aboral surface, you will find a structure, slightly off center on the disk from which the arms or rays extenda round structure which looks like a small piece of coral. This is the madreporite and it, like the spines, knobs, and plates is calcareous, that is, it is composed primarily of calcium carbonate as are the skeletons of corals. This device is an intricate series of canals designed to let water in, but stop debris; in other words, it is an elaborate filter. Why does a starfish need to draw water in or expel it? The answer lies in an incredibly elaborate system designed for locomotion, for attachment, and for food capture. Starfish have what is known as a water vascular system which controls hundreds of tube feet. Inside the central disk is a tubular ring which is connected to the madreporite. At each arm, there are two tubes that branch off from the central ring and run down the length of the arm. All along the length of these tubes are little bulbs or ampullae, rather like pipet bulbs, and attached to each bulb is a tube foot. In your preserved specimen, the tube foot will be contracted and the only way to appreciate this remarkable mechanism, is to observe a live specimen or watch some of the very fine marine cinematography in some nature videos. In these instances, what you will observe should surprise and amaze youit still has that effect upon me. The tube feet can extend to several times the length of the shrivelled artifacts which you find in your preserved specimen and they seem more like tentacles than feet. Furthermore, at the tip of each tube foot is a structure which allows it to function as a suction cup and so the ampullae (bulbs) control not only the extension and contraction of the tube feet, but also their attaching and releasing on a substrate or prey. Since these responses are likely largely a consequence of tactile phenomena, there must be some very sophisticated biochemical and biophysical reactions going on here and at an extraordinary rate for an organism with a primitive nervous system.
At certain places on the surfaceit varies from species to species, but the area around the mouth is a good place to begin lookingyou will find tiny, stalked pincers with 2,3,4, or rarely even 5 "jaws". These surreal devices are called pedicellariae and some of them can even emit a toxin. Pedicellariae help keep the surfaces of the starfish free of debris and kill planktonic larvae looking for a place to establish a foothold.
Another oddity about starfish is that many species have an eye spot at the tip of each ray. Starfish have eversible stomachs; they poise themselves over a mussel or an oyster and attach with their tube feet, using their arms to open the mussel or oyster shell just enough to create a narrow gap into which they can evert the stomach, surrounding the soft parts of the shellfish, digesting it, and then retrieving their stomach. When you dissect a starfish, or any echinoderm for that matter, examine samples of the stomach contents under the compound microscope. You will likely find protozoa, nematodes, algae, diatoms and other interesting odds and ends.
Let me mention one last extraordinary feature of starfish. In the early days of oyster fishing, the fishermen would find enormous numbers of starfish feeding on their oyster beds. To combat them, they created great "mops" which they would drag across the beds and to which large numbers of starfish would cling. Unwittingly, they were making their situation worse, for they would chop up the starfish and toss them back overboard. what they didn't know was that starfish have incredible power of regeneration and from just a part of the central disk and one arm a whole new starfish can develop.
Dissection is both a skill and an art. One must learn the techniques of working with the instruments, but it is also essential that one learns to see the relationship of the parts of the organism and come to understand how any particular part relates to the whole. The development of good abilities at dissection desponds upon practice and, above all, patience, You don't have to have expensive tools; you must learn how to use well the tools you make and purchase. If you have doubts about this, go to a major library and look at a copy of Jan Swammerdam's extraordinary volume with its remarkably detailed plates. Oh yes, the title of the book is The Book of Nature; Or, The History of Insects; Reduced to Distinct Classes, Confirmed by Particular Instances, Displayed in the Anatomical Analysis of Many Species, and Illustrated with Copper-Plates Including the Generation of the Frog, the History of the Ephemerus, the Changes of Flies, Butterflies, and Beetles, With the Original Discovery of the Milk-Vessels of the Cuttlefish and many other Curious Particulars. This volume was originally put together by Herman Boerhaave who compiled the manuscripts left behind at Swammerdam's death and published the work under the Latin title Biblia Naturae in 1737. If you look at the plates, you will see in the beautifully intricate drawings of this Dutch 17th Century microscopist, a wealth of detail derived from his remarkable skill at making sharp instruments and his patiencehe spent a whole month examining just the intestines of bees!
The specific prices which I have cited here are from the 2000-2001 Scientific Equipment Catalog of Cynmar Corporation, 21709 Route 4 North, P.O. Box 530, Carlinville, Illinois 62626; Telephone 1-800-223-3517; FAX 1-800-754-5154; E-Mail: firstname.lastname@example.org; Website: www.cynmar.com. I have no affiliation whatsoever with Cynmar except as a customer and I receive no financial benefit from citing them. I use them as an example, because it is a modest-sized supply house with a reasonable variety of apparatus, items that are mostly of very good qualityI have a strong disagreement with them about the Chinese microscopes, but they still put up with me. I have made eccentric requests for them to find items not in their catalog and they still put up with me. The two main reasons I have used their catalog are: 1) I have gotten good quality items at prices well below the large supply houses and 2) the people are friendly, courteous, and exceptionally helpful and in a world that seems to be working harder and harder to depersonalize us by treating us as a collection of identification numbers and credentials, I find the human touch very appealing.
Comments to the author Richard Howey welcomed.
The author's other articles on-line can be found by typing 'Howey' in the search engine of the Article Library, link below.
Published in the July 2000 edition of Micscape Magazine.
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