Microscopy On A Shoestring
by Richard L. Howey, Wyoming, USA
The combination of rampant greed, naivety, gullibility, and human beings’ catastrophically poor track record regarding predicting the future have led to a world-wide economic crisis of staggering proportions. As a consequence, virtually everyone is concerned about reducing costs, trimming expenses, and saving money. This means that the STMTWSFM (Society To Make The World Safe For Microscopy) must remain ever vigilant and become even more active, even aggressive. Amateurs, beginners and elders alike, can, with a bit of planning and ingenuity, find ways to trim costs and, at the same time, remain vitally involved in the study of microscopy and natural history. So, what I am going to propose here is a series of things you can do to save money and still engage in rewarding research. Most of these suggestions will be commonplace and rather obvious, but the propaganda of some scientific supply houses can act like a seductive siren song luring us into the wonderful high-tech world of equipment, supplies, kits, and convenience.
I have had colleagues in the biological sciences who never went out into the field to collect their own specimens and I always thought–where’s the fun in that? So, one of the things we need is collecting containers. For the time being–probably this whole essay–I’m going to focus primarily on aquatic biology. Plastic and glass jars appear in many households in abundance. Don’t throw them out. If you can’t use them for collection, you may well be able to use them for storage. Sometimes at yard sales, one can find a sack or box full of baby food jars for a dollar or less–these are a treasure. If you have friends who have infants, ask them to save the jars for you. A disadvantage is that glass containers tend to be significantly heavier than plastic and when you are hiking or cycling and add the water samples, things can get heavy rather quickly. So, save your quart and gallon jars and jugs for occasions when you take a car or truck to roadside ponds or streams unless you enjoy orthopedic therapy.
Another option for collecting water samples is to use off-brand, cheap, plastic sandwich bags. Take your sample, close it with a piece of twist tie, put it in another bag and close that one in the same fashion and you have the ultimate in light weight collecting and a fairly reasonable protection against leakage. If you find something too big to fit into a plastic bag–like Nessie, just give her a pat on the head and send her on her way.
Now, when you get all of these little plastic bags back into your lab, what do you do then? Well, that’s where all the jars come in. If you accrue an ample supply of baby food jars, then you can take a hammer and an awl and punch holes in the metal lids. This allows air circulation, so that your samples don’t get instantly foul and it also slows down evaporation so that you don’t’ have to constantly be adding water to them. If your sample is too big to fit in just one jar, then this is a good time to be doing some sorting. Don’t put too much vegetation or bottom debris in any single jar or it will foul quickly. At a discount store or a dollar store (they used to be called five and dime stores), you can buy a plastic turkey baster and a bag of plastic spoons of the sort that are used for picnics. You can use the baster to transfer the water from the bags to the jars and the spoons to transfer vegetation and detritus.
Two other sources of small containers for storing dry material: 1) prescription vials–I have enough to start my own pharmacy and 2) speaking thereof, if you know a local pharmacist, ask him or her to save their empty jars for you. Boy, what a job; one studies 4 or 5 years to learn how to take pills and capsules from big bottles and count them out and put them into vials and then when you pay them, they have trouble figuring out how to make change. Just Kidding. Over the years, I’ve found that some pharmacists know more about possible side effects, drug interactions, drug and food incompatibilities, and potential conflicts between prescription drugs and over-the-counter medications than many physicians. If the pharmacists seem reluctant to save the bottles for you–powdered residues of controlled substances that you might take home and sniff and all that, you know–just remind them that you are doing a noble civic service by recycling their containers, so that they don’t end up in landfills.
Sometimes, it’s desirable to have large wide-mouth containers for preserving either quantities of specimens like starfish or sea squirts or small fish or larger specimens. These can be either plastic or glass. For these you can ask the manager of a fast food restaurant or a cafeteria if they would be willing to set aside some jars for you. Some years ago, I approached the university food service with such a request and soon had an abundance of large containers.
If you are collecting in marine environments and are collecting specimens that are contractile or fragile and need to be preserved on site, then take along a good supply of Epsom Salts (Magnesium sulfate). Don’t bother with the purified stuff from supply houses which is considerably more expensive. Just go to your local discount, grocery, or drug store. This works quite well anesthetizing a wide variety of organisms which can then be preserved and brought back to your lab for detailed study. Never collect more material that you can use. If you do by accident, then give some to fellow enthusiastic amateurs who will not only appreciate it, but may at some time provide you with some interesting specimens in return. If there is a microscopical and/or natural history club or society in your area, you can schedule meetings when members can give, trade or sell for modest amounts, extra specimens, stains, reagents, small tools and bits of apparatus or even microscopes and accessories. The club can charge a small fee in order to advertise and encourage such exchanges. If you live near a college or university, you may be able to discover that they have sales of surplus items. Here, the university has such sales where departments have priority in purchasing and then later in the day, the sale is opened to the general public.
As far as nets and small grappling hooks for collecting vegetation are concerned, you can make your own as I outlined in a previous article.
Let’s assume that you have both a stereo-dissecting and a compound microscope. If you’re going to maintain samples or try to culture specific organisms, you are going to need dishes and perhaps an aquarium or several. Standard, stackable culture dishes are very convenient, but when even the more reasonable scientific supply houses charge $6 plus for a 5 ½ “ dish, that adds up very fast. Furthermore, these are thick-walled glass dishes, thus the weight also adds up quickly and the shipping becomes quite expensive. So, head to your local discount store, go to the kitchenware section and look for small custard dishes. These will not be stackable as culture dishes, unless you want a smelly mess, but just buy a roll of plastic food wrap and your problem is almost solved. You still can’t stack them but, you can cover the dishes to slow evaporation. If you’re handy at building things–I’m not–you can make some racks for the dishes to save space. Another possibility: these days there are all kinds of plastic storage cabinets, some with just shelves and others with drawers and with a bit of persistent hunting, you can often find something workable. Yet another possibility is to prowl around thrift shops, yard sales, and flea markets where one can find not only suitable storage cabinets, but a variety of other implements which can be adapted to use in your lab.
Something that always seems to be in short supply in my lab is dropper bottles. A few years ago, I happened on to some nice little amber ones (about 7 ml.) with little plastic droppers which means while they’re wonderful for lots of things, they’re not good for solvents. I found these online at American Science and Surplus and I think they still have some. Because of their small size, they are ideal for the microscopist who often needs only a few milliliters of a solution rather a liter or a gallon. However, I have a lot of solutions that are already made up and stored in containers without droppers. One day, I was in a dollar store and found packages of 200 six inch plastic straws for–you guessed it–one dollar. I bought, 1,000–straws, that is, not packages. So for $5 plus tax, I then had 1,000 finger pipets to use on those dropperless jars when I am making up slides of crystals to photograph with polarized light–a delightful addiction!
Online sites for surplus houses as well as some of the smaller customer-friendly scientific supply houses, hobby and craft stores can be a gold mine for the amateur microscopist. I must admit that I have an extreme susceptibility to good micro-tools. Mind you, I’m not talking about forceps that cost $200 or micro-scissors for $900. I do confess to once having paid $40 for a pair of micro-forceps and I saw that as an extravagance; nonetheless, they are a superbly crafted Swiss implement which it is a joy to use. I used to buy “micro-forceps” made in Pakistan from Cynmar Corporation. When you wish to or must buy from a scientific supply house, this company is almost always a good choice. Real customer service–human beings, not machines, answer the telephones–generally reasonable prices and items of good quality for the money with some inevitable exceptions. If I need something from a supply house, it’s usually the first place I check. With my passion for micro-tools and, for some odd reason, micro-forceps in particular–I’m sure Freud would have had a glorious time with that quirk–I recently discovered a wonderful site called Widgetsupply.com–hobby tools galore and micro-forcep heaven (except for the fact that they call them “tweezers”)–and this would be inexcusable were it not for the fact that the prices are terrific and the quality of some items, even though made in China–what isn’t these days?–is surprisingly good. Being congenitally (no, Virginia, that’s not a dirty word) suspicious, I initially ordered just one pair of the 5 types of forceps I like (under the listing of “Sharp Tweezers”) which stated that they were made in China according to Swiss standards. From my point of view, for $3.49 per pair, they are an excellent value indeed. You can also find on this site a wide variety of probes, scissors, dental tools and other useful items at bargain prices.
There are some other small companies which are also amateur friendly and they are well worth seeking out. However, since we’ve been discussing forceps, I want to mention another consideration. Forceps are often shipped with a small, clear, plastic band near the tips to hold them closed. More expensive pairs sometimes have a small plastic cone that fits over the tips and you can purchase small packages (a dozen, I think) from a large high-tech microscopy accessory company called Ted Pella. They have all kinds of goodies which will make you drool, but most of them are moderately to very expensive. However, these little gizmos are relatively inexpensive and–wonderfully, amazingly–this company has no minimum order requirement. These cones are useful and essentially designed to protect the sharp tips. The plastic bands are essentially useless in that regard and are easily lost and the cones are easily misplaced or stolen in the middle of the night by lab elves. The cheapest and most efficient solution is to go to your local pet store or discount store and buy a bundle of clear, plastic tubing used for aquarium pumps. You can cut the length you need, slip it over the end of the forceps and both the tips and your fingers are protected. This is especially important if you have sharp, fine-pointed forceps of the kind I prefer for delicate work. Now, if you lose one or two of the protective covers–no problem–you just cut some new ones.
Most amateur biologists perform dissections on a wide variety of organisms and so may require some other sorts of tools for these enterprises. Scalpels are, of course, essential. Certainly, these days the most cost efficient solution is to buy a #3 and a #4 stainless steel scalpel handle. On the Internet, you can find blades for these handles in about 10 or 12 different sizes and shapes. Furthermore, some sites offer exceptionally good discounts on boxes of 100 blades. Since these are surgically sharp, it is wise to invest in a blade remover or two. This is a small plastic box into which you slide the blade end and then have this clever little device remove and safely store your used blade. You need to exercise great care in putting a new blade on the handle. I hold the blade in a hemostat to insert the new one. Sometimes, you can also find excellent deals on the Internet for disposable scalpels. A few years ago, I got 400 of them for $20. It is essential to dispose of these responsibly. You are all familiar with those Christmas desserts that great aunts make from cement and wax fruit and pack off in colorful tins. Well, save the tins and discard the fruitcakes taking care not to do yourself a serious injury by dropping one on your foot. The disposable scalpels, when sufficiently used, can be placed in the tin and when it’s full, securely bound with duct tape to prevent any accidents for your refuse collector.
Micro-scalpels are a very different issue. What most scientific supply houses sell under this title are simply scalpels with small blades which are generally not very sharp. They can be quite handy for some things, but are not really very useful for micro-dissection. With a bit of practice using the surgical scalpels, you will soon discover that you can do remarkably intricate dissections. If, however, you wish to try your hand at slicing up some protozoa–I’m not kidding–then you can make yourself some micro-glass needles. Use the soft flint glass. You can make these with Pasteur pipets or thin solid glass rod, both of which are inexpensive. If you decide to go with the Pasteur pipets, buy the 9 inch ones which have a longer tip and are easier to pull. Heat them in the flame of an alcohol lamp fueled with wood alcohol (Methyl alcohol–CAUTION! very poisonous and highly flammable. Carry this out only in a very safe secure setting.) When the middle of the tip gets red hot, remove it from the flame and pull the ends steadily and vigorously in opposite directions. This is essentially the same procedure that you would use with glass rod. This is the same procedure I use when I want to make micro-pipets from Pasteur pipets. I use a metal tray to place them on while they cool after they are pulled. Frequently the “stubs” are very thin solid glass threads and they can be trimmed to a desirable length and use as glass micro-dissecting needles. A significant number of protozoologists have used such home-made tools to make incisions in Stentor, Spirostomum, Bursaria, Blepharisma, and Amoeba among others to study the regenerative capabilities of these organisms. If, however, you are an executive of a large banking firm, you can take a tiny bit of your bonus and buy a lovely pair of micro-manipulators (to supplement your economic macro-manipulators) for under $50,000.
Micro-dissecting needles are another item which you can produce in your own lab. The most convenient way is to obtain a couple of small pin vises which will close down to zero and get a packet of small insect pins. Hold a pin in a hemostat or small pair of pliers and snip off the small round head with a pair of wire cutters. WEAR GOGGLES! Using another small pair of pliers, you can bend the tip a bit to give you an angle if you like. The steel in these is very springy and the points extremely sharp, so handle them carefully. If you want something even smaller, you can purchase a packet of Minuten-Nadeln from Carolina Biological. They are about $17, but you get 500 of them. These are used for pinning very small insects like mosquitos. You can get a friend or two to share the cost with you and you’ll all have lots of ultra-micro-dissecting needles or pinned gnats and mosquitos. I strongly recommend using a pair of forceps to handle these as they are exceptionally fine and sharp and can easily get lodged in a finger or thumb.
If you want something not quite so small and springy, you can take standard dissecting needles and sharpen them to a very sharp point using a small diamond pocket hone of the sort fishermen use for sharpening hooks. These can be obtained for less than $5 and, with a bit of practice, you can also turn a dissecting needle into a micro-chisel which can be very useful. I have used them to scrape excess dried mounting medium from around the edges of cover glasses and also to remove sphaeridia from irregular echinoids.
Another essential for most microscopists is slides and also cover glasses. Let’s start with slides. For general use–and here’s a recommendation I almost never make–buy Chinese. In many cases, for example, when you are not making permanent mounts or doing critical microscopy at high magnifications, the inexpensive run-of-the-mill Chinese slides are quite satisfactory. I would also generally recommend that you NOT buy stocks of old slides on the Internet. Remember that glass is a very strange substance that is essentially an extraordinarily viscous liquid and interestingly, from the microscopist’s point of view, disturbing things happen to glass as it ages. I have encountered boxes of slides where the slides were stuck together in multiples and I had to pry them apart using a micro-scalpel. Also, older slides can be cloudy and sometimes pocked with minute chips and here, I am talking about major brands, ostensibly manufactured in the U.S.
If you do need some high quality (and moderately expensive) slides to do critical microscopy, then you may wish to consider recycling them by placing them in a wide-mouthed jar half-filled with 70% rubbing alcohol. This allows you to clean and re-use them as necessary.
Microscopical technicians (these are those little tiny guys that are a few microns tall) are adamant about cover glass thickness and insist that for critical microscopy one must use cover glasses of #1 ½ thickness, ideally 0.17mm., and you will notice on high quality objectives with a magnification of over 10x that engraved on the barrel of the lens is “0.17" which tells you that the manufacturer has corrected these lenses for that thickness and thus the cover glass becomes a critical part of the optical system of your microscope. The slides are not as crucial and a thickness approximately 1.0 mm. is satisfactory; it is after the light has passed through the specimen that it is crucial to have the proper thickness, clarity, and flatness in a cover glass. So, buy some high quality #1 ½ cover glasses and when you are using them for temporary, rather than permanent mounts, you can use a pair of your micro-forceps to remove them and save them in one of those handy baby food jars that is again half full of 70% isopropyl alcohol. When you need them, you can gently clean them. For routine, non-critical work, you may wish to buy some cheaper cover glasses and even some of a # 1 or # 2 thickness. For low power scans, these are often quite acceptable.
We talked about culture dishes, but not about culture media. You can buy a variety of different sorts from scientific supply houses, but they are not inexpensive. Many micro-organisms thrive in pond or artesian water to which 1 or 2 grains of boiled wheat has been added. Boiling them greatly reduces the number of spores and cysts that can quickly produce molds in a culture. If you can’t obtain wheat easily, you can take some whole wheat or multi-grain bread, slice it into small cubes and let it dry out. You can then crumble some of this into your culture dish, add water, wait a few days for a good bacterial growth to develop and then add some organisms. Another good food is rice grains and these you can find in virtually every grocery store except for the carnivore-only stores which ban all plant material and vegetarians. In this instance, I don’t recommend boiling, because you get a sticky mess. Yes, you’ll get mold growth, but take advantage of it. Actually the hyphae can be both helpful and interesting in their own right, because rice grains produce a sort of thicket in which many of our micro-beasties like to hide and feed.
A number of companies sell a product called Cerophyll which is dried cereal grasses (mostly rye, I believe), but it has gotten quite pricey. A substitute which works as well, if not better, can be produced in your own kitchen. Take a head of lettuce peel off outer leaves and place them in a baking pan or on a cookie sheet and heat them a low temperature in your oven until they are dry and crisply. You may want to make up several panfuls at a time. When they have dried and cooled, take a medium-sized jar, place a sheet of paper under, it and vandalize the lettuce, that is by rubbing it between your thumb and index finger, reduce it to flakes and powder over the mouth of the jar. When you’ve finished, you can take any spillage on the paper and by bowing it a bit make a little chute to transfer it into the jar. When you want to use it, sprinkle some over the surface of some clean filtered pond water or artesian water in your culture dish, let it sit for awhile to absorb moisture and then using a glass rod, stir it into the fluid so that as much of it as possible sinks to the bottom forming a nice layer of detritus, which is much preferable to having it floating around the surface obscuring your view when you put it under your dissecting microscope. After a few days, there will be sufficient bacterial growth, so that you can inoculate your culture with small flagellates and ciliates. If you wish to cultivate flagellates or ciliates that have chloroplasts or chlorophyll-bearing symbionts, then you must place your dishes where the will receive bright, but indirect light. This is a medium in which Colpidium thrives and it is a very desirable food organism to maintain for some of the larger ciliates.
Soil extract media are another culture material which biological supply houses will be glad to sell to you. Some of them even sterilize it for you, so that you only culture the organisms you are obsessed with at the moment. Heaven forbid that you should find out what’s lurking in that soil out in your garden. So, clearly this is another do-it-yourself project or as lingophobics in this meta-cybernetic age would say: a DIY project, which could just as easily stand for Don’t Injure Yourself. So, for starters, you need some nice rich soil out of your garden. If it’s frozen solid, then get a bag of potting soil and while you’re at it a small bag of manure and one of peat moss as well. You never know what bio-treasures you may find. If you can dig in your garden, find one of your neighbors’ young sons who is a fishing enthusiast and tell him that if he digs up your garden, he can have all the worms he finds for his next fishing trip and you get the double benefit of having some nice loose soil to use for the soil extract medium plus your garden is now ready for planting.
Most of us readily accept that there are lots of bacteria and molds in soil, but it rarely occurs to us that soil is also full of small ciliates, flagellates, and some very, very interesting amoebae, and nematodes, mites, and, of course, earthworms. There is in fact, a well-illustrated little book on soil amoebae by F.C. Page published by the Freshwater Biological Association. It’s a 155 page paperback, but I found only one listing for it at the outrageous price of $98.10, so you’d better head to the library.
We tend ordinarily to think of soil as just plain old dirt, whereas in fact, it is an extraordinarily complex environment filled with all sorts of nutrients and micro-organisms. Not only is your garden soil a micro-treasure trove, but when you are out collecting you should take soil samples from the banks of ponds and streams.
To sterilize or not to sterilize, that is the question. The easy answer is: do both. Divide the sample and boil one half in some pond or artesian water and simply place the other half in a jar and add water. You can judge for yourself which is the most desirable medium. Let the preparations sit for 2 or 3 days and then filter them. You can use laboratory filter papers–a slow and tedious process–or coffee filters which are quicker and not quite so fine-grained or a piece of nylon netting from a fabric store or, if you’re really casual and want very quick results, a few layers of cheesecloth. In any case, the point of the exercise is to get rid of the large bits of dirt and other detritus while retaining the nutrients and minerals and perhaps a few cysts. The chances are good that your samples will benefit from some additional nutrients to encourage bacterial growth as food for other organisms, but you need to exercise restraint regarding how much you add, otherwise you will simply get a foul-smelling mess that only the most low-life, disreputable micro-organisms will inhabit. A wheat or rice grain or two or a bit of dried shredded lettuce may produce a blossoming, blooming bonanza of bodacious beasties–experiment!
Some biological supply houses sell protozoan pellets. Years ago, I bought some from 2 different companies. I was not impressed and I felt a bit embarrassed about trying to take the easy way out rather than going out and collecting my own samples. A better and cheaper approach is, at the peak of the collecting season, to take some samples of sphagnum moss, Nitella, Chara, duckweed, and other aquatic plants and, when you get home, spread them out on some paper toweling, let them dry thoroughly in the sun and place them in jars with labels of the kind of plant and where you collected it. At Hutton Lake, in a Federal Game Reserve (yes, I called and got permission), I took some plant material which had already dried on bushes lining the shore. This lake is highly alkaline and everything is coated with a white deposit of alkaline salts. It is here that I found that strange little amoeba with acicular pseudopodia and minute scales, Raphidiophyrs, which will group together with a number of other members of its own species to form a temporary feeding net and then they separate until they are in the mood for another Lucullan feast. I have a couple of gallon-sized plastic bags filled with this dried material and whenever I get curious about Raphidiophyrs, I pull some of it out, pop it in a culture dish, add water and wheat and in a week or so–Raphidiophyrs! Such dried material has a special value when winter closes off access to our collecting spots. Believe it or not, although there are 4 major highways in and out of Laramie, there are sometimes snowstorms and winds which close them down for several days at a time.
If you have a strong stomach and constitution–and most important a current tetanus shot– you might also want to collect some dried manure–cow dung, horse manure, elk or moose droppings, etc. Even if it’s dry, I suggest you wear gloves. I have seen a cow pie (odd name–hard to imagine it as a pastry) in the water near the edge of a shallow which was white. That seemed very curious to me and so I took a sample to examine back in my lab. And what was this meringue on the manure?–masses of one of my favorite protozoans, the “micro-whale”, Spirostomum. The manure of ruminants consists largely of party digested vegetable matter and so provides an excellent source of nutrients for micro-beasties. And even something so lowly as manure can provide some nice surprises. On a number of occasions when turning over a dried cow patty on the prairie, I have found underneath, busily at work–what else?–dung beetles.
The point of all this rambling is that one generally needn’t spend money on culture media and nutriments. With a bit of effort and ingenuity, you can find all kinds of sources from your own back garden to your favorite collecting spots.
One final tip. If you are sorting small fossils, bones, or mineral fragments, ice cube trays are very handy and when you finish a session for a day, you can wrap them with plastic food wrap to keep the dust out. If you are sorting very small items such as forams, there are some mini-ice cube trays that have lots of small chambers and they serve quite nicely.
With a bit of imagination and ingenuity, one can maintain ones microscopic studies and at the same time cut costs. I’m sure that there are many other suggestions that some of you could come up with, so help us all out and write an article for Micscape outlining some of your ideas.
All comments to the author Richard Howey are welcomed.
Editor's note: Visit Richard Howey's new website at http://rhowey.googlepages.com/home where he plans to share aspects of his wide interests.
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