Inverted Microscopes:
Some Notes on David Goldstein's Article
(July 1998)

by Richard L. Howey, Wyoming, USA

David Goldstein presents an interesting account of the advantages and disadvantages of an inverted microscope which as a plankton microscope is incomparable and a delight to use once one adjusts to its idiosyncrasies. A couple of years ago I purchased a used Wild/Heerbrugg M40 inverted microscope. The condenser is brightfield, darkfield, and phase and there is a 4x scanning objective and 10x, 20x, 50x, and 100x phase objectives. The 50x and 100x are oil immersion. The binocular head has a built-in, slide-in rotating polarizer and a magnification of 1.5x, so that effectively I have a working range from 60x to 1500x. The objectives are designed for a cover glass thickness of 0.17mm which is no problem if one is using standard slides; you simply turn them upside down so that the cover glass is on the bottom.

Like David Goldstein, I did not have any specially designed containers and I didn't want to be limited to using only slides, so I decided to make some chambers of my own. I bought some plastic Falcon brand Petri dishes, 50mm in diameter which fit conveniently on my stage plate and allow full use of the mechanical stage. This particular dish has a tight-fitting cover and thus can be used to keep a sample for days or even weeks if one adds a bit of water from time to time.

To make the chamber, I take the bottom of the Petri dish, position a cover glass of the size I want to use and then with a sharp dissecting needle, trace around the edge of the cover glass. Then on each of the four sides, I move the edge of the cover glass in about 1/8 of an inch and mark again, so that I have a smaller square within a square or smaller rectangle within a rectangle depending on the dimensions of the cover glass. Finally one cuts out the smaller rectangle or square. Obviously one can also do this with circles as well, but the straight lines are much easier to cut.

I tried a number of different means of cutting. Small electric tools tend to generate too much heat and the plastic quickly melts and clogs the cutter or drill. I finally decided it had to be done by hand and I got some old tools from my dentist—the kind that are not for the squeamish. These are not your thin wire-like picks (which, by the way are very handy for picking fossils out of a matrix). No, these are the thick, robust, bone-scraping and cutting tools. One can put a very sharp edge on the cutting tip with a whetstone.

You then trace the line of the inner rectangle or square with the cutter over and over applying more pressure until you gradually cut out a hole of the proper size. This is a somewhat tedious process and each chamber requires about 20 to 30 minutes with pauses. The edge of the hole will probably be a bit rough and require a bit of trimming or one can sand the edges.

Select a number 1 thickness cover glass of the appropriate size and using a strong waterproof cement, attach the cover glass to the outside bottom of the Petri dish. Once it's dry, you will have a micro-aquarium. I have made up about a dozen such chambers. Some I use for freshwater material, some for marine samples, and others for preserved material. When attaching the cover glass, try to get a thin even layer of cement that seals on all edges. After this is dry, I usually add another layer or two of cement around the outside bottom edges to insure there will be no leakage. It is especially important not to let marine samples dry up in the chambers since the formation of salt crystals can gradually break the seal of the cement and cause leakage.

With the number 1 cover glasses, I can achieve excellent resolution even at the high magnifications of 750x and 1500x. In one of my little marine tanks, I found some specimens of Gromia oviformis which, when undisturbed, produce an astonishing network of filose pseudopodia. One of my friends, on first seeing them, remarked: "These organisms have got to be from some other planet. I've never seen anything so totally alien." The remarkable thing is that at 1500x one can see particles moving both toward and from the central shell like cars on a superhighway and furthermore the filopodia anastomose constantly altering the shape of the web.

The inverted microscope is ideal for observing such organisms. A stereo-dissecting microscope has insufficient magnification to observe some of the most interesting details. If you remove the Gromia from a culture to put them on a slide, you tear loose virtually all of the network of filopodia. If you are lucky enough to get one properly situated under a cover glass without crushing it (these organisms range from about 0.5 mm. to more 5.00 mm.), then you must patiently wait for the Gromia to extend their filopodia (and they are not particularly cooperative). In the meantime, the water is evaporating and with a marine sample on a slide, it takes very little loss of water to radically affect the salt concentration making it toxic to the organism. So, it is ideal to have a chamber which functions as micro-aquarium and allows, with the use of an inverted microscope, the organisms to be observed in situ undisturbed.

David mentions the cost as the major drawback of the inverted microscopes and indeed it does involve a significant investment. However, there is a relatively inexpensive alternative which gives quite good results. I described this in a brief article for the news letter of the Manchester Microscopical Society and I include it here for those who are looking for a low cost alternative for viewing organisms in a culture.

Comments to the author Comments to the author sent via our contacts page quoting page url plus : ('rhowey','')">Richard Howey welcomed.

Editor's note: Richard has written a Micscape article on the fascinating protozoan Gromia.

 

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