Inexpensive Water-Immersion Microscopy
by Richard L. Howey, Wyoming, USA
Although I have a very good stereo-zoom Olympus microscope, I am sometimes frustrated by the limitations of useful magnification. With 20x oculars, I can push it to 80x, but above 60x, I experience significant light loss. With a 2x auxiliary lens, I can go up to 160x, but at the expense of a great deal of curvature of field and color distortion halos surrounding the organisms. Use of the 2x lens also radically reduces the working distance and necessitates transferring organisms from my usual small culture dishes to Syracuse watch glasses. If I am examining new samples, this is not a problem. However, if I wish to examine the organisms in situ which have already established themselves on the bottom of the culture dish, I can do so only by disturbing them in the process of transferring them to a Syracuse watch glass. When trying to observe organisms which attach to the substrate, the transfer to a watch glass or a slide (for traditional observation with a compound microscope) often disturbs the very conditions which one is trying to observe.
Several years ago, I acquired an old American Optical
Microscope and a box of odds and ends of objectives and oculars
at very reasonable prices. As a consequence, I decided to try an
experiment. At the time, I was doing a considerable amount of
observation of Lacrymaria olor and an odd little amoeba
which secretes a gelatinous hemi-spherical "house"
around itself. This amoeba has filose pseudopodia. I have
subsequently discovered that this amoeba belongs to the genus Nuclearia.
In trying to get a good look at these creatures, I kept running
into problems. When I removed the Lacrymaria from the
substrate, they would begin to swim and take on their usual
active form. However, in the culture, dishes, they often anchor
themselves in the debris and some remain active feeders while
attached and others go into a "resting" stage. The Nuclearia,
when transferred to a slide for ordinary observation, were
disturbed and often distorted to such a degree that it was
difficult to observe them in a "natural" state.
To get around these difficulties, I equipped the old American Optical Microscope with inexpensive lenses about which I was not overly concerned should they suffer minor damage. Along with 10x oculars, I used 4x, 10x, and 43x objectives for immersion directly into the solution in the culture dishes. I typically use small culture dishes with a diameter of 2 1/2" and a working capacity of about 25 cc. These are convenient for handling when using a dissecting microscope and, in addition, they fit fairly comfortably onto the stage of the American Optical microscope.
I discovered regarding the 4x objective that the focal length is such that the lens does not immerse. The 10x and the 43x objectives do, however, usually require immersion in the culture fluid. Now, I do know that there are special water immersion objectives, but they are usually rather expensive, even when acquired used. I had no idea whether or not my experiment would produce images which would provide useful information.
The results exceeded my expectations, especially with the 43x (giving me an image magnified 430 times). I was able to observe Lacrymaria anchored to the substrate, but actively feeding. This is something I had observed in slide preparations which contained sufficient detritus, but only rarely. Also the extension of the "neck" is considerably more pronounced in the culture dish specimens which are unrestricted by a cover glass.
I was now able to observe the Nuclearia which not been possible before. In both instances, I was now able to observe the organisms undisturbed and notice behavior which I had previously not been able to see in detail. There are two major virtues of this technique: 1) the organisms remain virtually undisturbed and 2) when using this method, rather than the usual slide preparations, there is no flattening of the specimens as a consequence of a cover glass. When we routinely deal with slides, it is all too easy to forget that we are dealing with three-dimensional organisms. A short session with a dissecting microscope observing a large amoeba or Stentor quickly restores the proper perspective and vividly reminds us that we are not dealing with flat creatures, such as we see in drawings, but, rather, complex organisms that behave much differently in the semi-natural environment of a culture dish as against the flatland world of a microscope slide.
Cleaning the objectives carefully after each session is imperative! If one doesn't, a film will develop on the lens and produce a significant degradation of the image. At the time when I first undertook this experiment, I was also examining cultures of marine protozoa and again obtained very good images and information. With marine cultures, it is absolutely essential to clean each objective immediately after immersion. Saltwater is highly corrosive and can damage not only the lenses, but the metal housing. With care, the lenses will last indefinitely and provide a new way of observing organisms without elaborate and time-consuming preparations. Many texts on micro-technique recommend that amoebae be observed on slides by using a hanging drop method with a depression slide. The technique which I have described has the virtue of avoiding this tricky business of preparing hanging drops and allows one to see the organisms without disturbing them.
Comments to the Comments to the author sent via our contacts page quoting page url plus : ('rhowey','')">author welcomed.
Editor's note: read the Micscape article 'Tears of a Swan' describing the fascinating organism Lacrymaria olor.
Published in September 1998 Micscape Magazine.
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