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 1½ 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 1½ 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 Richard Howey welcomed.
Editor's note: Richard has written a Micscape article on the fascinating protozoan Gromia.
Published in September 1998 Micscape Magazine.
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