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Tuatara: Volume 6, Issue 2, December 1956

A Beginning in Microscopy

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A Beginning in Microscopy

The modern microscope is a precision instrument. Over a long period it has been developed to a high degree of mechanical and optical perfection; but the microscope is still only an optical accessory. Physically it enables the formation of an enlarged image. The accuracy of this image, its observation and interpretation are not properties of the machine. These are functions of the observer. Unless the observer is adequately trained in the correct manipulation of the instrument, in the observation and interpretation of the image, even the finest microscope is virtually useless and largely misleading. Instruction in the manipulation of the microscope can be given in three to five hours of carefully designed microscope drill which is directed as much to the safeguarding of the machine as to the elements of its use, but training in precise accurate observation and interpretation is a matter of a minimum of fifty hours of intensive disciplined study.

The strongest objection must be taken to the occasional use of the microscope by untrained observers. Apart from risk to the machine, there is failure to obtain a physically clear image and difficulties in the recognition of microscopic detail grossly hinder proper appreciation of the object examined.

In brief, the microscope is a highly technical instrument and unless technical training is a first aim, time spent in the use of the microscope is largely wasted. The observed facts, for which the microscope is employed, can be amply and better dealt with through the medium of the motion picture when the purpose is cultural and not technical training.

In the technical training given in biological courses at the university level, instruction in the use of the microscope has been most carefully developed and has had an influence on the design of such courses. Work with the microscope commonly commences in familiarisation with the mechanical and optical systems, followed by drill in the use of the instrument, using some stationary object (e.g. the cells of the ovary of the starfish), then moving objects (Amoeba, Euglena, Paramoecium, etc.) and the analysis of microscopic detail (e.g. the life-history of Monocystis). In this sequence, there is also a gradual approach to the facts and terminology of the morphology, physiology and life-history of cells and protozoans. At least fifteen hours are spent in such studies and this is followed later in the year by some thirty-five hours of microscopic work in histology and embryology.

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This is a tradition in technical training. It establishes no precedent to be used for cultural instruction in biology. The steps to be taken in this latter type of teaching may be and should be developed along their own and proper line with real advantage to the subject and the pupil, and an economy of equipment.

The following outline is intended for initial training in the proper use of the microscope. The drill must be made the subject of constant practice until the steps are established as an unvaried routine in the safe operation of the microscope.

The Microscope

Identify the parts as follows: the mount, consisting of a forked base, vertical pillar, hinge-joint, handle or arm (for carrying); the concave-plane mirror borne on the mirror-arm; the substage mount, bearing the condenser and iris diaphragm, carried on a substage focussing adjustment which permits movement of the condenser along or out of the optical axis; the stage; the body, consisting of a barrel (with draw-tube) carrying a revolving nose-piece on which are the low and high power objectives, and the ocular or eyepiece. The ocular, objective, substage condenser and mirror are brought into line on an optical axis.

The barrel is moved along the optical axis by the coarse adjustment and by the fine adjustment systems, operated by milled wheels on the arm.

Always watch the objective from the side when racking down with the coarse adjustment. The fine adjustment is used when viewing objects, and only for sharp focussing. The fine adjustment has a limited range and must be kept in the middle of its movement.

Do not leave the eyepiece out of the drawtube. This would permit dust to settle on the inside lens of the objective.

The microscope should be sketched and the parts labelled.

Operation of the Microscope

The following steps constitute a routine which must be minutely followed at all times for the proper use of the machine and to minimise danger to the object or to the mechanical or optical systems.

The microscope must be carried only by the handle and the base, never by the stage or barrel which may disturb the setting of the stage or damage the fine adjustment mechanism. The microscope must not be inverted.

Set the microscope squarely on the bench in front of the mid-line of the observer. This permits the convenient use of either eye. The machine should be vertical, not tilted. Watching from the side of the microscope to avoid striking the stage with the objective, swing the low-power objective into the optical axis. Rack the condenser up to the stage. Open the diaphragm. Collect light by the mirror from a steady source of adequate page 41
Figure by courtesy of Geo. W. Wilton and Co. Ltd.

Figure by courtesy of Geo. W. Wilton and Co. Ltd.

page 42 illumination (30 watt frosted or pearl bulb at one foot) and direct the light up the optical axis so that the surface of the condenser is evenly flooded with light. Rack the barrel down with the coarse adjustment so that the low-power objective is within 1.0 cm. of the surface of the condenser. Partially close the diaphragm.

View the field through the eyepiece and adjust the mirror to give a completely uniform illumination over the entire field. Check by opening and closing the diaphragm. Set the diaphragm at a comfortable light intensity.

The source of illumination and the microscope now form a single optical system. Neither may be moved without readjustment of the system.

Before examining a prepared slide, identify the thin cover-slip protecting the specimen which is mounted in Canada balsam. Gently clean the slide. Place the slide on the stage with the cover-slip on top. Centre the specimen over the condenser. Watching from the side, rack the barrel down with the coarse adjustment until the objective nearly (1.0 cm.) contacts the cover-slip. Viewing through the eyepiece, rack the barrel slowly up until the specimen is in focus. If the object does not come into view, repeat as before. To prevent forcing the objective on to the slide, do not focus down with the coarse adjustment unless watching from the side.

When the specimen is in focus, slowly alter the aperture of the iris diaphragm. Observe the change in detail as the intensity of illumination is varied. Resolve detail by use of the fine adjustment and by adjustment of the light intensity to the optimum for detail.

No two specimens or even two parts of the same field of a specimen are alike. Constant adjustment of the light intensity is an essential for the refinement of detail and as important as the use of the fine adjustment. Fatigue of the eye during work also demands occasional readjustment of the light intensity. To prevent undue fatgue it should be the practice to use each eye for short periods, not one eye continuously.

Examination under the high-power objective follows the above steps. The object is centered under the low-power objective, the barrel racked up with the coarse adjustment, and the high-power objective swung into the optical axis. (The objectives should be parfocal and swing into focus without racking up; but this cannot be relied on to protect the objectives from damage unless objects, cover-slips, etc., are standardised.)

Watching from the side, rack down with the coarse adjustment until the objective nearly (less than 1.0 mm.) touches the cover-slip. View through the evepiece and rack up very slowly with the coarse adjustment until the specimen is in focus. Resolve detail by use of the fine adjustment and manipulation of light intensity.

When work is completed, swing the low-power objective into the axis; remove the slide; place the microscope in a position where it will be safe and in no danger of damage.

When the observer has gained an appreciation of minute detail, he should set up the microscope as above, carefully focus under high power page 43 on a prepared slide such as a stained section of tissue to show clear cytoplasmic granules. The condenser should be gradually racked down, the diaphragm adjusted to provide optimum light for detail. It will usually be found that with the condenser slightly below the level of the stage, still further detail can be gained or the definition of detail improved. Likewise, the use of the plane side and then of the concave side of the mirror should be tested to show the effect on minute detail.

The microscope must be kept clean. Wash off any chemicals or sea-water, and dry. The eyepiece, field-lens of the objectives, the condenser and the mirror should be kept free from marks and dust. Canada balsam can be carefully sponged from the objectives or other parts with Xylol.

Keep the machine free from dust and always under cover when not in use. Do not grease or oil. Properly cared for, a good microscope will operate efficiently for five years and should then be overhauled. A poorly cared for machine requires cleaning and overhaul each year. Proper care and periodic overhaul will keep a machine in use for thirty to fifty years. Carelessness and negligence may destroy the best machine in a minute.

The ovary of the starfish: This slide is used as part of the drill, as an example of a simple cell and as a static object which can be readily studied while attention is directed largely to the manipulation of the microscope. The specimen is a section of the ovary, 10 microns in thickness, stained with iron haematoxylin for nuclear detail and counter-stained with eosin for cytoplasmic detail.

Study several cells under the low power and identify; the cell-membrane; cytoplasm; nuclear-membrane; nucleoplasm; endosome and chromatin granules. Sketch several cells. Label the parts shown in the sketch.

Examine and sketch a single cell as seen under the high power.

Euglena: This is a slow-moving protozoan and gives an initial training in moving and searching a slide and in following a moving object.

1. Fresh Material: Using a pipette, transfer a drop of the culture medium to the central position on a clean slide. Cover with a cover-slip. Locate a specimen of Euglena under low power.

Study several specimens and observe: (i) free-swimming in which the Euglena moves by using the single flagellum as a tractellum to pull itself along; (ii) Euglenoid movement in which the Euglena moves by creeping through contraction and extension of the body.

Study a specimen under the high-power. Identify: pellicle (which may be striated); thin layer of clear ectoplasm; endoplasm, containing the green chromatophores which may have pyrenoid bodies (the paramylum bodies); nucleus surrounded by a nuclear membrane; the flagellum (often retracted), when visible arising from the short gullet which leads into the reservoir; the stigma and contractile vacuole, both situated close to the reservoir. Sketch to show these structures. Label the sketch fully.

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A Simple Method for the Preparation of Permanent Microscope Slides

Parts of insects, small insects, section of dry wood, hair, fish-scales, a piece of feather, etc.

Kill and fix by dropping material into hot but not boiling 70% alcohol. Leave until cool. When cold transfer to 95% alcohol; leave up to five hours. Transfer into 95% alcohol in a watch-glass. Run in drops of clove-oil around the margin of the alcohol until the specimen floats fully supported on the clove-oil. Leave covered against dust until the specimen sinks into the clove-oil, or the alcohol has evaporated.

Clean a slide and cover-slip. Place a drop of balsam on the centre of the slide. Transfer the specimen with a minimum of clove-oil to the balsam. Cover neatly with a cover-slip and leave for the balsam to harden.

Clean the watch-glass with soap and water.

Other simple and useful techniques will be found in Tuatara, vol. V, part 1, pp. 12-21; vol. V, part 3, pp. 87-99; etc.

Useful Texts

1. Peacock, Elementary Microtechnique.

2. Guyer, Animal Micrology.

3. Chamberlain, Methods in Plant Histology.

4. Bolle's Lee, Microtomist's Vade Mecum.

5. Gage, The Microscope.