Archive for the ‘Microscopes’ Category

Information for students and teachers on how to work with microscopes.

Information for students and teachers on how to work with microscopes.

How to Work With a Microscope

One of the first requisites for the proper use of the microscope, is a thorough knowledge of its parts and an acquaintance with the optical principles involved. For this purpose the writer earnestly requests a perusal of the preceding pages, and is convinced that in cases where no previous knowledge of the instrument has existed, work will be .done with far more ease, in much less time, and with a greater degree of satisfaction. Ignorance of the instrument's capacity may lead to an idea that it is inferior and thus be the means of its final abandonment ; and in place of the anticipated pleasure there may arise a feeling of bitterness and disappointment for all future with everything connected with it. There are innumerable cases of this kind and they have induced a belief that it is difficult to acquire a practical manipulation of the microscope, whereas such is not the case when a limited time, properly applied, is devoted to it.

Working Table for Microscopy

A firm table should be used, preferably one with three legs, as this will always be firm no matter how uneven the floor is, and if it can be arranged, should be devoted to this purpose only. One with a round or square top of three feet provides ample room. Although not necessary, a table with a revolving top, provided with clamp, is very convenient, as with this two or more persons may make observations without changing their seats.

A very neat arrangement for a table-top is that suggested and used by Dr. J. E. Reeves. He places upon an ordinary table three or four thicknesses of white paper and upon these a plate of polished glass as large as the top ; this can be procured of almost any glazier at a low price. It is pleasant to work upon and will not soil.

As in almost all cities there is more or less continual vibration from wagons upon the paved streets, the writer suggests an effectual remedy. Take a thin board, say half an inch thick, of a sufficient size to receive the microscope ; fasten on the upper side at two opposite ends, cleats of 1 inch square and counter-sink into these through the board four spiral springs of such tension that when they bear the weight of the instrument, the bottom of the board will be about inch from the table.

Have the working table provided with drawers and arrange receptacles for the accessories, secure from dust, but at a convenient point to reach. When the instrument is not in use put it into its case or cover it in a manner so that it shall be free from dust. For this purpose a large bell glass is best.

Room to Conduct Microscopy

If possible to use a mirrored microscope, a room should be selected facing the north, as the light in this direction is most constant. It will prove a great saving of time if all or a portion of it can be permanently arranged to receive the entire working outfit. It should also be chosen with a view to its being free from disturbance.

Light for Microscopy

If using a mirror microscope as stated, the light from the northern sky is most desirable, and that from a white cloud is preferable to that from a blue sky. On account of its intensity, direct sunlight should seldom be used ; but if modified by a white curtain or reflected from a white wall it is excellent.

For lamp light an ordinary flat wick kerosene or student lamp is well adapted. The Hitchcock lamp, from its better combustion is still better, as its color more nearly approaches white. The ideal artificial light is that from an electric light. Gas light is not desirable as it is seldom sufficiently steady.

Position of Light for Microscopy

The relation of the microscope to the source of light is principally a matter of personal convenience if you are using a mirrored microscope. With daylight it makes little difference whether it is at the front or side of the instrument, although the writer prefers it at the front, as the manipulation of the object does not obstruct it ; but the lamp should be placed at the right or left side within, easy reach of the hand for the purpose of controlling it. The writer suggests that the beginner make it a habit at the outset to place it on the side of the instrument opposite to the unoccupied eye, as the tube then places the latter in the shadow.

Which Eye to Use with a Microscope

In a binocular instrument both eyes are used, but in a monocular only one is used, and it depends upon a trial which is best suited. A large proportion of persons are afflicted with astigmatism, often without knowing it, and when this exists it may be in one eye or when in both, may be to a greater extent in one than in the other. Its presence may prevent the eye from observing fine detail ; but whichever eye is found to be best suited should be used. When both eyes are alike it is sometimes advisable to change from one to the other.

It should be made a habit at the outset and strictly adhered to, to keep both eyes open.A little difficulty may be found to do this, as the eye which is free will probably observe the objects upon the table ; but as soon as the mind becomes fixed upon what it sees in the microscope, this impression disappears. After a time it will be found to require no exertion and will certainly add to the ease and comfort of the manipulator while working.

Order for Microscopy

Among the requisites for successfully prosecuting work with the microscope are a strict observance of the instructions, even if they appear superfluous, a systematic way of doing work, and cleanliness. Have a place for every article which is required, so that the hand may immediately be placed upon it ; after it has been used clean it before putting it aside ; keep strange hands from your apparatus unless you are assured that a knowledge of its manipulation exists.

Material to Use in Microscopes

Although the purpose of this manual is to be a guide to the intelligent use of the microscope and not the preparation or preservation of objects, it may not be out of place here to enumerate what every owner of an instrument should have at the outset. The first should be a book on objects giving proper instruction on their preservation. There are many of these, and all of them good. Next in order, slides, covers and labels are necessary. As covers are easily broken in cleaning, a larger proportion of them will be necessary.

A cabinet for slides, a large variety of which may be selected from, will aid in starting work in a systematic manner. Forceps and a small pipette are indispensable. For preserving objects, Canada balsam or damar should be purchased, while the other necessary material which may be gleaned from the instruction book is easily obtainable.

When it is intended to do section-cutting a good mechanical microtome, not necessarily expensive, should be obtained at the outset.

Classification of Older Microscopes.  Learn about the Jackson, Ross and Continental patterns of microscope instruments.
Classification of Older Microscopes.  Learn about the Jackson, Ross and Continental patterns of microscope instruments.

Classification of Older Microscopes

In the past, microscopes were divided into two classes : the Jackson and Ross models. While the latter was for many years very popular, particularly with the English makers, it was almost entirely superseded by the Jackson form, and with good reason. In the former the means of adjusting were provided, as near as consistent with the construction, to the body or tubes, whereas in the Ross they are placed at the back or more distant point in the instrument, thus increasing by means of the connecting arm the faults which might exist in the adjustment.

A certain form of instrument which was very popular and was called the Continental pattern, from the fact that it was made originally by the manufacturers on the continent of Europe, is a combination of both the Jackson and Ross models. Whereas, the coarse adjustment when consisting of a rack and pinion is placed closely to the tubes, the fine adjustment is placed on the arm and although being dissimilar from the original Ross in being higher, it nevertheless had the disadvantage of magnifying any lost motion in the adjustment by means of the connecting arm. Considering the fact that the Ross form alone is almost obsolete and many instruments of the present day are a combination of both forms, it appeared to the writer that their designations have lost their value.

There is another direction, however, in which microscopes are divided into two classes, which is of far more import ance, and affects their utility in a much higher degree. The writer does not know that instruments have been so classified by others, and knows that the subject has been given no important significance.

In the Continental form just mentioned, a short tube from 160.0 to 170.0 mm. (0.3 to 6.7 inch) is used, whereas in the English form, and this is largely followed in America, the length is from 8 to 10 in.

(216. 0 to 250.0 mm.). The short tube of the European makers offers no optical advantages, but is mainly used to contract the height of the instrument to as great an extent as possible, as this is the vital point throughout its construction.

At a meeting of the American Society of Microscopists, a committee was appointed to consider the tube lengths as well as other subjects to be mentioned hereafter and reported in favor of the adoption of two standards for tube lengths, 160.0 mm., or 6.3 inch for the short one, and 8| inch, or 216.0 mm., for the long one. The American makers have adopted these two lengths and we believe are generally following them. Practically, there were no advantages in one or the other, except, perhaps, in so far as the short tube might be considered advantageous, but optically this recommendation of the committee is far reaching, because an objective, particularly in giving considerable magnification, when constructed to be used with a certain tube-length, should be used with it only. When used with the other standard it will fail to give satisfactory results.

Microscope Blood Work and Red Blood Cell Counting. Equipment such as haemacytometes for counting.

Microscope Blood Work and Red Blood Cell Counting. Equipment such as haemacytometes for counting.

Microscope Blood Work

Haemacytometers

The haemacytometer is an apparatus for counting the blood corpuscles, and consists of a counting chamber, two mixing pipettes, and suitable optically plane cover glasses. The blood is first diluted with a solution known as " Toisson's" solution, for the red or white, or with a solution of acetic acid when a count of white cells only is being made. In counting red corpuscles a dilution of 1-200 is generally used, but in certain cases 1-100 may be employed. The blood is drawn into the pipette up to the mark 0*5 in the case of 1-200 dilutions, and up to the mark 1 for 1-100 dilutions. The pipette is then immediately placed in the diluting fluid, which is drawn up to the mark 101 above the bulb. Both ends of the pipette are then closed with the fingers, and the pipette shaken to ensure an even mixing, the glass bead in the bulb facilitating this. For white corpuscles, a dilution of 1-10 is employed and the other pipette is used. For filling the counting chamber, a drop of the mixture is blown out of the pipette, after allowing several drops to go waste, into the centre of the counting chamber. The cover glass is then placed over the cell. The drop of blood must not be allowed to overflow the platform into the groove which surrounds it, and the cover glass must be in perfect contact with the object slide, and all must be scrupulously clean.

The counting chamber consists of a plate of glass with an annular groove ground upon it. The circular portion inside the groove is ground and polished to a distance of 1 mm. Below the level of the plate of glass.

In the Thoma haemacytometer this portion is ruled with a diamond into squares l/400th of a square mm. Each in area. It will therefore be seen that the amount of liquid resting upon each square has a cubic capacity of l/4,000th of a cubic mm. The liquid which has been placed in the counting chamber is allowed to settle, and the corpuscles will therefore be in contact with the bottom of the cell. It will be found that it is a simple matter to count the corpuscles contained in each square. The usual method is to count, say, 100 squares, and it must be noted that in dealing with those actually on the lines, only those on two sides of the square should be counted, and this rule should be applied throughout.

The number of corpuscles in 1 cubic mm. Of undiluted blood is then obtained by multiplying together the rate of dilution, the number of corpuscles counted, the volume of each square (l/4,000th of a cubic mm.), and dividing by the number of squares counted. The above is the general method of counting the red corpuscles ; but in the case of the white corpuscles, as there are a very much smaller number of these, the method generally employed is to count the total number of the whole ruled area of the counting chamber, which is 1 sq. mm.

Cell Counting Chambers

There are other forms of counting chambers, such as the Biirker, Fiichs-Rosenthal, Breuer, and Zapperts ; the method of employment in all these is the same, but the ruling and also the counting are different in each case.

The use of a mechanical stage greatly assists the counting.

A simpler form of haemacytometer can be used which depends for its action on Mr. Rheinberg's beautiful process of making graticules and glass scales. A glass plate is photographed with squares in the pattern of a chess-board, so that alternate squares are tinted, although they are transparent. This plate is dropped into an eyepiece between the lenses, and by means of a stage micrometer the drawtube can be varied until a definite number of squares are equal to *1 of a millimetre in the micrometer. The chess-board glass plates are supplied with squares either 1/4, 1/2, 1, or 2 mm. In size. They are made to cover the whole field of view, or as a small block of squares in the centre of the field. The latter are to be preferred for blood counts.

The only other requirement is a 3 X 1-inch slip with a metal ring cemented to it which is 1 mm. Thick, into which the blood is placed covered with an ordinary cover glass. Suppose a 1/6-inch object glass is being used, a 1-mm. Chess-board plate dropped into the eyepiece can be made by drawing out the drawtube to the required position according to the eyepiece and object glass employed, of such an apparent size that nine squares, three each way, correspond to '1 mm., and the count of nine squares will give the number in a cubic tenth of a millimetre. If a 1/2-mm. Chess-board plate be used, then thirty-six squares, six each way, correspond to a cubic millimetre. The most convenient size to select will depend upon the class of object to be counted and the object glass that is used.

Due to the alternate squares being tinted, a count can be made with much less eye-strain than with the ordinary haemacytometer, and this method is preferred by some apart from the question of the cost of the apparatus.

Flatness Of Field Microscope.

Flatness Of Field Microscope.

Flatness Of Field Microscope

Although this depends mainly upon the objective, the absence of it may be owing to a faulty construction of the eye-piece. If it is so prominent as to be easily noticeable, and to the same degree with a number of objectives, it may be ascribed to the eye-piece. It must, however, be remembered that an absolutely flat field has not yet been obtained ; it may be closely approached by decreasing the diameter of field to less than its normal size.