I wish here to thank Mr. H. J. McKay, the museum locksmith for the skill with which he constructed the instrument. INTRODUCTION.

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1 A SIMPLE INSTRUMENT FOR DISSECTING MINUTE ORGANISMS. By J. P. HARDIKG, Ph.D. British Museum (Katural History) (Read Novewzber 16th, 1938.) THREE TEXT-FIGURES. INTRODUCTION. As far as I am aware no method has previously been devised for dissecting animals of the size of many small Ostracods and Copepods which are too small to be dissected with any precision by hand and at the same time are too large for elaborate instruments, such as the Chambers or Taylor micromanipulators. The new instrument, which is designed to bridge this gap, is very simple in construction and inexpensive to build. It is also very easy to manipulate. The point of each needle is controlled, not by a complicated arrangement of milled heads, but by the movement of a single knob in the required direction ; so that the needles are moved as simply as if they were held in the hand and with far greater precision. The principle of the instrument is that of the pantograph, which is an instrument for mechanically copying B drawing on a different scale. In the case of the dissecting instrument a knob is moved by hand and the point of the needle copies this movement on 8 much smaller scale. I wish here to thank Mr. H. J. McKay, the museum locksmith for the skill with which he constructed the instrument. DESCRIPTION OF THE INSTRUMENT. The complete instrument (fig. 1) consists of two dissectors mounted on a base which is attached to the stage of a dissecting microscope as shown in fig. 3. The brass base (fig. 1, G) has a piece cut out to take a microscope slide of the type with a depression in the centre. The centre of the depression comes in the centre of the field of the microscope. Although the space in the base is large enough for a complete slide, one end of the slide has been cut off leaving the depression and the other half complete. The reason for this will be explained later. Each dissector consists of a brass rod AB (figs. 1 and 2) with the mani- 2-2

2 20 Transactions of the Society. pulating knob A of some lighter material at the free end. The other end of this rod is forked and in the fork two flat strips of brass are hinged with small rivets at D and B. These strips are kept parallel to one another by two additional strips, one on each side, hinged at E and F. The distance between the rivets D and B is equal to that between the rivets E and F ; and the distance between D and E is equal to that between B and F. Each dissector is attached to the base by a ball-and-socket joint at c. This may be made from a bicycle ball bearing, softened and brazed to the strip BC and held in a simple socket drilled in the base as shown in Fig. 1 (b). A grip- I \ FIG. 1.-(u) (a) (a) Plan of the dissecting instrument ; (a) Section through the ball-andsocket joint. screw holds the dissecting needle N * in position in a hole drilled longitudinally into the strip DE. The screw should be at the side and not too large or it will be in the way. The needle should be adjusted so that its point is in line with the centres of the knob A and the ball C. The joints D, B, E, F, and C should all work smoothly but be sufficiently tight to support the weight of the dissector, a spring washer in one of the joints facilitates this adjustment. I have found the following dimensions convenient : AB, 5 inches (13 cm.); DB, Q inch (16 mm.); BC, 1 inch (24 mm.) and BF, a inch (12 mm.). If a microscope with a magnification of 50 diameters or more is used the needles can be moved freely over the whole field of view. * Suitable needles may be made by mounting fine tungsten wire about 0.06 mm. thick in rods of solder made by sucking the molten metal up a capillary glass tube. About 1 mm. of the wire is allowed to project. This is now sharpened on a hone of Arkansas stone. In order to obtain a sharp point the needle must be held at a constant angle to the hone. This may be accomplished by mounting the needle at the required angle (about 16 ) on to a glass slide with plaeticine, and sliding this on the stone, or if the tungsten is pure, and the end of the wire is held in a small gas flame, the tip will burn off leaving an exoeediugly fine tapering point. If desired the tip may easily be bent to form a micro-hook. Such needles while being sufficiently stiff are not brittle.

3 A Simple Instrument for Dissecting Minute Organisms. 21 Every movement of the knob in three dimensions is copied on a much smaller scale by the point of the needle. Outline of Geometrical Proof. -. Fro. 2. Let A (Fig. 2) be the position of the knob when the needle was adjusted so that its point lay on the line AC. Let the knob be moved in any direction to a new position A' then the point of the needle will move to a new position N'. Let B', D', E', and F' be the new positions of the points on the instrument B, D, E, and F respectively. Draw the construction lines AC and A'C, and also AA' and NN', the paths of the knob and needle point respectively. It is required to prove that NN' is parallel to AA' and that the ratio NN' : AA' is a constant for the instrument. By the construction of the instrument it is given that : DN is parallel to BC and that D'N' is parallel to B'C I.... and N is on the line AC :. A'S ADN and ABC are similar... AB : AD=BC : DN IV. and AB : AD=AC : AN The lengths AB, AD, BC, and DN are not changed by the movement of the instrument into its new position. :. Equation I11 becomes A'B' : A'D'=B'C : D'N' and D'N' is parallel to B'C :. A's A'D'N' and A'B'C are similar. :. N' is on the line A'C and A'C : A'N'=A'B' : A'D' =AB : AD (Same parts of same instrument) =AC : AN (Equation IV) :. A's AA'C and NN'C are similar

4 22 l'ransadms of the Society. :. NN' is parallel to AA' and NN' : AA'=AC : NC =AB : DB which is a constmt for the instrument. Q.E.D. We have only considered movement of the knob in the plane ABC, but the proof may easily be extended to show that the movement of the point of the needle is proportional and parallel to that of the knob even when A' is in some other plane. In addition the direction of the needle may be altered wjthout moving the position of its point by rotating the dissector about the axis AC. MODIFICATION FOR USE WITH A MICROSCOPE WHICH GIVES AN INVERTED IMAGE. The instrument as described gives a movement of the needle in the same direction as the movement of the knob. If, however, the positions of the needle and the ball-and-socket ball are reversed the needle will move in the opposite direction to the movement of the knob. With an ordinary microscope which gives an inverted image this gives the illusion of direct movement and manipulation is greatly facilitated. ATTACHMENT FOR FOCUSING. It is a great help when dissecting to be able to focus the microscope without taking the hands from the control of the needles. The method I have used with success for small objects is illustrated in Fig. 3. A lever is fixed to the milled head of the coarse adjustment on one side so that it projects over the edge of the table and is approximately horizontal when the microscope is in focus. A spring connects this lever with the back of the microscope stage. In practice a stout rubber band is suficient. The spring tends to raise the head of the microscope out of focus ; but this is counteracted by a thrusting rod attached to the end of the lever and resting on the worker's knee. With the ball of the foot on the ground the knee is easily raised or lowered sufficiently to bring the desired level into focus. TECHNIQUE FOR REMOVING APPENDAGES OF NINUTE CRUSTACEA. AND R'IAIiING PERMANENT MOUNTS OF THE By using the instruments described above all the 22 appendages from a single small copepod, for example, may be dissected off and arranged in order in hyrax * or Canada balsam under one coverslip in less than one hour. * Hyrax is made by Penn and Ruedrich and is obtainable from Messrs. Flatters and Garnett, London. Its advantage over Canada balsam is its very high refractive index, which makes staining unnecessary.

5 A Simple Instrument for Dissecting Minute Organisms. 23 In addition to the microscope and the dissecting apparatus and focusing attachment described above the following are required : (a) Two hand needles consisting of fine entomological pins mounted in light and slender handles and bent slightly at the points. (b) Two capillary pipettes. These are glass tubes about 5 cm. long and 2 mm. thick with a very fine capillary about 1 cm. long at one end. (c) Two smail specimen tubes shorter than the capillary pipettes, one containing pure xylol and the other benzyl alcohol. The capillary pipettes are placed in these and fill by capillarity. FIO: &-The dissecting instrument and focusing arrangement attached to a dissecting microscope. Benzyl alcohol is the most satisfactory fluid in which to dissect that I have tried. I am indebted to Prof. Cannon for suggesting it. It is a clearing agent which does not make the crustacea hard or brittle, and has the great advantage that it does not evaporate. The crustacea may be transferred to benzyl alcohol directly from 75 p.c. or 90 p.c. alcohol, there

6 24 Transactions of the Society. is, however, some danger of shrinkage and it is better to transfer first to a mixture of half benzyl alcohol and half absolute alcohol. Glycerine is not recommended as a clearing agent in which to dissect if a permanent mount of the appendages is to be made in Canada balsam or hyrax. The many transferences through the alcohols from glycerine result in a great danger of losing some of the appendages. As benzyl alcohol is soluble in xylol its use greatly reduces the number of transferences necessary. There is also a tendency for the contents of the crustacean to float out and foul the appendages in glycerine, and this nuisance does not seem to occur in benzyl alcohol. If it is desired, the chitin may be stained in chlorazol black (see Cannon, 1937) before clearing. The copepod, with sufficient benzyl alcohol to cover it, is placed on the depression slide in the centre of the field of the microscope. The needles are used in the same way as they would be if they were held in the hand ; one needle holds the animal in a suitable position while the point of the other is brought up to the base of the appendage and it is simply pushed off. Minute drops of benzyl alcohol about 4 mm. in diameter are placed in rows on t,he complete half of the slide by touching it with the benzyl alcohol capillary pipette. As each appendage is removed in turn it is placed in the appropriate drop of benzyl alcohol. One of the hand needles is used for this purpose. The required drop is brought into the field of the microscope by reducing the magnification or by moving the slide, it was to make the latter possible that half the slide was cut off. When all the appendages have been placed in order in the drops of benzyl alcohol the dissectors are removed from the microscope. The point of the xylol capillary pipette is brought to the drop containing the first appendage, some of the xylol flows out and the benzyl alcohol is washed away. The appendage is now picked up by touching it with a little hyrax on the point of a hand needle. The appendage is removed to another slide where it is washed with xylol until it is quite free of benzyl alcohol. A drop of xylol is put on a coverslip and a small quantity of hyrax is added to this. The xylol prevents the formation of air bubbles. The appendage is picked up again and put in the drop of hyrax. The coverslip is then placed appendage uppermost on a warm hot-plate, or close to the microscope lamp, and the second appendage is treated in the same way and mounted alongside the first. When all the appendages have been transferred to drops of hyrax on the coverslip and these drops have hardened (about half an hour is usually sufficient) the coverslip is placed on a slide in the usual way. Air bubbles are avoided if xylol is first put on the slide, sufficient hyrax is then added and wetted with xylol from a pipette, and the coverslip with the appendages is placed on top. If the crustacean has been previously stained Canada balsam may be substituted for the hyrax.

7 A Simple Instrument for Dissecting Minzlte Organisms. 25 SUMMARY. A simple dissecting instrument based on the principle of the pantograph, and an attachment to the microscope for focusing without using the hands, are described. A technique is elaborated for making a permanent mount under one coverslip, of all the appendages dissected from one small crustacean. LITERATURE CITED. CANNON, H. G. (1937).- A New Biological Stain for General Purposes. Nature, 139,549.

Caldwell's Automatic Microtome.

648 OALDWELL'S AUTOMATIC MICROTOME. Caldwell's Automatic Microtome. With Plate XLII. THIS machine has been devised to save labour to the histologist by cutting a very great number of sections suitable