liMiM 
 
 LIBRARY 
 
 New York State Veterinary College 
 
 ITHACA, NEW YORK 
 
 m 
 
 d66 
 
 Dolley, Charles Sumner 
 
 Notes on the methods employed in biological 
 studies. Compiled solely for the use of students 
 in the laboratories of the School of Biology. 
 University of Pennsylvania, I889 
 
CORNELL UNIVERSITY 
 
 THE 
 FOUNDED BY 
 
 ROSWELL P, FLOWER 
 
 for the use of the 
 
 N. Y. State veterinary college 
 
 1897 
 
 This Volume is the Gift of 
 
 ..Dr....F».. 1. Fish 
 
 Digitized by Microsoft® 
 
7^/^^ A r 
 
 ,4V /'■ \ — 
 
 NOTES 
 
 METHODS 
 
 EMPLOYED IN 
 
 Biological Studies. 
 
 COMPILBD SOLELY FOR THE USE OF STUDENTS IN THE 
 LABORATORIES OF THE 
 
 SCHOOL OF BIOLOGY, 
 UNIVERSITY OF PENNSYLVANIA. 
 
 BY 
 
 CHARLES S. DOLLEY, M.D, 
 
 Professor of General Biology, 
 
 PUBLISHED BY THE UNIVERSITY. 
 l8Sq. 
 
 uigirizea oy iviicrosonw 
 
This book was digitized by Microsoft Corporation in 
 
 cooperation witli Cornell University Libraries, 2007. 
 
 You may use and print this copy in limited quantity 
 
 for your personal purposes, but may not distribute or 
 
 provide access to it (or modified or partial versions of it) 
 
 for revenue-generating or other commercial purposes. 
 
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PREFACE. 
 
 When training large classes to use to the best advantage the 
 microscope and other scientific appliances, and to gain in a very 
 limited time a working knowledge of the yarious means and 
 methods employed in modern biological research, it has been a 
 consta.nt source of annoyance to be obliged to refer students to 
 a dozen or more different handbooks for the details of technical 
 apparatus or processes. To avoid this confusion, the following 
 notes were compiled for my own use in connection with the 
 laboratory work; they were selected especially for the needs 
 of this particular School, in which the Course in Greneral Biologj' 
 precedes tb.6 various Courses in Botany and Zoology proper, with 
 the intention of giving the student not only a knowledge of the 
 fundamental principles and phenomena common to aU living 
 things, and an acquaintance with certain typical plants and 
 animals, but also a desirable amount of skill in manipulation. 
 
 The rapid increase in the number of students, and my desire to 
 give as much personal attention as possible to each, suggested 
 the propriety of furnishing them with a set of printed notes 
 covering the field of microscopical technology as taught in the 
 Laboratoryj 
 
 As the following pages have been compiled during the last 
 five years without reference to publication, I am unable to give 
 proper credit without retracing the entire work, a task for which 
 I have neither time nor inclination; Free use has been made of 
 the Manuals and Handbooks issued by Fbl, Strieker, Yogt and 
 Yung, Schaefer, Heitzmann, Lee, Wilder, Cage, Comstock, 
 Strasburger, Bower and Vine, Shore, Poulsen, Behrens, Good- 
 ale, Bessey, Huxley and Martin, Marshall and Hurst, Howell, 
 
 (3) 
 
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Davis, Dolley, Dubief, Praenkel, Woodhead and Hare, Hueppe, 
 Sternberg, and a number of standard works on the microscope. 
 Each of these works has furnished its quota of valuable hints 
 or descriptions, and in acknowledging them as the sources from 
 A\ hich these notes have been drawn, I only regret my inability 
 to specify the author of each quotation. I am particularly in- 
 debted to my friend Mr. Edward Bausch for permission to make 
 copious extracts from his valuable little book on " The Manipula- 
 tion of the Microscope." 
 The notes are issued solely for the use of students of this 
 
 school and not for public distribution. 
 
 C. S. D. 
 
 School of Biology, University of Pennsylvania. 
 
 Philadelphia, Oct. 1st, 1889. 
 
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On the Construction and Manipulation 
 of the Microscope. 
 
 SIMPLE MICROSCOPES. 
 
 Purpose of the Microscope. 
 
 The Microscope is an instrument which magnifies objects, so 
 that we are better able to examine their structure than is possi- 
 ble with unassisted vision. 
 
 Kinds of Microscopes. 
 
 Microscopes may be divided into two classes — simple and com- 
 pound — tlie difference between the two being that with the 
 foiniej' the object is viewed directly, while with the latter a 
 magnified image is observed : while the first shows the objects 
 in their true position, the latter shows them reversed, so that 
 what is right in the object is left in the image, and when an 
 object appears to be moving in a certain direction, the move- 
 ment is in reality the reverse, and must be moved accordinglj- 
 to keep it in view. 
 
 Magnifiers. 
 Simple microscopes are usually termed magnifiers, and, 
 whether consisting of one or more lenses, always remain simple. 
 The most common are those with one or several double-convex 
 lenses. The shorter the radii (the more curved the surfaces) 
 are in these, the gTeater will be the magnifying power, and the 
 higher this is, the less of the object's surface can be seen at 
 once. Each additional lens increases the magnifyintg power in 
 proportion to its curvature. The distance between the lens and 
 the object, when this is seen most distinctly, is called the focus ; 
 at the point where the object is most distinct, the lens is said to 
 be in focus; when indistinct or blurred, out of focus. 
 
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Magnifying Power. 
 
 It is evident that a lens magnifies an object equally in all 
 directions; this is said to be in areas, and is the square of the 
 linear, so that if an object is magnified 4 times in the linear, it is 
 16 times in area. The commonly accepted term to express mag- 
 nifying power of simple, as well as compound microscopes, is in 
 diameters (linear).' A single lens of 1 inch focus magnifies about 
 ten diameters; one of 2 inch focus, about 5 diameters; one of J 
 inch focus, 20 diameters, and so on. In a lens of high magnify- 
 ing power, the focus is ordinarily about twice the diameter, so 
 that if a lens is J inch diameter its focus is about 1 inch. This 
 may, howeyer, be more accurately determined by projecting, 
 say a flame or window-frame, upon a white piece of paper; the 
 distance between the paper to tlie center of the lens, when the 
 image is most distinct, is its focal distance. When a lens is two 
 inches or more in diameter, it is usually termed a reading glass. 
 
 Using Magnifiers. 
 
 In using magnifiers, the lens should be held close to the eye 
 and such a position taken that the object will receive the best 
 illumination. In the lenses of equally convex surfaces, it is 
 immaterial which side is held toward the ej'e; but when plano- 
 convex lenses are used, the plane side should always be toward 
 the eye, as it gives the flattest field. 
 
 Aberrations. 
 
 Two factors arise which prevent the advantageous use of more 
 than about 2.3 diameters in magniners; they are called the chro- 
 matic and spherical aberrations. The first is the term employed 
 when the object is apparently fringed with color, predominently 
 blue and yellow; the second, when all but the central portion of 
 the lens shows the object indistinctly; these faults increase with 
 the magnifying power. In the case of a combination of several 
 lenses, they may be partially overcome by interposing an opaque 
 plate Avith a small opening', called a diaphragm, between them, 
 which cuts off the outer or marginal rays, or the lenses may be 
 made of a smaller diameterj An incision may also be cut into 
 the glass' equally between the two surfaces, when, from the name 
 of the inventor, it is called a Coddington. 
 
 Achromatism. 
 
 The most approved method, however, for eliminating these 
 appearances is by the use of one or two concave flint glass lenses 
 
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in connection with the double convex crown glass lens. When 
 the color or chromatic aberration is thus removed, the lenses are 
 said to be achromatic, and when both the chromatic and spheri- 
 cal aberrations are avoided, the lens is called aplanatic, and 
 is then said to be corrected. An achromatic lens, composed of 
 one flint and one crown glass lens, is called a doublet; one with 
 two flint glass lenses and one of crown glass is called a triplet. 
 The latter is the best form, as it gives the highest correction ; 
 such a lens (it is thus called from the fact that the lenses are 
 cemented together and act like one) may be held with either 
 side toward the object with equaUj^ good results, and may also be 
 held at quite an obliquity, without loss of definition ; this feature 
 is important, as it is almost impossible to give a lens a theoreti- 
 cally correct position to both the eye and object Avith the un- 
 aided hand. 
 
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THE COMPOUND MICROSCOPE. 
 
 As was previously stated a magnified image is observed in the 
 Compound jMicroscope. An>' two lenses, one of short, the other 
 of a long focus, placed sufficiently far apart, will attain this 
 object, and this was for years the method of its construction. 
 It was found that on account of the extreme magnifying power, 
 mechanical appliances were required to kee\> the lenses steady 
 during observations and at their proper distances, also that pro- 
 vision was necessary for adjustment and illumination. These 
 adjuncts were so necessary that the instrument could not be con- 
 ceived without them, and the entire apparatus is thus, bj' the 
 force of usage, called a microscope, and the instrument without 
 the optical parts, a stand. 
 
 Base or Foot. 
 
 This is the foundation of the instrument. It usually rests upon 
 three points (or should do so) and is of such a weight that it 
 keeps the instrument firm when it is in an upright or inclined 
 position. I 
 
 Pillar. 
 
 It is that portion %^hich is fastened to the base and may be 
 one or two, according to the construction of the stand. It 
 carries upon its upper end the joint or axis. 
 
 Arm. 
 
 This is connected with the pillar by the joint and supports all 
 the ^^■orking parts of the instrument. 
 
 Body. 
 This is the tube portion to which the optical parts are attached. 
 
 Nose-Piece. 
 
 This is an extra piece which is attached to the lower part of 
 the tube. 
 
 Society Screw. 
 
 This is a standard screw which is cut into the nose-piece, and 
 is called so from the fact that it was first established by the 
 
 (8) 
 
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Royal Microscopical Society of Loudon. Tt is also called the 
 universal screw, and is in Igeneral use in this country and Eng- 
 land; it has lately been adopted by some firms on the Continent 
 of Europe. 
 
 Objective. 
 This is screwed into the nose-piece and is called so because it 
 is nearest the object. It is the more important of the two opti- 
 cal parts (of the microscope proper) and upon its perfection the 
 distinctness of the image and therefore the value of the instru- 
 ment almost entirely depend. 
 
 Eye-Piece or Ocular. 
 It is called so because it is nearest the eye and is the remain- 
 ing optical part. It magnifies the image given by the objective. 
 This and objective will be treated more fully later on. 
 
 Draw-Tube. 
 
 This is that portion of the body which moves in the outer 
 sheath and which receives the eye-piece- Tt is provided for 
 the purpose of attainin'g variations in magiiifying power and as 
 a matter of convenience while A^orldng. 
 
 Collar. 
 This is a ring which is attached to the draw-tube and is 
 usually provided with a milled edge. 
 
 Coarse Adjustment. 
 
 This is a provision for moving the body quickly back and 
 
 forth for adjusting the focus approximatelyj It is done by a 
 
 sli<iing rack and stationary pinion or a sliding body in an outer 
 
 sheath. 
 
 Milled Heads. 
 
 These are attached to the shank of the pinion, which is re- 
 volved by means of them and are largely used to give sensitive- 
 ness to the movement. 
 
 Fine Adjustment. 
 This is slow moving and serves to get an exact focusj It is 
 attained by a fine thread, provided with a milled head, and acts 
 upon the body, either directly or by levers. This as well as 
 the coarse adjustment should be extremely sensitive and should 
 not have the least side or lateral motion. The fact that either 
 of them has it, is evidence of poor workmanship. 
 
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10 
 
 Stage. 
 This is the portion on which the object is placed for examiiia/ 
 tion. It is attached to the arm and may be either permanent! 3' 
 fixed, or revolving or mechanical, the latter by moving the object 
 by mechanical contrivances instead of by lingers. Metal and 
 glass are used in its consti'uction. 
 
 Clips. 
 
 These are two springs which are attached to the upper sur- 
 face of the stage and serve to hold down the object. 1 
 
 Mirror. 
 
 This is used for reflecting and condensing light upon the ob- 
 ject. As a rule two are used, one plain and the other concave. 
 The first gives a comparatively weak light, while the second 
 concentrates it and giA^es it more intensity. 
 
 Mirror-Bar. 
 
 This carries the mirror and by a sliding arrangement allows 
 the variations in distance of the mirror to the stage; it also 
 swings in a circle around the object in order to illuminate it 
 from any direction. 
 
 Sub-Stage. 
 
 This is a ring below the stage to receive various accessories 
 which may be required. It is sometimes fixed to the stage, but 
 in the best instruments it is separated from it and is provided 
 with an adjustment to vary its distance from the object. 
 
 Sub-Stage Bar. 
 This receives the sub-stalge and permits its adjustment. This 
 as well as the mirror-bar should be on an axis in the plane of 
 the stage, so that whatever position they may be in, relative to 
 the object, the distance from this to the sub-stage or nairror 
 does not vary, except when anade to do so. 
 
 Diaphragm. 
 
 This is a perforated, revolving disk, attached either to the 
 stage or sub-stagej It has holes of different sizes so that the 
 amount of light from the mirror may be modified. 
 
 Optical Axis. 
 
 This is an imaginary line which passes from the center of 
 the eye-piece through the body, objective, stage and sub-stage 
 to the miri'or. Whatever lies in it is said to be centered. 
 
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EYE-PIEOES. 
 
 Huyghenian Eye-Pieces. 
 
 This is now in general use, and consists of two plane-convex 
 lenses. It receives its name from the inventor, who first ap- 
 plied it to the telescope. The eye-lens is the small lens nearest 
 the eye, and the field-lens or collective, as it is also called, is 
 the large one nearest the objective. A diaphragm is placed be- 
 tween them, and gives a sharply defined field. This eye-piece 
 is also called negative, as its focal point is between the two 
 lenses (at the diaphragm) in contradistinction to a positive, in 
 which the focal point is outside of and below the field lens. 
 
 Solid Eye-Piece. 
 
 This was the invention of the late E. B. Tolles, and also be- 
 longs to the class of negative eye-pieces. It is called soli<l from 
 the fact that, instead of being composed of two lenses, it consists 
 of one piece of glass, which is cut to a cylindrical form, and on 
 the ends of which the proper curvatures are ground; the dia- 
 phragm is made by cutting a circular groove into the glass at 
 the proper distance between the two surfaces, which is then 
 filled up with an opaque pigment. 
 
 These eye-pieces are only made in high powers, as optical 
 glass is usually not of sufficient homogeneity to make low pow- 
 ers, and their cost would be too considerable, without a corre- 
 sponding advantage. For high powers they are superior to the 
 Huyghenian, in that they give a better illuminated field, as there 
 is less loss of light by absorption through the glass than by re- 
 fraction at the two additional surfaces of the eye-lens and 
 field-lens in the Huyghenian. 
 
 Periscopic Eye-Piece. 
 
 This consists of a triple eye-lens and single field lens. Its 
 predominant feature is a very large and flat field, with almost 
 all objectives. In this respect it has a considerable advantage 
 over the Huyghenian and Solid. It is positive and therefore 
 well adapted for micrometer work, as it is focused like a 
 magnifier, and its magnifying power remains constant, Avhile 
 
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12 
 
 with the Huyghenian it is variable, from the fact that the eye- 
 leas alone is focused, thus varying its distance from the field- 
 lens, and consequently the magnifying power. 
 
 Nomenclature. 
 
 The rating of eye-pieces was formerly, and is to a considera- 
 ble extent to-day, by lett^ers. This method, however, is arbi- 
 trary, as the letters of different makers have a totally different 
 significance, so that nothing like a standard exists. 
 
 Flatness of Field. 
 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 abso- 
 lutely flat field has not yet been obtained; it may be closely ap- 
 proached by decreasing the diameter of the field to less than its 
 normal si^e. 
 
 Size of Field. 
 
 Quite a general but erroneous idea prevails that the size of the 
 tube has an influence on the size of the field. Except in eye- 
 pieces of very low power, or with tubes of smaller than usual 
 dimensions, this is not so. It must be remembered that a Huy- 
 ghenian eye-piece admits of a definite size of field, and this is 
 regulated by the opening in the diaphragm; the same size of 
 opening is used in all of the same power, whether it is for an 
 eye-piece of a large or a small diameter. 
 
 The field of vicAV, or that which is shown of the object's sur- 
 face, is determined by the power of the objective and eye-piece. 
 
 Objectives, Classes. 
 Objectives may be divided into t\\'o classes, dry and immersion; 
 in the former no intervening medium except air exists between 
 the cover and objectis^e, while in the latter a fiuid is used to 
 connect the upper surface of the cover witli the front surface of 
 the objective. The use of immersion fluid has several advan tages, 
 the first of which is that the objective may be made to give 
 better ijei'formance, as Avill be explained later on; tlie second is 
 that more light will be transmitted, as there is less loss of it by 
 j-efraction. It must be observed that an objective which is 
 made to use dry can not be used with immersion, nor ~sdce versa. 
 
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13 
 
 There are two fluids in general use at the present time, water 
 ami homogeneous fluid. The latter expression means of the 
 same kind, and refers to the fact that the fluid has about the 
 same refractive and dispersive power as glass, so that when this 
 fluid fllls up the space between the two surfaces of glass, a ray 
 of light passes through the three mediums as if they were one 
 body. 
 
 Objectives are sometimes called powers, and in this sense are 
 divided into three classes : low, medium and high; Dr. Carpen- 
 ter classifies them as follows: low powers, 3 inch, 2 inch, 1^ inch, 
 1 inch, I or I inch; medium powers, x% inch, J inch, i^ inch, | inch; 
 high powers, ^ inch, ^ inch, j\ inch, ^V inch, -jV inch, ^\ inch, i 
 inch. 
 
 Systems. 
 
 Ad objective is said to consist of systems which may vary in 
 number from one to four and five; two and three are, howeveo', 
 mainly in use.^ They are the individual portions consisting of 
 one, two or three lenses, which, when more than one, are 
 cemented together and make up the objective. An achromatic 
 single system may consist of two or three lenses, and a three or 
 four system objective may consist of as many as seven or eight 
 lenses. The systems are called in their order : anterior or front, 
 middle and posterior. When one consists of two lenses it is 
 called a doublet; when of three lenses, a triplet. 
 
 The various features which must be considered as determining 
 the quality of an objective are : angular aperture, achromatism, 
 resolving or defining power, flatness of field, penetration, work- 
 ing distance and magnifying power. Although these attributes 
 may be considered separately, some of them go hand in hand. 
 The presence or extent of one necessarily involves or precludes 
 another. 
 
 Angular Aperture, 
 
 The angle which the most extreme rays, which ai'e transmitted 
 through the objective, make at the point of focus is called its 
 angular aperture, or in short its angle, and of all the qualities 
 in an ideal objective, this is the most important. The above 
 definition has its limitations, however. While in objectives of 
 proper construction it holds true, there are many in which it is 
 not the case. For instance, an objective may be so constructed 
 that it may transmit a considerable number of rays in excess of 
 those which combine to form an image, and it is evident that 
 these should not be considered as belonging to them. 
 
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14 
 
 As there are many objectives of the same power, but of differ- 
 ent angular aperture, there are again others of varying power, 
 but of the same angle. Other things being equal, it is the angu- 
 lar aperture of an objective which determines the qualityj It 
 is expressed in degrees, and is also spoken of as being wide, 
 medium or narrow, although this is indefinite and depends con- 
 siderably upon the power of the objective; while the angle may 
 be excessively wide for a low power, it may be narrow for a 
 higher onej 
 
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HOW TO WORK. 
 
 To Set up the Instrument. 
 
 Draw the instrument from the case by grasping the base, and 
 free it from dust. If it has a draw tube, bring it to its standard 
 length, which is indicated by a ring, by as little of a screw mo- 
 tion as possible ; if the draw tube is highly polished, its surface 
 will be best retained by observing the above preca'ntion. See 
 that the mirror and the largest aperture of the diaphragm are 
 in a central position — in the optical axis. After being convince<i 
 that the eye-pieces are clean, place one in the tube; then re- 
 move the objectives from their cases, and after first having 
 increased the distance between the stage and tube by means 
 of the milled heads of the pinion, attach the lowest power to the 
 nose-piece, by using both hands, being careful that it is as near 
 as possible in the optical axis while screwing it on. Then incline 
 the body by placing the left hand upon the bjise and drawiag 
 with the right hand upon the arm; be careful not to pull 
 on the tube, as it may prove too heavy a strain upon this 
 or the fittings. Incline the l^ody of the microscope imtil the eye- 
 piece about reaches the level of the eye, !^b that when an obser- 
 vation is made the position is as comfortable as possible; the 
 neck should not be strained; neither should the chest be com- 
 pressed. Next place the slide with a transparent object upon 
 the stage, by sliding it under the spring clips and get it as near 
 as possible in the center of the opening; for an object anything 
 near at hand, such as a piece ,of printed paper or cotton fiber, 
 will do. Watching the slide, adjust the mirror until it is seen 
 that the light strikes the object; incline the head to the level of 
 the stage, and observing the objective, rack it down to within 
 J inch of the object. Again placing the eye at ;the eye-piece, re- 
 verse the motion of the milled heads and observing the field 
 continue the upward motion of ;the body until the ima^e of the 
 object appears in view. 
 
 If the coarse adjustment does not prove sensitive enough to 
 focus easily, adjust by the fine adjustment by taking the head 
 of the micrometer screw between the thumb and firs,t finger and 
 move toward the right or left as may be necessary. 
 
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16 
 
 If the fine adjustment does not act, the screw has either come 
 to its stop or has "run out," and must be brought into action 
 again; the range of movement in almost all fine adjustments is 
 quite short, and constant care must be taken to keep it at about 
 a medium poiat. If the object is found not to give a full view or 
 is not in the center of the field, it must be moved on the stage, 
 but it must be remembered that a movement in one direction 
 causes an .apparent opposite movement in the field. At first 
 this movement will be in jerks, but after a little practice the 
 necessary sensitiveness of touch is acquired to give it more 
 steadiness. 
 
 Illumination. 
 It should now be observed whether the field is equally illu- 
 minated. Too much stress can not be laid on this point, as it 
 is one which is easily overlooked and is often the cause of con- 
 siderable mischief. If the light comes through a window a well 
 defined image of the sash is reflected by the mirror, and with a 
 low power objective this can easily be seen ; unless the mirror is 
 correctly adjusted the field will appear to be crossed by da,rk 
 bands. In the case of lamp light the flame is reflected and has a 
 similar effect. With high powers this fault is not so easily no- 
 ticed, and for this reason require the more care; proper resolu- 
 tion may in this manner be partially or totally destroyed. The 
 remedy is either by shifting the mirror or by varj-ing its distance 
 from the object. 
 
 Attaching High-Power Objectives. 
 
 As the difficulty of properly getting an object or a certain por- 
 tion of it in the field increases Avith the magnifying power, it is 
 a good rule to use the lower power objective as a "finder;" after 
 getting the point to be further examined in the center of the 
 field, remove the objective and attach the higher power, and 
 after following the procedure of focusing as with the low power, 
 except that the objective should be brought almost iu contact 
 with the cover, the point will be seen in the field or \A'ill be found 
 to be close to it. 
 
 This plan of focusing as suggested above is always a good one 
 to follow and is observed by many of the most expert manipula- 
 tors. In many cases however the focus is obtained without 
 this precaution, by watching the field as the objective is brought 
 down toward the object, but is often followed by disastrous 
 results. Almost any person who has used the nncroscope for 
 
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17 
 
 any length of tiir.e is without question aware that valuable pre- 
 parations have been destroyed in this way. 
 
 Camera Lucida — How to Use It. 
 
 The camera lucida is the apparatus by means of which draw- 
 ings are made^ There are various forms of these, but those in 
 most general use are the Wollastnn Prism and neutral tint glass. 
 The methods of attaching to the tubes are also numerous. 
 
 "When a satisfactory section or preparation has been secured 
 the student should make an accurate (Irawing of its essential 
 features^ The employment of a camera lucida insures correct 
 proportions in all parts of the sketch, and is always to be recom- 
 mended. Drawings made by its aid are conveniently designated 
 by the following abbreviated term, ad nat. del. It may seem 
 hardly necessary to caution students against obscuring any part 
 of their histological sketches by meaningless shadings; a few 
 clean and clear outlines suffice to express the chai'acter of the 
 preparation better than any attempts to give the effects of 
 light and shade." — (Bausch.) 
 
 " The use of slips of drawing paper of uniform size and the ar- 
 rangement of these under appropriate heads will render the keep- 
 ing of a systematic record of work much easier." — (Groodale.i) 
 
 "The procedure of worldng should be about as follows: Focus 
 upon the object and then incline the body, so that the center of 
 the eye-lens will be 10 inches from the table, ^.'o obviate re- 
 peated measurements, a standar<l stick of this length may be 
 used. If the instrument is so low that it will not allow the in- 
 clination of the body to an angle of at least 45 degrees when at 
 this distance, it should be placed upon a bux; or, if not too high, 
 upon the case of the microscope. I^ow readjust the ndrror and 
 attach the camera lucida from below and place the paper under 
 the instrument; look into the camera lucida from above, being 
 careful that the eye is directly over the center of its opening, 
 and the image will be found to be projected upon the paper. 
 Possibly, and very probably, it will appear faint. This is due 
 to the fact that the paper is almost as highly illuminated as the 
 field. To remedy this defect a cardboard shoulil be placed be- 
 tween the paper and light, so that the fomier will be shaded; 
 the object will now come out in strong contrast. Take a well 
 pointed pencil and follow the lines in the image. A little diffi- 
 culty may at first be experienced in seeing botli the pencil and 
 image at one time, but after a little practice this is <Jver-come. 
 2 
 
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18 
 
 It very often occurs that the pencil point can on no condition 
 be seen distinctly, but this is usually due to abnormal sight, with 
 which persons are often afflicted.' In these cases, the glasses 
 which are required for reading should also be used in drawing. 
 The difficulty is not experienced in the image, as this can be ad- 
 justed to the eye.'- — (Bausch.) 
 
 "In drawing with the camera, first take a trace in very faint 
 lines, then, removing the cameo-a, finish the drawing and put in 
 the details with the free hand, always maintaining a close ob- 
 servation of the object. Of every drawing made with the camera 
 the magnifying power should be noted at once, as weU as the 
 number or letter of the eye-piece and objective." — (Bower.) 
 
 Measurements. 
 
 " Microscopic objects are measured by means of the micrometer. 
 The eye-piece micrometer can be more rapidly used than one on 
 the stage of the instrument; and if its value for the different 
 objectives and for the length of the tube has been determined 
 accurately, it is usually preferable. 
 
 The values of the spaces in the eye-piece micrometer are ascer- 
 tained by comparison with known spaces on the standard stage 
 micrometer; fot example, if one space in the eye-piece micro- 
 meter corresponds to five spaces of the sta|ge micrometer, and 
 the latter has a value of one-thousandth of a millimeter, each 
 space of the former equals five-thousandths of a millimeterj 
 
 The unit of microscopic measui'ement is the 'micro-millimeter' 
 one thousandth of a millimeter. It is expressed by the Greek//.. 
 
 For convenience of reference the following table of compara- 
 tive measurements is given : 
 
 IJ. Inches. 
 
 1 , . . . .000039 
 
 2 000079 
 
 3 000118 
 
 4 .000157 
 
 b 000197 
 
 IJ. Inches. 
 
 6 000236 
 
 7 000276 
 
 8 000315 
 
 9 000354 
 
 10 009394 
 
 Inches. jj. 
 iweas equals 2.5399. 
 X(sW equals 25.3997. 
 t;J„- equals 253.9972. 
 
 One meter equals 39.370433 inches.'' — (Goodale.) ' 
 
 The Stage Micrometer. 
 "The Stage micrometer is a glass slide on which fine equidis- 
 tant paiallel lines have been ruled with a diamond. The dis- 
 
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 tance between the lines is marked on the slide; it is generally 
 either the ^J^ and ^-^^jj part of an inch, or the -jV ^'^'^ t-ots 
 part of a millimeter. The lines ai-e observed with the microscope 
 in the same way as the object, and their image can of course be 
 similarly projected upon a sheet of paper- and then marked dowii. 
 The distances between the lines being known, it is easy, by 
 comparison of the two markings to find out the distance between 
 the opposite points of the object. 
 
 The Eye-Piece Micrometer. 
 This is a flat piece of glass having a scale ruled upon it by a 
 diamond. It may be inserted between the fi.eld-glass and the eye- 
 glass of the eye-piece, either by unscrewing the eye-glass and 
 dropping the circular piece into the eye-piece, or, wlien mounted 
 in metal or rubber, by pushing it through a slot in tlie eye-piece. 
 The value of the divisions of the scale should be determined 
 once for all for each objective by observation of a stage microm- 
 eter. The divisions of the eye-piece micrometer are to be com- 
 pared with the magnified divisions of the stage micrometer. 
 Ihus supposing it had been found by examination of a sta^e 
 micrometer that with the high power objective and the tube 
 drawn out each division of the eye-piece micrometer was worth 
 T55ijiiich, any object which when viewed by the same objective 
 and length of tube took up three divisions of the eye-piece mi- 
 crometer would measure x/uff °^ 'sis of an inch. The advan- 
 tage of the eye-piece micrometer is that when its values are 
 once ascertained the size of an object can be read off at once. 
 
 Determination of the Magnifying Power of a Microscope. 
 The magnifying power of a microscope is determine<l by com- 
 paring the distance between the lines of the stage micrometer, 
 as they appear imaged upon paper (by means of the camera lu- 
 cida), when this is exactly ten inches* from the eye, with the 
 known interval between them. For instance, if, with the high 
 power objective and the ordinary ocular, the interval ofy^Vuth 
 of an inch of the micrometei- was represented on the paper by 
 a space of half an inch, this interval is magnified as many times 
 as theyxi'iititti of an inch will go into half an inch, tliat is to say, 
 500 times, and every other object under similar conditions is 
 magnified to a like extent. The enlargement thus obtained may 
 be determined once for all for each objective, the same ocular 
 
 * The ordiniiry distance of dislinct vision. 
 
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 being used and the tube being drawn out to the same extent — 
 say to the full length — in each case, and the scales may be made 
 repiesenting the intervals betA\'een the micrometer lines under 
 the different powers. For purposes of measurement it will 
 then only be necessary to compare the projected image of aki ob- 
 ject M ith the scale, which was made under like conditions, with- 
 out again making use of the stage micrometer." — (Schaefer.) 
 
 Test-Plate. 
 
 "Many microscopists who take an active interest in the 
 capacity of their instruments, supply themselves with a set of 
 test objects, of which Pleurosigma angnlatum is in most general 
 use, or with a so-caUed test-plate.' These plates consist either of 
 a series of bands of finely ruled lines ranging from 5,000 to the 
 inch to 120,000 to the inch (beautiful specimens of these are made 
 by Prof. AVni. H. Eogers, of Cambridge, Mass., and O. Fasoldt, of 
 Albany, N. Y.) or with a series of diatoms, upon which the mark- 
 ings represent certain divisions of an inch. The one of these 
 which is principally used is made by J. D. Moeller and consists 
 of a series of 20 diatoms. They are furnished mounted both dry 
 and in b;alsam, but the latter is more common. Below is a table 
 giving the numbers, naimes of the various diatoms and divisions 
 on their surfaces to 7550 inch. A specimen of Eupodiscus Argus 
 bsgins and ends the series : 
 
 Strise in t^^^tj- 
 of an inch. 
 
 1. Triceratium Favus, ELrbg 3.1 to 4. 
 
 2. Pinnulaiia nobilis, Elirbg 11.7 to 14. 
 
 o. Navicula Lyra, Elirbg. var 14.5 to 18. 
 
 4. Kavicula Lyra, Ehrbg 23. to 30.5 
 
 5. Pinularia interrupta, i^m. var 25.5 to 29.5 
 
 6. Stauroneis Phoenicenteron, Elirbg . 31. to 3o.6 
 
 7. Graminatopbora marina, Sm 36. to 39. 
 
 8. Pleurosigma baliicum, Sm . 32. to 37. 
 
 9. Pleurosigma acuminatum, (Kg.) Grun 41. to 46.5 
 
 10. Kiizscbia Ampbioxys, Sm 43. to 49. 
 
 11. Pleurosigma angulatum, Sm. . . 44. to 49. 
 
 12. Grammat<iphora oceanica, Ehrbg. ^G. subtilissima 60. to 67. 
 
 13. Surirella Gemma, Ehrbg 43. to 54. 
 
 14. Nilzscliia sigmoidta, Sm. ... 61. to 64. 
 
 15. Pleurosigma Fasciolii, Sm. var .... . .. 55. to 58. 
 
 16. Surirella Gemma, Ehrbg 64. to 69. 
 
 17. Cymatopleura elliptica, Breb. . . ... . . 55. to 81. 
 
 18. Kavicula crassinervis. Breb.^Frustulia saxonica Eabh ... 78. to 87. 
 
 19. Nitzschia curvula, Sm. . 83. to 90. 
 
 20. Amphipleura pellucida. Kg . .. 92. to 95. 
 
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 Cover Glass. 
 
 Thus far no attention lias been given to the use of the cover- 
 glass, although it is an important factor in reaching good results 
 In preliminarj' examinations of solid objects with low power 
 it may be dispensed with; but where fluids are used, whetlie 
 ^ith low, medium or high powers, it should always be used. A 
 drop or ^mall quantity of fluid placed upon a slide assumes a 
 spherical form, and, on viewing it with a low power, it will be 
 found to give a distorted field, and will cause disagTeeable re- 
 flections and shadows. 
 
 As stated before, medium and high powers have a compara- 
 tively short working- distance, and the front lenses will be so 
 close to the water, urine, blood, etc., that the capillary attrac- 
 tion will often cause an adherence to the front surface of the 
 objective; besides this, there is such a considerable depth to 
 the fluid that it obstructs the light, requires a great change in 
 adjustment for the various planes, and is usually in such vibra- 
 tion that a sharp focus becomes impossible ; by merely dropping 
 a cover glass upon it, all these objections are overcome. 
 
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CARE OF A MICROSCOPE. 
 
 Besides acquiring the ability to properly use an instrument 
 "with its accessories, it is important to know how to keep it in 
 the best working condition. It may be said without reserve that 
 an instrument properly made at the outset and judiciously 
 used should hardly show any signs of wear either in appearance 
 or in its working parts, even after the most protracted use; and 
 further than this, every good instrument should have a provi- 
 sion for taking up lost motion, if there is a likelihood that this 
 may occur in any of the pa'rts. 
 
 Especial care should be given to the optical parts, in fact such 
 care that they will remain in as good condition as when first 
 received. Accidental injury may of course occur to them, but 
 af a systematic manner of Avorking is followed and a special 
 receptacle for each part is provided, this may usually be avoided. 
 
 To Take Care of a Stand. 
 
 One of the first rules should be to keep the instrument free 
 from dust. 
 
 If dust settles on any part of the instrument, remove it first 
 with a camel's-hair brush, and then wipe carefully with a per- 
 fectly clean, old linen handkerchief, vsith the grain of the finish 
 and not across it, as in the latter case it is likely to cause 
 scratches; Keep the working and sliding parts absolutely free 
 from dust, as this grinds and will thus soon cause playj 
 
 Use no alcohol on any part of the instrument, as it will remove 
 the lacquer. As the latter is for the purpose of preventing 
 oxydation of the metals, it is important to observe this rule. 
 
 In using draw-tube impart a straight up and down motion to 
 it. If a spiral motion is given to it, it will cause scratches. 
 
 If it becomes necessary to lubricate any of the parts, use a 
 slight quantity of soft tallow or good clock oil. 
 
 To Take Care of Objectives and Eye-Pieces. 
 
 It is as necessary to keep these free from dust as the stand, 
 in fact even greater cleanliness should be observed. When 
 indistinct, dark specks show in the field, the cause may usually 
 be looked for in the field-lens,, although sometimes in the eye- 
 
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 lens also. The dust may, be removed with a camel's-hair brush, 
 but when this is not sufficient, use a well washed piece of linen, 
 such as an old handkerchief. From its line texture chamois 
 skin is desirable, but as it is fatty it should never be used until 
 after it has been well washed. 
 
 llie same method applies to cleaning objectives. Clean an 
 immersion objective invariably after it has been used, first, by 
 removing the fluid by a moist linen and then by using a dry 
 piece. 
 
 Keep the objectives in a place where they are not subject to 
 extreme and sudden changes of temperature, as the unequal ex- 
 pansion and contraction of glass and metal may cause the cement 
 between the lenses to crack. Also keep them from the direct 
 sun-lightj 
 
 Screw them into the nose-piece and unscrew by grasping the 
 milled edges. 
 
 Avoid any violent contact of the front lens with the cover 
 glass. Usually the latter suffers, but it is as liable to occur to 
 the former. 
 
 Order. 
 
 Among the requisites for successfully prosecuting work with 
 the microscope aire a strict observance of the instructions even 
 if they appear superfluous, a systematic way of doing work, ahd 
 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. "^ — (Bausch.) 
 
 General Rules for the Use of the Microscope. 
 
 "In using the microscope the following rules are to be care- 
 fully observed: 
 
 1. Examine every object with a low power flrst. Having 
 adjusted the eye-piece and objective, turn the mirror so as to 
 reflect the light up the body of the microscope; place the object 
 on the stage under the objective aad carefully lower the body 
 with a screwing motion till the object is about a quarter of an 
 inch from the cover glass; then look through the eye-piece, and 
 gradually raise the body till the object becomes distinctly visi- 
 ble. Focus accurately with the fine adjustment screw. With 
 the high power, begin with the objective close to the cover 
 glass, and then proceed as before. 
 
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 2. INever examine an object with the high power unless it is 
 prdtected by a cover glass. Take extreme care never to let the 
 objective touch the cover glass, and never to touch the face of the 
 objective or allow any dirt to 'get on it. 
 
 3. Keep both eyes open when looking through the micro- 
 scope. Also get into the habit of using either eye. 
 
 4. When examining an object, keep one hand on the fine ad- 
 justment and keep screwing it up and down slightly. In this 
 way parts of the object at differcTit depths are brought into 
 focus, and a much clearer idea of the object is obtained. 
 
 5. "With a high power use a small diaphragm; the amount of 
 light will be somewhat diminished, but the clearness and defini- 
 tion of the object miich increased. 
 
 6- See that the body of the microscope slides smoothly in its 
 tube. If it does not, remove it, and clean it by rubbing with a 
 few drops of olive oil. 
 
 7. The object may appear indistinct from dirt in any of the 
 following places, i.e., on the eye-piece, the objective, or the cover 
 glass. If it be on the cover glass the dimness varies when the 
 slide is moved; if on the eye-piece, it varies when this is rotated; 
 if not on either of these, it must be on or in the objective. 
 
 The eye-piece and the lower surface of the objective may be 
 cleaned with chamois leather or silk. If the objective is smeared 
 with glycerin, wash it carefully, then dry with a soft handker- 
 chief. Canada balsam, which sometimes gets on the objective, 
 may be removed by a drop of glycerin on the handkerchief. It 
 is, however, safer to leave this to an optician or to the demou- 
 strator, as the veiy small quantity of turpentine, getting inside 
 the rim which cariies the lens, may lead to the separation of the 
 two lower lenses from each other, rendering the objective use- 
 less."— (M. and H.) 
 
 Air Bubbles. 
 
 Very frequently air bubbles are formed between the cover- 
 glass and the slide, and beginners require constant watching to 
 prevent them from mistaking these bubbles for structural ele- 
 ments. They may be recognized by their spherical form, trans- 
 parency, and the uniformlj' broad, dark and sharply defined bor- 
 der. This characteristic border occurs in other air spaces which 
 are not bubbles. By the use of a rather l^hin glycerin, or a 
 mixture of glycerin and alcohol, air bubbles may be avoidevd in 
 temporary preparations. Plant cells must often be freed from 
 
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 air by soaking the sections for a time in alcohol, which, expels the 
 air more quickly if warmed by placintg a test tube containing the 
 alcohol and sections in a tumbler of hot water; Never heat al- 
 cohol directly over a flame. 
 
 Brownian Movement, 
 
 Minute particles, when mixed with water or other fluids, fre- 
 quently show a dancing motion when seen through the micro- 
 scope. This movement is kno^wn as Brownian or molecular 
 motion. Careful observation will show that this differs from 
 the voluntary motion of minute living organisms or cells, in that 
 the relative position of the dancing particles remains unchanged 
 in the Brownian miction; while with living particles there is an 
 actual change of place as "well as a trembling motion. 
 
 Mouches Volantes. 
 
 Freciuently when working A^ith the microscope, nxinute, brill- 
 iant bodies, separate or in masses, or like strings of beads or 
 little loops, move before the eye^ These floating bodies are tech- 
 nically known as Mouches volantes (improperly called Scoto- 
 mata), and have their origin either in the secretions that bathe 
 the cornea, or in the vitreous humor of the eye^ They are a nor- 
 mal product and need occasion no alarm. If they are a cause of 
 annoyance a few minutes closing of the eyes will generally bring 
 relief. 
 
 If, however, a permanent, motionless, granular cloud appears 
 to partially obstruct the vision (Scotoma), the eyes should be 
 given as perfect rest as possible, since this is an indication of a 
 congested condition of the retina. 
 
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Accessories in the Use of the 
 Microscope. 
 
 The Spectroscope, 
 
 The micro-spectroscope is intended to take tlie place of the 
 eye-piece on any ordinary microscope, and it is so constructed 
 that the spectrum of the light from the object under examination 
 passing through, the microscope is seen alongside the solar spec- 
 trum. The purpose of the micro-spectroscope is to apply the 
 spectroscopic test to the very minute quantities of colored sub- 
 stances.' It is from the position and character of the absorption 
 bajids that the coloring-matters of different substances can be 
 most accurately and scientifically compared. 
 
 "When a ray of white light, which has passed through a color- 
 inig-matter, — for instance, a solution of one of the coaJ-tai" dyes, 
 red wine, or a solution of chlorophyll, — ^is examined by means of 
 a spectroscope, certain dark bands, known as absorption bands, 
 are observed at definite places in the spectrum. 
 
 For convenience in examining the spectra of small ahiounts of 
 coloring-matters, a direct vision spectroscope attached to the 
 tube of a microscope is employed, and the coloring-matter in 
 question is placed in a flat- walled bottle or glass cell on the stage 
 of the microscope. The ray of light, which is reflected from the 
 mirror under the stage, passes first through the colored matter, 
 next through the objective, and lastly through the prisms which 
 compose the micro-specti'oscopic attachment to the tube. 
 
 In order to compare the spectra of different substances, a 
 second prism or set of prisms is often used, by which the spec- 
 trum of a second liquid can be projected by the side of that of 
 the firstj One of the combinations can also be employed to pro- 
 ject the solar spectrum (unchanged by passing through any color 
 whatever), and its constant lines (Frauenhofer's lines) can be used 
 for the determination of position of the bands seen in the spec- 
 trum of the liquid by its side. 
 
 The spectra of many substances have absorption bands of such 
 
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 constancy in position and appearance that they are justly re- 
 garded as characteristic.'' — (Goodale.) 
 
 "The following is a convenient mode of examining' a solution 
 spectroscopically : The tube of a microscope is withdrawn and it 
 is replaced by a glass tube, the bottom of which covers the open- 
 ing of the stage of the microscope; the sides of the tube must be 
 made opaque by wrapping round them a sheet of black paper; 
 the solution is then pouied into the tube, a microspectroscope is 
 introduced; the mirror of the microscope is to be sO' inclined that 
 it reflects a beam of light on to the bottom of the tube. The ad- 
 vantage of this method is that it enables the observer to vary 
 the thiclmesW of the layer of the solution to be examined." 
 — (B. and V.) 
 
 Moist Chambers and Life-Slides. 
 
 It is often desirable to preserve objects in the living state for 
 a considerable time under the microscope. This must be done 
 in a moist chamber. A moist chamber may readily be con- 
 structed as follows : A piece of thick rough cardboard is cut 
 to the size of the glass slide, and a circular hole is punched out 
 of the middle of it of such a size as to be covered by a cover glass. 
 The piece of cardboard is then soaked in water (or boiled in 
 water when pure cultures of l<\ingi are to be made), so as to 
 saturate it, and placed on the glass slide. A drop of water (or a 
 solution, as described below) is placed on the cover glass, the 
 object is immersed in it, and th6 cover glass is then inverted over 
 the hole in the piece of cardboardj Thus the object is suspended 
 in a drop of the liquid on the under surface of the cover glass. 
 Any loss from the chamber is prevented by occasionally wetting 
 the cardboard on the slide. 
 
 "The liquid to be used will of course vary with the nature of 
 the object to be observed."- — (B. and V.) 
 
 Warm and Cold Stage. 
 In order to study the effects of heat and cold upon living mat-, 
 ter under the microscope, it becomes necessary to use a warm or 
 cold stagej The simplest form of stage, the temperature of 
 which can be readily changed from a low to a high degree, or 
 vice versa, is a plain copper plate, of the usual size of glass slides, 
 having at one side or end a projection of about three inches under 
 which a Bunsen burner may be placed to heat the slide, or upon 
 which ice and salt may be placed to cool it. This copper plate is 
 
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 placed upon the staige of the microscope and insulated from it 
 by means of a piece of felt or cardboard The glass slide having 
 the object to be observed is then placed upon the copper plate. 
 
 Gas Chamber. 
 
 To study the effects of gases on objects under the microscope, 
 use is made of a slide upon which is a deep cell having two open- 
 ings, one on each side^ The object is placed in the cell and 
 tightly covered; gas is then passed into one opening, over the 
 object and out at the other. 
 
 I. STUDY OF LIVING OBJECTS. 
 
 (a) Macroscopic. 
 
 (b) Microscopic. 
 
 (a) All animals should, if possible, be carefully observed alive 
 before they are dissected, for by this means alone is it possible 
 to obtain a clear idea of the uses and mode of action of many 
 organs. — (M. & H.) 
 
 (b) For the best results in the study of li^ving tissues by means 
 of the microscope it is necessary to maintain the normal tempera- 
 lure and to surround the animal with normal fluid — a fluid ap- 
 proNJmately like that in which it normally lives. For protozoa 
 and other aquatic animals, the water in which they are found is 
 their normal fluid. For the blood corpuscles of necturus, frog 
 and man, and for the ciliated epithelium from the frog, artificial 
 serum made from egg-albumen, salt and wateo.* answers very 
 well. — (Gage.) (Vide Normal Salt Solution under Eeagents.) 
 
 For the observation of the more prominent points in the struc- 
 ture of plants, it suffices to moujit the sections in a di'op of water, 
 or, in certain cases, in a drop of alcohol. This is the only possi- 
 ble metiiod when micro-chemical observations have to be made. 
 
 2. STUDY OF LIFELESS OBJECTS. 
 
 (a) Macroscopic . Cursory Examination. 
 (6) Microscopic | permanent Preparation. 
 
 (a) General Rules for Dissecting. 
 Macroscopic Examinations. 
 
 "Before commencing a dissection, fix the animal doT^-n firmly 
 to a dissecting board or dish. 
 
 In fixing an animal with pins, stick them obliquel.y, so that 
 their heads do not get in the way or obscure the dissection. 
 
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29 
 
 Dissect under water, unless the animal is too large. Change 
 the water as soon as it gets dirty. A gentle stream of water 
 allowed to play upon the dissection is often a valuable aid. 
 
 Never cut away anything until you are quite certain what yoa 
 are removing. 
 
 Put the part you ai'e dissecting slightly on the stretch. This 
 applies more particularly to bloodvessels, nerves, ducts and 
 musclesj 
 
 In cleaning bloodvessels, nerves, etc., dissect along them, and 
 not across them; and avoid laying hold of them with the forceps. 
 
 The dissection is in many cases greatly facilitated by placing 
 the specimen in spirit for a day or so before dissecting it. In 
 some cases the dissection may with advantage be performed 
 under spirit. 
 
 Always keep your insti-uments clean and sharp. Be careful 
 not to blunt your fine scissors or scalpels hj using them for cut- 
 ting hard partsj 
 
 If you get in a muddle, stop, and wash the dissection 
 thoroughly under the tap before proceeding further. 
 
 Successive slices cut off an animal, or part of an animal, with 
 a razor are often exceedingly instructive; this is especially the 
 case V ith the mussel and snail, and Avith the brains of the pigeon 
 and rabbit. The specimens must be previously frozen or hard- 
 ened in spirit or other reagent, and the slices should be examined 
 in water or spirit; 
 
 Careful drawings must always be made, both of the living 
 animal and of all dissections and preparations. These draAvings 
 shouhi be made to scale. 
 
 Correctness of outline is of more importance than minute detail; 
 and the usefulness of the drawings is greatly increased by the 
 systematic use of diagramatic coloring. A separate color shouLi 
 be employed for each system of organs, the several part^ of 
 which may be indicated by gradations of tint. 
 
 As a rule, the more bulky organs, as the liver, should be 
 colored with dull tints, and the brighter colors reserved for the 
 smaller and less conspicuous parts. Arteries are usually colored 
 red, and veins blue. — (M. and H.) 
 
 The Cursory Examination of Lifeless Objects Under the 
 
 Microscope. 
 
 "The fluid in which a microscopic specimen is submitted to 
 examination is technically known as its medium. Chemical 
 
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 ageEts subsequently added for the purpose of producing changes 
 by which the chemical character of the objects may be recog- 
 nized are termed reagents. Some of the media, howcYer, in 
 common use produce characteristic changes in certain cases, 
 and might be as truly leferred to the latter class as several of 
 the reagents themselves. The substances in which microscopic 
 specimens are preserved are termed mounting media. 
 
 Media. 
 
 In all ordinary cases pure water is the best medium in w^hich 
 to place the object for examination. If distilled water cannot 
 be procured, filtered rain water or melted ice will answer 
 perfectly; In some cases ^ater produces an immediate change 
 either in the cell wall or in the contents of the cells.' For in- 
 stance, the superficial cells of the coats of many seeds swell up 
 at once when they are place<l in water, and lose their former 
 shape; on the other hand, important contents in the seeds of 
 many plants are dissolved immediately when the sections are 
 moistened. Hence, other media must sometimes be substituted 
 for water. Absolute alcohol is the most useful for meeting the 
 cases above referred to.- Thus, if a section .of a seed coat be 
 first examined in absolute alcohol, and the alcohol be gradually 
 replaced by water, as directed below, the changes due to water 
 will talie place slowly, and can be watched throughout. For the 
 cases in which the cell contents are suspected of undergoing 
 change from water, castor oil is a useful medium. If thought 
 best, this can be removed subsequently from the specimen by 
 alcoJtiol or ether, and the latter in turn may be Uiade to give 
 place to A\ ater, and the changes can be followed with certainty. 
 
 G-lycerin, either concentrated or somewJiat diluted with water, 
 is a highly useful medium, imparting a good degree of transpar- 
 ency to most specimens. It withdraws a pai't of the Avater of 
 the cell-sap, and in the case of thin-walled cells this is followed 
 by some change of form. 
 
 One medium may be replaced by another by the careful use of 
 bibulous paper. Good filtering paper is the best for this pur- 
 pose. If a littJe of the liquid which it is desired to place under 
 the cover glass be put at tlie edge of the cover, and the opposite 
 edge be then lightly touched with the paper, the liquid Avill be 
 at once drawn through. By successive applications of the same 
 liquid the specimen can be thoroughly washed without removal 
 of the cover-glass.'' — (Gcodale.) 
 
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 The Irrigation of Tissues. 
 
 If we wishL to observe the behavior of tissues when attacked 
 by reagients we run the reagent under the cover glass and 
 watch the result. Having placed a cover glass over the tissue to 
 be irrigated and in sufficient proximity with the cover glass to 
 commajid the presence of capillarity, we either place a drop of 
 the reagent we wish to irrigate with at one side or edge of the 
 cover glass and apply a small piece of bibulous paper to the 
 opposite edge of the cover glass, or we partially fill a pipette with 
 the reagent and allow it to descend upon the ed^e of the cover 
 glass, using the bibulous paper as before. 
 
 A large pill-box full of small triangular pieces of bibulous 
 paper should be kept on the work table whilst irrigating, and 
 with forceps, first one and then another corner of the triangle, 
 should be presented to the opposite edge of the cover to that at 
 which the reagent enters. 
 
 ON THE STUDY OF OBJECTS BY THE MICRO- 
 SCOPE IN PERMANENT PREPARATIONS. 
 
 (a) Fixation and Fixing Agents. 
 
 "The first thtag to be done with any structure is to fix its histo- 
 logical elements. Two things are implied by the word 'fixiag:' 
 first, the rapid killing of the element, so that it may not have 
 time to change the form it had during' life, but is fixed in deatli 
 in the attitude it normally had during life; and, second, the hard- 
 ening of it to such a degTee as may enable it to resist, without 
 further change of form, the action of the reagents with which it 
 may subsequently be treated. Too much stress can hardly be 
 laid on this point, which is the mOst distinctive feature of modern 
 histological practice; witiout good fixation it is impossible to 
 get good stains, or good sections, or preparations good in any 
 way. 
 
 Pure alcohol is not very suitable as a fixing agent, as it precip- 
 itates most of the salts of the sea water adhering to the sur- 
 face of marine animals, thus giving rise to a crust that prevents 
 the penetration of the alcohol to the interior (allowing macera- 
 tion of the internal structures to be set up), and also hinders 
 the penetration of the staining fluids. Besides this, it often 
 gives rise to coagulation of the perivisceral fluids that glue the 
 viscera together in a most undesirable manner. Therefore, if 
 
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 alcohol is used, acidulated alcohol is generally taken. But the 
 fluid most used is Kleinenberfe's picro-sulphuric acid. The 
 gTcat advantages of this fluid are that it kills quicldy, that it 
 removes the sea-salts from the tissues, and that it allows of good 
 staining subsequently. For special cases picro-nitric or picro- 
 hydrochloric acid is used instead. Osmic acid is used, but only 
 for very permeable tissues (on account of its very feeble pene- 
 trating power), and in cases where it is not important that a 
 subsequent nuclear stain should be easily attainable. Chromic 
 acid is also avoided as much as possible on account of its hin- 
 derance to staining. Combined with platinum chloride, however, 
 in the mixture known as Merkel's fluid, it is said to allow of good 
 staining, and is found useful In some special cases. Corrosive 
 sublimate is found very convenient, and is the fluid most used, 
 excepting picro-sulphuric acid. 
 
 Washing out is done with successive alcohols, water being 
 used only in case of corrosive sublimate, osmic acid, or the 
 chromic mixtures. 
 
 In some cases the simple application of heat is not only a good 
 means of fixing, but the very best. It will probably be found 
 that this method is very helpful in the case of minute structures 
 inclosed in very impervious cases of chitin or other impervious 
 material; such cases or capsules obviously opposing but an in- 
 appreciable resistance to the passage of heat waves. The method 
 consists simply in heating the objects to the temperature neces- 
 sary to kill the cells and coagulate their contents. This is done 
 by heating them in water or in a wateh giass.i It is, of course, 
 better, if practicable, to thrc^^' them at once into water previously 
 heated to the required temperature. It appears that a few sec- 
 onds' immersion in boiling water is not hurtful even to delicate 
 tissues. The method has the gi-eat advantage of aUo-ning of 
 good subsequent staining, and of hindering less tlian any other 
 the application of chemical tests. 
 
 The action of all the usual fixing agents (osmic acid, chromic 
 acid, alcohol) is intensified and made more rapid by employing 
 the solutions hot. 
 
 Alcohol. 
 
 Alcohol may sometimes be used boiling with advantage. 
 
 Alcohol is most usually employed for fixing purposes diluted 
 to a strength of o'J.^j per cent; This formula, Mhicli has now 
 becojiie classic, is due to Eanvier, who was the first to point out 
 its wide applicabilit}-, and tlie excellence of the results obtained 
 
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 ■by it. Alcohol of the strength of 33.3 per cent, is known in 
 France as 'Alcohol axi tiers,' which is the name given to it by 
 Eanvier himself; in Germany as 'Drittel alcohol' or 'Eanvier- 
 sche alcohol dilutus;' in Italy as 'Alcohol al terzo.', 
 
 Tissues should remain in the alcohol, as a general rule, for at 
 least twenty -four hours; they may be stained (with picro-carmine, 
 or, better) with some alcoholic staining medium. They must 
 never be treated with water or any aqueous liquid, except picro- 
 carmine or alum-carmine, or methyl green." — (Bolles Lee.) 
 
 Preparation of Absolute Alcohol. 
 
 Absolute alcohol is prepared in Ranvier's laboratory by adding 
 anhydrous cupric sulphate to 95 per cent, alcohol. Pulverized 
 cupric sulphate is heated to red heat in order to drive off the 
 water of crystallization; when cool the white powder is placed 
 in a wide-mouthed bottle, holding about a litre, and three-fourths 
 full of alcohol. The bottle is quickly closed and the whole 
 shaken. After standing a day or more — with occasional shak- 
 ings — ^it is decanted and the operation repeated, especially if the 
 cupariq sulphate shows much of the blue color due to the re- 
 assumption of water. As a test, a drop of the alcohol thus dehy- 
 drated may be mixed Avith a drop of turpentine on a glass slide, 
 alid examined under the microscope; if no particles of water are 
 to be seen, the alcohol is absolute enough for all practical pur- 
 poses. 
 
 To determine the percentage of alcohol, use is made of an alco- 
 holometer, a graduated tube, loaded with shot so as to rest verti- 
 cally in any fluid Capable of floating it. The alcoholometer of 
 Tralles is commonly employed in this country; it indicates the 
 volume per cent. 
 
 In pure water the instrument sinks only to zero, the lower 
 end of the scale. In absolute alcohol it sinks to 100, the upper 
 end of the scale. Mixtures of the two liquids permit it to sink to 
 various depths, and the number corresponding with the surface of 
 the liquid indicates the percentage of the alcohol by volume. 
 — (G. and W.) 
 
 Osmic Acid. 
 
 This is a volatile, stronig-smelling crystalline substance, very 
 
 irritating to mucous membranes. The breathing of the fumes 
 
 must positively be avoided. In commerce it comes in small, sealed 
 
 glass tubes, each holding a gi'amme or a half gramme of the 
 
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34 
 
 greenish, crystallized acid. It is used as a fixing and harden- 
 ing medium in aqueous solution of 1 to 500, or 1 to 100. The tube 
 should be broken inside a bottle containing the proper quantity 
 of distilled water by means of a glass rod. The solution must 
 be kept from the light, or it will be rapidly reduced. 
 
 "Osmic acid is best employed as a vapor, and its employment 
 in this form is indicated in all cases in which it is possible to 
 expose the tissues to the vapor. The tissues are pinned out on 
 a cork, which must fit well into a wide-mouth bottle, in which is 
 contained a little solid osmic acid (or a small quantity of 1 per 
 cent, solution will do). They remain there until they begin to 
 turn bro-RTij There is no need to wash them out before stainingj 
 Picro-carmine is a good stain to follow this process, which is 
 certainly, where practicable, the most elegant that has hitherto 
 been imagined. 
 
 When employed in aqueous solutions, osmic acid is used in 
 strengths varying from 1-20 per cent, to 2 per centj Solutions of 
 one-half per cent.i to 1 per cent, have been very largely used, but 
 the tendency of modern practice seems to be toward weaker 
 solutions and longer immersion. 
 
 Osmic acid stains all fatty structures perfectly and opaquely 
 black; it must therefore be avoided for tissues in which much 
 fat is present 
 
 Osmic acid must be well washed out before proceeding to any 
 further steps in preparation; water or glycerin may be used. 
 Notwithstanding the greatest care in soaking, it frequently 
 happens that some of the acid remains in the tissues, and causes 
 them to overblacken in time. To obviate this it is necessary to 
 wash them out in picro-carmine, or in 0.5 per cent, solution of 
 chromic acid. All solutions of osmic acid must be kept from the 
 light even during the immersion of tissues. If the immersion is 
 to be a long one the tissues must be placed with the solution in 
 well closed vessels, as osmium is very volatile." Stain osmi3 acid 
 specimens in picro-carmine- 
 
 "Chromo-aceto-osmic Acid Fixing Mixture (Fleming's formula) : 
 
 Chromic acid 0.25 per cent. 
 
 O^mic acid 0.1 per cent. In water. 
 
 Glacial acetic acid 0.1 per cent. 
 
 The best results (as regards faithfulness of fixation) are ob- 
 tained with this mixture when it is allowed to act for only a 
 short time (about half an hour). 
 
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35 
 
 Chromic Acid. 
 
 Chromic acid is employed in solution either in water or in 
 alcohol. 
 
 The most usual strengths in which it is employed in aqueous 
 solution are from J. to 1.0 per cent, for a period of immersion of 
 a few hours (structure of cells and ova)j For nerve tissues 
 weaker solutions are taken, l-50th to l-8th per cent, for a few 
 hours. Stronjger solutions, such as 5 per cent., should only be 
 allowed to act for a few secondsj 
 
 One per cent, chromic acid is useful for fl.King filamentous algae, 
 Nitella, etc., 12 to 24 hours. 
 
 The objects should be washed out with water before passing 
 into alcohol or staining fluids. Long washing in water is neces- 
 sary to prepare them for staining, unless an aniline stain be used. 
 
 Tissues that have been fixed in chromic acid may be stained 
 in aqueous solution if desired, as water does not appear to have 
 an injurious effect on them ; the acid appears to enter into some 
 chemical combination with the elements of the tissues, forming 
 with them a compound that is not affected either physically or 
 chemically by water. The best stain to follow chromic acid is 
 haematoxylin or safranin. But the previous washing out must 
 be very thoronJgh if good results are to be insured; it may talie 
 days^ 
 
 Picric Acid. 
 
 Picric acid should always be employed in the form of strong 
 solution. That is to say, strong solutions must always be 
 employed when it is desired to make sections or other pre- 
 parations with the tissues in situ, as weak solutions mace- 
 rate; the saturated solution is the one most emploj-ed. Objects 
 should remain in it for from a few seconds to tuenty-four hours, 
 .iccording to their size. For Infusoria, one to at most two min- 
 utes will suffice, whilst objects of a thickness of several milli- 
 meters require from three to six hours' immersion. 
 
 Picric acid should always be washed out «ith alcohol, as water 
 is hurtful to tissues that have been prepared in it. For the same 
 reason, during all remaining stages of treatment, water should 
 be avoided; staining should be performed by alcoholic solutions, 
 the only exception to this rule being in favor of picro-carmine, 
 which, probably on account of the picric acid contained in it, 
 does not appear to exert so injurious an influence as other 
 aqueous stains. 
 
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36 
 
 It is one of the adyantages of picric acid that, by sufficiently 
 prolonged soaking,' it can with certainty be entirely removed 
 from any tissue by means of alcoholj 
 
 Tissues fixed in piciic acid can, after the removal of the acid 
 by soaking, be perfectly stained in any stain. Mayer's cochineal, 
 alcoholic borax carmine, Kleinenberg's haematoxylin and G-ren- 
 acher's alcoholic carmine may be recommended. The most im- 
 portant property of picric acid is its great penetration. This ren- 
 ders it peculiarly suitable for the prepar-ation of chitinous struc- 
 tures. For such objects, alcohol of 70 per cent, to 90 per cent, 
 should be taken for washing out, and staining should be done by 
 means of Mayer's cochineal or Kleinenberg's haematoxylin. 
 
 Picro-Sulphuric Acid (Kleinenberg's Formula). 
 
 In very many cases it is advantageous to employ picric acid in 
 the manner suggested by Kleinenberig, that is, in combination 
 with sulphuric acid. 
 
 For the i-easons stated below, picro-sulphuric acid is not, as a 
 general rule, suitable for tissues of vertebrates. 
 
 Prepare a saturated solution of picric acid in distilled watei", 
 and to a hundred volumes of this add two volumes of concen- 
 trated sulphuric acid; all the picric acid which is precipitated 
 must be removed by filtration. One volume of the liquid ob- 
 tained in this manner is to be diluted with three volumes of 
 water, and, finally, as much pure creosote must be added as will 
 mix. 
 
 The addition of creosote is intended to obviate a drawback that 
 picro-sulphuric acid shares with osmic acid, viz^, that of oc- 
 casionally producing swellings in the primitive blastomeres. 
 (Mayer finds that creosote produces no perceptible difference in 
 the result, and has therefore abandoned it.) 
 
 The object to be preserved should remain in this liquid for 
 three, four or more hours ; then it should be transferred, in order 
 to harden it and remove the acid, into 70 per cent, alcohol, where 
 it is to remain five or six hours. From this it is to be removed 
 into 90 per cent, alcohol, which is to be changed until the yellow 
 tint has disappeared or greatly diminished. 
 
 Picro-Sulphuric Acid (Mayer's Formula). 
 Distilled water, 100 volumes; sulphuric acid, 2 volumes; picric 
 acid, as much as will dissolve. Filter and dilute with three 
 volumes of water, except for arthropoda, for which the fluid is 
 used undiluted. 
 
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37 
 
 Mayer directs that if the fliiid does not diffuse very rapidly 
 through, thick chitin, the larger arthropoda, such as insecta and 
 the larger isopoda, should be opened with scissors, and the body 
 cavity at once filled with the solution by means of a pipette. A 
 large quantity of the solution should be employed in all cases, 
 and only when the fluid remains perfectly clear should objects 
 be allowed to remain in the same change for any considerable 
 time. This is a point that is very important to attend to. 
 
 Washing out is done with alcohol of 70 per cent. "Warm alco- 
 hol extracts the acid much more quickly than cold, with which 
 weeks may be required to fully remove the acid from the chitin- 
 ous structures. I call attention here to what was said as to 
 washing out under the head of picric acid, viz., that washing out 
 should never be done with water. This is a most important point, 
 and one that is not sufficiently attended to. 
 
 The advantages of picro sulphuric acid as a fixing agent are 
 that it kills tissues very rapidly, that it has great penetrating 
 power, that it can be totally soaked out of the structures with 
 alcohol, leaving them in a good condition for staiidng, and, m 
 the case of marine organisms, that it effectually removes the 
 different salts of sea-water that are present in them. 
 
 It has some disadvantages. For vertebrata it should be used 
 with caution, on account of the swelling caused by sulphuric acid 
 in connective tissue. In parasitic Crustacea it also produces 
 swelling and maceration, and should be avoided^ Notwithstand- 
 ing this, it is, however, according to Emery, very suitable for 
 fishes, and for embryos of vertebrates generally, provided they 
 are not allowed to remain in it more than three or four hours. 
 For structures that contain much lime it is not to be recom- 
 mended, for it dissolves the lime and throws it down as crystals 
 of gypsum in the tissues. For such structures the picro-nitrii 
 or picro-hydrochloric acid is to be preferred. 
 
 Corrosive Sublimate. 
 
 Corrosive sublimate has of late been found to be one of the 
 best of fixatives in a great variety of cases. It kills and hardens 
 cells with great rapidity, and does not hinder the usual staining 
 processes. It may be used pure or in combination Avith other 
 reagents, as in the formula of Lang. When used pure a cold 
 aqueous solution is taken, and allowed to act on the tissues until 
 they are well permeated with it; they are then put for twenty- 
 four hours into weak alcohol, stained, and subsequently further 
 
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 harrleued witlL stronger alcohol if further hardening be desirable. 
 It is sometimes well to wash out with water for half an hour be- 
 fore passing into alcohol. Objects should be removed from the 
 fixing solution as soon as they have become soaked with it. It 
 must be remembered that the solutions must not be toucJied with 
 iron or steel, as these produce precipitates that may hurt the 
 preparations. To manipulate the objects, wood, glass, or plati- 
 num may be used, for pinning' doAA-n and dissecting them, hedge- 
 hog or cactus spines, or quill pens. Preparations that have 
 been fixed in sublimate should not be allowed to remain long in 
 alcohol, which makes them brittle, but after at most a few days 
 they should be stained and imbedded in parafiin, in which they 
 may remain until cut." — (BoUes Lee.) For plant tissues a rather 
 weak solution should be used. One-fifth of saturated solution, 
 i.ej, dilute a saturated solution with five times its bulk of water. 
 This "R-ill fix with great rapidity, say five to twenty seconds for 
 delicate cells, the length of time must be proportioned to the 
 thickness of the cell-walls. 
 
 (b) Preserving and Preservative Agents. 
 
 "A great many agents are used for hardening and preserving 
 tissues for histological purposes. Those most used are alcohol, 
 chromic acid and various salts of chromium, as bichromate of 
 potassium and ammoniumj Picric acid is also often employed. 
 None of them are equally good for all tissues, and in most cases 
 two or more of them are employed on each tissue; A perfect 
 hardening and preserving agent for histological purposes would 
 leave the structural elements with exactly the same appearance 
 they possess during life, color perhaps excepted. 
 
 "Fifty to seventy-five times as much liquid should be used as 
 tissue, and if the tissue is of a massive character, like liver, only 
 two to three cubic centimeters should be talcen. Label every- 
 thing. Put the tissues into the hardening agent as soon as pos- 
 sible after the death of the animal. 
 
 "An excellent method of hardening tissues is to inject the hard- 
 ening agent into the arteries going to the tissue or into tlie ducts 
 of glands. This may be done for all tissues and all hardening 
 agents by boring two holes in the top of a preserving jar, and 
 making a kind of wash bottle of it. Pressure may be obtained 
 by using a constant pressure apparatus or an atomizer bulb. 
 The connection with the vessel or duct may be readily ma<le by 
 a rubber tube and glass cannula. TMs form of injecting appara- 
 
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39 
 
 tus is especially adapted for corrosive liquids like picric and 
 osmic acid and silver nitrate, etc.'" — (Gage.) 
 
 As a rule, tlie specimen after killing and fixing may be trans- 
 ferred to 70 per cent, alcohol, and from that to 95 per cent, alco- 
 hol, in which they may remain until needed. 
 
 Corrosive sublimate is an exception to the general rule.' 
 
 Preparations that have been fixed in sublimate should not be 
 allowed to remain long in alcohol, which makes thejn brittle; 
 but after at most a few days they should be stained and imbed- 
 ded in paraffin, in which they may remain until cut. 
 
 It must be remembered that alcohol will extract the green 
 coloring matter, as well as resins and other substances, from 
 plant tissues. Some succulent plants fe.gj, monotropa) will turn 
 black in ordinary alcohol. A small amount of hydrochloric acid 
 added to the alcohol will prevent, this. De Yries' method is as 
 follows : — ^Kill the plant in spirit to which about 2 per cent hy- 
 drochloric, sulphuric or acetic acid has been added. The prepara- 
 tions are kept in this fluid for several months, and then trans- 
 ferred to spirit without acid. TJiis is removed from time to time 
 until all the coloring matter has been removed. The long stay in 
 the acid fluid does not injure the plants as they are just as useful 
 for microscopical purposes as freshi or otherwise preserved 
 organs. Even the crystals of oxalate of lime are not dissolved 
 by the mixture of spirit and hydrochloric acid, although they 
 are when the acid is mixed with water. 
 
 Dissociating Fluids. 
 
 Sometimes the elements of which a tissue is composed caimot 
 be separated without dissolving the parts which bind them. 
 When this is the case the tissue is cut into little pieces, such as 
 are used for teasing, and placed in some dissociating fluid. 
 
 "Concentrated nitric acid 77 cc.i, water 23 cc, a 20 per cent. 
 solution of hydrochloric acid also form an excellent dissociating 
 fluid for muscular fiber-ceUs and cardiac muscle^ 
 
 Place the tissue, the cells of which it is desirable to dissociate, 
 in 15 to 20 cc. of 20 per cent, nitric acid for three days. Then 
 pour off the acid and soak an hour or more in water, or 35 per 
 cent, alcohol. — 'Reichert's method.)" — (Gage.) 
 
 "Iodized serum is recommended by Max Schultze and by Ran- 
 vier as a macerating medium' for animal tissues. The amniotic 
 liquid of a sheep or cow is placed in a flask with some flakes of 
 iodine and frequently agitated for some days, at the outset the 
 
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40 
 
 serum dissolves very little iodine, but if an excess of iodine 1>& 
 kept constantly present, it will be found that after several weeks, 
 iodides are formeil, and a strongly iodized serum is obtained. 
 It should be a dark brownj Such a solution should be used for 
 iodizing fresh serum, by adding it in small quantities to the 
 serum that is intended for use. In general for macerating pur- 
 poses a serum of a pale brown color should be employedj The 
 manner of its employment is as follows: — A piece of tissue 
 smaller than a pea must be taken and placed in four or five cubic 
 centimeters of weakly iodized serum in a well closed vessel. 
 After one day's soaking the maceration is generally sufficient, 
 and the preparation may be completed by teasing or pressing 
 out; if not^, the soaking must be continued in a fresh quantity of 
 the solution.'" — (Bolles L©e.\) For dissociating muscle cells, allow 
 them to remain for 48 hours in weak solution of Bichromate of 
 Potash, (1— SOO). 
 
 Schulze's Macerating Fluid. 
 
 Several methods of using this important reagent have been 
 suggested. 
 
 (1) Place in a wide test-tube some pieces of chlorate of potash 
 and pour over them sufflcieut strong nitric acid to completely 
 cover them; then lay in the fluid longitudinal sections of the 
 material and warm over a flame till gas is actively evolved. Al- 
 low it to work for a few minutes, then empty the whole into a 
 dish o.f water, and carefully wash. Eemove the sections with 
 a glass rod into another vessel of water, and thence into water 
 on au object-slide; there they can be torn to pieces with needles. 
 
 (2) Put the sections in a tube mth an equal bulk of chlorate 
 of jiotash, cover with concentrated nitric acid and proceed as 
 before. 
 
 (3) Use one gi^amme chlorate of potash to 50 cc. nitric acid, 
 and proceed as above. 
 
 (4) Three grains chlorate of potksh and two drachms nitric 
 acid. Keep the sections in this, cold, for a fortnight. 
 
 After carefully washing in alcohol, the preparations as above 
 can be preserved in glycerin. 
 
 The cells become isolated in consequence of the solution of the 
 middle lamella. 
 
 In general, there is nothing preferable to Schulze's solution 
 in any strenlgth adapted to tlie special case; it must be remem- 
 bered that the slow action of a dilute solution gives better results 
 than the more rapid action of a concentrated one. If the section 
 
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41 
 
 to be examined is first subjected to the action of the macerating 
 solution of proper strength and then thoroughly washed, it can 
 be dissected at pleasure under a high power of a simple lens. 
 This method is always to be preferred to the ordinary one of 
 disinteigrating the whole specimen and obtaining a confused mass 
 of separated cells. 
 
 How to Tease a Tissue. 
 
 When it is neither possible nor convenient to submit tissues to 
 hardening or softening processes, which take time, counted by 
 days or weeks, we can examine in a rough and imperfect manner 
 the so-called fresh tissues by cutting or nipping off a minute 
 piece and tearinig it apar-t with needles: The process is called 
 teasing. 
 
 In doing so, moisture is frequently required, and is obtained by 
 adding fluids that will not alter the structures we are examining, 
 which up to within a few minutes or hours have been bathed in 
 natura,l or normal fluids within the organism; hence we imitate 
 this state of things as nearly as possible by using what we have 
 already alluded to as normal fluids. 
 
 Place the object upon a slide or watch ciystal, upon a suitable 
 background — ^i.e., black or white, transmitted light, or light 
 thrown directly down upon the object with a condenser or mir- 
 ror. Watch the object carefully by means of the lens of a dis- 
 secting microscope or a watchmaker's glass. 
 
 Take two fine needles, fixed in handles, and gently separate 
 the elements needed, keepinig the eye steadily fixed on the ob- 
 ject. When sufftciently dissociated cover and examine with a 
 hig'h power. 
 
 ON STAINING AND STAINING AGENTS. 
 
 " The chief end for which coloring reagents are employed in 
 animal and vegetal histology is to obtain a differentiation 'jf 
 tissue systems and a recognition of certain cell contents^ — ^that 
 is, a stain in which nuclei, or, at most, the nuclei and their sur- 
 rounding cell-protoplasm, are colored, whilst the formed 
 material of the tissues is left unstained. That is what the his- 
 tologist wants in the great majority of cases. He wants either 
 to differentiate the intimate structures of the cells by means 
 of a color reaction, in order to study them for their own salie, 
 or he wants to have the nuclei of the tissues marked out by 
 staining in the midst of the unstained formed material in such 
 
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 a Avay that they may form lanrl marks to catch the eye, which is 
 then able to follow out with ease the contours and relations of 
 the elements to which the nuclei belong; the extra-nuclear parts 
 of these being expressly left unstained in order that as little 
 light as possible may be absorbed in passing through the 
 preparation. Diffuse stains, or those which stain formed mate- 
 rials as well as protoplasm, are now more and more abandoned ; 
 for instance, eosin,which was once a favorite stain, is now but 
 little used, on account of the incorrigible difluseness with which 
 it stains. Except for special purposes, such as the dyeing of thin 
 membranes, which, unstained, would be invisible, or for certain 
 purely chemical ends, or for combination with a nuclear stain to 
 nialse a double stain, .diffusely-staining coloring agents are not 
 employed; 
 
 As a general rule, one, indeed, to which it is difficult to find 
 a plausible exception, all alkaline staining solutions should be 
 a/voided. Alkalies dissolve nuclein, or if they do not dissolve it 
 when very dilute, swell and distort nuclear structures, and are 
 frequently hurtful to formed material. Neutral or acid stains 
 should alone be used, and it will probably be found that better 
 preparations are obtained with acid solutions than with neutral 
 ones. It is most important to work with acid stains in all cases 
 in which it is desired to faithfully preserve nuclein formations. 
 
 In order to obtain precise stains it is important to operate on 
 tisiues that have been carefuUj^ fixed; or, if fresh tissue be taken, 
 that the staining solution should itself be a sufficient fixative. 
 
 It is desirable to employ alcoholic stains for objects that 
 have baen treated with alcohol; it will be sufficient here to 
 again call attention to the superior penetrating power of alco- 
 holic solutions. The histologists should never be without a 
 good alcoholic stain." — (Bolles Lee.) 
 
 " Of the whole class of staining agents, it may be said that expo- 
 sure to strong light diminishes the brilliancy of the coloring 
 they produce in the specimen, and in many cases completely 
 destroys it. In general, the staining obtained by allowing the 
 specimen to remain for a long time in a dilute solution of a dye 
 is more satisfactory than when a stronger dye is used with 
 haste.'' — (Goodale.) 
 
 The gi'eatest difficulty in the technic of staining lies in the 
 incompatibility of certain fixing agents with certain staining 
 agents. Thus chromic and osmic acid, two of the best fixing 
 agents known, are to a great degree incompatible with staining 
 
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 by carmine, the most trustworthy of staining agents, often ren- 
 dering the staining with carmine so difficult that it is better to 
 abandon it and employ some other stain. A few hints may be 
 useful. 
 
 " Haemaitoxylin is the best stain to use after chromic acid; but 
 some anilines give good results. 
 
 Cochineal may also be used after chromic acid. 
 
 After osmic acid, picro-carmine or alum-carmine, or haema- 
 toxylin. 
 
 But osmic acid preparations generally stain well only after 
 bleaching. 
 
 All stains take well after fixation with alcohol, or with corro- 
 sive sublimate, or with nitric aci<i. 
 
 After fixation with any of the picric-acid fiuids and due wash- 
 ing out with alcohol, all objects stain well with any of the usual 
 stains. 
 
 Carmine. 
 
 " Carmine, which is probably the most valuable and certainly 
 the most widely employed of histological coloring agents, was 
 first proposed as an acid in the examination of animal tissues by 
 Gerlach in 1838, since which time, notrndthstandinig the discov- 
 ery of numerous other substances which have proved most use- 
 ful in many kinds of research, it has held its place, with a firm- 
 ness which shows few signs of yielding, as the staining agent 
 par excellence. 
 
 It is not a definite chemical substance. As is well known, it is 
 prepared from the cochineal insect. Coccus cacti, by processes 
 the details of which are trade secrets, and which may or may not 
 vary in essential points. 
 
 The following statements are, I believe, true of all cai-mine 
 stains. Tissues to be stained must be freed from acids before 
 being put into the staining fluid. Overstains may in all cases 
 be washed out with weak HCl. (e.g., 0.1 per cent.) It is gener- 
 ally advisable to fix the stain in the tissues before mounting. 
 In the case of balsam mounting this is sufliciently done by the 
 action of the alcohol; in the case of an aqueous mount, acetic or 
 formic acid sJiould be employed, and the best way of doing this is 
 to let the mounting medium contain 1 per cent, of formic or 
 acetic acid. Formic acid is to be preferred. 
 
 The beginner may wish for some hints als to which of these 
 formulae he should choose. 
 
 If the tissue has been prepared [i^., fixed, hardened and pre- 
 
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 served) in alcohol he ought to choose one of the alcoholic solu- 
 tions ; or, if he takes the less advisable course of using an aque- 
 ous one, then it should be picro-carmine or alum-carmine. 
 
 Picro-carmine is perhaps the least hurtful to tissues. 
 
 Exposure to strong light diminishes the brilliancy of the color- 
 ing. 
 
 Whenever it is possible, alcoholic solutions should be pre- 
 ferred; they are less hurtful to the tissues than aqueous solu- 
 tions; more penetrating and generally more precise in their 
 action. 
 
 Grenacher's alcoholic borax-carmine may be recommended to 
 the beginner as being the easiest of these stains to "v\'ork with. 
 
 Alcoholic Borax Carmine (Grenacher's). 
 
 "Take a concentrated solution of carmine in borax solution (2 
 to 3 per cent, carmine to 4 per cent, borax) ; dilute it with about 
 txi equal volume of 70 per cent, alcohol, allow it to stand for 
 some time and filter. Acetic acid must not be added. Leave 
 the preparations in the stain until they are thoroughly pen- 
 etrated, and then bring them (without, first washing out) into 
 alcohol acidulated with 4 to 6 drops of hydrochloric acid to each 
 100 cc. of alcohol. They are left in this until they are thor- 
 oughly penetrated, and may then be washed or hardened in 
 neutral alcohol. 
 
 Preparations stained with carmine are best mounted in glycerin 
 or glycerin- jelly. Do not use carmine with objects hardened in 
 chromic or osmic acid. 
 
 Picro-Carmine. — General Remarks. 
 
 "Picro-carmine stained preparations should be mounted in 
 balsam, or if in glycerin this should be acidulated with 1 per 
 cent, acetic acid or, better, formic acid. 
 
 Picro-carmine is a nuclear stain with a tendency to diffuse 
 into formed tissues. If the preparations be washed after stain- 
 ing with water it is a single stain, the color of the carmine alone 
 disappearing; if they be washed quickly in alcohol it is a 
 double stain, the yellow coloration of the picric acid not being 
 dissolved out by the alcohol as it is by water. Of course the 
 washing with alcohol must not be ovei'done, or the yellow colora- 
 tion may be entirely removed. 
 
 Picro-Carmine (Pergen's Formula). 
 
 Five hundred grammes of cochineal (pulverized) are boiled for 
 
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 two hours and a half in thirty litres, of water. Fifty grammes 
 of potaJssic nitrate are then added, and, after again boiling for a 
 moment, sixty grammes of potassic oxalate; the ebullition is 
 maintained for half an hour. On cooling, the carmiae pre- 
 cipitates; it is washed severa,! times with distilled water. 
 These operations talie three or four weeks. 
 
 A mixture of 1 vol. caustic ammonia with 4 vols, of water is 
 then poured on the carmine, care being taken that the carmine 
 remains in excess. After two days the mixture is filtered, and the 
 filtrate exposed to the air until a precipitate is produced. This 
 is removed by filtration. A saturated aqueous solution of picric 
 acid is then added, the mixture is shaken up and allowed to set- 
 tle for twenty -four hours. It is then filtered and one Igramme of 
 chloral added for each litre of the solution. After eight days 
 the slight precipitate that has formed is removed by filtration, 
 and the picro-carmine is fit for use. 
 
 Carnoy finds this picro-carmine gives better results than any 
 other; it has kept for more than two years in his laboratorj' 
 without change." — -(BoUes Lee.) 
 
 Haematoxylin. 
 
 "The advantages of haematoxylin solutions are chiefly these: 
 that, when properly employed, they afford a most precise and 
 powerful stain; that their staining is not impeded by the pre- 
 vious employment of chromic acid or other similar hardening 
 agents ; and (as r^ards the alcoholic solutions) that they have a, 
 veiy high degree of penetration and are not hurtful to tissues." 
 — (BoUes Lee.) 
 
 Owing to the fact that solutions of haematoxj^lin rapidly de- 
 tariorate, it is preferable to use a fresh preparation each 
 time; Kleinenberg's formula is therefore chosen, and the student 
 will find the two solutions ready for use. 
 
 Prepare a saturated solution of calcium chloride in 70 per cent. 
 alcohol with the addition of a little alum; after having filtered, 
 mix a vol. of this with from six to eight vols, of 70 per cent, 
 alcohol. At the time of using the liquid, pour into it as majiy 
 drops of concentrated solution of haematoxylin in absolute alco- 
 hol as are sufficient to give the required color to the prepara- 
 tion of greater or less intensity, according to desire. 
 
 Saiall objects are best stained slowly with a very dilute solu- 
 tion. If it be required to dilute a solution already prepared for 
 staining this should not be done with alcohol, which may easily 
 
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46 
 
 cause precipitates to form on the tissues, but with the aboye pre^ 
 scribed solution of alum in calcium chloride solution. Over- 
 stains should be washed out with acidulated alcohol. J per 
 cent, hydrochloric acid may be used, and the specimens allowed 
 to remain in it until they belgin to acquire a reddish hue. The 
 acid is then removed by pure alcohol, which restores the pure 
 blue of the stain. 
 
 For large or impermeable objects immersion for days in a very 
 strong solution may be necessary for staining. Osmium and 
 chromic acid objects staui sufticiently. 
 
 A successfully prepared fresh solution should be of a violet 
 color with a decided touch of blue, and should not be at all red. 
 
 Sections stained in Kleinenberg's haematoxylin are to be 
 mounted in Canada balsam. 
 
 Aniline Colors. 
 
 A number of the aniline dyes are employed .with advantage in 
 microscopical technology. Their chief advantage lies in the 
 rapidity with which they stain fresh or hardened tissues. They 
 are not well adapted for permanent preparations on account of 
 their liability to fade. The stains, are, as a rule, removed by 
 alcohol, and care must be exercised in their washin|g. They are, 
 however, very useful in laboratory work. The following will be 
 used during the j'ear's work: 
 
 Magenta (Roseine, Fuchsin). 
 
 Dissolve 6 grammes of crystallized magenta in one litre of 
 water. Add 6 cc. absolute alcohol. 
 
 Dilute alkalies cause this color to fade completely; dilute 
 acids change it to brown or light purple. It is used for bringing 
 out the structure of thickened cell-walls. The sections must 
 previously be treated mth alcohol. It is also a good reagent for 
 corky tissues. When the section is staineil and then washed iu 
 absolute alcohol, the coloration is removed from all parts ex- 
 cepting the corky tissue. 
 
 Iodine Green. 
 
 According to Oriesbach, "the most useful of all aniline 
 stains." Dissolve 1 gTamme of crystallized iodine green in 35 
 grammes of distilled water. 
 
 A nuclear but frequently diffuse stain, valuable for the ex- 
 ceeding rapidity of its action, and for its striking power of 
 
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 marking out by staining in Tarious liues the different forms of 
 tissue. In generail bone and connective tissue do not take the 
 Ejtain, gland-cells stain most intensely and selectively, muscle 
 stains instantaneously and diffusely, but the nuclei are brought 
 out by their deeper coloration, the sarcolemma remaining un- 
 stained. It is useful for ganglion cells, axis cylinders, blood and 
 spermatozoa. 
 
 The objects are to be put in water a few seconds before stain- 
 injg. They stain instantaneously in general. They are to be 
 washed out in water and brought into glycerin, or dehydrated 
 in absolute aicohol akid passed through oil of cloves or anise 
 seed into balsam or damar. The stain is not destroyed by immer- 
 sion in alcohol for days. The preparations are apparently per- 
 manent in balsam. 
 
 Hanstein's Rosaniline Violet. 
 "This is prepared by dissolving equal parts of fuchsin ma- 
 genta and methyl violet in alcohol. It stains cellulose cell-walls 
 of a faint violet color, and lignified cell-walls red. It is es- 
 pecially useful for bringing out the different parts of bast, since 
 bast fibers stain a deep red, whereas sieve-tubes and paren- 
 chyma scarcely stain at all. The protoplasm is stained bluish 
 violet; amyloid s;ubstances, gums and nuclei stain different 
 shades of red, resins blue, and tannin foxy or brick-red." — 
 (Bower.) 
 
 Corallin (Rosolic Acid). 
 "A solution in 30 per cent, solution of sodium carbonate 
 colors lignified tissues, the callus of sieve-tubes, and starch 
 grains pink.'' — (Bower.) 
 
 Eosin. 
 
 A diffuse stain difficult to keep in ordinary mounting media. 
 Used mostly in connection with other dyes to give double 
 staining. 
 
 Prepare a 5 or 10 per cent, aqueous solution; add a few drops of 
 this to the watch glass of distilled water containing the speci- 
 mens. These may remain in the stain 'for ten or twelve hours, 
 when they may be washed out with water or alcohol. 
 
 Eosin stained preparations must not be treated with acids, 
 they should be mounted in neutral or, better, saline glycerin, 
 containing 1 per cent. NaCl, and charged with a little eosin 
 (othei-wise the color will diffuse out from the preparations); or 
 
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 in bals.am aftsr deliydratiiig with alcohol and clearing with clove 
 oil, both similarly charged with eosin. 
 
 Aniline dyes are especially adapted for staining bacteria. 
 Of these methyl violet, methyl blue and liismarck brown are the 
 most generally useful. Koch prefers the latter for specimens to 
 be photographed. Sternberg prefers methyl violet, as it does 
 not stop out the light so completely as Bismarck brown, and 
 hence allows the distinguishing of details of structure, such as 
 granules or endogenous spores. 
 
 Methyl Violet. 
 
 Take of pure aniline 5 cc, shake repeatedly with distilled 
 water. In half an hour 3 or 4 per cent, of the aniline is dis- 
 solved, the rest settles to the bottom. This mixture is filtered, 
 and, if not absolutely clear, must be filtered again. Prepare new 
 each time. 
 
 To 100 - 150 cc. of absolute alcohol add 20 grammes of dry 
 methyl violet, allow to stand several days, shake repeatedly. 
 
 11 cc. of the alcoholic solution of methyl violet is mixed with 
 100 cc. of the aniline water, and the staining fiuid is ready for 
 XLie. Add a crystal of thymol to keep it, and filter before use. 
 
 A preparation of methyl violet, especially useful for sieve- 
 tubes, is the following: Some of that substance is dissolved in 
 strong sulphuric acid, forming a birownish-green solution : on the 
 ■g'radual addition of water the violet color reappears. If a sec- 
 tion be treated with this fluid for a short time, and be then 
 yashed with water, it will be found that the ceU-walls, have 
 become swollen and transparent, that the protoplasjn has become 
 deeply stained, and the sieve-plates are very W'ell brought out. 
 Lignified tissues treated with this fluid assume a yellow color, 
 as they do when treart;ed with aniline sulphate. 
 
 Methyl Blue (Koch's Solution). 
 Take distiUed water 200 cc. Concentrated alcoholic solution 
 of metihyl blue 10 grammes. Shalte well and add 10 per cent, 
 solution of caustic potash 0.2 grammes. Allow the mixture to 
 stand for a day. It should give no sediment. Filter. 
 
 Bismarck Brown. 
 
 This takes the lead of all aniline dyes, in that it is not so easily 
 washed out. The preparations may also be mounted in glycerin, 
 which is not the case with other aniline dyes. It is also espe- 
 
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 daily adapted to photography. Weigert makes a saturated 
 solution with, boiling distilled water, or one in which there is 
 very little alcohol; after cooling, filter, set aside and allow to 
 settle. After staining, wash the sections for a short time in 
 absolute alcohol. Whole pieces may be stained at once before 
 cutting by using an alcoholic solution of Bismarck brown. 
 Baiimgarten used a concentrated (one per cent.) acetic acid 
 solution. 
 
 Safranin (Pfitzner's Formula). 
 
 One part safranin dissolved in 100 parts absolute alcohol; 
 after a few days 200 parts of distilled water are added. 
 
 An excellent stain for nuclei, b,eing purely nuclear. Stains 
 quickly. Works best with chromic acid preparations from 
 which the acid has been removed as far as possible. 
 
 Safranin is perfectly soluble in alcohol, imperfectly so in wa- 
 ter; stains tissues, in a few instants rose, nuclei more intensely 
 than cell-substance. Both fresh and alcohol-hardened tissues 
 are well stained; less well those that have been hardened with 
 chromic acid. Preparations keep well in saturated solutions of 
 (acetate of potash. As regards testing for amyloid substances, 
 the reaction is as follows: healthy tissues stain of a fine rose 
 color, those affected with amyloid degeneration of a fiery orange 
 yellow. 
 
 Double Stains. 
 
 "It is sometimes possible to color different parts of a specimen 
 with more than one dye; for instance, staining the fibers of the 
 bast green, and the wood of the same specimen red. The best 
 results are obtained by using an alcoholic solution of one of the 
 dyes and a aqueous solution of the other. 
 
 Double staining can also be effected by the successive use of 
 haematoxylin and an aniline color. By the use of two or more 
 aniline dyes, different parts of a specimen may be colored differ- 
 ently ; but as a rule all these effects are uncertain, and caainot be 
 relied upon for the positive identification of tissues. In general, 
 however, long bast fibers take characteristic colors. 
 
 The following combinations for double staining are recom- 
 mended by Dr. Stirling, and though originally designed only 
 for animal tissues, serve well with sections of plants : 
 
 (1) Osmic acid and picro-carmine. (2) Picric acid and picro- 
 carmine. (3) Picro-carmine and logwood (haematoxylin). (4) 
 Picro-carmine and an aniline dye. (5) Logwood and iodine 
 4 
 
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50 
 
 green. (6) Eosin and iodine green. (7) Eosin and Icgwood. (8) 
 Gold chloride and an aniline dye." — (Goodale.) 
 
 Rothrock's Double Statn. 
 
 The dyes are Woodward's caiinine and aniline green (or iodine 
 green.) The specimen (whether bleached by sodic hypochlorate 
 or left unbleached) is first saturated by alcohol, which hardens 
 it and causes contraction of the contents; it is then kept for a 
 day in a dilute alcoholic solution of aniline green. In a row^ of 
 watch crystals the following liquids are placed: (1) water, (2) 
 ^^'ood^\•ard's carmine, (3, i, 5,) alcohol, (G) absolue alcohol, (7) oil 
 of cloves. The specimen, taken from the green, is dipped for a 
 moment in w ater, then for ab ;ut a minute in carmine, then succes- 
 sively through the alcohols, in each of which it remains ten to 
 twenty ndnutes, except in the first, where it remains only long 
 enough to have the unfixed carmine Avashed away. Prom the last 
 alcohol it goes into oil of cloves (or benzol), where it should re- 
 main long enough to become perfectly transparent. It is then 
 to be mounted in balsam. 
 
 Diamond-fuchsin Iodine Green. 
 
 Make a solution of fuchsin and of iodine green in 50 per cent. 
 alcQihol; pour the iodine green into a saucer and slowly add to 
 it the fuchsin solution till the fluid has taken a distinct violet 
 color. A double stain useful in showing nuclear and cell divi- 
 sion, e.g., sections of anther, preserved in absolute alcohol, are 
 placed on the object slide in a drop of this fluid, w'hich after the 
 lapse of about a minute is run off by tilting the object slide and 
 sucking up with blotting paper. A drop of glycerin is then 
 placed upon the object, the sections arranged and covered with 
 a cover glass. These sections show the cytoplasma red, the 
 nuclear substance blue, the paranucleolus stained red. The pre- 
 parations ai'e exceedingly beautiful and instructive, though in- 
 ferior in sharpness of delineation to safranin and good logw'ood 
 preparations.'' — (Strasburger.) 
 
 Methyl Green and Eosin 
 
 (Calberla's formula alter BoUes Lee). 
 
 j\lix one pai't of eosin with sixty parts of methyl green, and 
 dissolve the mixture in warm 60 per cent, alcohol. 
 
 Sections stain in this solution in five or ten minutes; they 
 should be quickly washed in successive alcohols, and mounted in 
 balsam or glycerin. 
 
 In general nuclei of epithelia stain reddish violet or blue, 
 
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 nnclei of connective tissue green or greenish blue, the extra- 
 nuclear parts of cells being rose-red. Striated muscles stain 
 red, their nuclei green, smooth muscles green, and their inter- 
 cellular substance red. The cells of the ducts of salivary glands 
 stain blue, the secreting cells red, and the cells of the surround- 
 ing connective tissue green to greenish blue. 
 
 Haematoxylin Eosin 
 
 (Renant's formula after BoUes Lee). 
 
 Make a mixture of equal volumes of neutral glycerin and satur- 
 ated solution of eosin (in alcohol or in v^ater, according as pure 
 eosin or potassic eosin is employed). Add Boehmer's haematoxy- 
 lin (No. 94), drop by drop, until the Igi'een fluorescence of the 
 mixture becomes almost imperceptible. Filter, and you will ob- 
 tain a violet colored solution of haematoxylin eosin. Wash 
 quickly in alcohoL Mount in saline glycerin (1 per cent.) or in 
 balsam. (In the lattter case both the alcohol used for dehydrat- 
 ing and the oil of cloves used for clearing should be charged 
 ■ftith eosin). 
 
 ■Weigert's Method of Staining Tissues of the Central 
 Nervous System 
 
 (Gray's modification). 
 
 The specimens hardened in MuUer's or Erlike's fluid a re trans- 
 ferred directly (without coming in contact Avith water) to alcohol 
 of TO per cent. They are gradually dehydrated and finally soaked 
 in absolute alcohol for several days. They are then soaked for 
 one or two days in a mixture of equal parts of ether and absolute 
 alcohol; then transferred to a solution of celloidin on cork. The 
 pieces, still fastened to the cork in the celloidin, are inmiersed 
 in a solution of neutral acetate of copper (a saturated filtered 
 solution of this salt diluted with an equal volume of water) and 
 allowed to remain in a constant temperature of 30 or 40 C. for 
 one or tAvo days. 
 
 The specimens become pea gi*een after the copper treatment; 
 the celloidin more of a blue green. They may now be preserved 
 in 80 per cent, alcohol indefinitely. 
 
 After having made sections, Avhich must still be kept clear of 
 water, they are immersed in the haematoxylin solution,' the for- 
 mula of which is as follows : — Haematoxjdin (Merck's in crystals), 
 1 pai-t; absolute alcohol, 10 parts; water, 90 parts. Boil twenty 
 minutes, cool, and filter, and to each 100 parts add 1 part of a 
 cold saturated solution of lithium carbonate. 
 
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 The time for staining varies. In general, the longer the surer 
 the result; for cord sections, 2 or 3 hours are enough; in brain 
 sections, 24 hours are required to color the very fine fibers of 
 the cortex. 
 
 After staininig, the sections, now black in color, are differenti- 
 ated by imknersing in the following solution: Borax, 2 parts; 
 ferricyanide of potassium, 2 parts; water, 100 parts. Time 
 here varies; for cord, thirty minutes to several hours before de- 
 sired contrast between the white and gray is secured j In brain 
 sections, longer. Ko fear of spoiling the sections need be felt. 
 Fl'om this solution the sections are transferred to water and well 
 washed, then to 80 per cent, alcohol, then spread on slides and 
 dehydrated with 100 per cent., clarified with xylol or creosote 
 and mounted in xylol or ben sole balsam. If the steps in this 
 method are carefully followed out success is certain; and it is, 
 without exception, the best method for tracing nerve fibers or 
 demonstratinig nerve lesions^ 
 
 Clearing Agents. 
 
 "Clearing agents are liquids whose primary function it is to 
 make microscopical preparations transparent by penetrating 
 amongst the highly refracting elements of which the tissues are 
 composed, the clearing liquids themselves having an index of 
 refraction not gi'eatly inferior to that of the tissue to be cleared. 
 Hence all clearing agents are liquids of high index of refrac- 
 tion. The same substances have generally a second function 
 which consists in getting rid of the alcohol in which prepara- 
 tions are generally preserved and facilitating the penetration of 
 the balsam or other resinous medium in which preparations are, 
 in most cases, finally mounted. Hence, all of the group of bodies 
 here called 'clearing agents ' must be capable of expelling alco- 
 hol from tissues, and must at the same time be solvents of Canada 
 balsam and the other resinous mounting media. 
 
 It is important to note the manner of employing these agents. 
 The old plan was to take tlie object out of the alcohol and float 
 it on the surface of the clearing mediuni in a watch glass. This 
 plan was faulty, because the alcohol escapes fro)n the surface of 
 the object quicker (in most instances) than the clearing agent can 
 get into it; hence the object must shrink. To avoid or lessen 
 this cause of shrinkage, clearing is now generally done by the 
 method suggested by Giesbrecht, which consists in putting the 
 clearing medium under the alcohol containing the object. This 
 
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 is done in the following manner : Take a test tube, and put into 
 it enough, alcohol to contain the objects (a watch glass will often 
 do well, but a test tube is safer). With a ijipette carefully put 
 under the alcohol a sufficient quantity of clearing medium. Then 
 put the objects into the alcohol. They will sink down to the 
 level of separation of the two liquids at once; and after some 
 time they will be found to have sunk to the bottom of the clear- 
 ing medium. They may then be removed by means of a pipette, 
 or the supernatant alcohol drawn off and the preparations al- 
 lowed to remain untilwanted. The chief clearing agents are the 
 essential oilsj 
 
 Creosote as a Clearing Medium (Stieda's Method). 
 Einse sections in water, bring them on to slides, draw off the 
 water by means of blotting paper, and add a drop of creosote at 
 the side. \Mien clear draw off the creosote in same way and 
 replace with damar: cover. 
 
 Carbolic Acid as a Clearing Medium. 
 Best used in concentrated solution in alcohol. Clears instan- 
 taneously, even very watery preparations. As remarked above, 
 it is generally better avoided for preparations of soft parts which 
 it is intended to mount in balsam, as they generally shrink by 
 exosmosis when placed in the latter medium. For clearing sec- 
 tions imbedded in oelloidin use a mixture of Xylol and Carbolic 
 acid, equal parts. 
 
 Xylol, Benzol, Chloroform. 
 All three are good for preparing objects for balsam, but ai-e 
 not useful for preliminary examination media on account of their 
 great volatility j 
 
 Turpentine. 
 "Generally used for sections that have been cut in paraffin, 
 as it has the property of dissolving out the paraffln and clear- 
 injg the sections at the same time. If used for alcohol objects 
 it causes considerable shrinkage unless used in the thick- 
 ened state, a method which is much liked for some purposes in 
 Germany. Thickened turpentine ('Ferhartzes Turpentinol' of 
 German writers) is prepared by exposing rectified turpentine in 
 thin layers to the open air some days. All that is necessary is 
 to pour some turpentine into a plate, cover it lightly so as to 
 
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 protect it from the dust without excluding the air, and leave it 
 until it has attained a sirupy, consistency. Turpentine has, I 
 believe, the lowest index of refraction of all the usual clearing 
 agents; it clears objects less than balsam. 
 
 Oil of Cloves (Rindfleisch's Method). 
 
 "The first advantage possessed by oil of cloves over turpentine 
 is that it is miscible with stronjg alcohol. It is also miscible in 
 all proportions with Canada balsam. The shrinkage inseparable 
 for the use of turpentine is thus avoided on either hand, and the 
 introduction of this medium is probably the first important step 
 in the evolution of Lockhardt Clarke's original method. 
 
 Two important properties of clove oil should be noticed here. 
 It does not easily spread itself over the surface of a slide, but 
 has a tendency to form very convex drops. This propertj- makes 
 it a very convenient medium for making minute dissections in. 
 The second property I wish to call attention to is that of making 
 tissues that have lain in it for some time very brittle. This 
 brittleness is aJso sometimes very helpful in minute dissections. 
 
 Clove oil has, I fancy, the highest index of refraction of all 
 the usual clearing agents ; it clears objects more than balsam ; it 
 dissolves celloidin (or collodion) and therefore should not be 
 used for dealing sections cut in that medium without special 
 precautions. Notwithstanding the opinion of Schiefferdecker, 
 I consider this to be one of the best of clearing agents and very 
 valuable on account of the properties to which attention has been 
 called above. I think no work table should be without it. 
 
 Clove oil darkens with age. Dark samples should be rejected, 
 but so should very colorless ones, as these are generally adiilter- 
 ated. The most common adulterant is Carbolic acid (Phenol) 
 which may be detected by Fluckiger's test; — Shaive the suspected 
 oil with fifty parts of hot water, sloA\ly evaporate the aqueous 
 portion to a small bulk and test it Avith a drop of ammonia and a 
 pinch of chloride of lime. If phenol should be present, a green 
 is developed, which changes to a permanent blue. 
 
 Oil of Bergamot. 
 
 Schiefferdecker finds that this oil has many good qualities; it 
 clears 95 per cent, alcohol preparations and celloidin preparar 
 tions quickly, does not attack aniline colors, but the strong odor 
 is disagTeeable; it is as dear as oil of cloves, twice as dear as oil 
 of origanum, and three times as dear as oil of cedarj He con- 
 
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 siders its action preferable to that of oil of cloves, but, all things 
 considered, gives the palm to cedar and origanum. I think that 
 this is a very valuable medium, and though I do not agree with 
 Schieflerdet'ker in thinkinig its action superior to oil of cloves, I 
 think it should ah\a\ s be kept at hand. 
 
 Cedar Oil. 
 
 Finest cedar-wood oil is miscible with chloroform balsam and 
 with castor oil. Clears readily tissues in 95 per cent, alcohol 
 without shrinkage, and does not extract aniline colors. 
 
 Celloidin sections are cleared in five to six hours.' 
 
 Cheap but requires an inconvenient length of time for cleating 
 celloidin sections. 
 
 Cedar oil has, especially when exposed in thin layers to air 
 and light, approximately the same refraction and dispersion as 
 crown glass. It is the most widely used immersion fluid for 
 homogeneous immersion objectives. It is therefore probably 
 the bast fluid for examining objects with homogeneous lenses, 
 if it be wished to make such an examination previous to mount- 
 ing in balsam; I mean, the best fluid so far as regards the defl- 
 nition of such objectives, which are often unprovided with cover 
 Corrections.'- — (BoUes Lee.) 
 
 Caustic Potash. 
 
 If, in the study of vegetal tissues, it is not desired to observe 
 the details of structure of the protoplasm of the nucleus, but the 
 characters and arrangement of the cell-walls, the best clearing 
 agent is a solution of potash, either in alcohol or water. The 
 most generally useful is the 5 per cent, solution, made by dis- 
 soMng 5 grammes of solid caustic potash, in 100 cc. of dis- 
 tilled water. The alcoholic solution is made by adding strong 
 alcohol (ordinary methylated alcohol will do) to a quantity of 
 concentrated solution in water until a precipitate begins to be 
 formed. The mixture must then be well shaken, and allowed to 
 stand and settle for twenty-four hours; the clear fluid is then 
 poured off. For use a mixture of equal parts of this solution and 
 distilled water may be made. Goodale speaks of a moderately 
 strong solution of potash as "the most useful clearinig agent we 
 have" for vegetal tissues. 
 
 The clearing action of potash is due to the swelling of the cells 
 and their contents, so that they become more transparent; at the 
 same time it dissolves many of the granules in the protoplasm 
 
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 and saponifies the oil-drops. The swelling caused by the axition 
 of the solution in water is often too great, especially when it is 
 desired to see the cell-walls distinctly. This difficulty may be 
 got over by the use jof the alcoholic solution. 
 
 ''After a mass of tissue, for instance an embryo, has been acted 
 on by a solution of potassa until it has become translucent, it is 
 to be cautioHisly subjected to the action of am acid, preferably 
 acetic or hydrochloric, and then washed. A second treatment, or 
 even a third, may be necessary to make the object sufficiently 
 clearj Sometimes the potassa renders the tissues too nearly 
 transparent, in which case they may be slilghtly clouded by a 
 little alum waterj This process of clearing tissues was first used 
 by Hanstein in the examination of the tissues at points of growth, 
 and is of wide applicability.'' — (Goodale.) 
 
 After treatment with the aqueous solution, the sections should 
 be washed in distilled water, and after treatment Avith the alco- 
 holic solution in dilute alcohol, the sections, in both cases, 
 should be mounted ia glycerin. 
 
 Chloride of Lime. 
 -;Vnother method of clearing, which is especially recommended 
 for obtaining good preparations of growing plants, is to treat 
 sections with calcium chloride. The sections are placed on a 
 sliile in a drop of water, and are then covered with some dry 
 powdered calcium chloride; the slide is then warmed over the 
 flame of a spirit lamp until the water has nearly all evaporated; 
 a drop or t^o of water is now placeil on the sections, and they 
 are to be mounted in glycerin. 
 
 Putting on the Cover Glass. 
 "Grasp a clean cover glass of the proper size near one edge 
 •ndth the fine forceps, and, if necessary, dust on both sides with a 
 quUl duster. Without relaxing the grasp of the forceps, rest one 
 edge of the cover on the slide near the object and slowh- lower the 
 other till the cover is nearly parallel with the slide; then remove 
 the forceps, and the cover will fall into position. If this method 
 is followed, the cover glass may be put exactly where it is 
 wanted, anil there will also be avoided the displacement of the 
 object and the inclusion of air-bubbles." — (Gage.) 
 
 On Mounting Media. 
 "Dry specimens are to be preserved in shallow cells formed by 
 a thin ring of asphalt cement, shellac or white lead, allowed to 
 
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 dry nearly to hardness, upon which a cover glass fits flnnly, and 
 is retained by a second rim of the same cement. If the precaution 
 is taken to have the cover glass fit evenly to the first layer of 
 cen;cnt, there is little danger that the subsequent layer, which is 
 to hold the cover in place, will creep under it and into the cell. 
 Glycerin, pure water, calcic chloride solution, potassic acetate 
 and like liquids may be used as mountinig media in cells prepared 
 in the manner just mentioned, but made of greater thickness. 
 Care must be observed to avoid touching the upper edge of the 
 cement ring -n-ith the liquid ; and yet the cell must be completely 
 filled in order to exclude the air." — (G-oodale.) 
 
 Glycerin. 
 
 Glycerin for mounting specimens stained in carmine should 
 be acidvilisted by the addition of one per cent, of formic or acetic 
 acid. 
 
 Fi'i- closing glycerin mounts, the edges of the cover should 
 first (after having cleaned it as far as possible from super- 
 fluous glycerin) be painted with a layer of glycerin jelly; as soon 
 as this is set any of the usual cements may be applied. Long 
 soaking of the tissues in glycerin is a necessary preliminary to 
 mounting in all cases in which it is desired to obtain the best 
 possible preparations, and to insure that they shall keep well. 
 
 Glycerin dissolves carbonate of lime, and is therefore to be 
 rejected in the preparation of calcareous structiires that it is 
 wished to preserve. 
 
 Glycerin bottles must be kept well stoppered, as it is so highly 
 hygroscopic that if exposed to the air it rapidly diminishes in 
 strength. 
 
 Of the mounting media, one of the best is glycerin and acetic 
 acid in equal parts, boiled and filtered. It serves well for thin 
 walled specimens (especially in the lower plants). 
 
 Specimens of fresh cells or of juicy tissues which are to be 
 mounted in glycerin are best treated in the manner recommended 
 by Beal. The specimen is first immersed in weak glycerin, and 
 the density of the fluid is gradually increased, either by addinig 
 from time to time a few drops of strong glycerin, until it bears 
 the strongest, or by allowing the original weak solution to be- 
 come gradually stronger by slow evaporationj In this way the 
 softest and most delicate tissues may be made to swell out al- 
 most to their volume in the densest glycerin or sirup. They be- 
 come more transparent, but no chemical alteration is produced, 
 
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 and the addition of water will at any time cause the specimen 
 to assume its ordinary characters. 
 
 It is plain that mounts in any liquid must be liable to injury 
 from displacement of the co\er glass; but this can be partially 
 guarded against by fastening to the upper surface of the slide, 
 near its two ends, square pieces of pasteboard, a little thicker 
 than the cell itself. 
 
 Glycerin Jelly. 
 
 This is a mixture of glycerin with pure gelatine, it is liquid 
 at the temperature of boiling water, and solidifies again 
 on cooling. Any specimen A^hich is not injured by being slightly 
 heated can be mounted satisfactorily in the jelly, provided it is 
 first thoroughly saturated with glycerin. But this precaution 
 is by no means necessary in all cases. 
 
 A drop of the melted jelly, free from air-bubbles, is placed on 
 the slide (a fragment of the jelly can be melted on the slide if 
 pvef ei-red), the specimen placed therein, and the cover glass, pre- 
 viously moistened slightly on the under side with glycerin, is 
 carefully laid on and the preparation allowed to cool. When the 
 jelly is again hard, a varnish or cement ring may be placed 
 around the edge of tJie cover to hold it in placej Asphalt cement 
 is apt to impart to the jelly a dark tinge, which may sooner or 
 later s])oil the mount, and hence the colorless varnishes are 
 better. 
 
 The edge of the jelly may be lightly touched with a strong 
 solution of a chromate, for instance, bichromate of pota.ssium, 
 and exposed a while to light. This renders the jellj' insoluble, 
 and firmly sets it. 
 
 The following is one of the best formulas for making this use- 
 ful mounting medium : 
 
 One part of gelatine is soaked in six parts of water for two 
 hours, seven parts of glycerin are added, and one per cent, of 
 carbolic acid is added to the A\"hole. The mass is heated for fifteen 
 minutes, with constant stirring, and then filtered through glass 
 wool. All the ingredients must be absolutely pure. 
 
 The proportions employed in the above formula, but without 
 the addition of the carbolic acid, give a clear jelly; and it has 
 not been apt to mold, especially if the cork of the bottle con- 
 taining it be wrapped in a thin piece of linen, which has been 
 dipped in dilute carbolic acid." — (Goodale.) 
 
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59 
 
 Canada Balsam. 
 
 This is used either alone or in solution- In either case 
 the specimen must be free from water, and permeated with 
 some liquid easily miscible Avith the balsam. This is easilj'' 
 affected by first saturatinig the object with alcohol (beginning 
 preferably with dilute, and then using stronger), in order to ex- 
 pel all water; next placing the alcoholic specimen in oil of cloves, 
 turpentine or benzol, until the alcohol is in turn expelled. The 
 specimen thus permeated is transferred to balsam which has 
 been previously placed on the slide. Care must always be taken 
 to have the balsam free from air-bubbles. 
 
 When used alone, the balsam on the slide may be heated, to 
 drive off a jjart of its more volatile constituents, and the speci- 
 men can then be placed in the warm liquid. But this method 
 is not applicable when the specimen is affected by slight heat- 
 ing; it is best adapted to hard tissues, like woods and fibers. 
 Balsam which has been thus heated hardens on cooling to a good 
 degree of firmness. This firmness is secured with balsam used 
 without heat after a long lapse of time, during which the more 
 volatile matters have escaped. 
 
 When used in solution regard must be had to the effect of the 
 solvent upon the specimen to be mounted. Thus, it is impor- 
 • tant, for mounting stained bacteria-, to use Canada balsam free 
 from chloroform, a.s Orth and others testify that it removes the 
 color from the microbes. Baumgarten recommends equal parts 
 of Canada balsam (free from chloroform) and oil of cloves. 
 Xylol-balsam answers very well. Pure xylol should be used. 
 It burns with difficulty, and hence is much safer than naphtha, 
 benzine or turpentine. 
 
 Again, in making serial sections, where a cement of collodion 
 or celloidin is used to fasten the ribbon to the slide, a cloudiness 
 often appears which may be cleared up by using a solution of 
 balsam made as follows: Canada balsam, 25 cc. ; chloroform, 
 2 cc; clove oil, 2 cc. This preparation will be found as 'good as 
 any for all ordinary occasions. 
 
 HOVER'S MOUNTING MEDIA. 
 (a) For Aniline Preparations. 
 
 "A tall glass vessel with a wide neck is filled up to two-thirds 
 with gum-arabic, in selected white pieces. The vessel is then 
 filled up to the neck with a solution of 50 per cent, acetate of 
 potash, or with a watery solution of acetate of ammonia contain- 
 
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60 
 
 ing to esuch 30 grammes 10 grammes of caustic ammonia neutral- 
 ized by a sufficient quantity of acetic acid. The gum is dissolved 
 in a few days, if the vessel is often shaken, and forms a sirupy 
 fluid, which is filtered through thick swansdown — a process 
 taking about twenty-four hours. 
 
 (5) For Carmine and Haematoxylin Preparations. 
 
 This is prepared as above, excepting that, instead of acetate 
 of potash or of ammonia, a concentrated solution of chloral 
 hydrate, to which is added 5 to 10 per cent, glycerin, is used. 
 After some time this fluid may become turbid, and it is then 
 necessary again to filter it. 
 
 Preparations mounted in either of these fluids require no fur- 
 ther inclosing." (Strasburger, by whom these media are 
 strongly recommended.) They are partjicularly useful for prepar 
 rations of woods to be photographed. 
 
 Acetate of Potash Mounting Fluid. 
 
 This is simply a stronigly concentrated solution of acetate of 
 potash. It behaves much as glycerin, does not dry at the edges, 
 and is less refractive. Many aniline-stained preparations of 
 bacteria keep well in this solution : 
 
 Glycerin, 10 parts. 
 
 Glucose of commerce, 40 parts. 
 
 Spirits of camphor, 10 parts. 
 
 Water, 140 parts. 
 
 Mix and filter. The advantage of this medium is that it pos- 
 sesses the refractive index, 1.37, in the yellow ray, an index corre- 
 sponding to that of the substances composing sputum (albumen 
 1.36, saliva and mucus 1.34, pus 1.39.) — (Robin.) 
 
 Ordinary Canada balsam has a very high index, 1.53, in which 
 bacteria are poorly defined; colorless oils, for example, that of 
 rape or castor, are preferable, but their index is also too high, 
 1.48. 
 
 In the use of mounting media lacking tenacity and adhesive 
 properties the edges of the cover glass are usually painted with 
 some varnish of good quality. Those in best repute are: 
 
 Asphalt varnish, to be thinned with turpentine when too thick. 
 
 Maskenlack, a German preparation, thinned with alcohol. 
 
 Mikroskopirlack, also thinned with absolute alcohol. 
 
 Shellac in alcohol, tinged with some aniline color. If a few 
 drops of castor oil are added to the solution, it dries into a less 
 brittle finish. 
 
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61 
 
 Gold-size. 
 
 White lead (with oil). 
 
 It is a good plan to revarnish slides whenever the varnish first 
 shows any indication of breaking away. — (Modified from 
 Goodale.) 
 
 Cement for Glycerin Mounts. 
 Pure Venetian turpentine is to be poured into some melted wax 
 upon a water bath until a portion taken out on a glass rod be- 
 comes stiff at once and does not stick to the hand. — (Csokor.) 
 
 Section Cutting. 
 
 "Every student should be provided with a good razor and ren- 
 der himself expert in freehaml section cutting. 
 
 Many plant tissues are preferably examined fresh, and much 
 time is saved here by skill in the use of the razor. 
 
 Care must be taken to keep the object and the razor wet during 
 the process of cutting. When fresh material is cut, water, or 
 very dilute alcohol may be used for this purpose; but if material 
 which has been hardened is cut, it is advisable to use alcohol of 
 the same strength as that in which the material has been pre- 
 served. 
 
 To make a complete study of a solid mass of vegetal tissue, such 
 as a stem, it should be cut into (i) transverse sections, in places 
 at right angles to the axis; (ii) radial longitudinal sections, in 
 longitudinal planes including the axis ; (iii) tangental in longitu- 
 dinal planes which do not include the organic axis. 
 
 When a successive series of sections of an object is required, 
 a microtome may be used. 
 
 The objects are frequently so lailge that they can be held in 
 the hand whilst they are being cut. If they are too small for 
 this, it is convenient to imbed them in some substance. 
 
 The simplest method is to fix the object into a slit in a piece 
 of pith. 
 
 "Elder or sunflower pith answers the purpose well. Thin sec- 
 tions are best removed from the knife by a camel's-hair pencil, 
 and are to be placed at once in water or some other liquid. Ex- 
 cept in certain cases, water may be used as a medium for prelim- 
 inary examination of sections." 
 
 When the sections are to be made with a microtome, it is more 
 convenient to imbed in some easily fusible substance; by this 
 means also the position of the object is less likely to be disturbed 
 in the process of cutting. — (Bower and Vines.) 
 
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62 
 
 Razor. 
 
 A razor of good quality is the best cutting instrument: it 
 should be ground perfectly flat on one side. As the razor should 
 be drawn toward the body in freehand section cutting, the partic- 
 ular side to be ground flat vill depend on whether the person to 
 use it be right or left handed. The razor should be stropped to a 
 smooth edge, and the blade should be carefully protected when 
 not in use: it should never be left open on the work table, and the 
 blade should always be cleaned after use, since the acid juices of 
 plants are apt to corrode it. 
 
 'SATien cutting sections the razor is to be opened so that the 
 blade is in a line with the handle: The object to be cut may be 
 held between the thumb and first fln.gier of the left hand, while 
 the razor is gTasped firmly by the four fingers of the right, the 
 thumb being held firmly against the milled or roughened edge of 
 the shank of the blade. The edge of the razor is not to be rudely 
 forced through the tissues of the specimen, but a sliding cut is 
 to be made, thus using a considerable length of the edge of the 
 razor. 
 
 It ^dll be found convenient to have a glass of water (or weak 
 alcohol when resinous tissues are to be cut) on the work table, 
 into AA'hich the blade of the razor may be plunged immediately 
 after use; this avUI prevent immediate corrosioai^ and the sections 
 will be floated off without injury. In cutting sections of materi- 
 als imbedded in celloidin, stronger alcohol may be used in the 
 same way, or the section removed from the blade with a camel's- 
 hair brush : in either case the blade and tissue being cut should 
 be kept constantly wet with alcohol. 
 
 The success of work in the laboratory depends very greatly on 
 due care in the direction of section and on the condition of the 
 edge of the razor. — (After Bower.) 
 
 Infiltrating with Paraffin. 
 
 "At the present day most objects to be imbedded in paraffin 
 are previously infiltrated or thoroughly permeated with it so 
 that all the cavities of the tissues are finally filled with paraffin. 
 To do this : — (a) All the water must be removed from the tissue 
 by means of 95 per cent, or stronger alcohol, (b) The alcohol 
 is replaced by chloroform or turpentine by placing the tissue in a 
 wide-mouth bottle \\ith ten to fifteen times as much fluid as tis- 
 sue. After tweh'e to twenty-four hours the tissue is usually 
 thoroughly permeated with the chloroform or turpentine. A 
 
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63 
 
 longer stay in these fluids does no harm, (c) The tissue is 
 placed in a mixture of paraflin four parts and chloroform or tui'- 
 pentine one part, which is kept just melted, the temperature never 
 being allowed to exceed 55 or 60° C. The tissue should remain 
 in this from two to twenty -four hours, and then (d) it is placed in 
 pure paraffin kept just melted, and allowed to remain from tweh'e 
 to twenty-four hours. 
 
 Imbedding. 
 
 After a tissue is infiltrated it should be imbedded in pure 
 paraffin as follows: A small paper box, made of a strong piece 
 of paper, is nearly filled with melted paraffin and the tissue is 
 placed in it and quickly arranged, so that the object will be cut 
 at the proper angle by cutting at right angles to the length of the 
 box. As soon as a film is formed by the cooling of the imbedding 
 mass the box may be placed in cold water or in any cool place so 
 that the paraffin will cool quickly and avoid air-bubbles. It is 
 well to indicate on the box the name of the tissue and the end 
 where the cutting should commence. The tissue so imbedded and 
 labeled may be put away until one is ready to make sections of it. 
 
 Sectioning. 
 
 When one is ready to make sections of a tissue it is put into 
 the holder of a microtome. Trim the paraffin away from the 
 tissue so that only a thin layer supports the tissue on every si<le. 
 The appearance of the imbedded tissue when it is ready to be 
 cut is something like a bluntly sharpened lead pencil, the sharp- 
 ened end being square or rectangular in outline. In making 
 the sections, if the edge of the knife is made parallel with one 
 edge of the imbedding mass, consecutive sections will adhere to 
 each other and form a kind of ribbon. One may imbed in the well 
 of the microtome and not use the paper box, but it is not so satis- 
 factory. 
 
 Perfect sections should consist of only a single layer of struc- 
 tural elements. The sections may be preserved for an indefinite 
 time without deterioration; and they may be stained and 
 mounted at any time. 
 
 Fastening the Sections to the Slide. 
 In order that no part of the section shall get lost or disarranged 
 durinig the staining and mounting processes, the sections should 
 be fastened to the slide. 
 
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64 
 
 The method originally suggested for fastening sections to the 
 slide is as follows : Collodion is mixed with three or four times 
 its volume of clove oil, and some of this mixture is brushed on 
 the slide just before using it. The slide, after the sections are 
 upon it, is heated to 35° or 40° C. for a few minutes to drive off 
 the clove oil. After the clove oil is expelled tlie sections are to 
 be washed with turpentine or xylol to remove the paraffin, in the 
 same manner in which a photographer flows "developer" over a 
 plate. 
 
 Before the sections are washed with turpentine or xylol they 
 may be exposefl to the air without injury, but after the paraffin 
 is removed from them they would become desiccated if exposed 
 to the air. 
 
 Staining the Sections. 
 
 After the paraffin is removed from the sections by the xylol, 
 or turpentine, the slide bearing them is placed for a minute or 
 longer in 95 per cent, alcohol to wash away the aforesanl fluid. 
 The sections are then washed with water; The stain if it acts 
 within one to twenty minutes, is placed diiectly upon the sections 
 and the side laid flat. If a longer time is required to complete 
 the staining, place the slide in a jar containing the stain. After 
 the sections are stained sufficiently the superfluous dye is re- 
 moved by a gentle stream of water. 
 
 After the stain is washed away the sections are washed in 95 
 per cent,, alcohol to remove the water. 
 
 They are then mounted in balsam by putting upon them a few 
 drops of clearing mixture, turpentine or xylol. The clearing 
 is usually completed in two or three minutes, or even less. 
 
 When sections are cleared they can hardly be seen if held 
 over some dark object. If, on the other hand, they are held up 
 between the eye and the light, they appear very translucent. 
 
 As soon as the sections are cleared, the excess of clearing 
 agent should be removed with blotting or filter paper, and the 
 sections mounted in balsam. 
 
 Order of Procedure in Making Histological Preparations 
 by the Paraffin Method. 
 
 1. Dehydrating the tissues Avith 95 per cent, alcohol. 
 
 2. Soaking the tissues in chloroform or turpentine^ 
 
 3. Infiltrating with chloroform and pai'aflin or turpentine and 
 paraffin. 
 
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65 
 
 4. Infiltrating witb. pure paraffin. 
 
 5. Imbedding. 
 
 6. Sectioningj 
 
 7: Cementing the sections to fke slide with, collodion and oil 
 of clovesj 
 
 8. Eemoving the paraffin from the sections with xylol or tur- 
 pentine. 
 
 9. Staining the sections. 
 
 10. Washing out the superfluous dye with water or alcohol. 
 
 11. Dehydrating the sections with alcohol. • 
 
 12. Clearing the sections with the clearing mixture. 
 
 13. Mounting the sections in Canada balsam. ; 
 
 14. Labeling the slide. — (Modified from Gage.) 
 
 Imbedding in Celloidin. 
 
 The heat required for infiltrating and imbedding with paraf- 
 fin acts injuriously on some tissues, and some others cannot be 
 so well prepared by the use of paraffin as by the use of celloidin 
 (a solution of pyroxylin). If it is undesirable to remove the fat 
 from a tissue, both the paraffin and celloidin methods must be 
 avoided, and the sections must be made with the freezing mi- 
 crotome. 
 
 All the water must be removed from the tissue by 95 per cent, 
 or stronger alcohol; then it should remain from twelve to twenty- 
 four hours in equal parts of ether an;! 95 per cent, alcohol. It 
 should then be transferred to a thin solution of celloidin in alco- 
 hol and ether, and allowed to remain from twelve to twenty-four 
 hours. It is then placed in thick celloidin for a day and is finally 
 imbedded as follows: A cork of suitable size is selected, and some 
 of the thick celloidin dropped upon it with a pipette, the prepared 
 tissue is then placed upon this and more celloidin dropped gradu- 
 ally upon it, giving it time to thicken somewhat, finally when 
 completely covered with and inclosed in the celloidin the cork 
 and celloidin mass is placed in a jar of 80 per ceni. alcohol 
 where it should remain until it becomes opaque and of a uniform 
 consistency, when it is ready to cut. Then preparations can be 
 kept indefinitely in alcohol. 
 
 Cutting the Sections. 
 If a well microtome is used the imbedded tissue must be 
 >vedged in with a cork. A sliding microtome is preferable. The 
 cork on which the tissue is supported is fastened into the holder 
 5 
 
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66 
 
 of the microtome, the knife is made very oblique, so that a draw- 
 ing cut will result. The knife and tissue must be thorouighly 
 and constantly wet with alcohol of 70 ta 80 per cent., and the 
 sections, as they are cut, should be transferred from the knife 
 to TO to 80 per cent, alcohol with a camel's-hair brushy 
 
 Staining and Mounting the Sections. 
 
 The slides to be used for celloidin sections are lighty brushed 
 with collodion or celloidin, as directed for the paraffin sections. 
 The sections are transferred to the collodionized slide and dehy- 
 drated by pouiing upon them 95 per cent, alcohol with a dropping 
 tube, and tlien, after removing the most of the alcohol, several 
 drops of equal parts of ether and alcohol are added exactly as 
 for paraffin sections. The slide is kept in the air imtil most of 
 the ether and alcohol have evaporated — -until the section begins 
 to look dull — then the slide bearing the sections may be placed 
 in a jar of 80 per cent, alcohol, or several drops may be poure<i 
 on the sections with a dropping tube. The sections may now be 
 washed with water, stained, dehydrated, clea.re<l by the use of 
 a mixture of xylol and carbolic acid, and mounted, exactly as de- 
 scribed for par-affln sections. Sections may also be mounted with- 
 out dissolving out the celloidin. 
 
 Tissues imbedded in celloidin may be kept a long time, perhaps 
 indefinitely, in chloroform or 70 to 80 per cent, alcohol. The sec- 
 tions may be kept in a vial of 80 per cent, alcohol until one is 
 ready to stain and mount them. 
 
 Unless the sections are to be kept in serial order it is not abso- 
 lutely necessary to fasten them to the slide. If they are to be 
 in series the mass should be trimmed asymmetrically, and the 
 sections as they are cut must be transferred to numbered watch 
 glasses. The asymmetrical outline will enable one to get them 
 right side up. — ^(Gage.) 
 
 Sections with a Freezing Microtome. 
 
 For the study of many objects it is undesirable to subject them 
 to the processes necessary for paraffin or celloidin sections. 
 This is especially true of tissues in w hich the fat is to be studied. 
 In such cases the sections may be made with a freezing micro- 
 tome. The freezing microtome also enables one to make sections 
 of fresh or hardened tissues in a mininiuni of time, or when it is 
 desirable to treat the sections with silver nitrate or gold chloride. 
 
 A piece of tissue of the proper size, either in the fresh state or 
 
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67 
 
 after liardening, may be placed in the freezing microtome ami 
 frozen eitlier with, ether vapor or ice and salt, or ice and 95 per 
 cent, alcohol. Preferably, however, the tissue is first infiltrated 
 with gum arable for a day or more and then frozen in the same, 
 or gelatine may be used for the infiltration and also for surround- 
 ing the object during the freezing and cutting. If gelatine is 
 used, the tissue and gelatine must be kept warm during the in- 
 filtration. Alcohol must be removed from the tissue if it is 
 present before infiltrating and freezing in either gum arable or 
 gelatine; 
 
 In cutting the sections the knife should be cooled, and carried 
 directly across the tissue with a rapid movement, as for paraffin 
 sections. 
 
 As the sections are cut they may be transferred to slides, and 
 a drop, of glycerin added to each if they are not to be colored. 
 Or if gelatine is used for imbedding, etc., a drop of the desired 
 coloring matter is put upon the sections, and later washed away 
 with a gentle stream of water, and then a drop of glycerin added 
 to each. 
 
 If gum arable is used for the infiltration, etc., and the sections 
 are to be stained, they must be placed for half an hour or so in 
 water to dissolve the gum arable. They may then be transferred 
 to slides, stained, mounted in glycerin and sealed, as directed for 
 glycerin mounts.; — (Gage.) 
 
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SERIAL SECTIONS. 
 
 Serial Sections. 
 
 "For the investigation and understanding of emtoyological and 
 histological facts, only next in importance to the proper harden- 
 ing of the specimen is the method of serial sectioning and mount- 
 ing; for the parts may then be compared in the sequence they 
 occupied in the animal or organ, and the most delicate or loosely 
 connected part is preserved in situ. For serial sections the ob- 
 ject, whatever it may be, is properly hardened, and then dehy- 
 drated, infiltrated and imbedded, as described ; but in order that 
 the most complete and certain knowledge may be obtained from 
 the sections, it is necessary to know with absolute certainty all 
 the aspects of each section; and if the object is an embryo or 
 small animal it is desirable to Jvuow the position of various 
 sections with reference to the ends of the body or definite land- 
 marks. Hence (1) An outline drawing of the embryo or animal 
 should be made before imbedding itj This drawing should be 
 exactly natural size or some definite proportion ; as twice natural 
 size, etc. (2) The object shoiild be imbedded so that the sections 
 shall have a definite relation to the body axis, and to the three 
 principal planes of the body. 
 
 As the tissue is taken from the body it is arranged in a defi- 
 nite manner and pinned to a cork. This arrangement is stated on 
 the label which should accompany the tissue until it is finally 
 mounted. The aspect of a small animal or embrj^o may of course 
 be deter-mined by simple inspection any time before imbedding. 
 The sections should be made in one of the three principal planes 
 of the body. 
 
 When ready to make the sections, tlie distance the microtome 
 screw is out is noted, and when the slide is filled -ndth sections 
 it is again noted. The distance that the screw is raised in cat- 
 ting the sections on the slide is equal to the total thickness of 
 all the sections on that slide, or, in other words, the length of 
 the object cut in making the sections. The amount should be 
 put on the temporary label of the slide, and the same should be 
 done for each succeeding slide. In this ^\'ay one may know, by 
 comparison with the previously made diagram, the exact local- 
 ity from which each slide of sections was obtained. 
 
 (68) 
 
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69 
 
 If an enlarged model is to be matie the sections should be cut 
 of a uniform thickness. Born recommends that this thickness 
 be not greater than .04 mm. 
 
 Cutting the Sections. 
 After the tissue is properly imbedded and placed in the holder 
 of the microtome the imbedding mass is cut away so that only a 
 thin layer surrounds the tissue. This should be trimmed so 
 that it is square or rectangular in outline. The knife is set or 
 held so that its edge is parallel with one side, and then it is 
 earned directly and quicklj- across the tissue. If the imbedding 
 mass is of the right' hardness for the temperature, ribbons of in- 
 definite length may be made with the sliding or automatic micro- 
 tome. Short ribbons may also be made, after a little practice, 
 with a well microtome. 
 
 Placing the Sections on the Slide. 
 The sections may be placed on the coUodionized slide singly 
 as they are made, or the ribbon of sections may be broken into 
 segments of the proper length. In either case the first section 
 should be placed in the upper left hand corner of the slide, an<l 
 the others should follow as in columns of figures. The sections 
 are fastened to the slide, stained, mounted, etc., as directed above 
 for ordinary paraffin sections. 
 
 Slides for Serial Sections. 
 
 Unless the object is very small, slides 50 x 75 nrni., and rectan- 
 gular cover glasses, 48 x 58 mm., are reconmiended. The covers 
 can be more safely spread with balsam if they are laid flat. 
 
 Label for the Slides in a Series. 
 
 This should give the following information: (a) The number 
 of the series, (b) The number of the slide in the series, (c) 
 The date on the label belonging to the animal, (d) The name 
 of the object, (e) The number of the series of the first and last 
 section on the slide, (f) The thickness of all the sections on th** 
 slide, or preferably the location on the length of the object, (g,. 
 Ine thickness of the individual sections if a model is to be made 
 from the series. . (h) The thickness of the cover glass : 
 
 Series No. 5, Feb. 22, 1886. 
 
 Slide No. 1. 
 
 Transections of Necturus, 1 to 50. 
 
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70 
 
 Microtome screw raised from to 2 mm. \ 
 
 Sections, each .04 mm. thicli. 
 
 Cover glass, 16 mm-, tliick. 
 
 Serial sections may be made also by the celloidin method. 
 The arrangement of the object should be as described above. 
 The mass should be trimmed asj^mmetrically, so that one may get 
 the sections a desired side up on the slide and with' the desired 
 aspect toward the top of the slide. 
 
 The perfect sharpening of section knives requires great skill 
 and experience. A large, fine oil stone that has been flattened 
 on a surface plate with emery, and a strap of flue leather nailed 
 or pasted to a smooth board are desirable." — (Gage.) 
 
 Cleaning Cover Glasses and Slides. 
 
 New slides can usually be cleaned by washing in soft water. 
 Slides that have been used for mounting objects can be cleaned 
 by soaking them in the cleaning mixture for glass and washing 
 them in water. After the slides are well rinsed, take them singly 
 and by opposite edges, and wipe dry with a soft clean cloth. 
 (It is convenient to have in the drawer of the work table two or 
 three perfectly clean, fine linen handkerchiefs; old ones, very 
 pmch worn, are to be preferred.) 
 
 l^ew cover glasses should be put one by one into a cleaning 
 mixture for glass, and allowed to remain two days or longer. 
 Cover glasses that have beeai soiled in any way should be placed 
 in the cleaning mixture for a week or more. Finally rinse the 
 covers by a gentle stream of water till the color of the cleaning 
 tinixture is no more visible. In wiping the cover glasses, grasp 
 them slightly and by opposite edges between the thumb and in- 
 dex of the left hand. Cover the thumb and index of the ri^it 
 hand with a soft, clean cloth, grasp the cover and rub its two 
 surfaces. It is necessary to have the ball of the thumb and fin- 
 ger on exactly opposite sides in wiping the covers, or they wiU be 
 broken. When a cover is well wiped, hold it up and laak through 
 it at some dark object. It will be seen partly by transmitted 
 and partly by refiected lighjt. If it does not look clear, breathe 
 on the faces and wipe them again. 
 
 Handle the cover glasses and slides by their edges, or handle 
 the cover glasses with fine forceps. Do not touch the surface 
 with the fingers. 
 
 "As the covers are wiped, place them in pill boxes, and assort 
 them according to thickness. One can judge of the thickness of 
 
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71 
 
 a cover glass by looking at its edge. It is better, howerer, to 
 measure the tliickness of each, cover, as it is wiped, with Zeiss' 
 Beck-glass Taster, and place those of like thickness in the same 
 box." — (Gage.) 
 
 Seller's formula for cleaning microscopical glassware seems 
 to give general satisfaction. 
 
 K'ew slides and covers are placed for a few hours in the fol- 
 lowing solution: 
 
 Bichromate of potash, 2 ounces. 
 Sulphuric acid, 3 fluid ounces. 
 Water, 25 fluid ounces. 
 
 "Wash. with, water; the slides may be simply drained dry; and 
 the covers may be wiped dry with a linen rag. Slides and covers 
 that have been used for mounting either with balsam or a watery 
 medium are treated as follows ; 
 
 The covers are pushed into a mixture of equal parts of alcohol 
 and hydrochloric acid, and after a few days are put into the bi- 
 chromate solution and treated like new ones. 
 
 MICRO-CHEMICAL REAGENTS. 
 
 Besides the fluids which are used for hardening and staining 
 the tissues, a considerable number are employed which, on ac- 
 count of the characteristic effects produced by their action on 
 cell-walls and cell contents, may be regarded as chemical tests 
 for the various substances which may be present. The fol- 
 lowing are the principal reagents which are used in this way : 
 the mode of preparing them is also given, and some indication of 
 tlieir uses; but this latter subject is more fully treated in the 
 next chapter. 
 
 It is best to try first a very small amount of the reagent, and 
 carefully note its effect before adding more. If it is necessary to 
 increase the amount, draw a little through by means of a little 
 bibulous paper, as previously directed. Many reagents are slow 
 in producing their effects. Hence some time must be allowed to 
 elapse before one reagent is replaced by another, and it is well 
 in some cases to apply slight heat to accelerate or increase the 
 action; but this must be very cautiously done. 
 
 If one reagent is to be followed by another, attention must be 
 given to the effects which the reagents have on each other, or 
 npon the medium as well as upon the specimen. For instance, 
 small dark crystals of iodine separate from an alcoholic solution 
 when this is brought into contact with water. Eemoval of the 
 
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 cover glass is advised in all cases where one reagent is to be 
 washed out before the application of a second, or where one is 
 so delicate as to be disturbsd by it. Some parts of the specimen 
 are apt to escape action if the washing or the introduction of 
 several reagents in these operations is conducted Avithout lift- 
 ing the cover; but by the exercise. of great care both these op- 
 erations may be carried on successfully by the nse of bibulous 
 paper without removing the cover glass. — (Goodale.) 
 
 "Percentage solutions are made either by weight or volume; or 
 if the metric system is used, by either indiscriminately. Thus 
 1 per cent, acetic acid in water is made by taking 1 vol. acetic 
 acid and adding 99 vols, of distilled water; 5 per cent, potash 
 solution, by taking 5 grammes potash and adding to 95 cc. dis- 
 tilled water (1 gramme weight equals 1 cubic centimeter vol.) 
 
 A saturated solution can be secured by seeing that some of 
 the salt, etc., is always lying undissolved at the bottom. — 
 (Strasburger.) 
 
 I. ACIDS. 
 
 Sulphuric Acid. 
 
 This is used either concentrated or dilute (1 to 3 of water.) It 
 causes, in either case, the swelling up of cellulose cell-waUs, 
 starch-grains, etc.; when cellulose cell-walls which have been 
 previously saturated with solution of iodine are treated with 
 sulphuric acid, they turn blue. 
 
 Concentrated sulphuric acid dissolves cellulose and starch, but 
 cuticularized cell-walls and the middle lamella of lignified ceUs 
 resist its actionj It is used with cane-sugai" as a test for pro- 
 teids, and with aniline sulphate as a test for ligiiin. 
 
 Nitric Acid. 
 
 It colors cuticularized cell-walls and proteids yellow; it also 
 causes swelling up of cellulose and of lignified cell-walls. It is 
 useful for dissolving the crystals of calcium oxalate which are 
 frequently present in the cells. It is used with ammonia as a 
 test for proteids (xanthroproteic reaction), and with potassium 
 chlorate as a test for suberin, and as a macerating fluid. 
 
 Hydrochloric Acid. 
 "Used, with aniline chloride, phloroglucin, or carbolic acid, as a 
 test for lignin. By itself it turns lignified ceU- walls yellow; 
 
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 wlien its action is prolonged the cell- walls become violet, owing 
 to ttie presence of certain substances, sucL. as pMoroglucin, con- 
 iferin and pyrocatechin. — (Bower.) 
 
 " Pure concentrated acid is one of the most satisfactory agents 
 for the maceration of woody tissues. When dilute, it serves for 
 the discrimination between carbonates and oxalates, the former 
 dissolving with effervescence, the latter without. It must be 
 i'emem.bered ttiat acetic acid dissolves caibouatos, but not 
 oxalates. 
 
 This acid has been used by Pringsheim in the study of chlo- 
 lophyll grains, fresh sections of tissues containing chlorophyll 
 being exposed to the action of the acid for some hours. From 
 the grains minute spheres of a brownish color become nearly 
 detached, and these afterward appear as clusters of acicular 
 crystals. Hydrochloric acid is also of use in the examination 
 of some protein matters giving a purplish color, after prolonged 
 action. — ^(Goodale.) 
 
 Chromic Acid. 
 
 A strong aqueous solution of this acid dissolves ligniAed and 
 cellulose cell- walls ; cuticularized cell-walls resist its action, but 
 they become very transparent and may be easily overlooked. A 
 dilute solution brings out the stratification of cell-walls very 
 clearly. 
 
 Acetic Acid. 
 
 This is used as a dilute aqueous solution (1 per cent.). It 
 dissolves crystals of calcium carbonate; it causes swelling up 
 of cell- walls, starch-grains etc. ; it brings out nuclei very clearly. 
 It is useful as a corrective after treatment of a preparation with 
 potash. 
 
 Oxalic Acid. 
 
 An alcoholic solution is useful for removing excess of color 
 from too deeply stained tissues, and also for cleaning the fingers 
 of any accidental stains. 
 
 II. ALKALIES. 
 
 Potash. 
 
 This may be used either in dilute or concentrated solution 
 In water. The dilute solution is chiefly used for clearing prep- 
 arations, as ali-eady described. It causes cell-walls, starch- 
 
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 grains, etc., to s'O'ell up very much, and it dissolves proteid crys- 
 talloids, and most aleurone-grains. It gives a reddish color to 
 colls in -which tannin is present. It may be used as a macerating 
 fluid; when woody tissues a,re boiled in potash, the cells of the 
 vascular bundles become more or less isolated, for the lignin of 
 their walls undergoes solution. It dissolves inulin. 
 
 The concentrated solution is used as a test for suberin. When 
 sections of cork are boiled in strong potash, the suberin escapes 
 in the form of yellow viscid drops; when the sections are only 
 slightly warmed in potash solution the cuticularized cell-walls 
 assume a yellow color. 
 
 Potash is also used, together with copper sulphate, as a test 
 for proteids, and for various kinds of sugar. 
 
 Ammonia. 
 The solution in water is often used instead of potash for clear- 
 ing preparations, as its action is less intense. It is used, to- 
 gether with nitric acid, as a test for proteids, and with copper 
 sulphate as a solvent for some forms of cellulose. 
 
 III. NON-METALLIC ELEMENTS. 
 
 Iodine. 
 
 This is one of the most useful ol the micro-chemical reagents. 
 It is used in solution, in water, or alcohol, and in the chloride of 
 zinc mixture. " In some cases, as in certaui parts of Lichens, it 
 is necessary, to examine the effect of iodine alone, and it is recom- 
 mended that in such cases a minute fragment of solid iodine be 
 placed in pure water under the cover-glass at the moment of ex- 
 amination. But for all ordinary examinations, a solution of 
 iodine in water which contains iodide of potassium may be used. 
 
 I. Solution in Water. 
 
 Dissolve a small quantity of potassium iodide in the requisite 
 quantity (if water; then dissolve iodine in it until the liquid has 
 a dark sherry color. This may also be prepared by diluting the 
 liquor iodi of the pharmacopoeia. 
 
 2. Alcoholic Solution. 
 Dissolve iodine in alcohol until it has a dark sherry color. 
 This may also be prepared by diluting the tinctura iodi of the 
 pharmacopoeia. 
 
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75 
 
 Iodine stains proteid substances brown, cellulose faintly 
 yellow, cuticularized and lignifled cell-walls yellow, gum pur- 
 ple, starch, blue (only in presence of water). 
 
 Iodine is used as a micro-chemical test for starch and cellulose. 
 The blue color which it gives to starch, and the conversion of the 
 faint yellow color of a cellulose cell-wall stained with iodine into 
 blue when it is treated with sulphuric acid, are characteristic; 
 The cellulose reaction is also given with the chloride of zinc 
 mixture. 
 
 IV. INORGANIC SALTS. 
 
 Sodium Chloride (Common Salt). 
 This is used both in dilute (1.0 per cent.) and in saturated solu- 
 tion in water as a solvent for the proteid crystalloids. 
 The 10 per cent, solution is used for plasmolysis. 
 
 Normal Fluid 
 (Normal Salt Solution, Physiological Salt Solution). 
 
 Dissolve 6.75 grammes of sodium chloride in one litre of dis- 
 tilled water. 
 
 Gag'} recommends the following: — "White of egg 15 cc, water 
 200 cc, mercuric chloride .05 gram., common salt (NaCl) 4 
 grammes. This mixture should be thoroughly shaJfen in a bottle 
 with bits of glass to mix the albumen with the water. It should 
 then be filtered and kept in a cool place. The mercuric chloride 
 (corrosive sublimate) retards decomposition vdthout being in- 
 jurious to white blood corpuscles or ciliated cells." 
 
 Ferrous Sulphate. 
 Used in dilute solution in water, to which a drop of nitric acid 
 has been added^ as a test for tiannin. 
 
 Potassium Bichromate, 
 
 Used in dilute solution in water as a test for tannin, which it 
 colors dark brown; used also (in 1 per cent, aqueous solution) 
 for hardening tissues, also in a weak solution (1 part to 800 of 
 water) as a dissociating fluid. 
 
 Potassium Chlorate. 
 
 Used, together with nitric acid, as a macerating a'gent. 
 
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76 
 
 Nitrate of Silver. 
 For an interesting account of the reactions of living matter to 
 very dilute solutions of certain substances whicli are poisonous 
 when used in greater strength, see Leow and Bokomy. These 
 investigators use a dilute alkaline solution of argentic nitrate 
 in the discrimination between living and dead protoplasm; upon 
 application of the reagent the former turns black, the latter 
 remains uncolored. The solution is made by mixing 1 cc. of a 
 1 per cent, solution of the nitrate in distilled water A\ith an equal 
 amount of a solution containing 13 parts of potassic hydrate so- 
 lution, 10 parts of ammonia, and 77 parts of distilled water. 
 
 Chloride of Mercury (Corrosive Sublimate). 
 Mercuric chloride, or Corrosive sublimate, dissolved in fifty 
 parts of absolute alcohol, renders protein grains insoluble in 
 waterj Pfeffer recommeniis that the specimen should remain 
 in this reagent at least twelve hours. Dippel uses a dilute aque- 
 ous solution (1 in 500) to render visible the currents in the most 
 delicate threads of protoplasm (and for the demonstration of the 
 nucleus without affecting the other contents of the cell). 
 
 Copper Sulphate. 
 Used in very dilute solution in water; the blue color of the 
 solution must be only just perceptible. It is used, with potash, 
 as a test for some kinds of sugar, and for proteids. It is used 
 also in the preparation of ammoniacal solution of cupric hydrate, 
 which dissolves pure cellulose. 
 
 Fehling's Fluid. 
 For the preparation of Fehling's fluid the foUowinig directions 
 are given in Foster's "Practical Physiology:" 
 
 (a) Dissolve 34.65 grammes of pure crystallized cupric sul- 
 phate in about 160 cc. of distilled water. 
 
 (b) Dissolv-e also 173 grammes of pure crystallized potassic- 
 sodic taj."trate in 600 to 700 grammes of sodic hydrate, sp. gr. 
 1j12. Add (a) to (b) stirring well to cause a thorough mixture, 
 and dilute with distilled water to a litre. 
 
 Fehling's fluid should be fresh made when it is required, since 
 it decomposes on keeping; it will keep some little time if kept 
 in a cool place in the dark, and in completely filled, well-stop- 
 pered bottles.' — (Hoppe — Sevier.) 
 
 The solution (b) may be prepared, and kept for adding to (a) 
 freshly prepared when required. 
 
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 Before using a kept solution to test for sugar, always boil a 
 little of it by itself to see if any reaction will take place. 
 
 From 1 cc. of this solution the copper is completely reduced by 
 .005 grammes of gi-ape sugar. 
 
 Acid Nitrate of Mercury. 
 
 Millon's reagent, commonly called acid nitrate of meicury, 
 is best prepared, according to its discoverer, by pouring upon 
 pure mercury its own weight of concentrated nitric acid. For a 
 short time the action is violent; when it subsides a little, gentlj' 
 warm the liquid until the metal is completely dissolved. The 
 solution is immediately diluted by twice its volume of pure 
 water. After a few hours the liquid is to be decanted from the 
 crystalline mass which has formed, and it is then ready for use. 
 
 This reagent is more efficient when freshly made. 
 
 Albuminoid substances are colored red by this reagent when 
 in the cold, but much more readily upon the application of heat. 
 According to Millon, the reaction is due to the presence in the 
 liquid of both mercuric nitrate and nitrite. 
 
 This reagent has been employed for the demonstration of the 
 stratiflcation aud spiral striation of certain cell-walls.' — (Goodale.) 
 
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OUaANIC SUBSTANCES. 
 
 Alcohol. 
 
 Used as a solvent for various substances, such, as fats, oils, 
 resins, coloring matters, etc., and as a precipitate for various 
 substances. It has a peculiar action upon some proteid crystal- 
 loids. 
 
 "The air which occurs in intercellular spaces and in all dry 
 specimens is g'enerally removed with ease by the action of 
 alcohol, especially if a little heat is applied. 
 
 Alcohol is of use also in the preparation of some of the stain- 
 ing agents. 
 
 Absolute alcohol contains only the slightest trace of water. 
 Hence it must be used instead of ordinary alcohol whenever the 
 specimen is affected by water, as in the case of mucilaginous 
 tissues, crystalloids, etc. As a reagent for use under the cover 
 glass it is more satisfactory than common alcohol, but in keep- 
 ing it the greatest care must be exercised to exclude moisture. 
 
 Glycerin. 
 Only the purest glycerin should be employed in microscopical 
 examinations. The following are the most important of its many 
 applications : h In clearing specimens. 2. To cause* with- 
 drawal of water from fresh cells, the degree of effect depending 
 on the strength of the glycerin. 3. In the exandnation of pro- 
 tein granules. 4. As a test for inulin; this substance separates 
 sooner or later in the form of sphaerocrystals. 5. As a solventj 
 for iodine." — (Goodale.) 
 
 Ether. 
 
 Used as a solvent for wax, fats, resins, etc. ' 
 
 Cane Sugar. 
 The concentrated aqueous solution is used, together with strong 
 sulphuric acid, as a test for proteids. The thick sii-up is allowed 
 to act for some time on tissues containing protoplasm; a drop of 
 concentrated sulphuric acid is then placed on the object, when 
 th.e protoplasm will take on a faint rose-red color. The reaction 
 is uncertain. 
 
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79 
 
 A dilute (1 per cent.) solution is useful for mounting living cells 
 for obseryation under tlie microscope. 
 
 Alkanet. 
 
 The alcoholic extract, or, better, an alcoholic solution of al- 
 kanin, is used as a test for resin and caoutchouc. A fresh solu- 
 tion must be prepared on each occasion. 
 
 A simple method is that recommended by Strasburjger. Wash 
 a thin chip of alkaaet root in water to remove dust, place it with, 
 the preparation, and run dilute alcohol under the cover glass. 
 
 This, when applied to a thin section of a plant containing resin, 
 colors the resin drops a deep red in two or three minutes, but 
 does not serve to distinguish one resin from another. The col- 
 ored drops are dissolved in strong alcohol. 
 
 Alkanet tincture also stains protoplasm a pale rose-red, from 
 a quarter to half an hour being needed. It is often used to iden- 
 tify the ground substance of oil-containing seeds. 
 
 Phloroglucin. 
 
 Used in alcoholic or aqueous solution as a test for lignin. The 
 section is first treated with hydrochloric acid and then with a 
 solution of phloroglucin : the ligniiied cell-walls assume a bright 
 red color. 
 
 If phloroglucin cannot be obtained, it may be replaced bj' an 
 extract of cherry wood. ' 
 
 Twigs of cherry, rejecting the thin green parts, are cut up 
 into thin shavings, steeped in absolute alcohol for twenty-four 
 hours, to remove the chlorophyll as much as jtossible Tl'.e 
 shavings are now shaken free of the discolored alcohol and 
 steeped in a new supply, and allowed to remain for several days, 
 being frequently stirred. This Huid is then tiltered, and evapor- 
 ated down until a fragment of very coarse unbleached blotting 
 paper moistened wita it, and subsequently with hydrochloric 
 acid, quickly becomes a deep violet color. The residual fluid thus 
 obtained is brown and smells like camphor. Preserve in a well- 
 closed bottle. Reagent for lignin. 
 
 It gives a violet color to lijjnifled cell-walls, as it contains 
 other substances (especially pyrocateclun) besides phloroglucin. 
 — (IStrasburger.) 
 
 This is used in an aqueous solution. The specimen, subject to 
 the action of the solution for a few minutes, is transferred to sul- 
 phuric acid of specitic gxavity 1.2 (made by adding 1 part of cou- 
 
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80 
 
 centrated acid to 4 parts of water). Lignifled structures become 
 reel. — (Goiulalo.) 
 
 Phenol (Carbolic Acid). 
 Used, together with, hydrochloric acid, as a test for lignin. The 
 best preparation of it is its solution in hydrochloric acid. This 
 is prepared by dissolving carbolic acid in warm hydrochloric 
 acid, adding, whilst the mixture is cooling, sufficient hydro- 
 chloric acid t5 dissolve any precipitate that may be formed. 
 Lignifled cells, treated with this mixture and exposed to sun- 
 light, assume a bright greenish yellow or blue-green color in 
 consequence of the presence of coniferin. — (B and V.) 
 
 Aniline Chloride. 
 
 This salt is used as a test for lignin in cell-walls. It may be 
 used in solution either in water or alcohol, but the alcoholic solu- 
 tion gives the best results. The section is first treated with the 
 solution and then with the sulphuric or hydrochloric acid respec- 
 tively, or, better, the solution may be kept slightly acidulated by 
 one or other of these acids : the lignified cell-walls assume a. 
 blight yellow coloi-. — (];. anci V.) 
 
 Schultz's Reagent (Chlor-iodide of Zinc). 
 
 The simplest test for cellulose; cellulose cell- walls turn a blue 
 color inclining to purple when treated with this mixture; corky 
 and lignifled cell-walls turn yellow, protoplasm brown and starch 
 blue. 
 
 It is prepared by dissolving zinc in pure hydrochloric acid, and 
 evaporating the solution, on a Avater bath, in th.e presence of 
 metallic zinc until it has a sirupy consistence; it is then saturated 
 with potassium iodide, and then with iodine; a few grains of 
 iodine should be left in the liquid after it is poured for use. It 
 may also be prepared by dissolving 25 parts of pure fused zinc 
 chloride and 8 parts of potassium iodine in 8i parts of water, 
 filtering through asbestos and saturating with iodine. 
 
 On adding iodine to Schultz's solution till precipitation begins 
 a fluid is obtained which stains the cell- walls yellow, and the 
 callus of sieve-plates a deep bro\\Ti. — (Russow.) 
 
 Ammoniacal Cupric Oxide (Schweitzer's Reagent). 
 This reagent has the power of dissolving pure cellulose. Oxy- 
 hydrate of copper is carefully precipitated from the sulphate by 
 
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 a dilute solution of ammonia; the clear gi'een precipitate, sepa- 
 rated and washed, is added while still moist to strong ammonia, 
 in which, on slightly warming, it is dissolved. Upon cooling, 
 crystals of sub-sulphate of copper and ammonia fall to the bot- 
 tom. The filtered liquid contains only the ammoniacal cupric 
 oxide in solution. It must be kept in bottles of dark glass, or 
 in the dark.' It can also be prepared by digesting copper turn- 
 ings in an open bottle with liquor ammon. of the pharmacopoeia. 
 As it is very easily decomposed by light, it is perhaps best pre- 
 pared fresh when required. It is fit for use only so long as it 
 rapidly dissolves cotton-wool. — (Strasburger.) 
 
 This reagent possesses its chief interest from the fact that it 
 is the only liquid known in which cellulose appears to dissolve 
 without essential change in composition. It has a limited appli- 
 cation in the discrimination of the fibers used in the arts. 
 
 ON THE CULTIVATION OF THE LOWER 
 ORGANISMS. 
 
 For the study of the life history of bacteria, molds, mosses, 
 ferns, etc., various media are employed that furnish pabulum 
 suited to the case in hand, free from extraneous, harmful matter. 
 
 For the perfecting of this method of investigating bacteria 
 we are chiefly indebted to Dr4 Eobert Koch. In it we have a plan 
 by which we can select from the numerous bacteria found in a 
 diseased body, or in a putrifying or fermenting mass, those forms 
 which are the cause of the change; 
 
 The mucous surfaces of the body are always in healthy animals 
 the home of numerous bacteria: in a diseased animal, how are 
 we to find the potent factor of the morbid condition, how isolate 
 it from the forms which are comparatively innoxious ? Or, if 
 our attention is called to some milk which has turned blue, as 
 it sonaetimes does through the agency of bacteria, how are we to 
 select from the myriad of living forms, filaments, rods, ^c. — ^iu 
 short, all the forms which may have entered the milk from the 
 air of the stable or dairy — the one which has done the mischief ? 
 That this can be done seems scarcely possible; and yet it is not 
 so ditacult, providing proper care is exercised in the process 
 employed. 
 
 For instance, in the case of the blue milk, we take a' small 
 quantity of the milk (enough to stick to the point of a cambric 
 needle); this we mix with about a teaspoonful of properly pre- 
 pared gelatine, which becomes fluid al^ 35° C. In this melted gela- 
 6 
 
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 tine the {e^\- microbes which we have taken up on the needle be- 
 come scattered, and it is poured out on a flat surface, properly 
 protected from the air, and in twenty-four to seventy-two hours 
 we obtain from each microbe a separate pure culture — here a 
 mold-like patch, here a porcelain-like fleck, here a red, there a 
 gray or blue — all jjure cultures; We may now take from each of 
 these a particle, as we did from the milk, and make a new sowing 
 in fresh gelatine, and by acting upon some pure, fresh milk with 
 the various pure cultures, we find at last that those bacteria 
 belonging to the bluish patch are the special agents in the case. 
 After this plan of procedure — ^but not, of course, in so simple a 
 way — did Koch work out the " comma bacillus " of cholera, the 
 bacillus of septicaemia in mice, and others, the specific feerms 
 of pyaemia, erysipelas, pneumonia, etc. "It is only by such 
 monosporous cultivations," writes Prof. Lankester, "that Ave 
 can arrive at solid conclusions in reference to the forms and 
 activities of bacteria, e.g., as to whether one form can give rise 
 to progeny of another form when its food and conditions of 
 growth are changed; and again, as to whether fermentative pow- 
 ers can be lost or acquired in the course of generations derived 
 from one parent germ, but subjected to different conditions as 
 to food, temperature and oxygen. The method of gelatine culti- 
 vation devised by Dr. Koch places the means of following out 
 these inquiries in the hands of every microscopist." 
 
 Again, Bienstock, in connection with the statement that it is 
 impossible to establish the identity of two micro-organisms sim- 
 ply from the resemblance of their morphological characters, says : 
 "In bacterial observations, culture, and not the microscope, is 
 the important point. The microscope is principally only an 
 accessory checking apparatus. It gives exact information only 
 in the study of morphology; it is an uncertain guide, and, in 
 physiology, generally of no use at all." 
 
 Although many microbes seem to flourish in a solution of in- 
 organic salts, it is foimd preferable to have the medium in which 
 the cultures are carried on, rich in organic materials, containing 
 at the same time some inorganic salts. Successful cultures de- 
 pend upon the iise of a completely sterilized nourishing me<iiumi 
 and a pure sowing. These media may be either of a solid or 
 liquid character, each having its own particular advantage and 
 use. No medium can be prepared, however, A\hich will germin- 
 ate all forms of schizomycetes indiscriminately; e.g., micro- 
 coccus ureae will not gemiinate in broths, but in urine, while 
 
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the microbes of chicken cholera will (lie within forty-edght 
 hours if placed in a decoction of beer yeast, which forms an 
 excellent culture fluid for other forms, especially for filth, 
 bacteria. 
 
 A.— FLUID CULTURE MEDIA. 
 
 Broths made from all kinds of flesh, hare been used by various 
 investigators; these are freed from fat, neutralized, filtered and 
 sterilized. Decoctions and infusions of vai'ious vegetal substan- 
 ces are used under special circumstances. Sterilizing tlie culture 
 medium is accomplished ordinarily by the use of heat sufficient 
 to kill all germs oriully developed bacteria ia the fluid. It may 
 be accomplished ia bulk, and then transferred to sterilized cul- 
 ture vessels, or it may be placed in these, hermetically sealed, 
 or simply stopped with a plug of cotton, and then sterilized. 
 Solid or gvlatine culture media are made by adding to any of 
 the fluid culture media sufticient (5 to 10 per cent.) pure gelatine, 
 cr, preferably, -Japanese &ea-A\'ee<l glue, "Agar-agar," to give a 
 mass that will become fluid at a low temperature — e.g., 30 C. 
 
 Koch refers to culture media as follows : "In the ordinary cul- 
 ture of bacteria in a firm medium, the single germs must be 
 placed as far as possible from each other, in order that they may 
 develop entirely apart. The material containing the bacteria is 
 therefore placed in gelatine nourishing medium ^hich has been 
 made fluid, and in this they are distributed as much as possible, 
 and then poui'ed out, with the gelatine upon them, upon a glass 
 plate; the gelatine hardening quickly, the separate bacteria scat- 
 tered through it are fixed apart from each other, and each germ 
 can go on developing and multiplying until it becomes a mass of 
 IDure culture, visible to the naked eye, without being disturbetl 
 by or mixed with other kinds. The principle of the method is 
 to develop entire colonies from single individuals. It is much 
 more diJfioult to separate a number of different lands of microbes 
 on the surface of potato than by the gelatine process. In most 
 cases the separation of pathogenic from non-pathogenic bacteria 
 upon potato cannot be accomplished, because the bacteria of 
 filth igrow so much more luxuriantly on potato that they soon 
 overcome all others. Potato is therefore used as a nourishing 
 substratum for bacteria only when these have been obtained 
 from pure cultures, and it is desirable to know whether they 
 will subsist on vegetable diet or not. 
 
 The advantage of gelatine cultures consists in the ease with 
 
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 ■which different kinds of bacteria can be isolated and studied 
 through their various phases of development. When a test-tube 
 is used as a culture vessel for gelatine media, the mass should be 
 allowed to cool while the tube is inclined at an angle of 45; it 
 will thus, when stiffened, present a much laiiger surface for in- 
 oculation. Thus, after the sowing instrument has been drawn 
 over the firm surface from bottom to top, there will be a number 
 of colonies spring up along the line of sowing, and from any one 
 of these, material may be taken for new pui-e cultures. 
 
 Gelatine offers a good medium for the study of the develop- 
 ment of bacteria, since the germs may be sown in it when fluid 
 and then spread in very thin layers on glass slides, -nhich are 
 kept under a bell glass, and can be studied as they are with low 
 powers, or with immersion lenses by placing a thin cover glass 
 over the colony to be investigated. The great objection to gela- 
 tine culture media is that they cannot be retained firm at those 
 temperatures best adapted to the gi'owth of bacteria, 30 to 37 ° C. 
 A substitute which fulfills this requirement is Koch's Serum 
 Culture Medium. Serum of an ox or sheep is rendered as pure as 
 possible, and placed in test-tubes closed with cotton and rubber 
 cloth so that they are water-tight. These are for six days heated 
 daily for one hour at 58 ° C, by which process the serum is in 
 most cases completely sterilized. It is then warmed at 65° C, 
 until it becomes stiff and firm. After this treatment the serum 
 appears as an amber yellow, completely transparent or only 
 weakly opalescent, firm, gelatinous mass, and after several days 
 in the breeding oven must show no signs of developing bacteria 
 colonies. If the heat employed in stiffening the serum is over 75° 
 C, or lasts too long, the serum becomes opaque. To furnish a 
 large surface for culture, the serum is allowed to stiffen while the 
 test-tube is in an inclined position. For such cultures as are to 
 be studied under the microscope, the serum is allowed to stiffen 
 in a watch glass or a hollovi'ed-out slide. Upon this stiffened 
 blood-serum, which forms a firm nourishing medium at the tem- 
 perature of the breeding oven, the material to be investigated is 
 placed, and the whole kept at 37° or 38° C, until development 
 takes place, which in the case of the tubercle bacillus is from 
 twelve to fourteen days. Instead of blood-serum, Miquel's 
 lichen jelly is convenient, as it can be rendered fluid again, if 
 necessary, which is not the case ^^'ith blood-serum. 
 
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 Culture Vessels. 
 
 Ordinarily simple test-tubes closed with asbestos wool or with 
 cotton are used, but if it is desirable to be especially particular, 
 take a conical-shaped flask of goo<i glass with a flat bottom, also 
 a piece of glass tubing of a diameter smaller than the neck of the 
 flask and somewhat longer. This tube must be slightly pointed 
 at the lower end. Pack the tube three-quarters full of asbestos, 
 and upon the top of this place a pad of cotton; wrap the tube in 
 some cotton batting and press it firmly into the neck of the flask. 
 This apparatus may now be heated up to 200° C, until thor- 
 oughly sterilized. 
 
 To Sow the Microbes in the Culture Medium. 
 
 Only such instruments are to be used as have been sterilized 
 by heat^ Since hot instruments may kill the bateria, and, if al- 
 lowed to cool in the air, may be again contaminated, a plan has 
 been recommended whereby a number of cool sterilized wires 
 may be kept on hand. Take an ordinary test-tube and plug it 
 with asbestos, and pass through this plug a number of small 
 pipettes, each of which is. stopped with an asbestos ping, through 
 which passes in each a piece of platinum wire. Now sterilize 
 the whole tube in an oven. As required, one of these cool steri- 
 lized pipettes and wires may be removed and used. Thrust one 
 of these wires into the tissue the microbes of Avhich it is desired 
 to study, draw it out, and in the meantime having thrust one of 
 the sterilized pipettes through the asbestos pad into the culture 
 vessel, pass the wire through it into the culture fluid, gelatine or 
 blood-serum, as the case ma^' be, and the sowing is completed. 
 If the sowing is to be made from a liquid instead of a tissue, take 
 one of the sterilized pipettes without the wire, and, placing over 
 it a rubber cap, draw up some of the fluid and pass through 
 the asbestos pad as before. Then replace the cotton above the 
 asbestos. 
 
 To carry on the culture, it is best, with fluid media, to keep 
 them at a temperature of 35 ° 0., in a breeding oven. This is also 
 the plan for serum cultures, but gelatine must remain in the room 
 at ordinary temperatures, at which only do they remain firm. 
 Otherwise we should lose the advantages gained by the addi- 
 tion of the gelatine. 
 
 Where it is not necessary to exercise great precautions against 
 conta.mination, as in growing the various pigment bacteria, cul- 
 tures may be carried on under bell glasses by sowing the bacteria 
 
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 upon slices of boiled potato, turnip, on es;gs, or any similar nour- 
 ishing substances, as the gelatine or Agar-agar masses on glass 
 slides or in watch glasses. Place a piece of moistened filter 
 paper upon a plate, and another piece in the dome of the bell 
 glass; then place the vegetable, egg or gelatine Avith its bacteria 
 on tlie plate, cover and keep in a moderately warm place. The 
 colonies, especially in the case of pigment bacteria, will soon 
 make themselves visible to the naked eye. 
 
 Method of Cultivating Bacteria on the Slide. 
 
 If it is desired to follow the process of development of a form 
 directly under the microscope, it is done with the aid of small 
 m'oist chambers. 
 
 In the center of a glass slide is fastened, by means of Canada 
 balsam, a glass ring from 4 to 5 nun. thick, cut from a tube used 
 for organic analj^sis, and the cut sides properly ground level. 
 A thin cover glass, round, and of sufficient diameter to just cover 
 the ring without lapping the edge, is fixed on the upper side hj 
 three very small drops of greasy oil, to complete the cell.' In 
 order that the interior air may be always saturated by moisture, 
 a few drops of water are placed on the bottom of the cell. The 
 thinnest possible layer of the gelatine. Agar-agar, or blood- 
 serum is placed at the center of the under surface of the cover 
 glass, and this layer is afterward inoculated. 
 
 Permanent preparations can be made of these cultures at any 
 time by removing the covers and fixing the bacteria or molds 
 by the fumes of osmic acid, staining and mounting. 
 
 The following culture media have been found satisfactory : 
 
 1. Distilled water, 100 ccm. 
 White rock candy, 10 gr. 
 Taiirate of ammonium, 1 gr. 
 Phosphate of lime, 0.5 gr. 
 
 2. Liebig's extract of meat, 1 part. 
 Water, 100 parts. 
 
 3. Gelatine, 3 parts. 
 
 Pliosphate of aiiunoniuur, 25 parts. 
 AA'ater, 100 parts. 
 
 4. Meat extract, 1 part. 
 Sugar, 3 parts. 
 Water, 100 parts. 
 
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 5. Liebig's extract, 10 parts. 
 Peptone, 8 parts- 
 Water, laOO parts. 
 
 Pasteur's Fluid. 
 
 6; Distilled water, 100 parts. 
 Cane sugar (pure), 10 parts. 
 Ammonium tartrate, 1 part. 
 Ash. of yeast, 1 part. 
 
 The following is the simplest way of preparing Pasteur's fluid: 
 Keep dry in a bottle, ready mixed and finely pulverized, 20 
 grammes Potassium phosphate, 2 of Calcium phosphate, 2 of 
 Magnesium sulphate and 100 of Ammonium tartrate. For use, 
 dissolve in the proportion of 1 gramme of this mixture with 12 
 of sugar, in 70 cc. oif water. — (Strasburger.) 
 
 Cohn's Fluid. 
 7. Distilled water, 100 ccm. or 200 parts. 
 Ammonium tartrate, 1 gramme or 20 partsi 
 Potassium phosphate, 0.5 grammes or 20 parts. 
 Magnesium sulph. cryst., 0.5 grammes or 10 parts. 
 Calcium phosphate (tribasic), 0.5 grammes or 0.1 part. 
 
 Fluids lacking albuminoids are not suitable for the cultivation 
 of pathogenic organisms. In making broths, select as fresh meat 
 as possible, allow half an hour's boiling for each pound, and 
 calculate to have a sufficient water to yield, ultimately, one pint 
 of broth to each pound. When boiled, allow to cool, skim off 
 fat, and neutralize with carbonate of sodium, filter through, a 
 sterilized filter into sterilized flasks. If the broth is not clear 
 after once filtering, repeat; and if, upon standing, -a sediment 
 appears, decant or boil with the white of an egg and filter. 
 
 B.— SOLID CULTURE MEDIA. 
 
 (a) Miquel's Nutritive Paper. 
 Pour upon a large sheet of black or gray paper, boiling lichen 
 jelly, and spread it out uniformly, so that in the wet state it has 
 a thickDess of 2 to 3 mm. This done, dry it rapidly in an oven, 
 at 40°C. In a few 'hours the sheet is dry. It is thin, flexible, it 
 never presents cracks, and resembles altogether the photo- 
 graphic paper covered with bromo-gelatine emulsion, indented 
 
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by Dr. Maddox. This nutritive lichen jelly paper may be pre- 
 served indefinitely m ith all its qualities, it being only necessairy 
 to place it in a dry drawer. At the moment of makng the ex- 
 periment the paper is sterilized by being suspended in an atmos- 
 phere of vapor at 110° C. This is accomplished without its run- 
 ning, and there is scarcely any swelling. 
 
 (6) Miquel's Lichen Jelly. 
 
 Digest in one litre of beef broth 25 to 30 grammes of Irish 
 moss (Chondrus crispus), and pass the resulting decoction 
 through a sieve which retains the swollen leavesj After neutral- 
 ization and a short boiling, filter the broth through bolting cloth. 
 Upon cooling it forms a strong jelly. To obviate the loss of 
 broth which remains in the swollen leaves, Miquel makes in his 
 laboratory a lichen jelly with water; this he dries and adds to 
 broth in proper proportions, about 1 per cent. This nutritive 
 jelly possesses the following advantages: (1) It melts only be- 
 tween 55 and 60°C., which permits of the cultivation of such 
 organisms as require for their development elevated tempera- 
 tures. Ordinary nutritive gelatines melt before 30° C. (2) It re- 
 mains without alteration or losing its power of solidifying when 
 exposed to a temperature of 110 °Cj for rigorous sterilization. 
 Gelatine, on the contrary, is reduced under such conditions to a 
 turbid broth, which remains fluid on cooling. 
 
 (c) Blood- Serum (Koch). 
 Blood of a healthy sheep is drawn from the carotid artery, by 
 ineans of a canula into a flask, with which it is connected by 
 means of a rubber tube, the whole apparatus being first thor- 
 oughly sterilized. After standing twenty-four hours, or until a 
 firm clot has formed, the serum is drawn off by a sterilized glass 
 siphon, one end of which passes through the cotton plug in the 
 flask containing the blood, and the other through the plug in the 
 vessel intended for the reception of the serum. The serum is 
 then sterilized, and rendered solid by exposing it in properly 
 protected sterilized test-tubes to a heat of 32 to 38° C. for several 
 weeks. If prepared in this way the limpidity is not lost. 
 
 (d) Agar-agar. 
 Obtained from Euchema Speciosum ; it is used in the East in 
 place of ordinary gelatine for preparing soups and jellies- It 
 bears, without liquefying, higher temperatures than ordinary 
 gelatine. 
 
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 This substance occurs in commerce in bunches of thin,' 
 shriveled transparent strips. It is prepared for use by soalting 
 over night in salt water (1 to 6), and then dissolved by the aid of 
 heat. After being filtered and neutralized, it is mixed with some 
 nutritive material, 1 per cent. Agar-agar to the nutrient fluid. 
 Klein says that among all solid media he finds a mixture of A gar- 
 agar and peptone the best. He prepares it by mixing peptone 
 with the filtered Agar-agar solution, boils it repeatedly foir 
 thirty minutes at a time, and finally obtains a sterile transparent 
 mass, which remains solid up to 45 to 50° C.' It becomes liquid 
 at higher temperatures, and, in case of necessity, may be again 
 subjected to boilings Before considering it as perfectly sterile, it 
 ought to be kept, like all other materials, for from several days 
 to several weeks in the incubator, 32 to 38° O. If quite limpid 
 after this time it may be considered as sterile. 
 
 (e) Gelatine. 
 The best gelatine is cut up into strips and soaked in distHled 
 water over night (1 part of gelatine to 6 of water), and is then 
 melted, thoroughly neutralized and filtered through bolting 
 cloth. If it is not clear, it is boiled with some white of egg and 
 again filtered. The fluid gelatine is then mixed with half its 
 bulk of broth, peptone solution, or beef-broth solution, so that 
 'there is 1 part of gelatine in 9 parts of fluid, or 11 1-9 per cent, of 
 gelatine. This mixture is boiled repeatedly, and treated like 
 fluid culture media, as described above. It may be kept on hand 
 as a stock, either with the ad(Jition of peptone, etc., or as a simple 
 gelatine which can be added to any particular nourishing mate- 
 rial when desired.' 
 
 Method for Cultivating the Spores of Ferns. 
 
 Sow the spores on a piece of mo<ierately soft tile, laid in water 
 in a saucer, or kept upon a flower-pot or flower-pot saucer simi- 
 larly kept constantly moist. In a room it may be covered over 
 with a bell-globe. In this way all the early stages of develop- 
 ment can be well obtained, and it needs only to scrape off some 
 of the germinating spores day by day with the blade of a pocket 
 knife, and lay them in water or on the object slide, to be able to 
 follow the development. For full-grown prothallia for section- 
 cutting the spores can be well sown on a bed of cocoanut fiber 
 refuse, flattened down in a large flower-pot saucer, with a hole 
 in the bottom, or a seed-pan, and well drained, kept moist until 
 
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 toward the time they are needed for examination. Over-head 
 watering, if needed, can be given with a spray. 
 
 The quickest fern spores for cultivation are those of the royal 
 fern, Osmunda regalis.- — (Strasburger.) 
 
 Method for Cultivating Fresh Water Algae. 
 If onoe in possession of good Spirogyra material, you should 
 endeavor to preserve it in cultivation. This is effected best in 
 comparatively shallow vessels, whose walls are either opaque 
 or ai'e made opaque by means of black paper, as light falling uni- 
 laterally acts disadvantageously. The vessels must stand in a 
 light place, but be protected from the direct action of the sun. 
 Into either rdver or spring water, which is not too rich in chalk 
 (too haird) are thrown from time to time boiled pieces of turf 
 soaked in nutrient fluid. This nutrient fluid "ndll be prepared 
 suitably if we add to 100 cc. water 1 gramme nitrate of potash, J 
 gramme sodium chloride, i gramme sulphate of lime, J gramme 
 sulphate of magnesia, J gi'anune finely pulverized phosphate of 
 lime (of this last salt only a trace is soluble.) Under such cir- 
 cumstances the Spirogyra, and fresh-water algae in general, 
 thrive well. — (Strasbui-ger.) 
 
 Method for Cultivating Molds (Mucor). 
 
 If a piece of damp bread is placed under a glass bell- jar, it is 
 covered, even in a few days, with a thick felt of fungus threa<ls 
 (Mycelium), which almost always belongs to Mucor Mucedo, one 
 of the Phycomycetes. This fungus soon shows itself very lux- 
 uriantly upon fresh dung, kept in a closed moist chamber. 
 
 In dung-cultures the fungus occasionally forms zygotes (zygo- 
 spores), which present themselves as dark points. They can 
 usually be forced into the formation of zygotes (zygospores) in 
 the months of March and April, if the spores are sown in fresh, 
 flattened-out horse-dung. The zygotes are ready in fi'om eight 
 to fourteen days. At other times, in order to obtain the zygotes, 
 it succeeds well if the sowing is made in some drops of concen- 
 trated plum-juice, sterilized by long boiling, and then mixed with 
 10 to 20 per cent, of alcohol (not methylated). The sowing is 
 made on a cover glass in a damp chamber constructed of a glass 
 ring, and the object slide placed in the large plaster-of-Paris 
 moist chamber. — ,Strasburger.) 
 
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 Method for Developing Pollen Tubes to Show Proto- 
 plasmic Movement. 
 In a from 3 to 30 per cent, sugar solution, wMch contains 1 to 
 
 per cent, gelatine, most pollen grains put out tubes, in which, 
 protoplasmic movement is beautifully seen. The formation of 
 tubes takes place quite certainly and rapidly in 5 per cent., solu- 
 tion of sugar and 1 to 5 per cent, gelatine from the pollen grains 
 of Paeonia staphylea, and even fi*om Tradescantia when the 
 pollen grains are taken from freshly opened flowers. The most 
 valuable objects are perhaps species of LathjTus (e.g., sweet 
 pea, everlasting pea, etc.), in 15 per cent, solution of sugar and 
 
 1 to 5 per cent, gelatine. This solution must be freshly prepared ; 
 the sowing is best performed in a suspended drop in a moist 
 chamber.- — (Strasburger.) 
 
 Bern's Method of Reconstructing Objects from Micro- 
 scopic Sections. 
 
 Dr. G. Born is the originator of the following ingenious 
 method of constructing models of objects from serial sections. 
 By the aid of the camera, the outlines of the sections are trans- 
 ferred to wax plates; which are then cut out so as to correspond, 
 in outlines as well as dimensions, to the sections equally magni- 
 fied in all three directions. With plates thus prepared, it is only 
 necessary to put them together in the proper order to obtain a 
 complete model. The method is simple and extremely useful, 
 especially in investigating objects with complex internal 
 cavities. 
 
 An illustration of the method. — Use is made of three rect- 
 angular tin boxes of equal size, each measuring 270 mm. + 230 
 mm. + 2J mm. Sections should be made about -jV mm. thick 
 (never thinner than ^V mm). If we desire to construct a model of 
 an object from serial sectianSjgV^Hi. thick, which shall be magni- 
 fied 60 diameters, then the wax plates must be made sixty times 
 as thick as the sections, i. e., 2 mm. thick. 
 
 The surface of a plate, that could be made in a box of the 
 above-named dimensions, contains 62,100 G mm. and the volume 
 of such a plate 2 mm. thick would therefore be 124.2 ccm. The 
 specific gravity of common, raw bees-wax amounts to .96 — .97. 
 For use, it requires only to be melted and a little turpentine 
 added to make it more flexible. Thus prepared, its specific grav- 
 ity is about .95; and this number has been found sufficiently ac- 
 curate in all cases. The weight of the wax required to make one 
 
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 plate of the size just stated will, accordingly, be 117.99 gr., or, in 
 round numbers, 118 gr. The wax having been weighed and 
 melted, the tin box is first filled 1.5 cm. deep with boiling water, 
 and the melted wax poured upon the wafer. If the water and 
 wax are quite hot, the wax will generally spread quite evenly 
 over the surface; if gaps remain, they can be filled out by 
 means of a glass slide drawTi over the wax. As soon as the 
 plate has stiffened, and while it is still soft, it is ^\ell to cut it 
 free from the -nails of the tin box, as further cooling of the water 
 and the box might cause it to split. By the time the water be- 
 comes tepid, the plate can be removed from the water to some 
 flat support, and left till completely stiffened. Fifty plates 
 may thus be ja-epared in the course of a few hours. The out- 
 lines of the section are transferred to the plate in the following 
 manner: A piece of blue paper is placed on the plate, with the 
 blue siiie turned towards the Avax, and above this is placed a 
 sheet of ordinary drawing paper. The outlines are dra'W'n on 
 the latter by the aid of a camera, and at the same time blue out- 
 lines are traced on the wax plate. The plate can then be laid on 
 soft wood and cut out by the ai<l of a small knife; Thus a draw- 
 ing and a model of each section are prepared. The plates, when 
 finished, can be put together in the proper order, and fastened 
 by the aid of a hot spatula apydied to the edges. — (Amer. Nat., 
 1881.) 
 
 Models in Metal of Microscopical Preparations (Salenka). 
 To obtain a plaster representation of the brain of a vertebrate 
 embryo, for example, the outlines of the head, the external and 
 internal boundary lines of the brain are drawn on paper, from 
 specimens, with a camera lucida. According to the size of the 
 separate sections, every second, third or fourth is selected, the 
 drawings are numbsi ed, and then carefully stuck on cardboard 
 of the necessary thickness; the reverse side of the cardboard is 
 covered with glue. The separate figures are then carefully cut 
 out. Small strips for joining must then be left in the brain. The 
 different layers of cardboard are then glued together in their 
 proper order, and thus a case model of the head is obtained. Any 
 gaps or seams on the surface are filled in with plaster of Paris, 
 and then the hollow model, which is open behind, is filled with 
 Wood's metal, heated to about 75 ° C. When cool, the cai-dboard 
 is softened in luke-warm water, and then stripped off. The 
 model is then cut in two with a fret saw and the internal sur- 
 
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 face of the brain freed from the cardboard. Unevenness of the 
 surface and holes are easily got ri(J of Avith a heated needle or 
 knife, or by touching up with a stick of Wood's metal which 
 has been softened at a gas-jet. It is necessary to leave vent- 
 holes in the cardboard model. — (Jj R. M. S.) 
 
 Semper's Method of Making Dry Preparations for Exhibi- 
 tion or Class-room Purposes, 
 
 This method is especially adapted for the preservation and 
 exhibition of dissections. The animal, e. g., a mouse, is dis- 
 sected so as to show the desired organs. It is then hardened 
 by chromic acid; after hardening, it is removed and thoroughly 
 washed in waterJ It is then transferred from one to another of 
 a series of alcohols, until it reaches 96 per cent, to 98 per cent., 
 the object being to remove all the water. 
 
 Prom the strong alcohol it is transferred to spirits of turpen- 
 tine, where it remains until thoroughly impregnated, after 
 which it is simply dried in the air, and will be found to have a 
 soft kid-like texture, not easily broken. The various organs 
 can now be painted suitable colors and the preparation labeled 
 for use. 
 
 Frozen Sections. 
 
 Very instructive preparations are made by cutting sections 
 of the head or body of any form of animal being studied, after 
 it has been completely frozen. The animal when completely 
 rigid is rapidly cut up into tr-ansverse or other suitable sections 
 with a sharp wide-bladed saw — a rip saw answers very well; 
 each section should be placed upon a glass plate, washed with 
 a gentle stream of water, both surfaces freed from, hair and fat 
 by means of a soft bnish, and then placed in alcohol to harden. 
 Where necessary, the vicera can be secured by pins or needles 
 used as skewers. 
 
 Very instructive and permanent preparations can be made of 
 these sections, by Semper's method. 
 
 Preparation of Bones. 
 
 Free the bones from skin and soft parts, and place them in 
 stone jars or clean barrels, and cover them with clean water; 
 allow them to stand undisturbed, and protected from cold. 
 Change the water on the third day, and again on the tenth, to 
 avoid discoloration. Examine occasionally during maceration, 
 
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 and when the soft parts are found to separate readily, pour off 
 the water and wash with a gentle stream of hot water. Use an 
 old nail or tooth brush to remove the flesh, and, if necessary, 
 employ scissors, forceps, and a dull but smooth-edged knife to 
 clean some parts. After all the soft parts are removed, rinse 
 the bones well with clean water and place them on white paper 
 in a dry room. The vertebrae, ribs, etc., should be kept on a 
 string after cleaning, and the skeleton of each animal should 
 be kept in a separate box, properly labeled. A better and more 
 expeditious method is the liquid soap process. The cleaning 
 is done in this process by heating bones in a dilution of the fol- 
 lowing mixture: lain (or distilled) water, 2000 cc; strong 
 ammonia, 150 cc; nitrate of potash (saltpyter), 12 grammes; 
 hard soap, 75 grammes. 
 
 Remove sldn and soft parts, and place the bones in a vessel 
 containing water, i parts; liquid soap, 1 par-t. Prepare enough 
 of this mixture to completely immerse the bones. Boil in this 
 forty minutes; then pour off the liquid, and add a similar 
 amount prepared in the same way. Boil again for half an hour; 
 and usually the muscles may be removed by the hands, a 
 smooth stick, or a scalpel handle. After removing all the mus- 
 cles that come off readily, replace the bones in the dish, an<l 
 continue the boiling until the soft parts may be readily removed 
 with a nail brush. Care should be taken to avoid losing small 
 bones. 
 
 After all the soft parts are removed, it is usually l>est to boil 
 the bones for half an hour, in a mixture of equal parts of liquid 
 soap and water, to remove the last remnants of giease. Finally, 
 rinse the bones well ^\'ith. clean water, and lay them upon white 
 paper to dry. This is, hy tar, the best metho<i of preparing 
 bones: (1) The liquid soap saponifies the fat and acids in soft- 
 ening conuective tissue, (2) The bones come out white and free 
 from grease. (3) It requires but a very short time to prejjare a 
 skeleton or part of»a skeleton. (4) It is especially adapted for 
 sktills, as tire teeth are much less liable to fall out, and the gel- 
 atinized dental periosteum serves as a cement. (5) There is no 
 danger of blood poisoning (septicaemia). 
 
 To prepare skulls, the same processes are employed as for 
 other bones, care being taken in macerating to lay the slaiU and 
 mandible in the macerating dish, so that the teeth are upper- 
 most; then if the water is changed carefully, the teeth are less 
 apt to fall out and be lost. Teeth that have become loosened 
 
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 may be fastened in place with gelatine, or with white bees-wax. 
 — (After 'VMlder and Gage.) 
 
 Preparation of Natural Skeletons. 
 By a natural skeleton is meant one in which the bones are 
 held together by their natural ligaments. Such a skeleton may 
 be prepared according to either of the processes described above. 
 If the liquid soap process is employed, proceed as follows : boil 
 the bones in the mixture, as directed, until the muscles may be 
 removed without much trouble. It will be necessary, however, 
 to allow the boiling to proceed only to the i^oint where the mus- 
 cles will separate from the bones by using the hands, a smooth 
 stick like a scalpel handle or a dull knife-blade. The ligaments 
 will be found considerably swollen and somewhat softened. The 
 softened surface may be cautiously scraped off; then the prepa- 
 ration should be soake<l for 3 — 6 hours in a saturated solution of 
 arseniate of soda, to poison the ligaments and j^rotect them from 
 Dermestes. Then the part should be arranged as nearly as pos- 
 sible in a natural position, aud fastened with pins or striugs 
 and allowed to dry. The swollen ligaments will shrink very 
 greatly, so that what might have seemed a very imperfect prep- 
 aa'ation when moist will be excellent when dry. When the 
 specimen is dry, rough projections of ligament may be removed 
 with a sharp knife. — [Wilder and Gage.) 
 
 Disarticulation of Skulls. 
 
 Choose a young or barely mature animal for this preparation, 
 since the cranial sutures are liable to be obliterated in adults. 
 Prepare the skull by the liquid soap process; continue the final 
 boiling for half an hour longer than for a skull that is not to be 
 disarticulated. While still moist, the bones may be separated 
 by steady traction. 
 
 If a large skull is prepared by the liquid soap process, it 
 should be thoroughly softened by soaking in water two or three 
 days, or by boiling an hour. Then fill the cranial cavity with 
 dry beans or peas, force a cork tightly into the foramen magnum, 
 and place the skull iu water. The swelling of the peas will force 
 the bones apart. Macerated skulls should be treated as just 
 described, ,but they need not be boiled. — (Wilder and Gage.) 
 
 Method of Preserving Cartilaginous Skeletons and other 
 Soft Animal Structures. 
 
 As an example, the method of preparation of the skeleton of 
 a fresh Elasmobranch may be taken. The fish is eviscerated; 
 
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 the gills removed and placed in strong spirit, and the body- 
 plunged into water a few degrees below the boiling point. An 
 immersion, varying from a few seconds to a few minutes, serves 
 to so£ten the muscle and connective tissue to such an extent 
 that they may be stripped from the cartilage without injury to 
 the latter. In case of tiie gills, even a momentary immersion in 
 hot water is liable to cause a separation of the cartilages; they 
 are, therefore, best prepared in ,the cold after the ligaments 
 have been well hardened with alcohol. After the remainder of 
 the skeleton is cleaned, it may be put through the preserving 
 process at once or previously hardened with alcohol — the latter 
 alternative is the best, since it diminishes subsequent shrinking, 
 but it is not essential, and may well be dispensed with in the 
 case of large skeletons, for the sake of saving the otherwise 
 large expenditure of alcohol. It is always advisable to separate 
 the skull from the vertebral column, the pectoral fin from the 
 shoulder girdle, etc., as in this partially disarticulated con- 
 dition the skeleton is more easily manipulated, besides being 
 more convenient for future use. In the case of large forms, it 
 is also necessary to divide the vertebral column into pieces 
 small enough for the vessel used in the preserving process. 
 
 The various parts of the skeleton, with or without previous 
 hardening in alcohol, are then placed in "glycerin fluid'' of the 
 following composition : ■ i 
 
 Glycerin, 1 litre. 
 
 Water, i 1 " 
 
 Alum, 20 gxms. 
 
 Corrosive sublimate, 10 grms. 
 
 After remaining in the fluid until thoroughly permeated — ^two 
 days to a week according to size — ^the skeleton is transferred 
 to the following glycerin jelly: 
 
 Gelatine, 150 grams. 
 
 Glycerin, 1 litre. 
 
 Water, 1 " 
 
 Corrosive sublimate, 10 grms. 
 
 The jelly is kept at a heat just sufficient to melt it, in an 
 earthenware vessel (neither the glycerin fluid nor the glycerin 
 jelly should be allowed to come in contact with metal), over a 
 water bath, and the specimen is retained in it for about three or 
 four (lays. It is advisable to have vessels of various sizes, so as 
 not to xise more jelly than is absolutely necessary. For ordinary 
 
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 purposes, I use gelatine-glue instead of pure gelatine. Phenol 
 may be substituted for corrosive sublimate. 
 
 After removal from th.e glycerin, the specimen is thoroughly 
 drained, and placed in a dry room, protected from the dust. 
 Such parts as the vertebral column, the flns, and in most cases 
 the skull, may be left to dry without further care; but thick or 
 strongly curve*! structures, such as the jaws and shoulder gir- 
 dle, should be fastened out while drying with strappings of tape, 
 small wooden or cardboard supports, etc., as otherwise a certain 
 amount of twisting is inevitable. When no more shrinldng or 
 "buckling" is perceptible — it is generally advisable to allow 
 some Aveeks for this- — the specimen is varnished with a solution 
 of white shellac in strong alcohol. This should be done in a 
 warm room, as the slightest damp produces precipitation of the 
 shellac. After two or three coats of this varnish, pie cartilage 
 is found to have a dry and smooth, but not too glossy surface. 
 In mounting the skeleton, ,the best way is to support each 
 part separately on a light wire cradle, so that it can at any time 
 be removed for examination. If it is found necessary to articu- 
 late any of the parts, it is advisable to use platinum wire. 
 
 In preparing the chondrocraaium of Teleostei (e. g., Salmo), 
 it is again advisable to have recourse to parboiling; the mem- 
 brane bones can then be easily removed, and the cartilaginous 
 brain case, Meckel's cartilages and the branchial arches pre- 
 pajed as above. 
 
 Skeletons of earlier mammalian foetuses must be put through 
 the process in toto, the chief disadvantage of this method being 
 that the bones being impregnated Mith gelatine, never become 
 white. 
 
 In disarticulating mammalian skulls, it is a good plan to re- 
 move the mesethmoid and prepare it in the above method, 
 thus preserving an important part of the skull, which the stu- 
 dent, as a rule, never sees unless he takes the trouble to dissect 
 it out for himself. 
 
 This method is useful for the preservation of brains, hearts, 
 stomachs, intestines, etc. All these soft parts must, of course, 
 be first thoroughly hardened with alcohol or chromic acid. 
 Crustacea, Echinoderms, eitc, may be preserved so as to retain 
 their natural colors and flexibility. — (T. Jeffrey Parker.) 
 
 Small cartilaginous skeletons are very effectively mounted by 
 fastening them to glass plates with gelatine. 
 
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 Injections. 
 
 The object of injections is to render the bloodvessels more 
 apparent, and thus facilitate their detection. 
 
 The importance of a perfect familiarity with their position, 
 and relations cannot be overestimated from the surgical and 
 experimental standpoints. 
 
 A syringe is usually employed to force the Injecting mass into 
 the bloodvessels. It should have the following features: — (A) 
 Ample capacity, so that one syringe-full will fill the entire 
 arterial or venous system of the animal to be injected. Of 
 course this does not apply to large animals like horses. (B) 
 The piston of the syringe should fit weU and be leather packed. 
 (C) There should be canulae of various sizes corresponding to 
 the different vessels to be injec,ted. As soon as an injection is 
 finished, expel any remaining injecting material. Then fill the 
 syringe several times with clean hot water and expel it. See 
 that the canulae are thoroughly clean. Finally, unscrew the 
 top of the baarel and remove the piston; wipe it with an old 
 towel, oil the leather packing and return to the barrel. 
 
 "\\'hen wax, vaimish or gelatine injecting masses are used, the 
 animal or organ to be injected must be first thoroughly heated, 
 and should remain in water heated sufficiently to keep the 
 injection fluid throughout the process. 
 
 One cannot inject fishes or amphibia with wax mass, as 
 Hja'tl has well said, for the heat required to warm the subject 
 and the mass would cook the tissues. For them, plaster or 
 some other cold flowing mass, or glue which remains liquid at 
 a low temperature, must be used. — (Gage and Wilder.) 
 
 Injection Masses. 
 The most convenient mass is composed of the finest plaster of 
 Paris, stained with carnnne solution for arteries, and with 
 Berlin blue for veins. The masses should be used immeciiately 
 after preparation and before the plaster has time to set. Ap- 
 proximately the same volume of plaster and liquid should be 
 employed for ordinary injections. If, however, one wishes the 
 mass to fill the smallest vessels, the liquid should be increased 
 so that the ratio is as 1 — 2 or even 1 — 3. 
 
 Carmine Mass. 
 Measure out 100 cc. of the finest plaster of Paris and put it 
 into, a mixing dish that will hold about 400 cc. Add to this plas- 
 
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99 
 
 fer about 100 cc. of the carmine solution, (this is prepared by 
 grinding to a paste 4 — 5 gi'anunes of carmine, No. 40, in 20 cc. of 
 water, and then dissolving it in 50 cc. of strong ammonia. To 
 this solution are then added 75 cc. of glycerin and 500 cc. of 
 water. After shaking well, filter through fine flannel or ab- 
 sorbent cotton, and mix thorou^ly wi^h a wooden or porcelain 
 pestle. Finally, add slowly, and with constant stirring, a 50 per 
 cent, solution of acetic acid, until the color changes to bright 
 red, and the odor of the acid in the mass is quite perceptible. 
 An excess of aeid is less injurious than a deficiency. 
 
 Berlin Blue Mass. 
 Plaster same as for carmine. Add 100 cc of Berlin blue 
 solution (this is prepared as follows: Berlin blue, a saturated 
 aqueous solution, 500 cc; glycerin, 75 cc; mix the glycerin 
 and blue, and filter as for carmine. The glycerin preserves 
 the solutions and retards the setting of the plaster.) Stir 
 well; no acid is necessary j — ^(Wilder and Gage.) 
 
 Injecting Mass for Microscopic Work. 
 
 First procure some thin, clear, colorless French gelatine, in 
 sheets about 3 in.+ 8 in., with crossed markings. To 1 ounce of 
 gelatine add 10 ounces of water. Allow the gelatine to swell 
 for one hour; then place the kettle containing the whole in a 
 kettle of boiling water, and allow it to remain until the gelatine 
 melts thoroughly. Strain through previously moistened flannel 
 into, preferably, a flask. While yet warm and fluid, pour about 
 half of the gelatine into another vessel. Dissolve in the one- 
 half two grains of dry common salt, and in the other half, ten 
 grains of nitrate of silver. Should the gelatine become stiffened 
 by cooling, it must be warmed and kept fluid; When all is dis- 
 solved, mix the two gelatine solutions and shake briskly for 
 from three to five minutes. Add 10 grains of citric acid, and 
 keep the gelatine warm until the former dissolves. This is the 
 injecting mass, and is ready for use. If filtered first through 
 paper, the solution will be clearer, but this is not absolutely 
 essential. 
 
 The color of the injection mass in the mounted section is a 
 beautiful purple, and perfectly translucentj The differentiation 
 between arterioles, venules, and capillaries is perfect, and the 
 larger the vessel the darker the color of the mass. The citric 
 acid must be put in last, and metal vessels must not be uscvi, as 
 
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100 
 
 the silver salt will act upon them. The mass is not spoilt if 
 partly darkened before use. — (J. E. M. S.) 
 
 Process of Injecting. 
 For ordinary injections, the femoral or carotid arteries are 
 to be preferred. The vessel being exposed and separated by 
 carefol dissection, after lajdng back a triangular flap of skin, 
 cut a V-shaped opening in the vessel, with the apex of the V 
 pointing peripherad. Introduce a probe into the vessel, and 
 slip two stout threads under the vessel on the heart side of the 
 incision. The one is for tying the canula in the vessel, the 
 other for ligaturing the vessel when the injection is finished. 
 Both threads should be loosely tied in a surgeon's knot, so that 
 they may be quickly tightened. Now, remove the probe; select 
 a canula of proper size; make sure that it is not clogged up; wet 
 the small end and put it into the vessel, so that the injection 
 will be centred. To insert the canula, grasp one edge of the V- 
 shaped incision with fine forceps and pull it open. Now, intro- 
 duce the canula an li push gently with a slight, twisting motion ; 
 at the same time pull with an equal force in the opposite direc- 
 tion \^dth the fine forceps. As soon as the canula is properly 
 inserted, for about 1 cm., put the thread nearest the incision so 
 that it will press on the canula within the vessel, and then 
 tighten the linot. If there is no enlargement near the end of 
 the canula, or a glass is used, the thread must be tied to some 
 part of the canula outside the vessel. As soon as the canula is 
 secured in the vessel, fill the syringe with water or normal salt 
 solution; connect it with the canula in the vessel, and force a 
 little of the liquid in 'to make siire the canula is open and prop- 
 erly inserted. In connecting the canula and syringe, grasp the 
 canula with one hand and hold it firmly while making the con- 
 nection. Do the same in separating themj After forcing a 
 small amount of water into the vessel, separate the canula an<i 
 syringe; expel the water, and then prepare the mass as directed 
 above. Stir the mass thoroughly and fill the syringe; connect 
 with the canula, and hold this firmly in the hand as the mass is 
 forced into the vessel. Be sure that the vessel is not looped or 
 twisted in the least, but drawn peripherad just enough to 
 straighten it. Force the piston down steadily and continuously; 
 do not allow it to stop until the injection is finished. If the can- 
 ula becomes clogged, the resistance will be complete, and there 
 will be an entire absence of the elastic feeling which comes from 
 
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101 
 
 the distended arteries. One can tell only by experience when 
 the injection is finished; hut a slight incision at the point of the 
 body most distant from the point of injection is serviceable, as 
 the extravagation from the minute peripheral vessels shows that 
 they are being filled. As soon as the injection is finished, tie 
 the vessel with the thread provided for the purpose, draw back 
 the piston slightly, and cut the string holding the canula in the 
 vessel, and remove the canula. 
 
 Clean the syringe, canulas, and mixing dishes immediately. — 
 (After Wilder and G-age.) 
 
 Hyrtl's Corrosion Method. 
 
 Commercial mastic varnish is gradually evaporated over a 
 spirit lamp, or by other means, until it is of such a hardness 
 that it cannot be dented by the finger, and mth ditticulty by 
 the finger nail. The varnish should never be heated to boiling. 
 
 By means of a glass rod, allow a drop of the hot varnish to 
 fall in cold water; if this cannot be flattened out between the 
 fingers when cool, and only with difticulty after w^arming in 
 the palm of the hand or on the tongue, it is sufticiently evapo- 
 rated. To six parts of hardened varnish add one part of white 
 bees-wax. To color the injection mass, five colors are recom- 
 mended, and should be prepared at once, as they may all be 
 used in one preparation (e. g., in the injection of the lungs, 
 through the pulmonary vein and artery, the bronchial vein and 
 artery and the trachea.) For red mass, cinnabar; 'for blue, 
 cobalt or ultramarine; for yellow, light or dark chrome yellow; 
 for green, emerald green; for \^hite, carbonate of lead. The 
 latter holds more poorly tlian the others, becoming somewhat 
 brownish after heating. 
 
 To 24 ounces of the mass, add from 16- — 20 drachms of the 
 color; a little more than this for the blue and green. The colors 
 chould be rubbed up evenly in a mortar, with enough of the 
 fluid varnish to give a syrupy consistency, and this mixture 
 pDured slowly into the heated mass, while constantly stirring 
 with a small wooden spatula. To see that the mass has suffi- 
 cient color, allow a drop to fall on a sheet of paper and smear it 
 out in a streak with the finger; if it hides completely the color 
 of the paper, it contains sufficient pigment; if the paper shows 
 through, more color must he added. The permanency of the 
 color in the mass will be better insured by melting only so inuch 
 of it as is required for an injection; to accomplish this more 
 
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102 
 
 easily, the mass had better be poured into several small vessels 
 when first prepared. 
 
 The mass is warmed, preparatory to injection, over an or- 
 dinary spirit lamp, to a temperature just short of boiling, and 
 should be constantly stirred. 
 
 The method of injecting the varnish mass differs in no way 
 from that of ordinary injections; all tubes should be pro^dded 
 with stop cocks, aad the piston packed with pieces of felt 
 ins-tead of leather, as the leather shrinks from the heat at which 
 it is necessary to keep the mass. 
 
 By shaving off a thin slice of the surface of the organ to b^ in- 
 jected one quarter of an inch square, and keeping this area con- 
 stantly under observation during the injection, the time to stop 
 can readily be determined by the extravagation of the inject- 
 ing mass at the denuded spot. For a coarse injection, the slice 
 should be a thick one; for a fine injection, thinner. In injecting 
 entire parts (a hand, foot, head), or entire animals, the skin 
 should be cut through at the point most distant from the point 
 of injection. If this be done, there is little danger of the mass 
 bursting out of the vessels elsewhere. It is better to inject the 
 veins last. 
 
 For corroding away the fleshy parenchyma, concentrated hy- 
 drochloric acid is used. The organ is placed in a glass jar, of a 
 depth at least two inches greater than the diameter of the organ. 
 It is first rinsed with cold water, and then the cold acid poured 
 over it in sufficient quantity to float itj The gTeater the amount 
 of acid, the quicker the corrosion. The jar should be then 
 covered and labeled, and placed on the roof or some place in 
 which the escaping acid fumes will do no harm. From two to 
 ten days will be required for corrosion, according to the size 
 and density of the organ. At first the organ floats, but later it 
 sinks to the bottom. To examine it, draw off some of the acid 
 and touch the exposed surface of the organ; if it smears the 
 finger, it i^ ready to be washed. A fine spray or jet, made by 
 filling the mouth with water and blowing it through a glass tube, 
 or by the use of an elevated douche apparatus, is tlien played 
 upon the organ, and the corroded flesh carefully washed awayj 
 The preparation is then laid for two or three hours in clean 
 water and then dried. In case the organ has been left in the 
 acid too long, and the outer layer of the injection mass has had 
 some of its color removed, the bright color can be restored by 
 spraying very lightly with a mixture of spirits of turpentine, 
 
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103 
 
 three parts, and ether, three parts. The large vessels can be 
 brubhetl over AvUh this mixture. In case pieces are broken off 
 in tiie course of preparation, they can be fastened on. by touch- 
 ing the broken surfaces "with a hot needle ajid uniting while 
 melted. 
 
 To make the preparation less liable to be broken when used 
 for demonstration, it should be coated with a thin layer of 
 some tough, transparent substance. A thin solution of gelatine, 
 sprayed on in successive coats, answers very nicely. The 
 preparation is then to be mounted on a suitable base, to which 
 it is fastened with gelatine, and covered with a bell glass to 
 exclude dust. — (Hyrtl.) 
 
 Wood's Metal Corrosion Method. 
 The organ to be injected is placed in water, of a temperature 
 sufficient to keep Wood's metal in a fluid condition. The 
 melted metal is then injected by a gravity injecting apparatus 
 or injecting syringe; The injected organ is removed to cold 
 rimning water, where it is allowed to remain until the flesh has 
 macerated away, when it is washed and cleaned with a brush 
 and a very durable metal cast is secured. 
 
 Plastic Mass for Making Casts of the Cavities of Bones, 
 
 Shells, etc., etc. 
 This is a composition of glue and molasses, such as is used 
 by printers for ink rollers. It is liquid when warm, and 
 forms when cold a firm material, which takes a beautiful im- 
 pression of the surface with which it is in contact. It is so 
 plastic that it may be pulled out without injury from any over- 
 hanging depression, and yet so elastic that it will immediately 
 regain its exact form. — (Flower.) 
 
 Method of Preservation of the Brain, 
 
 Giacomini describes a process for the preservation of the 
 brain, which retains its form and color, and leaves it firm, and 
 yet pliable and easily handled. It may be freely exposed to 
 the air for any length of time, and does not crack on the surface. 
 It loses in the process only about ^Vof its volume, and this small 
 amount is not increased on being exposed. He says it is a true 
 preservation, not a mumiflcation, as are so many of the pro- 
 cesses hitherto published. His process consists of two stages. 
 In the first stage the fresh organ still enveloped in its mem- 
 
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104 
 
 branes is immersed in a saturated solution of zinc chloride. In 
 this it floats with a little of its surface above the fluid; and so, 
 while its form is not interfered with by pressure, it must be 
 turned two or three times a day, in order that all parts may be 
 uniformly acted on. If the subject has been dead for some time, 
 600 grammes of the solution may be injected through the caro- 
 tids under slight pressure, so as to give a firmness to the some- 
 what sof tish brain before its removal. After forty-eight hours, 
 the surface is hard enough to have the membranes removed. 
 Let this be done without taldug the organ out of the solution, 
 or, if it be taken out, let it be put into ^\'ater immediately, so 
 that it may the less lose its form by pressure. After having 
 been cleaned, let it remain in tlie solution tiU, as the hardening 
 proceeds, it begins to sink no longer, and then remove it. At 
 tliis stage it will be firm, slightly diminished in volume, the fis- 
 sures a little opened, and the color whitish, unless the mem- 
 branes have been left on too long, in which case the course of 
 the large vessels will be stained of a rusty color from the blood 
 pigment. Now it is immersed in alcohol of commerce for not 
 less than ten or twelve days, but it may be for an indefinite 
 period; here it sinks, and so must be often turned, to avohi de- 
 formity by pressure on the bottom of the vessel, and it is well 
 to renew the spirit two or three times — ^the oftener, the sooner 
 the process is finished. After the alcohol the consistence 
 is greater, the size a little less, and the convolutions some- 
 what closer together. Now comes the second stage. Let 
 the organ be immersed in glycerin of commerce, or with 1 per 
 cent, of caTbolic acid added. TMien first put in it floats, with 
 some of its upper surface above the surface of the glycerin, but, 
 gradually becoming heavier as the alcohol evaporates, and gly- 
 cerin is imbibed, it sinks deeper and deeper till it is just level 
 with the liquid — then it is to be taken out. In this part of the 
 process, neither surface, color, consistence, nor volume is al- 
 tered, but it becomes heavier. A brain should gain from 150 to 
 200 gi'ammes in from twenty to thirty days, according to its vol- 
 ume. Now set aside for several days, till the surface is dry, and 
 then cover it with several layers of gum mastic varnish, or, better 
 still, marine glue, diluted -with a little alcohol. This varnish is 
 not to prevent evaporation — -the glycerin does that — ^but is 
 simply as a protective against dust and injury. 
 
 Dr. Osier, formerly of the University of Pennsylvania, says: 
 "I have employed this method with the greatest success, and 
 
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105 
 
 can strongly recommend it to all who wish to prepare brains 
 for demonstrative or museum purposes. After varnishing, they 
 look like beautiful wax casts, and when I demonstrated the 
 specimens at the Medico-Chirurgical Society of this city, many 
 of the members, could scai'cely believe that they were not 
 artiflcial models. With the convolutions labeled on the one 
 side, and Ferrier's centers mapped on the other, they make 
 fine museum specimens — just fitting under the 7 J inch glass 
 shades. Such cortical lesions as superficial spots of softening 
 show remarkably well by this method. 
 
 "In the (ietadls of the process, I have accurately followed the 
 directions above referred to, though, where the organ is per- 
 fectly fresh and firm, I think it better tp remove the pia mater at 
 once and not wait for forty-eight hours, as recommended. The 
 glycerin should be good, otherwise the convolutions lose color." 
 
 The Slip System of Notes. 
 
 This system of note-taking, recommended by Profs. Gage and 
 Wilder, is so convenient and has proved such a satisfaction to 
 the many who have adopted it, myself included, that I most 
 heartily indorse it, and advise its use by all students who have 
 the intention of doing "serious scientific or literary work." - 
 
 "The essential feature of a 'slip system' is the use of separate 
 slips of uniform and convenient size. The note slips should be 
 of unruled paper, slightly sized, so as to take either the pencil 
 or the pen, moderately stiff, but not thick, and of the size of 
 the U. S. postal card." Some of these slips should be carried in 
 the pocket at all times, in the pocket book, or in covers, that 
 can be obtained of almost any stationer, and into which a pack- 
 age of the slips may be temporarily fastened. After a quantity 
 of notes has been taken, say at the end of each week, the slips 
 should be sorted and placed in manilla covers, twice the size of 
 a postal card. A rubber band keeps the slips secure, and the 
 subject of the notes being written plainly on the upper left 
 hand corner, by the folded edge, the covers and their contents 
 are arranged alphabetically, and are always ready for reference 
 or for the addition of new notes. 
 
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Digitized by Microsoft® 
 
INDEX. 
 
 Aberration?, 6. 
 
 Absnliite alpolin], preparaiion of, 33. 
 
 Acetate of Pota<li as a iDuuntiiig me- 
 
 dinra, 57, 60. 
 Acetic acid as a mounting medium with 
 glycerin, 57. 
 
 action on carbonates and oxal.itep, 
 73. _ 
 Acliromatisni, 6. 
 
 Acids as mlcrochemical reagents, acetic, 
 73. 
 
 clirnmic, 73. 
 
 hydrucliluric, 72. 
 
 nitric, 72. 
 
 oxalic, 73. 
 
 sulphuric, 72. 
 Acid nitrate of mercury, 77. 
 Adjustment, 9. 
 
 coarse, 9, 15. 
 
 fine, 9, 16. 
 Agar-agar as a culture medium, 83, 88. 
 Air bubbles, 24. 
 
 in intercellular .spaces, removal of, 
 78. 
 Albuminoids, behavior toward Millon's 
 
 reagent, 77. 
 Alcohol, absolute, 33. 
 
 as a fixing agent, 31, 32. 
 
 as a hardening agent, 39. 
 
 as a medium for examination. 30. 
 
 as a microcheraical reagent, 78. 
 
 percentage of, 33. 
 
 use of, 33. 
 Algae, methods of cultivating, 90. 
 
 fixation of, 35. 
 Alkalies as microcheraical reagents, 73. 
 Alkanet as a microcheuiical reagent, 79. 
 Ammonia iiir clearing, 74. 
 
 as test f(ir pioteids, 72, 74. 
 Ammoniacal cniiric oxide, test for cel- 
 lulose, 80. 
 Amniotic fluid for dissociating cells, 39. 
 Amphibia, injection of, 98. 
 Amyliid substances, stain for, 47. 
 
 reag-Tit for, 49. 
 Angular aperture, 13. 
 Aniline chloride, test for lignin, 80. 
 Aniline colors, 46. 
 
 best mounting medium for, 59. 
 Aniline sulphate as test for lignin, 70. 
 ,Anther sections, staining, 50. 
 
 A plana' ic lenses, 7. 
 Arm of microscope, 8. 
 Asphalt varnish, 56, 58, 60. 
 Axis cylinders, to stain, 47. 
 Axis of microscope, 8. 
 
 Bacteria, staining, 48. 
 Pase of microscope, 8. 
 Ba^t-fil.ers, siain for, 47. 
 Baumgarten's Bismarck-brown, 49. 
 
 Canada balsam, 59. 
 Beal's method for glycerin mounts, 57. 
 Benzol as a clearing medium, 63. 
 Berlin blue injection mass, 99. 
 Bichromate of Potassium, action on 
 tannin, as dissociating fluid, 75. 
 
 as cement for glycerin jelly 
 mounts, 58. 
 
 for dissociating muscle cells, 40. 
 Biconvex lenses, use of, 6. 
 Bi^marck-brown, 48. 
 Blood, staining, 47. 
 Body of microscope, 8. 
 
 to clean, 24. 
 Bones, preparation of, 93. 
 
 to make casts of cavities in, 103. 
 Borax carmine, 44. 
 Bern's method for models, 91. 
 Brain, Giacoinini's method of preserv- 
 ing, 103. 
 
 Parker's method, 97. 
 Bread, for cultivaiing molds, 90. 
 Brownian motion, "/&. 
 
 Calber'a's double stain, 50. 
 
 Calcium chloride as a dearing agent, 56. 
 
 as a mounting mediom 57. 
 Calcium oxalate, to dissolve in cells, 72. 
 Callous, stain for, 47. 
 C amera lucida, use of, 17. 
 CanaHa balsam, 59. 
 
 Cane sugar as a test for proteid.s, 72, 78. 
 Caoutchouc, lest for, 79. 
 Caustic Potash as a clearing agent, 55. 
 Carbonates and oxalates, means of dis- 
 tinguishing, 7-i. 
 Carbolic acid as a clearing agent, 53. 
 
 as a test for lignin, 72, 80. 
 
 (107) 
 
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108 
 
 Carminp, 30, 43. 
 
 triOrinling medium for, 60. 
 
 injecting ma.'-s, 98. 
 Cnrtilaginous skeletons, 95, 97. 
 Odar oil, 55. 
 Cc'lloidin, imbedding in, 65. 
 
 cuttina:, 65. 
 
 staining, 66. 
 
 mounting, 66. 
 
 bal.=iam mixture for use after, 59. 
 Cellulose, stain for, 47. 
 
 action of snlpliuric and of nitric 
 acids on, 72. 
 
 action .of ammonia and copper 
 sulphate, 74. 
 
 action of iodine, 75. 
 
 action of ohloriodide of zinc, 80. 
 Cements, 60, 61. 
 Cherry wood, for preparation of phlo- 
 
 roglucin, 19. 
 Chloride of calcium as a mounting 
 medium, 57. 
 
 as a clearing agent. 56. 
 Chloride of mercury, action on protein 
 grains, 76. 
 
 to show cyclosis, 76. 
 Chloride of zinc as test for cellulose, 80. 
 Chloroform as a clearing agent, 53. 
 Chlorophyll, test for, 73. 
 Chondriis crispus as a culture medium, 
 
 88. 
 Chromatic aberration, 6, 7. 
 Chromic acid, use of, 35, 36. 
 
 as test for llanified and cellulose 
 cell-walls, 73. 
 
 action on cuticiilarized and strati- 
 fied cell-walls, 73. 
 
 as a fixing agent, 32. 
 Chromo aceto osmic acid, .34. 
 Cleaning microscope and objectives, 23. 
 Cleaning mixture, for slides and cover 
 
 glasses, 71. 
 •Clearing agents, 52. 
 Clips on micrciscope, 10. 
 Course adjustment, 9. 
 Cochinial, 36, 43. 
 Codclington lens, 6. 
 Cocoanut fiber as nidus for cultivation 
 
 of fern spores, 89. 
 Cohn's fluid, 87. 
 Cold stage, 27. 
 Collar of microscope, 9. 
 Coloring drawings, 29. 
 Compound microscope, 8. 
 Concave mirror, 10. 
 Coniferin, detection of, 73, 80. 
 Connective tissue, stain tor, 47, 59. 
 Copper sulphate as a microchemical 
 
 reagent, 76. 
 Corallin, 47. 
 
 Corky tissues, reagent for, 46, 80. 
 Corrosion methods for anatomical prep- 
 arations, 101, 103. 
 Corrosive sublimate, 32, 37, 38, 76. 
 
 Cover glasses, 21, 24, 56. 
 
 to cle in, 70. 
 Creosote as a clearing agent, 53. 
 Crustaceans, Parker's method of pre- 
 
 servinsr, 77. 
 Crystalloids, 74, 78. 
 
 Cs'ikor's cement for glycerin mounts, 61. 
 Cultivation of low organisms, 81, 83, 85, 
 86. 
 
 of fern spores, 89. 
 
 of mohjs, 90. 
 
 of fresh water algae, 90. 
 Culture media, fluid, 83, 87. 
 
 solid, 86, 87. 
 Culture vessels, 85. 
 
 Cuticularized cell-walls, beliavior tow- 
 ard chromic acid, 73. 
 
 Nitric acid, 72. 
 
 Iodine, 7-5. 
 Cyclosis, to render visible, 76. 
 
 D 
 
 De Vries' method of preserving plants> 
 
 39. 
 Diaphragm of microscope, 6, 10, 24. 
 Diamond-fuchsin-iodine-green, 50. 
 Diatoms as test objects. 20. 
 Dissection, rules for. 28, 29. 
 Dissociating fluids, 39, 75. 
 Double staining, 49. 
 Doublets, 7, 13. 
 Drawing, 29. 
 
 with camera lucida, 17. 
 Draw-tube, 9. 
 
 Dung, for cultivation of molds, 90. 
 Dust, removal of, 24. 
 
 Echinoderms, Parker's method of pre- 
 serving, 97. 
 Elasmobranchs, preparation of skel- 
 etons, 95. 
 Eosin, 47. 
 
 Epithelial tissues, stain for, 50. 
 Kther as a microchemical reagent, 78. 
 Euchen)a speciosum as a culture me- 
 dium, 88. 
 Eyes, use of both, 24. 
 
 abnormal sight, 18, 25. 
 Eye-piece, 9. 
 
 Huyghenian, solid, periscopic, neg- 
 ative, positive, 1 1 . 
 nomenclature of, 12. 
 cire of, 22. 
 Eye-piece micrometer, 19. 
 
 Fehling's fluid, 70. 
 
 Fern, method of cultivating spores, 89. 
 
 Fibers, chemical discriminaaun of, 81. 
 
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109 
 
 Field, flatness of, 12. 
 
 size of, 12. 
 Fine adjustment, 9. 
 Fishes, injection of, 98. 
 Fixation, 31. 
 
 Fixing agents, 31, 32, 34, 3-5. 
 Fleming's fixing fluid, 34. 
 Flint glass in lenses, (i. 
 Floating bodies, apparent in use of 
 
 microscope, 2-5. 
 Fluckiger's test for adulteration of oil 
 
 of cloves, 54. 
 Flower's metliod for making casts of 
 
 cavities in bones, etc., 103. 
 Focus, 5. 
 
 focusing, 16, 23. 
 Foetal skeletons, preparation of, 97. 
 Foot of microscope, 8. 
 Fresh-water algie, cnltivation of, 97. 
 Freezing microtome, 66. 
 Frozen sections, 93. 
 Fuchsin, 46. 
 
 Ganglion cells, 47. 
 
 Gas chamber, 58. 
 
 Gelatine culture media, 83, 84, 89. 
 
 Giacomini's metliod for preserving 
 
 brains, 109. 
 Giesbreclit's method of clearing sec- 
 tions, 62. 
 Glandular tissues, staining, 47. 
 Glycerin, for examination of protein 
 gr.iins, 78. 
 as solvent for iodine, 78. 
 as test for inulin, 78. 
 as meiUum for examination of liv- 
 ing cells, 30. 
 as mounting medium, 57. 
 saline glycerin for eosin prepara- 
 tions, 47. 
 Glycerin jelly, for mounting, 58. 
 
 for preparation of cartilaginous 
 skeletons, 96. 
 Gold size, 61. 
 Grape sugar, test for, 76. 
 Grenacher's carmine, 36, 44. 
 Gum, stain for, 47. 
 test for, 75. 
 
 H 
 
 Hsematoxylin (as stain after chromic 
 acid), 35, 36, 43, 45. 
 
 with eosin, 51. 
 
 mounting media for, 60. 
 Hanstein's rosaniline violet, 47. 
 Hardening agents, 38. 
 Heart, preservation of, 97. 
 Heat as a fixing agent, 32. 
 Homogeneous fluid, 13. 
 
 homogeneous lenses, 55, 
 Hoyer's mounting media, 59. 
 Huyghenian eye-piece, 11. 
 
 Hydrochloric acid as test forlignin, 72. 
 behavior with proteids, 73. _ 
 witli chlorophyll, 73. 
 to distinguish between carbonates 
 
 and Sxalates, 73. 
 as macerating agent for woody tis- 
 sue, 73. 
 Hyrtl's corrosion method, 101. 
 
 Illumination, 16. 
 Imbedding in celloidin, 65. 
 
 in paraffin, 63. 
 Immersion, fluids, 12, 13. 
 
 objectives, 12, 55. 
 Incompatibility of staining and fixing 
 
 agents, 42. 
 Index of rei'raction of mounting media, 
 
 60. 
 Infusoria, fixation of, 35. 
 Injeetion, masses, 98. 
 
 process of, 100. 
 
 of bloodvessels, 98. 
 
 for miiTOBcopical work, 99. 
 Intestines, Parker's method of preserv- 
 ing, 97. 
 Inulin, test for, 74, 78. 
 Iodine as microchemical reagent, 74. 
 
 glycerin as solvent of, 78. 
 Iodine green, 46. 
 
 Iodized serum for dissociating, 39. 
 Irish moss as a culture medium, 88. 
 Iron, sulphate, 75. 
 
 Irrigation of objects under microscope, 
 31. 
 
 Joint of microscope, 8. 
 
 K 
 
 Kleinenberg's hgematoxyiin, 36, 45. 
 
 picrosulphuric acid, 32, 36. 
 Koch's culture medium, 84. 
 
 solution of methyl-blue, 48. 
 
 Lathyrus for study of pollen tubes, 91. 
 
 Lead, white, 56. 
 
 Lens, achromatic, crown glass, flint glass, 
 aplanatic, biconvex, biconcave, 
 plano-convex, Coddington, 6, 7. 
 systems of lenses, 13. 
 
 Lichen jelly as a culture medium, 84. 
 
 Lifeless objects, study of, 25, 29. 
 
 Life slides, 27. 
 
 Living objects, study of, 28. 
 
 Lignin, tests for, 72, 74, 75, 79, 80. 
 stain for, 48, 77. 
 
 Lime, chloride of, as a clearing agent, 
 56. 
 
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no 
 
 M 
 
 Macerating fluiJs, 39, 40, 72, 75, 93. 
 Magenta, 46. 
 
 Magnifiers, 5, 6 » 
 
 Miignifying power, 6. 
 
 determination of, 19. 
 Maskenlao, 00. 
 
 Ma_\er"s cochineal solution, 36. 
 Measurpment of microscopical objects, 
 18. 
 
 unit of, 18. 
 Meilia for examination of objects, 30. 
 Mercury, acid nitrate of, 77. 
 Metallic models of microscopical struc- 
 tures, 92. 
 Metric system, 72. 
 Mrtliyl-blue, 48. 
 Methyl-green and eosin, 50. 
 Methyl-violet, 48. 
 Microchemicid reagents, 71. 
 Micrometer eye-piece, 18, 19. 
 
 stage, 18. 
 Mic.ostopes, classes of, purpose of, sim- 
 ple, compound, 5, 8. 
 
 use of, 15, 23. 
 
 determination of magnifying power 
 of, 19. 
 
 care of, 22. 
 Micro-millimeter, 18. 
 Microspeclroscope, 27. 
 Micro pliotography, stains for use in, 
 
 48. 
 Milled he>ds, 9. 
 Millon"s reagent, 76. 
 Miquel's lichen jelly, 84, 88. 
 Miquel's nutritive paper, 87. 
 Mirror, plain, concave, mirrur-biir, l'>. 
 Models, Bom's, 91. 
 
 Salenlia's, 92. 
 
 Flower's, 103. 
 
 of Wood's metal, 103. 
 
 by Hyrtl's method, 101. 
 Moi.st chamber, '11. 
 
 Motion of objects under microscope, 5, 
 16. 
 
 molecular or Brownlan, 25. 
 Mold, cultivation of, 90. 
 Mounting, media, 56. 
 
 celloidin sections, 66. 
 Mouclies volante.'i, 25. 
 Mucilaginous tissues, 78. 
 Mucor mucedo, 90. 
 Muscle-cells, to dissociate,- 40. 
 
 stain for, 51. 
 
 N 
 
 Negative eye-piece, 11. 
 Nerve-tissues, stain for, 51. 
 Nitrate of mercury, acid, 77. 
 Nitrate of silver, action on living mat- 
 ter, 76. 
 
 Nitric acid, 39, 73. 
 Normal solutions, 28,75. 
 Nose-piece, 8. 
 
 Notes, the slip system of, 105. 
 Nuclear stains, 50. 
 
 Objective, 9. 
 
 classes, dry, immersion, 12. 
 
 attaching high, 16. 
 
 caie of, 22. 
 Ocular, 9. 
 
 Oil lontaining seeds, means of demon- 
 strating ground substance, 79. 
 Oil of Bergamot, 54. 
 Oil of cloves, 54. 
 Optical axis, 10. 
 
 Order, necessity of learning, 23. 
 Osmicacid, 32, 33, 34. 
 Osraunda rigalis, for cultivation of 
 
 spores. 90. 
 Oxalic acid, 73. 
 
 Oxalates, means of di-tinguish;ng from 
 carbonates, 73. 
 
 Pseonia stapliylea for pollen grains, 91. 
 Paratfii, infiltration, 62. 
 
 imbedding, 63. 
 
 order of procedure in, method, 64. 
 Pasteur's fluid, 87. 
 Percentage solution^, 72. 
 Pergtn's picro-carmine, 44. 
 I'eriscupic eye-piece, 11. 
 Plitzner ssatfranin, 49. 
 Phtnol as test for lignin, 80. 
 Phloroglucin, 72, 7.3, 79. 
 Physioloi;ical salt solution. 
 Picric acid. 35. 
 Picro-carmine, 34, 35. 
 Picro-hydrochloric, picro-nitric, picro- 
 
 sulphuric acid, 32, 36. 
 Pillar, 8. 
 
 Pith for imbedding, 61. 
 Plants, to prevent blackening in alcohol, 
 
 39. _ 
 Pliismolysis, 75. 
 
 Plastic ma^s for making casts, 103. 
 Pollen tubes, 91. 
 Potatoes as culture media, 83. 
 Pfisitive eye-piece, II. 
 Pota>ih, caustic sol., 55, 73. 
 • acetiite of, 59, 60. 
 
 bi chromate of, 40, 75. 
 
 chlorate of, 7o. 
 Powers of objectives, 13. 
 Preserving agents, 38. 
 Pringsheim's method for chlorophyll, 
 
 73. 
 Proteids, tests for, 72, 73, 74, 75, 76, 78. 
 P.\ i-ocatecliin, 73. 
 
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Ill 
 
 Razor, use of, 62. 
 Reading glass, 6. 
 ReagenlH, microcliemical, 71. 
 Refractive a.;enls, 60. 
 Keicliert's method of dissociating, 39. 
 Renant's doubJe stain, 51. 
 Resin, test for, 79. 
 stain for, 47. 
 Rindfleisch's metliod for clearing, 54. 
 Re i-aniline violet, 47. 
 Ro-eine, 46. 
 Rosolic acid, 47. 
 Rothrock's double stain, 50. 
 
 Stand, 6. 
 
 caie of, 22. 
 Starch, stain for, 47. 
 
 test fur, 72, 76, SO. 
 Sterling's double s-tains, 49. 
 Stomach, to preterve, 97. 
 Siiashurger's test for resin, 79. 
 Stratification of cell-wall, 73, 77. 
 Suberin, test for, 74. 
 Sniistage, 10. 
 Sugar, test fi r, 74, 70. 
 
 for pollen tubes, 91. 
 Sulphate of copper, 76. 
 Sulphate of iron, action on tnnnin, 75. 
 Sulphuric acid, 73. 
 
 Saffranin, 35, 49. 
 Sa'enka's method for models, 92. 
 Saline glycerin for eosin, 47. 
 Salivary glands, stain for, 51. 
 Schultze's macerating fluid. 40. 
 
 reagent for cellulose, 80. 
 Schweitzer s reagent for cellulose, SO. 
 Scotomata, 25. 
 
 Section cutting, 61, 63, 64, 65, 66, 68, 69. 
 Seller's cleaning mixture, 71. 
 Sem pel's method for preparation of dis- 
 
 seciions, etc., 93. 
 Serial secth-ns, 68. 
 Serum, ii dized for dissociating, as a cul- 
 
 tiiie medium, 84. 
 Shellac, 56, 60. 
 
 Shells, to take cast of cavity, 103. 
 Sieve-tubes, 47, 48, 80. 
 Silver nitrate, action on living matter, 
 
 76. 
 Skeletons, preparation of, 95. 
 Skulls, to prepare, 94. 
 
 di-articulation of, 95. 
 Slides, for serial sections, 69. 
 
 cleanlns, 70. 
 
 for cultivating bacteria in 86. 
 Soap solution for bones, 94. 
 Society screw, 8. 
 Sodium chloride as reagent, 75. 
 Solid eye-piece. 11. 
 Spectroscope, 26. 
 S|jermatozoa, staining of, 47. 
 SphserocryMals, 78. 
 Spherical aberration, 6, 7. 
 Spiral striation of cjlls, 77. 
 Spirogyra, cultivation of, 99. 
 Spores of ferns, cultivation of, 89. 
 Stage, 10. 
 
 warm and cold, 27. 
 Staining, after osmic acid, 34. 
 
 agents, 41. 
 
 sections, 64, 66. 
 
 Tannin, stain for, 47. 
 
 test tor, 74, 75. 
 Tea-ing, 41. 
 
 Thickened cell-walls, reagent for, 47. 
 Test-object and test-plate, HO. 
 Tradescantia for pollen tubes, 91. 
 Triplet, 7, 13. 
 Turpentine for clearing, 53. 
 
 u 
 
 Universal screw, 9. 
 
 Vision, ordinary distance of di-tiuct, 19. 
 
 w 
 
 Warm stage, 27. 
 
 Water, fur immersion lense.s, 13. 
 
 as medium, 30. 
 Weigeri's Bismarck-brown, 49. 
 
 double otain for nerves, 51. 
 White lead cement, 56, 61. 
 Woilaston prism, 17. 
 Wood's metal for casts and models, 92, 
 
 103. 
 Woody tissues, maceration of, 73. 
 
 X 
 
 Xanthoproteic reaction, 72. 
 
 Xylol and carbolic acid for clearing, 63. 
 
 Zygospores, method of obtaining, 90. 
 
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Digitized by Microsoft® 
 
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Digitized by Microsoft®