.1 IMPERIAL AGRI~ULTURAL ... RESEARCH INSTITUTE, NEW DELHI , BIOLOGICAL LABORATORY TECHNIQUE L1 ving ~eutral Ch.:'1mpy, Iron HaelIla toxylin Red ,L1 ving Scha.u:iinn I. H. Cp.jal-AoY3m"J. Ul traGe(ltrUuge1. Ch;tt!J.l!Y I. H. NR .;',.,---.-/.?" Sp81'ml1toeytcs of Heli.l: (j.~Jlel'''n. nA = Golgi apparatus. l\I = mitochondria. NR -~ neutral rOll granuleR. ARP = ltrl·hoplasrn (ehronlophobe part of (iolgi UPPUl'utllR). N ~ Iluelells. Tn th" lltst figllI'p the arrow indieatlls tho helwicHt pnrt of tho eoll. [P,onli.pi~,·e. BIOLOGICAL LABORATORY TECHNIQUE AN INTRODUCTION TO RESEARCH IN EMBRYOLOGY, CYTOLOGY AND HISTOLOGY BY J. BRONTE GATENBY B.A., B.Se., D.Phil. (Oxon.), M.A., Ph.D. (DuhL), D.Se. (Lond.) l'k'Ojc880.' (ij Zoology aad Comparative AJ'latoU1Y, 'l'l'initJl (follpoe, Dublin enirel'sitU; 11,Jliorury MetulJcr 0/ flu! Royul Societll of Nr'lI..~ Zertlillul; A[pnlbm' 0/ flIP ]fnyui Irish llradpJUlI. (UHf Fellow Of the Jloyal JUicl'o,st'opical Soc.ielJ/ oj Londsu. SOlneiilnc De))WllstJ'utor in lIistoloflJh U;z:{ord; SeriiOl' Demy, lUao(I(lltlt Collef/B. Ox/m'fl; Seniol' .rlssistunt in Zoology, fhliceI'sify College, J...rmdfHt; ',P('tlll'P" in CUto1orJY, London enil~e"l'oitll; Tllfll'el.U Seesscl Bellow, }'ale VIJirersif'h U.S.II.; PomJation VJtiN?fsitaire de ncl9iflue I..eclur'eJ', Loucllin. DJ'u8scl ... and Glumt. With 8 Illustrations J"ONDON J. & A. CHURCHILL LTD. 104 GLOUCESTER PLACE PORTMAN SQUARE 1937 50B6 111111~lInll IARI TO ,TAMES P]~'I'ER H.ILl" PREFACE Tms book has been written in a practieal mannE-r for two classes of persons, fust the University teiLcher or student who may desire a short and simple intl'orlncLioIl to the most recent and established pra,ctice in microtomy, and :secondly, the amatew' who may wish to make a t-;mall laboratory of his own, and is unaware that a good part of modern laboratory research is caniecl out \\ ith the simplest apparatus. Thus, many of the most modern methods have been adapted for smear teuhnique 'which cloCR not require fiection cutting. Having worked through most of the methods chosen for this book it is hopecl that t.he &tu,leHt or ~tnmteur worker will be ready to extend his illye~tigations t.o more specialized branches of microtomy, such as are t.reated in .. The Microtomist'iI \-"clemecum" and other Himilar works. The writer thanks his grrLdllate c;tndcuts, lY.Ii~s O. Aykroyd, lY.lr. \V. Boyle and .Mis~ R. Jones, for their helpful intel'eHt. The pl'Oufr-. wore alHo mad by Professor J. VV. Bigger, M.D., who made variOllH valuable suggeRtiolls. HOIIOOL OF ZOOLOGY, 'l'RINI'r¥ COLLEUE, DtTBLIN. TABLE OF CONTENTS ('lrAl'll' U 1. II. Ill. 1V. V. VL VIr. V111. IX. r\IrI Introduction. Laboratory Apparatus 1 Treatment of Living Cells, Saline Media, Vital Staining 17 Fixed and Stained Smears '27 Microchemical Tests in Smears Whole Mounts fiI1 Fixation Methods fi7 N~Butyl Paraffin, Dioxan and Celloidin Imbedding Alcohol and Hl Stains and Staining U\l Notes for Histology and Embryology Students, and Addenda vii 117 BIOLOGICAL LABORATORY TECHNIQUE CHAPTER I INTRODUCTION 1. In the first part of this book some of the modern laboratory equipment is discussed, Views on the best type of appfLrat.us to buy have l)een given, and some indication of the use and management of instruments, With reference to the vnJidity 1 of' preparations made by the methods for the cytoplasmic inclusions of the germ and other cells, it was felt that the student's early instruc· tion in this book shoulcl begin with cells in which t.hese inclmdons can be seen in the living, It is important that the student should be able to compare the living cell with that prepared on the one hand by the BOllin or Schaudinll and such methods, and on the other by established techniques for the Golgi apparCLtus_, mitochondl'h1, fat, and other cell inclusions. One of the most favourable cells for this purpose is the spermatocyte of Helix aspe1'sa 2 (see Frontispiece), which is common on the Continent and throughout the British Isles, and which has been intro, duced in various other parts of the world. Failing this mollusc, the insect germ cells, especially those of Gryllus 3 (the cricket) and Le_[)isma 4 (,the silver fish of kitchen 1 C. \V llJker and A llAll, PrOD. Royal Society oj London, Vol. 10 J, Hl:17, p. 512; C. Dohell, Parasitology, 1925, p, 47. 2 Gatenby, P-rac, Royal8ociety oj Lomion, Vol. 104B, 1928. 3 H, Herbert Johnson, Zeit. fur WiM. Zool., Vol. 140, 1031. • R. N. lIfukerji, J(YIlf', Royal ~Micro8Copical Society, Vol, 49, 192[1. I,ABOJ\ATOlW TEOlllHQl1E. 1 ! 2 IN1 JRODUOTION htl'dcl's), are very favourahle. Having eX!1millCd such cells alive, the student may then proceed to the making of stained smears. Some of the more useful and successful microchemical methods are given later, and the sectioncutting technique is described. Finally, a minimum of notes on histology is given. 2. Microscopes. Nearly all modern microscopes are gooel. If you ttre going to be a professional biologist or microscopist the best thing is to get one of the new binocular microscopes. Naturally a good man will do outstanding work with a cheaper and simpler model, but the newer binooular microscope is excellent. There are one or two points to note. First, it is nowadays possible to buy a stand to which either a monocular or binocular tube may be fitted. Purchase one of these. Secondly, do not buy a built-in mechanical stage unless you can afford two or three microscopes. Buy a body with a large square stage, upon which several types of mechanical stages lllay be clamped. You oan take off the mechanical stage and use the plain stage for staining, petri dishes, etc. It is better, however, to have another microscope for staining. r.rhis should have ird inch and ith inch lenses, and a No.2 eyepiece. You should buy a triple nose-piece at least. Very beautifully manufaotured, accurately aligning four nosepieces are made nowadays, giving you another fitting for a vcry low power of objective-that is to say, if you are going to use the arrangement of trd, ~th and l\rth lenses, which will be found most generally convenient. Some people like a higher dry lens than the lth, but this will depend on personal taste. A microscope should be used in a darkened room, with no window at your back or sides. If there is a window at the back you should have a dark blind on it. The 'window in front should also have a blind, or there should be a rectangular board 2 ft. X 2 ft. 6 in., clamped so as to cut. the direct JJ110R08COPES light from your face as you work aii the microscope. In a continental laboratory I have seen a black eardboard fixed on two legs fitting into lead holders, which could he shifted to the right position. For ordinary purposes these precautions arc unneeessary, bllt it is hetter to note that, for very difficult precise work, a dark part of the room should 1lt'~ chosen for the microscope. It is curious that this is so rarely understood. It has alrcady been mentioned that the combination of lenses which is usually chosen is the ~-rd, ·~;th and ·l2-th inch. If you wish to work on pond life, and whole mounts generally, it would be advisable to get another power lower than the ~rd. 1'ho T\-th or oil-immersion lens is a very high power suitable foI' cytology and protozoology, and is not really necessary at first. For eyepieces a X 8 would be suitable, though for expensive apochromatic objectives special compensating eyepieces are made. With low-power objectives the thing to look for is flatness of field, apart, of course, from the general clarity of the image. With oil-immersion and higher-power lenses there should be no coloured edge when a very small object is focussed, and it is much better to choose a lens with a good focal distance. Comparatively cheap oil-immersion lenses are now on the market, which give a really excellent performance, good enough for most research work. So far as the amateur microscopist is concerned, a great deal can be done with one power, a ird inch, and there al'C a number of cheap microscopes with ird inch anr11\-th in.ch objectives. These are quite suitable for work with whole mounts and sections oflarger organisms such as the earthworm. For these microscopes a substage condenser is quite unnecessary. 3. On the Care of a Microscope. If you own a valuable microscope it is likely that you will see that it is kept well covered eithel' with a good duster or special American cloth cover when not in use. You should put it back in its box if a glass or similar cover is not available! The l-z INrrRODuorpION moving parts of the mierm;cope should be oiled occasionally. If any parts seem stiff get some sperm oil, or " 3 in I oil," and oil it yourself. Never use, or force, stiff mechanical stages, stiff fine adjustments or nose-pieces. Forcing the latter eventually brings about inaccuracy of alignment. Lenses 1Lre ruined by being brought down sharply all a slide, or by being consbntly cleaned with xylol and a rough duster. If you have no lens paper, use a very soft cambric, or better, silk, handkerchief, and do not scruh the lens. A penetrating oil like xylol may dissolve the cement or packing of the various lenses inside the objec" tive. If immersion oil has been left on the immersion lens and has dried, screw off the lens carefully, rub oil off the metal part with a silk rag only moistened with xylol, then with lens paper (or the silk) just sweep the front of the lens itself clean, as gently as possible. Do not polish it. The nLg must not be dusty or the lens may be scraped. It is expensive to have a lens repaired. vVhen you have finished with your microscope see that the ~rd inch or lowest power is turned around towards the stage. Never leave the oil immersion in this position, and never remove a slide from the niicrm,cope without lifting up the oil immersion or tth lens frbm the surface of the slide. 4. Immersion Oil. The classical substance is slightly thickened cedar wood oil. It has the disadvantage that it must be wiped off the slide and lens after use, or it goes hard. Nowadays many people use methyl benzoate, which is rather liquid and somewhat smelly, but evaporates, and can be used on newly mounted slides. Liquid paraffin has also been used as a substitute for cedar wood oil. It is chel1p, does not harden, and can be wiped off easily at any time, but is not suitable for the most critical work .. Delicate stains are often mounted in immersion oil instead of bltlsam. 5. How to Use an Oil-immersion Lens. Put a small drop of oil on that part of the slide which you have just AllOR0800PE LAJ.1JP 13 been eXlLlllining with the f;rd or ·hth. Lower the oit imlllersion 011 to the drop, 'watching this ea-l'efnlly frolll the side until yun see the oil Just meet the lens, now lower very slightly, look into the microscope aml gmduaJly screw down with the fine adjustment until the object is focussed. vVllen you have finished always lift the lens fro111 the slide, tum it around and wipe it clean with lens paper 01' a clean .piece of silk. 6. Eye Strain. An older type of binocular microscope with one tube Rtraight, the other Rloping inwards, is dangerous for the ey£'s. Never use one. When using a monocular microscope do not shut one eye. Never nse an arc or Nernst lamp; th(;y are quite unsuitable. Either use an ordinary· fiO or 100 wat,t frosted hulb 01.' an inverted gas 1amI). U;ually the more expensive microscope lamps sold are saJe. Students often use t.oo bright a light. In a darkened corner of the room a 100 watt bulb is quite enough for most work. In Fig. 1 is a A o o L 40.",·m. , Fw. 1. MieroBcope lamp made fl'OIl"l H tin. A IlIHrks the original bottom of the ean, whieh has been bored to take the eleetrie fitting. '1'he hood in front has been solderorl on, ollcl vent holes hltve lJcet:l cut at the top of the ean. lamp made from an old tin can. Avoid long periods of work on red-stained preparations. Use blue 01' greenish screens on these. Choose gentian violet rather than safranin, and hoomalunl rather than carmine, if possiblc. 7. How to Measure the Size of an Object under a Microscope. In vertebrate preparations under an oil immersion a rough idea of the size of a body may be got 6 INTRODUOTION by looking for a red blood corpuscle near by and com· paring. The size of the human H. B. C. is 7-r5fk (mu) or micra" [L micron being Trluuth of a millimetre. The basis of more accurate measurement is a special stage micrometer which consists of a line a millimetre in lengt.h divided into 100 parts-that is to say, one division equals lOp. or 10 micra. These are prepared by a photographic process and mounted on 3 X I slides. If you are working with a camera luciela with certain known powers, eyepieces and t.ube length, you place the micrometer unde, each power and draw the lines on paper with the camera lucida, and measure ott the magnification with a millimetre ruler, and compute f()r all your powers. The size of One or more divisions of the micrometer is drawn on a stiff cardboa,rd with the eyepiece and object lens used in each case written beside, and this is kept for reference. Another way is to put the micrometer under your microscope, slip an eyepiece micrometer inside your eyepiece by unscrewing the top lens, and compare the number of divisions in the eyepicce with the size of the known divisions of the ·stage micrometer. Make a permanent note of the value of the eyepiece divisions under each combination of powers. If you cannot get a stage micrometer but have an eyepiece micrometer, make a thin coverslip preparation of your blood and knowing that a freshly-drawn R. B. C. is 7'5/L, make the necessary computation. This is, for most purposes, quite good. S. On Drawing a Preparation under the Microscope. The best way to do this is to have a }Jl'ojecting prism or piece of mirror fixed on the microscope tube pulled over horizontally, a very bright light, e.g., " point"o-lite " lamp (in a dark room), and to trace the image thrown on paper. Another way is to use a camera lucida, which consists of a prism and a mirror. The drawing board MIOR01'OllfE'S 7 should be sloped slightly towards the microscope. When using artificial light it may be necessa.ry to illuminate t.he board to the right degree. In daylight this is usually easier. If you cannot afford a call1era lucida, buy one of those squared discs of glass which fit inside the eyepiece, and use paper with squares of appropriate size, counting the number of squares across different parts of the organism and so getting the proportions correct. 9. Drawing Paper. ]'01' line block drawing, white paper (fashion-plate board) is good enough. Use India ink. For wash drawing, use a Bristol hoard "wit.h a slight blue tinge, and lamp-black water paint or Chinese ink. 10. Microphotography. The "vell-appointed laboratory has one of those excellent microphotographic cameras madc especially for the purpose. Elaborate ones cost over £100, which is beyond the ordinary person's powcr of buying. It is best to know something about ordinary photography before beginning microphotography. For yean:! we used a cheaper microphotographic camera which fitted iuto the aperture of the uJ)right microscope tube, and good results were got, but never so good as with the special heavy apparatus made for the purpose. This applies especmlly to low pow-er work. It is well for the beginner, as weH as the research student, to note that the India ink line drawing is more effective, cheaper, more likely to obtain puhlication, and to be copied in future text books. So, excellt in controversial matters, where a good microphoto may be a necessity, never use the microphotographic camera. Of course, if you cannot draw, or have at your disposal a laboratory artist, or if you are a very busy person, it may be necessary to use microphotographs. In some cases, as with tissue culture work, where the cell is spread fiat, a microphoto may be better than a dra"wing, but rarely in other cases_ 11. Microtomes. These are expensive precision instruments and there is really no cheap substitute for them. It may be mentioned, however, that for some classes of microchemical and cytological work, cutting sections is not really necessary. In other cases hand sections may do (§ 127). Most protozoology and some branches of 8 .lN'l'BOD U 01'! 0 N cytology actually are done with ntre recourse to the sectioning method. This is why it is very advantageous for the student to develop a good smear technique (§ 38). It is not difficult to advise on the purchase of a microtome. The Minot rotary micro tomes are the most popular, and some firlllR now supply freezing attachments, as well as clamps for putting the knife at a special angle for certain types of work. Buy one of these. Practically all classes of work can be done on a rotary microtome, but the sliding (sledge) types are especially useful in neurology and for cutting very hard or large sections. The microtome :;hollld be carefully oiled, and where fouled with paraffin wax, properly cleaned in xylol, and oiled before use. If this is done, the microtome will last for many years, with harel Weal', the sale upkeep being the sharpenillg of knives. However, nowadays razor blade holders arc sold, which arc quite goocl for orclinary work Even the clamps for the thin Gillette blades are excellent. Such holders can be made from a piece of iron shaped like a microtome knife. Another small rectangle of metal acts as a cover to the blade, which, projecting about 2 mm., is clamped firmly by means of two bolts threaded into or passing through the iron. The Lcitz Company sell a good clamp for Gillette blades, which we have used with satisfaction. I am convinced that a good clamp could be made of hard wood (knife shaped) with a shallow slot or indentation for one of the stiffer types of safety razor blades, covered with a metal clamp, and provided with thumb screws. It could be faced with metal at the ends where thc knife clamps into the holder on the microtome. 'The hest way to learn to use a microtome is to watch an expert. This, of course, applies to almost anything. To the person who has no expert to watch the first thing is to notice the safety catch which prevents the moving bar from descending on the knife. This should always be put into operation when you are doing anything with the block you are going to section. Otherwise yon may get a nasty eut yourself. So look for the safety catch. The second point is to sec that the safety catch is put out of action when you are going to cut sections, because if you turn the wheel suddenly it gives the OLA8SIVARE machine 11 nasty jolt; the lom;ening of parts in SOllle types of Minot rotary rnicrotomes is due to this treatment. 12. Bottles, Capsules, etc, These tend to be expem;ive. In lllany htboratories much of the glassware is provided with ground glass covers aml stoppers, Corked tubes and the cheaper variety of medicine bottles do for most routine work, however. What are extremely useful, and worth while purchasing specially, are small stelllier or petri dishes, with ground tops. These 111a.y range in size from Il to 5 in. aeross, and are indispensable for the general manipulation of material. \Vatch glasses are also very useful, and can be provided with squares of glass for covers. They can be hrought from watchmakers and hardware stores, and do quite well for coverslip smears, etc. For imbedding, washing, dehydrating, a number of short pieces of wide glass tube of various sizes are useful. Also some cloth gauze or butter muslin for tying over the ends. There should be a number of pipettes with fine and widely open ends, and rubber teats. For staining coverslip smears, the stender or petri dishes may be used, but for slides you wjll need some tubes, with or without ground stoppers. Corked or capped tubes a little higher and broader than a slide (3 X 1) 'will be suitable. These tubes are sold commonly in the 3t X llrr or 3t X It size, and can be placed in a wooden rack made by boring suitable holes in a piece of board, on t,he lines of a test tube holder. It is usually best to have larger vessels with ground glass stoppers for absolute alcohol and xylol used in the last stages of dehydrating, and for clearing preparatory to mounting. For' writing on slides one can either use Indian ink, a diamond, a piece of picked carborundum fixed in a harcl glass tube with sealing wax, or smear the end of the slide with thin amyl acetate celluloid solution. The slide is allowed to dry, the legend written, and ltnother 10 INTRODUO'PION layer of the solution quickly brushed over. The writing with the diamond is the best. Many people use labels, which, provided the end of the slide has been rubbed free of grease (with !1bsolute aleohol), usually stick quite well. It is a good thing to have a few drop-bottles. In some histological laboratories nothing else is used for students' benches. Another useful and ingenious aid to staining i~ Guu's staining Illate, which essentially oonsists of a plate slightly curved so that when a slide with sections is placed face down on the COIlcave face of the plate there is a small space left into which stains or other fluids can be run in with a pipette. Such a plate might be made from a flat piece of vnleanite or cellophane by warming and giving t~ necessary slight curve. With Gurr's plate the evaporation of the stain or other fluid is only from the edge, and not from the whole surfaco, as with the ordinary drop-bottle method. The method which is sure to find favour was suggested by Dr. A. C. Coles. Using a transparent plate it is possible with some stains to watch the progress of the staining. Something similar to this-a glass plato with a groove in ithas been used for years to stain large numbers of blood films at the same time. The slides are placed smear side down on the groove, and stain run into the latter. 13. The Arrangement of the Bench. It is always a good thing to have a bench for one's microsoope and lamp, a bench for the microtome, shelves for reagent bottles, and some place on your work table which can be cleared sufficiently to allow a space about 1 ft. square in which are spaced the symbols, 30%, 50%, 70%, 90%, 100%, written in chalk, or on labels stuck on the bench. When you are dehydrating you begin at 30%, or 50%, and put the dish or capsule over the proper figure, and movo • up each time you change the fluid. Some people use an oblong piece of flat glass with the figures written on paper underneath. The main point is to have some way of knowing what strength of alcohol the object is in. Alternatively, you may use the method of slipping in a OLEA.lUNa aLASS TVARE 11 piece of p~Lpel' with the grade written 011 it when you change the fluid. 14. To Clean Slides, CoversIips and Glassware. If t.he glass is in fairly clean condition rub wit.h strong alcohol (70%, or 90% with 1% Hel). If very dirty, soak overnight in 1 part commercial nitric acid, 9 parts ,vater, or in 5 gm. of potassium bichromate in 250 C.c. of 5% sulphmic aeid in water. Do not expose your hands too long to these fluids. Watch glasses, petri dishes, large dishes, Winchesters, etc., should be washed in hot water with Hudson's soap or Soapolio, and rinsed well in running water. Some people take coverslips from acid alcohol, through distilled water to 90%, into absolute alcohol. Each coverslip is held in forceps and flamed in a spirit lamp. For glassware to be used with living cells, use the bichromate sulphuric acid mixture, wash out at least eight times with distilled water. Even this may not be enough, as Oscar Richards has pointed out; soak overnight in distilled water. 15. Coverslips and Slides. Avoid very thin slides unless they are for some special pmpose. It is better to have good glass, but it really does not matter if there is a slight greenish tinge in theln. If you are interested in gum glycerine and similar whole mounts of pond organisms, etc., you may prefer to buy circular coverslips, as these can be very neatly ringed with a metal tube (§ 88). The thinner types sold are graded No. 1. They are necessary for very high power (oil immersion work), but if you are interested only in whole mounts, No.2 variety will do. They are cheaper and stronger. If you wish to make your own slides, this can be done by cutting 1 mm. thick glass into pieces 3 X 1 in" and rubbing the edges with emery paper. 16. Alcohol for Dehydration. The alcohol used ill laboratories is e.thyl alcohol. It is still universally used 12 TNTRO])UCTION for extracting the water from tislmes l1S '" preparation for mounting or sectioning. There are Rubstitutes nowadays, of which the most interesting is dioxan (di-cthylene oxide), the vapour of which is a cumulative poison at 1 in 1,000, and which has a slight and pleasant sIllell when concentrated, and hardly any at the toxic concentration. It will be interesting to see whether dioxan displaces ethyl alcohol. It has been stated that it is hardly likely that in the laboratory dioxan could reach the toxio margin. .]udging from the smell of carbon bis'ulphide, xylol or benzol in 1'ooms where imbedding is going on, I doubt the statement, and the hca,lth of a perSOll cOl1stant,ly using this fluid at from 56° to 60 0 C. in a small room with imbedding ovens is likely to be injured. If you have a small private hLboratory and imbed only occasionally it may be found convenient to use dioxan instead of alcohol. At all events, we recommend the ethyl alcohol method for the present, though the dioxan method is given below. The advantages of ethyl alcohol as a dehydrating agent are not evident except from the health point of view. The disadvantages are that it is expensive, slow, it hardens some objects too much, and it does not mix with paraffin wax. One should remember that all the modern aniline staining methods depend largely on the use of alcohol for passage to balsam, and the ·l1ew method with dioxan may mean certain readjustments for SOllle stains. The workers who are usually most dissatisfied with the l;Llcoholmethod are the entomologist, the embryologist dealing with hard eggs of both insects and Sauropsida, and the person who wishes to cut unusual objects like skin with developing hair or feathers. Other people are usually quite satisfied. It is usual to find two types of ethyl alcohol in the laboratory, one industrial methylated spirit, the other absolute alcohol. The latter is about 99'5%, the former about 95% or ALCOH()J~ AND OLJEARINCi 13 117% pure. The so-called methylated spirit sold by chemists and druggists is good for spirit lamps, and llreserving small animals if you are on holiday at the seaside-hut for nothing much else. The industrial methylated spirit varies consider[I,hly in purity and strength, and it is better not to usc it for making stains. It is all right for most other purposes. You dilute as follow!:!. Take the number of c.c.'s of 95% equal to the strength required in a graduated \w:lsel, and add di!:!tilled water to make 95 c.c. For example, to preparc 70%: take 70 C.c. of 95% alcohol and add 25 C.c. of distilled water. Absolute and 95% alcohol should he kept in bottles with ground glass stoppers. If you have a private laboratory it is well to remember that you can get a permit from the Com. missioners of Inland Revenue, 01' Customs and Excise, to purchase industrial alcohol at, a cheap price. 17. Other Alcohols. Another alcohol commonly used is methyl alcohol (if possible, acetone free and neutral). It is used for making stainR and as a narcotic. N-butyl alcohol cannot be used for clehyclrating in the lower strengths as it dissolves very little water and most of' the dehydration must be done ,·dth the aiel of ethyl alcohol. However, it does not harden tissnes. We believe the butyl alcohol method has been superseded by clioxall imbedding. Nevertheless, the method is given in § 137, and both techniques can be tried on clifficult objects. 18. On Clearing. When you place a piece of dehydrated tissue in an oil or liquid like xylol, benzol, chloroform, carbon bisulphide, etc., it becomes more 1 or less translucent and is said to be cleared. Except in the case of dioxan or butyl alcohol technique, this clearing process is always carried out before imbedding in wax or mounting in balsam. In one case it must be done because paraffin wax and ethyl alcohol do not mix, in the second because, while balsam and absolute alcohol do mix (Seiler's alcoholic balsam), it will be fonnd more satisfactory to use a balsam dissolved in benzol or xylol. Recently I have been mounting in dioxan balsam from 95% alcohol. 1 Refer to § 100. 14 INTRODUC'['ION The specimen of dioxan balsam I have appears t.o he going darker; and is still under trial. Of course, if you are going to imbed in paraffin by one of the newer methods explained in § 136, clearing is unnecessary, while the same applies to mounting in glycerine, gum glycerine, or Euparal (§ 82). Clearing previous to imbedding is a period when considerable shrinkage takes place, and also hardening. Caution is therefore needed. There are two good ways of clearing. When the material has been properly dehydrated pour away all the alcohol except what is enough to cover the pieces. Then add a quantity of about one-quarter the amount of alcohol left, of xylol, oarbon bisulphide, benzol, chloroform, or whatever oil or fluid you wish to use. Shake gently, cover, and you have the material in four parts of alcohol and one of clearing agent. After! hour add more clearing fluid, and so on till it is mainly clearing fluid. Then put it into pure clearing fluid for t hoUl'. The whole process can take 2 hours, but small objects can safely be done in 1 hour or even less. Another way much used is to take a tub() 10-15 mIll. in diameter, pipette in about 10 mm. in height of xylol, etc., then very cautiously add absolute alcohol, which will float on top of the clearing fluid. The piece to be cleared is lowered into the alcohol and stops just above the clearing fluid, where it clears slowly. After the two liquids have mixed the piece drops to the bottom of the tube, and should then be placed in pure clearing fluid. This method is not quite so easy as the first mentioned, but is WOl'th practising, and is much used by the chromosome workers. Now something should be said about clearing oils or fluids. If you cannot get good absolute alcohol you should pass the tissue ii'oIh 96% to methyl benzoate or less preferably carbol-xylol (add phenol crystals to xylol till no more CARE OF MATERIAL 15 dissolve), then to the fiui(l for imhedding. At the same time it will be just 118 convenient aml cheaper to dehydrate some 1)6%) fdeohol yourself, as described in the next paragraph. It will be assumed that the pieces can be dehydrated properly. Of all the clearing fluids used in routine work in laboratories xylol is possibly the best known, but in recent years carbon bisulphide has become the more popular. The reason for this is that it is more volatile, it can therefore penetrate better, and more freely mix with the wax passing into the piece of material being imuedded. It penetrates things like crustacea or insects better than xylol, and hardens them less. Use carbon bisulphide if possible. For very small penetrable organisms chloroform is very good. But for clearing before mounting in balsam it is best to use xylol or benzol, unless you intend to use Seiler's alcoholic lalsam, or the newer dioxan balsam (which is still on trial). 19. Dehydration of Alcohol. This can be done by heating blue copper sulphate in a covered porcelain vessel till the "water of crystallization~ is driven off. A bag of this is put in a bottle of 96% alcohol. It should be changed from time to time. 20. On Waste in the Laboratory. In large laboratories the old used alcohol is re·distilled. It should be collected for this purpose. Pieces of paraffin wax can be collected, melted • and strained. Osmic acid used for Weigl and Kolatchew can often be !Ised for making or rejuvenating Champy's or Flemming's fluids. Used xylol or benzol is not worth keeping except for taking out oil and grease stains on floors, etc. Old slides and coverslips can be scraped and treated with a mixture of used absolute alcohol and xylol, and then washed in hot soapy water. 21. On the Care of Laboratory Material. If you leave stained sections in the light they fade. It is very careless to leave valuable sections on the bench in the sunlight. When you have finished with the laboratory slides, put INTRODUOTION them bn,ck in a box, or at lef1st out of RUlllight. Never lenve your beneh with sta,in and aleohol bottles uneorked and petri dishes dirty. When stains dry in petri dishes they are often difficult to remove. After use, pour water into the dishes and leave in the sink. Never pOUl' wax into the sink. There should be a bucket for such things. Never make up stains in the gmduated measuring jttrs. Do not cut gritty animals with valuable microtome knives. Never underline or mark the Departmental Library books, and when yon leave the laboratory see that yonI' microscope is either put baek in a locker, if such be supplied, or eovered with a duster. CHAPTER II TREATMENT OF LIVING CELLS 22. On making Solutions. If you have no knowledge of chemistry the following may be useful. There are measuring jars for fluids graduated in cubic centimetres (denoted by the letters c.o., c.c.'s or mI.). Buy one of 100 e.(',. capacity, [mel if possible one of 10 e.c. Yon should never allow these jars to stand about with chemicals in them. They should be washed out in distilled wnter, or good tap water, after nse, and inverted in a stand. The various salts are weighed on a balance. For most purposes in a biological laboratory, it is sufficiently accurate first carefully to balance similar square pieces of paper on each pan (add fragments of paper to one side where necessary), and then to place the right weights on one side, and the chemical to be used on the paper on the other pan. Never remove chemicals from a bottle wifh a spatula which has just been ql8erl h~ another bottle. For instance, if a spatula has been used to ladle out corrosive sublimate, and is then put into the sodium chloride bottle, you are certainly going to have trouble with your saline media. Never put stains or chemicals directly on the metal pans uf a balance. When making up fluids in ,,,hich dry sa.lts are to be dissolved, choose a bottle of the right size and do the mixing in this and not in the measuring jar. Use this for fluids only. In general, keep all your reagents and fixatives on a dark shelf, espeoially away from direct sunlight. If you are working by yourself,. the local chemist and 17 18 TR1!JATMENT OF LIVING CELLS druggist will explain any difficultiel:l, or will make up any solutions for you. He ,vill also sell you empty Willch('ster bottles, and other suitable glasRware, Wash these as1jn § 14. If you live in a district where there is no university or college, remember that the medic!1} man has learnt to stain sections during his student days and could give you valuable help. If you are interested in biological science, in addition to your text books, get and read" The Microbe Hunters," by Paul de Kruif, and" Martin Arrowsmith," by Sinclair Lewis, 23. Saline and Artificial Media. 'rhe degree of acidity or alkalinity of a medium may have a marked effect on the organism or cell, and most of the reactions of the cells of an organism take place in a liquid environment very nearly neutral. The degree of acidity or alkalinity bears a definite relationship to the iOllS of hydrogen present in the volume of liquid. All aqueous solutions have a definite hydrogen ion concentration. The pH 1 (or hydrogen ion potential) of pure water is 7. Very acid. solutions have pH values approximating to zero, neutral solutions the value of 7, 'extremely alkaline solutions approximating to 14. The proper adjustment of the pH values of media may be vital for the growing of Protozoa, tissue cultures, etc., and should be carefully noticed by the student, who must learn the colorimetric method for determining the pH if dealing with organisms living in water, or artificial media. Certain salts are found in tissues which act as " buffers " in taking up acid or alkali without a corresponding change in the condition of the rnedi'l.('In. The two principal systems are the bicarbonate and the phosphate, the former NaRUO g, the latter NaH 2P0 4 and Na 2RP0 4 , and are used in buffe,ring artificial meoia, Special buffering tablets are now on sale at chemical warehouses, ready to use. III making up solutions of neutral red it will be found that in distilled water the colour is red, but becomes yellow in Hedon-Fleig Ringer, which has a pH of 8'8, and 1 See any text book of Physiology or Biochemistry. RINOER HI is suitable for molluscan cells. Neutrl11 red has a pH range of 6·5-8'4. Congo Red, a commonly used stain, is blue in the acid, red in alkaline range, pH range 3·5-4·7. The commonly used Ringer made from distilled watcr supplied in my laboratory has a pH of 7·4. Human arterial blood has a pH of 7·3, the blood of the common snail about 8, the hay infusion giving optimum growth of Paramoocium 7'8-8, and so on. Another conception of the greatest importance is that of osmotic pre881Lre. In the Hological laboratory its' commonest appliCf1tion is in the making of iso-tonic fluids for the examination or growing of living cells. For simple dissections for histologioal material, '75% NaCl (sodium chloride) for invertebrates and ,85% NaCl for vertebrates is sufficient. Years ago Ringer showed that both calcium and potassium salts were necessa,ry in order to keep the perfused frog heart beating. His solution consists of sodium, potassium and calcium ions in the proper amounts, generally nowadays with "buffers" added. To make up 2,000 C.c. of Ringer (that is, nearly a Winchester full) take that quantity of distilled water, and add 14 gm. sodium chloride, 0·6 gIll. calcium chloride, 0·5 gm. potassium chloride. This is a Ringer for coldblooded animals. For warm-blooded use 17 or 18 gm. of sodium chloride. Nowadays bicarbonate buffer is usually added, and if a sterile solution is needed, must be filtersterilized before adding to the sterilized Ringer j for the quantity (2,000 c.c.) add 3 gm. of sodium bicarbonate. Here is another fluid which is good for living invertebrate tissue mounts-known as Redan-Fleig fluid; it also is very useful for any work on living cells of terrestrial or fresh water invertebrates. Distilled wa.ter 1,000 c.c., NaCl 7·0 gm., KCl 0·3 gm., CaCl2 0·1 gm., NaRC0 3 .1-5 gm.; NI1 2HP0 4 0·5 gm., MgS04 0'3 gm., glucose 1·0 gIll. af yoli want a sterile fluid (not usually necessary) you ,cannot heat this, as it will partly disintegrate and its pH 20 TREATM10N'l1 OF .LIVING CELLS (8·8) becomes altered. Divide the constituents into four parts: (I) Nael, KOl, CaOl z, MgS0 4 • (2) GIllCQi,;e. (3) Na 2HP0 4 . (4) NaHCO J • (1), (2) and (3) in flasks plugged with eotton wool :tl'e sterilized in a steamer. The sodium hicarbomtte mllst be filter-sterilized. 'l'his is the really ollly satisfactory method. Hedon-Fleig is faintly alkaline, and in it neutral reel changes to a yellow colour. This does not indicate disintegration of the dye, for when taken up by living cells kept in HMon-Fleig, the segregated droplets are bright recl. Some other fluids which arc intended to be used with the living tissues or eggs of various other animals are given below. You willllotice that their salinity varies considerably. In the fluid which should be used for dio;secting marine urustacea for cytological or similar work, there arc 2:3 gIll. of sodium chloride per 1,000 c.c. of distilled water, whercas in mammalian Ringer there are only about 9 gm. per 1,000 C.c. 24. Clarke's Fl'lticZ jar Living In,,~ect Cell8. Distilled water 200 c.c., sodium chloride 1·3 gm., potassium chloride (}028 gIll., calcium chloride 0·024 gm., sodium bicarbonate 0·02 gm., monobasic sodium phosphate 0·002 gm. Holtfreter's Solution for Amphibian Cell8. Water 1,000 c.c., sodium chloride 3·50 gm., potassium chloride 0·05 gm., calcium chloride (anhyd.) 0·10 gm., sodium bicarbonate 0·20 gm. If desired storilize first, and add sodium bicarbonate afterwards. ~J1al'ine Crustacea Fluid. Water 1,000 c.c., sodium chloride 29·23 gIll., potassium chloride 0·75 gm., calcium chloride 4·44 gIll. lYIarine Mollu8ca Fluid. Water 1,000 c.c., sodium chloride 23·38 gm., calcium chloride 5' 55 gm., magnesium chloride 7·62 gm. Nicholl's Elasmobmnch l!'luid. Water 1,000 c.c., sodium chloride 15·38 gm., potassium chloride Q·8B gIll., calcium chloride 1·11 gill., urea 21·u gm., dextrose 1·0 gm.,., bicarbonate of soda 0'38 gm., acid hypophosphate of soda; (NaH 2P0 4 ) 0·06 gm. '1'188U1£ OULTURE ~l Artzjicial Sea WaleI' (Challenger Bepol't and Allen). Wat.or 1,000 c.e., sodium chloride 28 gm., pot.a:,;siulll chloride 0·77 gm., calcium chloride 1·25 gIll., magnesium chloride 2·55 gm., magnm;iulll sulphat.e 1-136 gm., sodium bicarbonate O·1l gm. Salinity about 35. Sea rYater R'ingel'. 0ne part sea water, three part.s dist.illed wat.er. 25. Examination of Living Cells. The common snail, H eUx a8pena, is nmv practiCfLlly cosmopolitan. In America it can he got from California. Its celb are very hardy and resistent to bacterial attack. '1'0 make a living culture of amcehooyt.es and epithelial cells :-wound a snail by thrusting a darning needle through its shell into the soft part of itH body. Leave it for 4 or [) hours. This preliminary wounding is not absolutely necessary, but it definitely raises the proportion of successful outgrowths. Remove most of the mouth part of the shell, pin the animal down on a piece of weighted cork and cut away part of its mantle wall (pulmonary cavity wall), RPC in Fig. 2, A, about 3-5 mm. SqlU1fC. It is a flat membrane. Place this on a clean slide, and with a sharp knife cnt into four smaller pieces (explants). Mount each explant separately in a drop of Ht1don-Fleig fluid (§ 23), covering with a small 20 mm. square coverslip. Leave in a covered petri dish, where the preparations will not evaporate. Do not use the hanging drop method, as this is not so demonstrative for thiR Rpeeial preparation. Examine next morning. In some cases the amcebocytes will have begun to emigra,te (Fig. 2, C), and in 3 days many of the preparations will bear a close resemblance to a Hoss Harrison vertebrate 1 tissue culture. Such preparati.ons will often remain alive for 3 weeks. 2 You should 1 For an excellent short account of vertebrate tissue culture, consult "Tissue Culture," by E. N. Wilmer, Methuen & Co .• London, 1935. 2 Gatenb~r ltnd Hill. (Juart. Jour. lYJ-icr. Science, Vol. 70, HJ34, and Gatenby, Hill and Macdougald, ibid, Vol. 77, 1\):~4. 22 TREATJYIENT OF LIVING CELLS notice that the slides, covers, scissors, etc., should be frce of chemical cOllta.millation (§ 14). With these preparaOVT A D F1G. 2. A. Outline of Hellx aspersr:t, showing at OVT position of ovotestis, at RPC position of pulmonary or mantle cavity. B. From a smear of ovotestis of Helix, showing sperm (SPZ), spermatogonia (SPG), spermatocyte (middJe) and spermatid (above). N = nucleus, M = mitochondria, GA = Golgi apparatus, YC = yolk cell. C. Explant (EXPL) of Helix, ~howing outgrowing cells. (By courtesy of the Editor of the Q.J.M.s.) D. Typical insect spermatid, showing nucleus (N), mitochondria (M), Golgi apparatus (GA), "yolk granules (Y-granules YG). aseptic precautions are not necessary. The slides can be left on the bench provided you ring them with vaseline. Just melt vaseline in a vessel, warm a pipette, tiOllS, Ol'O'lIES'l118 SMEAR 23 and with this place It wall of vaseline half OIl the edge of the cover, half on the slide. Note. Helix nemoraUs and Helix pomati{t are not so favourable for this experiment, their tissues not being so hardy as those of H. aspersa. Pieces of mantle cavity mounted in the blood of the snail often grow out quite well. Ordinary salt solution buffered to pH 8 is sometimes quite successful. Commercial buffer tablets from 6'8 to 8·4 are available. 26. Hanging-drop Preparations. If you try to examine a liquid preparation mounted between a coverslip and slide, you will notice that even although the oil used for the immersion lens be thin, the cells keep streaming about as you focus. This is caused by the lifting or pressing down of the cover. If you must use this type of preparation try the thinner methyl benzoate instead of cedar wood oil. In any case the hanging drop preparation is the one to use. Take some wooden matches and cut lengths to make supports for a 20 mm. square coverslip. Smear them with vaseline and make a neat square, upon which the coverslip can rest and be sealed. Place a small drop of the liquid to be examined in the middle of a coverslip of suitable size. Invert over the prepared square of matchsticks and press down. If the drop is too big, it will run on to the vaseline. Note. There are round glass rings which can be bought for making these preparations. Small size curtain rings or rubber washers do quite nicely. 27. Helix Aspersa. Ovotestis Smear. Dissect out the ovotestis of the snail (OVT in Fig. 2, A) and touch the middle of a coverslip with it. Invert the smear over a prepared cell either of matchsticks or a vaselined glass ring. Examine under the oil immersion. Both mitochondria and Golgi bodies (nebenkern) can be seen in the living spermatocyteR and spermatids (Frontispiece and Fig. 3,B ).1 1 Gatenby, Proc. Royal Society oj !-vndan, Vol. 104, B, 1928. 24 TRIiJAT1I:lEN'jl OF LIVING OELLS Since junior students usually are llot provided with an oil immersion objective, preparations of this type should A. Making It smear of bluotl. ctl). B. lvIet,hou of making It coverslip smear. C. Metal imbedding box madt) from two bent pieces of brass. ]1'10.:l. Le demonstrated by the teacher, who will possess the best apparatus, 28. Testis of Gryllus 1 (the Cricket) or Lepisma 2 (the Silver Fish). Dissect in a drop of saline (§ 24) on a slide under dissecting microscope with t,yO mounted needles and remove testiR. Tease slightly in the middle of coverslip and invert over glass ring or matches. Note in spermatids, Golgi bodies, mitochondria, acrosome, Y granules (for the latter use also Nile Blue sulphate, § 66). Given a critical light and good powers, this type of material is very favourable for studying the cytoplasmic inclusions throughout their entire cycle in the living cell. 29. Neutral Red. In the above preparations, subsequently lift the covers carefully a,nel add a very small drop of pink neutral red in saline or use prepared 1 H. Herbert Johnson, Zeit.Jur Wi88. ZooZ., 1.10, Band 1, Heft, 1931. 2 R. N. Mukerji, Jour, Royal :M'icro8copical Society, Vol. 49, 1!J2n. NEU'l'RAL RIClJ 213 coverslips (§ 31). The neutral red will become segregated in certain parts of the cells (Frontispiece). This usually happens in about 30 minutes. Note that the neutral red should be a pink solution, not red. The concentration is I in 2,000 to 1 in 10,000, but if yon add a little pink solution to ea,ch preparation, one of them is likely to be exactly right for observing the phenomena in about 30 minutes. It has ah'eady been mentioned that in solutions with a pH value above 7, the colour is yellow. Note. This is an important negative test for the Golgi apparatus. If some body or area stains in neutral red this is a certain test that it is not Goigi material. Hed globules often appear inside the Golgi apparatus, and thus mark its position. 30. Janus Green. The right conccntration of this dye for staining mitochondria vitally is less easy to determine, and the effect is often fugitive. The correct concentration is 1 in 10,000 to 1 in 20,000, and in a test tube the solution, so weak that the colour can hardly be noticed, will stain energetically. Stronger solutions are very toxic. As with neutral red, the addition of varying quantities of the dye solution to a number of preparations, to be examined at intervals up to 1 honI', will generally be satisfactory. You should notice, however, t,hat only certain brands of .Janus Green will work. Also SOllle specimens of Neutral Red are better than others. 31. Preparation of Coverslips or Slides for Vital Staining. Dissolve Neutral Red, .Tanus Green and Brilliant Cresyl Blue in plU'e absolute alcohol to 1% strength. These solutions, or diluted .vith absolute alcohol up to ten times, are thinly smeared on a warm cover 01' slide and allowed to evaporate. A drop of fluid containing the organisms is put on the slide or cover according to whether a,n ordinary or a, hanging drop preparation is needed. The organisms or cells should stain in from 5 to 30 minutes. Different cells or Protozoa 26 11REAT1I1ENT OF LIVING CELLS require different concentrations, but 1/10,000 IS about right for Neutral Reel. 32. Examination of Fresh Water Protozoa or Pond Animals. This should be carried out in water, not in the saline media mentioned above. But if you dissect the pond animals, this should be done in a saline medium. Examination of marine organisms should be carried out in sea water, their dissection if for cytology in appropriate salt solution (§ 24). 33. Toxic Examining Media. 'rhese kill the cells or organisms :(a) Methyl G1'een Acetic. Dark green aqueous solution with (H% acetic acid. This solution is suitable for examining Protozoa for class purposes. It stains the nuclei green after a few minutes, but is non-specific for chromatin. (b) Iodine Solution. Use a drop of Lugol (§ 71). A dark bl'O'wll colouration generally, but not always, means that the body is or contains glyeogell. This solution can be used for examining f::eces for amCB ba cysts. (c) Osmic Acid. One per cent. solution. A little is mixed with the organisms. 'These fine preparations last for a long time provided the osmic does not evaporate. A good general stain for neutral fat. Not specific, however, for this. (d) Formaldehyde. Four per cent. solution (§ 112). Usually preserves the colour of the organisms for some time. Toxic Vapours often used are those of osmic acid and formaldehyde. The wet smear is held over the uncorked bottle for a minute. Iodine vapour produced by heating a crystal in It small tube is sometimes used after staining in Janus Green (§ 30). CHAPTER III FIXED AND ST AINED SMEARS 34. The Validity of the Fixed Preparation. The following organic substances or inclusions are soluble in either water, acetic acid, alcohol, or xylol, or alcohol and xylol mixed: fats, mitochondria, Golgi material, glycogen, lecithin, cholesterol and certain other related substances. vVhen you fix or coagulate the tissue by certain chemicals, some of these substances are converted into materials less, or not, soluble in alcohol, dioxan, or xylol, but usually still in a state when their partial disintegration occurs during imbedding, dehydrating and clearing. Thus if you fix a smear of spermatids or Protozoa in alcohol corrosive sublimate (Schaudinn), wash in water, bring through alcohol and xylol and mount in balsam, you have remaining probably all the simple and conjugated proteins but nothing else (Frontispiece). Thus the pictures and descriptions of cells and Protozoa in most of the text books are mere skeletons, with all fats and carbohydrates raked out. Moreover, the ground cytoplasm and nuclear material left are nearly always net-like or thread-like in appearance, which is artificial (W. B. Hardy). This difficulty has largely been surmounted by the development of chrome-osmium fixatives and the formalin silver techniques (Frontispiece). Bichromate of potash and osmium tetroxide together (e.g., Champy) cause nearly all fats, and carbohydrates like glycogen, to become insoluble in water, alcohol and xylol. Delicate fats always tend to become dissolved even after long 27 2R .FlXED AND STAINED SMEARS t,reatmellt in mllllium tetroxide. Of all the Iixillg methods, chrome"osmiulH is the hest, and cells so fixed and rnmmted in (llycertne are morphologically as nearly identical with the living cells as is at present possible. The protoplasm of such fixed cells is smooth like that of the living ones, while the Vttrious cell grannIes are faithfully preserved. 35. Fading and Permanence of Microscopical Preparations. The object of making microscopical preparations is to procure a series of objects which can be studied under the microscope in a manner which willl)rovide as much information as ]Jossible about the nature, structure or microchemistry of the material. ]i'urthermore, it is desirable that such preparations be as l)Grmanent as possible. This meallS that the stain, if any, and the mounting medium must bc suitable. No preparation, however well made, will stand the direct Hunlight for long. All stains fade, and some mounting media become darkened, cracked or disintegrated by exposure to sunlight and heat. Of the common stains, the anilinc, such as methyl blue, the carmine, ·such as borax carmine, and the hmmatoxylin, such as Heidenhaill's alum h::ematoxylin, fade in the order given. Ehrlich's h::ematoxyIin, especially if mounted in acid balsam, is particularly liable to fade. Carmine and hmmatoxylin stains are usually quite permanent if kept in thc dark. Both osmic and silver slides are liable to fade or disintegrate. Stained sections should be kept in special cardboard boxes made for the purpose, or in slotted 'Wooden boxes with accurately fitting lids. 36. Tap Water, Tap Water Substitute, Alkaline Water. Some tap waters are alkaline enough to H blue" hmmatoxylin, and to wash traces of acid out of stained sections and smears. Others are not suitable, and in that case you keep a tap water substitute, which is merely dist.illed, or more or less neutral t.ap water with 0·2% to 0'5% sodium bicarbonate added. This is washed off with distilled water. S. G. Scott's tap water substitute is KHCO a, 2 gm., MgS0-t, 20 gm., Aq. dest. 1,000 e.c., with a crystal of thymol to prevent growth of moulds. Tap water substitute is not intended for "lvashing out after fixations. It would be unsuitable for this purpose. FUII'EAR 'l'JjJ(}!i.J,.\TIQUE 2ft 37. Acid Water, Acid Alcohol. Acid solutions aee llsed to differentiate over-stained smears and sections. The Rtrength is (}5% HCI in water or 70% alcohol. 38. Smears. The smear preparation does not call for the cutting of sections and may therefore he dealt with first. At the same time the making of a really good smear is not as easy as it may seem. '1'he smear pl'epaJ:atioll is a necessity in Hrematology, Protozoology and Cytology. Owing to the nature of the material in the tv,ro former branches of biology, the smear is a, necessity, and even in the last, smear preparations are almost exclusively used in laboratories where material like DroSOl}hila is being studied. The main requirements of a smear are that it should be made on a clean coverslip (No.1 preferably) awl that the material should be thinly spread. The reason for making smears on coverslips rather than on slides is tha,t the slips take up less space during preparation. On the other hand, they are fragile and not so easily manipulated as slides, and have a Wety of floating on top of one another in a dish. Some people prefer slides. Smears may be (a) examined fresh, (b) fixed and stained, (c) treated with a simultaneous fixative and stain mixed, (d) made on coverslips previously prepared by smearing with some stain (§ 31). The preparation of the slips for smearing begins with their cleaning. This may be easy, or quite difficult, depending on the state of the coverslips when bought. At one time coverslips during manufacture became covered with a thick layer of material which had to be removed by chemical means. Nowadays they clean quite easily, in acidulated alcohol (§ 37), with a chamois, or silk handkerchief, or ordinary duster. Careful people clean coverslips as in § 14, and store in a glass box with a ground top. The thickness of the coverslip is important, mainly 30 FIXED AND S':PAINED SMEARS because if they are too thick the oil immersion objective will not focus the SHl.ear, as the focal distance of many of these objectives is quite small, and if too thin the slip usuaJly breaks at the critical moment. Here again a good box of No.1 coverslips will be all that is necessary, as their manufacture, or sorting, has improved in recent years. Careful protozoologists have a special instrument for measuring the thickness of the coverslips. These are pushed between a screw and a plate, the thickness of eac:h c:overslip being read off, and unsuitable ones discarded. In general, such a precaution is not necessary and you can do quite well without a coverslip measurer. Yon should hit ve on your bench some bottles of graded strengths of ethyl alcohol (§ 16), some shallow petri dishes provided with lids or glass covers. In these the smears arc treated. The alcohols should range from 50%; 70%, 90%, to absolute. In the case of smears the 30% strength is not really needed. Of course, if you wish to be more modern, you may discard the alcohol, and use dioxan, which mixes with water, paraffin wax, balsam, alcohol, and all the oils in general use in laboratories, as well as dissolving celluloid. Dioxan should only be used for special work where the alcohol benzol methods are not so suitable, as for instance, cutting sections of Crustacea and Insecta. Then, in addition, it will be necessary to have a dealcoholising agent, which means an oil or clearing fluid which will mix with balsam (the mounting agent) and alcohol, the dehydrating agent. Now most people use xylol, but I think benzol (and benzol balsam) is better, for it becomes less acid. 39. The Smear and the Section-Differences. It may be noticed that· the smear may not stain as uniformly as the section. In explanation of this it should be remembered that the section is of uniform thickness and the material from whioh it has been cut was left at least a. day and a night in alcohol, whereas the smear is usually hurried and uneven. DIFFERENTIATION 31 In pracLioally all branche8 of cytological techni(IUe where sections are \Ised, it will he noted that not all pa.rts of the material are equlIJly advantageous to :;tudy. TIllis the enormous aggregation of cells of the sectioned material in a way gets over certain faults in fixation, and thence in staining. With the smear the fixation method and time have to be just right, for you have only one layer of cells and these become directly exposed to the fixing fluid. In general, the fixation times for smears may be shorter than those given for pieces of tissue. 40. Differentiating Stained Materials. Differentiation (i.e., the production of contrast by ext.raction of the dye from different parts) of nearly all st.ains is done in acid water or acid alcohol; other-s differentiate in alcohol, or in iron alum. The object of differentiating is to remove the stain from those parts where it is lightly held, so that contrast between these and the organs which ho1el the stain fast is brought about. It is the most difficult part of staining. Generally there are two ways you can stain an object: either use a very weak solution of the stain and let it be taken up gradually by those parts which have the greatest affinity for the dye-this method needs 110 differentiation and is known as progressive staining-or leave the object in such a strong solution that almost everything is deeply stained. You must then differentiate. This is called regressive staining. Both regressive and progressive staining can be done well with carmine and hoomatoxylin stains like Ehrlich (in which the alum is mixed ·with the dye). Generally speaking, you should remember that a section, smear or whole mount is going to be more transparent in balsam than when you look at it in water or alcohol during differentiation. It is a worse fault to under-differentiate than to over-differentiate, because if you bring the slides or objects up to xylol you can examine them before mounting in balsam. If they look all right, mount in balsam; if not, it does no harm to go :32 FIXED AND STAINED 8lYfEARS back and continue differentiation. Over-stained preparations which have Imen years in bahutlll can be dissolved off by leaving some hours or longer in warm xylol if the balsltID is thick, if not in cold xylol, and transferring i:mbsequently to alcohol and thence to the ditIerentiating reagent. 41. To make a Schaudinn or Bouin Smear. Smear the ovotestis of the snail, the testis of a mouse or rat, or, if you prefer this, use a mealworm or centipede, or other organism containing protozoan p<1rasites. Cut off one end of the body, and by carefully detaching a part of the othm: end pull out the gut, placing it on a, slide. Cut in two with (t knife, lift a piece with fine forceps and smear on a coverslip, beginning at the top and smearing with a zig-zag motion (Fig. 3, B). Large pieces of the gut should be taken up and smeared where they have broken frOID the main piece. Immediately drop face down on the Schaudinn's or Bouin's fluid in a petri dish. Leave at least !- hour. If the coverslips sink under the fluid, turn them smear upwards. For the Schaudinn smears notice that corrosive sublimate will corrode the metal of your forceps. Use matchsticks, or quickly wash your forceps under a tap and wipe. After t hour, pour away the fixative gently, and wash out the petri dish carefully with tl1P water, then add. 50% alcohol (for 5 minutes), then 70%. The Schaudinn smears must be washed in 70% alcohol (sherry coloured with Lugol's iodine, § 71) for an hour-the Bouin's smears shoulclremain in 70% for at least 1 hour. The point to notice is this-you cannot be sure to stain smears evenly by this method unless they have been in 70% for at least 1 hour. Where there are lumps in the smear it is better to go up to 90% strength for an hour, and the best resttlts are got by leaving the Stnea1"8 in ~O% overnight. In any case do not try to rush matters-it does no harm to leave these smears in strong alcohol for several days as 33 BOUIN SMEAR long as t.he petri dish is covered. Rcmember that you have akeady dest.royed everything except proteins, so the sojourn in alcohol does not matter (§ 34). When the smears have been in clear 70% alcohol for at lear-;t 1 hour (note therefore that the Schauclinn smears which have been in iodine and 70% mllst have been "\vashed for that period in clear 70% to get riel of most of the iodine) you may think about staining. The stain may be Delafield's, Ehrlich's or Heielenhain's h~-ell1atoxylin, alone, or followed hy Orange G. Take Delafield first, as it is the better of the first two for beginners. The stock Delafield is usually too strong, and must be diluted (take 1 part of stain and 2 parts of distilled water, or slightly acidulated tap water (§ 37) ). Cover the smears with stain and leave 10 minutes, when the staining should be complete. 'When, however, about 5 minutes have elapsed, it is a good thing to rcmove a coverslip, place it smear side upwards on the middle of a slide and to look at it under the microscope, See that the smear is kept wet with stain, or water, but elo not run the objective into the fluid while you are looking at it. With a ~rd objective, try to find some parasites (or cells), and see how they are staining. Possibly their nuclei are already a good reddish colour, and it would be the proper thing to bring the rest of the smears into water, while you decide what you are going to do. Water willl10t hurt them. If you arc not sure whether they are stained rightly, put one of the smears into tap water ill a larger petri dish, or just run the tap gently on it, or " blue" it, as the saying is, in alkaline water (see § 36). Then put it on the slide again and re-examine. It may be too dark, which lUeans the colour must now be extracted a little, or not dark enough, in which case all the smears go back to ..the diluted Delafield, and you wait a little longer. Now if the slides are too dark-that is, ifthe cytoplasm is too blue, and there is not a prop~r difference J.ADOR.!.TOIlY l'llCILNIQUE. 2 34 FIXED AND STAINED 8MEARS in Hhacle of colom hetween nucleus and cytoplasm, you POIlI' off the :.;taill and add acid water (§ :37) to the petri dish. A minute in the a,cid water will uHllally suffice. Then pour this off tl,nd place under tap water (or subRti· tute § 30) till blue. Now if everything has been done properly, the nuclei will be a sharp dark blue colour, and the cytoplasm will be grey or grey blue, and much lighter in colour than the nuclei. Notice, however, and this is important, that when finaily mounted in balsam, the preparations will be more tmnsparent, and the colouring not quite so opaque, so even if they look a little darker t,han desirable when in. water, all may be well. 42. On Counterstaining. The nuclei are blue, and it is desirable, but not necessary, to stain. the cytoplasm of the cells in It contrasting colour, say red or yellow. If you want to stain in a red dye, eosin is the stain sometimes taken, but I prefer Orange G. It is a more delicate stain, and easy to work. Saturate some absolute alcohol with a little Orange G. The depth of colour will be just right. Now transfer the smears to 70% alcohol for 5 minutes, pour off, and add 90% alcohol, and with two mounted needles, or forceps, lift or push aside each coverslip so that the 90% gets underneath each slip. Unless you do this at each stage, you may find thai; when you begin to clear the smears in oil, a little l)ocket of water lies under each coverslip, and your preparations are spoilt. After 10 minutes in 90%, pour off, leaving the coverslips in the dish, and with a duster remove the last few drops from the tilted petri dish, then add some Orange G in absolute alcohol, cover and leave 5 or 10 minutes. Pour this back into the bottle, and quickly add some absolute alcohol, move the coverslips about as before, cover and leave 5 minutes. Pour off alcohol, and add xylol or benzol. If the fluid goes millry the alcohol contained too much water. Take Some slides, rub clean with a duster, place a COUNTERS:f.1AINING S1fudl drop of benzol or xylol bn,lsam in the middle of the slide, take up t.he smear wit.h forceps, give fL quick flip to throw off excess xylol or benzol and rapidly place smear side down on t.he drop of balsam. The hLtt.cr should run under the whole coverslip-if not the drop was eit.hcr not big enough, or the balsam was t.oo thick. Be careful, howeycr, not to have the SlUeltr resting on a nlUch too large amount. of balsam. Set the slide aside for a few minutes, preferably in a ,varm place. Hit goes white, 01' is full of droplets 'when examined, you must have breathed on it, or on the slide prepared with the drop of balsam-or your absolute is not right. Note that if you use dioxan balsam or Euparal you lllay mount the stained smeal' from H5% alcohol, and ncednot take it tlll'ough absolute and xylol. If y01~ wish to stain in Orange G, you can mount in clioxan balsam after connterstaining and dipping in clear absolute alcohol. 43. Evaluation of the Method. Preparations made thwI give the appearance found in the pictures in recent manualR on Protozoology, and the older Cytology books. That is to say, a cell or protozoon with nearly all its internal organs except .nucleus and kinetic apparatus completely raked out. Such preparations are useful for identifying stages of Protozoa, but for nothing else. So far as the validity of such preparations is concerned, that is to say, the resemblance between the living animal or cell, and the end fixed and stained result, see § 34 and Frontispiece. On the question of nucleoli and chromatin, such preparations have been superseded by the modern "Feulgen nuclealfarbnng" method (§ 76), and give no precise information about chromatin. Notice. 'When it is said to " pour off" alcohol, say, of 70% or BO%, it, may be put back into the bottle from which it came unless it has been mixed with too much water or is badly stained. In theRe cases keep a waste alcohol bottle. It may be re-distilled or may be useful for some other purpose. 3G FIXED AND S'l'AINED SMEARS 44. Staining in Ehrlich's Hrematoxylin. Bring the smeai:s down to water, add some stain and leave 5 or ·10 minutes. When the nuclei are a deep red, differentiate slightly in acid wateI;' (§ 37), and afterwards" blue" in alkaline water (§ 36). Counterstain in Orange G. 45. Iron Hrematoxylin 1 Stained Smears. The smear, .. fixed in Bouin or Schaudinn, is brought to the stage def:lCribed ill § 41, where it is ready for staining and has been in 70% for at least 1 hour. Bring it down to water and wash away the alcohol (in distilled water a few minutes), then place in 4% iron alum for 1 honr, wash slightly in water (a few dips) and transfer to 1 % hannatoxylin in water, 01' Regaud's hrematoxylin (§ 147). Leave 2 or 3 hours. 'rransfer to water for a few minutes till the excess hrematoxylin has been washed away, then into 4% iron alum in which differentiatioll (extraction) takes place. After a minute or so, place a smear on a slide and examine with a -ird-in. lens. If the smear is beginning to look clear, it is better to pour off the alum from the others in the dish, and place them in water while you re-examine the other smear under the microscope. Remember that it is better to under-differentiate, as darker smears can easily be put back into alum and extracted to the right degree, but if you differentiate too much you will have to begin all over again, which does not produce good preparationsso proceed cautiously with the extraction of the stain, remembering that you can stop or slow down differentiatio.n by transferring to water, or by diluting the iron alum solution. When the nuclei appear blue-black, the cytoplasm grey, the differentiation has gone far enough, and the smears must llOW be washed under the tap for at least 15 minutes. Then counterstain if you wish; the method for Orange G, described in § 42, is good. Most people never counterstain iron alum hrematoxylin preparations. 1 Heidenhain. HEIDE.NHAIN AND LEISHJ.1IAN.;.Y 37 Note. The reautioll can be made more intense by using warm [.lU111 and sbin. 46. Evaluation of Method. Of COUI'He t.he method cannot be good as it stands, beeause the fixation is a poor one. The stain is one of the most beautiful and useful ill biology, easily eontrolled, the right colour for long hours of observation under the microscope, very intense, and. permanent except in direct sunlight. vVhen, however, combined with good .fixation, this stain is prolHlbly the best general cytological method. It is mueh used by protozoologists, and for the metaphase counts in chromosome studies. It is llot good for nucleoli. For these use Feulgen, Giemsa, or Mann's methyl bll1e eosin (which see). 47. Counterstaining in Eosin. The Delafield or other hrematoxylin smears stain well in eosin. When you have already stained the smear and it is washing in water, dip it for a short time in eosin (w:ttery or alcoholic) about the colour of red ink. 'Wash in water (or 70% alcohol for the alcoholic eosin) until most of the eosin has been extracted, transfer to 90%, absolute, 5 minutes each, and xylol. Mount in balsam. 'rhe danger with this stain is that beginners usually make it too dark and get a horrid red llless all over the smear. It is not a good stain, and if yon· must have a counterstain, Orange G is better. Counterstaining in Van Gieson. This is excellent for sections of vertebrate histological material (§ 155). 48. To make a Blood or other Smear Stained in Leishmann (Romanowsky, § 174). Leishmann's stain is: to a 1% solution of the best medicinal methylene blue in water add 0'5% sodium carbonate, heat to 65° C. for 12 hours and let it stand for 10 days. Then add an equal volume of 0·1 % solution of eosin extra B, let it ' stand for 6 to 12 hours, collect precipitate on the filter paper, and wash; wash with water until the filtrate .FiXED AND STAINED SMEARS 3R comes off nearly colourless; dry nnd powder. DiRsolve 0·15 gm. in 100 C.c. of pure 'In ethyl alcohol. If you got the correct eosin and methylene blue the stain will ''lurk, if not, it is better to buy it made up-there are numerous good dry and. wet Leishlllann stains on the market. In America a modification of this is called Wright's stain,l and is almost universally used there. 49. For Blood. Take some very clean slides (there is no advantage in using coverslips with. this stain) and make a blood. or other smeal.'. To make a blood smear place a small drop at one end of the slide and bring another I>lide against the drop as shown in Fig. 3, A. Push this slide along in the direction of the arrow. If the slides are dirty the smear is unsuccessful. Dry the smear (in the case of the blood Iilm) by waving it in the air, put it on the bcnch and add sufficient stain to cover the smear, leave 1 to 2 minutes, flood (not wash off) with about [) times as much distilled water, agitate the slide and leave for 1 minute. Tip off the mixed dye and water, blot by gently placing the film on a flat piece of smooth filter paper or blotting paper. You may either now mount in Euparal or balsam, or as is generally done, leave without a coverslip. "'To use, drop a little immersion oil (cedar wood) on the slide, and examine with an oil immersion. For gut and other not very liquid smears, the stain can be added immediately without drying the smear. Nuclei should be in shades of red, cytoplasm bluish, parasites blue with ruby-red chromatin. 50. Evaluation of the Method. A remarkably beautiful and elegant blood stain which is not likely to be superseded. Nearly everything except protein material is raked out of the cell. The stain fades badly in most cases, and slides without covers become smeared with. dust. It is recommended to mount in Eupaml, after blotting. For Wright's stain add 10 drops, leave 1 minute, add 10 drops of water, leave 1 to 2 minutes, wash off in distilled water, blot, mount in balsam. 1 (li~tilled C!HIWAIE-OSfilIU.JlI :P1iJCHNIQUB an This stain is useful for diagnosis of blood and blood disease!>, not for prec:ise Protozoology. 51. Re-staining Faded (Romanowsky) Slides. Remove coverslip, if any, 'with xylol (or warm absolutp alcohol in case of Eupaml), place in HO% overnight, bring down to water, place in 4% iron alum for 1 hom', wash quickly in water, place in 1% h:l'matoxylill for 3 hours, differentiate in 0·5% HOI ,mter. Wash under tap for 15 minutes, npgrade in aleohols, mouut in balsam. 52. Keeping Uncovered Romanowsky Slides. These should be kept in air-tight boxes if the lallOmtory is situated in a smoky town, as the film of sticky dust which sometimes get:;; on them is impossihle to remove. If the slides are to 1)0 stored for long periods they may be rolled up ill stnps of paper. 53. Carnoy Smear (§ 118). This fixfLtive is suitfLhle for al'tlll'opods, etc., containing impermeable protozoan cysts, and is the fluid to use for such things as smears of PScudo-llavicellre of Monocysts. Fix smears for at least 1 hour, then transfer to 90% alcohol to wash out overnight. Bring smears to 70% alcohol, transfer to 1% iron alum in 70% alcohol for 10 minutes, rinse in 70%, place in 1% hromatill in 70% alcohol for 10 minutes. Differentiate in iron alum solution, or hettel', acid alcohol 70% with 0·5% Hel. Wash out in several changes of 70% alcohol. 'l'his stain is Dobell's alcoholic luematin. 54. Evaluation of Method. Destroys all fats, lipins, and can only be regarded as a rough miero-anatomical met,hod. The result is not ilS good as can be done with impermeable cysts. ]'01' research work on cysts, cut frozen sections of unfixed material and transfer to a good fixative, such as Champy or Weigl. This will easily enter those cysts which have been cut open by the knife. Staining may subsequently be done in Heidenhain or :Regaud, or acid fuchsin. 55. Champy or Flemming without Acetic Acid Smear. These [l1'e t·wo ',vell-known fixatives which are definitely superior to Bouin 01' Schaudinn, but the method is more difficult. Smeal' as described in § 41 for Bonin, clrop 4n FIXED AND STAINED 8JJIEARS immediately on to a thin layer of Champy or F. W. A. in a Hat petri dish, cover and lmtVe overnight. It will nob hurt to leave the smears in fixative for 24 hours or even several days if well covered. Now here arises the diffic:ulty. You must wash out the fixative, but for how long 1 Generally, if the Slllears have been fixed overnight, an hour in running water next morning will be the right amount. If fixed for several days at least 5 or 6 hours, and for thick smears, overnight. In general, the over\Yashed smear showns chromatin centrosome and kinetic bodies better, the underwashed, Golgi bodies (invertebrate) and mitochondria better. Thus, if you are getting preparations somewhat like Bouin fixed smears, you l1re definitely washing out too long, or not staining long enough. If you cannot get the smears to stain in iron hmmatoxylin properly, they are probably not washed out enough. The stains to use are iron alum hoomatoxylin or acid fuchsin. In either case unless you are using chlorocarmine (§ 150) before you begin staining you must leave the smears in alcohol after they have come from the wash in water. They will stain, it is true, without this treatment, but not evenly or well. Here is a good plan for iron hoomatoxylin. Leave a day and a night in fixative, wash next morning for I hour, transfer to 70% till next morning. Wash in distilled water for 10 minutes, and place in 4% iron alum all day, and before you go In the evening wash the smears slightly in distilled water, and leave in Regaud's or Heidenhain's hromatoxylin till next morning. Wash off excess stain in water, and differentiate cautiously in 4% iron alum, diluted a little if necessary to slow up the extraction. After differentiation wash at least 15 minutes under tap or in substitute (§ 36), upgrade and mount in balsam. Champy and F. W. A. are often post-chromed in 3% bichromate of potassiulll for three days, then washed out aOLGJ APPARA'PUS S.MEARS 41 several hours, brought into 50% alcohol 30 minutes, 70% ovcrnight. They lllay be stained in Regaud'H hrematoxyliu (§§ 147, 1,18), or acid fuchsin (§ 154). Try the method if you find your Champy smears are not. succeeding as you would wish. 56. Gentian Violet Ita!; he en used with succeRS on Champy films fixed overnight or hetter for shorter periods (§ 10;1). It is a chromosome and nucleolar stain, and can be very nicc. The method is given ill § Ifj5. " 57. Evaluation of Method. This method is excellent for germ cell smears, Protozoa, etc. It usually gives fat as well, but not always, and to study the fat you must have recourse to special methods (§ OH). Uell granules are definitely very good by these methods. The method is valuable for spermatogenesis where whole spcrmatids or spermatozoa, are to he studied. One of the difficulties of the method is to get the time of washing out, just right. In this and in other similar difficulties, the propel' thing to do is to have a number of smears (or pieces) washing, and take them out at intervals. 58. Bleaching Chrome-osmium Smears. POl' nuclear and kinetic apparatns it will often be found better to bleach in hydrogen peroxide 4% in water for a few minutes. Then transfer to 70% for some hours and proceed ,vith staining. Thc botanists prefer washing out sueh material in tepid water for 2 hours. Some workers bleach in aqueOllR pink permanganate of potash solution (0,5%) followed by 4% oxalic acid. Goigi Apparatus Smears 59. The Aoyama (Cajal) Smear. This is a silver method and often gives very interesting results on smears. The difficulty in the formalin silver smear is a flocculent precipitate whieh often appears over the whole coverslip, not in cells, and which is apparently due to impreglU1tion of broken pieces of cells and coagulated proteid body fluids. 4:3 FiXED AND 8.'1'AINElJ S.MEARS If you make a good many smears Home are better than others. Fix a number of coverslip smears in Aoyam!L (§ 170) and take them out every 5 minutes up to 30 minutes. Dip in distilled water for 1 minute and transfer to newly made-up 1·5% silver nitrate in water for several houn, at 22° C. vVash slightly and reduce as described in § 170, wash in water, upgrade, mount in balsam. ~rhis method may show spinelle bridges and mitoehondria, and in some cases is remarkably specific for the dictyosomes of the Goigi apparatus. It is an interesting and valuable method. 60. The Weigl (Mann-Kopsch) Smear. Take some small-size coverslips (! in.). Make smears of insect or vertebrate gonads. Drop into Manu's oSlllo-sublimate (§ HI). Leave 30 minutes, wash several minutes in distilled ·water, transfer to a tube of 1 % or 2% osmic acid (Kopsch), cork ·well, place in incubator at 30° C. Leave 2 days, then examine. If the impregnation has not proceeded well, return to the tube for another day. Pour off after 4: days, fill with distilled water and put back in incubator for the night. Wash a few minutes in fresh distilled water again, upgrade, mount in xylol bals~1m. If you wish to stain nuclei, use a water solution of neutral red (water, 1,000 c.c.; N. R., 1 gm.; glacial acetic (Lcid, 2 c.c.). Stain from water, and transfer immediately to absolute alcohol, xylol, balsam. 61. Evaluation of the Methods. Rarely quite so good as the sectioning methods (§ 170). The Aoyama method is cheap, and worth trying, though definitely difficult on account of broken cells and coagulated body fluids. Sometimes alteration (usually shortening) of the times of fixation and silvering may produce better results. Reductioll (§ 170) of smears takes place quiekly and need not be llrolouged. What is critical in the Aoyama SlUear is the time taken for washing out after fixation and silvering. The animal spermatocyte is the best material for prac- lVliJIOL 8.MJiJAR tising the mcth()(l. UfiC Helix Of Mm; teHtis filllear;,; in ·which the Golgi apparatus iH well marked. The mallie methods are, of eoun:e, the beRt known to the cytology of the cytoplasm and can be remarkahly i:lpecific for the Golgi bodies. Interpretatioll of the smears is often diffieult. Your results should be compared with those got with seetioned material. CHAPTER IV MICROCHEMICAL TESTS IN SMEARS 62. Many modern microchemical tests work very well in smears. Of those given here, the fat tests are easy ~Lnd valuable. The Feulgen "Nuclealfarbung" method is altogether admirable, and easy to work. The glycogen methods, usually supposed to be easy, can be difficult in material other than mammalian liver. The lecithin and cholesterol methods work fairly well. The living l1totoplasm in some cells (which includes uni- cellular organislll!; likc Protozoa) may be extraordinarily lilllpid and clear of formed granules. In other cases the numerous granules lllay consist of starch, glycogen, fat" volutin or protein formations associated with certain metals and so all. Occasionally questions do arise as to the nature of such formations, and it is well to have learnt the more trustworthy of these methods. They must be used carefully, and the information they supply cannot in all cases he regarded as trustworthy. Note. If possible use daylight when judging colours in microchemical preparations. A yellow light is useless. 63. Fats. Some years ago we believed that the Weigl or Kolatchew osmic methods, followed by imbedding and sectioning in paraffin wax, would make it possible to study all demonstrable fat. It is certain, however, that the newly-formed fat of a more liquid and' delicate nature (e.g., in oogenesis), even though thoroughly osmicated, is unable to pass through heated xylol without being partly or wholly dissolved. Moreover, some histologists are sceptical as to the value of certain of the microchemical tests for various types of fats, and these test~ have had to be jettisoned. .Two tests we still give here, which are regarded with suspicion by some chemists, are the osmic 44 FAT 45 and the Nile Blue sulphate. The osmic method is useful for comparison with the Sudan, lH1d the Nile Blue is certainly still useful for m:trtl .~tU'i}lin(/, the impurity of the dye and itfl solution or segregation in three (lifferent colou]'s being an interesting ami llseful phenomenon in certain cases. For example, in the Y -granules of insects, no other method is so beautiful. In ad(lition it is known that in tissues, fats are pmctieally never pmc. hut [we a mixture of several kinds, so that the testR for the various tYI)es of fats were not of lllu(Jh use except in rare cases. Compare, however, a frozen section of ovary done hy the cholesterol method of Schultz, § 68. 64. The Osmic Test for Fats. Make a smear on tt coverslip and plaee it on a eh'op of 2% osmic acid on a slide. Leave in a petri dish for some minutes to honrs, examining from time to time. The true fats, phosphatides, cholesterin-fatty acid mixtures, and cholesterin, bla.cken or go brown in almost the order mentioned, ordinary tissue fitt blackening quickly. After fixation of tissues in biehromate of potassium (3% in water), true fat usually blackens easily. Myelin blackens easily in osmic alone, but not in chrome-osmic (Altmann's :fluid), while it if! said that cholesterol will not blacken in osmic alone, out will in Altmann's fluid. In certain Protozoa there exist granules of unknown nature ,vhich blacken immediately in osmic, but whieh are doubtfully of a fatty nature. Now the osmic test is a useful start, but must not be regarded as anything else but that. Proceed to the Sudan IV test. 65. Sudan IV Smears. Fix smear8 in formalin vapour as follows. Saturate a little cotton wool in strong formalin, place in petri dish, and warm. Put smears in . this, cover for 5 minutes, but do not let them dry. Transfer smears to a little saturated solution of Sudn,n IV 1 dissolved in equal parts of 70% alcohol and pnre acetone 1 Not Sudan III. Hi j}J[OROOHJiJJ.1IIUAL 'l'ES'l'S IN SJJiEAR8 (does not keep long). Leave 5 minutes in stain. Dip into 70% alcohol, \vash in water, counterstain if you clesire in Delafield or Ehrlioh, mount in glyoerine or Farrants. Fats bright red. 66. Nile Blue Sulphate Vital Staining of Fat (Lol'rain Smith). The above methods kill the cells, bnt it is possible to stain vitally fat granules by means of Nile Blue. Make c1, smear on a coverslip ancllower on to a drop of 0·5% Nile Blue in n01'mal saline; or place a small drop of Nile Blue on a ooverslip and tease a very small part of the tissue in it. Plaoe the coverslip smear downwards on a hollow-ground slide, or on a glass or metal ring. Seal with vaseline if necessary. The stain may take 30 minutes or longer to operate. This is an excellent method for insect germ cells, young ova, etc. It is recommended to try the method with thc testes dissected from a large caterpillar (Lepidoptera). It may be necessary to make the solution somewhat stronger, but for delicate work 0'5% has been found correot. It should, of course, be made up in saline. In recent years it has been usual to decry this method, but it is worth trying in conjunction with other teohniques. Nile Blue Sulphate for Fixed Tissues, etc. (Smith and Mail' Method). Make a saturated aqueous solution of the dye and add 0'5% sulphuric acid and boil under a condenser for 1 or 2 hours. To test whether the solution is right, pipette a little into a test tube with some xylol and shake. A fluorescent colour should appear. If not, continue boiling. Stain formalin smears 01' frozen sections overnight. Differentiate in 2% acetic acid, wash in water and mount in Farrant or glycerine. 67. Cholesterol. The Windaus-Brunswick Test is to smeal' or tease tissue on a slide, cover with a slip, run in a solution of 0·5% digitonin in 85% alcohol. Needles of cholesterol-digitonin (erystiLls) appear immediately if there is a good quantity of cholesterol, but not usually otherwise. The following test must be llsed as weU. 68. Schultz Chole~terol Reaction. Smear on coverslip GHOL.E8TEROL, GLYOOGEN ·17 and drop on to 4% forma,ldehydc (§ 112), Len,ve at lenNi. 30 minuteR. 'l'nll1flfer to a ~·5% solution of aqueoHR violet imJl alulll, (NHoIhS(\, Fe:l(SO,l)a, 24H 2 0, in an incubator at 37° C. for 2 or a dltyS. I-tinse in water for a. few minutes, Gently blot dry on slllooth filter paper. Place 011 a slide drops of a mixture of equal parts of glacial acetic aeid and concentrated sulphuric acid. Place prepared smear on this. In a few seeonds cholesteroleontainillg suhstances a})pear blue green, neutl'[LI fats yellow brown, and all nuclei brownish. The effect is fugitive, seetiollS hecoming dirty brown 'within ~. hour. 'rhe negative test cannot be taken as certain evidence that choleRterol is not present. This is a very nice method and usually works well on first trial with such material as mammalian ovary (frozen sections). 69. Glycogen. In the study of the metabolism of organs like the Ii vel' and placenta the various tests for glycogen have been much used. There is still a great deal to be done in the study of placentas by the modern microchemical methods. Glycogen or animal starch, as it, has been called, has usually been studiecl by the iodine method (Bernard) and Best's C!tl'luine. There is a third mcthod which consists in fixing material in Bouin's Huid with chromic acid (so-called Bouin-Allen), treating subsequently vdth chromic acid, and then with Schiff's reagent. By combining the three methods correct information on the glycogen content should be obtainable. The methods are not easy and need to be practised carefully. 70. The Iodine Gum Preparation. Used also by protozoologists to show paraglycogell. 'fhe basis is iodine, which is not specific for glycogcn, but stains a variety of other materials, as for example, protein granules in sponges, lecithin, etc. On thc other hand, if you use as a control a slide which after be,ing washed in u'ann u'aier has been spat 1lpon and left for 30 minutes in a warm place, the glycogen or starch is either washed away or digested by the pytalin, and does not aI)peal' after mounting in iodine gum. 48 llIIOROOHltMlGAL TESTS IN B.MEAR8 To malw iodine gUill, tlisFlOlvc 50 gill. of gum ambic in Strain through lllllslin. DissohTe 1 gill. of iorline and :3 gll1. of potassium iodide in a little water and add to the clear gum solution. To lise, make the smear Oil a slide, if very wet let it cvaporate a little, if not, put a strip of gum iodine on a coverslip and lower OIl to the smear immediately. The preparation is ready to examine in a few minutes. Two important modificatiollfl of the method should be tried. In the first YOIl fix in abRolute or !)(l% alcohol, and then mount. In the second YOll fix and leave for 24 hours in 1 % KOH in H6% alcohol. Thi~ method is useful for spermatozoa and protozoa hut gives disappointing results for conventional mammalian material. We do not recommend it for anything except protozoa, and the information it gives about spermatozoa is of doubtful value. The method may be tried on Sporozoa in gut of mealworm. lOll e.c. of water, which will take Home time. 71. C. Bernard's Iodine Test (Langhan's Method). This method is given here as used by J. W. Jenkinson for glycogen in placentas. Owing to the fact that iodine may stain a numbeT of other substances, among which ILre lecithin, amyloid, chitin, and almost any clense protein granules, it is absolutely necessary to use the warm water and spit tests as controls. Actually the whole section is over-stained in Lugol, and after mounting the glycogen is mahogany brown. But, while this method is good enough for demonstrating glycogen in mammalian liver and placentas, it is not so clear or beltutiful as Best's carmine, which I recommend in preference, nor can any of the three methods given here be used on invertebrate material without careful controls and after careful practice on mammalian liver. The method is as follows: fix in absolute alcohol, or Cal'lloy, see that the pieces are properly dehydrated, transfer to xylol, and imbed in wax. Fix sections to slide with 50% alcohol with a few drops of Mayer's albumen (§ 128), using the alcohol instead of water. Drain and dry. Bring to 70% alcohol through xylol and absolute, and stain in Ehrlich or Delafield OLYCOGE.N 4H diluted with the same volume of absolute alcohol, wal-;h off ill 70% alcohol; it is not, lleeesfmry to "blue." Stain 5-10 minutes in Lugol, dehydrate in absolute alcohol stLturated in iodine, clear in oil of origanulll, and monnt in xylol or, better, origanum balsam. These preparations fade badly after 10 years. In organs containing large quantities of glycogen like the liver, you can stain in ordinary Ehrlich's lUl3lnatoxylin and blue in tap water substitute (§ 36), but; the method given above never allows the sections to get into less tlmll 50 %alcohol. Note. Lugol solution is 2 gm. potassium iodide, iodine 1 gm., water 100 C.c. 72. Best's Carmine. This techniqne has been used for over 30 years, yet its explanation is Rtill unknown. It is not specific and must be used with the warm water a,nd (or) spit control (§ 70). Fix in alcohol 96%,01' absolute alcohol, and preferably imbed in celloidin if sections are to be used. If you have imbedded in paraffin wax (in which case you pass the material immediately from absolute alcohol to xylol and wax) it will be necessary to cover the sections with a film of celloidin, which prevents the loss of the glycogen. This is done as follows. Remove wax in xylol, bring hito absolute alcohol, and leave overnight in 1 % celloidin in equal parts of ether and alcohol. Drain and allow to dry partly, then harden the celloidin for a few minutes in chloroform vapour (in a petri dish with a few drops of ohloroform on one side). Bring into 90% to \vhich a few drops of chloroform have been added (to prevent solution of celloidin) and thence into 70% alcohol and finally water. For smears fix in absolute, leave in thin celloidin overnight, and proceed as above for sections. You must permeate the smear with celloidin or the glycogen will escape. In both smear and section you can now stain in hallna- 50 .MIOROCHENICAL TE8'PS IN S~~fEARS toxylin of Elll#eh or Delafield (§ 143), l1lld thcn wash well in rUllning water. Best's carmine lllade up H"; folluws : carmine 2 gm., potassiulll earhonate 1 gm., potassiulll chloride 5 gIll., distilled water llO e.c. Boil gently for a few miuutes, cool. Add 20 C.c. of strong ammonia. Keep in a well-stoppered bottle in a cupboard. The solution may not remain active more than a few weeks. Stain for 5 minutes or longer in 2 parts of' stock Best's carmine, 3 parts strong ammonia, and 3 parts pure methyl alcohol. After staining, differentiate in alcohol absolute 80 parts, methyl alcohol 40 parts, water 100 parts, till no more red comes out (about 3-D minutes). Wash in 80% alcohol, lLhsolute, clove oil, and Canada balsam. Glycogen bright red. We have found this the most satisfactory method. 73. The Picric Acid Glycogen Smear (H. Bauer). 'l'hi8 is an interesting microchemical method which is worth trying in any material yon may be studying. Smeal' and fix for about 3 hours in Bouin-Allen, which is ordinary BOllin's fluid to a 100 C.c. of which 1·5 gm. of chromic acid crystals have been added. Then bring the smears directly into 4% chromic acid for 1 hour. Wash 5 minutes in running water, then treat for 10-15 minutes in Schiff's reagent (§ 78). Afterwards rinse in three changes of 80 2 water, prepared by adding to 200 C.c. of distilled water 10 C.c. of a 10% solution of anhydrous sodium bisulphitc and 10 C.c. of N.RCl. (The S02 water prevents the reformation of fuchsin.) Wash in running water, stain in Delafield if you wish, upgrade, mount. GJycogen is reddish violet, but so also ml1y be starch, cellulose, tunicine, and certain other materials. This smear should be used in conjunction with Best's carmine, iodine gum, and the spit control (pytalin) test. 74. Lecithin Test with Smears. Romieu's iodine test for the lecithins has been llsedlately in cytology. Lecithin is soluble in hot alcohol, and incompletely or hardly soluble in acetone. According to Romieu, with iodine, slightly hydl'OZy8ed lecithin gives a mahogany 01' reddish violet colour, which is iodocholine. Whatever chemical value the test may have, it certainly gives interesting results on smears of mammalian sperms, showing a definitely located region at the back of the sperm head (post-nuclear body). As far back as 1904, LEOITH.IN 51 Defiamlre fixml material in formol (4%), hrought it into acrtone, which dissolved fat hilt not lecithin according to him, and after ,Yashing, staoined tlw lecithin in osmic acid. When neces:;;ary to section material, Romiell dehydrates in aectone, imhed;; in wax acetone, removes the wax from the scctioIl~ in a closed jar of warm acetone (in the imbedding oven) : proceed as below for SJlle~11'S. The srnear also is fixed in for mol and brought into warm water to dissolve glycogen (sections cleared of paraffin wax ill acetone as ahove are t.rcated in exactly the same way with warlU water). Mild hydrolysis in 10%, HeI is now C<tl'ried out. The smears (or sections) are warmed in the acid tm fuming just begins (or hetter the acid is warmed previollsly), and then put ....·ithont washing into the following Lugol: potassium iodide 8 gill. ill 150 C.c. of distilled water, to which is added (i gIll. of iodine. Dip quickly into distilled water and mount in glycerine. The colonr i;; fugitive. The critieal point appears to be the degree of hydroI~'sis. This method used in conjunction with the acetone osmic techniquc probably gives information of valuc. 75. Material to use for the Glycogen, Fat, Lecithin and Cholesterol Methods. Undoubtedly the proper material for beginning on the glycogen technique is the liver of it well-feel rat or rabbit. Fix in alcohol anelleave the pieces overnight, and thin hand sections can be made with n, razor next day. The point about the liver is that the glycogen will usually be found in the cells immediately grouped around ~L blood vessel in it very characteristic lIlftnner. Note that for Best's carmine the pieces must be left overnight in thin celloidin, next day put on slides and stuck by exposure to chloroform vapour. The simple smearing of the sections with celloidin is not enough. After the very characteristic flaky lumps of glycogen have been found in the liver, both with the Best and the iodine or Bauer method, a good thick smear of snail (Helix) ovotestis can now be tried. Very characteristic sphere:;; and flakes of glycogen can be stained in the nurse (yolk) cells of the molluscan ovotestis. In these cells the yolk spheres are yellowish in colour, and the glycogen lies 52 N10ROOHEJ.lHOAL TESTS IN SMEARS amongst them. The smear of snail ovotestis is also excellent for practising the Sudan IV method, the yolk grannIes of the nurse cells taking the stain avidly. The hest material for practising the Schultz cholesterol reaction is frozen sections of (formal fixed) mammalian ovary, where both neut/ral fat and cholesterol will stain side by side in different colours. For the lecithin test, the smear of ovotestis of Helix Illay be tried. The Golgi apparatus dictyosomes go brownish black. 76. FeuIgen's Nuc1ealfarbung Test for Chromatin. This is an accurate and valuable test for chromatin, a,nd has displaced the older methyl green acetic test, which was of restricte(l value (§ 33). It should be pointed out that :Feulgen's method is a stain as well as a, test. The preparations arc valuable for chrom.osome and nuclear studies, and are permanent. The method is well worth learning. It is true that there is still a certain amount of dispute about its complete specificity and its ability always to demonstrate chromatin. It usually works well at first trial, and on any type of smear or section containing nuclei. Feulgen's Nucleal Reaction in Smears. Fix in corrosive acetic (6% sublimate in water with 2 C.c. of glacial acetic acid per 100 c.c. solution of sublimate). Other fixatives can, however, be used, except Carnoy sublimate alcohol mixtures (Gilson, etc.) and Bouin. Very good preparations can be got after Champy or Flemming • fixation. The type of fixation used is closely related to the time needed for hydrolysis and may be fairly critical,l Too long a sojourn in HCI weakens the reaction. Hydrolysis is carried out at 60° C. in an acid solution prepared as follows. Add 82·5 C.c. of HOI (Sp. G. 1'19) to 1 litre of distilled water. Leave for optimum period (4-15 minutes), then dip into cold acid and rinse in distilled 1 For corrosive acetic material it is 4, for Zenker 5, for Fleming 16, Bouin-Allen 22, Navashin 7, Regaud 14 minutes. Ji'EULOEN TEOHNIQUJj} 53 water. Stain for Ii hours in fuchsin sulphurous acid (Schiff's reagent) prepared as in § 78. Wash excess st.ain off slide ill three washes of 80 2 water, to prevent reformittion of fuchsin. If 80 2 water is not at hand it may be prepared by adding to 200 C.c. of distilled water 10 c,o. of a 10% solution of anhydrous sodium hi sulphite and 10 C.c. of N.HCl. Wash in distilled water and counterstain if you wish for t minute in Light Green (0·5 gm. of Light Green ill 200 C.c. of alcohol 70%). Pass to 90%, absolute, clear in xylol, ,tud mouut in balsam. Material containing thymonuoleic acid is reddish violet to dark violet, plasmosomes green (in Light Green). 77. Vitamin C. The researches which led to the elaboration of !L cytological technique for this vitamin were initiated by A. von Szent-Gyorgyi in 1927. Adrenal gland cortex treated ""ith AgN0 3 solution rapidly blackens. A crystalline substance" hexuronic acid" was isolated, and finally identified with vitamin 0, partly with the assistance of the Americans, C. King and W. A. Waugh. In 1932, Moore and Ray showed that in scurvy the silver nitrate reducing property of the adrenal disappeared. Giroud and Leblond showed that this vitamin was localized in cells in connection with the mitochondria and ({algi apparatus, and the questions thus raised are of prime importance to cytologists. G. Bourne has carefully investigated the cytological technique for this vitamin and recommends the following procedure: .Fix tissues in a solution of 5% AgNO s, to 100 c.c. of which 5 C.c. of glacial acetic acid has been added (modified solution of Giroud and Leblonrl). For Protozoa, etc. smears, a few drops of the solution are added to the liquid on the slide with the cells. Examine after ten to fifteen minutes. Or better isolate organisms in centrifuge, treat en masse with solution, wash in Ilure glass distilled aCl. dest., treat in photographic "hypo." Wash several times in aq. dest. upgrade in ethyl alcohol, clear, imbed and section if necessary. The best results are only obtained by ca?-rying out impregnation in dark· neA~8. This is important. Finally, it should be mentioned that vitamin 0 may exist in A reduced form, reacting readily with AgN0 3 , and B IH J.1flC'ROCHEMIOAL TES'P8 IN SMEARS reversihly oxidizE,d form ]lOt, ;;0 reacting. Tlw orrlinary teehlliqllf' Hilllply denlOllstrates forlll A. To get over this difficulty A. Boul'llo expmler; pieces of tiHrme to vapour of glaeia,l acetic acid for some miIlutml, cuts thin slices and Ie aveR in atmosphere of hydrogen bisulphide for fifteen minutes. This converts form B into forlll A. All trace of hydrogen bisulphide nUGst be rem.oved. This is dono 1:n vacuo until most of the HoS has heen suckeu ant of the tissue (15 to 30 minutes). The se~tiom; !Lre then exposed to a strong Htream of nitrogen gas for H5 minutes. Before concluding that vitamin C is not present the hydrogen bisulphide method should be used. Actually there may be litt,le difference between the various methods given when one examines the slides. This is recognized widely as a valid method. The vitamin hecomes black with silver nitrate and is founel in the form of scatterod granules. 78. Schiff's Reagent. Dissolve 1 gm. of powdered basic fuchsin (and prcferably not acid fuchsin) in 200 c.c. of boiling distilled water. Cool to 50 0 C., then filter. Add 20 C.c. of N.RCl, cool to about 25° C. and then acId 1 gm. of anhydrous sodium sulphite. S02 is given off and the liquid slowly turns yellow. Allow to stand for 24 hours at least, keep well stoppered in the dark. Should the fluid become red or rose instead of yellowish, discard it. 79. Volutin. Fix smeal' in Schaudinn's fluid, wash out in 70% and iodine, bring down to water and place in a 10% solution of methylene blue for 10 minutes. Rinse in water and treat with 1 % sulphuric acid for ! minute, upgrade \1nd mount. The nucleus and the cytoplasm decolonrize, but volutin remains coloured. Volutin is found commonly in Sporozoa and Flagellata. CHAPTER V WHOLE MOUNTS Unstained 80. It is mmal to mouut many types of Inscets, Polyzoa, slllall Aruehincla, Kithont. staining. The mouuting medium often m;ed is Pa/'J'ants' med-iu'ln or one of the improved modifications. Here is a good one: Distmed water 50 e.c., gum arahic 4l) gm., glycerine 20 c.c., ehloml hydrate GO gm. Dissolve the picked gum in the water cold, \\'hil~h may take some time, then add the chloral and dissolve wit.h gentle heat, then add the glycerine and filter t.hrough cambric in a hot fUllnel. Another we11known fluid is that of Berle-se: Distilled water 20 c.c., chloral hydrate 160 gm., gum arabic 15 gm., glucose syrup 10 gm., glacial acetic acid 2 c.e. Pnri'sjlnicl for mosquito larvm is : 'Vater 10 c.c., gum ambic (acacia) 8 gm., chloral hydrate 70 gm., glycerine 5 c.c., glacial acetic 3 c.c. Dissolve in a water baHl at 50 0 C. in order named, strain through three or four thicknesRes of muslin. When using any of these media it is best to kill the la.rva or other animal in hot water or 10% acetic acid. Alcohol 01" formalin spec'imen.s shmlld be washed out well overnight in di8h~lled water 01' 10% acetic ncicl. Place the objeet on the slide, a.dd a drop of medium, put on coverslip, and warm a little. Set aside to harden for a few days, then ring with asphalt, Apathy's cement, cellulose varnish or ordinary Cnnada balsam (in xylol). 8l. Dioxan balsam appears to he excellent for mounting insects and similar object;;. Drop them either alive or from fixative (see §§ 114, llH, IHI) into dioxan or 95% 111cohol, and mount Ilext morning in dioxa.n balsam, made WHOLE liOUNTS by dii'isolvillg dry Canada balsam in dioxan to the thickIless of honey. 82. Euparal. For ~O% or absolute alcohol specimens. Transfer the objects to a drop of euparal and cover. Will prohably he superseded largely by dioxan balsam. 83. Glycerine Jelly. Dissolve 10 gm. of best gelatine in liO C.c. of distilled water, mix in 70 c.c. of pure glycerine, and add 0·25 gm. of phenol crystals. These operations should be done on a water bath, or in a beaker in a dish of hot water. Mount objects from glycerine (§ 86). Of the three methods above mentioned gum glycerine medium clears the least; dioxall balsam the most. 84. Canada Balsa~. This may be dissolved in absolute alcohol, benzol, xylol, 01' dioxan. The so-called neutral balsam supplied by certain commercial houses goes as acid as xylol balsam after a time in the light. Make up your routine balsalh in benzol, not in xylol. If you wish to mount stains which are prone to be fugitive use thick cedar wood oil. This hardens after a time. Canada balsam should be thin for sections, and thick for whole ~unts, but about the viscosity of honey does for most purposes. It is recommended to keep balsam in a bottle covered with black paint or paper, and never to allow' it to remain in sunlight. There are special balsam bottles supplied by the commercial houses. Some people put a strip of plasticine around the neck where the loose cover comes on the bottle-this to prevent sticking and to make an air-tight joint, which is an excellent idea. Cork stoppers are useless for balsam. If the-balsam lid sticks, cautiously warm it in a flame. 1'0 use Canada balsam for unstained whole mounts, dehydrate in absolute alcohol, clear in cedar wood oil or beechwood creosote, wash out in xylol and mount in balsam. 85, Other Resinous Media. 8anclarac, Camsal and other resinous or oily media have been used by Gilson for BALSAM AND GLYr)EIUNE 57 making monutauts. Euparal is rather difficult to make and it iR better to buy it. Here, however, is a " neutral monnt.ing medium" which can he made in the laborn,tory, ltnd i;:; an excellent substitute f(ll' euparal in every case: Denham's Sandarac Camphloral is 2 parts by weight of crystalline chloral hydrate ground in a glass mortar with 1 part of " flowers of camphor." If any difficult,y is found in getting the last few crystals to dissolve the mixture is left in a warm place for 11 few hours. 'fhe liquid is then filtered. The salldarac solution is prepared separately. Selected crystals of sandarac gum are dissolved in excess of isohutyl aicoholllt (jOD C. to make a solution thin enough to filter easily. It is then shaken up widl animal charcoal, and filtered several times through paper, after which it is evaporated to ~1 thick SYTUP, using a eondenser to retain the alcohol. One part of gum is mixed with two of camphloral, and incorporated by warming and stirring. The product should have a refractive index of 1·485. Objects may be lllounted from 70% alcohol. Euparal is cOlllIllercially made in much the same way, the gum being dissolved in eucalyptol. 86. Glycerine Whole Mounts. Fix in weak (4%) formalin (§ 112) for se'veml days. Bring into water overnight. Very gradually bring into 50% alcohol, by dropping frmn time to time a little alcohol of this strength into the tube of water and leaving for hours until mixed, shaking slightly now and again. Upgrade gradually for days to 90% alcohoP Place a layer of glycerine at the bottom of a tube. Pipette in a similal' layer of 90% alcohol and ether (9; 1), and transfer the objects to it. Leave uncorked till the alcohol evaporates and the objects fall into the glycerine. Mount in glycerine, or glycerine jelly, ring 'with asphalt, etc. (§ 88). To mount 1 Somo people begill by fixing tho organisms in 91)% akohol. This would be alright for hard insects amI such like. TVHOLE JlfOUN1'S in glycerine jelly, warm a little on a slide, transfer object from glycerine into this and cover. Ring after l1 few days with cement. 87. Mounting in Balsam or Glycerine, etc. Students often use too much mountant. You should notice tha,t the space between the coverslip and slide, even with a 20p, section, is very small, Use a small drop of mountant, and if it is not enough, place another small drop at the edge of the cover and let it run in. If you wish to cement a glycerine mounted slide this calmot be done if it is messy. Moreover, it may be difficult to use an oil-immersion or other high power lens with short focal distance on such preparations; and they dry very slowly. When mounting sections, put a thin strip of balsam on the coverslip and lower one end first on the slide gently with forceps, a pin, or sharpened matchstick. 88. Cementing Glycerine or Similar Wet Slides. TV'ith Canada Bal8am. Some people mount in a fiat drop of BeI'lese or Farrants, and leave away from dust and without coverslip till dry. Then put xylol or benzol balsam on top and cover with a coverslip. This would not do for glycerine slides, which must be covered and ringed or cemented with one of the following media, or ~wjth Canada balsam. Peter Gmy's jJ.{ediutn. Four parts anhydrous lanoline, 8 parts resin, 1 part dry Oanada balsam. Melt together, stir. This forms a solid mass on cooling. For circular cover glasses, dip piece of metal tube, right size, into molten mass. For square covers, use broad, bent needle in holder. Amyl Acetate Celluloid Cement. This is made by clissolv~ ing some (food hard celluloid in amyl acetate. The solution should be thin enough to spread with a soft brush. It may be used for brushing on the end of slides instead of labels. Allow it to dry, write legend, let it (Iry, brush over another layer of cement. It can be used for ringing s'rAINED IVHOLE llIOUjYTS :jfI glycerine and gum mounts instead of other oil remellts. In all eases slides should be grease free. ApathY'8 C'e-!nent for Olycerine }rlolln{8. Bqual part,s of 60 0 C. paraffin wax and Canac11t balsam. Heat together in a porcelain vessel till the lllass becomes golden and no longer emits vapour of turpentine. To use, warm, and apply with glass rod or spatula. An excellent medium. Paraffin Wax. Useless for permanent slides. 89. The Stained Whole Mount. Two methods are given here-the Borax Carmine and Mayer's Acid Hmmalum. It is rate to see a really good whole mount made by students. The fault most commonly noticed is 'Undel'dilfeTentiation. Fix in corrosive sublima,te acetic (§ 108) or Petrunkewitsch (§ I HI) only if the object is rather impenetrable. The main point h.; to use cOlTosive sublimate as it gives bright preparations. If the animal haR to be amesthetized this must be done first (§ 02), or it may be held between a slide and coverslip and the fixative run in with a pipette. When fixed, which will depend on its size, bring into 70% alcohol. (In any case no harm will be done by leaving it in fixative overnight.) Add iocliue (Lugol, § 71) to the 70% till sherry colour (light brown). Leave several hours, and if it decolourizes add a little more iodine. Next morning pour off and add more 70%. Leave for several hours, or as long as you find cOllvenient. Pour some borax carmine or acid luemalum into a petri dish just to cover the specimen. Leave overnight at least, then pour away the stain and begin differentiation. For the carmine stain use 70% alcohol with 0'5% HOI, for the hremalum use water with 0'5% HOI. Leave all next day 11lld night; when this period has elapsed the stain ghould have been extracted from cuticular structures like skin or membranes, and only left in the internal organs and glands. The rest of the animal should be pink (carmine) or reddish (hremalum), and it should be possible to see GO WHOLE MOUNTS :mmething of the structure. If it is opaque, it is better to leave it a little longer. On the other hand, if it has got rather pale after the first day in differentiating fluid it is best to place it in non-acidulated 70% overnight and ra-examine next morning. If differentiated rightly, place the luemalulll preparation in alkaline water till hlue (§ 36). Upgrade slowly to 90%, absolute, and clear in xylol, or if your absolute alcohol is suspect, in methyl benzoate or beechwood creosote followed by xylol, mount in balsam. It may be necess(try to make glass feet to support the object under the cover. Rernembel' a8 the balsam drie..'1 the cover will press down on the pl'epamtion. Note that if you use dioxan balsam, you mount from 95% alcohol, or from dioxan if you have dehydrated in this. 90. Staining Chrome Fixed Material in Carmine Stains. This cannot be done successfully if the smears, sections or pieces have been taken to alcohol. Always stain in the carmine first, just after washing out in water. 91. Treatment of Formalin Specimens. Organisms got in the field are often fixed in formalin, and with ordinary technique it seems quite hopeless to get good stains in either acid hffimalum or borax carmine. Such materi!11 may be rejuvenated by being left in 4% hydrogen peroxide or pink solution of permanganate of potassium for all hour or so. The former solution is the better of the two. Small ll1edusre stain nicely after 1 hour in either solution, but larger or more impenetrable objects would have to be left longer. After rejuvenating leave overnight or longer in 70%, or 90%, alcohol before begin. ning the staining. 92. Anresthetizing. Narcotic substances which can be got easUy· are menthol, methyl alcohol, chloroform, chloreton, chloral hydrate, and chloride and sulphate of magnesium. In addition, cocaine hydrochloride or hydrochlorate" stovaine, urethrane, hydroxylamine, eucain, AN/ESTHETICS iiI morphia, and curare have been used, but are not so easy to buy. Cocaine is rather indispensable and is the basis of ROllsselef's solution (does not kcep well) : Cooaine 2% aqueous solution 3 c.c., methylated spirits or ethyl alcohol of 90% 1 c.c., distilled water 6 c.o. For Protozoa use 10% methyl alcohol, or Rousselet's solution. Oorri'8 solution, which is important for both fresh water and marine animals, is: Methyl alcohol of 96% strength 10 c.c., water (fresh or sea water accorcling to organisms) 90 c.c., chloroform three drops. For fresh 'vater organisms only, add O'(l% sodium ehloride. .Menthol is used for larger marine animals; jW'It sprinkle over surfaee of water and le~we for from 12 to 24 hours. Chloral hydrate is used in the same WH,y, but acts sooner. .Maynesiurn sulphate (33% aqueous), added gently in water, acts in about l hour on actinians and sueh marine animals. For slllall animals in general: Place them in a little water in a very clean watch gla,ss, allow to expand and quieten, add the narcotie gingerly. Go slowly or the animals contract. Try thc following in this order for Protozoa, Rotifera, and such like: 10% methyl alcohol, Rousselet, and Cord. For larger organisms, magnesium sulphate and ehloral hydrate are recommended. 93. Peter Gray's Technique for Rapid Permanent Mounts of Fresh Water Organisms. (Time 5 minutes.) Prepare some pipette drop-bottles, as used in chemical laboratories, and fill one each with 70% alcohol, 40% formaldehyde, glacial acetic acid and basal fixative solution (which is picric acid 1 gm., corrosive sublimate I gm., 100 C.c. of 95% ethyl alcohol). Have also a stoppered drop-bottle of ether, some Mayer's albumen (§ 128), pipettes with rubber teats, writing diamond or carborundum pencil, a staining trough of 70% alcohol, strips of filter paper, clean slides and several 2-in. specimen tubes in a stand. 62 WHOLE MOUNTS Before beginning examination of ,1> collection of pond organisms clenn slides by rubbing with acidnlated alcohol (!)5% with 1 % acetic acid) and drying with duster. Plewe n small drop of Mayer's albumen on centre of eaeh slide lwd rub down with alcohol cleaned finger a patch 1 em. in diameter. Now prepare the necessary fixatives from the pipette drop-bottles as follows: for Protozoa, basal fixative 10 drops, ether 3 drops, glacial acetic 2 drops, 40% formaldehyde 5 drops. For heavily c1lt'icnlal'ized forms, basal fixative 10 drops, ether 1 drop, glacial acetic 4 drops, 40% formaldehyde 5 drops. For deNcate lal'vm, basal fixative 10 drops, ether 2 drops, glacil1l acetic 1 drop, 40% formaldehyde 5 drops. These are made up in the 2 X t in. specimen tubes and placed in the stand. They are unstable, and are all modified They cause Mayer's YocOln-Duboscq-Brasil fluid. albumen to become sticky, unt,il treated with alcohol, when it hardens. Examine your collection under a dissecting microscope, pick up an animal with a pipette in as little water as possible, and drop it in the middle of the patch of albumen. Draw off surplus fluid, leaving' sufficient for the animal to swim naturally. Some forms are surprisingly sensitive to either the glycerine-albumen or the sodium salicylate in it, and fixation must be hurried. Watch until the animal is in a normal position and place a large drop of fixative on it. If in the subsequent streaming the animal passes outside the albumen patch it must be gently put back with a fine glass needle. The animal must now be watched tlntil the d1'oplet of jl1lid has evaporated 80 as to show clearly the outlines of the object. Now flood gently with 70% alcohol. If this has been done properly the animal is now stuck in a natural position in the albumen. If very small, ring the organism with the diamond, and write a reference number or name on the slide. Now transfer to staining trough of 70% alcohol. Drain slide, wipe around B},fALL OROANIS.1.1JS 63 quiekly, put Oll heneh, add I drop of Ehrlieh's luemntoxylin. Leave 30 seconds for rrotozOtt (Paralllwcium), H minutes for miracidinlll, run off excess stain with !JO% alcohol, never uater, hrieHy flush with 0·25% He1 ill 70 % alcohol, wash uff with Seott'r,: tl1P Witter substitute (do not use ammonia. for" blueing "), stain quickly in eosin (0·5% in 50% alcohol), rinse off in 95% alcohol, deftr in terpineol, mount in balsam. You C[tIl 1ll0l1llt in euparal or dioXlLll balsam if you wish, in whieh cn,se do not clear in terpineol. This method is not very successful for larger organisms like insect larvre, which should be trented as described ill § flu or § 80. 94. Free Living Organisms like Protozoa and Rotifers. For those large enough, treat as described in § fl3; for smaH flagellates and ciliates, SUlear a drop of the medium containing them on a coverslip, kill in formalin or osmic V!L"{)OUl' for a few minutes, now mix a small drop oflVfayer's albumen with the liquid on the cover, and drop smeftl' downwards on to the fixative. If there was not too much water on the coverslip, the organisms will stick. Some workers use gelatine water. It is a good thing previously to concentrate the organisms in their medium by mmms of a hand centrifuge. 95. To make Fixed Protozoa and other Small Objects Adhere to Slide or Coverslip. ]'01' unfixed objects, which are to be mounted and stained in Ehrlieh's hwmatoxylin and eosin, see § 93. For fixed objects, bring into water, prepare slides with albumen, piace objects in very little water in middle of albumen, remove excess water with pipette, leave for a short time until albumen and water mix slightly, flood gently with 70% alcohol. Alternatively, mix ohjects \vith albumen, smear on cover, [Llld drop into 70% alcohol. For objects in absolute alcohol place drop of thin celloidin on slide, mix objects in it, flattening the drop, harden in chloroform vapour. Note that unless strong WHOLE .MOUNTS (j·1 alcoholluls a little chloroform mixed with it, the celloidin bccomes dissolved. Should you not wish to use alcohol, the following method is indicated. Cut 2 gm. of sheet gelatine into small pieces, place in flask, cover vvith distilled water and soak several hoUl's. Make up to 100 C.c. with distilled water, warm to 60° C. Filter only if necessalyl Add a crystal of thymol. Take a little on the small finger and smeal' on a slide. If the objects are not too wet they can be placed all it immediately. If they are wet excess water must be removed. Now fix the gelatine in a warmed slide bottle containing a piece of cotton wool soaked in commercial formalin. Success depends on allowing the objects to mingle with the gelatine, which must not be diluted too much before fixation. 96. Whole Mounts of Crustacea such as Asellus and Gammarus (from Fresh Water Ponds). These crustacea are good examples of rather impermeable objeets for making whole stained mounts. The secret of success is not to hurry matters. Soak well in 90% alcohol after fixation, leave 2 or 3 days in carmine, and longer in acid alcohol if necessary. It is always a good thing to prick impermeable objects with a very fine needle to aid penetration of fluids. 97. Whole Mounts of Nerve Endings in Muscle, etc. These interesting preparations show the palmate or dendritic nerve endings in muscle. Take a strip of muscle from a newly killed frog's leg. Try to get thin sheets. Soak in fresh lemon juice until they become transparent, that is, for about 5 or 10 minutes. Wash in distilled water, bring for 20 minutes into 1% aqueous gold chloride, wash in watcr slightly, bring into 50 C.c. of distilled water and 2 C.c. of acetic acid. Expose to light for 24-48 hours. These prep,al'ations can be mounted in glycerine and water 1 £01' This is often used to CO!1t old lantern slideR for making diagrams projection. 81 L TORR 1JljJ /(RU,YA'I'IO;..Y (1 ; I), IlIlt. are not permanent. Here iR a method which gives permanent preparations. Take It thin .sheet of fi'eBh muscle, suak in 1 ~~\ gokl chloride tin' 11 !e.w hou1's in it dark cupboard. Place on a piec'c of filter Tlllper and gently mop up solution from outside of piece. Transfer to tube of 1 % formic acid, leave in direct SUll all day in winter, 01' in hright light in summer. If the solution goes browll change the acid. Wash ill distilled water for a few minutes, place on a slide, mop np water with filter paper, add glycerine and cover. 98. Silver Impregnations of Fresh Tissue. Kill a frog and remove it flat piece of web from hetween its toes. \-Vash ill distilled water, stretch on a Rlide, remove eXCel-\f; ,vater with filter paper, add some (1'5% (or weaker) aqueous silver nitrate, leave in bright sunlight unt.illight hrownish and the cell walls are stained. The speed of the reaction depends largely on the brightness of the light. In summer many preparations have impregnated in less than 30 minutes. Now wfLsh in distilled water, mop up with filter paper, re-stretch the piece, and harden with a drop of 90% aJeohol. Stain in Ehrlich if you wish (§ 44), upgrade and mount in balsam. Permanent. Try this method on mesentery, lung of frog opened out, cornea, etc. These preparations are remarkable for the clarity with which they show cell walls. The method is worth trying on fresh smears of germ cells and Protozoa (Silver Line organs). In these cases the silver mlty " come down " on various parts of the cell. 99. Cartilaginous Skeletons of Small Fish and Embryos. Fix in alcohol (90%) or Carnoy and wash out well in alcohol of 70%. Stain for a week in 0·25% methylene blue in 70% alcohol acidified with 1 % hydrochloric acid. Differentiate in 1 % hydrochloric acid in 70% alcohol for about a week Cartilage remains blue, all other tissues colourless. Upgrade, mount in balsam or leave in clearing LAROIlO\TOl\Y TEOHNIQUE. 3 WHOUf] J.110UN'I'8 Huid, next. §. Both t.hionin and tJnluidiu blue llHty IJe m,erl in the sallle way. 100. Oil for Clearing Embryos, etc., for Observation Whole. 'I'he t'eeol\lmeurlcrl fo;uhstallce .is Wintergreen Oil (clear first ill benzol). Nowadays methyl benzoate (clear straight from ahKolute) is being used also. CHAPTER VI FIXATION METHODS 101. Cutting Material for Cytology and Histology. It is generally to be arlyised to avoid_ orushing the material by using sci!':sors or a hlunt knife. vVhile this is good advice, expcrimentK carrieel out in crushing glamluhtr tissue did not have the had efforts expected. In any case buy a packet of sflJet,y mzor blades, and llse ono for (mtting thin slices of mo,terial for fixing. The:-:o hlades can alE;o hc used in H spot·ial holder lUI' cutting paraffin seotions (§ 1:31). 102. Dissecting and Killing Material for Cytological and other Similar Work. The hest way is to cnt off the head of an insect OI" snch arthropod anel pull its viscera out into a drop of fiXative. The necessary organs are then found and Beparated under a dissecting microsoope or lens, and put into a capsule of fixative. The main point is to get the material into fixat·ive as soon after the interruption of the blood stream ns possible. If you are working on animals like rats, kill in coal gas, not chloroform or ether. Never use these, as they tend to alter the cells around blood vessels. 103. Vivisection Acts. You may not cut, inject. or otherwise experiment on a living vcrtebra.te without, H, vivisection licencc in Great Britain and Ireland and in certain other l'Olwtries. If you do so without a licence, and publish your work, you are exposing youmelf to prosecution: H, vivisection licence may therefore be necessary; hut you can vivisect any animal without a vertebral column. li7 FIXATION ,METHODS 104. Fixing fluids practically all consist of mixtures of i()l'IHaJill, acet,ie [1eid, pioric acid, chromic acid, bichromate of potassiulll, mercuric chloride (sublimate or COTI'osive sublimate), ()smium tetroxide and alcohol. No modern fixative consists of one of the above substances alone, with the possible exception of formalin (in saline), which in some eases may give a remarkably good fixation. Acetic acici nl1(l alcohol have a destructive effect on certain lllateriab ill the uell, and are never used when the most fl1ithful fixation is needed (§ 34,), Altmann'::; fluid, which consists of bichromate of pot,i1SSiUlll and osmium tetroxide, Wl\S invented nearly hnlf a eentuJ'Y ago, and since that time no real advance in cytologieal fixation has becn made. The Goigi ap7 rmratuR methods of Cajal (formalin silver) and Kopsch (o~miurn tetroxide) twe not to be. regarded a~ advances in fixation technique, so ll1ueh as in post-fixation technique. The fixatives mentioned above have very different efJeots when brought into contact with bioehemieally isolated globulins, nueieoproteins, lipoids, carbohydrates, or with egg white. Some of these fixatives do not precipitate or otherwise change all these materials, as for instance acetic add which only fixes llucleopl'oteins, or corrosive sublimate and picric a;cid which htwe no apparent effect on fats. Osmic acid does not precipitate proteins, but it changes them in a definite manner without producing the same effect as alcohol or corrosive sublimate. It is the . most valuable tixative known, and it is strange that it should have figured so little in the technique of proto. zoologists. Alcohol anci corrosive sublimate are energetic coagulants and produce the well-known net-like protoplasm seen in .all the older pictures of cells. It is agreed nowadays that in metazoan tissue cells, the nets are art.ifacts. They do not allpear ill good chrome-osmium PRNRTRA 7'lO..V OF FIXA'J 1/TE'S nn fixed cells, LUlCl arc be!:it. seen in Curnoy or Schaudinn fixed cells. Aect,ir' :wirl is an intereHt.ing subr4ance in fixation. H d()p~ not prel"ipitate ol'{Hnnry proteins ill t·jw weak 8t1'(~ngth ill which it is used in fixatioll. but. it, dUCH fix nn('l~o" proteins, and in this aml t.his alone its virtue lies. In the case of bichromate of potasBiulU the degree of acidity of the solution is of considerable importance, hut bichromate should be used with or after chromic acid fixatives fbr lipoid cell organs if one wishes to get a good stain in hrematoxylin or acid fuchsin. In this and other cases the mere fix~1tilln is not the whole problem. This is why Champy is superior to Flemming without acetic in cases where an intenEe stain of various protoplasmic granules is desired, and why pm;t-chromillg has to be resorted to especially after Flemming fixation. Flemming, of course, contains only chromium tl'ioxicle and osmic acid, and while it gives an excellent fixation, it does not always prepare the ground properly fO!' staining. The problem of penetration rates of fixatives is, in some cases at let"st, closely related to thc added effect of the osmotic pressure of NaOl, which may be used with such mixtures as Flemming, Ohampy and corrosive formol. There is at present no explanation of this, and the beneficial effects of NaOl are even denied by some recent writers. While there is so much work to be done on penetration and chemistry of fixation of comparatively simple extracted organic compounds like globulin, nucleoprotein, glycogen and so on, the problems presented by the fixation of cell aggregates and their products by the mixtures like Ohampy (chromium trioxide, bichromate of potassium and osmic acid), Regaud (formalin and bichromate of potassium) or Zenker (corrosive sublimate, bichromate of potassium, acetic acid) are immensely more difficult. Do all these constituents of a fixing fluid penetrate a piece of tissue equally, or does one constituent enter faster than 70 F'IXAT.IO;),T JJE'l'}j(JDS the otllers '? It seems that n, substance like acetic acid, such as in Flemming's formula (chromium trioxide, acetin a,cid and osmic acid), does leave the others behind, and they follow much more slowly. Whence the statement t,hat acetic twid helptl penetration. This is to say, the IlIlClei deeper in the piece of tissue look hetter than in .Flemming without lleetic. because with the latter fluid these deeper layers of cells I'(;>main for longer unfixed in any \ray. Not all fixatives penetrate at the same ratl~. Some like osmic acid are very slow, others like corrosive sublimate and alcohol comparatively fast. Some swell, oth0rs shrink tissues, and the good general fixlttive Huch as BOllin's picro-forrnol-ar,ei;ic gives a balanced result, the tellllency of one fluid to shrink being counterbalanced by the eiIeet of ,ulOthel' constituent of the fixative. It should never be forgotten that in discussing the etleets of fixatives there are t,yO possible criteria, one, the actual effect of the fixative, two, the finaJ appeara.nce of the cells which, having passed through ethyll.1lc:ohol and xylol, are now ill balsalll. Corrosive sublimate and picric acid, for example, do not injure fat or lipins, mitochondria, Golgi bodies, etc., and such cell inclusions appeal' in eells fixed in these substances when examined in water, after fixation. This can be shown hy fixing smears of insect or molluscan gonads. But neither corrosive sublimate nor picric acid is able to prevent the solution of fats and lipin-like bodies during the dehydration, clearing and imbedding. When it is said that corrosive sublimate and picric aeid do not fix certain cell inclusions, this is all that can be meant. 'When, however, you use bichromate of potash, osmic acid and formalin, various cell inclusions are brought into a condition which partly 01' wholly prevents their subsequent solution in alcohol and benzol, for example. If you fix in. corrosive sublimate or picric acid, you can (subsequently) postosmicate or post-chrome, and bring about this desimble change in the fatty cell inclusions. This is BOP IN';') FLU}D "j l the explanation of Sehridde's method for fntH amI mitochoncll'in. When you come to the f01'1na]in ~ilver methods, the matter is even more ('ornplieatcrl. Da FallO'::; method (('obalt nitrate formalin, followed hy Hilver llitmt,c, (mel reduction of the latter) certainly demonstrates a part of the sperm head claimed by some observers as being lecithin, even though the material has heen passed through alcohol and xyl(lL Nor is the usmic af'id method for the Golgi apparatus llnderstood. It is snppm;ed hy SOllle to ('c)llsist in tho reductioll of Of:;(\ to a lower oxide. Chrome-osmium iixati\'es are said to, and npparently d.o, convert glycogen into a suhstmlCe insoluble in w~lter. AU fixatives should be usccl intelligently hl eonjundion with microehemim1 tests 011 smerU'H (§ 34 et .seq.). 105. Amount of Fluid to Use in Fixing, and Size of Pieces. In l!'ig. 4 is a, ('overed sten(ler dish (seale lllHle1'neath) containing t,vo pieces of tiSS1W. The amount of fluid ttnd the size of pieees would he suitable for Bonin, --=corrosive' sublimate, :Petlrnnke\vitseh, C~aruoy" Zenker, a.nd Susa" hnt \Vl'Ollg for Champy, :Flemming-wit,hout-acetic 40,,,,,,,. RllCl such fluids of low penet-m.tiug FIG. 4. StendOl' dish eontl1ining power. Moreover, there is another fixl1tiY(l and tWl) serious fault if this diagmm is suppieues of tisRue. posed to represent Champy fixation-far too much fluid is being used, it is wasteful. Osmic add, whioh is a part of Champy and such chrome-osmium fluids, is very expensive. Cut the pieces very small and just cover them ,vith chrome-osmium. :For Aoyama and similar formalin :d11'e1' nitrate method::; it would be ll11wh hetter to cut the pieces shown in Fig. 4 into four. In this ease the !LlllOunt of fluid is right. 106. Bouin's Fluid. This fluid is ll1uc:h used for chromosomes and fixi1lg smail arthropodB, worms, ete" for geneml iii ---=: 72 jI'/XATIO.;_\, J.llltTHOlJS anatomical purposes. It is nut 80 penetrating as Camoy or Gilson-Petnmkewitseh, which do equally well for most purpm3Ps. It ,vill not give fat or cell inclusions generally, hilt is nowacIays used for glycogen (§ 7:3). Unlike some other well-known mixtures, it, is just as good for vertebrate tissues as fiJr f1mall invertebrates. It. does not tend to forIll preeipitates in tissues as does corrosive sublimate, it need not he ,vashed out well, does not make tissues brittle a11cl is the best all-round anatomical fixative. l~xcept for ehromm,omes and kinetic apparatus it is useless in cytology. Its formula is, picric acid, saturated aqueous solution, 75 parts, good commercial formal 25 parts, acetic acir! 5 p~1rts. ,Vash out in alcohol 50% or 70%. Fix from 12 to 24 hours. Wash out overnight in a good quantity of alcohol, but, aR mentioned, it is not necessftry to get rid of all the picric acid. It dissolves out later mainly in the sections while staining. Here is an important modification used both for chromosomes and in microchemistry: B01lin~Allen, which is the above formula, to 100 c.c. ofw·hich, warmed to 38° C., you first add 1·5 gm. chromic acid crystals; then 2 glll. of urea. For fixing, warm the solution to 38° C. Eor glycogen, etc., do not bother to add the urea, or to warm. Lithium carbonate washes out picric acid, if you wish to do this. Add just a few drops of aqueous solution to the 70% alcohol containing the pie cos or animals. Another modification of Bouin's fluid ill Dobell's Alcoholic Bouin. Saturated solution of picric acid in 90% alcohol 15 parts, formol com. mercial 5 parts, glacial acetic 1 part; add one or two drops of chloroform just before use. Wash out in 90% alcohol, to be followed bv Doball's alcoholic iron-hrematin. It is more destructive, b~lt more penetrating, than plain :Bouin's fluid, "'-... is us~fnl for cysts (see, however, § 53), hut is otherwise a poor ~lve. 10~nker. 'rhis is a good fixative, definitely better than Bou~r most purposes, anel really gives quite promising preI;-arations when the acetic acid is left out. ZENKRR AND CORR08IVB AURTIC 7:3 It is 2! gm. of bichromate of potash, in 100 o.c. of wn,ter, to "which 5-8 gm. of corrosive sublimate and ,} c.e. of acetic acid have been added. Fix 24 hour.s, "wash in running water for mORt of the following day, transfer to 70% alcohol with iocline (sherry eoloul'), and bring after" severnl hours iuto 90% alcohol. Tissues stain hrilliantly after this fixative, iron alum hWllULtoxylin and U nua's methylene blue and eosin are particularly good. This fixative has been modifien ~lS follows. Leave out the acetic acid when the stock Zenker is made up. Befilre fixation pour 5 C.c. of formalin into a vessel and {ill up to the 100 c.e. mark with Zenker. 'rhis fluid iR ('allt~d H elly, Of Zenker-Jormol, iLIld is definitely hetter than plain Zenker. It is mllch better for geneml histology as it preserves a good part of the granules in ceils, giving mitochondria, and Dlany of the details of glandular secretion and germ cell format,ioll. In Helly, material may be left for 12-24 hours. Afterwards wash under the tap overnight, transfer to 70% aleohol with iodine for several hours, and then into 90% alcohol. Helly or Zenker are specially indicated for vertehrate material, and could be used for experimentftl or descriptive work on such subjects as the l11ammalian <:estrus cycle where uniform results nn a large amount of materif1l are ·wanted. Bouin is also used for such problems, and is simpler, but not so good for cell pictmes. Note that many routine methods in pathological laboratories are standardized for Zenker or formol mft terial. 108. Corrosive Sublimate Acetic. Twenty-five or thirty years ago this was the best-known fixative, leaving aside sllch mixtures as Flemming. Corrosive aoetic Rubsequently beettme displaced by Bonin, which is better and less troublesome. But there is OIle dass of preparation which iH best llHLcle from corrosive fixed l1HLterial-the earmine or hremalum whole mount. There is a number PIXAl' /oN METHODS offol'l1mlre for thif; fixative. A general one is the saturated Roll1tioll of corrosive sublimate in water with 0·5 l%-1 % glaeinl aectie acid. Flome people use up to 5% acetic acid. Small piecei'> of tissne, flat worms, pieces of LU111,bricus and similar objeets Jix in about 2 hours. Afterw[l.l'(lH wash the pieces for SOUle minutes ,,,,ith tap ,Yater, and then acld 70% "'ith severnl drops of Lugol's iodine solution (§ 71) to make a sherry colou!'. Leave some hours. If the iodine clecolOlll'izcs you mllst add lllore. Then bring into cIear 70% for seveml more hours. It, never hurts to leave such ohjects in 70% or 90(% alcohol. Then p1'OooO(l to stain as described in § 89. If you want to make soction8 it i8 far hetter to use Bouin's fluid. One slight modification of the ftbove eorrosive acetie iH called Kai8er'8 .tll.dcZ. 109. Heidenhain's Susa Fixative. 'Water RQ e.c., corrosive sublimate 4·1) gill., sodium chloride 0·5 gm., triehloracetic 2·0 gill., I()rmalin 20 c.e., glacial acetio 4 c.c. 'Wash out in 90(1'0 alcohol with iodine, not in 70%. Mnch used by histologists. Gives brightly staining preparat,ions, and as there is little deposit of sublimate, washing out in iodine can often be left, out. It might more often be used iU8tead of ordinary corrosive sublimate. 110. Schaudilln's Fluid. Two parts saturated watery corrosive sublimate, absolute alcohol one part, acetic acid 0·5%. Some people use as much as 5% acetic. The sine qua non of protozoologists. It is a destructive fluid! You ,,,ash ont in 70% with iodine (§ 71). 111. Mann's Fluid. Freshly prepared f-mlution of equal parts of 1 %osmic acid, and corrosive sublimate (sat,uratecl in water, with 0·9% sodium chloride added). Exclusively used as fixation preparatory to post-osmicatillg for Weigl's method (§ 172). 112. Formol, which is formaldehyde gas (40%) in water, is often a trouhlesome subst:1lloe. The commercial fluid ('ontain8 traces of formic add. It may become [1'0 R JIA LIS more acid by subsequent oxidation, and some eOlUmerc·ial specimens of formalin disintegrate so much as to he liBeleRS after It short time. It, is recummended to buy the be8t fiJI'malin, itlld to keep it neutral (pH about (j'4) either with ('aleiu11l carhonate, powdered and tied up ill muslin, and left at the bottolll of the jar. or to neutralize the diluted strength you arc going to use with borax. Add borax till a good red colour is shown with phenolphthalein indicator. The best strength is 4% formaldehyde in water, to whieh if'; arMed 0'75% NaCl. In case this ltlay not be elear, you (uhl no parts of 1.iJatc)' to 10 IJal'i8 of the conunercial j'01'lnalin (Jannal) liquid [LIlli rli:::>;olve in it, tJl(J 0·75 gIll. of Nael (f(mllOI-saIine). 'l'his giVCB "10% ti)l'lllOI" or "formalin," or .JJ% formaldehyde. When one reads "10% formalin" in the literttture, it. almost alwftys means 10 parts of the eommeroialliquid and 00 of water. 'rhe strengths used in Cajal, Dlt Fano and Aoyau1l1 silver nitmte methods vary from 4% to G% formaldehyde gas. Thin slices of liver, left three or four days in 4% formaldehyde, washed out in 70% alcohol and stftinetl in iron alum hromatoxylin, sometimes give very good preparations of lllltoehondria and bile ducts. Formalin is often combined with corrosive sublimate (sublimate formol, 1 part formalin, 3 parts saturated corrosive sublimate) and with bichromate of potassiulll (Formal .:Jhlllel' or Regaud). Sometimes (}75%-O'9% salt is added. It is vwrth trying. 113. Regaud's Fluid. Very much used nowadays for mitochondria of vertebrates. It, will give fine preparations of invertebrate neurOlle8, provided the brain or ventral cord has been dissected out. It il:! a mixture of an oxidiseI' and a reducer and is theoretically irra,tional. Nevertheless it works. Regaucl used it for his studies in spermatogenesis of the rat and got the best results by mixing 3% aqueous bichromate of potassium, 80 vulumEs, ,(i F'/X.-ITI(LY MRTHOD8 with j'OI11111Cl'cinl fOl'malin, 20 volullIes. He fixed for -I days, changing the rlisintegrated flnid every clay. This is lmneCOHf!ariJy long. Fix overnight in Regaud, mordant; Ii II' :1 claF: ill j,iehrnmate, dWllgil1g if the Hui(l goeR elundy. Wash out in rUllning water fur 2{' hours. nehy~lmte slowly. Stain sections either in acid fuchsin (§ 154), iron alum hmrnatoxylin or Regaud's special lu:ematoxylill (§ 147). 114. Kahle's Fluid. An alcohol formol nml- much uged. It is 115% ethyl alcohol 15 parts, forlllol 6 parts, glacial acetic 1 part, distilled water 30 parts. Bring into 50% aJcohol after fixing overnight. A penetrating fluid good fiJI' hlBlllUlulll whole mounts, etc. It is useless for cell inclusions. A good general histological fixative. 115. Altmann's famous fluid is equal parts of 5% aqueous potassium bichromate and 2% aqueous osmic acid. Some workers use weaker potassium bichromate, as for instance Bensley, who recommends 2·5%; t,he ~weakeI' biehromate is an advantage, and it may be helpful to add 0'75% sodium chloride, as advocated by C. Z. Young, for such fixatives as this and the two following. 116. Flemming with and without Acetic Acid. '1'he formula used nowadays for chromosomes is 1 % chromic acid 15 parts, 2% osmic acid 4 parts, glacial acetic acid 1 part: with 01' without 0·75% sodium chloride. Very small objects or pieces are fixed for from 1 hour to 24 hours. The points about fixation periods are that complete penetration must take place and that the hardening effect mnst be carried on even after penetration if you wish to use a watery stain of long duration. For eXQ,mple, if you intend to use gentian violet, complete penetration is all that is necessary, whereas for iron alum hrematoxylin it is more favourable to fix .for a longer period. You will be using the Flemming acetic formula for chromosomes, not for cytoplasmic bodies, and there is no object in leaving the material too long in the fluid. After fixation ]I'LE3lMLva AS]) C'lJAMPY 77 wash out in running water for at least as long as you have fixed if the pieces are of close tissue, e.ff., mammalian testis. Botanists prefer to ,,,[((o;h out for 2 hours in tepid 'water, changing the water every ao miuutcH. For Flemming-withnut-aeetic-ac:id, long fixation if> indicated beeause tho cytoplasmic granules which you will wish to study stain better after prolonged immersion, follovi'ed by a careful washing out. Now if you waHh out too much of the fixative, the stf1ining il:! not intense, heing nuclear rather than cytoplasmic. If you wash out too little the I:-ltaining may he a failure for hoth partH of thc eell. The best way is to lUINe a number of small piect'H of material, transferring one ur two pieces at intorvals during the washing into 50% aleohol. '1'he proper stain is iron alum hWllmtoxylill (§ 145) used for the timel:-l givell in § 148. 117. Champy's Fluid. This is a modified Flcl1lmillgwithout-arct.ic-acid, or Altmann, whirhever way fllle prefers to regard it. It. is a Flemming with hiehromate, and giveR a more robust Rtain with hromatoxylin than (loes Flemming-without-acetie. The same result ('an he got, by fixing in F. W. A. Itud post-chroming. Pix as long as 3 days, and wash out slllall pieces overnight. The proper stain is iron alum hromatoxylin (§ 145), and the washillgout period may be rat,her critica.l (§ 121) so far as good staining of eell inclusions is concerned. It is admirahle for small invertebrates like Archiannelida, and for sponges. Its penetrative power iR increased by adding 0'75% NaCI, in the case of testis and similar material. The formula is 7 parts of 3% bichromate of pot!tssium, 7 parts of 1% osmic acid, 4 parts of 2% osmium tetroxide. 118. Carnoy's Fluid. This is the most penetrating fluid known, and is indieated for cysts, spores, eggs with impervious shells (Ascaris), ha.rd insects like small beetles, and so on. It is a destructive fiXative but has a limited application for chromosome cytology. Glacial aeetic 78 FIXATION .JIBTHOD8 [wid 1 part, absolute HIcohol 61Jarts, ohlorofol'1ll 3 parts. Leave till penetrated (scveral hOurR) , wash out in 90% alcohol overnighb. Another formula (Carnoy Lebrun) contains l'(wl'osive suhlimate, anrl does not keep long. H is equal parts of the three ahove-mentioned iluidR, saturated with suhlimate. 119. Mecuro-nitric Alcohol of Petrunkewitsch. Les!'; rapidly penetrating than Carnoy, but also good for small insec.ts, cmstncea, etc.. vV~1ter 3110 c.c., [Lbsolute alcohol 200 c.c., glaci'1l acetic no C.c., nitric acicllO o.c., suhlimate to satumtion. After saturation ill corrosive, filter. Undergoes Home disintegration after a time, but still works well. 120. Key to the Use of Fluids. To make sections of sUlall inve'rtebrates such as Polyzoa, flatworms, sponges, the more delicate Crelenterata, small embryos and ohjects are (lCCe88ible to the fluid, use ChamI)Y overnight; Rtain iu iron alum hrematoxylin, acid fuchsin and methyl green. Champ}, is quite useless for vertebrate embryos, e,g., human ernbryos. large lumps of tiHsue, 'whole mounts, and impervious objects like insects. No piece of tisRue should be more than :3 llllll. thick. If possible prick or tease the objeets with needles after they have been put in the fluid. For 'whole mounts use cOlTosive acetic or alcohol. For vm"iebmte tissues generally, use Zenker-Formol or Hegaud, or if only good anatomical fixation is needed, use Bonin or Susa. The same a,pplies to the invertebmtes mentioned above. For carmine or other whole mounts of vertebrate tissues (e,g., injected material) use corrosive acetic or Sus a if possible. For impervious things like inseots use Petrnnkewitsch or Carnoy. Stain in Borax Carmine or Acid Hremalum. Insects like aphids do best if pricked with a fine needle after fixation : they wash out better and stain hetter. ~\\'hich WASH ISU OfT1' i!1 ,For uhrolllusolIWS begin with p.lltill HOllin Hnd imll lucmatoxylin. You may later try ]i'lelllTlliug (with aceti,~ acid). Navashin (plants), and, (If ('OUl'SO, the Heoto-cannine I"lmear. To :mm up: the heflin'll!"/' l'ihould U:;l' Bonin for sections a,wl corrosive sublimate acetic for whole mounts. 121. Washing Out Fixing Fluids. 'rho beginner is usually puzzled as to what to do "bout this. As it rule after chrome fixatives material lllust, be washed uucler a running tap overnight, provided they hiLVe been fixed at leaf:;t overnight. This applies to slwh l1xatives as Regau!l, Zenker, ReIly, Altmann and _I<'l('mming. Fixativell not contaiuing chrome salts, hut having eorrosive sublimate, must be washed out in 70%) alcohol, to whieh enough Lugol solution (§ 71) has been added, to make R Hherry colour, Fixatiyes containing picrie acid should he gut into 70% !tnd transferred to 110% fairly SOOll. \Vith fixatives eontaining tl'ic:hlomeetie :'LCid pie'ees of vertebrate material :-;hould go immediately into tlO% alcohol. Carlloy material (without corrosive :-nlbliuHtte) gOOR fitl'itight into 90% or absolute aleohol, or the formula with (;01'1'0::;iV8 sublimate into 90% with Lugol llS above. In the case of picric acid fixation, water-soluble picmtes form (though this has been denied). 'Whatever may be correct, it, is true that, solut,ion of part of the ground cytoplasm takeR place if Bouin materia.l is left too long in ,vater or watery stains. But this is not all, especially with the chrome-formalin and chrome-osmium fixat.ive". Th(~ fixatives nULY contain some substance necessary to morda.nt the stain--in plainer terms-to make the stain bite deeply into certain part,s of the cell. 'This apl)lies both to iron a.lum hrematoxylin and acid fuehsin staining of mitochondria of a.Il animalH, and to the Golgi bodies of invertebrates. Thus, if you wash out too mueh the subsequent staining is unsuccessful. 122. Method of Washing Out. This is usually done under the tap, and presents no problem if the pieces are prXA11lON JlfErpHODS large. With it rubber band attach a piece of ll1uHlill on top of 11 pet.ri dish and plaee under the t~tp. For a, large number of pieces which are to be kept, l:Ieparate. cut some f;hort lengths of wide glass tube, tie muslin at. both ends with the pieces and their label (written in pencil) inside, place in a short, narrow j,Ll', and leave uncleI' a good stream of water. It is well to see that the stream is adjusted before you leave at night, and that the ,vater is not turned off overnight, as Homctimcs happens. For numerous small objects \,...hioh are not very valuable, wURh (lut uuder tap, using finer muslin, or sill{, hut for vnJuablc objects wash (Jut with distilled water in a petri dish. Remove the water from time to time with a pipette. Small object;; obviously wa;;h out more quickly, and call be done properly in a few hours by this method. If impermen.ble, wash (Jut in tepid distilled water. rl'he bot!1llists usually prefer to wash out their Flemming fixed material in tepid water. The water is changed every 30 minutes, for 2 hours. This treatment is quite good and might be tried more often by zoologists. It lllay have to be used if t,he water supply is turned off during the night. CHAPTER VII PARAFFIN DIOXAN N-BUTYL ALCOHOL AND CELLOIDIN IMBEDDING 123. The Paraffin Wax Imbedding Method iR almm;t universally used. The fixed and dehydrated material is passed into an oil or other liquid which clisHolves paraffin wax and finally into meltcd pure paraffin \vax, which permeates the tissue. Heat mUilt he URed to melt the wax and Qil wax mixtures, and this nee:essitates some form of irnbedcling hath, \vhinh can he cheap and primitive l1ud yet satisfactory (Fig. 5, A), or one of the spccin1 copper or wooden incuhators made eSIJccially for the purpose. Recently a vcry interesting incubator has been dc!:dgned hy Craig-Bennet 1; it costH five or six shillingi:l, and is shown in Fig. 5, C. The advantage of the \vax imbedding method is that it is cheap, easy, and best for subHequent section cutting. On the other hand, it causes shrinkage, hardcns the material somewhat, distorts thin embryologieal and other membranes, and is unsuitable for the study of fatty granules. In l'eeent years the general method has hcen much improved, ((t) by using instead of the higher dehydrating strengths of ethyl alcohol .'lome other alcohol i:;Qch as butyl alcohol, (b) by adding ceresin to the wax to l)revent crystallization on cOO~illg and to toughen the mass (§ 124), (c) by imbedding in dioxan (§ 136), (d) by combining the dioxan and eeresin methods. vVhile these improvements are decided advances, they do not get over the fact that the wax medium may cause distortion of l In the C.B. incubator only the top layer of the wax melts, thus ensur· ing that the pieces being imbeaded do not o,,:crheat. 81 PA R.t [1'/1'0,' j)/()XAN E'f'c. j,hill 11ICmilrn·lIeK --whcr(m~ t Ill' celloidin method i'i1l)l]Jorh.; t.lw memhrane!:'. r~OlUpal'p, t(ll' lm.;t-anee, l,mphihinn __ ~N ~==;~=""~"=='=~" A P ··-6R -- co i¥--:'1o/-----./ B B '·H loow. c -MS /MT • T4 FIG. 5. A. Drying plate, or pdmitive imbedding plate made from Rh"et of {'opper (P) bolted. at N to four threade!l l'()(l~ (BR). Height ean be adjusted by loosening bolts. B. Imbedding small objects in a silver paper COllO (CO), supp01't,eel ill it stonder dish with a blook (B). C. The Craig-Bennet Imbe<lding Bath. ('ollsisting of H. tlll'e.,,ply woollen box, with a lift-up front hingeel at H, vent; holes in top at V, IOU watt lump nnll flhade (MS). tlmw tray (MT) of wax-filled tubes (T.J-), 8witdl at, S. flex F. _Hf1at.f; up ill ] 0 minutes. (By (:Ollrtesy of the Pmsident HIHI Coundl of the Hoyal Mif'roflcopi"al Soddy of LondoIl.) embryo sections cut from celloidin wax imbedding, or from plain wax. The former show all the incipient spaces and membranes in their natural positions. But for all IMBElJDINO general purI)oses the \vax methorl hils come to stay, and suffices. The main filUltS to avoid, and pnints to attend to itl wax imhedding are: (a) '1'0 see t.hat tIl£' material has heen properly dehydrated and is permeated with oil lwfore being placed in the fil'f'.t oil wax hath. (b) To Hvoid uuneeessa,ry heat in the pl'OCef'B. (c) To infiltrate fl.lowly hut thoroughly, judging the t.ime according to the sizes of the pieces or animals heing imbedded. In the case of (a) the Wf~X will not penetrate if t,Ile oil has not permeated the material previously, and in the end the hIllel\: or animal ma.y fall out of the 'wax when being cut, or may have 80ft patches in it whieh will prevent cutting. This is extremely important" and It common ±:'lult. It is, of eOlll':o:e, traceable back t.n absolute alcohol cOllt.amina,ted with too much water, or to hnrry in dehydrating. In the rase of (fJ) yon will make Home tissnes like stone by overheating them. It is had for yOUl' knife. (c) Olwionsly a large piece of' It testif' or gland, rather impermeable material at best, or an art,hropod with only'the mouth, anus or other poreH through whirh the wax mixture may penetrate easily, needs careful infiltratiol1. A frog's eggs manifestly, or a sumll organ like a fi:og's kidney, will be penetrated more quickly. There are no, definit.e rules, t,rial and error will be your guides, but this much may be SfLid : if the pieces of tissue or embryos are no bigger than 10 mIll. square, 1 hour on the top shelf of the imbedding bath in equal parts of carbon bisulphide and wax, followed by 2 hours in pure WILX OIl the bottom shelf, will imbed most, t.hings suecel:lsfi.dly. But if you are a cytologist. you Hhould not try to fix pieee~ as large as 10 111m. square, and if you are a histologist, cleanly-ent sliceH 3-5 mm. thiek Hhoulcl be enough for most pl1l'poses. Using such thinner pieees 1 hoUl' in the first mixture <tud 1 hour in wax is enough. Some Htudents, however, lw.ye enriolls i<leaR on dehydmting, whieh Illllst PARAFPIN DIOXAN ETC. he dOlle properly for successful imbedding. They take a row of graded alcohols ill petri dishes, and leave pieces of tisfme 5 minutes in each. This is useless. Now, assuming that you have cleared the pieces as descrihed in § 18, you now proceed to imbed them. If yon are working in it laboratory there are likely to be some p(':tri dishes possihly ncar or inside the oven, whieh have previously .been used for imbedding. You transfer your nmtel'ial and enough oil I to eover it into one of these, seeing that it is free of water. The next step depends on a number of things. In some laboratories there is kept a " IRt wax oil " mixture, which reany consists of the old WiLX I1nd oil poured a;way from previous imbeddingR. If this is cold, it will lJe possible to remove some from its dish with a sCl1lpel fwd [LCW it to your own capsule. The fLmount, you 11<1(l does not matter very much, but it should not be too lit.de, and you want to be sure that yonI' pieces of tif'Rue are not pushed high and dry above their oil. "When this is done put the dish on the top shelf. Before going away you must Ree that the next bath of pure wax is ready; it has got to he melted. Possibly there is ,1 metal dish of "pure wax," already melted, in the oven. Regard it with suspicion lintil you are sure that it is pure wax. Dip a dry knife cir match into it and cool under tall. If it goes properly hard it is pure wax. If not, you will have to chip some hard wax into another dish and put it to melt in the bottom of the oven, 01' possihly melt it ca?ltiously over a bunsen. After ! hour, pour off the melted liquid (wax. and oil) from your clish with the objects, and pour on the pure melted WI1X. Uthe first clish of paraffin wax and oil after i hour contains lumps of unmelted wax, you must put the dish in the bottom of the imbedding oven and wait longer . .Tl,le oven certainly is not at the proper temperature, and is not suitable for imbedding. Going hack to the first 1 l\'[(,I'C11y it ('OIlVElllil'nt tt1m) for whatevor wttx Bolv(1ut you aI'C uBiug .. IMBEDDI~YO step, if there is no wax oil mixture ready for you beside the oven, you simply chip some (ll'Y, hrrrcl wnx into the (liRh with the oil and ohjeets ami ipt it llW!t, 011 the top Hhelf. On the othel' hawl, if there iN nwllerl wax oil mixture ready in the oven YOll :-;honld place yOUl' !lit'h and ohjects, with oil, on the top shelf' and warm it before pouring ill the wax oil mixture. If you are :mre the WILX oil mixture is about half-antl.half: you can pour off yom' own oil before adding the mixture. ·When an hour or so ha,s elapsed, and inspection has shown that the pure wax in your imhcrlding capsule is liquid, you may proceed to eool the wax as follows. Take a flat piece of glass 3 or 4: in. square, rub it over ·with a little glycerine on one l:;i(le and p!tWli two pieces of flnt metal together to make it cell, aH shown ill Fig. a. These pieces of metal should he about 20 liun. high amI 40-00 mm. long, with 15 111m. at the end bent around at right angles. They should be about 2 mm. thick anel perfectly flat. Yon can make Heveml from pieces of Hat hrass so as to give different sizes. Now quieldy pour the wax into the mould, and with 11 pair of forceps warmed in J;I, flame fLrrange the piece or pieces as you wish-say in a row or closely placed together; remembering that the sections must be small enough to go on !t 3 X 1 slide, the closer together the better. It is a good thing to have a bunsen or spirit lamp burning nearby in case you are too slow and the wax hardens before you can an'ange llmtters as you wish. By heating an old scalpel and dipping it in the mould you can keep the wax melted till the operation is concluded. Now breathe lightly on the top of the wax till a film forms, then gently lower into a dish of cold water. If a long string of wax shoots up to the tal) of the water the film has not been made thick enough. Leave for at least I hour, till hardened throughout. 124. Wax and Temperatures. In warm climates, or in Kli PARAFFIN nJOXAiV E'l'C'. summer. it. will be necessary to use a harder wax than in eolde~' climes or in \vint~l'. '1'he usual melting poillt i:; 52° C. Due to the work of Higgs, "\Vaddington and Kriehel, we know that t.he addition of ceresin, which ii'1 f1 vcry hant wa,xy substanee, giveR the cooled wax a fine, hard, miero-crystalline texture. In fact the hardness can be controlled largely by adding more or less ceresin. If YOll are just. doing ordinary work do not bother about ceresin, but if you wish to out such objects as feathers in skin, very yolkS eggs, 01' insects, try ·wax ceresin mixtures. ·Waddillgton says that even 0'5% ceresin is advantageous: in pmctice you wnuhl use higher ratios. EspinaHse haR used successfully equal parts of "\Voolworth eandles, paraffin wax 52° C., and ceresin for cutting bird skin (1ncl developing feathers. If yon are planning to cut very hard objects try ~c()lllbilling the dioxan (§ 13G) and ceresin wax met.hods. 125. Cutting Sections. There are two types of Rectioll {Jutting, one ea,sy, the other difficult. The first consists in merely getting some sections of material like a gland or genitalorgrLll (histology). The other kind is serial section {Jutting in which all the seotions must be cut the same thickness, and none lost (embryology, etc.). The first thing in this latter type of section cutting is a good microtome-that is, a rigid instrument which will cut consecutive sections the same thickness. Not all micl'otomes will do this. We will take the first and easy type of section cutting, sa,y of a piece of liver or testis. The material has been imbedded as described in § 123. The trimmed block of wax CH'ig. 6., A) cuntaining the piece of tissue is to be fixed on the special table (Fig. 6, B) which clamps in some way 011 the microtome bar which moves the block towards the knife. The table is removed from the microtome, and if the ribbed surface is free of wax some of the latter is melted on to it with an old scalpel. rrIils makes the CU1"P];..Yf/ SEC'l'/()X8 K7 :-;eat for the block. You now trim the latter. The iil'l.;t thing to notice iR that the block mtl:st not Il(, longer than 1 in. on the side \vhich strikes HlP knife. and it, shuuld be le8s. hecause, presumably, you arc going to mUltnt thc' sections 011 a 3 ;< 1 slide, an(l you lI1U~t not !llwe t,he H AEl}'" ;. l.:~ _ D ~ L j ~B m K 1:-O(OICI:I~ G o rc?1 xUx sect.ions as broad as an inch or t,hey GI1111l0t then he flattened easily, using routine methods. Inspect the hlock and ta,ke one side whieh halO a good depth of wax as the siele which is going to be stuck on to the microtome table. TIlis process is done with a hot knife and there must be enough wax for this. Having determined what side i:-; to be used for this purpose, trim it slightly first, of alL and ~tlbseqU!mtly lJegin to trim t1w PARA]i'Ji'lll DIOXAN E'l'U. other sides carefully. Do not attempt to cut too deeply, just shave a little at a time, using a stiff safety razor blade or a good 80al}le1. At the end you should have tL block as shown in Fig. 6, A. Fix this on to the microtome table with a. hot knife, and dip into cold water. Inspect it and Hee that the two sides which come down parallel to the edge of the knife are pa,l'allel to each other. If they are llot parallel, the ribbon of sections will turn to the left or right QlS :;;hown in Fig. 0, M. In :Fig. 6, E, presumably the block is to be fixed on the table with the hot knife at X. But there is not enough wax here for the purpose. Compare with Fig. 6, A, which is correct. In Fig. 6, H, the block has been cut too long. It will bend or vibrate too much as the knife strikes it, or it will break. Fig. 6, I, shows what should be done in the case of a long object; there is plenty of support here, ,wd the lower part can he trimmed clown later when the upper part is cut. In :Fig. 6, ,J is a chain of three sections. There are two faults here, hoth causeel by the fact that the block has not been cut properly cubical and parallel. Fig. 6, 0, is one section with the part X wasted; this should have been trimmed off the block. Fig. 6, K, has white lines in it, caused by pieces of grit having turned or broken the edge of the knife. The end result is often what is shown in Fig. 6, F, where the sections have split completely. Fig. 6, L, seven sections cut on a defective microtome, or on a microtome in which the wax block was insecure or the knife blunt. Having st/uck the block on to the table by running a hot knife between A and B in Fig. 6, you are now ready to cut sections. Put the safety catch in operation on the microtome, and fix the holder in place, hut do not. screw it tight until it is pushed in far enough to be Btruck by the knife when you release the safety catch and lower the moving bar towards the knife by gently turning the handle (in n Minot microtolne). When the moving hal' is let SERIAL BEUTIOH8 down, hring it up until the wax block ean be moved HIl against the edge of the knife. See that the sideR are parallel to the knife edge. Raise the moving hal' to it" upper position and let in the safety eateh. Nnw damp up the table holding the wax hlOl:k. On all modem miemtmnes there is f!ome indic:ator fur thickness; set it to 1O/-L, and begin tll1'lling the microtome wheel until sections are cutting. Continue turning untit the whole face is being cut. Stop awl brush off the sections already cut. Ul-le it pointed brush, muil':!ten it now in your mouth, cont,inne turning the wheel, and try to catch the first few :;e(Jtions by putting the hrush under them, and by genMy lifting them just above the knife, you Rhould get a chain 01' ribbon of sC0ti0l1B. RoaneI' or later, and mmally sooner, the ribbon broaks, and the piece adhering to the brush must he laid on a sheet of foolscap. If the ribbon keeps breaking yuu mnst inspeet the knife; it may be at the wrong Hlope. If the hlock clears tho .i<nife below the edge as it descends the fault lies ''lith the wax block. It is too hard, and must be faced with softcr wax. Turn the ·wheel up and put in the safety (lateh. Melt, a fragment of softer wax on a scalpel and gently face both upper an.d lower sides of the block; see that the knife is just hot enough. Now try again. TJsuaUy this ,..·m cure the trouble. If the sections keep curling up, it uSUl111y means that the knife is hlunt. 126. Note on Serial Section Cutting. The microtome should he of a rigid. pattern and the bolts which clamp t,ho block holder and the knife in position well screwed up. The block will have been carefully trimmed as in Fig. (I, A. If the microtome eutt:> altel'l1ate thick and thin sections (Fig. 6, L), or sueh varying sedions fairly dOl:'e together, the hlocK if' loose, or the knife is not damped, or the mechanism is out of order, unoiled, or it may want cleaning. which can be done in xylol. If the mechmdslll is fouled with 'Nax it obviously will not, work properly. PA HAPFL\' DlOXAN ETO. On the other hallfi. you should note that if yon out a rib hun at a qnick lJa~e and slHldenly stop or slow down, the next He(:tion will. be thinner because the wax block hal'; warllled and expanded ·whi.1e it was being struck by the knife, fwd in the interval cooled. Any differences in temperature will show in the sections. Therefore, if you have a good ri1hon coming off, and have got to the stage whell yon are going to Rtop, do so gradually, and when you begin again the fi1'8t sedioll will not be so different ii'Olll the otht,l's. In all enRes turn the handle evenly and ;;teadily. Sometimes t,l18 ribhon hecomes electrified, and ilies to\\'anIR, and sticks on to, objects nearby. Various elallol'ltte methods to de-eleetrify the ribhons have been sngge::;te(1. The l)eRt method is to go home fl,nd try again next day. 127. Cutting Sections without a Microtome. By putting a piece of jhcrl tissue taken from water or 50% alcohol between dderherry or such pith, fairly good section;; can he cnt "ith a RhM'P razor or a new safety razor blade. ]'01' histology these spctiOlHl will do quite well. Put into a l)etri dish of 50% tdeohol or water and select the thinnCloJt for subsequent treatment. For staining, dehydrating, etc., the seetions are stuek on !t slide with lVIayer's albumen or gelatine (§ 95). }tuh H smaU drop on the middle of the slide, press the section down on it with it cigarette paper or JitMe fine filter or blotting paper, :flood gently with 90% alcohol which 'will harden the albumen anclmake the section adhere. The slide can be htmdled quite sllecessfully if you are gentle with it. 128. On Mounting Sections. 'Vax sections are stuck on to a 3 X 1 slide by glycerine !1ml albumen (Mayer). If you have no glycerine and albumen at hand, use distilled water instead, in which caf!C the sections do not stick so seem·ely. To make glycerine and albumen, l'emove white of egg from yolk, add an equal quantity of glycerine, stir up with It domestic fork so as to break the large lumps of albumen, add I gm. of salicylate of sodium dissolved in a little wo,ter (this is to keeJ) the material sterile-yon can use im;tead a crystal oHhymol dropped in the store bottle). MOUNrfllNO SIiJOl'JOX8 Bl When thoroughly stirred up, filter through a coal'se filter paper. This will take days., and the funnel must be covered. To use, allel 10-15 chopi':! to 50 C.c. of distilled water (or good tap water), Rtir. Take the brush you have used for the sectioning, dip it in the mixture and flood. the slide. Rub the brush backwards and forwards and see that the slide is properly covered with liquid. Now, when putting a length of ribbon OIl the slide you must keep in mind what size of coverslip you intend to use later, and remember that the ribbon stretches when flattened by warmt.h. 'rllUS yon will havc to cut the ribbon just a little shorter than the coverslips you are going to use. Suppose you are mounting a large series of sections by this method; having determined the desired length of ribbon, you can count the sections in each length, ·which is tedious and sometimes clifficult. I ahntys use a matchstick impaled in the middle on a mounted needle (Fig. 6, G). The stick is previously .cut the right length. The tYlle of scalpel to use for cutting the ribbon is shown hl Fig. 6, N; it should be sharp. Pick up each ribbon length with the moist brush. When the ribbon (or ribbons) is in position c(lliliously warm the slide over a flame, tilting and agitating the slicle from time to time, so as to keep the warmth evenly distributed. The sections will begin to stretch out and flatten. When they look quite flat, set the slide on the bench to cool for a moment, then cautiously tip the extra albumen-water off the slide. Place ill a warlll place to dry. 129. Drying Plate. This can be made of an iron or glass plate on four legs (Fig. 5, A), underneath which is [L tube with an aperture for a row of very small gas lights. The plate must not be so hot as to melt the wax. Some people use an electric bulb in a flat glass-topped box for this purpose. This may become rather expensive. .:\Jl()tilcl' way iH to put shelves near the hot-water cisterll. Tn summeI' it usually Hufliecs to placc the slides end up ,~gaim;j:, the h,tl',k of the h(1)(·h. On a hot, l)late they Itre' rlry next morning. 130. To Deparaffin Slides. The dried Blicle iH yently warmed over a flame (without melting the wax), (a) to remove moisture, (1.nd (b) to enable the xylol to dissolve t.he para:lfin more easily, and then dipped into xylol or henzol for a few minutes. When taken out it is dra,ined, and it::l back "\viped ,,,ith a dm;tel': It is then ilipped into tH) %OJ: absolute alcohol, and is ready to pass down through graded alcohols to the stain bath. If at any time you are not sure on which side of the slide the sect.ions lie, hold the slide sideways towards the light. 131. Microtome Knives. For students these ha.ve been largely sUllcrseded by safety razor blade holders, which will do all that is necessary except cut valuable or heavy material such as large embryos, etc. Some people buy a heavy straight ground razor (difficult. to find nowadays) and use as a microtome knifc. These and the heavy microtome knives can be sharpened in the laboratory, but the process is not easy, and certainly cannot be done properly by an unskilled persall. :iVIicrotome knives generally need re-grinding after cutting anill\als containing grit-e.g., tadpoles or worms, and such animals should be fed on meat, onion or potato, or some such soft material, before being lalled for sectioning. There are on the market microtome knife sharpeners consisting of ioul' surfaces mounted on an oblong body with ,11 handle. Each side has either a stone or a treated leather surface. You begin on the stone and stroke the knife on succeeding finer sides. Mier practice you may learn to sharpen a knife properly. 132. Thickness of Sections. For general purposes, lOp, is the right thickness. This for two reasons; firstly, it is thin enough, and suits the times given for staining; secondly, it is a good ullit for measurement, or in case you intend to make a model in embryological work. For cytology (chromosomes) you may have to cut sections which will contain whole cells and be no thicker. This IMBEDDING SMALL OBJEOTS (13 will depend on the size of the cells. For the cytology of the cytoplasm and such work, very thin sections (3-5IL) are usually cut. 133. Handling Protozoa and other Small Objects. This can be a tiresome business, especially if you have only one small object to be cut in a special direction. For a large number of Protozoa, embryos, or eggs, the task is easier. If you have no hand centrifuge, you can usually manage as follows. Kill the animals in a test tube or centrifuge tube, using a fixing fluid like Kahle's fluid (alcohol formol) or Cal'lloy in which they are almost certain to sink. Of course the killing can be clone in a stender or petri dish, and the individual organisms picked up in a pipette, and, as a matter of faet, most things will sink in the usual fixatives. After ihatioll is complete, remove from the test tube as much of the fluid as yon can with a long pipette fitted with a rubber tea,t. Now add the washing fluid, be it 50%, 70%, alcohol (for Bonin, etc.), or water (chrqme fluids), or 1)0% (Cal'lloy with sublimate). When the organisms settle, remove the liquid agnin and replace with fresh water if further ehanges are necessary (chrome-osmium, etc. (§ 121)). Finally, earry the organisms up to the liquid to be used for imbedding (carbon hisulphide, xylol, etc.). Now prepare a lead foil cone (from the" silver paper" from cigarette llackets). Take a piece of round wood, like, but somewhat thicker than, a lead pencil. Sharpen it to a smooth point, so that when a sUlall circle of foil is pressed on it, and taken off, you have a neat "watertight" cone with smooth sides. Prop this up in a stender dish with some small stones or pieces of metal (Fig. 5, B). Now pipette the organisms with the oil into the cone. Remove excess oil and add cold soft W~tX and place in oven. Leave 20 minutes, with a warmed pipette remove as much as possible of the oil wax mixtul'e and pipette in hard wax. Leave 30 minut,eH. Change the hard wax, PARAPPIN DIOXAN E'1'O. aftor ] i'l minutes if you think it necessary. Remove from the oven, and gontrly run cold water into the stender dish to eool the paTaffin. Hemove the foil, and you have the organisms on the top of the wax cone .. Now take a nullo of hard wax about the size of ,1 piece of loaf sugar. Cut a little hole in one side just big enough to take the tip of the eone, which you cut off and dry on a filter paper, if wet. v\Tith a, hot wire melt around the hole you have made in the cube and drop in the tip of thc cone. Run the wire al'olUHI it just enough to melt the outside of the COlle. Allow to cool. Trim the block for sectioning. Anotlwr method much u;.;ed, but not m~arly so good, is to hrillg thn ol'ganisl1m into a sml111 quantity of 70% alcohol. Take It uuhc; of fb:er11ll'11in or liver, awl in a smooth part of it malw n hole, and pipette tho organisms into the hole. COVN the hok with a drop of egg albumen, and gently lower the hlock into ()O~'{J or ahsolute alcohol, whioh will coagubte the albumen ILnd sE-al tho opening. Some people simply tangle up the objects taken from 7()% in It drop of albuDlen and cover tht> alhumen with 00% alcohol. Anot,her method is to flatten it drop of albulUen on a slllooth flat piece of liver 01' twain. Pnt the object from water on it and remove excess water 'with a pieee of filter or blotting llaper. Now gently flood with 70% 01' 90% alcohol. . Now snppose you wish t,o orient a small object so that it can 1)6 cut in a definite direction, you may use a modification of the above method. Cut a neat slice of the liver or prefemhly brain (taken from 50%) alcohol), and fashion it into a thin rectangular plate. The objeet (also from 50%) if; lllaced on this moist phte at right angles or parallel to the shorter sides, as YOll wish. A little egg albumen is run elll gently, and when l1Jl seems to 130 well, the capsule is cantio11sly flooded with 00% alcohol. When you are subsequently trimming the block fOT cut.t.ing you will know which dil'eut.ion to cut. Fur ohjects a,bout as big as tadpoles orientation can be done fLt the end wheu the hot wax is poured illto the L'lELLOll)JX .~IN'/) ()()~ nil mOlild. UHe hot needle:,;, and keep rt Hc~dppll't'ad.v io hea j, in ease the wax starts to (iool too fJOOll. 134. Frozen Sections. ThesE~ are IllU,ric hv freezing fresh or fixed (usually J(H'lllalin) lllHterinl ill C()2 gill';, ar~ much used in :Pathology labomtories, and are not really very necessary except f()l' certain work, such as microchemistry. Even here the teased preparation may do. The advantage of frozen sections is that perfectly fresh unfixed material may be cut immediately without imhedding, a useful and quick \yay when diagnosis of a tUlllOUl' must be made in a short time. Suppose, however, you wish to Htndy ovarian cells by microchemical methods, the frozen section must be used, and simil~H problems sometimes arise. Descril1tion of the method is outside the scope of this book; refer to the "Microtomist's Vademecum," or some such book. Note. vVhcn buying a freezing microtome Fl.ee that it I;; provided with a kllife-eooling attachment. , . 135. Celloidin Technique. Celloidin (gun cotton) if! soluble in alvohol and ether, and is hardened by expmmre to chloroform. 'rhe object is bl'Ought into absolute alcohol, thence into a tkin mixture of dried cel10idin in ether and alcohol (I : 1), then after a few hours placed into a thicker mixture. After several hours (01' better, days) the celloidin mixture, which should be just thin enough to pour, is ti11ped into a paper mould and the latter placed in a properly covered vessel containing a fe·w drops of chloroform. The vapour stifl'ens the celloidin, which can be then removed from the paper mould, trimmed and transferred to 70% alcohol containing a fe·w drops of chloroform. Or, the block may be tmm;fened to chloroform just after the hardening in chlorofor111 vapoUl',· and paraffin wax subsequently chipped into the vessel. Imbedding is clone in the ordinary way, chloro,form being the medium instead of xylol. The eelloidin method is not very lUuch used for ordinary WOl'k It PARAPPfN n!OXAN E'PO. may Imve to he learnt. for Rpecial l'efiea.rch. ~Refer HlP "Miel'otomist'r; Vade-llIE\(·.\lm;' 01' f'ome such hook. 136. Dioxan Imbedding. Dioxan is !l cumulative poison in I in 1,000, musing leeions of the lungs, liver and kidneYR. It is quite ?lnsuitable for C0118tant use in 81nall 'I'OOUt8, especially when it is heated during imbedding. However, knowing its poisunous qualities it is possible to me it without harm. It. is a very remarkable solvent., dh;solving 01' lllixing freely with water, alcohol, xylol, p[tmtIill wax, celluloid and balsam. It will dehyclrate material, "\',hich can 8uhf:'eqnently pas!:> straight to l)araffin wax f(JI' imbedding. It causes 110 apparent shrinkage. Materials dehych'u,ted ill dioxan ean be mounted in dioxan Imlsam. It therefore does away with the necessity for uRing xylol or similar dearing agentR. After iixatioll in Bouin, Formol, Carnoy, bring straight into dioXllll; after COl'l'osive sublimate fixative:; it is better to wash out in 130% or 70% 'with iodine, hut some people add it crystal of iodine to the dioxan. Chrome fixativeH are washed out in water, and then transferred to dioxan. Dioxan mixes gently with water, and after three changes of dioxa,n during the day, leave overnight in pure dioxan, or use it dehydrating jar of dioxan. This is a wide-mouthed short jar with a good ground glass stO}lper. A piece of gitUZC is cut in the form of a star, and the points are bent over so as to make a table. This is thrust into the bottle. A small bag of calcium chloride is arranged underneath the gauze, and dioxan is poured in. Pieces to be dehydrated can be put on the gauze with It pair of long forceps. 'rhis is the cheapest way of nsing clioxan for dehydrating. Subsequently the pieces arc put in a 1 : 1 mixture of' dioxall and wax (30 minutes), and pure wax (1 hour) according to sizes of pieces. Yon should notice that all modern staining methods are designed for alcohol dehydration, and it may be necessary, tu B[T'PYL ALonHOL !l7 if you u:-:e diOXltll, to modify tJw times of clifff1l'cutin.t,icnl in Nome eaHes. 137. N-Butyl Alcohol Imbedding. Tn l'e,tlize the hardening effed of ethyl aleohol it is unly ncnpl'sa.I'Y to leave a piece of liver in it for 2 or :; dttys. N-butyl alcohol has no such hardening effect, but it absorbs very little water and part of the dehydration must be done in ethyl alcohol. 'The most recent method (Stiles) for this technique is as fullows: From 30% ethyl alcohol the tissue is placed in a mixture of D parts of 45% ethyl and 1 part of N-butyl alcohol. After about 2 hours, place in an 8 : 2 mixture of 63% ethyl and butyl alcohol (2 hours), then in a 6·5 : :3·5 mixture of 77% ethyl and butyl alcohol (4 hours), then in a 4'0: 5'0 mixt,ure of' 90% ethyl and butyl alcohol (6 hours to a day) and finally in i1 2'0 : 7·5 mixture of absolute ethyl and butyl, where it should remain overnight. Now the tissue is placed in pure N-butyl alcohol w·hich should be changed twice during the course of several hours. Tissue may remain in N -butyl alcohol for days without deleterious effects. To imbed, tissue may l)e taken from N-butyl alcohol and placed directly in melted paraffin, but it is better, perhaps, to use an intermediate step of 2 parts of paraffin and 1 part of butyl alcohol, leaving the tissue in this (in a closed vessel) for 20 hours. Then the butyl alcohol may be allowed to evaporate, or the tissue can be placed in pure wax. In any event a long period of infiltration is essential, 4 01' 5 days not being too much. N-butyl mixes with dioxan, and so far as I am aware the possibilities of combining the two techniques have not yet been tried. It seems likely that the N-butyl technique has already been superseded by the dioxan, but the results of the two methods will have to be tried out side by side byentomologists . • 138. Storing Paraffin Blocks. This is often done on strips of paper 01' thin card. The bottom of the trimmed I,ADOIlATORY'fECIINIQ.UE. 4 !IS PARAFPJR DIOXA.N ]I}'l'C. bloek as it haH emne off the microtome is put on the paper and <t hot knife held a.t the haek till the ,,,ax melts slightly. On (~oolillg the numhel' (11' description of the hlock is written beside, ~1nd the whole eoUection pinned on the wall, 01' kept in a drawer. ' CHAPTER VIII STAINS AND STAINING 139. Stains and Impregnations. Tho staining substances which you will have to learn to use are carmine and hrematoxylin, known as natural dyes, and a nnmber of synthetic coal tal' dyes-neutn~l red, Janus green, methyl blue, methyl green, nigrosin, orange G, eosin, acid fuehsin, gentian violet, Sudan IV a,nd Nile blue. There are many others which it will be unnecessary to mention here at length: Methylene blue, s[~fmnin, hasic fuchsin, uurantin" light green, toluidin blue, thionin, alizarin, tl'ypan blue, etc., are very important stains which the beginner need not use. The most important dyes are carmine and hrematoxylin, and maDY of the best known formulre contain one or the other of these substances. Carmine is the ground-up (hied bodies of the cochineal insect, while hrematoxylill is the ether extract of logwood, a product of South America. The extract is dried, dissolved in water, and the hrematoxylin is crystallized out. The other dyes mentioned are derivatives of the hydrocarbon, benzene (C6Ho)' What happens when a section or smear is stained ~ There are two main theories of dyeing, the chemical (Ehrlich, Dnna, Giemsa) and the physimtl (A. Fischer, Bayliss). From the fact that in double and triple stains, differentiation is simply got by extracting (and recovering) one or other of the dyes by some solvent like alcohol, it seems that in most cases no chemical compound is formed by dyeing, but the phenomenon is one of adsorption. It would seem that no staining method can be speciiic~ ~I.' 100 STAINS AND STAINlNO nor is there actually any that is. It is only the microchemic,tl met,hods like Fculgen's technique which approach complete specificity. Yet the electivity of some staining methods is surprising and naturally has given support to the chemical theory. l!'or instance, acid fuchsin for mitochondria in properly fixed and lllordanted material, Weigert's elastin stain, or methyl green for chromatin are all remarkably elective, but other things may be stained-but not many other things. 140. Silver and Osmic Impregnations. These, especially the former, are of tremendous import,ance in histology and oytology. Silver can be made regularly to impregnate anyt.hing except fat and chromatin. The process is nearly always akin to a photographic one. The silver salt is absorbed and held by certain bodies or surfaces, and on subsequent reduction shows as a brown or black impregnation. Careful examination of a series of under- and over-impregnated silver preparations will show that a given body may be impregnated throughout with silver, 01' the effect lllay have bec:ome an enc:rusting one. In the same way osmium tetroxide (OS04) is reduced to a lower substance, possibly hydrated OS02. At all events, in some caseR, this "stain" may he made to stream out of a sec:tion by treating with turpentine in much the same way as alcohol 'will differentiate certain coal tal' dyes. At the present time the exact nature of osmic staining is unknown. 141. Stains that Need not Ripen. In this book it iR presumed thll,t you are working in a laboratory in which ripe stains are to he got. If you are working by yourself, and want to start staining at once, here are some general stains which can be used immediately: 1Jlann's methyl blue eosin for all usual purposes, especially for sections, but not whole mounts of invertebrates or embryos. It tends to fade in acid balsmn, but it is a, beautiful stain. MUiJer's Acid Ham~aZ.llm, a transparent, delicate yet HiEJJIA'POX YLJN 101 energetic alum luematoxylin stain, very useful both for sections and large embryos or whole mounts. Boma: carm'ine, 1:1 good general stain for crnstacea, any ·whole llloU11ts, anel nlfoio for staining in hulk (as also iH MILyer's above). Bon1x carmine and Mayer get better after a litHe keeping, sa,y 8 weeks or a month. In any case it is recommended to make up and ripen· some Delafield and Ehrlich, '",hich will be ready later on \"\'hen wanted. Heidenhain's and Regwud's hromatoxylill, and Leishmanll or Giemsa, lllay be used immediately they arc made up. For chromosomes, gClltia.n violet can be used immediately. 142. How to Use Stain Jars. When a slide is being passed from ja.l" to jaI' it should be quickly drained into the jar it came from, and excess stain or alcohol ruhbed from its back with a dean ahmrbel1t duster. Keep the stoppers or corks on the _jars when not in 1{Se. Hrematoxylin Formulre 143. Delafield. Solution A. Ammonia alum, saturated aqueous solution, 400 c.c. Solution B. Hrematoxylin 4 gm. Ethyl alcohol (9(i% or absolute) 25 c.c. Mix A and B, and let it stand exposed to ai1' and light for 3 or 4 days. A large beaker does for this. Solution C. Glycerine 100 c.c. Methyl alcohol 100 c.c.; to be added to the other, when it is ready. Allow the mixture to stand until it becomes quite dark. Transfer to a well. stoppered bottle. It lasts well and improves with keeping. It, is a strong stain, and works well ,,,hen diluted by half or one-third with distilled or acid water (§ 37). 144. Ehrlich. Water 100 c.c., strong alcohol (95% 01' 96%) 100 c.c., glycerine 100 c.c" glacial acetic acid 10 c.c., hrematoxylin 2 gm. Dissolve the hrematoxylin in the alcohol, aeld the acid, then glycerine and water. 102 8'llAIN8 AND 8TAININ(-I- Add potash alum to excess. Ripen in an open beaker or open flask in bright light unW a dark red. Pour off into a well-stoppered hoUle. 145. Heidenhain. A 0'13% solution of halmatoxylin in distilled water which improves on being used. Do not throw it away when it gets black, as it is then staining hest. 146. Mayer's Acid H<Emalum. Hffilllatoxylin 1 gIll., diRtilled water 1 litre. Dissolve and add 0·2 gm. sodium iodate (NaIOa) and 50 gIll. of alum, dissolve and filter. Add glacial acetie up to 2%: This keeps well, it stains fairly satisfactorily at first, but is better later. 147. Regaud. One gmm hffilllatoxylin in 10 C.o. of absolute alcohol, added. to 10 c.c. glycerine and. 80 C.c. distilled water. Much used nowadays instead of Heidenhail1. 148. Times for Regaud or Heidenhain for Cell Granules. Leave all day in 4% or 5% iron alum, rinse in water and place in the hrematoxylin in the late afternoon. Differentiate next morning in 4% or 5% alum. Neve~" for example, leave overnight in alum and 2 or 3 hours next day in halmatoxylin. Carmine Formulre 149. Borax Carmine (Grenacher). Carmine 2 or 3 gm. 4% aqueous solution of borax 100 C.c. Boil for 30 minutes. Cool, add equal quantity of 70% alcohol. Allow to stand for a few weeks, shake now and again, filter. 150. Hollande's Chloro-carmine. The student used only to borax or para-carmine does not realize that some carmine stains can be very intense and rival even the hrematoxylin techniques. Undoubtedly one of these stains is chloro-carmine of Hollande, which stains mitochondria in frozen or ordinary sections, and is useful in OARMINE 103 'smears or whole pieces. Place 5 c.c. pure hyclroohlol'ie acid in a porcelain dish, add gradually 14 gm. of powdered carmine, stirring constantly to lllake a doughy mass. Allow to digest for 24 hours; add 250 c.c. distilled water, stir, and bring to the boil for t hour. Filter and make up to 180 C.c. with distilled water, then add enough 75% alcohol to make up to 200 c.c. Smears or sections are stained for from 2 to 24 hours, rinsed in distilled water or 30% alcohol, transferred to 3% iron alum solution, in which they become black, subsequently decolourizing slowly. 'When differentiated, rinse in 1 % pyridin solution, wash under tall for 10-15 minutes. This stain is highly recommended. If you are using reel stains like this, and safranin, constantly, place a blue screen in front of Y01U' microscope lamp. 151. Aceto-carmine. 'fhis is the stain now so much used in the genetical laboratory for the rapid study of chromosomes by, the smear method. Though fairly permanent preparations may be made, it is really not good for this purpose. Schneider's aceto-carmine: saturate boiling 45% acetic acid with carmine. This strength of acetic is taken because it dissolves the largest proportion of carmine. 152. Belling's Iron Aceto-carmine. Add a few drops of ferric hydrate dissolved in 45% acetic acid to some Schneider's aceto-carmine nntil it becomes bluish-red, but without visible precipitate. Then add an equal amount of ordinary aceto-carmine. When using both the above stains with large teste8 (e.g., locust), you first smeal', add enough carmine (and not to excess), covel' and W~1rm gently; set aside for a few minutes. Ring with vaseline in a warIn pipette if necessary. For very small testis (e.g., Drosophila 01' Asellus) dissect out, add drop of aceto-carmine, cover, leave for short time, and gently crush by moving coverslip. 'Varm if necessary, ring 'with vaseline. STALNS AND 8JlAININO Coal Tar Dyes 153. Mann's Methyl Blue Eosin. Mix 36 c.c. of 1 % aqueom; solution of (water-soluble) eosin, a,nd 45 c.c. of 1 %methyl blue, and 100 c.o. distilled water. The solution should be neither red nor blue, but purplish yellow green. Not all eosins are suitahle. It stains instantly, but may be left up to 24: hours. Mann used the stain as folImn;: Fix in IJicric corrosive sublimate (I gill. picric acid, 2 glll. tannin in 100 c.c. saturated corrosive). Btaill sectiollfl or smears for 24 hourfl. Wash in water, transfer to absolnte alcohol 50 C.c. with 1% NaOH in absolnte :3 or 4 drops; sections go red. Wash quickly in absolute alcohol, transfer to water for 2 minutes, then bring into water slightly acidified with acetic acid. They become blue; dehych'ate in absolute and mount in non-acid bal~alll. Good results will be got as follows. Stain sections or smears 15 minutes to hour. ·Wash in water, transfer to nO% for a few mimltes, then absolute and mount in balsam. It will be found that one of the two dyes is more soluhle in alcohol or in water than the other-depending 011 fixation and time of staining, and you must try to keep bot,h stains in the smear or section by observing such details during differentiation. Note that the stain is good for all fiXations except chrome-osmium (unless bleached). t C. Dobell stains for 12 hours, then differentiates in a weak solution of Orange Gin 70% alcohol, then passes rapidly into absolute, xylol and balsa,m . •J. Ford recommends to stain for 3 hours at 37° C., to wash in water and to treat with pure formalin for 5 seconds, then to wash in water and to continue as above according to Mann. This method is particularly vain able for Negri hodies. Mount in cedar wood immersion oiL 154. Altmann's Acid Fuchsin. This is definitely a very fine method with which beginners often have trouble, traceable to one of two faults in the cll-se of failure. Firstly, lllOrdanting is not, right, secondly, the stain is not right. eOA L 'PAR j) Y]I}S 1O;} Altmann'8 fl1lid is l,)otassiUlll bichromate 5 % (Bensley Ul-lerl 2t% strcngth, which is better) amI osmium tetruxide 2% equal l,)arts. Fix for at least 24 hours up to 2 or 3 rIa,ys. Alternatively you may fix overnight in Flemmingwithout-acetic lwid, Champy, formalin, or Altmaun, aUfl then transfer to 3% bichromate for a days. Hegaud's technique is given in § 113. All these methods consiHt of lllordallting, either in the cold, or on the top of the incubator in bichromate. \Vithout this the fuchsin will llO"b "bite." After lllorclrLnting, wash out overnight under the tap (see § 122). Now this direction is quite right for pieces of firlll tissue like mammalian testis or liver, but may be too lllllch for a well separated locust testis, into which water penetrates more easily. A few hours ill the morning does for such material. As to the stain, there are good and llad fuohsins. The latter are due to lack of eontrol in sulphonation, and they do not all dissolve in water to the same degree. Altmann's specimen dissolved up to 20%, and so does the American sample we have used. Some British specimens dissolve up to 10% or 12%. To make up the solution, first shake up some drops of aniline oil in excess ,vith water. Filter, and add 10-15 gm. of acid fuchsin. Now this stain works hetter after a, few days, and it goes bad after a few weeks. Bring the Bectiolls or smears to water, drain and flood with plenty of aeid fuchsin, hold in forceps cautiously over bunsen or spirit flame, moving it backward and forward till the stain steams, but does not boil. Keep it hot fiJI' 30 seconds to a minute and then set aside on bench to cool. Wash off with distilled water; the Hections should be dark red. Dil,) quickly into methyl green (1 % aqueous), drain and transfer straight into absolute alcohol, then xylol and mount. If the stain does not" bite," it may be better to t,rea,t seetiolls for 30 seconds before st.aining with fL 1 % soilltion uf permanganate of potash, followed by 5% hili STAINS AND S'l'AINl.NG solution of oxalic (wid for some time. Then wash well in water. Now if the Rcction or smeM' is over-stained the next, one should be differentiated in picric acid (96% alcohol saturate(l with picric acid 1 part, water 2 ·parts) after being washed in distilled water. The methyl green is left out. This method is easy to work with vertebrate material, but is usually difficult with the invertebrates. It fades, being reel is trying for the eyes, and has no special advantage over iron alum hrematoxylin, which we recommend in preference. 155. Van Gieson. This counterstain is much used in Histology fLnd Pathology lahomtories, and may be especially recommended for connective tissue, after Heidenhain or Regaud (§§ 145, 147). Remember that picric acid differentiates these stains, and so you slightly under-differentiate before counterstaining. After differentiation in iron alum wash out well under the tap, add the Van Gieson for 3 or 4 minutes, wash in water, transfer to 95% alcohol, then dehydrate and mount in xylol. Some workers pass through origanum oil from the absolute alcohol into xylol. To make Van Gieson, add to a saturated aqueous solution of picric acid sufficient saturated aqueous solution of acid fuchsin to give a deep gamet reel tint. 156. Safranin and Light Green. Safranin has no advantage over gentian violet,; it is red and therefore more trying for the eyes. It was used greatly a quarter of a century ago. With light green stain it makes a pretty combination. Use Babes' fluid, which is equal l)arts of saturated watery and saturated alcoholic safranin. Stain till you get a good deep red. This mav he an hour 01' so for corrosive fixed material 01' overnight for ehrome-osmium. After staining, quickly upgrade, and counterstain in -;\-% light green in clove oil. If the result is too red, leave the next slide longer in 90% alcohol f111d clove oil. After the clove oil, wash off well in xylol, mount in benzol balsam or, preferably, immersion oil. v T.S. EARTH W()IU~[ 107 157. Piero-nigrosin is a. mixture of 1 pa.rt of saturated aqueous solution of nigrosin w'ith 9 parts of saturated aqueous picric acid. After staining in horax or chlorocarmine, bring sections down to distilled \vater, then into the stain for 5-10 minutes. WI1sh in tap \Yl"1ter for a minute, then pass rapidly through 70% and 90% alcohol, etc. 158. Piero-indigo-carmine is 2 parts of saturated watery solution of indigo-carmine, 1 part saturated picric acid in w[ttel'. Use after carmine, safranin, or magenta. Both this and the above stain give very pretty preparations. 159. Transverse Sections of Earthworm by Borax Carmine and Piero-nigrosin. Put a clean live earth WOJ'Ill in a flat tin box lined with slightly moist filter or white blotting paper. When Hoiled replace with cle,:m paper till the worm is scoured. If you wish you may put SOllle small pieces of onion in the box. When scoured free of grit drop the worm into corrosive sublimate acetic (§ lOB) and leave for at least 5 hours. Take out, wash in water, and with a safety razor blade gently cut 5 nun. lengths of the intestinal region. Do not crush the worm. It would be quite suitable to leave the whole worm ovel'llight in fixative before cutting. Wash the pieces in water for 15 minutes and transfer to 50% alcohol for 1 holli'. The sublimate must now be extracted for several hours in 70% alcohol coloured light brown with iodine (Lugol, § 71). If the liquid decolourizes add another drop or two of Lugol and leave an hour or so. Leave now several holli's in clean 70% alcohol (overnight is best). After this bring the pieces into borax carmine and leave a day and a night. Wash the pieces quickly in water and then upgrade at least 1 hour in each strength, beginning at 50%; .leave overnight in absolute alcohol in a wellcovered capsule. Change for at least I hour next morning into fresh absolute alcohol. Pour off most of the absolute and add an equal quantity of carbon bisulphide or ·xylol. 81 JAIN8 AND S'PAINING I,cavo 1hon1', then pour off and add pure carbon bisulphide or xylol for the same time. Imbecl in wa,x as describecl in § ] 23, and section. 'When yon have made sections these Rhould be dark red if the borax carmine is "biting." If they are only pink the Cn,l'lnillC is not, right. In this case you can extract the borax cnrmine in acid alcohol and stain in Ehrlich and eosin (§§ 44, 47). However, if the nuclei are a good bright reel you can cont,i.nne with the original method. You now bring the slides down through xylol and absolute or 95% alcohol to 70% alcohol, and dip them in acid alcohol if the carmine is too cIal'k for a few moments and examine under the microscope. The nuclei should be deep red, the rest only pink. Wash off in distilled water and stain in picronigrosin (or picro-indigo-carmine) fol' a few minutes. They should stain deeply. Wash quickly in tap or distilled water, upgrade a minute or so in each strength, then llenzol or xylol and benzol balsam. Note. Try also with taupoles (feel on meat till grit scoured out), using the same times, bnt after fixation the tadpoles need not be cut into pieces. Chromosomes 160. There are three main methods used nowadays. The aceto-carmine smear technique, the gentian violet, and the iron alum hrematoxylin. The fhst needs no fixation provided the undiluted stain is used. 'l'he latter Ileed careful fixation. 161. The Aceto~carmine Smear. Carmine becomes dark if mixed with iron salts. Thus if you tease the preparations with steel needles, a trace of iron produces an iron carmine. This is done purposely in Belling's iron aceto-carmine (§ 152). If you do not wish to darken the carmine, use nickel or glass needles for smearing. Dissect out the gonad of an insect, remove practically all salt CHROMOSOMES lOll solution or liquid with filter paper, and proceed as dir0cted in § 151. 162. With Fixation. For subsequent staining in gcntitLll violet use Flemming's strong formula with acetin acid, fixing smears for 2-4, hours, slllall pieces 2-·24 hours. \Vash out either in running ,vater overnight m' in tepid tap water on top of the paraffin oven. Change the water every i hour for 2 or ,3 hours. Upgrade very Riowly from 30% to absolute, and dear by the two-layer method deRcribecl in § 18. Some people elmtr first in cedar wood oil, then in benzol or xylol. Smears are upgraded to 70% or 1)0%1 umlleft overnight. It luts been recolllmended to nse the Flemming 011 iee and to add 2% urea before fixing uULtcrial. Large teste;; like those of locusts should be gently spnmd so that t.he tluid gets to all park;, and pieces of vertehrate material lllU::;t he small. A really good Flemming gentiltll violet prepamtion i8 superior to anything else for clarity ttnd beauty. 163. A modified Flemming with formalin im;tcad of osmie add is Navllshin'.'1 Fl1dd, whinh hag heen used recently by botanists. Ch1'01nic acid 1% in water 15 parts, commercial formalin 4 parts, acetic acid I part. It is essentiltlly a botanical method, _the ordinary Flemming being more suitable for animals. It is really excellent for root tops. Try those of the domestic pea, slitting them longitudinally before fixing. Stain in Heiclenhain, hlEmal1Ull or gentian violet. 164. Bouin, Bouin-Allen. These picl'o-formol-acetic fluids are famous for animal chromosomes (§ lOG). Fix . material (l,t, 38°-'1,0° C. for 1 or 2 hours. Dehydra,te carefully from aO%, a day in each; or by the drop method, which is to begin by putting the material in a little water in a tube, ltncl to arrange a slow drop into it from a. bottle of 70% alcohol, and later from a hottle of HO% alcohol. 110 STAINS AND S'I'AININO This material stains bmmtifully in hromatoxylin and with care in gentian violet, as well. 165. Gentian Violet (Violet Rosaniline or methyl violet). This is an intense violet stain much used by chromosome workers. Fix preferably in Flemming acetic for both animah; and plants. or Navashin for plants. Bring slides (or smears after a sojourn in 70% alcohol) into water, and then place in 1% gentian violet solution boiled and filtered for 3-10 minutes, according to the age of the stain. Wash in water and mordftut for about} minute in 80% alcohol with 1% potassium iodide and 1% iodine. Dip quickly into Ofi% or 06% alcohol, then quickly through absolute to clove oil, in which differentiation takes place. Wash in three changes of xylol and mount in xylol balsam. Picric acid can be used with this stain as follows. Mordant 10-20 minutes in above iodine solution, rinse in water, stain 15 minutes in 1 % gentian violet, rinse in water, transfer to iodine solution for a minute, then 95% or 96% alcohol, then flooel with saturated solution of picric acid in absolute alcohol, followed rapidly by a wash in pure absolute. 'Pass to clove oil in which differentiation takes place, then wash thoroughly in xylol (at least 15 minutes). This is an excellent method. For Bouin fixed material, after staining in gentian violet, mordant in iodine 2 minutes, rinse 2 seconds in absolute alcohol, 15 seconds in 1% aqueous chromic acid, 10 seconds in absolute alcohol and differentiate in clove oil, wash off well in xylol. 166. Heidenhain. Objected to by most chromosome workers because it is too opaque, and is really only suitable· for animals like moths with small or few chromosomes. Mordant sections or smears ([tfter sojourn in 70% alcohol) in 4% iron alum, for several hours. Wash slightly for a few minutes in mnning water, transfer to 0'5% hromatoxylin for twice as long as in alum. Wash off exees!> OOLOf APPAR.-JTUR 111 l'taill in wat.er, tntnsfel' to 4% iron alum j,(J rlitferentiate. If t.his happens too quickly, dilute the alum by half. vVhen diffel'elltitttion is finished, Wm-dl uncleI' tap for at least 10 minutes. Upgrade, mount in balsam. The stain becomes more transparent if the times ltre shortened, but material left a long time in chromeosmium does not stain so deeply as docs that fixed in Bouin or Navashin. 167. Mayer's Acid Hremalum is no\v used, as it is more transparent. Leave for at least 1 hour in a good specimen of the stain. Differentiate in acid water, wash well in running tap water or in ta.p water substitute. 168. Nucleoli and Chromatin. Use Feulgen's method counterl3tained in light green. Golgi Apparatus 169. For the Goigi i1ppa.ratus four methods may he learnt. These will covcr the whole ground. Theyare (Cajal) Aoyama, (Cajal) Da Fano, Weigl and Kolatchew. 'fhe first two are formalin silver, and are cheap; the latter, osmic, and costly. The osmic methods give a more faithful fixation and some at least of the fat, the formalin silver are less trustworthy so far as morphology of the Golgi apparatus is concerned, and give no fat unless used as in § 171. Fat can, however, be more relilLbly studied in Sudan IV, Farrant or gum glycerine mounts. 170. Cajal-Aoyama. This and D,t ]1'ano are modifications of Cajal's method. The three fixatives for these methods may be given here: Aoyama\~ Fluid is cadmium chloride 1 gm., neutral commercial formalin 15 c.c., distilled water 85 parts. Da Fano's Fl1lid is cobalt nitrate 1 gm., formalin 15 c.c., water 100 c.c. GajaZ's Fluid is uranium nitrate 1 gm., formalin 15 c.c., water 100 c.c. The fluids are essentially the same except for tlle "mordanting" substance. I have found that STAINS AND 8'1'AININO A( ,yCtllJa if{ t.lw h('st, 1d' the t.hree and definitely I'cmmllwnrl it,' F'(lJ' Illalllllwlian gOllads. Ifl)'llHdin is fL PUOI' fixM,ive. ,;(1 ill (ihis 1'11(1 hegins badly. Befter 'I'P8uff8 'll!iII fll' got fly add in!! fl'o/ll (I·7ri'XJ to X(/('/ to fllMI' .I'llitlN, ((lid by 8ee.i'ilf/ tha! the,{Oi'iHali-1l ,is nelti)'al-ized with calciuln c({'I'bonctte. 0'0';:, Excellent fixatiolls of other glands or material will be got with these iixativeR. 'The propel' time to fix is :3 or 4 hours for small pieces. 'Wash in t.wo changes of distilled 'water for n, minute, tra.nsfer to newly made up ] 'i)% silver nitrate in water fill' 10-15 hOLlrs at warm room temperature in the shade or :\,t 22° C. in an incuhator. Rinse pieueH again for ] minute in distilled water, i1ncl transfer to reducing fluid (see helow) for at least ;) hOllrs. \Vash under running tap for a few minutes. Upgrade from :30% alcohol, imbed in paraffin. Mount seutions withont toning or staining. If you wish to tone this can be done in '5% gold chloride, after bringing the sections to water, but is really unnecessary. For a countel'st,ain, dip into MaIm's methyl blue eosin for 1 minute, then water, then into 90% alcohol, absolute, xylol and mount balsam. Safranill and light green (§ 156) are sometimes used as a counterstain. The above times are for Aoyama, but cIa well for Da Fano. In the latter method the tendency ill recent years has been to use the fixing fluid with 0'75% salt for only a few hours, not overnight as DR, Fana advised. Da Fano used the silver bath for 24-48 hours; note that Aoya1lla advises only 10-15 hours. Reduce at least 5 hours for Ua Fano. These silver nitrate preparations are not absolutely specific for the Golgi apparatus (what method is '?), but in metazoan cells there is usually no difficulty in finding which structure is the Golgi apparatus. The method has been used and abused by some protozoologists, who have not hesitated to identify any blackened granules as a Golgi apparatus. 1'he Aoyama and Da Fano methods a.A. A}U) 11'..11' 8,]'.41117 II a will give very intel'eRting I'm-mIt,; wit.h f.:J1WltI'R. hut, h('.re tile teehnique it-; definitely not flllKy (~ IlH). RedllC'inr/./1u irl iH hydrnqu iUolle 1 ~ gill., formalin I;') (1.('., distilled wa.tm 100 c.e., Rm\inm :-;nlphite (Ni gm. After some weeks this fluid umy beeOllle Ilsete~s. It is Htraw yellow at first, and later goes hlack Ifor an unexplained reason old silver nitrate solution does not work sometimes. vVhen starting new work it iH better to make new reducer, new silver solution, and to look to your formalin. 1'he latter should definitely he of the best grade. 1'lote. If YOll intend to hegin rE's('arch work on the Goigi apparatus of Metazoa, you should lise the silver and osmic methods side by Hide. For some unknown reaS011 the osmic mpthods 011 oecasiolls entirely fail to impregnate, and it i::; neoessary to have some control on them. In the CUKe of tIle Goigi aI)paratns of Sporozoa, the osmic methods arc the best, the silver methods rarely giving ii, proper impregnation, though I have seen excellent silver preparations of HpOl'OZOitll Golgi bodies. 171. Combined Goigi Apparatus and Fat Stain. A successful method is the combined Aoyamu (or Da Fano) and Sudan IV. You prepare either smears or frozen sections by, preferably, the Aoyama method. The piece of tissue cannot be imbedded in wax as the upgrading in alcohol to xylol removes the fat, so frozen sections must, he cut, and the thinner the better, or the material may be teased. After the Aoyama smeal', or the block of tissue has been brought into reducing fluid as described in § 5H, it should he washed in water. The smear goes immediately on to the fat stain, whereas the blook of tissue is sectioned, and then st.ained in Sudan IV, or a smail piece is first stained in Sudan IV and then teased up in glycerine and water (1 ; 1). In both cases the lllountant is glycerine, glycerine jelly, or Farrants (§ 80). Exactly the same can be done after the Weigl or II t 81'.-1 INS _./ NIl 81'AININ(( l\.nll1tdJew methods. Ymt stiLin ill Sudan IV and monnt. in Farrallts, gly"t:'l'in8, or glycerine jelly. "In l)l1th thu silycl' nitrate Hud the oRmic method the Golgi Ilppnmt,w,; is blaek, alld fat, iR 1'0(1, hut ill some osmic pl'cparatiollH the (;oiour of the fat is hl'owniRh red rather thrLll the bright 1'or1 of the formalin silver preparations. 172. The Osmic Acid Golgi Methods. Osmium tetroxide it:! one (If the mORt expensive substances known. While it is (1, good thing to learn these methods for Metazoa, the oumbinecl Aoyama and Sudan methods will really be fmfficient if skilfully used, There are two main osmic methodR, (l) prefixation in corrosive osmic of Mann for a few houl't:! lilltil l1elletratecl, (2) prefixation overnight in any ohromc-osmiull1 fluid without acetic acid. Subsequently the Ill£ttel'ial \vashed out for an hour 01' so for Mann fixed pieces, but overnight and thoroughly for c'.hrome fixed pieces. The explanation of this is that the presence of excess chrome salts in the pieces of tissue inhibits the osmic reaction, whereas the corrosive sublimate in Mann's fluid has no such effect, You must see that in both methods the pieces of material are small, After washing out transfer to I % or 2 % aqueous osmic for several dttys ttt 30 0 C, or thereabouts. It does not matter very much, only at room temperature the reaction takes weeks, at 30° C" dttys, Many people use 2% osmic, but I % does well enough, and this strength is being used by most workers nowadays. After 3 days break a small piece of the ma,terial in glycerine water (I : I) and examine nnder the oil immersion. If the Golgi apparatus is black, pour off the osmic acid and leave the pieces in distilled water at 30 0 C. overnight. Upgrade and cut thin sections in wax, Counterstain as in § 60, is Mitochondria 173. For vertebrate material fix in Regaucl (§ 113), stain in Regauc!'s or Heidenhain's hrematoxylin, or acid RO J}[A NO JVSK y lUi fuchsin (§ 154). For invertebrates, same stains, but fix in Champy 2 or 3 clays, wash out overnight, or fix in Champy or Flemming-without-acetic acid overnight, mordant 3 days in 3% potassium bichromate. Methylene Blue 174. Polychrome Stains, with Special Reference to Methylene Blue. If you wish to work on glands and blood ii'om a general histological aspect, or Protozoa and parasites in' tissue, there are three methods of great importance. One you have ah'eady tried is J~eishmann or "\iVl'ight for blood smears. Two others are Giemsa, and polychrome methylene blue, with or without emlin or some snch eouuterst,aill. Methylene hlue solutiolls when left standing clisilltegmte slightly, producing lo·wer homologuefl such as Azure A and Azure B, whieh are oxidation proclucts. Tn Unna's polychrome methylene blue these lower homologues have heen produced by hoiling with potassium earhonate. Unna's stahl is methylene blue I gm., potassiulll carhonate 1 gm., water 100 C.c., but it needs some months to ripen. I give also Goodpasture's acid stain, which can be UHeu immediately it is made (formula from Mallory and Wright). Methylene blue I gm., potassium carbonate I gm., water 400 c.c. Dissolve and boil for 30 minutes. When cool add 3 C.c. of glacial acetic acid, and Hhake until the pl'ecip~tated stain is redissolved. Boil for 5 minutes until the solution is concentrated to 200 C.c. On cooling it is ready for use and keeps well. Use as follows. Stain in 5% aqueous eosin for 20 minutes with heat, if found neceESt1l'Y. Wash in water, drain, and add polychrome methylene blue diluted I part in 3 of water. Wash in water, differentiate in 96% alcohol. Giemsa, Leishmann, Jenner and Wright are all Romanowsky stains. Roma,nowsky nearly 50 years ago lIG S'llAI NB AND 81TAINING first used combined eosin and methylene blue. Eosinate of methylene t'lcpamtes out in various granules and cell elements in a rellHuknble manner, producing a polychrome effect exceedingly striking . .Tenner, LeiBhmann tLnd \Vright are eosinates of ]loly" fihrOllle methylene blue dissolved in methyl alcohol, which must be lleutral and free from acetone (though this has reeelitly been questioned). Giel1lsa is another stain like the above, hut containing eosinate of Azure II. All thciie staiml are hetter bought than made. 175. Giemsa Method for Paraffin Sections. Fix in Zenker for 24 hours, make paraffin sections 3-5f.l. Remove sublimate in iodine, to he followed by 0·5% hypo (sodium hyposnlphite) to get rid of all iodine. Wash in diRtiIled water. Stain for 12-24 hours in Giemsa's liquid stain, 1·21) c.c., pure methyl alcohol 1·5 C!.e., 0·5% sodium hiearhonate 2 drops, distilled water 50 c.c. Differentiate in 05% ethyl alcohol, olear, mount in thick cedar (immer" ['don) oil, as balsam is too cwid (even the "neutral " balsam !). If t,he cytoplasmic strnctures are too blue, omit the sorliulll bicarbonate, or mordant before staining in 2·5% potassiulll bichTOmate; if they are too red, treat l'e(1tiolls before staining in 1 % potassiulll permanganatc, washed out afterwards in 5% oxalic acid, then distilled water. For some tissues IT. Ford recommends washing in water after staining, rapidly flooding with strong formalin, washing off in water, then differentiating in strong alcohol. Mount in immersion oil. CHAPTER IX NOTES FOR HISTOLOGY AND EMBRYOLOGY STUDENTS 176. Glands. (a) For superficial histological study use Susa, Zenker or Kahle fixed material stained in a hrematoxylin and Van Gieson, or Mann's methyl blue eOSIn. (b) For cytological work, Aoyama, Weigl or Kolatchew, Regaud, and the various microchemical methods (§ ti2). 177. Connective Tissue. Zenker, Susa, Formalin, followed by hrematoxylin and Van Gieson for collagen fibres. For ela8tic :fibres a good method is that of'raellzer. Fix in Bonin, Susa or Helly. Bring sections to 70% alcohol and transfer to Orcein 1 gm. in 100 C.c. of 70% alcohol, with 1 c.c. of hydrochloric acid. Leave for 1 hour or longer if necessary, wash quickly in distilled water and pass directly to 96% alcohol, and watch under microscope until the elastic fibres stand out a clear brownish red. 178. Cartilage. Fix in sublimate formol (§ 112), stain in safranill or Mallll's methyl blue eosin. In the borax carmine and picro-nigrosin method the cartilage stands ont well. 179. Muscle. As for Glands, (a) and (b). 180. Bones and Teeth. '{'hese cannot be cut without decalcification. Fix in sublimate formol (§ 112) or Bouin (§ 106). Transfer to a good bulk of 5% watery solution of trichloracetic acid at least overnight, until deca,lcified. Pass straight to 1)0 % alcohol. Here is a good fluid (C. E. Jenkins), ·which fixes and decalcifies at the same time: Hydrochloric acid 4 c.c., 117 liS H [8'1'0 LOO r glacial acetio a O.c., chloroform 10 c.c., water 10 c.c., ahsolute i11coho1 73 c.c. Immersc tissue in at least 100 times its volume ofthe fluid. Leave at least 24 hours for I-1!lULU pieces, larger objeots will take several days. If ill doubt change the fluid and leave longer. Wash out in 90% alcohol. Tho sections stain well hy ordinary methods. Central Nervous System (Brain, eto.) 181. Nissl gran'llles, fix in alcohol, Zenker, or conosive sublimate acetic, and stain in polychrome mothylene blue and cosin (§ 174) or 1% watery toluidin blue for several hou!'l';, differentiating in alcohol. Golgi Bodies (§ 101)). Spinal ganglia or cerebellum. Mitochondria. Hegaud aeid fuchsin (§ 154), or alum. IW:~llIat(Jxylin (§§ 145, 148). A:ris C!ll'ind(?t" Dendrite arltZ Ne'uroglia Sta-ins. The methods chosen here do not need a freezing microtome, or imbedding in celloidin. Ranson'8 Pyridin Silt'er Stain (modified Ramon y Cajal ,J\lIethod). Fix pieces of variable size in ahRolute alcohol cont!1iniug 1 % strong ammonia for 48 hours. Rinse in distilled water for ahout 2 minutes. Put in pyridin for 24: hours. 'Wash in many changes of distilled water fol' 24 hours. Transfer to 2% aqueous silver nitrate at 35° C. in a dark cupboard for 3 days. Rinse in water and reduce for 1 day in a 4% solution ofpyrogallic acid in 5% formalin. Upgrade, cut paraffin sections. This ,method shows various fibrillar structures, often cell bodies. ~Myelin. Make paraffin sections of formalin fixed brain, and stain in Regaucl's hrematoxyJin (§§ 14-7, 148). Axis Oyl'inder and Dendrites (Zeihen's Gold and Sublimate Method). Fix small pieces of fresh tissue in equal parts of 1% gold chloride and 1% corrosive sublimate for at least 3 weeks, but preferably even up to 3 months, by which time they will have become reddish brown. CLASS MA1'E.RIAL Il!1 Cut hand sections with a razor, pass into Lugol's solution diluted with 4 volumes of ..vater until differentiated. '~Tash in distilled water for Heveral hours, dehydrate and mount in balsam. In various specimens of tissue, both medullated and non-medullated nerve fibres, nerve cells awl neuroglia are stained. It is advisahle to start this met.hod some weeks before you will need the pieces. Trial specimens can be cut and. examincd ii'om time to time. 182. On Fixatives to Use when Collecting Material for Cytological and Histological Study. Whore the llluuhel' of bottles nnu~t he cut down to minimum, take AOYUUHt and Bonin-Allen and treat snbsequently as follows :Aoywrna. (a) Oontinue with method for Golgi apparatus. (b) Post-chrome as for Regaud. (c) Cut hmld or frozen sections and stain in Sudan IV f(_)1' fat. (d) rrest for lecithin and cholesterol. Bouin-Allen. (a) Cut sec:tiollS for ordinary histology stained in hrematoxylin and eosin, etC'. (b) Prepare seutions for ehromosomes by Heidenhaill, Mayer, and gentian violet. (c) Feulgen. (d) Glycogen (Bauer). As a third fixative, Mann's fluid for the Weigl method is recommended. The best general fixative in which materieL1 may be kept till desired for use is Bonin without aoetic acid. This refers to marine material espeoiaJIy, though some things harden rather much. When back in the laboratory the material should be washed in water for a few minutes and tl'ansferred to 70% a1c:ohol, and parts of it picked out later for st,aining in Mayer's hremalum, or for sectioning. 183. Recommended Material for Class Purposes. General. III l'pring l)olleet slllall swimming tadpoles, fee(l on I110ttt, I :.!O HI8TOLOGY and fix in corrosive acetic and Bouin, store in 70% alcohol. For any time of the year use scoured earthworms, .1rlitocllOndl'ia. Fix Rlices of mammalian liver in (a) formol-saline for 4 days, upgrade, section and stain in Heidenhaill's alum hrnmatoxylin. (b) Regaud, stain as above, and by acid fuchsin. (c) Altmann (Bensley's modification), stain in acid fuchsin and alum hrematoxylin. Guinea-pig testis. Fix in Regaud and salt Ohu,mpy (very small pieces). Stain as above. In spring and SUlllmer fix Helix ovotestes in salt Flemmillg-without-acetic or Champy. Stain in alulll hrematoxylill. Golgi Apparat11s. Fix t pieces of Helix ovotestis in Mann's fluid for 2 or 3 hours, wash in distilled water for 1 hour, transfer to 1% osmic acid for 3 days at least at 30° 0., or for 3 weeks at room temperature. Fix dorsal root-ganglia of rat or young cat in same way. For silver method (Aoyama) use similar material and mammalian epididymis and slllall intestine. For good invertebrate neurones nse ganglia of Helix. Chromo8ome8. Fix root t,ips of the d01llestic pen (preferably), bean or onion in Navashin's ,tiuid and stain in alum hrematoxylin and gentian violet. ' For chromatin, fix in eorrosive sublimate l1ncl stain by Feulgen's method. Collect testis of Salamanda or Newt from June to August. Fix very slllall pieces in Flemming with acetin for 3 hours, section and stain in gentian violet or alum h£(lmatoxylin. For mammalia, fix cavy testis in Bouin-Allen, and stain in Mayer or Heidcnhain. In sumlller use locusts and grasshoppers. :Fix in ]'lemming with ac:etic and stain in gentian violet. Fo?' Aceto-cannine Smears. In winter practise on testis of Drosophila or AseUus, kept ill warmth for a few (lays. Tn RUmmel' use early instal'S of hemipterous inseets, UE.iV'l_lRIl? [TOES 1::!1 and other insects like locusts (8tenobothru8 virid-ulIlR, the grasshopper, is useful). Fm' JJlic1'ochmnical TC8t8. Use smears of Helix ovotestis and Cavia testis for trials of Lecithin, Glycogcn, and Cholesterol methods. Use also l11alllll1ali~tll material, sudl as liver. Protozoology. For Golgi bodies use whole intestinal tracts of mealworlll containing grcgarines, fixed by the Weigl (Mann-Kopsch) method. Try also centipedes, a,nd if possible, cephalopods for AggregM,a, in whieh there is an excellent Golgi apparatus. Ultra-centrifuge ciliates and flagellates (next §). 184. Centrifuges. There are two types of centrifuges commonly found in biological la boratOl'ies- ·the lutnd centrifuge and the electric centrifuge giving up to 5,noO revolutions per minute. '1'he latter has been used with some success in rcsea,l'oh wOTk on eggs, hut recently the development of the nltra-centrifuge hy J. 'V. Beams, A. J. Weed and E. G. Pickles has marked the heginning of a new epoch in protozoology and cytology. This is an air-driven centrifuge and necessitates a source of compressed air, at least 50 1bs. to the square inch, preferably up to 80. The plants used for motor tyre inflating, found in garages, are suitable, if the reservoir is a good size. In Fig. 7 is a diagram ofthe Beams ultra-centrifuge. The rotor, TI, is accurately turned on a lathe out of a rod of duralumin, brass or steel. It is top shaped, and provided with grooves all the sides as shown in Fig. 7, B. The solid rod is accurately turned out hollow inside (X) and provided with a screw (or catch) lid (L) which is screwed at 8. The lid is not absolutely necessary as liquid does not spill when being eentl'ifnged. It is hetter to luwe a lid. Objects to be centrifuged <11'e put into t,he cavity X. It does not matter whether t,he rotor is balanced, as it will revolve quite well with a piece of lead put inside. The stator i<3 H, piece of t~\'l'geI bore brar;s tul1ing, into which the HISTOLOGY 122 crater and tubes, H, TT, are screwed or welded. On the sides of the crater are up to eight staggered holes, arranged so that the air issuing from them hits the grooves OIl the A B FIG. 7. A. Rotor and stator of Beams Ultra.centrifuge. B. Bottom of rotor, showing grooves. C. Looking down into erater, showing eight staggered holoH and tube aperture in middle ef (·rater. L = lid. S = thread of serewNl lid. TI = rotor wall. X = cavity of l·otor. G = grooves on rotor. H = hole in stator crater. V = cavity of stator. TT = tube running through stator ehamber and opening below. rotor (G), thus making the latter revolve rapidly. The size of the tube, TT, which must open below, is, in the case of the centrifuge we possess, 3 mm. in diameter, and in some commercial makes there is an arrangement for controlling the airstream ,,,,hich is sucked up by the BEAM'R CENTRIFUOE 12:1 revolving rotor, and which seems necessary for the proper working of the apparatus. The air source is introduced to the tuhe on the right of the 8ta,tor. '1'he centrifuge is opera,ted in a box with double sides, filled with sand. Sometimes the rotors burst. For laboratories where any sort of work is being done on the structure of Protozoa or metazoan cells this centrifuge is a necessity. It can be made by any mechanic used to working a lathe, and absolute accuracy in the specifications given in Fig. 7 is not necessary. For example, the number of staggered holes on the crater is not critical so far as we know, nor the number of grooves cut in the rotor. The tube, TT, is necessary, but here again a certain latitude in size of bore is possible. The centrifuge makes a piercing whistle when in operation. It can be turned upside down when in operation without the rotor falling away from the column of air. ADDENDUM I 185. Directions for the Use of this Book. Begin by reading Chapters 1. and II. Then start fixing and staining smears according to the directions in Chapter III. Next make some whole mounts as in Chapter V. This may be done before learuing thc staining in Gha,pter III. if desired. After some acquaintance with staining and mounting, the fixing and imbedding of piecoR of tissue of the earth'worm or of tadpoles (§ 159) may be tried, reading Chapter VII. If yon are working by yourself, directions for making fixatiVes and stains will be fonnd in Chapters VI. and VII. The microchcmical treatmcnt of smears and sections found in Chapter IV. should certainly be attempted last. In connection with imbedding and sectioning, the notes on histology, Chapter IX., may be used. Some suggestions for material to be l1sed at different times in the year hl1ve been given in § 183. . 186. Amateur Microscopists living in isoll1ted places, who may be interested in newer developments in microscopical science, and who wish to join a Society, are recommended to write to "The Secretary of the Royal Microscopical Society of London," B.M.A. House, Tavistock Square, London, W.C.I, England. This Society issues a Journal which prints suitable and original contributions from microscopists. The .Fellowship is open to foreign as well as British subjects; revicws of recent advances in microtomy as well as in general Botany and Zoology are published periodically. 187. Writing a Paper for Publication. A few short remarks may be useful. It should be remembered that the modern biological journal is now approaching the stage which the chemical journal had arrived at some years ago. That is to say, a bulky thesis has little chance of obtaining publication. Papers must be short, with few, preferably Indian ink, illustrations. The first tIling is to look up l)revious literature on the subject in hand. Unless the latter is something quite new, the previous literature is certain to be fairly large, usually very large, and often in foreign languages. Thus you 124 125 must know German and French, unless you haye friclldR wIlO will translate for you. But even Germans and II'rene}mwIl, 'who are not specialists, will often bc unable to uuclerstnml technical papers in their own language, amI it is better to learn the language necessary yourself. You must read these papers, and make notes on the ontstrmdillg pointR of iuterest,. Usually, if you write to the authors titenlHelves, they will send reprints of their recent papers. These translatiom; made and digested, you will have some knowledge of the existing state of the subject. Some of the authors may be negligiblp and may be left out, some will be masters of their ;mhjcet, and you must concentrate on their work and technique. If you can get to an international congress you Illay cven meet the authors themselves, and see their preparations. Sometimes these are worth examilling, and always you lerun some· thing even from the occasional poomeRS of tl1eir ,yolk Yon see the standard, at any rate, which is an education not to he got from books and l)amphlets. On writing your paper there is little I call say. A carefully balanced short review of the IJrescnt state of the Bubject, as you know it, precedes t,he original observati.ons you have made. These should he written up in proper sequence and clearly, and at the end of the paper you discuss other people's work in the light of your own results. Then follows a Hhott summary of results. This summary is an absol7ite lwcessit!l. It should be short hut must include all outstanding results, for you should remember that it is very helpful to three classes of persons, the foreigner who may understand English badly, the harassed general teacher who is trying to store in his brain facts which may be useful for his students, and the specialist who wishes to see at a glance what yOll have been doing. At the end of the paper is the bihliography, which should be aH short as possible, but should include the recent outstanding papers, as well as the original papers written by the perSall who initiated the special branch you are investi. gating. ADDENDUM n 188. Books recommended for further study :"The Microtomist's Vade-mecum." Tenth edition. Edited by ,J. Bronte Gatenby and Theophilus Painter. J. & A. Churchill Ltd., 104 Gloucester Place, Portman Square, London, W.l. . " Precis de l\Hcroscopie." Sixth edition. By M. Langeron. Masson et Cie., 120 Boulevard Saint-Germain, Paris. " Taschenbuch del' Mikroskopischen Technik." 1:3 Auflage. By B. Romeis. R. Oldenbourg, Munich. " Histochimie Animale." By L. Lison. Gauthier-Villars, [i5 Quai des Grauds-Augustins, Paris (VIe). " 'rhe Cell." By E. B. Wilson. Columbia University Press, New York. "Schaefer's ESllentials of Histology." Edited by H. M. Carleton. Longmans, Green & Co., :39 Paternoster Row, London. " Recent Advances in Microscopy." Edited by A. Piney . .J. & A. Churchill Ltd., 104 Gloucester Place, Portman Square, London, W.l. "Recent Advances in Cytology." Second Edition. By C. D. Darlington. J. & A. Churchill Ltd. (Deals with recent work on chromosomes and heredity.) 189. On Buying Laboratory Material. Many of the firms mentioned below issue illustrated catalogues, and some also distribute lcaflets on staining methods. You arc advised never to buy dyes or reagents with patent nameR, as research of any kind must be carried out with materials, the formulro of which are known. The science of biology has been built up hy men who have cheerfully published their discoveries and formulm for the benefit of all. 190. Firms Supplying Laboratory Apparatus, Stains, Chemicals, etc. Great Britain. Flatters & Garnett Ltd., 309 Oxford Road, Manchester (all sorts of chemicals, dyes and apparatus, makers of Gilson's "Eupal'al"); the British Drug Houses Ltd., Graham Street, City Road, I.Jondon, N.1 (makers of many sorts Ot dyes); The British Dyestuffs Corporation, 126 ADDJENDtIM I J J 27 Ltd" 70 Spring Gardens, Manchester (important makeJ's of flyes--Loudon agents a,re Baird amI Ta,tlock, 14 CrORs St,rcet., Hatton Garden, E.O,1 ); G, T, Gurr, I3H New King's Road, S,W,1i (maker of all stain;; and dyes, etc,); G. Holt & Co., 4H Kingsv""oo(l :Road, Brixton Hill, S.W.2 (agent for Griibler) ; Hawksley & Sons, 83 Wigmore Street, Cavendish Square, W.I; ahd Charles Baker, 244 High Holborn, W.C. (both firms are agents for Grubler, and for most microscope and accessory apparatns makers), For incubators, ete., for imbedding and tissue-culture work, Charles Hearson & Co. Ltd., 235 Regent Street, W.l. United Staie8 of America. Eimer and Amend, 205-211 Third Avenue, New York; Palo Company, 90 Maiden Lane, New York; Edward Pennock, 3609 Woodland Avenue, Philadelphia, Pa.; General Biological Supply House, 550H Kimback Avenue, Chicago, Ill.; The Will Corporation, Rochester, N.Y.; Paul Weiss, Hl20 Arapahoe Street, Denver, Oolorado; Natiolml Aniline Corp., New York. INDEX Nfl/c. Thp nmn]Wl'R r"t'el' Aceto-carmine formula, l:i I Acid alcohol, :;7 Acid water, :17 Addity ami allmlinity (pH), :23 Albumen for slides, 1:28 Aleoho[ (lothyl), l(_] "\lc'ohol (how to dehydl'l,te). 1n Alcohol. IllL'thvl, 17 Aleuhr,( N-IlIIh·1. 17 Alkaline water,' :!li Altmann '5 acid fuchsin method, 154 Altmann '8 fluid, lUi Amyl acetate CPJlleJlt, 88 "\me"tlll'til'H, H:! Aoynma fluid, 170 Aoyrnua, Hillel\-}', 5(1 Apat.hy·s "elUent, 88 Axis t:ylimloI'R, 181 t(l paragraphs, not to )"luges. I Cal'lloy Lebrun formula, IlK Citrnny smpar, i)j Oart,i1agp, 178 (,);n·t.ilaginnlls Hkelr·tOIl", !ll) Capsules, J:! l!"lloiciill tC('hniqul', Jail Celllliohl cement, SK CelUents,81l Centl~al Jl('rVUlIs sy~t"lll, 181 CeIT'sm, 12:1 Champy's fluid, 117 Champy, Hmea!', 55 Chloral hydmt.e, 02 Chloro-carmine formula, 100 Cholt'sterol, tl7 Chromatin (se.e .Feulgen). Chromosomes, ltlO et 8[1[. Clarke's sliline fluid, 24 Class material, HI:{ Clelll1ing glass, l·t Clearing, 18 (!Icaring emhryos, 1011 Cocaine, U2 Conneetive tissne, 177 Oord's solution, !J;! Corrosive sublimate acetic, lOS COllntt'r"taining in O.-angl· G, 42 CoversliJls, preparation f'H' vital staining, :n Urustacea- sltline flui(}. ~·l Clltt.ing ser,tiollR, 1:25 BaIKlIm,8·l Bauer's glyeL)gen methud, n BOlLIng' c:ent.l'ifuge, 184 I1elling, ,weto.mli·mine, 16:2 BellRlc;y'8 modification of AHmann, 115 BerleHp's medium. SO C. 11m'nard, iodine tp8t, 71 Best's carmine, 7i ]3Iea.t1hilll,( ohromo.osmium, 08 }lones and teeth, ISO Borax carmine formula. 141) 110mx elll'JIlim' whol,' mount., 80 ]~ottles, 12 . Dl'call'ifientioll, ISO Deflandre's test. for lecithin, 74 Delafield formula, 14:1 Dcla1ield, staining ill, 41 Dendr-ites, IS1 Denham's sttlldarac medium, 8il Diamond for writing on slides, 12 Digitonin, 67 .. Dioxan balsam, 81 Dioxlln imbedding, 1:~6 Bouin's fluid formula, lOU ]~Ol1ill smear, 41 Brusil fluid, n:l Cuuad" halslllll, 8.J, Carbolxylol, 18 C,L1·bol'undum pencil, J:2 Carnoy formula. II Il 128 INDEX Drawing paper, 9 Drawing unde,' microscope, 8 Duboseq fluid, 93 Dyes, 18!J et seq. Earthworm, transverse sections, 15ft Ehrlich's hrematoxylin formula. 144 Ehrlich's h:ematuxyJin, staining in, 44 Elasmobranch saline fluid, 24 Embedding, 123 Eosin, counterstain, 47 Euparal,82 Eye strain, 6 120 Reidenhain hrematoxyJin formula, 145, 148 Reily, 107 Rollande, chloro-carmine, 150 Holtfreter's fluid, :U Imhcchling, by dioXllll, IH6 Imbt'dding ill wax, 12;{ Imbedding, N·]mtyl alcohol, 187 Immersion oil, 4 Impregnation, fresh tissue, 98 Iodine, for glycogen, 70, 71 Iron hrematoxylin, 45' Kahle's fluid, 114 Farrant's medium, 80 Fat, combined Aoyama. and Sudan, 171 Fats. 68 Feulgen's test, 76 Fixation images, validity of, 34 l?ixing fluids, 104 Flemming smear, 55 Flemming, with and without acetio, formulre, 116 Formol,ll2 Formol-saline, lJ 2 Formol sublimate, 112 Fresh tissue, silver, 98 ]!'rozen sections, 134 Gelatine solution, 95 Gentian violet formula. 165 Giemsa formula. 175 Glands,176 Glauber's salts, 92 Glycerine jelly, 83 Glycerine, whole mounts, 86 Glycogen, 69 et seq. Golgi apparatus, 16!J Golgi apparatuH, osmic methods, 172 Golgi apparatus smear, 59 Goodpasture's methylene blue, 174 Gray, Peter, medium for cementing, 88 Gray, Peter, mounting ponu organisms, 03 Hanging drop preparations, 26 Hedon.Fleig saline, 2:1 LABORATORY'fEClllllQIJ11. Langhan'H glyc'ngcu test, 71 Lecithin trst, 74 Leishmann blood smear, ,18 Living calls, examination of, 25 Lugol's solution, 71 Magnesium sulpht\tc, 92 Mann's fluid, 111 Mann, methyl blue eosin formula, 158 Mayer's acid hremalum formula, 146 Mayer's albumen, 128 Measurement under microscope, 7 Menthol, 92 Methyl benzoate, for clearing, 100 Methyl green acetic, 33 Methylene hIlle, for cartilage, 90 Microchemical tests, 62 el seq. Micrometers, 7 Mierophotogmphy, 10 Microseopes, 2 Microtomes, 11 Mitochondria, 173 Mollu~c[1 fluid. 24 Mosquito Jarvie, 80 Muscle, 179 Myelin, lRI Navashin's formula, 163 Nerve endiugs, 117 Nervous system. 181 N eutmlmountltnt, 85 Nile blue sulphate, 66 Nissl grullllles, 181 INDEX 130 Oil of Willtcrgl'('Cn, 100 Osmic method fol' Golgi bodil's, 172 Osmic test, f[Lt~, (j'1 Osmotic pressure, 2:3 OxuJic acitI, 08 J'arailin WI1X imbedding, 12:1 1'[1raglycogen, 70 PcrrilangulU1te of pobsh, b1l'l1oh, 58 Petrunkewitsoh fluid, lUI pH,23 Picric chromic glyoogen method, 73 Piero-inc1igo-carmine, 158 Piero-nigrosin. 1.57 Polychrome methylene blue, 174 Pond life, Pete!' Gmy's method, Progressive staining, 4U Plll'i'~ medium, 80 ua Hmoll!', Oal'lloy, 5:J Smmtr (Sehltudillll or E(min), 41 Smears (living), 27 Smears, stained. :38 Solutions, how to make, 22 Stains, lao et se']. Stemler dishes, 12 Sudan IV, (jfi SUjJm-vital RtfLilling, 2!J-31 Suaa fixative, lOll Tadllclcs, sectioning, 1511 Tap water substitute, 3() Teeth, 180 Toxic examining mcdi[t, a:3 Trichlor[lcetic aokl, 180; and see SUSI1. Ultra-centrifuge, 184 Unoa'a stain, 17-4 Razor bh1da holdlll'R, II Reduoing fluid formula, 171 Regaud's bichromate-formal, ll:1 Reguud'a hrematoxyliu formula, 147 BegressivD staining, 40 ltomieu, lecithin, 74 Bousselet's Rolution, \)2 Van Giesoll formula, 155 Vital staining, :20-31 Vitamin C, 77 Volut\n,71l Safranin !tnd light grl'on method, llitl Salt solutions, 2:1 Nanrll1r1tc, 85 Schaudinn's fluid formula, 110 Sehaudinn SI\1O[ll', 41 Rd!iff's reagent, 78 Washing out, 121, 122 Weigl method, 172 W dgl smear, eo Whole mounts, 80 el seq. Windaus-Bnlllswick te3t, 117 Wintergreen oil, 100 Wright, bloou stain, 48 Schneider, aceto-carmine formula, 151 Sc:hultll, cholesterol, 68 Y oCllm fluiu, 0:3 RN~ watcr (artificial), 24 Reetioll (Jutting, 1~5 Hil vel' line orgaIls, !J8 Small ohjects, handliug uf, 1:1:J l'HlNTEII J~ GREAT JllllTAl~ Zenker-formel, 107 Zenker formula 107 Ill' 'rnE WIUTt:FJUARS I'RERS !.TD. J.oXIiOS AND TOSUlUDUE HAWKSLEY & SONS LTD. Distributors for Great Britain of c E sc MeR PES British Made by Cooke, Troughton & Simms ltd. Illustrated List on request SECOND·HAND MICROSCOPES by Makers of repute REPAIRS to Microscopes and Objectives, Microtomes, Optical Projection and other apparatus. Estimates submitted MICROSCOPE LAMPS, DISSECTI NG SETS, ETC. Hremacytometers, Hremoglobinometers, Sedimentation other apparatus for Blood Diagnosis and NEW STUDENT'S HIEMACYTOMETER (BRITISH MADE) Solid glass counting chamber, 2 cover glasses, 2 pipettes, complete in case £1 • 7 6 • Manufacturers and Importers of Scientific Instruments Contractors to H.M. Govt., L.c.c., etc. 17 NEW CAVENDISH STREET, LONDON, W.l 'Phone: WELbeck 3859 'Grams: "Diffract, Wesdo t London ,. LEI fREEZING The handy and convenient instrument for the Laboratory Automatic feed of the ~pecimen Cuts down to 5 microns accu rately Very small CO 2 consumption Knife cooling attachment for cutting fatty tissues, placenta, etc. USED AND APPRECIATED WORLD OVER THE E. LEITZ (London), 20 MORTIMER ST 0, W.I MICROSCOPES for General Use also DISSECTING MICROSCOPES Simple and Binocular LENS HOLDERS Several patterns Wood Block Dissecting Stand ES-Rockin g , Rotary, Bench M .CROTOM • and Hand Models F& G H Bench" Microtome Vertical Staining Jar GLASSWARE-Slides, Covers, Staining Jars, Balsam Bottles, etc. CABINETS and Boxes for Storing Slides DISSECTING INSTRUMENTS-in sets and loose The Universal Oil for Immersion Lenses SOLVAX, II EUPARAL EUPARAL "Vert" Prof. GlJson's process A special Solvent miscible with weak alcohol and also dissolving paraffin wax FLATTERS & GARNETT LTD. Scientific Instrument Makers, etc. 309 OXFORD ROAD, MANCHESTER 13 Laboratories and Works-Fallowfield HEARSON APPARATUS for the M ICROTOM 1ST PARAFFIN OVENS PARAFFIN BATHS SLIDE DRYERS SLIDE CABINETS EMBEDDERS DEHYDRATORS STAINING RACKS STAINING WELLS Paraffins, Slides, Covers, Stains, Media MICROTOMES-Reichert Sledge, Rotary and Freezing HEARSON RO;Y-ATOME-a new type rotary MICROSCOPES-"all makes in stock Please request boo'<let-" Hearson Microscope" Catalogues and lists free on application CHARLES HEARSON & CO. lTD. Sole Agents (or Reichert Microscopes 27 MORTIMER STREET, LONDON, W.I A.ND AOCESSORIES R 'S STAINS FOR BIOLOGY - - are used and approved in the principal laboratories .---------Stains, Mounting Media, Bacteriological Sugars, Immersion and Clearing Oils, H ion Indicators, Filtered Paraffin Wax, Dissecting Instruments, etc. Price Lists and Literature on application GEORGE T. GURR 136 NEW KINGS 'Phone, RENown 2463 ROAD, LONDON, S.W.6 Telegrams: "GURR, RENOWN 2463" NOTES NOTES BOOKS ON ATU CIE L CE Published by J. & A. CHURCHILL LTD. INDEX TO SUBJECTS Analysis. Bacteriology Biochemistry Biology. Chemistry. Hygiene PAGX PAGlil 2-7 12 Microscopy 15 Pharmacognosy and Materia . 10, II Medica . 10, II Pharmacy 8 Physics . 15 Miscellaneous . 13 14, 15 2-7 9 London: 104 Gloucester Place, Portman Square W.l A CHEMICAL DICTIONARY Containing the words generally useu in Chemistry, and many of the terms user! in rclatcd sciences. By IKGO \\7. D. HACKH, A.iH .. F.,\.r.~:., F.RS .. \., Profesoor of Chemistry, Colle"," of Phvsicians and Surg-eons. San Francisco. "'ith tile C(Jllaboration (If jl;I;11>; Gr'llit, i\LS!'" I'h.D .. 'F. I.e. S~coJl(i Edition. Large S\'o, 1030 PP, Many lllustration5. O\'el' 100 Tables, 455. (I93~) ALLEN'S COMMERCIAL ORGANIC ANALYSIS \Vrittcll by specialists Oil each SUbj,'ct. Fifth EllitilHl. Edited bi' C. c\I:;:\iSWORTH ilIITCHELL, D,Se" ilI..-\., F,LC., Editor of Tlic ,.j ualyst; Consultillg Chemist, London, S. S, SADTLER, S,}:1" and E. C. LATHlWl', A.B" Ph.D. Vol. 1. IntmLll1ctiou. _·\.kohuls. Yf'a"t,! all!1 Allit·:', its Napilt.hylanlinci=., t.2UtIlQlIIW and AeriLlinc Da::'C5. Malt amI Malt Liquors. 'Vinc~ and Potilhl~ Spirits, Nelltral Alcohol lk~ri\'ativc~, Sugal'~1 i G Figlll....!s. 3~S, Sturch and it~ Isnmcri(lc~, PapN ;'tnt! PulpTesting. Alipbatic Acitb. :'::[\.~ pp, Sy!]. 103 l"it{urc.3. 3~~. (19:':;1-') I Fi.;:wi Oils, FLlt5 iinti \\"ax('s, ~rt'('i,tl aud MrtllOci3. Butter Fdt, Lard, Linl;rcli Oil, IIi~b('l' Fatty Acids, SrMps, layCt~r[n, Wool F:.lr 1 \Vor,l Greas~, Suint, nl:!,;ra~, SWl'ui 8\'Q. ..!.~ Fi~ltrl''';. IIyJ[f)C;ll'b'TI1~, THtllll11'Il:>~ T111:' S~'nthe[ic D,·,~-.:Htltf5, ulJ;- J;p. 3""Q. _\uall,.'sis of 5 Figures • ['£9.:8) \'ul. \'11. Gl'll('[;li Introrlw:tiOll tl) the :\lk:lluid:o', \-c'gt:t;J.ble .-\l[,:aloid:;, AC11nitc, El'll"' berilil~. Cati'c:,-jIlt~, Tea d.nd Coil~e, ,Jnd\Otlt\ Alkaloid;;, L'Oi:uiue, i.'m'('f:l. <lud Choeoiatl:, Opiuln ,\Ikakli(h" Str~·!·h!H.'~ Alkll,)iLls, Tohacco and i\icotinc) Trr,piHL: .-\lk,ll(Jitl::. IJS.1 pp. II FigllTt:S, 3~!:l· (10'1) \'01. lIT. (!~)27J .~~S. Vol. II. 8'::0 pp. .M.lt(cr~, PYl'i(llne, pp. ,5\'(J. Co]orirw:trr, lJyes and CnlonriniJ Cnlouriltt;' ;,ratt~r5, Character~ :\lcohoL::.. Yol. VI. 712 Naplith.l' ,'" (19 29) (lud itii Derh··ati .... t.>:;, Anlhr<l,l'ene and it:-:. AS~(Jciilles, Phenols, Aromatic Acilb, Ca.llk Add Vul. VIII. (allt'U5id._:s awl Nt)ll-Gluen::iiual and it~ Allies, Phthalic Acid and tlw Phthaldoti, : Biller Prjncivh-~, Enzyu\f:~J Putrduclioll Bases, Mmlf'1'1l Expln:::iws. j.P! Pl'. C\'I.I. 3~) Figlln~ii. :\nil1l.tl Ba51'';, Auill],ll AClt):-:, CY;lUog(~n Com3"· ([9';) [1U1l11l.1i\ Prntdll:-, Digl~~thln ProdllLt~ of Proteins. k~llt; 7720 rp. Vol. IV. Spcf'b,l Charadt::fs 01 E~~elltial Oil:;, Resins, IlHIi~·Hubtll:r, Guttapct'('ha, H<1/,1l<1 ;wd r\Ui('d Substallces, Con::.titnenB lIf Es~Cntjill OUs and ;\lHec1 Sub:;tauc'cs, GNI~t;J.l Ch;H':t~'ter,; and Analy::.is of Esspntial (lib, (ljH pp. ,S,·o. 325. (l~~u) 35 Fignn s. l!~130) J::;-;. Vul. IX. rnlWil1R ut l'hiJlt~, ProtPirJ5 of Milk, Milk auLl :\IlI1 .. 1'wt'lllct:i, Meat an[l ~re,\l Pl'odw.;t::,. t1;:0 pp. ;5 Fib'llrt::-, 3~.:), (It)3~J \"ul. X. AIlJlllUilll.li,i5 H.L·llhJl{idUilL or cmu its Dl!rivatives, ~cll'l'oprotein~, Struc.Llu·al l'rotc;in;;, Lxanlillnlioll o[ Food~tutf:; fl"'( VitaUlins, 'flIt! lIornllme'i) The Idclltitlcallon of Vul. V. TOllmius, \\'riting, StalllpiIlg, Typint:, "{arking and Printing Inks, Amilll'~ and Ammo~ £limn Bases, Analy~is o[ Lea tIH'I', Cololl1'illg' Matters of Natural Origin. 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(I9.}'l) ELEMENTARY PRACTICAL CHEMISTRY AND QUALITATIVE ANALYSIS By FRANK CLOWES, D.Se.Lond., and J. BErWARD COLEMAN, Assoc. RC.ScLDublin. Seventh Edition. Part I. General Chemistry. 258 pp. Post 8vo. 65. Illustrations. (1920) ELEMENTARY ANALYTICAL CHEMISTRY (F. CLOWES and J. B. COl.E:MAN) 12th Edition revised oy C. G. LYONS, 1\[./\., Ph.D., l.ectUl"er in Chemislry, Bradford Technical College, and F. N. APPLEYAHD, El.Se., F.[.C., Ph.C., Head of the Pharmacy Dept., Bradford Technical College. 2.;6 PI" Grown 8vo. 6s. (I93-f) Oils, Fats and Fatty Foods. Their Practical Examination. A Handbook for Analytical and Teclmical Chemists. The Chemistry of the Proteins and its Economic Applications. By DOROTHY JORDAN LLOYD, D.Se., F.1.C., Director of l(f's['arch, British Leather Manufacturers' Research Association. and AGNES SHOnE, A.I.e. Sl'conu Ellitioll. RCllrly (,arly in 1')38. By E. RICHARDS BOLTON, P.T.C., F.C.S., Consulting Technical Chemist. \Vith Chapter on Vitamins by Professor J. C. 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