- _______________,.. ___,__ _____ ___,,_._~,~~ ~~-==~~·~~---.--.~--~ STUDIES I IT PLlllTT HISTOLOGY, CYTOLOGY, !1ND SYSTE!.1.!~ TIC BOT.t\ITY. UAJOR ~HESIS IN BOTANY . presented by F. for the dogreo of Master of Science. VIRG I 1TI ...I\. POLYTECIIlTIC I1!STITlTTE . Jtme 1, 1911. - . .. .. ,_ - -· • • • ----STUDIES -IN PL.A.NT--- HISTOLOGY,--------- CYTOLOGY, .1.ND SYSTELil1TIC BOTANY. INTRODUCTION. This paper is designed to give an account of the work done by the writer in a oourse of Plant Histology and Systematic Botany for the degreo of llastcr of Science. The l!laterial has been. gathered from a num- ber of sources. Besidoa the booka ·named in tho bibliography, which is found at the end of this pnper, various periodicals such as"Science"and the "Botanical Gazette" have been consulted in looking up special points. This discussion naturally falls under three heads: first, ~.~iorosoopic Teohnique; oocond, Histology and Cytology, and tl1ird, Syster.iatic B otany~ Tho first two divisions are very closely related and might well be considered together but for convenience of treatnent and olcurness, it is thought best to separato them. #2 • • PART I. MICROSCOPIC TECHNIQUE. While it is not possible in a discussion of this length to ·go into details in describing the various steps used in the histological laboratory, tho more important points will be briefly noticed. It may be said, however, that all point.a are of prime importance in the laboratory. The overlookins of a single apparently uni~portnnt detail ~ay be the cause of absolute failure. Carefulness and ·o1eanliness must be the con- stant watoh,vords of those w:ho would succeed. A description of the various pieces of apparat'l1a is unnecessary and will not be attempted. The one most important piece of apparatus- if there is such a thing- is the compound microscope. In fact, we can not oonoeive of histology or oytoloGY without the compound microscope. The first compound microsoope was r-ade about 1600 , and it has been continually improved sinoe that time. ~he improvement of the microscope han l~ept po.oe with the work in histology and cytology. The microscope reveals a field as rich and varied ao tre most enthusiastic investigator could desire, and the re sult ·a of the last half cent1iry show that the field has not been neglected. ~IBTHOD USED. ~he paraffin method was used in ~ost of the work done, end the discussion of the various steps ta1ron applies to this oet11od. It is the most used of all the methods of the histological laboratory. By the use of the rotary rniorotoma, or even the slide microtome·, very satisf)actory work can be done. • KILLING A]ID FllI1TG. The first step in the preparation of material is the killing. The object of killing i' to bring to a sudden and complete stop, the life process. By doing this, we can observe the cells as they actually were at the instant they v1ore lrilled. One other step is necessary, l1owo11er, if we are to see the result of the killing, that is fixing. The fixing so hardens the cello that they retain their original condition .through all the subsequent steps. With animals, and sometimes with plants, one agent is used for lrilling and anothe1.. for fixing. l1B a ri1le, only one agent is used for botl1 processos with plants, since they have r. o foe ling in the sense that animals have and this is less troublesome. A number of ~gents aro en:ployed; alcohol, chloroform, formalin, c'orrosive, sublimate, and various ncido aro the r..iost c o~on. A stock solution of the following formula; Chromic acid c.rystala 10 g. Glacial acetic acid 10 o.o. Water 1000 o.o. was kept for diluting to the proper strength as needed. The amount of dilution necesnary must be deter~ined in each case by experiment. It may be desirable to change the proportion of chromic and acetic acids. Chromic acid causes contraction and acetic acid causes ·swelling. Anothar formula is; (1 peroent chronic acid 250.0. A-(1 percent acetic acid lOo.o. (Water 55c.c. B- 1 percent oa~ic acid lOc.c. ... The osmio acid io added to the mixture A as it is needed for use , s i nee the +vWO d o n o t 1reep vre 11. tope+her· · o · v · • It gives good results where ... II·.i4 one wants to study·mitosio. Flening's triple stain gives its beat results when used after it. The should bo removed from tho nato1·ial and it should be cfit into o~all piecoo before it is placed in the killing fluid, so that it will bo penetrated quickly. · The material should romain in the fixing flu~d fro~ six to twenty four hours according to the ohnractar of the tissue. WASIII~IG. The next step in this process is the re~oval of tho killing and fixing agent which if left would interfere with subsequent stepo or leave precipitations w11ich wo11ld bo rni~leo.ding. The best v1ay to do this is to leave the matorial in a vesoel under a slowly running faucet of water over night. HARDE.?TING .AND DElIYDR.'lTING. Tho fixing agent, as already stated, begins the hardonins but this must be oontinuod as soon ao the washing is comploted, end since the material must also be dehydrated, it is passed thro1lgh a series of alcbhols of different strengths. From tho water, the ~aterial should go to 15~ .• where it should remain from 6 to 24 hours. It should then be loft for the same length of time sucoossivel~T in 35 %, 50~0 , 70%, - 85%, 95%, and 100% alcohol. This finishes the hardening and the dehydrating proce3s. As far as hardening ie concorne~,the 70% alsohol perhap~ hardens the ~ate­ rial sufficiently but it must go on through the 100% alcohol to co~plote the dehydration. CLEARIJTG. After the material is completely dehydrated, the alcohol must be This is done by the uao of sylol. Other agonto may be used, but this is most o cmunonly employed and perhaps, it is the best. Lilre the :/1 5 11 • hardening and dehydrating procesoos, the change from alcohol to ~lol should be gradual. From the 100% alcohol the material shonld roraain from 6 to 12 hours successively in 3 parts 100% alcohol and 1 part xylol, 2 parts 100% alcohol and 2 parts xylol, 1 part 100% alcohol and 3 parts xylol. The material should now go to pure xylol where it should remain till it is clonr. 'Thio time will vary greatly with the material. INFILTR~\TION. The embedding also is n gradual process. Tho first step is to place a pieco of paraffin in the bottle of zylol. Thia should not be larger than tho xylol will diosolve. The bottle containing the xylol paraffin mixturo should bo kept wnrrn over night. From horo tho material goes to soft paraf fin ( 43° C. ) for from 2 to 12 hours, depending upon the oharucto~ of tho tisoue and its size, from which it is put into the harder paraffin (52° C.) for the name or a greater length of time. Tho temperattrre of the bath io l:opt abot1t 1 ° above this at 53° C. filiBEDJJING. Tha material is now ready to imbed -in paraffin in which it may in~efinitoly. The harder grade (52° C.) is used for thio purpose. Paper trays of the aize requirod by tho amount of material to be imbedded Phoy should be placed in a tray with wax in the bottom near the temperature of the paraffin. A layer of paraffin, deep enough to coyer material io now poured into tho tray along with the material or it be put in with a JE,ir of wnrrn forceps. A warm needle is useful in within the paraffin. Water should be poured around tlie paper trayo at once and, as soon as the paraff in hardens ouffic i ontly to bear the weight of !t, water should be poured on top of tho paraffin. If the paraff in cools slowly, it doos not nako good ribbons when it is cut. .,r.,l~ 6 I/ SECTIONI:tTG. Cutting may begin as soon as the paraffin cools or it may be delayed any length of time desired. !f a good riobon of sections is to oe secured, we cust watch several. points. The microtome ~ust be in good order, the temperature must be· right, and the bloclr :rn11st be n perfect rectangle. The microtome should be well oiled and t~e knife sharp. The temperat11re of the room in '\v!1ich tho cutting is done should bo neither very hot nor vary oold. If the blook is too- warm, it ~ay be cooled by placing it in cola water for a shert while; if too ·cold, ones breath or thumb upen the knife a few minutes ~ay make the sections ribbon as de- sired. Unless the block is cut a perf ect rectangle and all points strike the knife sinultaneoualy, the ribbon will be crooked and this causes much waste of space on the slide as well as being very annoying. Practice is j necessary to be able to mul{e good ribbons and even this sornetiI!lco fails. material is uaually cut betwcon 2 and 10 "If f' thick. FIXIITG TO SLIDE Tho length of riobon or ribbons to be put on the slide will upon the size. of the cover glass. The slide should be cleaned perfectly with alcohol, and thon a small drop of albumen fi~ativo should be spread evenly upon it with tho finger.