To understand cytotaxonomy

L2. l,f/{.figr,/AN/r.4/snr.8?.1 99.-u1w*sq._w, Definitio Ehcre ate tDro worda in cytotaxonou{ir: ., . cYtologXr = etudlr of the cell - tarcouoqr = gcience (laws) of the claseification cytotaxong = taxono3r (cLaeeificatioa) by neans of the cell chafectcm Notions of cytoJ.ogy (d etructure of tlre celil vegptaL ceLL = oba€rtatl@; under nicroscope, of a fragnGnt of thc interaa.l nembraae of an oionls sca-le: x uithout co.loration (in a drop of water) x rithout coleation (in a @ eaccharia eoil.utio) x after colonatio, by the tlRouge Neutretf (Anneur L). = observation of a parameciun, under nicroecqtc: x rithout coloratio x after coloration by metlrylene blue, by ttre rlRouge Neutrett, by todo-d-otiscd uater (Rnncoc t). ia both caBG6, we distinguish: nenbrare, cytoplaem, nuclcue, vacuolea (Fig. I and tr'ig. 2). (b) gtructure of, ttre aucleus Ia a nuclaus, ue d:istiaguieh: mabrane, nucleoplasme, nucleolrrsl chrmati.ne (chroosones). Fig. 5, (c) etructure of the chromosme Oo a chrmoeone, we distinguish: - primary coastrictioa and eecondarlr conEtrictj.on 8rn8 = short arm (S) and long arn (t), right arn and lef,t arn. eatellite centrcmene - ahrooeor (clrrmosomic filsnente ] aerie of gaua) 8i.6. 4, FiB. 5r fig. 6. (d Notlon of cargotype Each epecies hae a speoifi,o nunber of, chromoaomeB rhich ie geaerally patr: - 2n = 8 tor eeps$1g}gp-ogg_t-ei. - b a 46 ton &-eSLrS;1p. - 2n = 5 ton Sjnrrliun dannogun. *" - aninal ceLL .! * I) l{hen the perirs of chrmoso@es are aunbered. ia a ce}l, frm the l.o6eet to the etrontest, this constitutee the speciea carlrotype. Fig. 7, Fig. 8, tr'Lg. 9. lfe diEtiagu:ish ordinary chrmoomee (autosoes) and eexual chrcrroEoroa (heterochrmosooeE). Fo(' nan, therb are l* autoEmee + 2 heterochromosmaa (x ana r). (e) Bhe chronoa@es, support of heredittrr. G.l The ceII diuision ltitose (Rie. 1O) It ie th.is kind of division uhich ensrlrea grotrth and healing. It Leade to the formation of the .giant chromosoee (pac$tcne e pol4yteae chrmosones) in the ceLLs of the ailk gr-and6 of {tpterousr drrriag the dlvision, the chronosmic f,ilanents unspiral to na:cXruo (feaetAenind but donrt eeparate after splitting (ttrictreatng)r and the cytop}am doeentt ctivide ia tuo parts. $leioee (fig. It) It ia thie kind of di.rrleion which ensurets the aemral rcproduc- tlou = 2 ce]-le with n chrmosmea (ganeteg) unite (fecondattm) to g:ive oue ceIL rith 2n chromosmee (ornrn). e.2 The seoc determiration (FiS. 72) e.3 Heredity linked to autosmes (exanpLee) dminant cturacters = x fttzzy hairs x dark skin x blood groups AB x positive rheeus recessive ctraracters = x albinisn x dreponocytos5.e x bLood group O x negative rheeus s.4 Soc Linked heredity (ocanplee) clraracters Linked to chrmoaone x = x haeuophilla x coLor blindaees r. characters linked to chronosome y = x ichtyoeLs or hyetrix gravion (poncupiae dar). 3. ftre si.Erliun cartrlottrrpe (d $rc E:--eggLo_qg cmglor epecies chromosone urmber is: ps = 6. [trat ig to salr 5 chrmosmes-in ] pirs, atmbered I,r Alr IIf r fron the longest to the shorteet. ,(b) In the 8j€rt nuc.Leus (rig. U) ot the ell,k El-ands, the chroosmes are lu an haplold nunben (n = 51 r as ill the escual cells (ornrla and, eperaatolOo), and they can be as L5o tines longer ttlan in the ondrnaqr cclle (graat chrooemes). (o) lftre etraracteristica of each chrmosone ares 4. tfiorouonphoLogical charactere oa the chrmosoues (d Xho chrouoameg have been d:irrided lato IOO segnente, nunbered froo I to 1OO eegnents 1 to It2 correspond to chrmoooue I (t to 20 f,or IS and p]. to 4a fon fr) - eegnente 43 to fZ comespond to chrmoeome lt (l] to 5j f,e II{i and 55 to 72 tw II,L) - segnenta 71 lo 10o conrespond to chromoefiie IIl 0ll to 8O tor ttts aud 84 to IOo fon IIIL). Each se8nent ie ctivtded in ponticrs called A, Br c, D, E, F, and Ln cach portioa, the traneveneaL baads are nunbered. (b) Ia ttre.[.-*S-o5gg, (conplex) speciesr g:iant chrmoeomea, we dietinguigh peroaneat micrononphotr ogical characters: Aa orpanded region (Uc) utrich includes the controere (C) o, chronosone I; the nucleolar organizecr (UO) on IS in aegnent l9i the Bing of BaLbiani (B) and ttre doubLe bubbLe (aU) on IIS, in eegueate 5] and J2 reepeotively; the para Balbi^ani (p) oa IIt, irr segoent 5J. the bLister (u) IILS. ftre briater and the doubl.e bubbre are Errchrooatic sectione. theee permanent characterE alrsry the recognigron of the chrmosouea aad their *iffenent Brnsr Fig. J.4. 5. 4- Ihe inversions the inversions are 18Oo rerrersala of one or Beverel fragmeats of, chr@oames, af,ten breat<ing at two poi^ots. Fig. Il. ftrey are ideatified by arm and nunbened in series, takjrtrg Do acaount of the apecies, Conveutional rmiting: - fixcd inveraLoae = written ln italicel IS-1r IIII-I+ - floati"ng inversions: rrittea in vertical characters: I[,-Il Ih25 - iatereBeclflc inversions = yritten on the left of the chrmosmes - intraspecific i-avereLoog = dri.tten on the rigfrt of, the obrmogonee t hmoa6rgote inversionn = Irtu?/? t' IJT--4r6/4.6 - beterozygote irversions: 1,5-/2t TIJ*3/8 - unltlple iaveraione: IIL-4.6.7, lI'fi,-2.6. CoLlectlng lanne in carnoy Fon gtatmeu! see Annex 2t Chrmoeamee stai.ning for etatemen{see Annoc 3. Rea&ing. Identificatioa of ttre speoies Ue have to recogniee the follotriug specific j.aversions: 5r 7. B. S. ganctirauHk chrmosme I r chromosome II : IS-ArE- JL-Ar It-FQ : ILS - 6b, Ils - ?r IIL - llr IIL - 6r IIIL - Ar Iff. - Dr LIL - Xr tlJ.r - 7. ohronoqme III : IIIL - CrC2r IIIL - B (ttre triple Lnversion a€qu€lrce 4.L?.2 i.s always i.qLied). s.e:gPgr:g/F.-.fl r$Apeqgy'S...qgEgXHlg/S-..-qLeSLeI'gIlE3 chronosmel ! IS-2r IS-1, f,L-1r'IL-Z' fi-35 chrmoeme II : IIL - cr IIL - 8r III, - , chrooeone III : llt - br IIIL - ?r (ffff' - 2 is af.ways inplied). g* egf gsofl S/S.. Jgle.rF q chronoeomel : IS-2t IL-J2 chronos@e II : IIL - 8, IIS - b chrmosme III 3 ILI CS Cf, Anaor 4 and Anne* 5. /t itmrvrrlnot ns y1o;law c;[";f-rr*e Yllra C. ,( Fi>. L c" tt*f J o nl "-.*Qc- F;x- & ce dJ,. *i6JFJc i. I t I I Flc. 9t -- Dtvrlrs NoYAUx au B:Blos. , Scnsiblcmcot, m€mc grossisccmcat'ActB;ooyiur pcu'ri{r&ur{i, -,, & larvcs d'Oursin; C, noyou tr& srueutt d'A+Uopodc,(bl&lliunt intcftioal d'Oairtu atcllvt); D, noyeu dc hrvo &,Saho.o&q-cctluh du foic; E,racior,d'Allimn aap1, (odgioal)- .,- F 0' 3 ''** *#li#" ..;$ ' l l'r, :- ,-., !l .". . ,':]Ir,';1.'fl-rii_r :.r:' . !. *1fl;..' . .r: r'&i\ ;i;iI eji;ii $;j: r-' -i ffi/ol"'aO),' Frc. to7 - st-rrr:}r.r, D'h.* cHnouosouE tr DE sEs coNsxrTUANI.n. |lrsprrc de .Schratlcr. Cp,.constri&ion prinrairc; C., .*rro er";Cr, coo5trtition sccoodaire; Clr,_chromon€nw; Ma, mattix; peipllicrlc; f, setcllite. ,4 I c F,c, + - Srrucrrrlr scutx - '' 'TregE Du csf,oro6ortB(Daptts ltcitz] C- cindtochorc; C0, chrmomirc; Co, conStrielion secor&irc; ll, h6t6rochronatine ; ^&f, rnatricc. bL. f "r.an3 l ..,,&j ^4N,a'i, Nfl -T-ln ,f Fi6. 6 I'F_iI f-',V"q*z -',i-i! I-. l-i r7-lr U-3 7_ t2 ;8rl-[Fm_ 'l . .l!_=*l5 -X-I-[n-]r- -in-0rlf- *l-IhJtr : --bit} r-r r-t't- B- tt 13 .-I'I,^ lx. -r-r:lil .. i& ,, =.$itpryf" "ii;,;, "i"t r"rsplce xurel'ri ,Chcz l'tloriunc af6 cfromoqomcq dom r X a r 'Y. ,; It- 20 2t-a F*, ,l - Cenrorrrr NotM,tL oexg I'rsolcr HUltltN& Chcz b Fcmmc : 46 chromosomcs, dont z X. F;6'9F,t. + {fi--[f-flt-trhltr ,li' It-u: *.I tt *'- U*.. < J*!a Appi', Y*.tn!' l1" "trri^ rrc- , -.r h''*."Lr*a la rto' a\Pt'i'Jto 4.o . - /^ ,u, ,l'roe-(e^ A-t^r ) 7I . -/q, t ('ieot(en I.^.,f ) @- ?{.t.*bL- ,,-.. ^h -l*f ,Lar6^ rfrrutl 9..r.^ t(p*or l- \,\, f 't^. n, 5f p -^.1"'- ^ ol.a <. U.r-Q.i - fu- ' I I &iqrah'".r.,prtlirrc d- J,r fl(-,-.r^friln t no.- en\^i laa f;:;*:fi -h )r'vll!trr J.e. Znl..f^ . #fi-l# Fa ".rqG-XX AOt-rnu xY Un ac^( Lrp" .c- tr*rr-^-eYerl L ,4-* Jc- - i,hea ):- Fy Pu xy Ga*g..6 ., ! ! i -.t .r. n .lo.Aio n '' FoV XX P,4. XX Fi ilo --5"F xY Got.5ea Fi 6 .7?, * /to A'**,- I va .a F. n da Aex oa I -"1 " PTATE I rl firc. rzE - tloyeu oe Br,r.arltxlt ' Glafldcs sativeica dcCbinnau plwttt,(d'apes Brbi.oi). f.6. 43 h [' I,, bI 12r4r678 t 2, 9 t0il t2) ,r-Jr5*r.r** 9 toll 6 suAlEffi,i;58{+- *f,F I I F, A. 4+ i.h ;- 1.t! t-. I :. j ct -t ."t Pb ll {l li Itri' ,i. iJ ,,,tTuDE Du cpMpLExE sb4aLIaM.DAMNosaM EN.AFRIQUE DE L'ouEsr - I Billc pb : parahrlbftrni. ilLo-{ -pb 3 tIih Nilc Sirba Soul>rc I tlaodirrna Frc. 5. - D€termination des 6 cytorypes par comparaison dc l'extr€mit€ du chromosom€ ll. I hornoz]gote ou hetCrozygote. D'apres Yertnr (comm. person-) cett€ inversion chez Yah serait !i6e au sexe, les fcrnelks €tant hornozygotes et les m6tes h€t€rozygotcs pour IIL* (rappelons que chez << Yah >> il y a fusion cctllronEfiqtE). Pour la paire Nilc-Sirba on nolc lu pr6- sence $ur le bras long du chromoeome ll d'un complcxe d'inversions gue nous avons interpr€tC conune forme de 3 inversions successives llLcr, llLq et IILCT. Chez Nile, consid€r€ cornrne le ptus proche du standard Bille, Vruu"u; pcnse que cette inrrrsion coraplcxe est egakrn€nt li€e au scxe. Elb est standard pour les fenrclles ct h€tdrozygote pour les mihs. Chez Sirba oir elle n'aurait plus de relation avec le sexe, elle est hotnozygote. Enfin pour Eandama- Soubrd I'inversion lll, est homozygote chez Bandarna et standard chez Soubre. Au cours de notre 6tude de la r6gartition geographique dcs cytotypes qui fera I'objet d'une publication prochaine, nous avons trouv€ de trds nornbreux h€t6rozygotes I I L -r -,/ I I L.-,i- ? ce qui laisse pcn- ser qu'en fait Baa&rna et Squbr€ fornrent un seul et n€me cytotyF€, dl} rnoins en C6tc d'lvoirc. Le chrormsorne Ilf (Eg. 6) donac quelqrcs infiorrna- tiors supphmentaires. ll est staadard chez Bilh-Yah; chcz Ban&arn:a-Soubr6 il possEde lcs inversions fixcs lllLl, IILI et lllLr?+2; chez Nile il possede I'inversion fixe IllL3 et ctrez Sirba il possedc de plui I'inversion lllh A l'6tst hsnozygote (raremcni Mdrozygorc). Cettc nr0rne invcr- sion se rctrouve ir t'6tat tret6rozygo{c chca Nik. Ces diverses inversions lixes ou &lerminantes nous permeltent d'etablir rxre cle de deterrninatbr des c)rtotypes. I lnvcrsions lixcs - lsr + lLJ. . , Bitlc-Yuh - llLrs hdtdrozygote i - llLrx lromozygtltc. . . i . Yah (mdile) Yah (ll'nrelle) !!r 6 Coh O.R.S.T.O.M., sdr. Ert. md.l. et Porasitol., vol. Xlll, no 2, 1975 : 85-98 93 cYtrotN(olroMY Annerc I 0bscrvation of cel-Is ,;-.,",,:.,'fi.Ii'l;Silternal r.renb:'anc of an onionrs rxicl scale (oboervation af,ter colo- '''l;trl j ;nation witir tlrc :iouge llcutre) .i'- ,r',? Put thc r,ie::bra.ne ir.to 0 15 fJt :louge iieutrp lor a feu niJtutes E Rinse r,rith cLlstilled water pui it LsL,,rce:r blace ancl sIide i:: e drop of 4dil'saccherine sol-uti. on , t- 2. Parameci,;.1 : (ob$c:'vatiotr Iodo-ioui:cu '",'rrt..rr = 10 1 10c aiter coloration vrith iodo-iodized rtater) ; of potassiun iodide * of iod.ine crrl of distilletl uater I aI. ClfpTrl x,r!6r\^y A,^ ,.e--tr. l.pr:tgil 1b Collection ancl .t i:cation o:.' .r.,arvae 4. On rccel1:t of larvac for c;'toIo.gica1 idcntificettion thc Carnoyrs ehould be changcd r-rgaln. ('liris is bocernsc alcoirol antt acctic acid.quLck3-y rcect to fo::rn cthyl acetatc '.'rh1ch hardens thc chromogomcs andthus untlL the larvae,-::.c coupletcl.;. fixc<l, it is clesirable to replacethe. ftxative. ) . 5. l?hen thc larvae &rc rcceivecl by thc cytotaxononist, dctails of'the Locatlon, detez sairplc size, con.litlon (parasitizerlr-sizc; ;;")- should be recorded, attcl cach samplc numbered on the record. sheet and on the tube, and storcd in thc rcfrigerator until exanination. once tho larvac havc bccn exnminccl , illc appropricrtc ntrnbor on 6icsheet can be tlcl:ccl off , rvith any comments There is ;, conciccrtrblc .'r;cunt of con-uroversy rcgarciii:g ti:ocolrection and f ixation of .:. da44osun s.1. larvae io:. cirtologicar work. Several authors rccoffiEe-usc of ice to irunobilize thelarvael which are tncn oirene(l, blotterl and plurged into carnoyrsf lxative (ounuar lgTz, c. r/ajirnc -:,crs. conm. ).- "oirro, -"o"r,"r i:ref er ' whole larvae to be p}-rngecl i-trto Carnoyrs, anci tire fi:cative c:i.:ngccl af ter 1o seconds, ancl a6ain af ier 10 minutes (n.,r. Irost I i)crs. "o"*. ) .Ifgw_ever, f have fonnd no ed,vantage in using larvae disseclecl in thefLeld, in fact they often give inferior resr-rlts due to 1iie si:ecdand dtfflcult cond.itions und.cr which disscction takes p1ace. l-,orthis reason the following procedures are recommendcd. 1. 'Before going into ti:c fie1d., tubes of absolute alcohol shoul-dbe prepared, ancl a mcrlc ml.tle on thc tube sl:o..,,ing the vortule of gracialacetic acld to be pcld.ed.':i rlie tubes sholrrd be put lnto an ice box(4-O tuues containerl in a polythonc bil)r-or " vride raolthecl theruosflosk. A snaIl, tightly sioppererl botire of graciar acetic acitl shouLd be kept separertc - put on ice the acetic acid freezes tiuickly.A oeparate container lvitir preparcci labeLs should be icept vrith theacetlc acid. 2' In the fie1dl the ftppropn;.rtc volumc of acetic ...cicl is :rd.declto the tube of alcohol im:::eciiately 1,yi6r to plunging. the larv;.e intothe fixative. rf necesser;y', piecLs of vegetation containing lo.rverecan be ptrt 5-nto the tubc. A 1abe1 ivitlr loca.tion, date, end if possible, site co-ordinates(thcsc can, of coursc, bc srrpplicd i;;";) r-i" prt in ilrc tr-rbc rzhichis then rcplaced in titc ice-box. Tirc tubc's containing larv*c s}:ou1clbe kept as ncar to 4cc as possibrc for at lcast ) days, or prcferablyuntil thcy are scnt for idcntification. 1. On rcturning to il:c i,abora.tory or field basel thc Carno;,rs should bc replaccd rvith frcsir fi:cati-vc. Fixation is clirectly relate<l to the ratio of larvae/fixative, and for this reason not morc than about 5o larvae/tube (25pt. rixaiive)is recommendeC. * See ;\ppcndix 1c for details of fixative. I' 6. on examination of tlie sanples it is advisable to record thc lnverdions used to irlent:fy t::c species, as vicll as the ot::cr heterozygoue and floating inversions, and thc nurobcrs of larTac parasltlzed by ilertithiirls, llicrooporiCia ancl CoelorlJrtes (-etancLard sfreets havc blcn made for il:is purposc which can bc raodifiecl if requt rea). the record s5,eets can thcn bc icept in ring binders in order of rl.\rer syotems and rlatcs of samples and can be easiiy referred to. Statnlng There are several reethods of staining polytenc chromosomes(see Darllngton and T,a Cotr-rr 19761 )unbar, 1972), howcver only 2 ncthods will bc describcd. herr-:. 1. Feulgen staining (see Annc:l for c-lctails of solutions) ' this is the nethod used r.,rlten bul]r staining is requircci. '. niIc1 hyCrolysis Ln lO/, I{yCrochloric ec:-d pr,-iperes tlie chromosomes b1- exposing thc aldehyd.e Sroups of cicsoliy-sli6: r i';ilich ti-.cn rcact vritil tttc stlin' Iiydroiy:sis is- the most critic.. I st.;p, a.::i cl'!n bc ''- sourcc of st;:in failure. For fresh r:;tcrtr.t] in gooC cotrditio:--, ? ilinutcs hrrctrolysis at between 60-65 oC i s acletlltatc . :lcvlever, if tllc uatcrial has been in fixative sone tine (sevcral montirs) ti:e hydrolysis tin:e ccn bc extended, or the tenpereturc raisecl siightly. *}rocedure Remove larwae from Carno;'rs and wash in distilled water, pour off the disttlled vrater, blot ttu ltr,\lae and put into 1"o/' dJ]- at GO-6:roC for Z ,i""i""-(,i"p""aing on the eondilion of the material). furter hyclrolysis quickly pour off iiCI and add dlstilled I'ratcr, wash for 1-2 mlnutes, r6tore iarvae and blot before putting them into a smalL quantlty of ieulgen Etain in.a sma11, ttghtly stopperecl bottle. The statn nust !e ltept in the darkl (enpty filn cassettes are vely usefuL for this purploc). fJta"inigg tine dcpends on ti:e roeterial: fresh and reared-nalerial nay neecl only It'hoursl whereas Zii-J hours ts necessary for most other naterial. $tainlng timc can be cxtended for 4-5 houig, but nith incrcasir:g time in the stainl t5o condition of the chromosoroes deteriorates. Ylhen stalning is judge<i to bc adecluatol the stain is pourecl off and the la:rrae washc<l 2-j tines :-n tap water. Prenaration of Slidcs The sallvary glands Lirc disscctcd out iu 601" o"otic acirl l::d reraoveil to a clear, "i:.a" rvith a clrop of 6Ofl zcetic rciC' 3-4 larvac can be put on a slide for disoection, and it is often better to leave the larvae f or a f crv seconcls to sof tcn in tl:e acid. Iffarvachavcalotoff|'tboc]y,orthereisalotsillr attached to the 6;1r-nCs, BO/'acctic acicl can be used' Acleaneoverslipislowereclontothesalivary_gIandsrcare- ful1y avoldlng alr bublles, and the slidc and. coverslip gently blottcd' trror caees when there is fat body or siLk gently vra:mi'n6 the slide over a sBirlt laup beforc squashing rryiII heip to sprearl the chronosomes' t oo 'spread, the chromosomcs the coverslip can be gently tapped rvith apenelI or dissecting necdle o.nd flrcn squashed. uy appiyin!-prcssure with the thunb or first and second flnfer. CarL nust- be tiken notto move the eoverslip durlng squashlng, The slide sliould then be ring.ed' wtth rubber solution to avoid clcssi'cation; (tfre mna nscd. torepalr bleycle tyrcs, }ocaIly ava,.ilabIe ln thc narket). severalslldes can then be preparerl Lefore examination unrler the coropowr4mlcroscope. fhe slides can a.Iso be kept in the frldge for severalhours before examinati-on, or for pcrnnancnt preparatlon. 2. Orcein Stainlqg orcein staining often gives better preparatrons than those stainodwtth feulgcn, but it is very slour and. onty ina:-viclual laryae can bcstalned. llowevcr, it is a'uscful ,;.rtcrr:ative f or snall sa:mp1esand, also to counterstain fa.int feule;en-stalned larvae. Procedure larrrae are taken fron carnoyrs and put into a crop of 60,lr,Acetic acid for 1-2 rni-nutes. fhe glands are then dissected out toa clean sI1de with 6Or',.1 .,'.cetic acid and gentiy warmed. over I spiritLanp. the acid ts then blotterl off and a drop of lacto-aceticorceln ad'ited. [h9 slide is gently warued iron trne to ttnne, afterJ:9- rl"ytee the staln rs bloitoa otr ,-eo% acetrc acta added and thesilk and fat bocry removerr from the uirr."nry glands. fhe acid isbLotteel off and i gTo, of orce:.n added. +itlr 2_5 minutes the orceinls removed, 6$, acLd. aaaea ana ire-;;;;r";;es squashed as describedabove, Penqenept Preparati ons There are-tvro principal metl:orls of nalring permanent preparetio:r,s.lhe fi.rst rnethod involves qulekly rrcezing ih" sridc in riqurdnltrogen or d.ry ico. (lr,*--^ii,f6-,: c.,.,n bo Iroocr: in a clecp frceaelbut froeztng n6y not bo qurclr orouBh to prrvpnt r10!rt,E,,tian) , Bhrcovers3.tp 1s quickly prized off rviih a yLzor bladc ;;ii-;;;i; cover_srtp and sl-ide prungecl into absolutc arcohol for up to 2 mi-n...rtes.[he chronosomes are thus frozc:: in placc, and the alcoho] carn rep]aoethe aqueous Acetic acid solrition. ?hc srlce and coverslly, are removed, fron tire alcohol, clrained., anci a d.rop of Eupo.rol lut on thesIlde and tlre coversli!: rcplaced. speed is essential to preventdesslcation and shrinicage of the chromosomes, ancl it 1s iiportgnt to ensure that the aIcol:ol is rrbsolutc - as In the tropics alcoholqulckly becomes hydrated. the second method is Lrseful vrhcn dry lce or liquid nitrogents not avallablc, but gcne-raIl;, 6i.res srides of inferior quality. ?he slldes &re put 1n a petri d,ish and eoyered. with Acetlc -aloohol (Carnoyrs ttxitive) *n,1 teft overnight. [he acetic aleohol sLowly repLaces the aqueous sorurtion. The iext day the sride is removed, blotted ancl the sl-ide and. eoversltp pasoed throug5 alcohol ae descrlbed above. $ehednle of fixativc and stains FtxatlvbiT,1. CarhbytE fixatlve (r\cetic alcohol). ii!' 5 Parto'' absolute alcohol 1 part,.$lacial acetic acicl' Carnoyls flxativc should bc freshly preparc'd prior Statns )- FeuLgbn (Leuco uabic fuchsin) . Ingredients g. baslc fuchsin sms. f,-S,O- ;,.( z > 10'cc r0l scr to use. 1 2AO cc,, tli.sttlled lvater, r lFroced.ure IIo*-t ?00 cc d.lstilled water to QOoC, add 19. basic fuchsln. CoSi:lF9 60oc, adtl 2 gnis. I(aS205. coq?.lto 5ooo, add 1o eic tof tict. Shd'ke'weLl and keep ini a ti6htly stoppered cont:.iner in the frlfrge for 24 hours, erfter whicli bleaching should. be conplete. .-ir:,_t.. Att'd*.,about 0.5g. of activated charcoal (decolourising ca.rbon) t1,',"lfibe etatn and. shalce rvelI. ; iLl,.,J i.6... fdIfr{r.,rapldIy to reno,ve charcoa.l r:nd impuritr-cs. ihe resultnnt s!a{a should, be tr colourless 1iqr,.irj, hc''.revcr pocr riua}ity Fuolietn may rcsult in a ye.liorv ol: orangc solnti:,:. If ti'ris is th'e ease, add no.re cli;r:icoal ,..nrl. filicjr rS.^i::. ."'rre nust be t;"iien to ftller or-rt aI.,l. tr.iqes of charcoal fron tlic sr)1Ltt1on. i 7. Keepl J.n a welL otol-rpercd bottlc in the rcirigcrator. I I lacto-aeot{c Orceln {:' Stock HoLutlon .,iI g. Orbeln 25 ss-.gLactal acettc acid 25 cc 85rt l,actlc acid thLE oan be.,|rgpt tn a rveli otopporcd. bottlc for long perlOd.s:'ofl t1me. For countersttlntng Seulgen-treated chromosoues, it can be'ueed dlrec!Ly. However, to, Orcein-stoining )- part of the Stock ieolutlon shduldl Le dtluted wtth J parts 5a,4 aceitc aci<i 1Y ,,t - $ .ii , 5 :{, ir.t .lri,#+ ,*.+l ,t' ''li .|} ,d' 'b t 'lii s -:t , _t' si{',}, ? ,ii l1*d rls si .s|:i $& iB *. :,7 ., i! rtl # $ * CYTOEiri(OlI01ff Anncx J Staining of silli glands clrronosomes 1. Pour the carnoy awaY 2o Rinse the larvae tirith disti].led trater! 3o Dny the larvae on hy6iellic papcr, then tlo Put thc larvae in L0# chlorid acid at J r:rinutes or. Ll ottin6-irctr,or' 6ooc for f rnitrutec ir, . tubesl heating with tirernlostat 5. Rinse l'rith. distiLled trater: J tines 6. Dny the larvae (hygienjc paper, tlen blotli*6-pcrpcr) ?. Put the larvae in Feuli;en for i hours. In rrell drir;tl ,-,,.,ir!-l The Feur6en stocl'. is alr'lays out of li6ht (o"rt< bci6) aricl it'rr;' freezexl" B. Rinse tle Larvae vrith tap water. Leave tircr: in tire l-i:r;t trrr' dissection of the silli glands. Dissect (extraction of sill;. glancis arrcl c11'ot.io$ones) j,,i !,C., . r:'.tic acirl' Set the chromosomes (the BaLbiuri nuclei) bettueen bl;t.."t, lrlri'r;Lirie i:r a drop of orcein. 11. Crush under blottin5-Pi:Per. i.i.i..:oIyse 'u i:tls. .,'r; in ,' until 9. Lo. s r\ -{ o -l x o z o ,I lst btt r + a B Hl{(a- F b Hd i, : I xo =o @o =F a H v,Hoz a, tp a+ o ooPF o o oo c)oPP o o a+o4 P. o F' ln a ats(D5 a a a c)oI5 d .I tg s+ o o xg P.p oo a 2, o (D xg P. (Dp a a a a a zo P.9,op rt P.|-t P.oB a ClToTnxoNoMrE A ^v^?tsc, 5 C//R lnrvO.94,/y? Sct(8rc €/v s'€l 4,t1.1 <, c, : *: ,} .1 lH at,. wao/arcxo/rz page 1l FTC. {. DIAGRAII' br CANTOS .,0f 7 a- ,vt i7 L,^^i_", @"t) ,,\iii! r'l- I I { .j