ANTIMITOTIC EFFECTS OF TERRAMYCIN Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Stella Marie Wilson 1954 This is to certify that the thesis entitled Antimitotic Effects of Terranwcin presented by Stella Mario Wilson has been accepted towards fulfillment of the requirements for Master—Le- degree in luau—(cytology) Mega/e4; Major professor Date W— 0-169 - .Mgr—hfi._ -' k a. AI‘ITIMITOTIC EFFECTS OF 'l'Srizlixl‘i'fCIll By Stella Marie Wilson Submitted to the School of Graduate Studies of hichigan State College of Agricu ture and Applied Science in partial fulfillment of the requirements for the degree of PLiSTIJl C ’13 C’) C ‘2 H l"“ C 1 [*1 Department of Botany and Plant rathology School of Science and Arts Year 195M .Approved by -//fl€'gég/Q4é7y~v’ i'... ['1 Cu on, . “pl; n93 l 1 fl“ l‘. r! / A \4 used 5 ea mane u of 1 Stella Marie Wilson The Eisum test was used to determine the cytological effects of terramycin-HCl. The root tips of young seedlings of Pisum sativum were treated for eight hours with concen- trations of 10 ppm. to 200 pm.; and for short term treatments or 15 minutes With looand 200 ppm. and for 30 minute: with 600 and 800 ppm. followed by a recovery period in nutrient solu- tion. A quarter-strength modified Hoagland solution was used as solvent. Cuttings were made at the beginning of each run and at spaced intervals thereafter. Slides were made using the Feulgen squash technique and were microscopi- cally examined and scored. It was observed that the mitotic index decreased with increase in time and concentration. Early prOphases disap- peared. Late prophase showed an increase in proportion to total mitotic cells and was characterized by contracted .figures and reversions. Post prophases were the least ef- .fected. Abnormalities were of the "spread" and "scattered" types. It was concluded that: l) terramycin-HCl has an inhibi- tory effect on "antephase"; 2) interferes with center and kinetochore movement in prophases; 3) may have a slight des- innictive effect on the spindle; h) prevents cells in mitosis at the time of treatment from dividing again. No mutagenic action was indicated for this antibiotic. 331291 ”' nu: .E.if +h $1.01 CYE'vlC‘Lav' u V v'. n ' wees, ‘ .nt GOES ‘ . ss-‘ \- DLblia mar‘le WllSOll 2 Terramycin-HCl differs from colchicine in respect to cytological effects: 1) the former inhibits "antephase," whereas, the latter stimulates "antephase"; 2) the former does not have much effect on post prophase, whereas, the latter does; 3) the former inhibits effected mitotic cells from dividing again, whereas, the latter does not. The cytological effects of terramycin-HCl does not differ from those of other antibiotics studied thus far. There is some indication of a correlation between cytological effects and antibiotic action of the antibiotics. I ‘ ‘ "than-1. r-A' . l.’-'.&. .. . s“. { h'.~-I "A “a. .... __J BIBLIOGRAPHY Auerbach, C. 19h9. Chemical mutagenesis. Biol. Rev. 2Q: 355-3910 Bowen, C. C. and G. B. Wilson. lQSh. A comparison of the effects of several antimitotic agents. Jour. Hercd. g§:2-9. Hawthorne, Mary E. and G. B. Wilson. 1952. The cytological effects of the antibiotic acti-dione. Cytologia ll: 71-850 Hyypio, P. A. lgSh. The effects of colchicine upon the mechanism of mitosis (Unpublished Ph. U. thesis, Michigan State College.) Levan, A. 1938. The effect of colchicine on root mitoses in Allium. Hereditas gg:h7l-h8o. Tanaka, N. and S. Sato. 1952. Effects of Streptomycin on the mitotic cells of Tradescantia paludosa. Cytologia yuan-133. ‘Wilson, G. B. 1950. Cytological effects of some antibiotics. Jour. Hered. g;:226-231. and C. C. Bowen. 1951. Cytological.cffects of some more antibiotics. Jour. Hered. ggz251-2FS. ATTIEITOTIC EFFECTS OF T735£h¥31fl by Stella Marie Wilson A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER CF SCIENCE Department of Botany and Plant Pathology School of Science and Arts 195M ACKNOWLEDGELLETS The writer wishes to extend her sincere thanks to Dr. G. B. Wilson, under whose direction this research was conducted. His guidance and patience will long be remembered. To my colleagues, thanks are extended for their friend- ship and aid. To Mr. thilip G. Coleman, is extended thanks for his photographic work. Appreciation is extended to the All College Research Committee for defraying many of the research expenses, and also to the Naval Research Grant Assistantship which made it possible for the writer to conduct this study. vvov II- F r. .......Bar 61.31.»... 8-12.13. u... .% lukbhkfi INTRODUCTION .. ................... ....... . .......... LITERATURE EVEN ....... . ........................ .. . MATERIALSANDHETHODS ..... .............. A. Experimental Procedure B. Treatment C. Cytological Examination OBSERVATIONS .. A. Classification of Aberrations EB. Continuous Treatment 6 . Fifteen Minute Short Time Treatment and Recovery D. Thirty Minute Short Time Treatment and Recovery II. Summary of Observations TABLE OF CONTENTS DISCUSSION...........O.O.0.................O.........O........... A. Nomi Cycle 3. The Effects of Termmycin-HCI C. Terramycin-HCl as Compared to Colchicine D. Cytological and Antibiotic Activity of Antibiotics SUMMARY ........ ....................... . ......... BIBLIOGRAPHY ...... . ......................................... .. PLATES .. ..................................................... coco;- 10 12 13 13 15 22 31 37 41 ill #3 u? a? 50 52 55 . I .lHfl lll.|l.| I:|I.lll lllll X|llllalll| i \ll ... . u DH fl|\ .\‘ (JU . 2L an ... W WA " n x (H r . f4.“ . CU A .K wt. .. i n flit 2.4 fl. . u. A . a . u w M fiv , u .H» s . 3.. a. . ta Lu .. av A u u v .- I . Mu. “A; «1‘ L CV |. L MAW w A .o A 10 .md aw: an in TD in; . [\‘r‘ vs IIWThK)ULY)1L:Aa The primary purpose of the research for tgi: thesis VI ' V" J- " l ‘I .V" .‘.\' :" . "2 . ‘. ‘. n ‘1. s ‘ ‘ "1‘ '. h -: '1’," I "£4,tns dcteimnnu> LLC L_prdlt0tlb (unfects Cl c;11argrflal. A preliminary investigation of the effects or this antibiotic was made by G. B. Milson and C. C. bowen (lffl), Tmld' the Q Allium cepa test. At that time, it was determined that the general mode of effect was the same as of aureomycin, acti- dione and streptomycin, but a detailed analysis was not made. Terramycin was introduced as an antibiotic in 1950. It is produced by Streptomyces rimosus. The structural formula of terramycin is as follows (from Peterson and Strong, 1953): H- \C/ \C/ \C/ \C/ A E5 6 'c') H H Terramycin - C22H24N209 lll|ik ll» Ll] .I. ‘ ll l .l . l «A awh r a O . u r v .. r, Cy . . L l . . I u r I V H . r - \V s u L a la n. A .r I A I . 7 . . .. h . L . . a Al .. M Ta 4 t A 1A . . L . I . rv n. . A A ... Irv. .1 (Q by he . w... . 1 «a 8 Lu Cw V. a; 1 . a. \v . .ru r: ... I. ‘ A . .. V v 2 v . o l . "t! \ . l \ . . 1 n ‘l« vii . _ file .L R H L 1 .. Q. ‘ film N P. Y. W I w: v; a .. V? n A.“ m. b . ....H a). a l e r: r: .7. “C l. 4. .s a a... L» . .5. Cu . u a. .A 3 . . s . . Ii . v :2. Cu C.» a J n. t . "d Ce 5 l e nu 1.. Q» LL , . re. .3. at l .15 . . a . .4 ~«4-N..§tr. 2!. tan. . Pure terramycin is highly insoluble and is amphoteric in form. However, the sodium and hydrochloride salts of terramycin areruadily'soluble. The dry hydrochloride salt is very stable and shows no loss of activity after prolonged n [’0 ‘ storage at 2) C. Terramycin and its salts have a broad antimicrobial Spectrum and are effective against aerobic and anaerobic, Gram-positive and Gram-negative bacteria and certain of the rickettsiae. They have been found to be a very effective treatment of some virus diseases, pneumonia, and certain venereal diseases; and have shown some premise of tuberculo- static activity. Terramycin is readily absorbed by the blood stream following oral or paranterial administration. Complete elimination in the urine is realized within one to two days, V. .4..A$~l v . G) C 11 pl.) ,4 ‘—¢ e‘\‘a- - .lcg Tf‘v—I I .4. Kl) L. G. Nickell (1953) has found that treatment of seeds of cucumber, corn and narrow—leased sorrel, with con- centrations below 25 ppm. added to the soil, increases germination percentage and growth rate. In contrast to tfiege results there is a report from Italy by D. Della Bella and C. Gabellini (1951), of reduced root growth of Lupinus albus when treated with 10 ppm. in aqueous solution. Also, from Brazil by M. Mateus Ventura (1952) comes a report of mark- edly inhibited growth of roots of Zea mays. However, in this case the higher concentrations of 50 to 1500 ppm. were used. Viewing the wide scOpe and use of terramycin, it seemed warranted to make a more complete analysis of the cytological effects using the Pisum test. (C. C. Bowen and G. B. Wilson, '19su>. V. A. l :Fc ‘ llc~ .K- 1 ) (f' fu- ._‘ (7 LITERATURE REVIEW The first report of cytological effects of antibiotics was made by G. B. Wilson (1950). The antibiotics Penicillin G, Streptomycin, Neomycin, Circulin, Endomycin, Actidione and Streptothricin were investigated using the Allium test. Peni- cillin G and hndomycin showed very little measurable effects; Neomycin and Circulin showed few mitotic deviations, but were highly toxic; the others showed "C-mitotic" effects at the toxic level. In all cases, some "reductional groupings" were f‘ound. In 1951, Wilson and Bowen reported on the cytological effects of Aureomycin, Terramycin, Streptomycin and Chloro- mycetin on Allium root tips. All were capable of inducing mitotic aberrations of the "C-mitotic" type. Cytological and toxicity thresholds were close and recovery was not re- alized, with the exception of Chloromycetin, in which recovered cells were normal. Levan and Tjio reported on Penicillin in 1951, in which they used the Allium test and concentrations ranging from 0.1 1 to 20 MIU (million international units). The stronger concentrations of 10 to 20 MIU induced lethality. "C-tumour" was produced by concentrations of 0.7 to l MIU, and "C-mitosis" by 2 to 5 MIU. In addition, a low and erratic frequency of radirimetic effects (pseudo-chiasmata, attached and free 'U “‘ 'r ‘3'): u. u..| L 1 m 1' K lit “A w fl» ~,J . «\H [xiv \f'l fragments, and stickiness) was reported. From these results, they concluded that Penicillin had some, but very weak, mutagenic activity. Hawthorne and Wilson (1952) reported on a further in- vestigation of Actidione. Effects were: a decrease in mitotic activity correlated with time; fluctuation in the relative frequencies of stages correlated with time and con- centration; toxicity at concentrations producing "C-mitosis"; and general effects of contraction, "split" figures, and stickiness. Tanaka and Sat3 (1952), reported an investiga- tion of the effects of Streptomycin as observed on Tradescantia paludosa, using the concentrations of 25 ppm. to 1000 ppm. Effects reported were: contraction, "C-mitosis", stickiness, fragmentation of the bridge and fragment type, micro-nuclei, faint-staining prophase chromosomes, and clotting at the sub-lethal range. They concluded that the action of Strep- tomycin was mutagenic. Bowen and Wilson (l95h) reported a further investigation on the cytological effectsof Actidione, Streptomycin and Chloromycetin, using the Pisgm test. Actidione was determined to fulfill the D'Amato requirements of a "prophase poison." Scattered metaphases were not considered to be "C-mitotic" and the term "akinetic mitosis" was proposed to describe them. Streptomycin was similar in effect to Actidione, with very little margin between cytological and toxic concentrations. Chloromycetin was the least toxic, exhibiting prepropnase inhibition of mitosis t sublsthal levels. In recent years, there has been some controversy on the terminology used to describe cytological effects. "C-mitosis" was the term used by Levan (1938) to describe the modification of the mitotic cycle y colchicine, In its strictest sense, "C-mitosis" includes spindle suppression, and a delay in kinetochore cleavage. "C-mitotic" substances are poly- ploidizing agents, by definition. Since Levan introduced the term, it has been used to describe the effects of many other substances, all of which are not necessarily polyploidogenic, although they may cause spindle suppression, chromosome scat- tering and other effects associated with colchicine. D'Amato (l9h8, 19L9) has shown that both prOphase and preprophase poisons may exhibit effects which resemble "C-mitosis." D'Amato and Avanzi (l9h9a) stated that true "C-mitotic" sub- stances are those which do not interfere with the entry of interphases into prophase. Substances which cause prophase poisoning, prevent the entry of cells into mitosis (D'Amato and Avanzi, 19h9 b). It has been recently shown by H"Vp10 (195u) that colchicine, in addition to causing polyploidy by suppressing Spindle formation, also affects interphase nuclei at the stage called antephase (Bullough, 1952) and causes the appearance of scattered figures through the failure of an organized movement of prOphase chromosomes to the prometaphase clump. he pointed out that although polyploiov and scattering of chromosomes may be found at the same time their ordighl is quite difiexrnngznni that scatfix “in; leac I-“ to the formation of micro-nucleate cells and not polyploid cells. MATERIALS AND ELTHODS A. EXperimental Procedure The source of meristematic material for the experi- ments conducted was the root tips of germinated gisum sativum var. Alaska seedlings. The seeds were furnished by the Ferry-Morse Seed Company from their 1952 and 1953 stocks. This strain has proven to give a fairly uniform mitotic index and "normal" mitoses, rendering it favorable for mitotic studies. The seeds were placed in distilled water for a soaking period of six hours to hasten germination. The soaked seeds were then placed in paper toweling, moistened with distilled water for 60 - 72 hours for a germination and growth period. At the end of this time, seedlings with roots of approxi— mately three to four centimeters in length were selected for treatment. This operation and all subsequent procedures were carried out at room temperature (about 2h0 0.). The root tips were suspended in an aqueous solution , for treatment. This was accomplished by the use of 250 ml. beakers as containers. Support was given by one-fourth inch ‘ mesh metal grids which had been coated with paraffin. ll .‘90 “;“k!¥.-.‘£\!“fllx Luna v‘ i V3.4!“ I cht'va' --_—._.____ . ___..-r rm 0‘ - f‘ .5. ‘ .,.\. u . ,‘d’ nr-I‘ n~\r”6-~ a “(42,. VP“- it 2:16 i €— .' \' . ‘P‘Y':.“‘(-‘ .-I\-‘b‘-‘-‘d‘ f 9..“ F a“: ‘2 I‘L- ( C f (f v' f The solvent used was a modified Hoabland solution as developed by Huskins and Ltienitz (lQhB). The composition of one liter of full strength solution is: .095 gm. Ca(N03) ’ MHzO .129 gm. NHuNO3 .180 gm. M 80“ ° 7H20 .13h gm. KHZPOLL .007 gm. KZAPOM - 3H20 A quarter-strength nutrient solution was made up for use at the beginning of each run from stock solutions of the individual salts which.were kept under refrigeration. The terramycin used in these experiments was the HCl salt donated by the Charles Pfizer and Company, Incorporated, 11nd bore the control number WBt527S28. A solution was pre- jpared at the beginning of each experiment. A solution of strmnmgconcentration was made and then diluted to the desired ccnlcentration in parts per million. Refrigerated stock solu- ticnus were not used due to the formation of a brown preci- Pitéite of terramycin which formed by hydrolysis of the salt, in isolutions which were kept more than #8 hours. At the time of treatment the 250 ml. beakers were filled witll 220 ml. of the required solution. This included the con- centration of terramycin - HCl to be tested dissolved in quaITter-strength nutrient. Fifty to sixty seedlings could be Sdaspended on the grids of each vessel for immersion. :l A - ’3 v i .. ‘i‘dv \ V “.2" -c 'l~'-.¢‘A.;' & 0 (Ah n-rq n!‘ but It... .0. . v r, A. ‘W t v -‘-4 "'m\ c. A 3 n.- ..Y‘. n ,a. I , H“ VA -u.»,‘) A a"L"" 5'“... q‘V‘lU \ym'v ., r .- _.' . b..\& V‘.‘,. ' n.0,‘L ul‘vdJ . ' fl vnaro“- L A Q 10 During the treatment period aeration and agitation was supplied by air bubbled through glass tubes with small Openings. The air supply was from the laboratory outlet, and was passed through a charcoal filter to remove any for- eign material. Four root—tips were collected at the beginning of each experiment and were used as zero hour controls. All subse- quent cuttings included four root-tips from each test vessel and control according to times required by the mode of treat- ment. The root-tips were placed in three parts 10C percent ethanol to one part glacial acetic acid after each collection for fixation under vacuum for one to two hours. A hydrolysis ,perdcd of nine minutes in]illKfl.at 6000 followed fixation. ffhe Feulgen reaction was used for staining and slides were nmuie by the squash technique. The slides were then micro- sccuoically checked before being immersed in 95 percent ethanol for 12 to 2b. hours for dehydration. A small amount of fast greérn.had been added to the ethanol as a counterstain. The Slicles were made permanent with Diaphane. B. Treatment TIhe experiments were planned with the goal of reaching a dOEBage threshold at which effects and aberrations could be strudied. The first trial runs were set up using continuous .-,.-v' ox}, u “v,..74.. <- 'k“ l . . ‘4~ .\~ . ‘ O. ‘~.c- I‘ .4 . v..u J. .- n -\ ~«-p> but .u..; a L--. 'r" b, . . » we duh-l C... ‘ I A'grm # uV.c ‘U c I've", ~ T ‘vUn‘ . ‘ ‘f‘ ) "WW‘ h "' ~ -A. In ~ . “P +. Q. —V'- ‘ q"“$ at. new . ~ f‘ n- "“Ve-avr’c P' r- . h F. ‘“ t" .t' v 4» ~ P Y‘. .. _, ""Ay’_.'-Q ’ V v1 “‘4. C‘- .H ‘ t C. l “, ulul L.' . . r”~ x. run-h v,. ‘1 av ,‘ .. V ll treatments in aqueous media. Fifty ppm., 100 ppm., and 200 ppm. concentration of terramycin ° HCl was used with collec- tions at zero, one, two, four, six, and eight hours. At the end of treatment, the roots were thoroughly washed and placed in moist paper toweling for recovery. Upon examination of the slides, it was ascertained that these dosages soon reduced the mitotic index to a point where it was difficult to study aberrations. Therefore, another run using the lower concentrations of 25 ppm., and 10 ppm. was set up according to the same schedule as before. At these concentrations a decrease in mitotic index was not realized, but there was a scarcity of affected figures. To try to overcome these difficulties, it was decided to use short term treatments, followed by a recovery period .in.quarter-strength nutrient. One hundred ppm. and 200 ppm. (nancentrations were used,with treatment for 15 minutes. ITne roots were then thoroughly washed and transferred to quatbter-strength nutrient. Collections were made, at zero h011r'(control), 15 minutes treatment; one-quarter, one-half, OHGB, two, three, four, five and six hours recovery. Fol- lonning this method of treatment, there were few aberrations and! recovery was realized within a few hours. It was then decided to use the higher concentrations of 000 tand.800 ppm. and increase the treatment period to 30 mlnlltes. Collections were made after zero, fifteen and ~ I L, w.»- Jné‘t V" nu-.. ' ‘ n l.... y.-.a c.~ r. 1 v armi’ o-- "HY , - 5-.» I. , All ' ;. U¢- a...._. 1 1 I 7. 'h a We». ‘;f‘1 , .\ V1,. thirty minutes treatment. The roots were thorou hly washed J L) V0 C‘ and transferred to quarter-strength nutrient for recovery, with collections at one-quarter, one-half, one, two, three, VA four, five, six, seven and eight hours. C. Cytological Examination The slides were microsc0pically examined using a 90 X oil immersion objective and 12.5 X oculars. Lighting was supplied by a ribbon filament lamp, using a type B green filter and a type B orange filter between the light and microscope mirror. Quantitative analysistmxe made by examinations 50 'units apart on the horizontal scale. At least three planes ‘were checked. One thousand cells were counted to determine 'the mitotic index. All mitotic cells were classified ac- cnording to phase and type of aberration until a total of cure hundred was reached. Any abnormalities seen in resting nuclei were also noted. Data taken from examination of the slides, was kept on SCClre sheets. These were used as a basis for a quantitative analysis of mitotic effects. l Fm"- A ! ab. 21 u—v-r—nn¢“cmb my... .uyu. ,‘a -. Eroptase To f a categcries A plate c1 ities is CBSZRVATICHS A. Classification of Aberrations Prophase To facilitate analysis prophase was divided into four categories: early, mid, and late prophase; and pro-metaphase. A plate of photomicrographs of the main types of abnormal- ities is included at the end of this thesis. (Plate III) Early prophase. No abnormalities were noted in this category. Midgprophase. Reversions were the main type of abnor- These are characterized by a relaxation of the A few spread con- mality. coils and a general diffuse appearance. fiégurations were also seen, with the chromosome threads loosely arranged within the nuclear membrane. Late prophase. The greatest number of aberrant cells were f0'LJ~ITLd.i,nthis stage. They had chromosomes of varying degrees or (bontraction and reversions. Contracted figures were classi- fiedl as "slightly contracted," "moderately contracted," and "severely contracted." The slightly contracted figures are r9°C>gnized by a thickening of the chromosomes; the severely °°n1DPacted figures are of metaphase length and scattered ..... 1% I m .. . . V _. . . ‘ i .. (C by w . Be . c .e - . o. q I . . v I. v .1 .0 . . r . a Q. a “J C» L 4 .-v V; fife We a.. t . . 1 . I. v. a A...» .3 ~ l nG n; In .x .. CL L» F. v i on Q» S. 3. pH. L. o. C .2 as 9.. L. r. .2 O V. 3 Q5 WA. nxL IO v h‘ Au; PC 7 A o v ._ r» n. >U Lb L. .Fu 5... e 0. »a FL 1& L; L . .. a .4 a -h v.\1 H . Fl.“ ~M r N .K\ AV FC I s n \ «IV cu fa . .vfi . ,v I. t o. o n A. n u ‘.U putl‘ v e Y... Q» at n.» CL ‘.L .¢ 4. ‘1 u ‘ PC .Kh ah . a ~ .. 11L around the periphery of the nuclear membran ; with the moder- ately contracted figures in an intermediate category. Rever- all types .L C. sions were observed in normal late prophases an of contracted figures. Pro-metaphase. No abnormal figures were observed in this stage. Post-Prophase Post-prophase was classified according to the traditional metaphase, anaphase and telophase. Metaphase. Laggards, slightly scattered figures, severely scattered figures and reversions were found. Lag- ‘gards are chromosomes which sit apart from the rest of the chromosomes. In the slightly scattered figures, the chromo- sonms are grouped towards the center of the cell, but the a plate. In severely scattered icinetochores do not line up on ’d throughout the cell. Q fiigures the chromosomes are widely spre Truese are distinguished from the severely contracted figures ifil late prophase by a separation of the chromatids except at the kinetochore region. A few reversions of the severely scsittered figures were observed. Anaphase. Abnormalities were classified a laggarcs, es, spread figures, scattered figures and ges are strands of chromatic material between the rv 37- ' the Hm" , me 1gvv-A,.y w flr“,‘\,-v\t A», i 9‘ . J...» u ‘ U \ r~-. ,. a, «1‘» v. ll-_,.'\‘_‘ ‘ ‘. ~ ‘1‘} r .. ‘¢ -‘v.vh . b.. x g 511‘.“ ~14. _‘ v ‘k‘ s . ’ ‘9 (nr&.» 1. ye ch‘hg“ .r’- a" o-..‘ a H .J.‘ 3Y~ . . . u ..e ‘ "'r. “"22“ - d‘.c~"(“u- 3 ' o. “The? ‘jj Q "N "flu ‘JL. l .‘N... ‘ “I. '\ :eqerzr ‘ A .F “c. 15 separating chromosome groups. These may be due to sticki— ness of the matrix or to entanglement of chromosomes with trabants. Spread figures are typified by two groups of .chromosomes, in which the chromosomes are somewhat shortened and without a close orientation of kinetochores towards the poles. In the scattered figures, the chromosomes form only one grOUp, with sister chromatids side by side. Reversions of scattered figures were occasionally observed. Telqphase. Spread figures and bridges were the types of irregularities noted. The morphology is the same as in anaphase, with the exception of a telomorphic appearance. Interphase Pycnotic and binucleate cells were observed, but a quantitative analysis of them was not made.- B. Continuous Treatment General Observations A deviation from the normal condition was observed in the (development of a root system in the recovery material (Pliite I). After a recovery period of six days the material Whicfla had been treated with 200 ppm. terramycin ' HCl had no snide-roots and was shorter in length than the control. ThOEhe which had been in 100 ppm. had an occasional very short i r...‘ . -J v “'3 ‘HO Q ail-IA.“ rum V.nli-‘l"ll‘ I h' I' o Canci-7.,F"\ bolv- “"" NY." .y I L“".'“ I\‘ ’1‘. Y “a vbi “pHLA ~- .VUL4~7 a“ l ‘ r ‘F"'. -- -‘- M...» a.“ . .35..“ l' i‘ui'.. . h.‘ ,V- n~-- :nta , , “CUP: -.Y. . ~' - 4;. “L. ‘ ,A- a vaCx .. , \ fi,. m". u" v» u ..“A. tr; ’- lu» I y" Q‘Ct . ‘\- “J'¢IS T P IP-H‘ . v I... ryv lo side-root above the region of immersion, and none in the new growth region. They, also, were shorter than the controls. After a ten day recovery period, the 50 ppm. treated roots showed only an occasional small side-root above the immersion region and a few short side-roots from the new growth. Those from 25 ppm. treatment, had only an occasional side-root above the immersion region and had several side- roots in the region of new growth. The main roots were shorter than those of the controls. The 10 ppm. material deviated from the control only in a lack of side roots in the immersion region. Analysis of Mitotic Index In text Figure l, a comparision of the mitotic indexes for the various concentrations used is shown. Ten ppm. con- centration of the antibiotic was not graphed, as it follows tflxa same trend as 25 ppm. concentration. At 25 ppm., no sig- nifszant increase or decrease was realized in the mitotic index. The mitotic index of material treated with 50 ppm. Showned a very slight increase at the first hour and then de- CPeELsed gradrelly to zero at eight hours of treatment. At 100 jppm. treatment, the mitotic index dropped steadily for the .first four hours and then tapered off to zero at eight hOUJPS. The mitotic index of material treated with 200 ppm., dz“)pped abruptly to zero within two hours of treatment. III“ C u .Ennnm WN aneaadeua maosauadoo ameeu capes“: mo assessed» .a museum axes a: m .r m .H . rm “H1. 111.. 111. w . /// 1’ II.II. . .111 111 11.111 11.111/, /// // //.///// ..oo~ //. // room road T can rcom 1cos 1 com 311:0 ooo'or / sasowln so Hasunn .I I] l u 11 r N‘ . A ”J ‘ ML 1 til. I- . I c~ flit I 1 1 ' .. .. #1. C. . . n . ,C . . Hr. 1r. “ . .1. .L .C A. rc .C .0 11M .W ”a 1 1 .1. LL WA r . . . V1“ . . .i. O .1 . no A (L . 1. Cu ‘ . :C S on u... LVN W1. A A A 1 1 1 I .1 n . I. t I. a .1. 71* «J -\. n1,¢ Si «N . "I r. O MNV A as t Ye IAI‘ 7.“ £1»: bu .. I C. o» I o . V . \ no. 1. 1. Ll“ (L Yo CU . AL VA 1 r! .L\ «1v 5» . .v v.1; . . . #111— ..a C» {L . C Auk c he A V 1' w .. rC 1.. r u A C {1‘ .1 . .14“ + . a L :C . N u .i e as i . L1 3 . nu. . . a A 1.. f r s .. . .m. fi a... ,1. e . 1. 1. . - 1mm" 0 3r“ .9 :1. LU . u. 1. 1|. 1.5.14.1...»5“... .151! its: 41. . ‘ udflh‘d 1' l’: w L! 18 Analysis of Abnormalities as Compared to the Total Number of Mitotic Cells In this analysis of continuous treatment the concen- trations of 50 ppm. and 100 ppm. were used. The concentrations of 25 pnm. and 10 ppm. did not show enough aberrations and in 200 ppm. there were insufficient data. The absolute number of total prophase figures in 50 ppm. treated material, decreased with duration of treatment as is shown in text Figure 2a. The proportion of these figures which was abnormal increased with treatment. At the end of two hours, no pro-metaphases were observed. Early prophase” dissappeared rapidly and were no longer in evidence by the end of four hours. Also, the total number of mid-prophases decreased gradually during treatment. This stage showed re- versions as early as one hour, with the proportion of rever- Sions to normals increasing gradually during treatment, until at tdie end of six hours only a few normal figures were left. The preportion of late-prOphases to total-prophases iner’eased gradually during treatment, as is seen by a com- pariJson of text Figure 2a with text Figure 2b. The first hour? of treatment showed an increase in the number of normal latEr-prOphases, with a few contracted figures and reversions. At irwo hours the number of contracted figures, the degree of At con1::I:'~1rs.ction, and the number of reversions had increased. foul? hours severe contraction was in evidence and reversions anoaaeena usesnapsoo ".amm on Senna.“ ”38. no 333.3» .8 8&3 page . m o mum mrImmununuunntlttltttlllwr Mr, L a 1111tiuuu ”1,1llll -IHIIII.111I|1|.1H.I.IIII 1 \ \hi“ IIIIII111 I!!”Iw1l-1hu\1 . r co." . 1 com 3332.3” mo nee—uh 30a 11 1 I 1 II e33 awakened no 30.852 H.309 lllll 1 com 330 we hang Haven. 1 o3 8.9398 2.3 no «333.9; :5 chew: axon. T OOH h 08 $3995 each we soaueaug .oN earn axes — p p F — A I OCH .1 com 1 com 00¢ s'rmo OOO‘OI / sssonu so am nflfl‘l ’nErt "Ico‘.J pu Ur . "CA; r “a ,J \ Tere to annual .31 v. wtw 1r ‘ a.& l "’15 A. J r‘a‘.‘ Amid. s . were mainly of contracted figures. By six hours no normal figures were found. Text Figure 2c shows the analysis for post—prophase. There was an increase in absolute number the first hour, followed by a gradual decline to zero by the end of eight hours. As treatment continued, the proportion of abnormals to normals increased. These consisted mainly of slightly scattered metaphase figures in the first hour of treatment, which were replaced with severely scattered figures by four hours. Abnormalities in anaphase and te10phase were mainly of spread figures, with occasional bridges in both normal and spread figures. A few scattered figures in anaphase, and reversions thereof, were also noted. In.all three phases laggards were found during the whole course of treatment. The picture in 100 ppm. treated material was generally the smne as in 50 ppm, but the effect was more pronounced as is shown in tmxxt Figure 3. Again the absolute number of figures de- creas ed, but more rapidly than in 50 ppm. .Also the occurrence and proportion of abnormalities was egreater. All figures were abnormal after four hours of treertnwnt. Both early-prophase and pro-metaphase disap- Pearwed within two hours. Mid-prOphase had virtually disap- pearfisd by the end of four hours of treatment, with the majority beirng of the reverting type before that time. escapees aaoaagnoo “.399 ooa 3933A Haves Mo uoauoaadb .om enema axon. 333053” mo popes: Haven. .28 35qu «a tense 339 uaaoo M0 9355 H309 3.9398 33 no nowadays» .pn enema. axon. w OOH . CON 1 com [00+~ Sundown poem no 333.3» .om enema axon. 1 00.... .. cow 3m OOO'OI I sasomm so new 22 The preportion of late-prophase to total-prophase was greater than in 50 ppm. Contracted figures and moderately contracted figures were in evidence by the first hour of treatment, as well as several reversions of normal figures. By four hours, nearly all figures were ef reversions of moderately and severely contracted types; and by six hours only reversions of these contracted figures could be found. Post-prophase also showed the same general trend, but to a greater degree. Severely scattered metaphase figures were found earlier and in greater proportions. These severely scattered figures and reversions of them were the only type found in the later hours. Otherwise, the picture is the same as in 50 ppm. treatment. C. Fifteen Minute Short Time Treatment and Recovery Analysis of Mitotic Index A comparison of the mitotic indexes of material treated Witti 100 ppm. and 200 ppm. is shown in text Figure A. The mitotic index of material treated with 100 ppm. shows a ShaJfip increase at the end of 15 minutes of treatment. One- quaJPter to two hours recovery showed fluctuation. At three houI‘s recovery the mitotic index dropped, and then again went up lit four and five hours recovery. The 200 ppm. treated matfierial did not show as sharp an increase at the end of 15 wowuem bambooom use pneaumepe means“: 3 £33 039:: no .8323» .: enema thee .DHN 0 p run a m . N H 'H'N trN den 8.... 1l11.11 and 03 9325689. mo psalmlv _ 00H com com 00.: com 08 00b com com OOOH s'nso OOO‘OI / sasomm so am minutes treatment. There was a drop in the mitotic index at one-quarter hour recovery and a small increase at one-half .hour recovery with a steady decrease until four hours of :recoyery time was reached. The mitotic index then increased until it reached the normal state by six hours. Innalysis of Abnormalities as Compared to Total Number of Mitotic Cells The absolute number of total-prophase figures in material trwaated with 100 ppm. for 15 minutes, showed a fluctuation frwnn 15 minutes treatment to one hour recovery. The total numfloer of figures then decreased gradually until a low at three hours was reached, followed by an increase, as is shown in text Figure. 5a. The proportion of early-prophase was never very low, but reackrsd.the lowest frequency at three hours, and the highest at Ikyur and five hours. Pro-metaphase varied very little durirug the course of treatment and recovery. Mid-prophase also showed little prOportional variation, with the lowest Propoqftions at four and five hours. Reverting figures ap- pearedi in this stage, with the highest frequency at the end 0f trenatment and one-quarter hour recovery, followed by a gradual decline to the end of the recovery period. TFhere was no marked increase in the proportion of late- pr‘ophase to total-prophase, as is shown by a comparison of 25 text Figure 5b with text Figure 58. The majority of abnor- malities were of the slightly and moderately contracted types. Reversions were found in a small prOportion from 15 minutes treatment to three hours recovery and were of normal and contracted figures. Post-prOphase analysis is shown in text Figure 6. The total number of figures fluctuated during 15 minutes treat- ment and one-quarter hour recovery, than climbed gradually to a high at two hours of recovery. There was a drop at three hours recovery followed by a gradual rise to five hours of recovery. The prOportion of abnormal cells was low, reaching the highest proportion at two and three hours of recovery. Abnormalities in metaphase were of slightly scattered figures at two and three hours recovery. Spread figures were the main type of abnormality observed in ana- Phase and telophase. Some laggards were observed in all Post-prophase stages. The results obtained from treatment with 200 ppm. for 15 minutes, followed the same general trend as 100 ppm. treatment, as is shown in text Figures 7 and 8. With 200 ppm. the initial increase of total prophase at the end of tret‘itment was not as great as in 100 ppm. A decrease at one‘quarter hour recovery was followed by an increase at one-half hour recovery, and then a gradual decline to a low @3an bobooom .23 3253.8. «333: mu ".55 03 3939:» H308 go 533.3» .3 enema axon. .5 m a m N H a a m 0 pl . L ? bl b n ''''' H’Isldo’IIII-lolo\\\\ IIIIII \ \ x \ I OOH / / \ 3328£ mo spasm H33 I . .I. .II . I 1. r com 325 302.4 «o 9352 3.8. - I: In .I v aaaoo mo hope—an .398. i pecan—sous no unalml .. com 8: 8m r o8 omfisfim 33 no using .pm wear“ an: . 02. . c2 u cow. sum OOO'OI / sasona so am cornea rebound use newsman. savanna ma “.899 03.. gunman «com me “333."; .w enema anon. .93“ a m N .n $.mwo pl} J-I - L? b F» llll/l/ \ // \\// \ // \\ / \TOOH / \\ / L‘V pace» sous mo can . 3:5 258$ no Spas: H39.“ I. l I I I nice we ~35: H38 \y con r cow uoo: s'rmo ooo‘OI / sssomm so am 28 at four hours of recovery. At five and six hours recovery there was an increase in the total number of prophase stages. As in 100 ppm., early-prophase stages were always found; however, the decrease in number was more pronounced with the lowest point at four hours of recovery. Pro-metaphase stages were found at all points, except three hours recovery. Reverting figures were noted in this stage with the highest proportions at one and two hours of recovery, followed by a gradual decline to six hours recovery. Late prophase showed a greater decrease in proportion to total-prophase than in 100 ppm. There was a sharp in- crease in this stage at one-half hour recovery and then a gradual decline to six hours recovery. More contracted figures and more severe contraction was seen than in 100 ppm., and was most evident at two and three hours of recovery. Reversions of normal and contracted figures were found with the highest proportion at one to three hours recovery. A graph of post—prophase analysis for 200 ppm. is pre- serited in text Figure 8. Again, there is a similarity to 100 ppm. treatment. The total number of figures varied only slightly to two hours recovery, after which it dropped to a lOW'point by four hours, followed by a gradual in- crease to six hours. As in 100 ppm., there was always a 800d.proportion of normal figures present. The highest cornea" hobooom .23 383602. «255: ma Tana com guinea .5309 we nowadays» .ux. cauwwm anon. ovum w. m. N mi NP H- .W .W .4. VIIIHHIIIIHI II.” HIIUIhHIIIIILI/ A / // .H .IIILV / / \\ / / I l a l \V\ IIIh\ 3930.5 3.3 no dogmas» AK. 0.3m: anon. - r P p r F Till I I I I'll: IIIIIILIIIIHI/ \1\ I I l / I II I II. I .Il\\\| II I I IIIII \ oqoamuobom no pong H33. I . III I 'I 4 azoo Hen—noun: no 93852 Haven. lllll pace—ado?“ v a o o no .309 S o a papa H no can.“ 00H com com 23 com com OOH com com 3mg ooo‘or / 3530mm so Harm venom Eobooom and 325689 nephew: m." Tana com ouflngm «mom no nouusfiue> .w 0.2%: anon. .3: m s m N H m x. w o p’ L p P b b r b .III'IIIIIIIII'IIII‘vl'III/ ..| / \ / a / \‘I‘IIIIII/ \ I 1» III / \ < s 1 \l/\ j aeoapwaa we deal: «:8 Hafioafi no .HopaaH H33 IIIII nHHoo mo .Hopapu H38 OOH com com 00.: s'ruo OOO‘OI / sssomm so mama 31 proportion of abnormalities was realized at one-quarter hour recovery with a gradual decline in frequency thereafter. .Abnormal figures were the same as in 100 ppm. treatment, with.the exception of the aspearance of more severely scat- tnared metaphase figures at one and two hours recovery, and their maintenance in subsequent recovery hours. D. Thirty Minute Short Time Treatment and Recovery Analysis of Mitotic Index A comparison of the mitotic indexes of roots treated with 600 ppm. and 800 ppm. concentration for 30 minutes, followed by a recovery period of eight hours is given in text Figure 9. Six hundred ppm. treatment showed an increase at the end of the 30 minute treatment period and a decline to one hour recovery. At two hours of recovery there was a slight increase, folfilowed by a gradual decrease to a low at six hours recovery. ‘At :seven hours recovery there was an increase, again followed by a slight decrease at eight hours of recovery. The 800 ppm. treatment did not show an increase at the end of treatment, and exhibited a gradual decrease, with hourly fluctuation Until a low was reached at seven and eight hours recovery. cornea .3233 can escapees moves“: on . 0 .uoHaH 0303: .3 333.3» a charm an a N H m m m- a. o W m +~ 'M k p if p F . .E a N. . L . r r 2: - com I\/ II II. \/ I I / // \ \ x/ T can / \ / / \ / \\z /\ / / \ \ / / /\ \ / W 03 / / . / . 1 com / . x \ \. s r .. x n o8 / / . / / 8s .93 com IIIIII com .55 000 com V r 22583 .3 Elm] 30H snap ooo‘OI I 3350th so am 1M ,. dfiiguififlhl [1w "Li. I Ju rli IL. 33 Analysis of Abnormalities as Compared to the Total Number of Mitotic Cells A graphic analysis of total-prophase figures for 600 ppm. treatment is presented in text Figure 10a. The number of figures increased to a high at the end of the treatment period, followed by a sharp drop to one hour recovery. There was an increase at two hours recovery followed by a gradual decline to six hours, another small increase at seven hours and a decline at eight hours recovery. As the number of total figures drOpped the proportion of abnormal figures increased. Early prophases were always noted; however, the fre- quency showed a sharp drop‘oyone hour recovery and was very low until six hours recovery, at which time there was a gradual increase. Pro-metaphases disappeared at two hours recovery and did not reappear until seven hours recovery time. The frequency of mid-prOphases realized a sharp drop within one hour of recovery and was maintained only in small numbers thereafter. The proportion of reversions, in this stage, reached a high point at one-half hour recovery and remained fairly stable until six hours recovery when a slight increase was realized. There was a marked increase in the proportion of late— prophase to total prOphase during the duration of the treatment, as is shown by a comparison of text Figure 10a with text text Figure le. By one hour recovery the proportion of late-prophase had showed a sharp increase and continued to rise until five hours recovery, when it started to de- crease slowly. Abnormalities were of contracted figures and reversions, with the most severely contracted figures becoming more predominant with duration of time. Text Figure 11 shows the analysis of post-prophase for 600 ppm. treatment. There was a gradual decrease of total figures to four hours recovery, after which the total number remained fairly constant to the end of the recovery period. As in prophase, the proportion of abnormal figures increased with the decrease in number. Abnormalities in metaphase consisted of slightly scattered figures until one hour re— covery, after which they were replaced gradually by severely scattered figures. A few reversions were observed in the later recovery hours. Anaphase irregularities were of spread figures and scattered figures. Bridges were evident and were due to matrix stickiness or entangled trabants. Spread figures and bridges were found in telophase. As in the other treatments, laggards were seen in all post-prophase stages. Total prephase analysis for 800 ppm. follows the same trend as 600 ppm. treatment, as is shown in text Figure 12. There was a gradual fluctuating decrease in the total number of pronhases, reaching a low at seven and eight hours recovery. .eoauom humbooom can engage conga: on “:53 com 839on H38 mo 833.3» 63 8:th tee wflOdeOFGN MO 809% 309 uHHoo Hen—Hound Mo HOE—8h Haven. lllll «HHS no .Honasn H308 «seasons 33 no deflate» .SH 253m puma 00H CON oom 00: co: . 8m ‘- -oom gangsta no vain r 00H . :oom _. con s'rmo ooo'ot / sasomm so am weapon bobooom use unofisona mousse: on Team 000 caveman amok no soapsang anon dances. mo pong deuce «HHS mo .Hopasm H33 .HH 8%: tea pqosasoaa .«o .93!de 00H com com 09: s'rmo OOO'OI / sssom 10 am 37 The frequency of early-prOphase figures dropped quickly at the end of treatment and early hours of recovery, reach- ing nearly zero at four and five hours recovery. They also followed the same trend as was seen in 600 ppm. treat- ment. From one hour recovery to five hours recovery, late- prophase was the main class of prophase figure. After five hours the proportion of late prOphase began to decrease gradually. Abnormalities were of the same type and followed the sane trend as was noted in 600 p‘m. treatment. Post-prophase effects for 800 ppm. are presented in text Figure 13. Absolute numbers, prOportion of abnormalities, and types of abnormalities followed the same general pattern as was seen in 60C ppm. treatment. E. Summary of Observations 1. Continuous treatments reduced the number of side roots in recovery material; the degree depending on the con~ centration. 2. The mitotic index showed a decrease in all treatments. depending on the concentration used and length of treat— ment. 3. The absolute numbers of total prophase figures decreased in all cases, varying with concentration and length of treatment used. The greatest proportional decrease was deflom bebooom eds escapees.“ mouse“: on “.amn cow 8938a H33 mo 3:33» .HaH 933» use Ham 5 a. L is: m a La m .H w w a we. N.’I1PIII.U.II.I.’ .\\.. I I I! I. I . \ \ 'I // o a r \x\ 2: \ c I a I\o\\ \ T cow / /\ I com ueoamaobom no sunfish H.309 .l . .ll . v cod «HHS Hnagfl mo Spade H38. lllll /\ - 8m «38 no .HopaHfi H38 - com 29303 33 no 333%» £2 BHaHh anon. WI“. r 2: r com I com pawn—packs Mo dun.“ f .00.: s'ruo OOO‘OI I snow! 10 seems dogma. bassoon use gaseous «eased: on «.55 80 3.3%.:— puom no nowadays» .ma chum: axon. 2338a no can anon Hgodpd mo hops—fin H309 IIIII .28 no “252 H309 I OOH /. 8N - com '00.: srmo OOO‘OI / sssomn so new \TI to in early prephasc and pro-metaphase. Mid-prOphase also showed a preportional decrease in figures and a rise in reversions. Late-prophase figures showed a proportional increase to total-prOphase figures, with duration of treatment or recovery. Abnormalities were of contracted figures and reversions, with degree and preportion of contraction and the proportion of reversions increasing with time. A correlation of abnormalities with concentration and length of treatment was observed. The absolute numbers of post-prophase figures decreased in all treatments according to concentration and length of treatment used. The proportion of abnormal figures increased as the total number of figures decreased. Ab- normalities in metaphase consisted of scattered figures, with degree and proportion dependent upon time and the mode of treatment. Anaphase abnormalities were mainly of scattered and spread figures. The abnormalities ob- served in telophase were mainly spread figures. LlSC*:SION Normal Cycle In order to understand what has gone wrong in an ef- fected mitotic cycle, it is first necessary to know the normal mitotic cycle. For the sake of convenience, the normal process of mitosis has been classified into several stages or phases. However, it must always be kept in mind, that these terms are nothing but names given to certain stages in a constantly moving and changing system, which results in the formation of two nuclei and cells from one nucleus and cell. During the process of mitosis, the chromosomes go through observable morphological changes and movements. Interphase is diffuse and granular in appearance. At pro- phase, definite Spiralled threads can be seen. At the be- ginning of prophase the kinetochores are arranged around th old telophase pole and form a hollow Sphere. As this stage proceeds, the chromosomes shorten and become thicker. Simul- taneously, the kinetochores move towards the equator of the nucleus. Usually before they reach this point, however, the chromosomes move to the center of the cell. They are then in the usual pro-metaphase arrangement of an irregularly shaped clump. This centerward movement appears to be associated with disruption of the nuclear menbrane. While in pro-metaphase, more contraction and un- winding of relational coils is realized. It is possible that the spindle, which was probably formed sometime during prophase, may become functional and have some influence along with the kinetochores on the movement of the chromo- somes to the periphery of the equatorial plate. This movement results in the formation of the metaphase plate. While at the metaphase plate, the kinetochores cleave. This is fol- lowed by a mutual repulsion of sister kinetochores. The forces of the spindle then come into Operation and the re- sulting configuration is called anaphase. The sister chroma- tids move apart, with the kinetochore leading and the arms trailing behind. Telephase is the name given to the stage when the chromosomes reach the poles and begin the morpho- logical transformation towards interphase. During late ana- phase and in telophase, cytokinesis occurs and the nuclei are separated into two different cells. This involves, among other things, the loss of matrix and some relaxing of the chromonema coils. This account of mitosis is concerned only with those things which can be observed. Schrader (1953) has reviewed many of the theories of mitotic movements. Most of them are highly theoretical and can not even be proved with present techniques. Therefore, not much would be gained from a dis- cussion of them in a study of this sort. The basic theory, followed in this research, was the one proposed by Wilson, Hawthorne, and Tsou (1951). It states that there are at least two components of mitotic movement; a dipolar cyto- plasmic orientation, and a nuclear component inherent in the chromosome or its kinetochore. A simpler theory, such as this is, is much more readily and easily used as a basic working hypothesis. The Effects of Terramycin-HCl The primary cytological effects of terramycin-H01 are on prophase and "antephase." Therefore, this antibiotic may be classified as a "prephase-poison" or better still, a "pre- prophase—poison." That movement of chromosomes from prephase to subsequent stages is stalled or prevented, is shown by the early increase in the proportion of late-prophases to total number of dividing cells. As time and concentration is increased, this effect is even more noticeable, as indicated by the graphs in text Figures 2, 3, S, 7, 10, and 12. Chromosomes which are in early-prephase and "late- antephase" at the time of treatment, probably never proceed farther than late-prophase. This may well be due to an in- hibitive action of terramycin-HCl on the center and kinetochore ML movements. The movement of the chromosomes, which is realized, may be caused by forces of contraction and torsion within the chromosomes themselves. That they are incapable of forming a pro-metaphase clrmp, is clearly shown by the early disap- pearance of this stage. The morphological changes are not interfered with, except for a possible slowing action. here- fore, he continued morphological changes along with the in- hibition of movement, can be considered to account for severely contracted and reverting chromosomes in late-prephase. The degree of contraction at the time of reversion, may be related to the advancement of prOphase at the time of treatment. This conclusion is drawn on the basis that the number of contracted figures and degree of contraction increases with time and also shows a correlation with concentration and length of treatment period. "Antephase," according to Bullough (1952, p. 1L5), "is undoubtedly the most sensitive phase in the entire course of a division." The action of terramycin-HGl on$antephasefl is a preventative one. This is clearly illustrated by the reduction and subsequent disappearance of early- prophases in division, during the early hours of treatment in continuous treatment, and in recovery following short time treatments. The failure of new cells to enter into division accounts for the reduction in the mitotic indices as is shown in text Figures 1, h and 9. That recovery from such inhibition LLS may occur is shown by the later return of early-prophase interesting to note, that in the U) during recovery. It i short time treatment of 15 minutes with concentrations of 100 and 200 ppm., the increase in number of early-prophases and the mitotic index is realized within three to four hours, which is considered to be the normal period of time a cell requires to go through mitosis. The cytological effects of this antibiotic on post- prophase are not as pronounced as 6n prophase. The slightly scattered metaphases may possibly be considered as normal and due to a slowing action on the movement of chromosomes fromthe metaphase plate, thus making it possible to see them. Or, they may be considered to be the result of a partial impairment of the spindle. Likewise, spread anaphases and telophases may be considered as a normal condition, made ob- servable by slow movement. On the other hand, a partial im- pairment of the spindle and kinetochore repulsion could ac- count for the spread, ball-shaped groupings. Severely scattered metaphases probably come from chromo- somes which were in late-prophase at the time of treatment. This is indicated by their arrangement within the cell and their later appearance in treatment and recovery. This type of cell rarely enters into anaphase, and usually reverts l situ. Theoretically, this should lead to four-stranded chromosomes. It may be assumed that these cells, as well as as other cells undergoing mitosis at time of treatment, are incapable of redividiny. This conclusion is made on the basis that polytene or iour-stranded chromosomes have never been observed in recovery material. The lack of side-roots in the immersion region of roots subjected to continuous treatment, may be considered as further evidence. However, the exact diSposition of these cells is not known and can only be speculatedmat the present time. The bridges and fragments, which were observed in ana- phase and telophase, were of the type reported and pictured by Tanaka and Sate (1952), and Levan and Tjio (1951). A close examination of an equal number of control slides, showed that there was no significant increase in occurrence or kind. Therefore, it does not seem possible to conclude that such bridges and fragments are the result of treatment. It is more likely that they are caused by an entanglement of chromosomes with trabants. Also, there was no evidence of ring fragments or translocations which are cormnonly associated With radiomimetic chemicals such as the mustard gases (Auerbach, 19119) . Such fragmentation as has been noted does not seem to be of a degree or kind which would warrant classii iCEtiO-l 01 this antibiotic as a mutagenic agent. ll :1 i I , ,4; ‘I fly Fl .Lln. i‘l'uW r, F» .EIHDJH , . x a Eh‘ M7 Terramycin-HCl as Compared to Colchicine The cytological effects of terramycin-hCl are quite different from those of colchicine. Colchicine has a stal- .ling effect on pro-metaphase and metaphase, which ultinately leads to unipolar anaphases and telOphases causing poly- ;flxsidy. This early, complete disruption of spindle function is; not caused by terramycin-HCl. Colchicine stalls anaphase and.prevents cytokinesis, forming binucleate cells. Terramycin- IKIL exhibits only a very slight effect of this type, as is ixuiicated by the appearance of only a few binucleate cells. The; severely scattered metaphases from colchicine treatment £3) through anaphase and telophase, and multinucleate cells are ;formed. bellowing terramycin—HCl treatment, severely scatrtered metaphases revert in sitg and do not lead to multi- nuclmeate cells. In contrast to terramycin-H01, colchicine has la stimulatory effect on "antephase," rather than an in- hibilzing one. Further division of colchicine-effected mitcrtic cells has been observed in recovery. There is no eviC€ance that mitotic cells effected by terramycin-HCl re- GlViJie in recovery material. Cytological and Antibiotic Activity of Antibiotics Tflie cytological effects of terramycin-HCl have followed tum; SEUne pattern as shown by all the other antibiotics which us have been st. died by the cytology arm)? at Iuichiban State College. These effects in review are: a high percentage of reverting; prophases; a prevention of the movement of prophase chromosomes into post-prOphas-e and. a pile-Up of late-prephascs; . , 1 ' . "II _J'J-’.j .$- -\- :1 1,‘. ‘. ~ “’. (V. : " ‘ ': ’ e. L ..-l-ll-.-x..e.a.1!_, Rifth JJ '.~ ‘.«_I‘.ii.0«:_’e resulting, .Ln 3 10578 Lilla, {In of the mitotic index; the occurrence of several scattcre metaphases; and failure to find four-stranded chromosomes in recovery material. This would seem to indicate that there may be a direct correlation between antibiotic and cytologi- cal effects. It is possible that the action of an antibiotic on sensitive bacteria is due to an inhibiting effect on the division of the bacteria. Such failure of reproduction would lead to their death. The only difference, which has been found through the CytOlogical study of the action of antibiotics, is ii the concentration level at which effects and toxicity are realized. In Connection with this relationship, it should be noted that Actidione shows the most toxicity as an antibiotic and anti- mitOtic. It is the on y antibiotic, thus far studied, which is 1500 toxic for use in medical treatments. The concentrations Of Actidione, which show cytological effects and toxicity: are much lower firth for the medically accepted antibiotics. In this connection, Dr. G. B. 'wilson “as noticed a direct C“wrelation between antibiotic and cytological toxicity ra). " o o o figes’ insofar as they had been studied. If these Observations 1; *n—mu should orove to be valid, it would seem to ‘ toxic effects of new antibiotics could preliminary cytological screening tes ’49 indicate that be subjected t. i any.“ i» :_“u_.m3’ SUMRARY l. The Pisun test was used to determine the cytological effects of terramycin-HCI. Root tips of Pisum sativum were subjected to eight hours continuous treatment with concentrations ranging from 10 ppm to 200 ppm; and for short time treatments for 15 minutes with 100 ppm and 200 ppm and for 30 minutes with 600 ppm and 800 ppm, followed by a recovery period in nutrient solution. 2. The mitotic index decreased according to time and concen- tration. Early prephases disappeared and there was a pile-up at late prophase. Scattered and Spread figures were observed in post prophase. Continuous eight hour treatment inhibited side-root development. 3. I (1' was concluded that: terramycin-H01 prevents "ante- phasé'from entering into mitosis; may have a slight destructive effect on the Spindle; interferes with cen- ter and kinetochore movement in prephase; prevents cells in mitosis at the time of treatment from redividing. No mutagenic action is indicated. ho The cytological effects of terramycin-H01 and colchicine are different in several re:pects: a) the former has an inhibiting effect on "antephase," whereas, the latter i.‘_‘._--s__-- u *7 A 51 is stimulating; b) the former does not seriously effect post-prophase, whereas, the latter does; 0) the former inhibits effected cells from dividing Y“ again, whereas, the latter does not. The cytological effects of all antibiotics, thus fa studied, follow the same pattern. There is some indi- cation of a correlation between cytological effects and the action of antibiotics. BIBLIOGRAPHY Auerbach, C. 19h9. Chemical mutagenesis. Biol. Rev. 2Q: 353’3910 Bowen, C. C. and G. B. Wilson. 195h. A comparison of the effects of several antimitotic agents. Jour. Hered. 45:2-9. Branion, H. D. and D. C. Hill. 1951. The comparative effect of antibiotics on growth of poults. Poultry Sci. 29; 793'80 Bullough, W. S. 1952. The energy relations of mitotic activity. Biol. Rev. 27:133-168. D‘Amato, F. 19h8. Ricerche sull' attivita citologica di ‘ alcuni composti organici con particolare riguardo alla colchicine-mitosi e agli effetti tossici. Caryolgia ;: u9'780 . l9h9. Preprophase inhibition of mitosis in root meristems. Caryologiall:lO9-121. andgm. G. Avanzi. 19u9 a. Studio comparato dill' attivita citologica di alcune essenze. Caryologia 1: 175-1930 . 19h9 b. Prime contribute alla conoscenze dell' attivita citologica e fisiologica delle sostanze di crescita sul testo Allium cepa. Caryologia 2:31-5h. Della Bella, D. and C. Gabellini. 1951. Action of principal antibiotics, alone or in combination, on root development of Lupinus alba. Boll. soc. ital. biol. sper. 21: l90h-7. (Chemical Abstracts he) Hawthorne, Mary E, and G. B. Wilson. 1952. The cytological effects of the antibiotic acti-dione. Cytologia 21:71-85. Hobby, Gladys L. et a1. 1950. Antimicrobial action of terra- mycin in vitro and in vivo. Proc. Soc. Exptl. Biol. and Med. 11:503-110 EA...“ 53 Huskins, C. L. and L. N. Steinitz. l9h?. The nucleus in differentiation and development. Jour. hercd. ia:oo—77. Hyypio, P. A. 195a. The effects of colchicine upon the mechanism of mitosis (unpublished Ph. D. thesis, hichigan State College). Levan, A. 1938. The effect of colchicine on root mitoses in Allium. Hereditas 2g:h71-h86. and J. H. Tjio. 1951. Penicillin in the Allium test. Hereditas. 21:306-32h. fk Nickell, L. G. 1953. Antibiotics in the growth of plants. Antibiotics and Chemotherapy l}hh9-h59. Peterson and Strong. 1953. General biochemistry. Prentice Hall. Regna, P. P. and I. A. Solomons. 1950. The chemical and W physical properties of terramycin. Ann. N. Y. Acad. A01 0 52:229'370 Reynolds, w. M., E. H. Weber and H. o. Luther. 1951. Effects of terramycin on chick growth. Antibiotics and Chemo- therapy $3267‘73' Sieburth, J. No N. et a1. 1951. Effects of antibiotics on intestinal microflora and on growth of turkeys and pigs. Proc. Soc. Exptl. Biol. Med. l§:15-18. Tanaka, N. and S. Sets. 1952. Effects of Streptomycin on the mitotic cells of Tradescantia paludosa. Cytologia ll: 12h-133. Venture, M. M. 1952. Action of antibiotics on green plants I Inhibitory effect of terramycin on growth of rootlets of germinating Egg mays. Escola agron. Ceara, Fortaleza, Ceara, Brazil. Pub. tec no. 3B. o pp. (Chemical Abstracts fig). Welch, H. 1950. Absorption, excretion, and distribution of terramycin. Ann. N. Y. Acad. Sci. 51:263-265. Wilson, G. B, 1950. Cytological effects of some antibiotics. Jour. Hered. 41:226‘231° 51+ and C. C. Bowen. 1951. Cytological effects of some more antibiotics. Jour. Herod. I2:251-255 h. Hawthorne and T. h. Tsou. 1951. Spontaneous ’ 1V1. . o u 0 " _ . " nduced variations in mit051s. dour. nered. 42: f: r di 3-1 f‘\ I1 C‘ a 1 5 ‘11!!! figure 1 PigmeZ hoot (evelOpment of seeclings, after treatment with terramycin-H01 and six da s of "paper towel" recovery. A.- Treated with 200 ppm for eight hours. B — Treated with 1C0 ppm for eight hours. C - Control. Root development of seedlings, after treatment with terranycin-HCl and ten days of "paper towel" recovery. Treated with 50 pom for eight hours. U3 21> I - Treated with 25 ppm for eight hours. 0 I Treated with 10 ppm for eight hours. C4 I Control. ‘5 ~ v. n J“ l—eov. Lac \o .r\) ‘11 10 11 ’3 C. \ fl FLA"; is l L “V‘sLI‘w‘ 1. q ~ 111bmi'f.)il£'.f;6:5 7- ‘] ‘ PAW ‘ harly prophases Late prophase Contracted late proghase, promotaphase and.early prOpLass l Contracted late prophase Prometaphase Prometaphase with lagging chromosome Pronetaphase and early anaphase Early metaphase and prometaphase Early and late prophase; early metaphase metaphases Early prephase and anaphase Telophase with lagging chromosome division of the scale represents ten microns. nr-fi. I ‘ _ ‘ . ’V'JIJ éiwsa PLATE Ill LPILCTS oi ‘1‘s1-.:;m;..1c1;4-;ic1 us new; swivm Concentrations and type of treatment are indicated at the \L) 15 16 bottom of this page. Reverting mid-prophase; (1), l/h hour recovery Reverting late and mid prophaSes; (2), 1/2 hour recovery Reverting slightly contracted late prephase; (2) 9 L hour recovery heverting moderately contracted late prophase; (2), 7 hour recovery Moderately contracted late prephase; (3), L hour recovery Severely contracted late prophase; (2), 7 hour recovery Reverting severely contracted late prophase; (2), 5 hour recovery Slightly scattered metaphase; (1), l/h hour recovery Severely scattered metaphase; (1), 1/2 hour recovery Reverting severely scattered metaphase; (3), h hour recovery Anaphase With bridée due to entanglement of trabants; (2), 3 hour recovery Anaphase with lagging chromosomes due to entanglement of trabants; (1 , l/h hour recovery Spread anaphase; (2), L hour recovery Spread anaphase; (l), 1 hour recovery Spread telophase; (3), 3 hour recovery Teloptase with bridges; (3) , 1 hour recovery (1) Mitoses from 200 ppm treatment for 15 minutes (2) Mitoses from 600 ppm treatment for 30 minutes (3) Mitoses from 800 ppm treatment for 30 minutes Each division of the scale represents ten microns. 57 Arum-”“3““: F-C’ a.