'a,.: ■ y Some Methol •'>■ •»P4|'»b W4 TABLE OF CONTENTS. I Introduction: the nature of the problem; some advan- tages of toads as experimental animals 1 II Materials. Methods of collecting and keeping toads 2 A. Scarcity of animals during the autumn 3 B. Florida toads 3 C. Collecting during the spring near a breeding pond. . 4 D. The outdoor cage without water supply 5 E. The flower-pot type of cage 6 F. Use of the large aquarium 6 0. The toad pen in the greenhouse ; methods of feeding. . 6 III The water content of the toad 8 IV The absorptive force exerted by the toad's skin JO A. Previous work on the frog's skin 10 B. Description of the osmometers used 11 C. Methods of recording results 13 D# Experiments with toad's skin 13 E* Conclusions 17 V Respiration apparatus 20 A. Apparatus for obtaining pure air 20 B. Apparatus for determining CO 2 by titration 22 C. The respiratory chamber. 23 D. Experiments with the titration apparatus 25 E. A modified biometer for animals as. large. as toads . 26 F. Small respiratory chamber for insects, etc 31 VI Summary 33 VII G-raphs, plates and tables 35 VIII Bibliography A 9 ^ X'l . '.;J . . , . . j^f.7 i'4 . I • : ‘iC 1 ■■ f ‘ I I ■; 1 --V I V I Introduction Hibernation has been studied by various workers, and much attention has been given to hibernating mammals in the hope of finding some internal physiological explanation for the phe- nomenon, More recently emphasis has been laid on the condition as observed in insect pests and its bearing upon methods for their control. A great many phases of the subject have not yet been studied, and especially is this true of certain ecological relations to external environment as opposed to the internal physiological side of the problem. It is my purpose to study the phenomenon of hibernation from an ecological point of view . with the hope of throwing new light on some of the economic or purely scientific problems involved, especially as regards this condition in cold blooded animals. With this in vie?/ it seemed best at the outset to choose an animal for experimentation large enough so that various d-eterminations such as body temperature, blood analyses , etc . could be made if desirable. The common toad,Bufo americanus Le Conte, seemed to fit these requirements very well, being a land animal during the greater part of its Ilf e-history, cold blooded, accustomed to pass the winter in a state of hibernation,and large t *.woJ,,|jeNr a?f; '7^\)^|if.*‘X4cf.' { ,.*‘ • • y — lf\ >jrtvf ,- ‘•Ivl'iJ flyl'". ’'iV , •' * yc;‘ >r Vi B ,*I M " f) Li 1 * i»ol :fr®c o v-^,;ifo ■1 C-W«. ;l Cfh7)> >1 vXri I‘ut#i^/> fT/' ' tS: ,£^''^'t *»» 4>Jt< i?iwr «> 4i5Wi.-j ^ ■Ail x3iV}., m., ■/ 'X . ■ . . • ■■' • i. ^ ^ ■ 1/,^ i •- o^tt • t.* rj^btocf *»X;/o^- fp? _*?*** '■ ■ Vk^k ' ’■ ' /•» ‘■^*’ . ->‘V-'. ;•/ ^Vv .. ‘ x‘:‘!? v.-x« ' ,’• ■.■ f"kr' p i^f■^ ■^■.‘*. *'. -t 2PtT?”7 I 'P* , AfcjVVk; , ; ■A.-riJ^cvS^ „■' / .V ' t: ' •>.: j?rv<" - 2 - enougji to admit of easy determinations of body temperatures, blood analyses, etc. Accordingly, the first part of the paper is given over to a study of various methods of keeping toads in captivity for experimental purposes. In the course of this study, the importance of the water content of the animals became evident (Part 3) and led to some preliminary investigation as to the regulatory mechanism involved, the results of v/hich are dis- cussed in Part 4. The remainder of the paper is occupied with descriptions of apparatus for studying the carbon dioxide output of the animals together with some preliminary experiments v/ith these. The data so far gathered is not sufficient to warrant the drawing of any new conclusions regarding the metabolism of hibernating animals, but such data will be gathered later when the apparatus and methods here described have been used to a greater extent. II. Materials In the fall of 1919, I began my work on toads by attempting to collect specimens in the vicinity of Urbana,, 111. At that season of the year I foimd tliem to be very scarce, and after two or three v/eeks of persistent searching I had procured only tv/o or three toads and a few snakes. Later observations indicated that the toads must have been in the vicinity, but a.t that season they were widely scattered and well hidden so that there were no definite places where they could be collected in '■t; , -S!- ■m. ' , i^<>0 In vh 4« 'i > S i&xn A|WfM9 I ’ '' ".i V ■• V* ■ •' f I ' f i)'*? ^rfi .cKfT 6o|lVj? ^ ffi i}!)^i ’i awyl*;;$.V 'to 5^ » f lKt 'to- a'> icl ■ . 'j£o JouV vit’o • t>of!^Snoc:|t4 V a , ft *J.:ii3^' t.rir: v*i^.JL llax;i ^W\)r c4 ^oX ; fi X«^S: Jf -nio ti*:j* tct ’h5‘.C'jT5!rr t^JT .' ^viov/ij: oa rw be-Xfyot.o. ' jrff 'lo 7o|»r4X^’io'i A' {%Jt 'i5#ai^ ■ • r- i* i *,• t^ r.r ‘ f -V * 4 fT /f t i if 11^ rib'll* CiJ* uJiiHSJtVJ* c r^4 4 ^ jV o, 4 taa ^‘iV’xt>fi.+ .-j til tw aii. .oCtU^.* -’? Ik5 » * ‘f i^' I O •»] .,.m'‘, -i . .' . ^ ' r%*. r^c ^ , 9X&i ’^#XX/i‘: ti? ^li ^ .. ’ ■* ■' '‘, ,t < . .A i. .ftAiXxX g ';XXii;oXv oftt. fft f\{it04^Xt>^4ili XooXiOfr ti? ;}«■ •lu:' ,^i?r»4Jocv vi^or o ■«<»-*? 6* ^ • , ■• . ^ - -^' ■ ‘•■^* ' ' '^ i ■' .w^^r/i %ot B t^ns pifeisc^ rto ‘:rV'J ^ ■ ■" *!!S’ . ' ' ■ . • — ■ ' -.oJ , r >4t. Sinrf .'^SicrX&iv',; n^j rtl *ioai(. ^oi/sa otaos X 7^ /«£^-it:rssa^^ •j«' -*>1:42 5, k -t ^ J .-' ri’ ! .. .f «' 4 -3- numbers. Accordingly a dozen Florida toads were procured from tlie Chicago Biological Supply House. They were shipped in a screen-sided wooden box half full of oak leaves, presimably from their native habitat. The toads and leaves were transferred to a wire screen cage ( 14 in. by 10 in. by 8 in.) and placed in the Vivariimi green-house. The cage was placed on strips of v/ood above a water table and a continuous stream of water was kept flowing under it to keep the air moist. The temperature of the room was about 68° F. About the middle of December, upon examining the cage, I found one toad dead, and I then observed that while the water ran under the cage continually, the oak leaves immediately surround- ing the toads were quite dry and brittle. I lowered one end of the cage into the water and about an hour later found all the toads congregated in that end and several of them swelled up to a considerable size because of the amount of water which they had imbibed. This experience served to emphasize the importance of a readily available water supply for keeping toads in a healthful condition, and to meet this need better, I removed the toads to a new location. I returned the animals to the screen-sided box and placed this, screen side down, on top of a galvanized iron tank of water. The rest of the tank was covered by a board. By this means I hoped to obtain a suffi- cient degree of saturation of the air in the box to keep the toads in a healthful condition. The whole enclosure was placed on a brine coil in a cold room (temperature about 11° C. ) in jsrsat.:: r ? .n f . r <«'S*rt *,\ (» 'r' .'^ *‘;I- "^li c,^<^oirtt' ^ 'nv-.-^ : O t/ 'I V- tjV* ;J i\'< t« t csYfc^r. -riVUii * r-, %\f^. t ^ ^ii # s?> ' . J« *’■' ' t ' lAir *lfTir^X «£H‘'4»'’ il^' --“ " 'JB ■ » - ."VT »,'i’i?*''t' i.4.> nl i*j * az \.rt^ * V .i».‘ *fri Mj ii}it> 1}‘. . v:”^ •*■ ■* ^ A-'orft uC'C'y It '• - »■>'. x»iii‘*-i'" . '»ijjm>ii-.r.-^<3*;7 s > ■ ■ . - -- ^ ■ ‘ ,, , ♦ * V. ■ '. ■ ^ n ■i >t' ' i.oX3 v^v '^'* r ifji <3v/il;*4<-i) «‘*'4'.jf ■ '^ • • ' » ’ * ■■•pr^'* : • 'f. ^ ii> f,<. ■•ja*. ^ ‘.'ifri-^til TJLjr 0N> ir |[’ t\r ,u ,* t tV.'uT? ^ / ' * ^ i^di V ^ ^ * frij. " r>:** a .tr 1 t»n^' II-. ,*it i'i*o^ ^;iol f f' » ii J i-, ur 1 ‘V J ^ - t . ; ' ■ uio , \ ' Oi iJO J ■* ''-A'' V*.. .* *u\'i tuo- : li.i^ .r ] iT-vyi^m tfc» ib? 'f v'i- .'i v', - tra X . Dr ¥-^ Hi- v ^ V>1 .<;» ?ic JiiUtJca oiU 3v J/« 0 Xi ] .•>v' r 1 9'jpii* &-:’i? w l/o7,i$a 'J,52^z t<^ Tifid^ dSlui/x^iB *iXiJ&4JJt *43 j ^ wiAC; I Won d»i'’ Ilf’ :Oi' i>lXui3iii& |)(S(fit^,^|i'X^ ’- . '4ff'*tr f 6^-'' . t Cfucf r.o 5^i5 /ji«./?*i« ffi&Ui Yijiil 6off^r vXiQk 'Ik • * . '* ■ ‘ ♦ ■ ‘ • ' ’ • *1^’ r'v •#* t‘* ■‘■.ccfj /|.J ‘tja -tW^ * 1^1 ^ ■i ■ 'X * ! '- ' J ^ * ' 1 K .-*' O'? 0 X 4 twn' B'j;'r . ■ ^- \nu9h‘ . iti; ', siif/f ■ , ';’too^S 772:^^»r-f4 ‘- ’ I ■-■ • . •' • i-q 1\:* , ■•. X: ' '' ■ ' ' ?* 'I:,?fw: ,VWD4j*^ cm s^ym ‘ ‘ , - ’ . * ^ i* ■ ' ' "’S ■' ■ ri} <>*•„■ '^/x;-. r,:fr^lKi ■ |Jbi»Tr ^1.. .vVi^cio ,Ar > ' i V c(^*.|| |^‘ • ^fJr'iriftuTl ri:o ftf -n; #)pnjiJ\^..^:' fffiii *yi“. 0 J ^ V i4 Vf4i , • ' ^ ft ‘ ■ .. ' -. iiiBR « # A ^ i*>)'flr:^i/» l/sToO a J:/c.it iM f.^i^ '6J0^rr-oo f^Sr vi.ia’d' ft (f-^.irt:/^^ t^/p J ovy o^diir .'t\yo. »wvL'.ifcir *-«jetitr{>^*rf^ * r'' ••*■■" * ' ^ w ^ fi / ' , ? 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In this way about 50 specimens were collected and brou^t to the vivarium, where several methods of keeping them alive were tried, as follows - Three toads v/ere placed in a screen cage ( 12 in. by 12 in. by 12 in. ) and this was placed on the surface of the ground in a sheltered corner on the north side of the vivarium. No sunlight ever reaches the spot and the soil is always moist. A shallow* depression was dug in the soil and the cage was placed in this and banked up about two inches on all sides with moist earth. A few leaves were placed in the cage. Although the bottom screen of the inclosure was all that intervened between the toads and the moist surface of the earth, and the air in the inclosure mi^t be expected to be reasonably moist and cool, nevertheless a fev/ days later the animals were found to be stretched out on the cage floor, too stiff to allow ready move- ment of the limbs. They were removed to the laboratory and held under a cold water faucet and two of them responded by a very slight movement. Repeated drenching v/ith water completely re- vived these two and they were apparently as healthy as before their confinement. The third toad did not recover. Apparently the moisture content of his body had been reduced below the min- imum. The few leaves contained in the cage were found to be par^ tially dried up when the animals were removed. The foregoing observations would seem to indicate that the toad must be able to come into direct contact with moist soil, perhaps to burrow in it in order to maintain the proper n • ,t , ■ • . . ,•■• . « , X 11 cnovi :f* oi \.'w- .#4,! -.‘A >x " .Hftfxa.ti^fx »4i /.', .. .. ^ V- > ,. ■ ' w „■* ■ • W ‘ LJ ■,• - ■■ , ■ • . , , . J iihru'Tj.^. Su'x rVn ..JWlTtiiiiiv .fi i 4* f>#/ >ii j ‘■5 '■ , ■ ■' " c^tf¥ 1 «|icv r;if.N'-.'4? , oi ^ilft , ftrf.* lo n,ki^ 1x9 lor,(11 / C''*’ . P\1 . * A ^ ^ ^ Iti »■! r. .•», i-.M.oxi-' iio It.,.':', 4* lU ;j'l|ir^ C*4^rl.. . |/ rr.* S>^i. 5u> ^ ^'/t5 ^i^lj-..' 4 j ■ '* ’ ' ' '"-X* " [ ' ^^:v aj.44 r^-; -^r:7 tfs* J L./' „ '■ •fl.® ■ *^ ■'' I "" J », o. tPil hAiff, ' U ,* ' '. I '* rija: n« n*^vU.f ®T‘^ !l tir; //C >r ii4 a?.4pi^' fjJW . ‘ _ . ‘^1 o.* D%j^ .rt'TKi**: & v 4 > |A 0»,V4:>- o«i ,'rto/v» e-u-.r^ ii 'vfc xf0^4lvX4Ui' '^>- ' M .‘.ri>L-Vjt r. Sih^ . » 4 * : . I . . V . - '.A *) ! « l^ “ J Ifirxr 4:. iiijr ioa'-ti d /O i^ cmi x>o^«s 1 t ■ .•' '.I I '/ I « 1 ►■■ ■•'. "'S''. * • 'M ' |f.iv -9^ vl^' I'j 'i*;? tf/iw ^ ’ I . <1 •► v' . _ '^ .'" r ” ‘ » ■ . ,»^ .V V.:, .- a-.'^', ''• -4 4fe ^ M ■• i > . • . " . ; .V V..: . . j ■ . r . ‘ ' . .> fi\m ^ '-v/n P/t5 WcCfftf -»44^ W"' ^*' ., ^ , * ''•'* i- - -'•* yx /■ *T 4 'i -r ‘ ', *i •• Si •«-,st ^ 05> bvi\cA o’xtv.., ofiij rJi .;» .oJL- /■:':• « ' 'h! ’-4 ^v**, ' I ,■« . ' ' ,’, , *i, r r •■ '... J.' . . ., y. J- ., . M^J ;^$viL-frit,i; n [j^itw ' cuu' aul ^i*i4 _. *1 ■ j 7 * 1 . ^ ' ,- jT t ■ ^ t 00 tifjf'fft 5^f 0 ? it I ifl 4i », Clift 1^ y A -' ' ./ j, ; _ . ■ ,ut..’. .*. . hIi aw w ». ij-,. ^ •> ■ ,..• ■ . ,s . r ' ' ■: mil - 6 - moisture content necessary for health. This conclusion is fur- ther borne out by experiments with another type of cage as follows: Several flowerpots were prepared for use as inclosurss; {See Plate I, Pig. l) ; each was filled 2/3 full of soil, pro- vided with a wire screen cover and set in a saucer of water. The soil in the pot soon absorbed a large amount of water and became of the consistency of v/et mud. One or two toads v;ere placed in each pot and the covers weighted down to prevent their escape. In this type of cage so far as I have tried it, the toads live in perfect health. Usually the animal burro7/s backward into the mud and sometimes completely buries itself out of sight by this means. When dug up the toads were always fat and plump and v/ide awake. Apparently their surroundings of wet mud furnished just the right kind of environment for them. A number of toads were kept in a large aquarium tank with about two inches of water in the bottom and provided with scattered bricks and boards for them to climb onto. Here they remained apparently in good health. Several occupied a pile of v/et soil in one end of the tank, burrowing deep in the mud, fre- quently in pairs, the male clasping the female. As a means of keeping the toads thru the summer, I arranged an inclosure in an open screen-covered room connected with the green-house. A corner of the room underneath some tables v/as fenced off with vrire screen two feet hi^. A pile of black dirt was dumped on the concrete floor in the middle of the ttixCT .-^".Litmii t«it xt«0V |- li'J. ‘ ■ , .;. K V iv^'liP t’.'/ f'cri.'tj^a v*.<^ Ji/fli •rfl.'ij jI * t '*^W ' v^- • i; 'it . .-jc ■: '-iC-': ■- Mif 'L ? 0O>MV. ' 1 4r“rcVbe ‘’* - '••T *' . -X . --I J iH'y C\;. «-i * ¥'.9'* ‘ rfj)- f»'. '’(, i'’i J-- a“’ .:V *♦»;' ,i; ^^lTC • . I *. -i 1 o» 'rc) nc^3 A^fU oT ^' . ' ’ ’ ''t, ' ■ ' ■ > t. * .V ' t . , . ^JXJ|!^4 ^Q!‘V;%a ni i f .-r ’■■'•■ ,1’' i'Vl , i)i-i i wit^ ^w« n-i-''-' aoifl ■ t ■" . " ' ■ ■• ma x-Lt u.4?t :,i /iC-r?Wvr -- : * v : ,.;*■<> ••n^^ I • t'J- '‘*ol p*flv ,•*#:» iwVa.',l^ W(# ^ ityii-.iii/* .1 ^.nX i<^3 lE^ i#W3tx;,t • ‘ ** ' ' 4 ■ p^, , -»i^r wf 41'i' cm^ 'Jrao^i ^i v iif k'.. ' . - / . _. •■ . • ' / k' 'f • ;1 ‘^ ’- ;v.t 'jrsal' - .y^io .’ar^Xo u# coirtil seV biui, BS?oA<<.6&'isJ7««i» I? ' ; *^' ' ” , , ^ .. ^ ‘ ‘ .'in f>|'f^' 4- bo)tp:T/<:0 1' ii ' ■■ '.*■ . ‘ ‘ . ■” ■■ 1 ' K --si't ..^4A‘T; flJJ Xli', bt^ Xc> Ufiit hith .li IX^ J'&l.t ^ ',"■ .,,- ■;" ■ ' '*' ' - ■ -' •^♦£vi5i^j»'V-6.‘f.^ £?ritj OlAif . 5i^,t. fil v-i- . • • ,* ;v. , ■* ' I , A/x*iJ ^ibikuv tn> J5 xa / .1 •■'v • i d;i a o-J^r «;jcr./3cnt>»ticd .x»W/vfiXiyiiyi;9 ft 3 ' ? z u 6 irtLi>;Vv^j 'A*<; t. h 4 &'Jff 4 i «. X'V"^* o^iWijpu tn*?X >»?V«oidr. «w nX ' . ttmu r^/'" ■ I?;’ dst ie ei^-cm ca , .Xior* Wtxf^ " ^ -c^^r.*'..- :« '' .oJ^sItra of 'XsWPfSft^*;’ #>.+:! r t- ^ V .-J^i’ 1 *» »*tJ" ;*, 0 ' ^{^j 3 ttut> Cki o*/i \7 tWDCO.If *^* •' '' 7 TlX llfcijC^ ’iO »<».a a^ 4 v^Tt«l ^V^v. -4 Vli in U. t'- ^•'K I (\I- . 1 - . Jifj *y% SS^J^t'fo 1^ “•*'’ .t^.'l^f'f*^ Cfti4 *y;ii\ •>iftiA flf:!*r / t*tfi- •.-+.#/*•♦ rW^ ' ■ 'Vi ii.#n- « Okfif n''--*< iiQ^laW ’^Q Oy;i{;m oif4, fei. f«Ai, ^iji^ j|l '• - , » i! «!.'• "^ r ' :>fU cnC* c-l irS^iiv* If. > 1 ® ili ri*\ «f!i2;/ '.-*stf r,.ib b.V Qd!jb^u^ « T’* • l^t«|ifc: ^^).». 0 :^ »j?? . ’.'^l^l« >Ya ^t 4 *tiV;A? r}ilP£j A eiflt c# lo 4^0 ^/iX^iXJX i< 3 ^ fc ^ ,y “ ‘■^ ^ •liVt^ gggaV^V.; ' 1 ^ 4 l»'^ - 8 - supply of food for the toads for some time. Such a feeding de- vice seemed to he very successful, hut the supply of insects ohtainahle at that season proved to he inadequate for the thirty animals and not many of them were in good condition when cold weather set in. All died during the early winter. Death may have heen due to the cold weather rather than to the lack of feeding. The toads had crawled under the hox in the center of the cage and burrowed a little in the shallow earth there, hut this furnished only a scanty protection against the severe cold. Furthermore, the freezing of the soil rendered the moisture in it unavailable to the animals and this may have heen a factor. Experiments described earlier in the paper have indicated how im- portant a factor in the toad's environment is contact with moist soil at least during the early summer. Presumably the same prin- ciple holds good for the animal during hibernation, and a condi- tion of physiological drought hrou^t about by cold weather and lack of protection may be fatal. III. The Water Content of the Toad During the spring of 1921 some experiments v/ere per- formed with a view to detemining the minimum water content necessary to the life of the toad. A number of males were col- lected from the breeding ponds and placed in cages in the labora- tory without water. They were v/ei^ed at intervals and the loss in wei^t recorded until a few of them had completely dried up 'T' *1 r ' f * ‘ ^ • • '• I • I '■ I Ml mi^z ‘ V'k' S .SJIK' •e^*‘s «6 jio/ tvst* xol tivot *'V xJL,4:? -sMr 0^f‘ 'c6aii6>'S'^T?D .-i^' * • * . '* '■ ' V . • !rt«!i nciiiiujti.B ftotv, ni titair tarl/ lo t.>«« sort *«« jiXifejiltia ' ' I ;|jiix.'t lik .r.i Waj \ . In iruoi -^1 »' ««»<> W. ' , •',?•' -^j ' ■■ • ' ■r a •taOi-'io -ji'j itl Toa nni letetli Sf/irtit. liOdojVa.'iy •.f.-.nJ^ual Rt •'*'*' •rt' «.■•»•"» *«XX4*fti orfj nl rl.>j:.i .1 nsiS ' ^ ^(TN ' •■ r ■' ! .' vn n&f 0^.7 ’^i’L'.^sr- ^ \Xr,c^ lf£»;?Qi4i*Si. i ^ jpi pt^ .^1 .:jcc To %4fi,n©ott o.{^ '■".?«Tosa ^ rs«tf tuta eiAiiii- ^lis, cp ?i -ii ««< i>M.iol6ai -ijitfaii o^S nt ..t- hrtfiaattnh u3r4&..oasS *'‘-. ‘ * '■■ '1 .fli ^ ivUiU li^hr r ^rof-»»f.x>vnti n'6A<^v a.l.^ «?. -hi'**/ VSTSV. r,/* 5-' .V ' 'wag . ’?P =-Xi^itta i).‘* 0’ ,r -i ./i OQiz;; ■ miiri' ^Cia T''-:Vaw- r 4 oa x<^ ;r%jcnfr- Xm^lsci!oin.\;r^ltf aoii*;'i • I ^VOT ©fi: T o .Tf»^ .?r o/il' .rxt - d • '/... *. 14 ' i’j ^ - • * “r \ < 0‘i!>5r f'.+nei^ixoqpco oflOR Xsex lo o^i£^ ‘ '6.Q%, while that of the animals which did not survive was 46.5^. Those which revived when given access to the water increased their wei^t by 48.9 gms. on an average, practically doubling the average weight of the survivors at the end of the drying process. (49.9 gms.) The average wei^t of the dead animals was 36.1 gpis. The above observations and experiments have emphasized the importance of readily available moisture in the environment of the toad, and while the experimental data is limited, it neverthe- less seems to indicate that a toad can carry a relatively enormous amount of water in its body. Perhaps this capacity for retaining moisture may be a factor in enabling the toad to live on land away from the immediate vicinity of the ponds. We have seen that in a comparatively dry atmosphere such as would obtain in the laboratory the animals lose moisture and may die in the compara- tively short period of 52 hours, (in one experiment five toads died in 41 hours). This fact may perhaps be correlated with the . n Ti or:. I*; 0.-9 ftSJf «idT .'fiOt^-fjb^, ' Sv ■“ '■ fc. ■ ■ . , ■ ■ *,Y'. - 4rfc»‘er v„* iv* oi^ jSrwoxXr^t^ rtt)r,r ’ • , • '^ *< '- , *3 " nlrU,-xis4>w l^i» «*'*.. ij- cj!9S-*:c»^*j_ &£|jjt 'vtf'ff 1;/j , --.rjJlHr o,:: r.i t\i6:i ptfolyt, Ju hKtti.4^:^ bii.'' -^tf *X^w '■ -V If/ t^i>^ rirti^vr V./ ^*oX tfe.‘,,*r :p f v(W fik.£^iu« tfdf to o-.-i.'* \ »«' ?^t».:^*x^ yrl^^?C.a^’• .y^tt'ivlV^ zSf.--.w b->rr{:^ dot:fw ijnc.VP .•>-.' v.-m ofX>?i-a' J pu yic ‘^X4^ ac «r0 .!>;;;* . . . ^ ^{C| cii^UJaSitt*. m? ,,.ai.fj,o- -{Us^i.-Wii. .‘(r^jj-tteii' ^itviLT*) 9^x*y* “iC ’.viy; S.Vfj,: .’tn.trir sraxW '”1 ' . e-^V'ri-sntjxo' b.ia r ’ Ic t>v^ m 5i» :>ii4£«y .^o -r.*.y *wr«fr XaJ4iL>!!::i-lw qX'^*'.JT' ,yj*o tfoj. ‘ii Xh'ul j4 :, HI. aii^-edi I:s»3k/oo sX^ fli' ^3.^4j ’^'ix .Vi?^vV.i^ <^#:^^no sTi-t c.^ ^niJ;ci>^9 ni !Xcc.&«V‘t «tf’ 67A4^or;' .a2.«oi |V'n t.o' rf-xojfe - 10 - reactions of the animal to gradients of evaporating power of the air as shown by Shelford (1914) and indicates that these reac- tions tend to keep the animals in their optimum environment in moist situations. The question as to v/here in the toad's body the water is taken in and where it is retained has not been answered satis- factorily. I have never seen a toad drink in the ordinary sense of the term and it seems unlikely that any appreciable amount of liquid is taken in in this way. When a toad which has absorbed a large amount of water is handled roughly for a few minutes, much of the water is expelled thru the anus and possibly this might be where the water is taken in. A more likely place, how- ever, would seem to be thru the skin by some sort of osmotic action. Possibly it is stored in the lympth spaces beneath the skin and passes from here thru the wall of the bladder, when the animal is disturbed, or it may be stored directly in the bladder. These points have not yet been studied. Some preliminary work has been done on the skin. IV. The Absorptive Force of the Toad's Skin Reid in 1890 studied the osmotic action of the skin of the frog and reached the following conclusions: 1. The normal direction of easiest osmotic transference of fluid thru the living skin of the frog is in the direction from the outer toward the inner surface. ,t* -u ix:rr^.;r^ JLi.rl.tn ejt; .1, f ■"V* ■/: f • f ^ ■> 1#^ fli stA6«\/io-£vriD‘iipTL'jij?^ ■* ' • »',.* r ' > ' ■ .a ^ •■ ■ ■ • ^1 ^ « ^ 'V ^ Xj. ' \: , . ; 7 . ■ ; ■ ‘ • ''tiJiuEta j'^i'cfl I ‘#<* «>« r:-i ,it t^\xat3*rL ,rtn ■ t. t w * i • ' - - '»'V Mi*»3 riifr'f M -« «i. jliiStJ. », »iiriiir i ft .. V ^ . ,. ’ / ., , " 4t; 1 ; ii ^ rt,ur .^nr ciS.. til ri j[ r . vo‘ :ii4v -^^.ws -x?/; xA- -' ni ^ ^ux axx*cat»9 -iv- r t.'K -^..wioa ;t;,>.x ^,tj tor^ ''■" ■ "'al^ r-i. J ifw n<:i n -ki-C ' kj r J ,lnr.A^ O -.J lo /lev f> w* irrx^f ^>Ttva .7st ijsf il e^.1 >i.«»*-tio i*»t«3( W v« ri TB .ftwru/Aifey.! i«*t«ii^!; i ,- j{%ov Y-z ititrx^et-r ^ P ,6 y«f(«io; #i» ;6orit.4»:i fin« *ortY siU? Oii.o»)i4'ocjti6 .ii-ji^or, ,£* '^. ■ . (»iv'o«-io r4 »jf g„t ,ftf •*« ^-,iX »(U «TU« xJ. Ijfc * ,1* \ ^ • V •' j '^* ^ • t, / ^ ^ r-a- art. T ^.oos'niid eit? jbiiiw »£4 xjwi' .*do_ __S'S; ■• ... . - 11 - 2. The transfer of fluid in the above direction is intimately associated with the condition of the tissues. Condi- tions or agents tending to depress vitality diminish the transfer in the normal direction whereas stimulants tend to augment it. Reid suggests the possibility of a definite absorptive force exercised in the living skin which mi^t explain some of his results and he records three experiments in which his dis- charging osmometers showed a definite action taking place thru the skin when the same solution was placed on both sides of the skin, thus eliminating the possibility of osmotic action in the ordinary sense of the term. It seemed worth while to determine whether these same principles applied to the skin of the toad with its more terres- trial mode of life than that of the frog. For this purpose, I devised a simple type of osmometer shown in Fig. 3, Plate 1. An ordinary Harvard staining Jar A, is provided v;ith a two-hole rubber stopper B, in which are inserted two glass tubes, C and D. The lower end of tube C is flush with the lower side of the stopper, while tube D extends about inch below. To prepare the apparatus for use, stopper and tubes are removed and a small piece of skin,E, is placed over the longer tube (the inside sur- face of the skin being placed next to the glass for example). The skin is drawn ti^t and sealed in place with sealing wax F. The Jar is now filled level full with the solution for the ex- periment and the stopper is inserted in place. This forces some of the liquid up into tube C, and if the apparatus is properly (px - -iapitE-jr •XT- W . i> t '^c, «0X>^4^ «.*iw " >'^3*^.1.* P.;^ rtni,aa^ .^i o« n4n»£ijpXJ-r; sAM^0‘i ‘ f.'m. ;u.-.;^-4 :.*%i« ,f'.“.tir 'Ctisuf jairi: oa: r»i - bXJ;< .fcrXa^ >i ?t^Yrf:?‘ OA*: r^o-i f»xt A-ri crfirKiK^ stj> ‘ ., 4 ^^ «,tx^niit m feci^f ; 0/U -Ja «p ^14-^9X^ -.;r ooijui^p *>jcf ••■■•.f-ra ^/.{T jX^ i\tif >0 IftSOr-ft ->,Jj^Jfc;U-i>.tO « • • ' • « ^ ' o-liM «»;’,?'• rt^^^^t*)4ji» or/X'Jwr.'-i.^ cit) ^ X r**:v ' sH*en»Mi i,I.4^jV _ . ^ 4 . = ," ^ ^ ■ . ' *v J viXOC! 0^4 *>.V Vior IX. .'^i^; ..rfi- CX '«r- i ,^yi>rrir.f: oW e-' t. ,&,vS k^j. >l v:Am ^ n»w*ia .H»rC>? AX‘!;r' 4 'j?’>||Aftl>^veA « ttJi#' V»lv;v 0 -».!r srX ,i ^*t.;rTL«K ttk-^ - ente W 'lAliir. /U. iti-dJui la otU» 5^>rr: ^fiX rOi^ xX/Kwri .,jjb ? aJraX ■ y‘,^ vT . fCXpd i(6i*f0^ac6 eifj^ XXii^^^ o-j , *^^vlv »{i. , 0 i»y icl MX -T<#tl ©bXc.-il 04iX;^ rcfi ‘.WP m ’C -fb© if>4w flX i^X^e^br44ia(%ij wjaTb tX-;?i 5 t« wi-X"; -tsatff riea' amutfip- ai/* lO iv-^iXi^^ jr»7!»i .hsxiJtt woa .V '^ >> •>_.«} ^ e^t^j 9f-/5? .#arf<(., ni- if*" ^.- 111 \ ./ 'rMt ' ■.'■0&S.I ■ ' . jJ'.'V.'"’"' '"•^'• iJ “-'■ It'. HLt <-■? - 12 - made, no air "bublDles will remain in the jar. The same liquid or one of different density is now poured into the top of tube D to a convenient hei^t and the levels of the two colimms of liquid adjusted until they are the same. The apparatus is now ready for use. It is set aside and readings are taken every few hours by measuring and recording the heights of the two columns of liquid. If osmotic action takes place thru the skin from the outside to the inside, the liquid in tube D will rise and that in tube C will fall; if from the inside of the skin toward the outer surface the level of liquid in tube C will rise and that in D will fall. Thus we have a means of measuring both the direc- tion and the speed of any osmotic or other action which may take place. The tubes used in these experiments had an inside bore of 7 mm. and were of uniform size. Each tube was flamed before using to remove any trace of grease which mi^t affect the cap- illary action. Tubes having an inside bore of about 7 mm. were found to be most convenient for several reasons. If smaller ones were used, it was found to be difficult to pour the liquid down into them in adjusting the level. With a tube as large as 7 ram. in diameter, a pipette may be thrust down into it and the level of the liquid adjusted by means of this. On the other hand, if a large-ended thistle tube is used, the piece of skin becomes limp and stretches and sags in such a way as to make the results inaccurate. • ‘ail I ^ ■ V“' * j ’ ' . , ■ ; . . — ;-i j| < io ^.NJ ‘O^c^x Ai>^c;o< 5 Vrni fciz-V'i»c^ «,'* /''•'•■ '*3 - cr?» 9#sy .vx«« eaj’^^* tt^M bnsi^fy,j >iiuhh f ' ■. ' ■ » '.> . ■ ' ■ V ‘ n *V A.tr r> ... J — A i. . . ' . ‘ ^ T-*- V’xi^vn rj.if^ ->t-> © Jf.A ,x it .t>«v _i* _ Yu* .•< crwt.' ^)r»^ 5.:fJ-u/|w^r ’.•:<( »nv^K. '. rar-i . 'tt'utr it .'iifnii *^0 »■«.•■- Jjc,: ft MV) rri i**WW •£» . •<» OJ oj;.*>f!'r^ al\- ‘i’i '^m-laaxx. 7! iJU«5 JtWw .0 *<)« nl ■ \-'iii& lirt.. f>e;-i lllw.s f^ivt Hi i/Af>|,i;p,>6 Cfty^jT f;i,' e^>;4i7{i^»«/o ♦ 6 . . * ,, .aXjkVXii^. a.ni V' ’ ir *, Iv F o^nXc V J?) » r.' ,ii- aicdd flii ni‘ S in'Tj ■ a-iCiirf »MKX^«Vr;iAi» doia .txia mMim .lu ww •Ma ^«r 7 to' ...o pi , ini. ttu' iii^ljlr .•f.iii'tw, enj«--i^ it? »3»it;J ’^(le PVPiUftS. oi"^ 5 ^Hiai/. aio« .etx V iu-'a-ioft piJoj,/ iv atliy^d '«ajj/(i /. 8*1*4 t*»Ul TI ■ ;».■.. fc«t*ir*i -...- l*ia-S»»i«*;i«M, i.£' or teftol =.11.. ,j«, „j ftX^^IXipi t, P^-OJ, Jirw.ot ..ov M MOo'^OHa:',': 1' aJ!.; i-.w.JJ .oiftf .aolor^Jt±,,;fi;|. ^ T-j!| I t6!Uc 9(U n« ^ ^.«iir :o a,K^ Vi la^sut^'t iiti'eti a.-a -^o, JaraX-^J | : lU^ i« 0oO-lj,-0 otful^oiloX/W 6p),n«,-iBiyil Ijf 1^ *. ‘ * * vji04ii*,i fj Xl,*acii j - ' -ll^ c - ^ 1-1 1 » \i‘i -13- Results with this type of osmometer were recorded on the experiment sheets as shown in Table II. Two columns were arranged for each osmometer, one marked (Column 4) referring to the tube which was closed by the piece of skin, the other marked - (Column 5) for the open tube. The heights in milli- meters of the two columns of liquid were recorded at intervals in their respective columns. The difference between the two columns between each two successive readings was then computed (Column 6) and the number of hours elapsed is calculated (Col- umn 3). Results may be recorded in tabular form, showing the maximum difference between the two columns during the course of the experiment, as in Table III. This, however, does not show the rate of action taking place at various times during the ex- periment. This may be shown by plotting the results on coordin- ate paper, representing on the ordinate the number of millimeters by which the hei^t of the positive column exceeds that of the negative column of liquid and on the abscissa the time in hours. Graphs I to IX illustrate this method of recording results. Table III and Graphs I to IX give a summary of the experiments which have been performed to date. In the experiment represented by Graph I, the main reservoir of the osmometer and the negative tube were filled with distilled water, and the positive tube was filled v/ith a solution of higher osmotic pressure (5^ cane sugar in distilled water). Two osmometers were used, one represented by curve x on the graph, being arranged with the skin placed in the apparatus with the ,trT4T ' V'' *'*■ -IT-W •.O. aeroo attuKotl O.t'V n,1Wi/.lrKl*T .n sxi:Hi€(v^«X5?o2^;d li tJ'U'O.'T “its -xJ-OBw/ft ftir: r MfJ'H : .mA 0“' r.,w>v..4c,it w ^..3 e-Xujf^ . E ,ci,xr. lix. 'oxj-t .-a ». . I _- -X£> <.fllUli ^0; f , f [|. rtq, a,;siiiih-r. B„,a flO ai«r.p»-. 4MJC-0 .irxjJBO, ,, .=•; -i-i Ml 0,«;j 6 J& ft'it-pA •»* its i..« hUfPtt---i,0'Vmt.O» r s, --ii > -.vta « o# 4 bp»>tn «lw*i “ i ' ' . * ' ‘ •'•J®>-'»^i,i»«i.»li9i? »y.ff K-- o«J .1 ri«“-tv.jc# nj>^ f . ^*y ?>t« w ■9^1/j;; /• .■ ■ ■• .if 'lii.S ^ ^r iJOrt-rtyno w 71 '. j ♦' » ' I# t * V' ' .* , . "^ ( ,_...•• .-, " j -i ■ ' • ■ ■■ |?1' %0i.?vXo, ,, p:r'xw .69Xli*.. _3«y 9'’ srXfXSpp. , OSl..'.ti«ig i if , . (w.* naUUAt, a..o ».) .ri^Xy .I,*; B* . ^ **»^,.»«*aw »„c ,09 0^- 9,.,. p^4«ao.liBO o^.f' .■P*) '^^>r Di.Ni-.vC99, oiB ,tfX, J.r/ ■'iii V >! M - 14 - outer surface down; the other represented by curve y arranged with the inside surface of the skin down, thus placing the li- quid of greatest density on opposite sides of tlie membrane in the two parts of the experiment. In other words curve x repre- sents results with the cane sugar solution of the inside surface of the skin, curve y with the sugar solution on the outside surface of the skin. A comparison of the two curves shows imme- diately that the osmotic action took place readily when the sugar solution was on the inside of the skin but not at all when the solution of greatest density was on the outside of the skin until late in the experiment, when the action ceased in curve x and began in curve y. If we assume that the tissues of the skin had died at this point, then the experiment would seem to corroborate Reid's conclusion regarding the frog’s skin, namely that the normal direction of easiest oxraotic flow in the living skin is from the outside toward the inside. Graph II illustrates a similar experiment in which the fluid of greater density was some water expelled from the bladder by toads when handled rou^ly as above described. Presumably this liquid was of a greater density than distilled water, altho this has not been proven. The general trend of the curves is similar to that of Graph I, but since nothing is known of the nature of the liquid used, no definite conclusions can be drawn from the experiment at the present time. A number of experiments were performed using the same liquid on both sides of the membrane. Three liquids were used » * *r - " 'W I \a - - .1 I ^ ’ ll 4i “j ' ■ - <*>1 »»>J n ^ ., ■ V f f : -i * 0 -.i.^ •.‘iig.-C^ k -» i n 4« «. 4 A" ■* ^ ri#. ^ib‘Jp::iiSf} ti^i rrri .!> , *Wt"t pr-i:\r>'r„^iiir • . ■';'f nf ^.^(ina/'r.KWP ircli tt) c»‘Ti-5 o4-^-;f»ifj -■■Ai.t: sSUj^ -,. .-OJ-JV/,.'. vntJi# nwp ^^# .lt#Jtf iiMu|5?l.£>nA« ’', ^ * ii., ■ I t “ "~‘ •’» V- ■' f *«i * ..JiB Kli ’■ r ^ _ , . . . ,- . t'^?,i:;« f> 1»1 «: jp >/i i%r.j 'E^ip,*'4rib 4 ^ ^ur. V.. . V-., <- !<• r;yr ‘ '•'' ry*f. s '^^'€^4# ft frill ’...i,^ n"^tvA tpk* t ' ■ ^ 4i '5,0'' . or* >r;i0’x**^e’2 hcts'xit'tlqk I e.*? ftf v.,n micAt^iu ' .* ' 7 ;*‘|l , if"/- t ^cvC'# i .1 © iJ r.i: «^iia»:'rt4'g:p *? ^ je:s.t^^i’XI UI rl/A4i'cO ■.. is;>!>arrf er-f-i-nn^ V>r:el.-;a m*aW, »*r V^itra.© tpJjjs,,' 1 & tvoiir-g^ 3>t^rinMtt ggesi at uoS x^ f3^:f^' " , »xiJ .' ■>/ 5 eXr I ^ f^' -.t Kjr ns-r-tuo ^’'■^Skk J f>r'fj^\<. pj ,ia-j,on to i«ctr' ■--■ -*■ .'.i.- . . ,. ! - Iffl * > _ , -4, ^4'^-' l^5*T<{ f>i4r >) t0AtjLto?rt?) -j S'" ' , * •*-y^.,?:>» S.4w »i -x ► ,[ ’.i . n O'fi n;X' aAivj^oinoa .i^^arlr* {au^.^*, biv'^ '■* *. ^ .tnm^ 0 -rv* aa oMe ^-.: tUiKf - 1^, cit.5 -*nrfo jif?r n/;? ■ &a /jr-;*. j,c*a /a->:c r ;, lift- ^ M’.t Hal f. fit f -V It .;b!-iitt n<>jja« oijU ’lA ^{^as^l■•1i. » »rt; *.‘J»‘'i:-.t .is .Kri tt k;i 1o-j(»A/tCb<.; »j{# “ _ r>pn^ 9 'i. B.>. (• 4 >;n.o»i>-^^ffl. iitiropa .■.Jij.-ao'i rf!r.ji'..-il:a» »"i;i «iJi> v; £«ij! *iv-t'l .nioir^y/cki «i^*j rA «.-} 2 : o o •#<*;> -M aj: ^ Qf» ,". A .5 - fX- ..io,^ .Alt o:,rf«T /j % 4 »JtXii 5 o #• ^ vii^ nt -rl:lt W t»o*»aoP^^ r-tUj . U( V^/lPWirzaq;^ ^ .-/tod XXI ‘I '. • i ri VI ri ni^Si , tV^«fA 3 l* |tr. mi i>nr> +*w-s 'r__. .... ovjir in Hi .tAttf ©liHv ,i.mtsJc.ii ic >idijjcf axij «%[^^! A ,J» '■ ‘ :' •'* ' .->/*fi'r ■ ■ .^i- -16- of the body, the action being strongest thru the back skin. This fact is also shown by later work. Possibly this difference may be correlated with differences in the structure of the skin, the presence of poison glands on the dorsal surface or some other factor not yet studied. Graphs V, VI, and VII show the results of an earlier experiment in which the skins of three toads were compared as follows: Skin No. V was taken from a normal toad. No. VII was taken from a toad which had been allowed to dry up and die, and skin III was taken from a partially dried specimen, representing roughly an intermediate stage between the other two. Results indicated that the absorptive force varied markedly with the con- dition of the animal when killed. Unfortunately however, the skin for these experiments was not taken uniformly from the same part of the body, that for the alcohol and chloroform being de- rived from the back and that for the Nad from the side. Hence, in the li^t of the later work indicating that the skin from different sources differs in its absorptive force, this experi- ment needs careful corroboration. This was begun in experiment 9, Table III, in which a partially dried toad was used, one which had lost 31 gms. in wei^.t. Skin was removed from the belly side and from the back and the two compared. Results were quite striking as shown by Graphs VIII and IX, chloroform accelerating the action when skin from the belly was used, whereas alcohol produced a strikingly similar result in the case of the back skin. What the signifi- ■>» ,1 r 1 * * r.x/Jt': if... ■>Y^ inf4 :r^4>^^ciicc aoWoi ©iiW ,|h; (Juc'it>^ # ■ ' * '•s ^ »■ ; .^(a* e.v -.o 0-uC;.«-ii!; 9.J . ’• ’ < ' r " V, * ‘iv otiifi)lj,f\ta ^ \& f^J»uyQ-t #a- av.x>r.^ ,r>/::* V r.-. Ct6i ioSiVa^,. t.6td, „t m.. •**• 1 ^ ^ -1 ^ . V» . '- -' ^cr .; » V Ji..:2fh H-crorl f!0X«4 urv 7 ' L-Uf; ' .6;'. N^.p-Tti <>J' ^‘■'■^^;^^/.'t<-~^‘<.^.i .-aicV - nif D-a \xie.o:.^ -Xiri't-v ftOTJU-t f»vi.* ,%oti./ii,o/;i 7ni^^^4S^il),Ti • | ^ ,.. 'XT-'ffW' .3f*;ri,y Oii&\XiO-r^ vXh^miflb h itln A-:^: » > • .... » ^ 4LCA ^vV ;x5^i4 I' i«r:oC.Xr b/ta * ^rJT .-A,., BCTi-J ■„, .ClO’tl Iti‘>*'i .nftif .tcs-’4* Ifc- Ik,... .... ... '2 ■rxo'tl ^iU lt3(. X?|n 9Xi:5. d®* r:^ • ’>raB fe X " ’■ ' .* , » ' '♦ ‘ Olf ' Ztj'X^'l Jjf) e/^ ■i rtdir,x ‘TX V a«w J 3tr>aX D;t TC^7'X: SikvxSigtex cj^ X pi.mr, J P*»^^ iSv .. ai..^xo^itv 0Oi^oM.,.0ix^ hJiX^^'nilooof m-Q^qpXKn . .xtbu^ IfXJ^ '1^, "■■•i*; ■ _ ^ ^'. .., *' *■ ': --*■• *^>. * .i'-‘j’‘ ''.j . • ■^--— „ V. , , " ^, ;'j}^ . ':«■■ - _-,.''liii._. .... ^ VJ- . ,t- V UB -17- cance of this may cannot toe stated at the present time, tout in this connection it 7^ill toe noted that in experiments 2 to S. (Tatole III), where skin from the toelly was used, chloroform was the only solution which showed siny noteworthy results. As regards skin from a normal toad in contrast to that of one partially dried, Graphs IV and IX may toe compared and show striking differences in the results in the different solu- tions. Conclusions The data so far gathered cannot toe considered as suffi- cient to warrant the drawing of any definite conclusions. It is merely suggestive. As mentioned atoove, Reid ( 1890) as a result of his work on the frog’s skin, suggested the possitoility that some positive atosorptive function mi^t toe exercised toy the liv- ing cells of the skin which would explain his results. In other words, he put forward the hypothesis that the living skin exer- cised some force comparatole to a force pump and th^t this force ceased with the death of the tissues. This seems so striking an assumption that very careful work must toe done and a great deal of data gathered toefore it can toe considered as proven. I merely wish to call attention at this point to certain aspects of my experiments so far that seem to fit into his theory. In Graph I as toefore mentioned, the osmotic action seems to go readily from the outside surface of the skin to the inside, tout l>uava.yta stf \9 'Isihjo | ^ ^ ^ W •- -V '*.'**^v 11 .s rj t? «■ 'biu A»-^c=c-. f-; ^:, A'fr - V'l ,<• .^C ri rJ oj«;if.t'-»* '■ - .ilaf-^.i'orria '"> _ .a' '■'- ■■■*' .. vv;:'^ tti " ‘ 'I ^ t . / -; f^ ' 7 ■■ ';t >1 » . *i W- oO ^ ^ »•’■;•' tyi ;• i a f • *i,aek: ov: »rr*it) ofC’^- ^:. . 'VVW .A .*'ij 4 r^ ..$ ^ .'f;.:i^-^ ^' •■*■'•* ^ i»p»iri'«»- M i^taj •««{:#», w-: 9 »'iw<'» ^ •Ji/.'a f.l .;*>U.«5S;t,aUJ iTi/a:j;j.f, (ioigs, ^.- 'J '--«Ka -a-.: J..avu «.;,V «{"*«/,» av.^ 0^. ^ j,,,"-/- ^VoW '''" • ., 1 ■•, , •■i * * ,7/^ nJ/41 tk .r I *•* ’- ?nto:iw -5 ‘ . .io^ 'o-i, ) 1®* - T'"' « t^Vcy.'jr'i jIc. ** ifKk. w* ^ . 'k. ._ : *t’ r- ncVfli^tBV sA (WO (fi B'to'tB- *(.-«., '.'J,a rJ tiler SIX VI -* • , ‘ * , • . • -*. d 4r . t. s^-, i.wpe :.foSM?HJ(^.«» ftioi»rf „-.r , ?^«| ... : ■ . . J, .. •: - . V « ■ fA fS. - ^ >*^ . 3 >ii 9 « .^, 0 ? ,?«|» 4 l^e 4 tf».»V' «#V ' •% -18- not at all from the inside surface toward the outer one until late in the experiment. At approximately the point where the curve X ceases to rise, the curve y "begins to rise. This would be quite in keeping with a theory that the tissues had died at this point and the absorptive force exerted by them had then ceased. Hence curve x ceased to rise. The skin represented by curve y also having died at about the same time now acted as any non-living membrane and osmotic action took place toward the liquid of greatest density. V?hy curve x should fall to nearly zero is not so readily explainable, but bubbles in the liquid confused the readings in this part of the experiment and hence perhaps the curve should not have gone so low. i>lo bubbles formed in the other half of the experiment to confuse results. Another point worthy of note is illustrated in Table II. At the bottom of the table is a transverse column headed ^ increase derived as follows. If we glance down Column 4, for example, we note that this column actually rose only one milli- meter in hei^t, and a number of other columns did not rise at all. In the chloroform part of the experiment, the positive column rose 2.5 mm. The difference in hei^t of the two columns was due then in a number of cases to a lowering of the column in the negative tube, not to a rise in the positive side. At first thought this would seem to indicate that no ’’absorptive force’’ had operated, that the difference between the two columns v/as due perhaps to evaporation in the negative tube. The sli^t rise in the positive column mi^t have been due to temperature I wrf T ,j TTa -B/* . -rrxfcTiX; =T Aafl flii i i '*■"• '"V .' 4 •‘I:-..^ ?5M^ .sd ^l >.; It , r ^ 3S^0c'^ DValfo' ' iTT . •* . ^ SM i' ir i; p ,>ni^ £>^-*. r'T.-t'ij- e*x' j.ni jr . nf'-'f.r I) .ft ’V,1 ndioI; s>r4rg'4<*dtf* aiU irvi^ •eiiKl . lSl< ;r - •Js:r. 0 't«t.vii . ,* 4 »i 43 ^ c ->ft.;e^> x d*nwv % 3 K?M 6 . « 7 . V f s. S,.-..4 »o.-5 •'''■f'* 91'- -Tied' ». t»i‘i< sniv-.i oa: x..«?tia~'- ‘-5^ y * lUC k w w wwjT f*;rjt-7 :L'V 3(f. j -* ■ V , , ' .'- v'-j f -. . L '. ..* '■' •■ '■ cj ii.a .'•i^J..•■ X * j , y •” '7 '- - «. • . S.^\ . ;tu. -^ty 4>i,<*pli; rti C.;iO,C»..#ti . - :.r: ,'. /• J Otl/f 1 -n ji. rM.' al, „>> %-^tfb;.. «»»..; >;i'A,-i an ■ iX jjii^A * .■---»:, uc'-. t>Ui«ri. : »,in,;i/j|jjj J ^ - . <-'. ^ T <. 4 . n .^liL ^ •"^■?iy Q^. 'jRtairU flJl ' ' i ‘ '•r:rf«t ./C u: ,«y;:f.n UiUa JaraVS ^ * ‘ tf « r.iujjXcci’ a *..Ci ,r« ni» ;i* ' . r.'s ,.l)ficV/Xt»tr, Itifli'fv 'H 7'> fiWn i A. Cii I P/.r^*Xwcj #!a .fiyj «i.£ ■- -f , ., ■ ■ ' -1 ■* - , .^^ItJ oyxc^itoq r./ •DtfJt'l 04 q«# iii ^l 1 ' - ■ . > . -■ . ' ^, ' '9vX^0,uoX^'' an, ,4 <;■) iiXeyn ;ac£'i? as*' ns^frjeij r>ort,xl %**|i)Va.I« 6-.f 4.a>tot;' |.tfiil,tv>.'.ftriY .arfui svi^j^n a* <«i lua^itsssyo o» si<-,rfv»« -nh’iJi'w .•vr -■• 'a ^ ‘ ‘ ‘ ^ '■. *« . . * ■yj _ j* ^f"t I ■ ’ v 1 ^:i .1 A ¥ . . i »-.* w |'rf i iy . /! 1 . which unfortunately was not well controlled. But an examination of other experimental sheets in the series shows that in a num- ber of cases a definite rise in the positive column took place. These observations are summarized in Table V. It will be noted that in Experiment 9 A, the chloroform solution produced a def- inite rise of 6.5 mm. This could hardly be due to temperature althou^ this point needs to be tested carefully. If now the results are due to evaporation in the negative tube, then tlie question arises as to why evaporation did not lower the liquid in the positive tube equally. The two tubes unfortunately were not always of equal hei^t, the greatest difference between them in any osmometer being 32 mm. In most cases they were nearly equal in hei^t and in several cases the shortest tube was the one in which the lowering of the liquid did not take place. It seems very unlikely then that unequal evaporation was responsi- ble for the results obtained. If now we return to our working hypothesis that the skin exercises a definite absorptive function, this phenomenon might be explained thus: Evaporation takes place equally in both tubes, but as fast as the liquid evaporates in the positive tube, it is re- placed by liquid drawn in by the absorptive force of the skin, and in some cases this liquid drawn in exceeds the amount lost by evaporation and the level of the column of liquid rises. Thus the coluxnn in tlie negative side would be lowered by evaporation on that side plus the liquid removed to the positive tube by the absorptive force of the skin. '■tasrt -ci- y -T *- •• v' I * f > i- aeJ.^'. 0 >.' r.rai'r,a »vi#iiOQ; {,y kt ja tt | »; tfsXi'i; a.< XX /s? ,(„1 •■ siouV «x ■"■ V npi** c«o f ;n*. >e nA ^ ' 0i> -e PA'l^btpu-' (tSf fi'C 07.ri|f ft' Q r> , A . ♦ ‘r «,, ,- suyA-v^ri fc.-rao „■-„ a.V^>c"oA^!!^nJ kltMh'i.f 4 ,i_ «. ii _ L ^ '’ •< ■ '‘'f' ,’ ' ' 'i ico tit .'ia£54*-. 7^;^ ci -je;^W*Lpi f "7^''’ ti.L- ,t-. fi'-x;w;4 j| txjijvo-oj ^XaojJ:.ny nV.eeX.<«vp^Pl£ .aiM' m ' ■ -•■■ • ' ' ■■ ' ...s ^:»««T „:ti -la eiw b,;a 0- xd^ jf. ..!iu(| ©itsrU/ ^w- us^txfQOoo 'ipa &^^ cn^^si . .»•>’■ c, .i> T.0 is0 ci j^t>rd ." .mi{tC’V;y<^o fi-'- fudm>%r»i%ii^i;|eXn4 ^«isot3 p-Y>'i> Lur >s,d .-. ,¥niv ni''.i-'» ^4;%..*^ * ,rc<|:; 7. . foi:! otifi \^ 'Si !>^riAlPa b % r^^-vt . . n * } uiukI . \, b ■sc^i^trtsJb 12' r A — ^ t t H ’* ■ • .V „ \ ,. il , r*.- . i!. 45r. n i> f'tfuiiuiq,. yrViU ^PO\tfcii^iT;5^ ^ #» 3niinl*5 ff< '4(fatr*-pv !j*v, -r :*o'l sna. .•,() diki V. S f , ■■ .Wrr-Jt Ri?‘pixDr U' ir/uu^'i ^ aX. .OCi^‘'j. B' Ml '> ’ ,/ ■ > ® ' *1 ’ i- '^ ’§V o,T.y ori>. '\i/io- -^vX^yni aJtrf!!® F A^J- I Ot'oXtjfeV ^ i^;Ul^^r>QD irC'^'X lo ,‘5Xr? YJI/* Ov.ii» lf9 ■ , J -A» IS vjj' .6.^X^Anu o^'i ;fs;i5 ^rt^t^*i^o{^ mi tiU o^f fi1 i1/^i JuT J^lf X»pjtf|*3U:.Jir«^ ’ .jr H 9XlJ-0'!f /ft^nv ;j a ■ j.. . , , * ' •' "^ f" ' . ,>J(tJTSi tl ^ ti_m .KOait \t} frtlS' 'i Xo Xii-' 0 btiA a^^oco^iiiid^ 6k A 04 UM. ■ H'^v » • ' . . .' ,» ■ X‘. % - 21 - are closed and C and E opened, so that the suction from the pump exhausts the air in bottle B and tube R and creating a partial vacuum in the bottle F, thus drawing the NaOH from bottle H thru the tube G and into the bottle F. Bottle H is thus emptied and is in turn filled with air drawn in thru tube I (screwcock N being open and K closed). This air has been sucked thru the series of alkaline towers J, in which process it is bubbled thru the solutions of NaOH contained in them and thus freed from any COg it mi^t contain. Thus as the suction continues, bottle F is gradually filled with NaOH and bottle H is filled with CO 2 - free air which is now available as a supply for cleaning the Biometer. Ihen bottle H is almost emptied of its contents of NaOH the suction pump is stopped, screwcocks E and N are closed ti^tly and the supply of air is ready for use. Part of the NaOH from bottle F siphons over into H but this soon stops if everything is tight. Usually this air is forced under pressure thru the Biometer and the method of procedure is as follows. Screw- cocks B and E are opened to allow the air to enter bottle F and then screwcock K (previously closed ti^it) is opened. The NaOH in bottle F now siphons thru the tube G and down into bottle H. The pressure in bottle H forces the CO 2 - free air contained therein thru the tube P into bottle L where it is bubbled thru weak NaOH again as an added precaution and then passes out thru tube M and into the Biometer. With the bottles F and H of the proper size ( 10 gallons) a continuous stream of COo - free air may be forced out thru tube M for more •.TT- C okutf si.'y ireni ncj j^we »i« >3»r« o» ,UB.e*qo #» f jOna fc88©l|' is® «■• irjt n Jtr»» f tJjJvttJtl gin erJ j}ow«et>v,i K a«> 30«ie «!» .7.^0* *r..* I' •i.6» u a oijjoe .V 8XJ. >r «a.' mbx p orfl, ‘ * ^ *1 4»ocarv»‘t^) I* 8tfu?f n| TI* pi e t ; n<-yu i»A*- lia «ixn' j *■ . . ., - ^ - ' . i^oI', p*:jXvCi!J el H i,j[ iauXi't C.jt- 5 I * ' . i '' -fc " ^ \ia*\te A. 8^ cl /i^aiw j.lJ tirr'i : 1A-.»4*!^4'«?o- ,« t1i . to ®fl^!opo^ QtOfc ,J 8a-3_r,j£ «i BOiaoii* HOija IL •■ “.1 «'%» _ Oi^l ^o'lx^u .ftiifxi -64«*z' «i TfU lo 'li no^ii F^ias iiKf S o^ffi ^ K? C\g M- .>'C.iOXC scbxuf ftj >f^ -. '-.^'V:.iOXC SCbxuf 8l :tkr. 81 .ewoIXo: OA 8l 1®. 6^rlj<>i4 ciV •> , '' « ' ^1 ’ ' .»■/., w ^ ^ r , 5-.aix p;^4l oi4JM! bX i’1 m^ ^A-/f trsds ,^1 11 ' ‘ -M ■ A .*’'*» I / &X1.® C(f oJifU irw^xb’ hlxik ’ ■" - I ^l'' :■ s’ , ^ • V - ' i ' ' 'tJ AW Ofi! i>o« M »4W(T00 :, 'saoXijig, OX ) tislB OKjo** *b I, 3 604 t' n^XJ^o’ . ^ . ■■ ■ , . • -80 t 'i virm ‘^05 U 8rfji/? ifim' Aaoa^ji 0(f 93rxl:\ vOO to me%/i .r*^\ • ^ * ‘. y ■■■- r ■ i'« • - ■ ' t^i tfJTL ' ■>|B' ,■ >; ■ ,, ^ »fl - 22 - than an hour. The time required to refill the hot tie H with pure air is also about an hour. Titration Apparatus for Determining COg Plate 2 represents an apparatus for the determination of CO2 by titration, applying the principle used and described by E. J. Lund (1919), namely, the titration of standard HCl against standard Ba(0H)2* The apparatus is used as follows. Standard Ba(OH)g from bottle A is admitted thru tube B into the side-arm burette C, and is titrated against standard HCl similarly admitted to the burette D. When the two burettes are filled screw-cocks 1 and 2 are closed. Pinch-cock 3 is now opened and a known amount of Ba(0H)2 is admitted into the leveling bulb E. This is now diluted with distilled water ad- mitted thru tube F and the apparatus is ready for use. Tube G is connected to the respiratory chamber ( to be described later) in which the animal is contained, and tube H leads thru the dry- ing tube I to the suction puimp. By means of this suction pump on the one side and the pure air being admitted under pressure to the respiratory chamber on the other side, as will be des- cribed later, a current of air is kept passing thru the tube G and bubbling up thru the solution in the bulb E. The COg con- tained in the air unites with some of the Ba(0H)2 to form an insoluble precipitate of BagCO^ and the excess Ba(OH)g can be can be titrated with standard HCl. After the experiment has ■ f n; ir V 4xxi«* ' 03 fe*^-?i#.7>ik*r t{I iifc '><. . ... 1 ^ 3‘^J-id*L.ooX'r ti' iJt # y^ii/g V,' e '*^ ’■ »■ atJ-XiU-riN, a/>, i..‘j^ ,v L ’.'.:r^AXUy:'>i e>3 I? rr-3S3i%oi:of* ^:o' |^Cp *)i o ' o-j^,a.'jV^rv^>u4 * ' i i^:. f A i: »tj^r tif MCI 1 c ( ^ • v3 - i / V n. *" ^ - w - • 9X ^ ,-;Uico-if;orri^ «^ot oliS ?>ts i' tita^4 B dXr^^^.'li ofi3 tc &iWc^ o t^wp otja -^'« in.'sr I’- .tr »rt *il mxy: .V -,r.ii^l>X ’ ■ . ’j ' ,^ • • •»«» ^ n'tfp^ .e^u^'ial ffi er^.rj^ tv’c VriU 'i ^tfus iJ:ui^M4lei iaftdi^oa^ll ci; ) ftSt3*6X h»j^^rmi& jttf' * ^ ' N .2 ‘-V bn« iW'UM | * ■ ' • - . • - . . - j ■ I ^rwq noit^sne nit.^ \c cii^d'a .^tmfq niEJ6:qtt9t;™n3l o^ 0 K’l ^Ja lo t/tf^-ii/'o Votf'lVo !f J * ' "J “• ' I* -itoo ^00 eit* hX ox€J e%rf3^^« V cr(0t o3 c*(HC;)w^ i© euaoQ »»jJiiuk/ >xi4f %fU hi Doi.'iai ' *i -, ■ , ■‘•‘A -' * “ < U3 ;/wo, i.( so)^. •ftfPidsa'ifi I , «- ' ■ .• i ;« ‘ ■«ifri ii*urJ£■ ■ * ^ . . .- ^ tt JlL< .»IF . ine^ =>as'-,- 7 awjjw M ii nnxy ji •23- "been continued for an hour or any other period, the suction pump is stopped, screw-cock No. 5 is closed quickly so as to catch % all of the solution in E ( this can he done if tube G is of rather small size) and the solution is ready to titrate. A few drops of phenolphthalein indicator are admitted to the bulb from the thistle tube K and the solution is titrated with standard HCl from burette D. The HCl used represents the excess Ba(0H)2» i.e. that which has not united with CO2 to form Ba2C03. By sub- I traction the amount of Ba(0H)2 used is determined and from this figure the amount of CO 2 which has united to form Ba2C03 is cal- culated. This represents the amount of CO2 in the air breathed out by the animal in the chamber during the experimental period. The Respiratory Chamber The respiratory chamber for use with the apparatus is shown in Big. 6, Plate An ordinary bell- jar L with the tubu- lature (atM) near its bottom, is inverted inside a battery-jar N N and covered by an ordinary desiccator cover 0 0. Application of vaseline around the edge of the desiccator cover and the addi- tion of a weight of some size on top will usually make an air- tight connection. A wire cage P P containing the animal experi- mented upon is placed inside the respiratory chamber. The bat- tery jar N N outside the chamber is kept filled with water which may be of any desired temperature and may be siphoned in from a reservoir above. Thus the temperature in the respiratory chamber r ^ • 4 ,. * ■ ' ^ .! ' * \ ^1 4 I . ■*■ : ' . ; ^ . ii ..vtfft: .nal^w»K s*f^V' r li H&di9 xc latiiT iie xo'^yUBUcd^nob 'lyi^t^ti i Mz'ihO lij uci tTtriiclo d *eIT >l^?'’^--•4i.^Hrpi «3t 6- c -^.o 0 li r-ac© r^o 9it^:t ) L hi tioitf Ir'jtflt tr; /X /i 9i iittfs.rifit i>n* ^uitg: moy’'- iSUrf rj Li*ttflfi8fsm rrr« ‘Xt' frfsI^ihqXOiWW "t P ,#:i ’_ (• 'i- h$j&^ li Mi r.ir*uJCo9 9'y t>rm^ P . »tsj « rfoltii ■ rt*li fito*/'* tyrtu iibhii*r.t\*?f nt r .i ' ' .'1*- > V l.Tii v T^ oi -:v:o .?i ^rOOy-at' iirica 'Of Joo lo .ThzfOfi*; ^h!f rxua'i.'X - . ^- - '■■ ' ' * .•r'4 >'-*Si-H^*cc ’li^T #vfT^ ff.l vi>.? *tc '£•'.• ' J .«7^''Xi.'0 . i . :-pjSi ivtix'* <>r” M J. • vr.i rtl, »>?» .iftf Jap ;‘..r ' ) -.-f 1^1 3AC*»*x^X^/i t^ r>: Sc. x xc^'Jk^'k x%pxM.,\iiiBpx oisT.^ -ucTw^ S ii- iiJiyf 4.ir^.f,-l;^acr ’c !/ gifoiity. fiA {>4^^:.i*T ,d- it KWOft^ a . '• ■■■ • * '"■• „ ' <•■» *^'-v ^ oi.'’ ;*i bJJ ww? (a >i?) 0 -i^r^vjpro iii* .y.<3' Xic>*rgrc>o; . |^ Ibpu 010 bni »f(J t- 0Sl-l>ci/c<'£# «N3Uf4iiv«xo' i!' -^liK njfi xXX-'iVf w 1?0^ n/> dria, och# ij lo^hoi^ ' '• - . <• ■ - ■ ^ f . ' '• -4^^ 0^T \-Todri»nu xwjril4iee% e-^ e^.iehl ' 1 * ’ i ' - ' . ■ " --! ' fjA ||^: ^ at vh«-nuxU\)i} xnh ‘to ’c»S) /j *^ . '■ ' ■ .- ' ’ ■f'l' ' ' ’ 50ji«Df:oy»<-Aoa^:'i:|^x.fti> oitj- til -iji ,- . ■ ‘ . -i"-^ r r.4S' . . j" '‘^' ' ' ‘ /..k ■ *“ ' ' ■■. . .■>'1-^*^ * -24- may be kept constant or may be gradually changed according to the needs of the experiment. The chamber is used as follows. The wire cage P P containing the animal is placed in- side the respiratory chamber and the cover 0 0 is vaselined and placed tightly in position, being held down by a wei^t if necessary. CO 2 - free air under pressure is admitted thru the tube 0,, passes down into the water jacket and around thru the glass coil R R, thus being cooled or warmed to the same tempera- ture as the air in the chamber and finally enters the chamber thru the inlet at M. Here it circulates around the cage PP and passes out thru the tube T at the bottom, and thence thru the tube G into the titration apparatus. Since CO 2 is heavier than air, practically all of the CO 2 given off by the animal will sink to the bottom of the chamber and pass out into the titra- tion bulb without difficulty. Por certain experiments it might be desirable to regulate the pressure inside the respiratory chamber, in order to avoid subjecting the animal to either a hi^er or a lower pressure than that of the atmosphere outside. This may be done by regulating the screw-cock controlling the inlet of pure air on the one side and the suction pump on the other side of the apparatus, and as an, indicator of the pressure inside the chamber a manometer may be attached to the tube S. The manometer used consists simply of a long glass tube bent sli^tly in the middle as shown in Figure 7. The end of the tube A is attached to the tube S of the respiratory chamber. End B is left open. Inside the tube at C is placed about one cc. <•> *^5; 0.' '■?*' *‘* . *-?vO,C* 0)^ 9/1 £|*1«U ^ i TO'Pl^fU’ ^^fl? ^rUt'^Ty^ x>rti I /. rr‘-^- •" 'j; ^iviis,*f ®i gou .^v. n*ut , ^ ' ' Ij;. . Ji b£9d ^h£^ .ec>Uiit\aA V ' . ^ ■ ■ «>- »l cTu^iioTo Tt.-* s*^‘^ - 203 od^ '/*^'xfy birt P'vf? ip/ ift/ T (J/t| itwcir 09®n»pf - .a9.^ 4^4-9 «.ft^ fU *ri* tf-j^WactsiO; ii Aa«.F /i i# /tijri, 'Xi^.;':^ - '” ^ *? ■ ■ ’ _ /K'** 1 " ‘ , a 9 -L,. in.b'y s^P**?*; / » i^ f'^'j ^ c T ■ 0 durq,‘>u:n ai.^spltev^^o /ti«4tPb *i«l^ .K^XablXliti A/*f V'TOdaTico^X nfj tiOlpiJt f> 14 iK-rXlrt^©*! Si4^i©#-i> 00 *3 'ra/(3‘X© f»rf4 ‘»o 64 a-^/fTQ ,fi: A> n ^’i3/fqaci54ji /w/W* , O"-^ j >' tp , ■* Om 4 . S*l/JXoi7f {130- ikOcvn-f'ev^pr o/X 4 ^2<4 tinol> cdT t4c(T^^^ ,- 04j 4 «6 CU>i4fWa ©r.4^ i>Aii Af//« f»/i6'0/f3 /tO ti o 4©xSl ; •■ _' ' ‘ „ .. ' I ' , J 'Ic tiii 3 *. i>iV, , otto t:»jAc .8 edu4 .or?4 p4j a©i4oison®«r 0 «h'l«rfjj , JcMtf ftcfttj 8o«Xs, >,rje/ 4 I'o >.vt<^j, 8#>itiaat; ikofitr wMooaSI. <,rf#'?ii ntff 'tti J>cm '«(rr .? «i r!W<-.iB o* rti .■tatfci,;^ ^t:^m■^h■KPt 'gtlJ■ lo g v»s^ «f» 0 } I«^x 4 ji '4 A >1 ' «av *f^ ‘ '•9 «V-Skb/ J«ctto tnoiicj oi- 3 it «)W- »«s mini .»t),», n*jf pi ft, ■■ ".:s, •■ ^. ■■', . v-,fi-jA.lifl| '■WptrsJiWE nw-tfl i -ri f ' tr^ • ’(“•■t; It 7 a > fk< - 25 - of Ellison’s draft gage oil. If the pressure inside the respira- tory chamber increases, this drop of liquid will be forced over toward B and if the pressure decreases it will be drawn toward A. By adjusting the air inlet and the suction pump, the drop may be kept at the center C and the pressure inside the chamber will remain constant at the pressure of the outside atmosphere. Experience seems to indicate that ordinarily there is very little danger of changing the pressure within the chamber enough to in- fluence the metabolism of the animal, but with the manometer attached, the pressure is easily adjusted and this possible source of error is eliminated. Experiments with the Titration Apparatus Table VI gives the results of some preliminary exper- iments performed with this apparatus. The CO2 output of several toads is given together with other data and calculations which mi^t be of interest. The results are given merely to illustrate the use of the apparatus. The wei^t of the animal used should always be recorded, together with the length of time during which the animal was kept in the cage, A record of the number of cc. of Ba(0H)2 which were neutralized, i.e. which united with the COg in the air sample to produce insoluble BaCOj serves as an index to the relative amount of CO2 produced per toad. From this and the time, the final calculation, given • in column 7 may be made, namely: mg. of CO2 per kg. of body wei^t per hour. rjt^±2?5; iiaBarau -OS- --ni; s\. «e,n:} o»(.* ’lx bnu a btiwbj j - , ;auq ;ioi;*&;/e lufiT ibit# , ai 'lU j>fU j ^j| . .©t^it^?;»curt3 pJ-iaicft axf4 ’t?' 0':Mi7aoT:T a_tr wU' S'iw i* -04 ,2ii5WfB Oitf ot3jKM*tj 0;Jt 84ii- *ti^ca-oxw.B .^ii :. t ai rrs M^4 * a. trc i tcd*4» : axJ’iacOii xSivai} tti ,4^ftdoi44* >1 *r< > ‘ * ■’- -* '• W|| - 1 .-•Xfjqr.s p^ua Tlo «4fMB0*x n.si* d^vi^ IV-^XdviT " ^ nJ:f 4 d4Xt>.: s>cl,'<3u ^ ^ iiOx;f» «4ToX4^£ifj>x:«> x>fof *i4/,x, xstto d: i-y «X 4»o4 ■,&4r.54»«XXi 04 x-iAiftra = ;.4t 1^4* oif 4dglm ao ^4iW car ..* ;x 5 ?wtIjwS :.aiiuc <«.;« av,0.4,m.t.'x,r» ,U1, TcajiBo, >c6torcx cc .?3 ao 190'euii e.-tt \0.1X9C>k A Jta na .’tjcjl ■>,« XAiLa: »d» ^ ofiS r.-tl.i-*' bsrbay .o -i .beail-sxjfofl .ST?-*" ds^JUs/j^C^’oiae' lo :»e ac e^^*i cO&^l® flJViUosoa 0»('!,0I5 of .V'l^-S ■ itJt fid: ab^(g 09 «j| flKia? 'ibcnt beoxA<«(l'jOO a« j/uuKos svUia.i oa» oA .yAcai 1 • CBIB Cii-.«eri8 ^ .J>. A , S.-00 a.o t?'^i > -26- In column 8 this figure has been divided by the body weight in each case and the results so far as this table is concerned seem to indicate that the CO 2 production bears no direct relation to the body wei^t. In column 3 some notes on the general condition of the animal at the time the analysis was made are given. These twelve analyses were made during the fall of 1920 using animals which had survived the summer and were being kept in the outdoor enclosure at the Vivarium. About the middle of November, the last of the toads died and no more experiments could be performed. Numbers 11 and 12 are analyses of the same animal on two succes- sive days when the vitality of the specimen seemed to be v/aning. The animal died soon after, probably on November 15. A marked decrease in the CO 2 output from this toad is noticeable on the second day. In these two experiments the temperature was con- trolled by siphoning ice-water into the outer jacket of the respiratory chamber. Outdoor temperatures on these two days were 3.5° C. and 5.0° C. respectively. The temperature in the water- jacket was kept at 4.5° C. and 5.5° C. respectively. In experi- ment No. 10 the period used was 20 minutes instead of one hour and the number of cc. of Ba( 0 H )2 neutralized shows a correspond- ingly lower figure. The Biometer Plate 3 represents a modification of the Biometer described by Tashiro in 1917. As originally designed by Tashiro the apparatus was equipped to accommodate only very small animals ."aji^sSRi ^.— ••ftCl* m r . ... . • ,k ‘ :^\rf. Ol .rriV.ifjT, >^Ocu^ (tfj' x.i{ i:«ptf .Bf U CJlriJ - o^(I ->(X'' Qtii ts^tiylbetX 04 ,D t 2 ^ 1 :i!tu<\Q >^(i 4 f<0 o*4o/t oacif ^ fifi:;^Xoo itl, o(£4 * ■ ■- ' ' f t. o, :!' .i:avt situt^y. ficf* ftJt^V;£Jk'Ul oKl? o>l4 ' A\Ciua4fta* W4^ t I «t;,4tifili!i, V'$9X 'ta «>;-> ^^4a> i*i 9 V Ott-ixX^^ ^Vif)w4 h — - ft * ' l*JlfO »iP4: .7l Iv^'i:' QOlOC DT'*f? w ^Uiii i 4 h #44 boVit, u« ikvif .ilc itfW ' '.J rt44 ,'L Bwt'vrl* *v .ftiOiioviV «i4W- 4 j» ©^»»oioA€| j* &9 Oil 0*’' w'tCi^r on a f ib ©^o4 u.'W X0r4cj^i^‘ >» 3’.- ^ cy 4 no i wi .--a;/ 5 . t© r*ii^iArc.M:o XI - .irtf, ^ c-^ >v3ti/3*«r4** ».U lo x41i *^jiv tvojit jsvio. * r * • ■ ‘ "a ■ t '! tilths ^ioi X .^L *f.iUu'’Vpl~ m( \t>< oo4r, b*i'b axiX ■fc* • p >:> ®i^4 fio 9,^Jrniii4fui el *t)V.^ oinU civt; .te t4ij(jBb ow4 ^0 6ia*To44ii**<'^iro4 %oot>4u0 ,7fs^iseiin V,ioJ ^ -Tni).ivy fiX tU/4«!;so5Ra?4 OitT .'c-LC“ViJ ss:^liyit -Ix'-^ixo ,nl ^ J\J 4 o 4.^031 uiti^r . in Off »no "iOt^oJeiji iVewfiJi'.^a ti^rr bii(^ -^aoi^vc. *.44 Ci .oft inocT '■• t‘ I ' - - ' I _ ’ . -DnaciftfTXorv * ili^-Dn’o 44e^I>rx4jL’ec .;.ai u ** i ' ' I "'klJp ♦ -*'#. I'tX* .r\ ‘I ft '^^fiJOoIS Xo flti^^til\it> 6 hi M aOAPX(.r9*X • [' oxXrix^T »i.3 if)^l««uv vrU^lUsiXG B/. .mx t-iiiCe#:Y x.fi' .'t Xr> , 0^ •XOg^'^al OOTI • . ■ ' ■ ' . ' .'j/'jjca c»*Kf ^od^.: *U-r. . ^ ■ . '^ t. ' ‘y. V- '-a r.^r ^ Lcr :>AP» t.!T . ^ I ” . ■ •'■ -i "' *4^ c*-*cy ii >»'' C^:o *A .4^t»n>*|f(li^ ^'tHfJ*l^?fl^ n;jj.N: ,«sr • iw- ^ 4:«/Ji*kavi^ »• Ia»or a,, “Xf^tscuf^' -i-t. iyc^: jr>frn#V: .r- j* iTv? ‘heiff j;giJ: 4.,* :u W •»« V < ^ , iV'-^ ^ itOJ ,^<1 *tc£ A(f ti« -iea* •♦ .Ovtv ;-a • «4,^ ’ic'i %u fui> -I alt if»r(v^ la cri]^ Cy'J^ v»‘4i«ft<'<(©u4£a,- I -’ -I *.‘v ’ ~£L V^» P.| e-tp^^. *0^ .aiWJfcxtrQdi^ 'rc^J^ritJrl ^ p 4 ln:tiQal c^ tt.il^.^8 o Uif. aai; iW A I 1 ^ .b"-, . ,. , . , v: , c ..aOO'ljtSu iia-X' 01 /iif titl-f inUx ssEr-rasij 'jX* Sail - kOO od¥ ■-■' <-•'■ r ■•, ■■ ■' ■ ■'•*■>• '■.'' ■ '■ ' »■ ■. '2 • . 'i g,- .1 t^9m'x‘-^ jrtoJaUqfw BTj o#.t_i V a«tiu op's at6t liiui ,,.C Wsi. ,2 10 aopx^utf, e^y J05 ! 'bx"’bXiIT .T orfuJ eW ’irrp» Jop POnV^J -**.%«*-*. *^11- ... * . .. .r>polh£^^ rfOBfi ne‘ !V^ptiS XIOthi tot^vCi O ^'^.T u6 hoiTuuv t/oj ?*i »‘. ’ ,ji iDooqel# • * ■ ‘ * i et'iX h.-T>‘. ,2 jyL'o SL rca''^^sil grtlwolt TtiS -> yr. - v^ - <>9K ^ .saCt’-V- f " oJ -'ioVoXX# rX^tgriiCtfo fc.T4 c;i j?j!>ooqbX.« no/iT . ;iii tvJt d* Iw'* ? ■ ^. ■•“fi'rt'o pt^, ^ “'o oti rtrfi'Sky tx ^ 2^s>oDq&x»- xftm-t\rtii^‘ /, ^XCT't? j’-' j;.ft o »4'ui intdl nxtr* 9H'i»Xt v'/: V '^ . iL' ^ *i;io * ;^oo o.i^ bvt^ bvifcio ?i a ^ce>qvi»»{ tdx 0 ^ P^‘ .^■^.'X i)rtit XX'<; UpnftX'i ^Xit.U'<5’Xv>*iJ tJWl .t’Jto * - ‘ 61; -T 3Cot‘!>-wi&T0« 1>.*J/ ncXi^ » 6,ir «x & v5/*.WoT;>»fei^^"'.eoi# • oei oJ ^•x>bi>4i r;:lw V xm 54-»V ^*>iieXB-£ eX 31 o^^o.^cT\- .t '^r ^5 iBiu'i^x v*^o*r&*j oiCT .£ o^rioo^oXa tc-'JXairirX ,Y XXi.1 bS d 'f ftl) I(,'/itJ B.isJ cX € ioooqo^ o^ ,0' «<5u>, 9rtX «:.-:x £»a ’ftw'Cw'Xj# otf ous^xloo 'cti:cie» ^j, j ^ iX X^-^fcoIo t 02 i i»l b" 4OQO*^’0ijod ( aifitj bxva afe- oor»oXe 1 | > oKX Xxo^ . ^a:u 6 ’u«Xt >0 XXi /5 Y j/girdei . I .4 T®^J" > I I . c|b«fihq^' f»t)Tl X- BVS0I jK* b Xbcl'^o^ a' P S- :. ,.; '*/.r\''' f ' ' .• * .»v"-^ --^ 4 m o&8iiii al' D' -fioXe «orCr *3 oxxix lc;leV?x; rlXI^ r !' ‘ i . 'HH' i . ’rt:£X,4yo g^XTi«>qo ijjd*! Vtc Xi/Ks 'og oe ®i ft*j?ooo -ji if5QV^; ’ ’•^ ' ‘ ' . 'i) 'v *-u r^pT: .ij.TSv, ;:oiX iooq ct Zapiliin. pt^'X, anJlgiJ'X/yoo ^ *l mi*>0 rAX owilo* ;• ., ■*' \it ’*" • , J7 \.. , ' vurf^*.X<- rui: yrxm »t i4an> oftj ' '-i(i;f<»7!J{V *Wff' ■* ' ■ 5'’^'- '^' ' ' 1 . ‘ < . ' uJtiL4.Xl ,.r*: _ - - i'fr'i * ' 'll III* lil'>»i(i(^ lTllMl>»»i| ^ r ->«*|i^. ) r, . -29- minutes or any other period of time and then a sample of air is removed and analyzed. The method of analysis is as follows: With stopcock 2 turned obliquely (i.e* entirely closed) and stopcock 3 turned so as to connect I with V, screw-cock 6 is opened and the mercury contained in tubes V and I is allowed to flow downward thru 6 and thence into the respiratory chamber. The vacuum at first produced in tube I is later replaced by air which bubbles past the falling mercury. Thus tubes V and I are filled with air from the respiratory chamber, i.e. the air we wish to analyze. Now close screw-cock 6 ti^tly and open screw- cock 7 and raising the mercury funnel N if necessary so as to fill the lower part of tube V with mercury, thus compressing the air contained therein. Next turn three-way stopcock 3 in such a way that tubes I and V and burette J are all in communication. Mercury burette N is now slowly raised and K is simultaneously lowered so that the air held in V under pressure is forced over and into J. When V is completely filled with mercury up to stopcock 3, turn stopcock 3 so as to break the connection between I and J. Now adjust the mercury burette K so that the height of the mercury in K shall be the same as in J. This adjusts the pressure of the air in J to that of the outside atmosphere. The sample of air drawn from the respiratory chamber is now contained in burette J under atmospheric pressure, and ready to be tested. At this point we should briefly review the general principles of the Biometer as described by Tashiro - The air to be analyzed is introduced into the chamber E. Then a half- Bi Tia. B iritic Iw boi*tt>N^ 'it'S^o Vffj t '^^ ©.fii: 1/tvl a; r ■ ' ^ 5 r • ■ ' .1^ viCXo .-Na 4 ^XoUP'lX %' )(V04}^0Jff ^ b Jtikoo-Wf icui fi/iw 1 iai^^co Z)»f\Tiud‘^»-C [► SnYrcLX\^‘ Hi 1 b/fA V D» /fui jhi f>r>«j t,vfMSa x^tjoi9M, hOJi ^c>cr»tfQ^ai; . *. i^j*r!o mU' aonr*® ># bttid I utfit tf*u .'Ttiirol) wol- o^^ t 9^ifj li'i t t)air4>iq auitio*:v ofIT i:*rT .Vturyricydytf rtaldv ' a-^> mb'! hBdiXt “'* 0 H if ii ' at oio \iroSl >6^ (faXir v‘ c4. e*r'o« ti Ji Xtr(.7#-£ ’^<,..'1 y,y^^n hm ito^oc , ■ ' ^ i pj^ '^f^ibutizqmoa BiftH ,-^i'tj'jvfr. 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'■.*■.• j . * ■‘i ©iiv .9-:94^q«oaxU' nhin^Lwyptii^ tQ Jjir!3 / f. ,^X 4 | 4 C;o’Sq 6 l 5 ;©ifq|| 9 i»^i» 0 ^S/!ffiU ^ nl " Ift^©^5sg \^-i«XTtd‘bXi|t)de. -aw,, jrUuq- eXdj;,. ^ -viort X f:©/i? prt,^ iioai/lib'xd-itl Jt^o^'cl^AfiA, 9 VS iritis fT^’.jCJ to ftoliu-Jk^ 1 . ■ - ^ -iwr* Iwii O.J n 1c f^.ucrv) 0. ilo,6ox;o ^>^. , a , , ;. 4 ■ --^V- 3 '< ^' ■ ■' “•f ' ' k%'^x\z t^\ t.~-Xn: t\ ,0j C x$, lijXJi?* vvai-Xi<>co hiiih^*XQ nfc #Uo • ' -V , .-4 '^0,j V*' j £4 j^\tjcS^ .i •ft'.'Wi'a til mta^ T)n*^ ,C ija it oj. cc-s^"5j^uo 1^ firmer Diinii sti^ n a i&^Juiado ni tU ofiJ *«Jk^ ‘ - '' ' •, *-Xf -veer* 1.. c^,^iqrlt>v'tq- hijdlaItjMfl utitfprlitn ^'jtr-J ocftjr».f«f^6> ‘iwVj hr/; 3; .nVi'vf i*^iw “ 0 U * •XCU. .n. (iCKfitioxf Xit^ .iOXiv lo, J iiuti#i3^.iS/;o'X f*' »' » , , '* . f: /. '’sf ** 'A o:r :^i>S5TO; ^oj X ,.x lU jvi?^lu£i ;- Ji^ ec* ..• o^ ti -■ f- - ,/*f»w^f " c£ ia lUj.ttf k^%f .■'•'^I'p y^flXovdo*!.' .li. v:TJ .jjOO lo " •Oi'T "O. i *:i,t 0^ . ®«-{MXo\ «i jn. i-H;/«*i/(:;^ ed^, o4iii fivtt lo ♦j9;4AX‘^ s’’" -.:rs5i^-ixi ’^i«4iiacT i)m~, X, M) io-.q fv/t? »I howXaJ'dco voi? o-Jt y \ ■- -^ ' - "it n > rroS.-r.., cs-u* ce f- ^IcvoqQXv .tt» ftir4rl\t€r'iv/e»X: ai ^'*cpqr^a. asuj roK J^f.aaS»4 ■ -*s?woX fjci^.o S^' qy woX\ tij ^(Hdhe ^c4la ot 93 C3' S "5(c’co(H)»a.ocpXftv,,ti: rl^o^X' > .-■4 ... . .• r*.'. } . I 1 dt.'iif iu..); ... iStf*'C’i Pi.^;»ft4Tq^ -»i WtoXIai • ,»t teofew^a ax oa-wr^joii/^ fsi •DMi;^‘.i)i2jft:f& ftarys.4 ..f-4 H/.. . . i . / ' B <*auix.o Ti'tvi^aK irfl *p >»,6"o.:? -31- J. Slowly lov/er burette G and simulta-neously raise burette K until the mercury column in J has risen a certain number of cc. This number represents the number of cc. of air introduced into chamber E, Now close stopcock 2 and very carefully open stop- cock 5 until a drop of Ba(0H)2 appears on the end of the tube at D. Close 5 ti^tly and wait 10 minutes, for the appearance of the precipitate in the drop. If at the end of this period no precipitate of Ba( 0 H )2 is discernible with a hand lens, the same process may be repeated, raising burette K and lowering burette G, thus introducing a few more cc. of air into the cham- ber E. This process is continued until sufficient CO 2 is intro- duced with the air to produce a precipitate of Ba 2 C 03 on stand- ing 10 minutes. Then the number of cc. of air introduced con- -7 tains 1.0 X 10 grams of CO 2 . Knov/ing the capacity of the respiratory chamber and the number of cc. of air in it which contain this amount of CO 2 it is a simple matter to calculate the number of grams of CO 2 given off by the animal in the ex- perimental period. This is usually calculated in terms of grams of CO 2 per kilogram weight of animal per hour. Use of the Small Respiratory Chamber Figure 9 shows a small respiratory chamber for use either with the Biometer or with the titration apparatus. Such a chamber mi^t be useful for studying the CO 2 output of insects r r • JJ 'n. -AC, - •j ■V# .’iijt^'T s»kJijU>4 tfri iifT ' r?; -c-c:*?* 'ciS7.ffojfc 48ff£o. Hz cc:U» j- - <»0*s5T!»f>a*r •“ .'i '^L Jirs* Jbr-^ c .'! 4" *■> ' ' & .e ®! .«* i ^ HX . >.n? p. 9 -? M -^p ,W 1 <• • o , ■ .♦» (if - fiTxW. o^Virr^»^»aih ;ji in Oif .s _ , - » ' '■ i' Vi : ii*. 'I aj'- i ,oi»^4o©iaff' oi ij<»ti.,!l;j.-.cti »i oai^oo’i(i> ^trtT .IT offtf j ' *. ""1 -.'3r*ria .no oX .^ t w -{TOO <3uovt.()*tXj i: tXa ’Icr- .^.'^ ic Tfi''/ r.J av5' fvj * 4 ; !»:;<, ic* »/; .- il^OnX .rO:- 1 < c^tr '"or ijC^^ •.nvTis'i ‘■f'3‘'V tiX ti-- To .’>0 Ty. *4Qt’*«wa 6f<^ |ji*x4 T 'irtnilj; T^^v^i^,*ri(28o.n ? 9XjaXoyX£;o, o;^ a uf »<;bO Ty oiai .tiyX'Soo A I- 'i -xo TtX- X/^Mirx- £>J^ x( 1 ^ aorxV i*- •?'-.”’ic «Jlf ncsif'A^ 5«r:'a,t t^is§Jiti ’ wl.tC , ^ ' - t V • — * < i .rtycfC junBOii* te V r ' -M^jp ^ 'lat^a^dO Vi I (^pfl X \rif To ftoU aoXJ^nxii o;lt ^(XiV *f^ ^iat^vUC.B^ wiUr^ ’ 30‘r^Wt;^^ e< w^%Xxff xptfw^/oV 3 E/^, ' ' -• , , .'.'k^ -'‘ - ' — ■- 5a_^ jr ’ " » ' ^’, 1 .'. -32- or other small forms which would not require the large chamber shown in Figure 6. To use it in connection with the Biometer, the proced- ure is as follows: Place the animal in the small screen cage C, which is fastened to the glass stopper B’, and insert the stopper in place. With stopcock 8 open, lower the mercury burette F* until the height of the mercury column in the chamber A* is low- ered into the tube D’. With stopcock 3 properly turned raise mercury burette K until the level of mercury in J is at Z. Close stopcock 8 and leave the apparatus for the desired period of time. Then remove a sample of air for analysis as follows: Turn stopcock 1 so as to connect chamber A’ with the tube U’. Turn stopcock 2 so as to connect tube U’ with I. Thus we obtain a continuous passage from chamber A’ to burette J. Now by simul- taneously raising burette F’ and lowering burette K a portion of the air in chamber A’ is drawn over into burette J. Turn stop- cock 2 so as to sever connection with chamber A’ and the air sample in J is ready for analysis as previously described. To use chamber A’ with the titration apparatus, turn stopcock 1 so as to connect chamber A’ with tube U and connect stopcock 8 by rubber tubing with tube G (Plate 2, figure 5). CO2- free air is now allowed to pass thru the temperature control coil R R (Plate 3) up thru tube U and stopcock 1 into chamber A' , and out thru stopcock 8; thence into the titration apparatus, where the method of analysis is the same as that described previously in connection with the large chamber. 1 ' ■ . /; ' TOW -< . *’ . '■' ' -.' - Ty i'rfcr**\0 flj ‘1 *X ^i3U. tfXiJ JW 6iOtl f* d ^0 >..i ■ . ^ ^ j T -' V i ir* »_ #■ i" ft ^ • -i !_■>»>■’ ’ 4 , i?t owoeiB j f > • 4, - ■> - • ‘^' ir»J02- ©.’J ?/o4 Jo©imc<> U »i eaX^ wi , *r j . :Qj T' « Wox-s^ « x^fti o/ ,«©ac ^ ••‘jrjtiici ''•••'-• * ■* ‘ ■ ' ‘'•‘^' j r -hC-l‘ 4^ *A x-KfMr t>ifS> ^i. H /lU’C: TiX^"aX?»T J .. |©;( otif ikJtZvK\ cal^ ^-srthfS -r^ /iv^ XV f of,oVj 't' ;iyX© #rrjT i/roXi«'t ©4. xiii io o«cfAIfc"x^a* ix-ruico ocf cfe TX ^oo/^oia M •'._ .A-..,... . . ’ ^ . . ■ - ■ = ■'•■■t ' .' w , ^ ai^c'o t'f npfff .1 ' ij oiri Jc*©^jntfu ©X Cr. 6a, S a^oip'oijfc^rjj ft uVjH • -i.jL/ri^ Vfor, .u'©iy^x*:;> cX <*j. aioxt ^Cu&k,*^ mfOURli c ^6 i ^o noiyioz ^pun6ttU (^uji ^Xm\30x\JB!^ - 3 o;a a-ri-T .1. oi^i ir.v* /n»*Tp .ai ■ -v, lia arij • * I * ■ ’ '*#■ ' ■ ^ ' ' * , xl>‘ o:U bn- '-ii Te»tfi^'»c- dS iv nc?t4'^bac«'iii t;^r^v: ©i+ nf oz $t :iyoo «r ' ' ‘''•' ' ■' •’ 'v ' ‘ ’ ' V * *' '•■jy ^ 'ilXir^, . ^CUrMX'J ©cm o?' •'* ^ -'X- j p' ^oacLioo bff.k Cf ti*, ^ : ^o»oaoo od, q5V“0H X'^aCbaOX© '^ .'U .©Ty^jX*^; i>^* iri? xotfcfA'X ^Aa^ oJ'ni XAj(5lf.o4od6' /)«a U i/xfe -»5Jtr 4c’ iiVsl . - X ■ - ■ ■ < " • ■ t^iiSn'tAqqf^. aAiJ cxtctX .uon^iLd ;> ** \t5l f 'V w>^ ■* 4'^ 4 A' .JV ‘ 'i ei n--: 1 • ’ ' ‘j — ■* ’ j* iMUntfUm^ xLciv^tn^Jt e'ir"4'<' tx 9*ldiv i.v$ ‘ X«rt>nx*0i34?a li ,4X^c^ t:iu \fd >3h«wot^ti» : /l kL. . . * « • * .' •> Uo«t 4»rf ^lK»A *5it^«j>V£ei Bf{j ’‘! t l||o i i Jq^^#w4 jt ■ b .pjB jf iiir ,ijiicu a 6 ? jeio^iov^o^ 1 i'»' r rw r- j-' t- .' .^u fWi’tr „ ' ’ 'jj '^■ dcf oj t>o^r,oq<^0' rfv.«$6 trifl]»'u osi^Xi^ XJTj* ^ jailJ ^'fs>vp4: fti- Xo Ayra XXoT" a^oqrx^o/t «i ^CAi, g&aoT • ■' .. ■ ?■ ■* ■ GOI^ni vr^t.i;. i. f ‘ L ,' .■ 1 i*« * , ■ .“U ■*‘J Vi iia .is 0^ V^ 00 1 0x53 Q Ti J'ox oa cf . .jft* -i9n"Mipwf: ,^cif .m k ^ . -34- A. Toads which have partially dried out rapidly talce in water when placed in a vessel of it. B. Toads kept in mud usually remain plump and healthy, "being somewhat swollen "by the amount of water imhihed. C. Preliminary experiments on the absorptive force of the toad’s skin seem to indicate that: 1. In the live skin osmotic action goes on more read- ily from the outside of the skin toward the inside. 2. VJhen the same solution is placed on both sides of the skin a current frequently flows thru the skin from the outside surface of the skin toward the inside indicating that some sort of absorptive force is operating apart from the usual osmotic action of an ordinary non-living membrane. 3. This absorptive force is stronger in skin taken from the back of the animal than in that taken from the ventral surface. 4. The absorptive force varies with the solution used and with the water content of the animal at the time when the skin was removed. Acknowled^nents My thanks are due to Dr. V. E. Shelford for the original suggestion of the problem dealt with in this paper and to Dr. Shel- ford and others of the Department of Zoology of the University of Illinois for many helpful hints and suggestions during the course of the work. t .. ' ' . . , • , ' T nJt t'Aisi aiJ-art* rr * ..^i to Xeid«V i fSjt ’ ’ ’ I hi .QssjJfqt nlnars>"2 xirmumf /li *4.f> J.nt’fitirs -jiS nt-ircm J lei jsTol pvB^ortO'' ti-i^ rio W tcacx.-^ xi^x^ “ • / -toxjfn fTt^ CO noi:J*/ oi-:r;jrflj^, ulia' cVjt 'V.:? al .1?^^ ^ v If «. Mi*,-i* fs.id.3i *i::i o.t #'e>oa ciafB • w , jj^i. ' l^Ut f ' %i 0,. 1 fil9 ‘4^Zi f- :. j^ - ^ - aXo« fi/tat# 3^.'> a^{H x?i>ffc »r^r xrid,}' tvoi'. 7 .; itilfli b£|,?' s.vU 5>i>v*ij^ /Yi'^n- or>/- lo 4t,Xg:f /,: ■’ 'ibo^IV-*^ £>rl« Mr ;. iB _>0 iTCUfiL tliwfc’ If i-VAf •, ^i.'Ji.‘^;rl» i i ." 1 i‘:> J f pj: r pi/rlHjo i,: tni ' *wji"v ‘ t,. ■; 'V '- trv Ch aoiim ■ " * ' ' . * 1 ^ Oj^iti^ gl/p itX at ooTful »js ittxifi, V 5 P r „ ^■’' J1> Ifrf? “6.^J *ta i •i?va>irir. Lt :s^i«\ xpJai 6^il» o'-ff ^4Xr jaJLXav i>o*soi *».4iT .. r — * j . • * ‘ ^ ' * *r . •■ * *>:’j v4^ ^^ipu> or.j to o.t'fJ' /L..*vf Jbiiaa ■ ^ *.'■ ' .^pv.orfljt fiertp 9^5%c o4w OBcifr iMaJtg > / it / I ■ I . og^£i«^«rpnlDA .;fi b;tr tqI fe'xo'J-XpAC^' :V^ .•:^.X' c,i riimrii- rJl ' .Td 0+ ai rt^lw t X6 ^ #. > , ■■'•'. . ,, ■.-*•, ‘ -1 -J -i ic "la i^olCrStK io-'ifjociijrti^, fn<9fifc f>r... .'loV- ^ - 4 ♦ . • ^ X ,t . • * I /o*f*iapo Sjtiiyft aaolJ’o4ig^<^ ^ri>i.BJn4!il;£i(iBXr*rj& «Xoox:.f I 1J “ '■ ■ ;.■ >■ . ’ '‘■'•'•.'■J . ’■ bn0ip iJflAil.'l lSi4 -I' ; .T ^ ^ ' * L. -3 5- Graphs In the following graphs, figures on the ordinates indi- cate the number of millimeters by which the column of liquid in one tube of the osmometer exceeded that in the other tube. Fig- ures on the abscissas represent time in hours. Graph 1. Showing the effect of ordinary osmosis thru the fresh toad's skin. Skin taken from the ventral side of a toad. cane sugar solution on one side of the skin; distilled water on the other. Curve X shows the effect when the sugar solution was on the inner side of the skin; curve Y when the sugar solution was on the outer side. Graph 2. Ordinary osmosis. Same as graph 1 except that in place of the sugar solution some water expelled from the bladder of a toad was used as the liquid of greater density. Graph 3. Shov/ing effect when the same solution is on both sides of the skin. Different effects -with different solutions. Skin removed from the ventral surface of the animal. Graph 4. Same as graph 3 except that the skin was removed from the back of the animal. Graphs 5, 6,«&:7. Use of the same solution on both sides of the skin. Skins taken from three different animals which I varied in the relative amounts of water contained in their | [ bodies. Graphs 8 & 9. Use of the same solution on both sides of the skin. Specimen partially dried before removing the skin. (Loss in wei^t - 31 gms.). Comparison of skin taken from the ventral surface with that from the back of the animal. V t ?;W: , jTM . ’■'‘V<#.^ T .i ,/ii ^ii;i>XA io ratuXo© ^£^•^ aJjin 1 ■ -:.,ix .oq'jjS i<» 'J^o cl:C jii ©<»lx» 3 er)T^’ 3 -wi\it;t^i 4 l>f a "io » ^ -5 * * ‘ - ••• .aijoc nl /p»a»"Uif**r tEr.'aa.^ml:« o*rv d/t5 1c siji’»i 0.^0 .*c to;?*' ft;i^e’'tf *V < - ‘•i' ‘ ^ »' *'i'-v 9;ii i^i'ajp Xoa'He i»rvX ?j*rc/ • 'I C n • ’ y ;t:i fc| ' eiij' u *:‘f9 X9i9^^6ar fto «X aoXlAi:l(. slj^, inclift .£ iiiipntx^ * ' • * ’ " . '* ■■ ', . ' • ^ ' '' 5 ' ‘ a.” t .i 5 ir/ c a c^-T^vxp ircnM'HC .'t:£iffi dfi: Ic -’'; . x® ri. •> '.w/- .. ) V ^ . /A jK|^ . *■ .isiijftiftA erfw 1 u T.fi’tJjtdv btfVC'j^u r* ». 4 '''IjlfwS .' .■' , ^ ... - ’ ■ -'; ••; .-..'.-i >/ iip'/piitt-'i %i,x. (tlM Xitii. C «> aa/;a l^*V' ' • '' , H * ’ ^ '• '4#ajL- .li^ifiv jloArf tifiijB ’ ’ i8fei mi 'io n^sht ric n;oiti/iOf> frK#o .V / >-) ul nsiqz^& rn^y I- ' ... I *.;. v .^- -' onX^itvT09 ’^6' Q^:fu/oi03 gix iJoXiiiV' % i '• iX'»v ■.a-*--?**! . ^ , Bfc. . » ^,. ”• ■ yl'' .x:/>‘c «*;C to *vo5 :-. eaowi ; 3 >,i:vo/m >^ r' '■ ■ "'' i’ * ' *g ■’■''15 r ., o^T4 «fc-iy. fiistti .( .eto Ifi - X-rtelrpis 'tii-T^ c ' ' , ’ ’, ' / »• V I ■*- ■ ■ ■*■ ■* ' -s •X#4^iiW to JjtktSn'fY — ';'~' fyAT i' ~ 4 ..'~ *.* 'Y%' ~~^ ~" * . .. ^ I. " F^r v\ Jl OToHfV\ sv 3-X3 w/ nr»w “40- Plate 1 Plate 2 Plate 3 P la tes Apparatus and equipment. Pig. 1. Feeding cage; Fig. 2. The flower pot type of inclosure; Fig. 3. The osmometer; Fig. 4. Apparatus for obtaining CO 2 - free air. Apparatus for determining CO 2 by titration; Fig. 5- The titration apparatus; Fig. 6. The respiratory chamber; Fig. 7. The manometer for regulating the pressure in the respiratory chamber. The biometer. Fig. 8. The apparatus as modified to accommodate animals as large as toads; Fig. 9. The small respiratory chamber for insects, etc. «tj '5 in . i ^ I 4-1^, hi / ;ff<-«oIrirri, lo > '. - ,3 - *5oV j.--> • ^. {’i^i i^|g I! g , f) f \<»*I:bu«> t.tJQi|4 tjp ;*/ .'* ' L| ■' , ■ ’“- -J ‘ ‘^''-Vt ■ 1, 1^ v'lo^^ n. o«<^ • * 1^ t ^ ‘-'li«tlinr : •» \ ■ir ' ’ O'l, »1 V M I ■■T I ^ i "' ‘‘ iKW^® . >•■ •:'™ ’siP '■« .fw' if’A'Q' tf':'.*,'- .: ti ' '• v» ~ ■■■■ '■"' - .'.'■Jf. ‘t '- "^.wT ''V. 4 '’ :' jE"' % *' 1 ■ 4 ^'T-T ?| 1 1 ♦T' k M xr9: m r ^■ V • ii c')l -(> ■ f*‘p ; v.V , . *’ ■'■‘PI 1 '**; :i\ \R% sf •‘•J l H tjiL* iv; thi yj' *r *J • • > V/ p« ^ ■ ' I f < f*' ft) *i I. L ji, 1R1** 1 ItN'. ■ ipr, % 7t< * ■'■ /Vi 1 Hi 1 ^0 tel >. 2 » > J '■>& a XHi > s& ■t /.^V i ; A v!l s: fft'.'H ■t- % ■> ^ k*' mr^ ..iiW^ 14 ^ / V-»- nHj ' V’ J i ^ wikiii -44- TABLE I Determination of the water content before and after drying, after being replaced in water, etc. Max. Min. Aver Wt.in gms.at begin- ning of experiment 99*0 52.5 71.4 Wt. of survivors after 52 hr s. drying 60.2 41 .0 49.9 Loss in gms. after 52 hrs. 39.5 24.3 30.7 ^ of weight lost 58.0 27.0 42.0 Wt.of survivors after drenching 133.2 80.9 98.5 dms, of water taken in after drenching 73.0 39.9 48.9 % of weight lost by 8 survivors 42.0 27.0 36.8 % of weight lost by 11 dead animals 58.0 37.0 46.5 Weight of 1 1 dead animals 62.4 21 .8 36. 1 — r • • — - - t' !! 1 ! 1 M s\ H I J5»r..''r-Tr. • 1 r\t\- • Y'^'i ’■V-\J4 1 l! I -Vi ii ■ n m •\ i ir -45- TABLE II Showing the method of recording readings on the osmometer. Height of the two columns of liquid were measured in millimeters at intervals. Columns in the table marked indicate tubes covered by skin, those marked indicate open tubes. Differences between the two columns are recorded under "diff." Date Time Hrs. • 6 ^ NaCl 5 ^ alcohol weak chloroform (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) ~h — dlff . & — • dlff. ► + diff. 5/6 10:30 P.M. 68.0 68.0 00 83.5 83.5 00 71.5 71.5 00 7 9:30 A.M. 1 1 .0 68.0 60.0 8.0 81 .0 71.0 10.0 72.5 7.0 2.5 2:30 P.M. 5*0 68.0 58.0 10.0 76.0 65.0 1 1 .0 72.5 68.5 4.0 8:30 6 . 0 68.0 57.0 1 1.0 73.0 58.0 15.0 73.0 68,0 5.0 8 9:30 A.M. 13.0 68.0 56.0 12.0 65.0 52.0 13.0 73.0 68.0 5.0 1:30 P.M. 4.0 68,0 55.0 13.0 62.0 50.0 12.0 73.0 68,0 5.0 8:30 7.0 68.5 53.5 15.0 58.0 45.0 13.0 73.0 67.0 6.0 9 8:00 A.M. 11.5* 69.0 50.5 19.0 57.0 41.5 10.5 73.0 67.0 6.0 2:30 P.M. 6.5 68.5 49.5 19.0 48.0 38.5 9.5 73.0 66.5 6.5 8:30 6.0 66.0 51.0 15.0 44.0 37.0 7.0 73.0 66,0 7.0 10 8:30 A.M. 12.0 61,0 54.0 7.0 40.0 34.0 6.0 73.0 66.0 7.0 12:30 P.M. 4.0 60.0 53.5 6.5 39.0 32.0 7.0 73.0 65.0 8.0 7:30 7.0 58.5 53.0 5.5 37.0 30.0 7.0 73.0 65.0 8.0 11 8:30 A.M. 13.0 57.0 52.5 5.5 34.5 27.0 7.5 72.5 64.5 8.0 2:30 P.M. 6.0 56.0 52.0 4.0 33.0 25.0 8.0 72.0 64.0 8.0 8:00 5.5 55.5 53.0 2.5 32.0 25.0 7.0 72.0 65.0 7.0 12 8:30 A.M. 12.5 55.5 53.0 2.5 30.5 23.0 7.5 72.0 66.0 6.0 8:30 P.M. 12.0 54.0 52.0 2.0 28.0 20.5 8.5 71.5 65.0 6.5 13 8:30 A.M. 12.0 53.0 51 .0 2.0 27.0 18,0 9.0 71.0 65.0 6.0 Increase in mm. over original height 10 00 2.5 0 Maximum difference 19 15 6.5 ♦ - A* r - 46 « TABLE : III Results of experiments on how this force varies witl solutions used ,etc. In ai: placed on both sides of tl Condition Source An Date Exp. Sex of of ( no. animal skin in 1921 -May 2 partially dried belly oui 3 normal It 1 4 II tf i] 5 II li OU' 6 ij 11 i] 8 M black OU' 9A partially belly OU dried 9B partially back dried ^ belly 1920 IV normal B back May C " partially A belly III dried B back C " A belly II completely B back dried C " (dead) -46- TABLE III Results of experiments on the absorptive force of^ the toad's skin, showing how this force varies with the condition of the ahimal , source of the skin, solutions used, etc. In all experiments here shown| the same solution was placed on both sides of the skin. 1921 - May- May Condition Source Arrangement Exp. Sex of of of skin no. animal skin in osmometer 2 partially dried belly outside down 3 normal tf It II 4 » ft inside down 5 It if outside down 6 II n inside down 8 It black outside down 9A partially belly outside down dried 9B partially back If rt dried _A belly IV normal B back It ft C " partially A belly III dried E back ft II C " A belly II completely B back If It dried C " (dead) ;j Millimeters of difference between the two columns of liquid. Condition A B C of skin ■ max. effect max. effect max. effect when used ln.6 ^ NaCl in 5^ alcohol in weak chloroform fresh i ! 0 1.5 1.5 It in water ! 0 i 1 .0 22.5 10 min. i 1.0 0 1 .0 - fresh ti 1 4.0 1 .0 7.0 1 1 1 0 1 .0 1 .0 It A fresh i 19.0 I ^-5 15.0 6.5 E NaCl 3hrs 4.0 C " A fresh 1 .0 i 35.0 B NaCl 3hrs tt It i ■ i 40.0 c i 1 7.0 2.5 fresh It 1 ' t4.5 ' . 24.0 36.0 35.0 12.5 37.0 1! 83.0 8.0 ] -47- TABLE IV Showing the difference in the osmotic force exerted by skin from a normal toad as con- trasted with that from one partially dried; also between skin from the belly side and that from the back side of the animal. Fig- ures represent maximum differences in mill- imeters between the two columns of liquid in the osmometers used. Condition Solutions Skin from Skin from of animal used belly back Experiment no. 5 8 NaCl 4.0 19.0 Normal Alcohol 1 .0 15.0 Chloroform 7.0 6.5 Experiment no. 9A 9B Partially NaCl 3.5 1 .0 dried Alcohol 4.0 40.0 Chloroform 35.0 2.5 TABLE V Showing actual Increases in the h( liquid columns ; In various experimi Solution Experiment numbers 3 4 5 6 8 9A 9B .6 % 0 0 0 0 1 . 0 1 .0 0 NaCl 0 0 0 1.0 0 0 0 5 % 2.5 0 0 0 0 t.O 0 alcohol 2.0 0 0 0 0 0 0 weak 2.0 0 1.0 0 2. 5 6.5 1.5 chloroform 00 0 0 0 0 0 - t I •V '• \ . \ . . w • : t t f '* •. f . ‘ - j •• I < It j t . '4 . Pi <« t/X4 - 48 - TABLE VI GarDon dioxide output of twelve toads as determined by the titration apparatus Date No. Condition of toad Wt.of toad Time iin ©c N /100 Mg. OOp per kilo CO2 per kg. in gms. min. Ba( 0 H}p per hr. divided 1920 10/30 neutralized per hour by body wt. 1 Active 73.2 65 11.89 ^ 32.69 K' 29.30 .446 2 50.0 60 6.66 .586 3 48.0 60 10.55 48.35 1 .007 4 Female, sleepy but 60 9.84 i - .279 not hibernating 88.0 24.60 5 Active 68.0 60 9.63 29.53 .434 11/1 6 Female 1 18.0 60 11.38 21 .21 .179 7 Sleepy, not in hiber- nating position 39.0 60 9.43 53.19 1.363 5 8 Female 109.0 60 15.37 31.02 .284 0 Sleepy, in hibernat- 60 9.02 1.180 ing position 41.0 49.40 10 73.0 20 3.08 27.84 .381 13 11 Hibernating position moved when touched 40.0 60 8.71 47.90 1 . 197 14 12 Same, stiffen but still alive 40 . 60 3.69 20.29 .507 » T. -r: SC- '•u *’n j = luV ,.Vi„c ■« ■ .^jL , : t i<- - nii *'t &&V \ 0 l .' H <> . I H It* - ■> »'■ ? t. ^ (i S ^t' " ' *~^’^ CCCr* ' ’ ’•■ STafi"' •■ ■ T ’’MW i»i; :i- ^ 1 ‘ Xiii*'; *.<. -^\Tf;’ iv jll f'„ ■ ' p.A.’ .r'T. r r ■- k* f 1 # S ' , ' ’ - r. c^i’i(yvm 'tf iv . • ' ! .; 4 ! Ju • * » X ^ ^ ♦‘^tisii“ ft r" i. ! \\ T -iT s— t r*.. ~)^-, r wiitf > - 49 - Bitliography Brooks, Eleanor S. 1918. Reactions of frogs to heat and cold. Amer. J. Physiol., 46: 493-501. Deckert, Richard 1917. Do the Fowlers toad and the itoerican toad inter- breed? Science, N. S. , 45: 113-114. Dickerson, Mary C. 1908. The Frog Book. Doubleday, Page & Co. Hill, A. V. 1911. The total energy exchanges of intact cold blooded animals at rest. Jour. Phys. , 43: 379-394. Holms, S. J. 1906. The Biology of the Frog. Macmillan Co. N. Y. Kro^, August 1916. The respiratory exchange of animals and man. Longmans, Green «S: Co . N. Y. Lund, E. J. 1919. A simple method for measuring Carbon Dioxide produced by small organisms. Biol. Bui. , 36: 105-114. Miller, Newton 1909. The ianerican Toad. Am. Nat. , 43: 641-668. Porter, W. T. 1906. An Introduction to Physiology. University Press, Cambridge, Mass. r . * .r. tonnoIX m ‘ / / ' - *4V t. i ■ 3 - . .:4* u >"L1 6J lO , ' ' ' . "3 w ' . * . ■ t i'» • . T M 'f^oM , tlOb‘otiCi' <•*. *-r1',: » % JL' ' , I ltt .lo^fioO cKt\ 3CS A ;-*^3 ,.Ivt .«,c;i^. .firt9Xcw?<^!i^ £X4i.i *■■' ' ^ Ji i' t li ^ A>; -50- Re id, E. Waymouth 1890. Osmosis experiments with living and dead mem- branes. Jour. Phys. , 11: 312-351. Robertson, T. B. 1917. Suggestion regarding the mechanism of one-sided permiability in living tissues. Science, N. S. 45; 567-570. Tashiro, Shiro 1917. A Chemical Sign of Life. Uni. of Chicago Science Series. Treadwell, P. P. and Hall, W. T. 1915. Analytical Chemistry. Vol. 2. John YiTiley & Sons, N. Y.