C II ¥ 0 1 1 1 O f , ( } { ; Y

V o l , 3 0 N O . 5 , 1 9 t 1 7

-- , , . . . . . --- , , ~, , tHa i , I I O T l l ; ' E I I P H I L O D I N A S I U I ) I E S O N I I I E S U R V I V A L 01)" . . . . . . ,

A F T E R F I I E E Z I N G A N D T I I A W I N G ~

J A M I .,S K . I'~ 0.1:, 1t LL R

DeparZ.mc..t o/ Biologi(al St.ru.cture, Um.ver,~dy o/ li . , sh~ ,q to , , S S , " . -1t •asttu*fl.ton. chool el Medicine, ,..e.attlc, " . " '

It~ is gene ra l ly ,,meei}ted tirol; ee r t a in r o t i f e r spec i e s , , ' " !' 1., . " , l)a~l,u, .uar5 ]3dellouJe,~e, a re gu i l e re- s i s t n n t l() e l l v i r o l u n ( : I ~ l n ] s t r e s se s such as lie,'tt, cold, and desieea.l ion. ~ M u r r a y ~ re-

' " (..:tt,un a r c t i c s - , , ' , , l ) o r t ed that; , o ' , " ~l)CCms e::t~: be re- pe:~tedly f rozen arm t lmwed w ith(n~t~ d a m q g e in the ,~(;ti,.e" " v st, t i c " " , . T h e m o s t hardy., free, z ing

s " - , , , "-' shown b y ro t i f e r ~ )e(,i('s which re. . l~I. , t l l ( . ( . 1~

]laVe inili;~lly ~mder~one dt~sie(.'.alion e i (he r t.hrougl~ natt~ral p rocesses or in the l,tl~.)r, tto~3,.

j , r l r , g l , , ( , q t r w ]{.ahm" not. on ly showed .hat, ,,,..,..,,...,.,,.,I

ro t i f e r s cotlld s ~ r v i v e f reezing a n d t lmwi~g but, r ecogn ized lhe ' } " ' ' e , mHmtan( . . . , df cooling r a l e in i lm s ~ b s e q u e n t s~r B, al of l he an imals . His w o r k sugges t s !hai; s low cooling is SUl)eri(n" lo v e r y . . . . . . . ralf id cooling a l t h o u g h (hese e'..a~'..lv st.udies inel~Med no a t ( e m I ) t to de te rmin( , lhe l .eml)era- t u r e - t i n m re la( ionshi l )s d u r i n g freezing. Bee-

. ) 1 . " q~tcrel I r e s e m p l o y e d the teehniq~m of a (ha - " ¢ T * " ' [ t l , t t , ( , e l . a l n b,.ttie d e m , t g n e t i " t" za . t on to show , ' , "t,

( " ~ t ) "

( ' ~ ' ' " ) - . e " ' >~ (1 ..... m(.,~)c'd J~ lh (hna ,m(l Ph ih )d ina Sl)...(.,u .... can s u r v i v e t,(.ml_eratu~es" ~' ' ' ' " a l ) l ) roaeh ing ,d.,,..olut(.; ~ , z e r o .

I ) e s p i l e t.hese i n t e r e s t i n g s tud ie s i l l u s t r a t -

ing t.hat, ro t i fe r s can be , success fu l ly f rozen whi l e m a i n t a i n i n g v i ab i l i t y , . there l-m,. b(.c,n l i t t le or no r e c e n t v, ,rk. . exploit, tag these o rga - n i sms for. e ryob io log iea l e x p e r i m e n t a t i o n .

T l i e r o t i f e r c an . be eonsMered: , ar t . o r g ' t n i s m

cons i s t ing of seVerM t i u n d r e d eel ls , ..pcch.l~zcd • . -" i , 2 ; - .q for the~..mos(i p~:irt, i n t o - d i s t i n c t t~ssues a n d

o rgans , i nc lud ing n e r v o u s , m ~.~cutar, d iges t ive ,

a n d excretory" s y s t e m s . - I n : th i s s e n s e , ro t i f ers &fie , g r e a t l y f rom mi iee l lu l a r : o r g a n i s m s such

¢ - ", ; ' - ~ ; ' " i , r 3 ¢ * ~ " as y e a s t : ) r . sperma. tozo~ whmh..ha.~e l.ccn dea l t wit l i :,.S0 s n e e e s s f i i l l y - in.. c r y o b i o l o g y , : . . N e v e r t h e - l e s s , : e.ertain - ro t i f e r .~ , " ' ~ ,." ,.pc(,m~. e,,n be-h:indled

. ; , ~ ~ . . . . almosl~ ~as :ensi ly as s i n g l e cell .5. ,~tems, =with r ega r ( I ; t o : !~ inH) l i c i t y of c u l t u r e , : u,se of . : : sur- r i v a l testS, ere,

l l e e c i v e d : N ' o v e r n b e r 15, J :mu. " " " .... th . . . . tflOlTlt I Scu..nce • SUl)t)ort.c'd by grants fl'om o N" " ~ , ' "', , ,

Foundn t i0n : (GB:a587) a.nd£flie S ta l .e of Washing- • . - o , ) " - , ,

lo l l Init.l,tI.:lX,{. I ~ 1 ]!'IIIi(t.R}r ,~" ' ,~(.mn title R ( . ~ a r c h .

t ~ 1 : J.~ )s r e p o r t will s u m m a r i z e a t ( . empts to ob- IM~ m,"rx.imun~ s u r v i v a l of the • ' ~ , ' , ~ o t l l e ! .Iihilo - d inn b y i n v e s t i g a t i n g tim effeet:s.ol ' :~ rqnge of eocding ra tes a n d g lycero l e o n e e l l i , r a t i o l - i s oll

~,tl)~l~3..

~".[A'rEIt~:~LS AND ~'I'ET.~tODS

f ' / t i lo(l i ,a acut. icor, is odiosa ( M i l n e ) , bl ind 1 1 ( ) ! 1 Cr 1 ; " . , ,, . " ~ 1 1 ~ vtl.riet.y, wns used t] '. , h . o t t th~s s t ~ d v .l c

c u l t u r e of this or~zanism is aehieve(I l~y.:etn- ' t ~ ' 'a' I I) loying the bne(e r ized b a k e d lett~u.e medi~im

or ig ina l ly dese r ibed bv S o n n e b o r n ~ fo r . .Pa : r a - " ' " w ) . ; r , ,;a" ~ r m e e i u m c u l t u r e a n d ,qdapted to I . t . , e r .... 1.3

~ { % I * Birky.Z M o r e ~.eentl.,,., a c o m m e r c i a l plan(; t}re, l)ttration. . (CcropiDl" " ' L,d}or, t t o l . ~ c , . , ' , ' ," ,.~, K.:tnsas.

C']ty) has be(m Slfl)stittlte(l for the b::dce~l le t tuce . A e r o b a c t e r ae~og6tes" "~ ' inocula te( / in to n u t r i e n t , a re a l l owed to mt, t f l . ) , l t e t t t " ' ' . . . . ')q°(", ,., for

pe r iods of 2.,t. . to 48 l',rs a t wtf ich, t ime the bae te r i a l s u s p e n s i o n is 1.12(.'(1 to feed the r o t i f e r c u l t u r e s . . F o r f reez ing e x p e r i m e n t s , " ro l i f e r s were . s u s p e n d e d . i n the S o n n n e b o r n mediun~ wit hou~ b i t e t e r i a l . add i t i ons .

C o n t r o l l e d f r e e z i n g w a s c a r r i e d Ol.lt ill £1. c o m m e r c i a l :I, mdc.. ].)1.-,3 u m t . T h e .... tand,.tr.t

ffu>d .,.t,. sh own Linde" s p e c i m e n .ih0Ider w a s . - m 0 d : ~ " , s .. i n + " " " 1 ,1gure 1, l m m t l n l v : I)eea~tse. of..- a. :des i re to ' " ." ",. • r r , , , ' e . • a - ' _ g r , ( h e n t , eon- m m m n z e e, eomct , I J.al f r e e z i n g

v ) , " - - , -- . "I'll( • > : 1 ' ~ ' , ~ si(le.rat(ionS in the q. ecru,ca,., ._ ..-..ro...1.f(:.l,.. w e r e eolleeted."..in S m a l l d rop le i s : (Mxm(. 0.05

: . r .

e e ) - - . I n c h w e r e . p l a c e d o n - : : ( i - m m : : d m m e t c r - g ~ . i , " . . . . . - , . : F : . ~ " . . ~ ~ . . . . . . . : : . ~, : "

(0 .20 m m th ink) c o p p e r d~sk:,. I h e dtsk.~ were p l a c e d . i n dep re s s ions o n : t h e p l a t e ( P ) . s h o w n m .I4.~gure i..:-A lead b!oek. i (L) was a d d e d t o t he . . . . . u n i t : (..():. iiic. r@se...~. '.. tile. ., flea. .t.:. ca. -l:)aei.. .. tv-~ . . . . . o f : t.he s y s t e m " to .i a~ po in t , w h e r e eooling -.rates r a n g i n g

• " ' ] : 0 :: ~ . i . . ' . . '. ~ " " - . . . . . . f r o m . C p e r r a m . t o . 10 C p e r min c()illd be h a n d i e d . b v t h e . c o n t r o l l e r ; c i r c u i t ; , .The differ- . • I ' . . . , " . . . . " ' . . ,

entm~. t h e r m o e o u p l e . ( D ) -was ~ c e m e n t e d m i " ? , . I) lace o n : : t l ! e ; : ] ) l a i e a n d was c h e c k e d , for re-

Im.bfl~t,. i n t l i e / - f o l l o w i n g . w a v . : A . / t h e m f i s t o r r } . ~-; , ,_ • .. , • -.. , , . . . . : I) o ) e '~as .m~ )la.nled m a .d to let, of V,tseline .. . . . . . . . . . . I - ,. . • . P , . .

a d h e n n g to: . a : . e op tm r ; . . d~sk - spee~men holder . T h e d i s k - w a s , e : " " ' ' , 1, - , , I ) Ia .ed o n t h e e o o h n g . I ) a t e a n d

392

I{.OTII)'I';It ];'RIi',.EZIIN(, ANI) 'I . ' I tAW[NG " " ' 393

:ll]oxved to cool al, variotts rttt(',q from l'OOm I.e,rnl)eralt~re Io ,tt. oll t .--()0 (., (:)v(;r t i l ls r:tnge, the th(,.rmistor (emp(:;rgthtre (Yelh.)w Springs

o ~ ': ?' "e ; ° C Model-t.~+SI.,) ,tgt :.ed to within 1 with the

Ft(;:l.. Ti{e Linde. 13F-3 spe(dmen.hGMer.-.was modlfie([ to-:hoM:Small sp<:cihm,~ disks .as shown in t, ltt,,.s" hgut(: ..... l.h(. lead blo(k . (L) u. nc .( ........ at3 .tp g ,,c. p r o l ) e r h e a t ca.l)a(:~ty eh,'.tractertst:le.s. S p e e i m ( , : t disks are plmm.d, in depressiOns!in t he plate:_(P ) t,o Which :the differe.IitiM I hermoeou pie. (D),L;has been eemenl;ed..About ~z aet;ual siz6.

Brown recorder ~ "' " mmmn ng llm:-Linde tlmr- moeot tp[e.

A lypie~l cooling tra.ee, is shown in Fi,cure '2. 'l"i~,~e is lflOlled ml the ord-btat($ at 5-rain in/erv~ils b('twoell h.or izmi . ta l l i .nes , rA lng lle.- rio(I of :~l)l)roxlmtttely 5 rain oe(~urs at{+,~ :,(!tivalion of l lm soId~dh.l eolilrollii~g liquid-~i- tro~en flow an(l is followed t:)y a. liilear t, ime- teml)erntut'e, i)]oi hn, ving a slol)e of ">- e.o--, ,. , , ,~,,) .C, 1)(..1 m i ~ A s " "e, + . ~l~(t~.,,t.t(.(l by l:lw m~rve, lhe ~nslrh- m,..n,, is s~tt~h.,d, to mnxim:fl cooling (over- ride) ,tt nl)l)t.mm~,ttel.x --a() C. This cooling

'u,,, ', loll m e d in the. major iW of the p t ( .,,(]t~l¢ w a s : ' ( •

a l(~nl)erat~re of exI)erimen~,~. Aft.e.r r e a c h i n g ' " - --I()O°C, the san~l+)les were tn ,ns fe r red in . 2 to ;3 see from tli(; Lin(te uni t to :t, smnll liquid nilr()~en,eooh.)(t vessel eonlaining ]iq!lh:t Freon

+ ' 1" ~ o "* r ~, 12 nl: :~ ternl)er:tture of ,~t.)ou, --:1o0.(.,; l h e aninmls in. tlmse shor l - i e rm exl)erintents re- m,t.~u(d at final ].ow t(m~l)ertitttres :i -toial of lO to 15 thin l.)(~f()re th,+.m'ing. Wi th t h e exee.l)- tion+ o f o n e e x l m r i m e n t w h i c l l J wi l l b e (:lis-

eu~s(.~l l,~ter, all Sl:mcimens were .quickly thawed-t.)y 1)Itmging t h e eopl)erdis l~s into de- l){'ession " ' , " : " .... r':" " ~ "- tO 3 : m l + o f n u - (hsh(;s . ( . : o n t , t u l l l l g o , . . . . . . .

t r ient me(t i tin+- n.l:. r()om temp.c'ra turei,:.-l{ eeovery . , . , . ' ' r " ~ , ' " ' " ~ " f ro th :freezing was s(.'..ored-mtc o+col)~C.,dl..,,...m

1.,... t O h i ' s , n fl:et, tliese, depression dishes i~q 24 +t . j

:{ha.wing. As 1 . t o 3 d a y s are...:reqi:fired for

hatcl i ing .to: o c c u r af ier . . t lm ~:l:iyinta of au eg,:,,, {hii:!;e is litlle ' l i k e l i h o o d thnt: ..this:.+..faetor

. ~ , . . - : , . - . .

woul(l d is turb the mmfl)crs..:ot ,~mm,tls c o r n (.d, " Xl • '~ n o f r e e . : :t : i Smcc :' e g g s . w e r e iised ..il he : e mr -

N

+,£9~

Fm 2.: This:fi.mlre i llustrates a recot~c[:of :a-.:fr~.c~ng • . ! : : : " , :. : : : : . . : . : ~:.,:::' o ' : " ~ :. ~ : : % : , . : ; : : i ' * ~ .: :<L - ' ' : . ' :+ , , ' : : ' : " : : ~ ~

run.+ Hm2.zontal l~:nes: arc sI)aced,at ::a-ml~ ml;ervals. A.n ~:inl~al-la~ I)et]md :0f: app~oxn!.mtdY 5 m m l s followed: by: a.lmear:::rel,'d.mns!nl):vduch gtves::~ .:s!ope, m::tjns)nstance,:,0.f:,3.a,:~/Per rain, :A t, ! ii,:' tern l Jei:a.tute : o f : a liiJ i :ox in iaLe iy :: ~ 5 0 , ! O i::: £ :! ]ii .~he r::.: ebb l in ~!(i',{t:;, ~ re fl (iiiie.,d i n a i i : i i i e rOase l i i t t h e S] o P e 0 f l t li e ::.eu r y e a t .thit~t, p o i n t:.

094 J. K: KOEIILER

ments, egg matulatmn would also first I mve to proceed t,o completion before.laying. Counts of total popula~ti0ns, made immediately after thawing correlatedwell wath those mad~ 1 day 1,1.ter: Glycero]: ' t l 'eatment induces :t::non. active or dornmnl~ state in :these .... anmm]s, ~ " •thus, this, t ime period: ( 1 8 .:hrs) was requi red to allow, essentially fuli recovery o:f survivois;' and simplified differentia.tion between li~ing and dead animals.

Each point on the survival curves represents count~ of a t .least~ 100 and in many eases 300 to 400 a mnmls, thus, st~at,~stm'd wtriations de- pendent on sample size '~.re estimated to be well. below 10%. Duplicate counts: of the same populations by several individuals indicated that counting erorrs of 5 t o 10% were common.

" " " " 2"." } ' . " An arbH~a ~, but pro.mbl5 conservative,-, est,i- mate of 20%: is placed on the over-all precision of the.survival da~a.

E X P E R I ~ { E N T A L I ~ E S U L T S

The widespread and successful use of glycerol a s a ery0phylactic agent suggested.a series of experiinents aimea "at determining the physio- logical i effeets o f this reagent, on Philodina. Samples of anin.mls were t reated with glycerol solutions of Vaiqous concentrations and viewed microseopiealh,. T h e addition of solutions having glycerol concentrations greater t h a n 3

C : n • or 4N wa~ accompanied by anini t ia l , rather violent, contraction of theaniinal , resulting in a rounded configuration. Tin the higher con- cei{trat i0n ranges: : e m p l o y e d : ( 1 0 % ) , this

.... stmpe :~ s ...... " ' . . . . . . rounded . . . . . . "a . main ta ined: lndelmltely while, in glycerol.: :~%~q~i~T/<the flower i; glycerol eoneentration:s ~i(1: to ~: 3%)/itl~ere was ::a partial recovery: 0f n0rmal~. -form and motii!ty. When treated animals, were<again returned to nor- mal nuilrient, :~they.:,gi;adually resumed i: their el0ngated form:;:and :sWimming .movements. These~results, are summarized in:Figu re 3.

Tllei:pi'ocess Of::recovery ifrbm glycerolation was miirk~d:by;~consistent Set of:morphologi- cal ~ctla~gis gnumerated: below.

I.iTli6):i:}!medialporti6n of the animal swells out it-/a> pear:.shape} i Ttie foo t pr0 contracted internal state at a,bout;the same:time: (Fig i 3c),

2;':!:The:!footi:£tthehes:t0: ~ ttie glass substrate and:tl~e animM remains 'at: / this locus until recovery:b:: co mi~lete:~i:Elongation ::Continues to- g~the r witii: brief: musCular::spasms.

3. Elong,~tion is essenti:dly.complete before the f " m,tl phase which involves eversion of the head or coronal segments from tlie body ca:vity.

Tlie ent i re recovery process has t~ duration of several hours, varies from one aninml to anotlmr, and is dependents on the !glycerol con- centration used during pretreatment:, Re- covery fronl glycerolation can be semiquanti- t,tted ,rod is d0curnented in I~ Igure ,t. ~I,tns figure shows that plot t ing :the percentage)of motile animals (per cent recovery) as a,:funetion of time after i'eturning to normal. :mediunl yields "~ s moot]l curve. Essentmll3 complete re- covery is actiieved after several hours. Treat-

1" ' ¢ i n g t h e same. anmmls a second time with glycerol al~lmrbntly Ires no beneficird:or del- eterious effect; on :the course of recovery. AI- tlmugh glycerolconcentrations of 10% or less seem : to ;have ~no permanently harinful effects on Philodirm, t r e a tmen t : w i t h 20% glycerol Solution resulis :.in >100%< lethality. The eft, o:t,t, of glycerol incubation time beff~re .freezing on survival:: was : :not. investiga:ted : i n ehis Study and was maintained,at,' a Value of 3 to 4 hrs in experiments discussed below.

Glycerol concentration and cooling rate were t h e two factors followed most extensively with respect to t he survival of Philodina ~fter freezing, and thawing. A • " , ~' ,.: : t, ypmal sum ~ al pro- file is shown.in Figure 5, whicti flhstaa.tes re- covery as ~ function of cooling rate using 1..0% glycerol pretreatment. Maximum recovery ~ is seen to lie in the Cooling rate region of 3 to 5°C per mm,::which gives a yiekl of : 3 0 t o 35%. T h e v e r y slow r a t e o f 1°C " per, rain-is appa.rently n o t : v e r y effective, and very rapid cooling : (scveraI hundreddegrees per see) re- sul ts : : in-death . Exten~mns .of these expen-

- t , glycerol: eoncent:rations are men.,s t o other Shown:in :Figures 6::and 7, It ea:n b e s e e n t h a t rotifers ::can survive freezing without iglycer' olation (black:- triangles, Fig. 6 ) b u t that the yield is rarely ab0ut :10 to :15%. Pretreatment with: 0.1% glycerol :giVes: survivals :! not~! signifi- cantly different from::the nonglycer0latcd e0n- trois. Wit h. {m!. iner.e,'ise, in gl3¢cerol,! up: t02 .5 %, survival is dramatieally i::improved. Fifty o r 60% recovery i s : a t t a i n e d . b y once again us- ing cooling r a t e s o f 3 ;:to. 4°C per min. .Very r a p i d cooling.: n e v e r yielded viable rotifers ir- respective of tlie' glycerol concentration em- ployed.

NO extensive experiments were carried out

, ' ~ " ? ~ ~ ~, ":~r, - "t " ' ~ 7" " RO,I I]. ],RI)R]~]~,,ZI-N G AND I H A ~ ING 395

Fro. 3. ' Glycerol trea.tment of.,.Plulodma :results .ln:-..t.:charactenstlc: set,~:ofi~:morphologlcal '~ ' ' ' F . . . . . . -~ " • ~ ' " '" " " " ~ ; " ' " " ' : ~ : ~ " ~~' : , " ' ' . . . . ' " " ' : ' . ' " ' : " : ' " " . . •. : ' ! ' ' ' , ' " , " ". " ~: ~::°-'~, : "

changes, A,. :['he normal elongai~ed, ammal ,coi ona an d foot:extended, x 17.0..B. The: t, ypicaliy contracted a n d nonmobllc : form o f the.. animal after. treaf~ment :i~vith:; a, :5%i/gl~cerol: solflti0n

3Jb ,l K .K.OLHMsI{

on the effect of tha,wing rate on surviw:ll However, on .one ' occasion, several disks were allowed to tba.w in the Linde unit. a ra, i;e Of a b o u t 1 °C p e r irii:n ~tll¢.l compared with the

; • . . •

si:nndard i'apid tliawing procedure; The sur- vival Of .the i: slow ' t h a w animals w a s lO-fold lower th' ln that o f those thawed ral) id lv.

Pretr, eatmen~ invo lv ing h i g h e r glycerol con - centrations, namely, 5 and 10%, a s seen in Figure " ' - . . . . " " ~, yields a s o m e w h a t puzzhng picture with respec t t o survival. :Rather t.htln obtain- ing a m a x i m u m i l l " " ~ ~rr" : the cooling ~a te r%:on of it t ¢ * *

3 to 4°C per rain, a milfimum results. Max> m u m • recove ry w i t h these p re t r ea lmen t s is

Recovery of Philod!na after Glycerol Treatment 0 : 3 hours ifl gtyce~o!

°. =. Recycled offer r2 4 hO~ r S

80J ° o

e, so

"~ 40- -

zo' °

0 , , 2 - 0 ' ..... ' ' , , ' , , ' F - f - - r - - ° l - - , .. 0 40 60 80 I0(" 120 20 hours Time (rain.)

I,hci. ,ti T h i s figure indicates the, time course of recovery after treat°neat with 1. ~t (7.2%)glycerol for 3 hrs.

Recovery o f Frozen Philodino using P r e t r e o t m e n t

of 1.0% .Glycerol

40-

36~ 5 2

2E ~L~

=" 24,

"" 20-

1~

8; °

4-

o:

T 1

I

i Io Cooling rote~(,°Clmin.)

/==",O- Very rapid

cooling

70-

60-

50 L

40- b Qa

ta a, 3 0 -

qa

!0-

T , Recovery of Phi lod ino.of ter i Freezing" Prelreatmenf with

] A Glycerol ! o ::,.~o/o

• ' 0 . I %

• " N o ' p r e ! t e o f m e ~ l

T

b 0

It

• T .:r

2 4 6 Very ropid C O0 ~ i n g Cooling rate.(~'Clmin.)

I.I(;. 6. '1"lie surrival of Philodina as a timer°on of i cooling ra te r i s i n g various pretreatmevts. ,

Recover~ of Frozen PNiodino afler Pretreelmenl wilb GiyCe~ol

GO ~ T, t . , ! X

bS'o~

.. - ! \ :J & t ~ \

"°i Z.xZ \\ t - - - a - r V : Mx,,

i 2 3 4 5 6 7 8 ~ Very rop,~ d COoling r,~le (.°C/rain.) cooling

Fro. 7. The survival :of Philodina as a function of cooling rate wid~ high. glycerol concentrations u~d in pretreatment.

seen to occur a t the extremes of ' the :rates ob- tainable b y controlled cooling, Tile 5% pre- t r ea tmen t generally results in higher Survivals than the 10% pre t rea tment .

Figure 8 shows tha t maximum s u r v i v a l is a.tt,uned by p re t r ea tmen t with 2 / t o 3% glyc- eroI when freezirw. .~ is. accomplished. _ at rates_ of 3;5 t o . 4 ° C per min: Of course, this p l o t represents just..one of a family of curves hav- ing a ,.ensltave dependence on cooling .rate.

Fro: 5 . :The survival of Philodina as a flmction Th'e p a r t i c u l a r : combinat ion o f conditions of: eoo!ing rate u s ing 1% glycerol pretreatment (2 5% glycerol 3 ° T h e b a r s on]?igure 5. ' "~'~ • .5 to 4 C per rain in cooling 6,-and t .epresent probable " estimated errors ~md:no~:spread i , . rate) Shown here has given fhe highest re- Onepoii i t equals one experiment, n actual data. covery r a t e t h u s far achieved (50 to 60%).

-. f" "~ ' :~ " I r "T ' t RO 1 IIi ]:tAg FREEZINCI AND TttAX,~' IN G 397

Recovery of Philodina o i ler Freezing ol

3 .5 Io 4 ° Per Min,

60 .

50 .

40- b

k a

"~ 30-

2o-

10-

0 ! Y. 3 4 5 6 7 8 9 I0

Glycerol concentration (%)

;~ , ] I(,. 8. Pooled data f,'onl various experiments showing t..lm dependenco of survival on glycerol concentration in a restricted cooling ratc. range.

q'ABrW .L ,"~L . l ~ l J 1 "¢ * r ~ U R ~ , I V A L O F P I t l L O D I N A z?kI~"It.Et{, I . ~ O 1 , V

v ' ~ 4 "~ r * * " - i ( . . [ E i M P E R A I U R E S T O R A 3 E

• c l r * ",r ,..torage in liquid N~ after freezing in 2.5% glycerol, cooling rate of 3.5°C per rain.

Storage Total Animals Recovery

days

0 2

10 20

1.34 264 428 133

%

62 49 56 49

The survival of the animals .subjected to such "optimal freezing" and subsequent storage in liquid nitrogen is shown in Table 1. The varia- tions in percentage recovery with storage times are probably within t h e limits of experimental error, leading to the conclusion that storage for 3 weeks, has produced no appreciable decline in viability.

D I s c u s s i o N

The assertion that active nondesiccated rotifers can withstand freezing a n d thaw: ing '~''~'" has been largely Substantiated by the presentexper iments . Recovery even under the most, favorable cooling rate conditions is generally below 20% for animals frozen with- out protective additives, .~.ssuming that

' " f r st, raw,hi Sonneborn medium h a s no inherent protective quality, of its own. This figure, aI- tlmugh modest, is rem,l, rkriible in itself when we realize that the specimen is a complex organimn possessing highly differenti'tted tis- suc s y s t e m s w h i c h are notor ious fox'. their l'lck of regenerative powers? The addition of glycerol at~ even quite low concentration (1 to 3%) enhances the ability of Philodina to withstand freezing to the point where 50 to 60% recovery can be realized. This degree Of survival has thus far been achieved only for unicellular organisms such as yeasts; micro- organisms, red blood cells, and the spe rma- t ozoa of certain species. At tempts to freeze and thaw viably-tissues and organs o f higher anilnals, not to mention the alfimals per se, have met with only minor success. Smith ~ has discussed some of the problems inherent in freezing highly organized systems. Among the most important of tlmse problems is t l ie fact that the many different cell types present in a given organ may have quite different optim't for parameters such as cooling rate. Thus; one can achieve the survival of a specific cell type wi th in the organ, but generally not the organ as a whole. I t appears that in the case of:the rotifer the various cel ls that make up the several organ systems of :the animal are simi- lar enough, with respect; to freezing tolerance, to achieve sur~ival for the animal as a whole. Obviously, the minute size of the specimen (50 to 150 ~,) also has a beneficial effect when

CO S" :~ '~' one . n l(lex,:, s u c h f a c t 0 r s a s thermal ~radi- ents during freezing and thawing.

It is of some interest, to examine the present ~'~,f ~.t results in the light of ..'~.za.zur~ r e c e n t hy-

pothesis concerning freezing damage .particu- ( • , ~ r " larlx ~l th respect: to the Cooling rate factor. I t

must be realized that ' M a z u r s considerations pertain primarily to unicellular rather than

. • . . . . .. ~ - . . . ( ; ~

multicellular orgamsms. The rotifer ~1~ .uffi- • l y " p l "gt cmnt sm~ er, however, that w e ! m It ex-

tend t l rx C~) erag rpu rposes ~of the, 1 eo "s v e fo :~ dis- cussion and al)I)roxHnatc the annnai no more than the sum of-:its eelhflar :.:parts..Ac- cording to Mazur's flmory, a lnaximum in survival should occur at relatively slow eoof ing r a t e s where the combined effect o f ex- posure to high ionic concentrations and in- t racelMar ice nucleation is mininml.i At higher cooling velocities, survival, falls off be- cause of intracelMar crystallization and rises

398 J .K . " ~ . . . . ' .KOIsI:IL]:~I~

again only ~tt, extremely rapid coo)ling rates where only minute crystals or I)ossibly vitrifi- cation occurs. A m.'.tximum of the sort pre-

* I " d~¢,ted for low cooling rates does occur in the present work, ,at least, in the ease of low giye- er0l coneentra:tion I)retreatnmnt, namely, at the value of 3 t o 4 ° C per rain. It:. is somewhat surprising.{.h~t such. a rela, tively sharp maxi- mum should occur w i t h i n the r ' t the r : restricted range studied (1 to 10°C per min). Very rapid freezing in. these studies was uniformly un- successful in producing viable animals. One can only speculate that, the rates used in rapid, freezing were effective in produeing cellular, damage due to ice crystalliz~rtion a.nd

the life span a n d rei)roclucl:ive e:~pacity, Pre- limiimry studies suggest t im t, frozen animals live as iong q, nd have -is many proge.ny as nor- real control individua, ls and, therefore, sup- port, the hypothesis that survivors of freezing and thawing are not merely alive, ibut ap- proach normality in many respects.

~ U M M A It Y

The surviv,d of the rotifer P l i i lod inaaf te r freezing and th'twing has been examined under a variety of experimental conditions. Partieula, i' emphasis was placed on obtaining maximum surv iva l by employing the mos~ advantageous combination of protective :tddi-

tha.t: even higher ra tes are necessary t o pro- tive concentration and cooling rate. Glycerol duee, in these organisms, a state apt)roaching is an effective cryoproteet.ive agent in this vitrification.

Tim:rot i fer , being up to several hundred microns i n s i ze , . i s l a rge relative to tlie m~lg- nitude of single cells, It.. may be that sig- nificant, nucleation and ice growth can be avoided in structures of this size only by extremeiy high cooling velocities, if at all. The: Cooling rates investigated obviously rep- resent a very limited sampling of t h e tota, l range, but they are sufficient to. suggest that the present work correlates in a positive way with t h e ideas expressed in :Ma, zur's hypothe- sis."

Undoubtedly, one o f the major sources of variability in survival is ::the lack : of uni- formit:y in ro t i f e r age and ..physiological

" : • • . • :1 .

s ta tus :of ~the cultures. ])esp~te efforts to ex- clude ~ er~, y o u n g : a t d ~mv okt. animals: from the-freezing: experime:trts;, the samples still rep, resented a considerable spread in age and physiol0gieal state.

1-totifers. represent one of tim cla:~smal ex- . " - , ¢ . " : . - ] . , ¢,, r J ~ amples :of . cell coI st,~.ne3 among aniinals.

With:: t he exception of: : enibryonie tissues, cell turnover resulting froin:i division is . unknown in ,:these-::animaIs. This. fact,: p.trtially explains the ~ hiek of regenera:tion in injured or stimu- lated : t issues. . i¢.. suggests :that, recovers/ from freezing~:i:!i::imust " result: from tlie surxfival iof: l a lge fract, lon o f the. cel l which *: initially make up t h e rotfler mgan s3,,.temsl The number of cells wh~ch~ are : expendable after an insult such as •freezili~g: is: unknown; however, i t :may- be po~.~lble t o ans~.er this:~que~tl~ "¢~n indirectly.. by investigating parameters more sensitive • : ~ ' 7 " * : than .... ur~ lvaI,per se. TWo: such parameters. are

system at, concentrations as low as 1% and ex- ertJs a maximum protective influence tit :l con- centration of 9 t.o 3/v. Glycerol solutions in the range of 5 to 10%, result in somewhat anomalous survival curves as a function o.t cool ing r~te, although survival at, these eoneen- tra, tions is moderately lfigh at t,he extreme,." s of the "slow cooling,' ra,nge. Treatmeng of r0tifers witli a 20% glycerol solution results in Cle.lth;

• . . , ¢ , . , thus, i f is possible that sublethal.toxin effects

play a.. role at lower glycerol concern rations. Cooling rate opt, ima appear in the region of 3 to 5°C per ra in witll deviations occurring a,t higli: levels of ~"lyeeroh, tion.

Survivals:of the order o f 50 to 60% can be routinely alchieved by employing tile most suitable pretreatment and cooling rate. Ani- mals frozen in this manner have~ beens t0 red up to 20 da.ys a,t liquid ni trogen telnperatures wit l i0ut a : noticeable decline in viability. I n addition: to manifesting normal swimining and feeding habit,_, survivors of freezing appear

' " ' : ~ : . r - [ ¢ " " to e n j o y a.: n o r m a l hfc ~pan and :repr, oduetive i ¢ t - r cap,tell.X...,

• " i ~ t " i" " REFI!,ItEN Cti,S

1. Becquerel, P. La. suspension de la ~vie ".mdessous de 1/20 ° K absolu .par demagnetisation adia- batiquede l'alum d e fer dans le vide. le plus

x, ' R. Aca 1. bc. (Paris),~..,_ 1.- ,.61-,63, elc,e. C . ' r ~" i o~ .,o . 9 1950.

• " 7 . S " . .. 2. B i r k y , C . "vX ,, Jr. ~. tu(hes on the physiology and genet,ies of.th6 roJ;ifel', Asplanchna. I. Methods and ph3,siology. J. Exp. Zool~, 155:: ..~3-292, .1965.

3. Hymen, L. In The invertebrates, . . . . vol. III, o.hap-. ter 13, :pp .d48 . !" .19 , .MeGraw-E. i l l Book Com- pany, Inc.; New York. 1951.

' . , .

4, Mazur, P,. C':mses of injury m frozen and

ROI1. LR FREIsZI.NG AND Tt tAWING 399

t imwed cells, Fed Proc., o., . , , . . . . . . ~-,/: SupI:, 15, S 1 7 5 - S 1 8 2 , 1 9 6 5 .

5. Mt~rra.v,, J. Arctic rotJlers." . Proc, Roy: Phs.,~. Soc,~.',~ ' ' • 1 1 . 1.r.}10, Edinburgh, ~" "

6, I lahm, (]. ]?i ..Biologiselm und physiologist.Ira Beitrage zur ken.this der Moos farina,. Z. Allg. Physiol., ..0. 1-34,192,~.

7. Snii(.]i; A. U. Prol)lerns in freezing organs and

their componm~t cells and tissues. Fed. Proc., O~ : Supp. 15, Sl96-$203, 1[}65.

8. Sonneborn, T, M. Methods in the gem:.ral biol' og3r find g.,met!cs of Paramecium aurelia, J. Exp. Zool., I18: 87.-148~ 1950.

9. Vor0ntsova, M. A., and Liosner, ]]. D: Asexual prop,tgal:.ion a n d : : regeneralmn. Pergamon Press, London, 1960.

The ,tulhor expresses his thanks t0 Miss Dora. Beth Parsons for her skillful ' :~ "J ' * ~ J " , '~* ', "¢ h I tcchmcal asmstancc ~hroughout, {this work. Drs. ~dward :Roosen,Itunge and

N. ]3, EVerett of: the Depar tment of ]?;iological Structure m.'tde cri t ical read ' , ' . , . : • : .

ings of the.manuscripl;and supplied many-valuabh~ suggestions.