- Email: [email protected]
28. Studies on the mechanism of freezing damage in bacteriophage T4B
17
ABSTRACTS cost thermoelectric cooler include size and simplicil.y of cooling unit, precise control of cooling rates, simple adaptation for hyperbaric environment or concurrent perfusion, portability, and o:~s{? of operation. 26. Rapid C o o l i n g by Means of Pressurized Liquid Nitrogen2 ~ A. P. MAC].~I~NZI~, C. .I~:RO~:~Er~,* aND B. J. LuYm' (American Foundation for Biological Research, Madison, Wisconsin). PrEvious determinations of the relative efficiencies of various refrigerants more or less frequently employed in the rapid freezing of biological materials yielded the following sequence, in which the refrigerants are listed in order of decreasing ef'fcetiveness: a) propane at -180°C; b) freEzing nit,rogen a,b--210°C; e) ].~'reon 22 at --150°C; d) isopentane at --150°C; e) liquid n~trogen at --196°C; f) liquM helium II at -271.5°C. Thus, li{luid nitrogen at --196°C ranks rather low in the soriEs. But. according to the principles of heat transfer in boiling liquids, the rate of exchange can be considerably increased if the boiling liquid is subjected to pressure, the rate being approximately in proport.ion to the density of the vapor in the l:,oiling interface, whether the process be at the "film" or the "nucleate" boiling stage. To apply this principle to liquid nitrogen, the following appara.tus was set up. A copper cylinder, mounted vertically and partly filled wit,h liquid nitrogen, was equipped with means for sample onl:ry and with pressure gauge, safety valve, etc., and partially immersed in a Dewar flask• The liquid nitrogen within the cylinder was then pressurized either with helium, in which case the liquid nitrogen could be maintained at --196°O or below regardless of the total pressure, or with nitrogen gas, in which case the temperature of tho liquid nitrogen becomes a function of the pressure. t}reliminary tests show that the apparatus, when ln'Essurized to 100 psi, permits higher: cooling velocities than attained by any of the methods previously employed. 27. Lethal Effect of Ultraviolet Light on Bacteria and B a c t e r i o p h a g e s at Low T e m p e r a tures, hi. J. ASHWOOD-SMITI-I (MedicalResearch Council Radiobiological Research Unit, I=Iarwell, Didcot, Berkshire, England)• The survival of Escherichia colt B/r :WP2 after irradiation with ultraviolet light (2537 .~) has been studied over a temperature range o f +22~C to -269°C. In the presence of ice, the lethal effects "Supported by the Office of Naval Research, .on~racL NON R 2437(00).
of ultr'lviolet irradiation increased by a factor of 7 betwEEn I°C and -79°C. No increase in sensitivity to ultraviolet irradiation could be demonstrated in supercooled solutions or in glycerol glasses at --79°C. Studies with bacteriophage T~ dEmonstratEd a similar but less pronounced increase in sensitivity to ultraviolet irradiation at -79°C. . At temperatures as low as --I96°C, the lethal effects of ultraviolet irradiation on E.coli are less than at --79°C. This latter result may be explained on the basis that reactions which involve "ch'~rge transfer" have different temperature coefficients from reactions involving "excitation energy migration." Evidence from experiments on low temperagure irradiation of E.coli t ! C R (this strain lacks the enzymes necessmT to excise thymine dimers) and Ta phage is interpreted to indicate that .~t new lesion, not thymine dimer, is formed at low temperatures. 28. Studies o n the M e c h a n i s m of Freezing D a m age in Bacteriophage T 4 B 2 ° STANLEY P. L E I B O 17 AND PZTZI~ MAzuR (Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee). In an attempt to understand the destruction Of biological function by freezing and thawing at an organizational level intermediate between the macromolecular and cellular levels, bacteriophages T4B and T4BO~ have been exposed to a variety of freeze-tliaw treatments. Phages T4B and T4BO~, osmotic shock-resistant mutants, were suspended in distilled water or in various solutions and w e r e subjected to se~,eral combinations of cooling-warming rates i n the range o f - - 2 ° C to -:780C for 2 to 50 min. Survival was assayed by standardmethods.-No: dainage to phage :T4B occurred, a t s u b z e r o temperatures in the absence of ice formation. When ice was present, the osmotic shock-resistant mutant, T4BO~, was found to be much less sensitive to rapid warming than was the shock:sensitive f0rm, :T4B. ttowever, when T4B and T4BO~ weresubjected.to slow warming, a :treatment tliat :eliminates osmotic shock, the survival of the. two Strains was parallel: Increasing lengtlis of exposure a t - - l { C , ~ 5 0 C / o r - 7 8 ° C had little or no additional effect on:survival of T4B t h a t had been frozen rapidly ~and warmed rapid!y. :However, the survival:::of iT4B :exposed to -200C and -~30°C decreased with increasing lengths of exposure.. Wlien T4B phagessuspended in osmolar s01utions o f KNO~, N a C l i o l ; C s C l ~ were: rapidly cooled to a n d rapidly warmed f r o m subzero temperatures, the lower t h e eutectic point of the suspending solution;the lower ttmsurvival. • "
}O
-.
O
Research spon.ored m pal t }> the:U. S. Atomm Energy Commission Under contract witti t h e :Union Carbide Corporation and in part b y U . S..:Public H eel t l i Serv i ce I.~"ello wsh ip 1=F2' A:i- i9,981,91A1, ~TU. S: Public HeMth Service :Fellow.
18
A 13STFI AC.'TS
On ll~e Imsis of (hose resul(s, it is eonehule(l th.'~(; n(, h m s ( ( w ( ) factors qre Ol)ernl,ive in ['n~tsing freezing (lnn.~g:e to phage '~P4I)). One of (l~ese is the osmoli(, shor'k to wl~i(.l~ the phage is s~I)je(:te(l when r'~l)i(I]y warme(I, nn(I, tlmr(,fore, r:~I)i(ll3" (lilu(e(l, fron~ (h(> frozen sI:tt(,. 29. Effect o f C o o l i , l g a n d W a r m i n g Veh)('ily on Ihe S u r v i v a l of F r o z e n a n d Thaw('(! Yeast. TM P).:'rEt~ M,.~zt:~ :t.,,'D ,IaXU:~.: a. Se~.~D'r* (Biology ])irish))), Oak Ri,lge N , li())m] l,abor:)t.oD', Onk Ri(lge, Tennessee). O l l e of iJle I~oI'(' intl)()rlun[. ('ollSC(lHences c)f c(),)lin,:. velov, ily is (o (h:termim, whether inlra('elhfln)" wa((,r freez(,s within ('ells (a. usually leth:d provess) or flows out: of (:ells :m(I freezes exle)'n,lly (nn often inn,)(:uous pro('('ss)..If cooliL~g is )'api(1, the formc, r occurs; if it is slow, the latter oc(:urs. Cnleulntio)~s in(licale thai; f(.)r l.h(, yenst:. Saccharom!lCeS cere~,islay, inira('ell~la.r f)'eezing shouhl occur if the cooling velo(i.ty ex('eeds I ° (o I0°(,' per rain, an
'SR,esearch sponsore(t by tlm [7. S. Alomi(,. E n e r g y Commission under cent(a(:(: wi(h the [_Tniol~ Ca rbi(h: Corl )oral ion. '"SUl)l)orle(] in I)'~rl ])y Gran(s CA-0,E)53-(}6 and CA-03S45-08 from (h(: Na(ion')l Cnn(~er Insli(ule an(I Grant I;:-111 F fro)n (he Ame)'ic'm Cnnee.r Society.
.A.UTItt:, E. (I,EEXI.: (Soutl) Jerse.y Medical Iles,,:Iv(:h F'oii~i(ht(,ion, Cam(h.;n, New Jersey). Tile nee(1 for n mmlimml)s <'uil.un:: line of chick oral)rye fil)rol)lasls h:is l-)econm more a(:ul~e with t I.~ in(.,rt,tse in the ttse of el)iek c,m b r y o (.issues in l)ioh)gieal res(,nrvh :tn~I l~r()du('lion. A.I; the pres(mI: l illt(' ( h o s e w h o tlse ('Ili(.:I~ ( : m l ) r y o etl]i:ures as n I'es(.mrell ()r l)ro(]tlvliort le(']tlli(llie ill IlS(; l'eLurn, r e -
l)('n(edly to (h(' ('ml)ry()na(.(,(I egg bee'l)tse, lhe cell ("ulttlre c:m~o( I)e cnrri~'(l in series for more thgm 4 It) 6 l):lss, g('s..,ks nn ,tlternative to :m estnb[isI,:,(I (:l~ick (.ell lhw, we stlggest, t..hnt n ('ons!.:mt s)II)l)lY (.)f at cl~ar,('ieriz(:(l chi('l{ cell can be m,(le av')ih)l)le by f)'eezi))g )nany "~)))l)))]t:s of p)'imnry ('ell cul(.tlres in li(ll)i(l nitrogen. Cells qre fully reeoverqI)le, can l)e sul)('itltxu'ed for at. ]enst, 6 pnssages, and ("fill ].)O ("]ltlrtl(.'.ieriz(,(l ill v)lri()tl:S..' w a y s tllld showl~ 1o
be fr('(, frOlll t)()ill lnierol)i:,l nnd viral exl;raneous agen(s. In Ill(, e<,ll }milk fncility at. lJle 8ottlh ,lersey Me~li(.nl l{c.~(,ar()h Fmu,.l.'tt
flueni, (Imy w(,r(, lWl)sinize(l 'rod SUSl)ensions eon(:tining 2.5 (o 5 × 10" veils per ml were prepqred. l ) i m e l h y l su]foxMe was added to a final eoneenit',lion of 5f:,;-. Cell suspensions were distributed in(o 1.2-ml ntnlnlh,s nn(l frozen in liquid nil.rog(-,.n in the Linde BF-I freez/ng ~mit.. These amr)ules :~re s(,ore(I in li(lui(l uilrogen. Several lois of (,issue Imve been frozen, nn(l nml)t~les of each lot. hn.ve l)(?en l.est.e(l for vinl)ilit,y and grow(;]~ upoll, recovery from sto)'nge. The suscel)libility of these tissues to .Rous saree)ha, vosim)htr st.omat;it, is, ,and het'l)es simplex vimtses Ires been studied. Excellen~, viability nn(l gr()wlli iil)on r(,('ovory w e r e observed in ;5 out of 7 h)ls. Tile vi,ll)ilii,y of the cells reeovere(t from li(0fid nilr()gen was 8;5 to 9;5% by t r y p a n b l u e (lye exclus/on. Cult.ures prepared from (:hese sust)e.nsions grew to confluency (m each of 6 successive ])'1:SSgl~es.
31. C y t o l o g i c a l Comp.'irison o f [ l u n m n K i d n e y Cells Cultu red f r o m Fresh ;Ind F r o z e n - T h a w e d Cell S u s p e n s i o n s . T. J. MAtaXlX aND V}:I~NOX ]L P)~mn" ( D e r m r m m n t of Research P a t h o l o g y , Ge.orgetown Unix'ersily School o f M e d i c i n e and ] ) e n t i s t r y , V~:nshingt.on, D. 0.: and the Tissue Bank DelmMment, U. S. N a v a l M e d i c a l School, Be( hesda, M , rylnn(t). Cells ()f hum:)n ki(Ineys from fel;uses and infants were (lisl)erse(I l, 3, e n z y m n i i c digestion. Freshly l)rot't'ss(~(l (,ells were p l a n i e d in ('.ulture; aliquots of cell suspension were frozen in liquid nit.).ogen wi(ll (limelhyl sulfoxide ns prot, e¢'tive agent, Cell
ABSTRACTS cost thermoelectric cooler include size and simplicil.y of cooling unit, precise control of cooling rates, simple adaptation for hyperbaric environment or concurrent perfusion, portability, and o:~s{? of operation. 26. Rapid C o o l i n g by Means of Pressurized Liquid Nitrogen2 ~ A. P. MAC].~I~NZI~, C. .I~:RO~:~Er~,* aND B. J. LuYm' (American Foundation for Biological Research, Madison, Wisconsin). PrEvious determinations of the relative efficiencies of various refrigerants more or less frequently employed in the rapid freezing of biological materials yielded the following sequence, in which the refrigerants are listed in order of decreasing ef'fcetiveness: a) propane at -180°C; b) freEzing nit,rogen a,b--210°C; e) ].~'reon 22 at --150°C; d) isopentane at --150°C; e) liquid n~trogen at --196°C; f) liquM helium II at -271.5°C. Thus, li{luid nitrogen at --196°C ranks rather low in the soriEs. But. according to the principles of heat transfer in boiling liquids, the rate of exchange can be considerably increased if the boiling liquid is subjected to pressure, the rate being approximately in proport.ion to the density of the vapor in the l:,oiling interface, whether the process be at the "film" or the "nucleate" boiling stage. To apply this principle to liquid nitrogen, the following appara.tus was set up. A copper cylinder, mounted vertically and partly filled wit,h liquid nitrogen, was equipped with means for sample onl:ry and with pressure gauge, safety valve, etc., and partially immersed in a Dewar flask• The liquid nitrogen within the cylinder was then pressurized either with helium, in which case the liquid nitrogen could be maintained at --196°O or below regardless of the total pressure, or with nitrogen gas, in which case the temperature of tho liquid nitrogen becomes a function of the pressure. t}reliminary tests show that the apparatus, when ln'Essurized to 100 psi, permits higher: cooling velocities than attained by any of the methods previously employed. 27. Lethal Effect of Ultraviolet Light on Bacteria and B a c t e r i o p h a g e s at Low T e m p e r a tures, hi. J. ASHWOOD-SMITI-I (MedicalResearch Council Radiobiological Research Unit, I=Iarwell, Didcot, Berkshire, England)• The survival of Escherichia colt B/r :WP2 after irradiation with ultraviolet light (2537 .~) has been studied over a temperature range o f +22~C to -269°C. In the presence of ice, the lethal effects "Supported by the Office of Naval Research, .on~racL NON R 2437(00).
of ultr'lviolet irradiation increased by a factor of 7 betwEEn I°C and -79°C. No increase in sensitivity to ultraviolet irradiation could be demonstrated in supercooled solutions or in glycerol glasses at --79°C. Studies with bacteriophage T~ dEmonstratEd a similar but less pronounced increase in sensitivity to ultraviolet irradiation at -79°C. . At temperatures as low as --I96°C, the lethal effects of ultraviolet irradiation on E.coli are less than at --79°C. This latter result may be explained on the basis that reactions which involve "ch'~rge transfer" have different temperature coefficients from reactions involving "excitation energy migration." Evidence from experiments on low temperagure irradiation of E.coli t ! C R (this strain lacks the enzymes necessmT to excise thymine dimers) and Ta phage is interpreted to indicate that .~t new lesion, not thymine dimer, is formed at low temperatures. 28. Studies o n the M e c h a n i s m of Freezing D a m age in Bacteriophage T 4 B 2 ° STANLEY P. L E I B O 17 AND PZTZI~ MAzuR (Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee). In an attempt to understand the destruction Of biological function by freezing and thawing at an organizational level intermediate between the macromolecular and cellular levels, bacteriophages T4B and T4BO~ have been exposed to a variety of freeze-tliaw treatments. Phages T4B and T4BO~, osmotic shock-resistant mutants, were suspended in distilled water or in various solutions and w e r e subjected to se~,eral combinations of cooling-warming rates i n the range o f - - 2 ° C to -:780C for 2 to 50 min. Survival was assayed by standardmethods.-No: dainage to phage :T4B occurred, a t s u b z e r o temperatures in the absence of ice formation. When ice was present, the osmotic shock-resistant mutant, T4BO~, was found to be much less sensitive to rapid warming than was the shock:sensitive f0rm, :T4B. ttowever, when T4B and T4BO~ weresubjected.to slow warming, a :treatment tliat :eliminates osmotic shock, the survival of the. two Strains was parallel: Increasing lengtlis of exposure a t - - l { C , ~ 5 0 C / o r - 7 8 ° C had little or no additional effect on:survival of T4B t h a t had been frozen rapidly ~and warmed rapid!y. :However, the survival:::of iT4B :exposed to -200C and -~30°C decreased with increasing lengths of exposure.. Wlien T4B phagessuspended in osmolar s01utions o f KNO~, N a C l i o l ; C s C l ~ were: rapidly cooled to a n d rapidly warmed f r o m subzero temperatures, the lower t h e eutectic point of the suspending solution;the lower ttmsurvival. • "
}O
-.
O
Research spon.ored m pal t }> the:U. S. Atomm Energy Commission Under contract witti t h e :Union Carbide Corporation and in part b y U . S..:Public H eel t l i Serv i ce I.~"ello wsh ip 1=F2' A:i- i9,981,91A1, ~TU. S: Public HeMth Service :Fellow.
18
A 13STFI AC.'TS
On ll~e Imsis of (hose resul(s, it is eonehule(l th.'~(; n(, h m s ( ( w ( ) factors qre Ol)ernl,ive in ['n~tsing freezing (lnn.~g:e to phage '~P4I)). One of (l~ese is the osmoli(, shor'k to wl~i(.l~ the phage is s~I)je(:te(l when r'~l)i(I]y warme(I, nn(I, tlmr(,fore, r:~I)i(ll3" (lilu(e(l, fron~ (h(> frozen sI:tt(,. 29. Effect o f C o o l i , l g a n d W a r m i n g Veh)('ily on Ihe S u r v i v a l of F r o z e n a n d Thaw('(! Yeast. TM P).:'rEt~ M,.~zt:~ :t.,,'D ,IaXU:~.: a. Se~.~D'r* (Biology ])irish))), Oak Ri,lge N , li())m] l,abor:)t.oD', Onk Ri(lge, Tennessee). O l l e of iJle I~oI'(' intl)()rlun[. ('ollSC(lHences c)f c(),)lin,:. velov, ily is (o (h:termim, whether inlra('elhfln)" wa((,r freez(,s within ('ells (a. usually leth:d provess) or flows out: of (:ells :m(I freezes exle)'n,lly (nn often inn,)(:uous pro('('ss)..If cooliL~g is )'api(1, the formc, r occurs; if it is slow, the latter oc(:urs. Cnleulntio)~s in(licale thai; f(.)r l.h(, yenst:. Saccharom!lCeS cere~,islay, inira('ell~la.r f)'eezing shouhl occur if the cooling velo(i.ty ex('eeds I ° (o I0°(,' per rain, an
'SR,esearch sponsore(t by tlm [7. S. Alomi(,. E n e r g y Commission under cent(a(:(: wi(h the [_Tniol~ Ca rbi(h: Corl )oral ion. '"SUl)l)orle(] in I)'~rl ])y Gran(s CA-0,E)53-(}6 and CA-03S45-08 from (h(: Na(ion')l Cnn(~er Insli(ule an(I Grant I;:-111 F fro)n (he Ame)'ic'm Cnnee.r Society.
.A.UTItt:, E. (I,EEXI.: (Soutl) Jerse.y Medical Iles,,:Iv(:h F'oii~i(ht(,ion, Cam(h.;n, New Jersey). Tile nee(1 for n mmlimml)s <'uil.un:: line of chick oral)rye fil)rol)lasls h:is l-)econm more a(:ul~e with t I.~ in(.,rt,tse in the ttse of el)iek c,m b r y o (.issues in l)ioh)gieal res(,nrvh :tn~I l~r()du('lion. A.I; the pres(mI: l illt(' ( h o s e w h o tlse ('Ili(.:I~ ( : m l ) r y o etl]i:ures as n I'es(.mrell ()r l)ro(]tlvliort le(']tlli(llie ill IlS(; l'eLurn, r e -
l)('n(edly to (h(' ('ml)ry()na(.(,(I egg bee'l)tse, lhe cell ("ulttlre c:m~o( I)e cnrri~'(l in series for more thgm 4 It) 6 l):lss, g('s..,ks nn ,tlternative to :m estnb[isI,:,(I (:l~ick (.ell lhw, we stlggest, t..hnt n ('ons!.:mt s)II)l)lY (.)f at cl~ar,('ieriz(:(l chi('l{ cell can be m,(le av')ih)l)le by f)'eezi))g )nany "~)))l)))]t:s of p)'imnry ('ell cul(.tlres in li(ll)i(l nitrogen. Cells qre fully reeoverqI)le, can l)e sul)('itltxu'ed for at. ]enst, 6 pnssages, and ("fill ].)O ("]ltlrtl(.'.ieriz(,(l ill v)lri()tl:S..' w a y s tllld showl~ 1o
be fr('(, frOlll t)()ill lnierol)i:,l nnd viral exl;raneous agen(s. In Ill(, e<,ll }milk fncility at. lJle 8ottlh ,lersey Me~li(.nl l{c.~(,ar()h Fmu,.l.'tt
flueni, (Imy w(,r(, lWl)sinize(l 'rod SUSl)ensions eon(:tining 2.5 (o 5 × 10" veils per ml were prepqred. l ) i m e l h y l su]foxMe was added to a final eoneenit',lion of 5f:,;-. Cell suspensions were distributed in(o 1.2-ml ntnlnlh,s nn(l frozen in liquid nil.rog(-,.n in the Linde BF-I freez/ng ~mit.. These amr)ules :~re s(,ore(I in li(lui(l uilrogen. Several lois of (,issue Imve been frozen, nn(l nml)t~les of each lot. hn.ve l)(?en l.est.e(l for vinl)ilit,y and grow(;]~ upoll, recovery from sto)'nge. The suscel)libility of these tissues to .Rous saree)ha, vosim)htr st.omat;it, is, ,and het'l)es simplex vimtses Ires been studied. Excellen~, viability nn(l gr()wlli iil)on r(,('ovory w e r e observed in ;5 out of 7 h)ls. Tile vi,ll)ilii,y of the cells reeovere(t from li(0fid nilr()gen was 8;5 to 9;5% by t r y p a n b l u e (lye exclus/on. Cult.ures prepared from (:hese sust)e.nsions grew to confluency (m each of 6 successive ])'1:SSgl~es.
31. C y t o l o g i c a l Comp.'irison o f [ l u n m n K i d n e y Cells Cultu red f r o m Fresh ;Ind F r o z e n - T h a w e d Cell S u s p e n s i o n s . T. J. MAtaXlX aND V}:I~NOX ]L P)~mn" ( D e r m r m m n t of Research P a t h o l o g y , Ge.orgetown Unix'ersily School o f M e d i c i n e and ] ) e n t i s t r y , V~:nshingt.on, D. 0.: and the Tissue Bank DelmMment, U. S. N a v a l M e d i c a l School, Be( hesda, M , rylnn(t). Cells ()f hum:)n ki(Ineys from fel;uses and infants were (lisl)erse(I l, 3, e n z y m n i i c digestion. Freshly l)rot't'ss(~(l (,ells were p l a n i e d in ('.ulture; aliquots of cell suspension were frozen in liquid nit.).ogen wi(ll (limelhyl sulfoxide ns prot, e¢'tive agent, Cell