(From the Department of Bacteriology and Office of Naval Researck, Task V, University of California, Berkeley)

Transcription

1 THE EFFECT OF SODIUM CHLORIDE ON PHAGE FORMATION BY STAPHYLOCOCCI AT ELEVATED TEMPERATURES B~ J. FONG AND A. P. KRUEGER (Frm the Department f Bacterilgy and Office f Naval Researck, Task V, University f Califrnia, Berkeley) (Received fr publicatin, September 1, 1948) It has been demnstrated previusly that staphylccci which have been subjected t rapid grwth in an xygenated medium at 36 C. fr 2 hurs and then reduced t a resting state by washing, resuspending the cells in Lcke's slutin, and maintaining them at 5 C. fr 2 hurs, pssess the ability t prduce an immediate six- t tenfld rise in phage activity titre when added t a phage-cntaining slutin (1). This bservatin was regarded as evidence that actively grwing cells cntained smething which n cntact with active phage particles was transfrmed int phage, Attempts have been made t define cnditins under which cellular reprductin wuld prgress nrmally while phage frmatin did nt ccur either because f the destructin f this factr r the inhibitin f its synthesis. Experiments carried ut several years ag shwed that when a mixture f phage and grwing susceptible staphylccci in rdinary nutrient brth was kept at 42.3 C. the bacteria grew withut inducing the frmatin f phage. This was interpreted as being due t inhibitin f the prductin f the phageaugmenting substance (2). Mre recent investigatins have shwn that bth the "activated" cells prduced by the rapid grwth f staphylccci under favrable cnditins and the phage-augmenting prperty f these ceils culd be quickly inactivated by suspending the bacteria in distilled water and expsing them t 44 C. fr a perid f 15 minutes; at the same time it was bserved that 1 ~ NaCI prtected the "activated" cells against thermal destructin and preserved their ability timmediately give rise t an increase in [phage] when brught in cntact with active phage (3). This bservatin suggested the pssibility f prviding an envirnment fr the phage-bacterium reactin in which 1 ~r NaCI might be expected t prtect the "activated" cells thereby permitting bacterial grwth and phage frmatin t prceed at temperatures nrmally inhibitive. The riginal purpse f the present investigatins was nt attained, fr it was fund that 1 ~ NaC1 interferes with the srptin f phageby susceptible, actively grwing bacteria, and thus prevents phage frmatin. EXPEB r~r~.ntal METHODS The fllwing terms are used thrughut this paper: (a) [phage] = cncentratin f phage/ml, expressed as activity units; (b) [phage]0 = initial cncentratin f 279 The Jurnal f General Physilgy

2 280 ES~PECT 0F SODIUM CHLORIDE ON PHAGE FORMATION phage/ml, in activity units; (c) [phage] f = final cncentratin f phage/ml, in activity units; (d) [phag%t]0 = initial cncentratin f phage/ml, in plaque units; (e) [phagepl]f = final cncentratin f phage/ml, in plaque units; (f) [bacterial0 -- initial number f staphylccci/ml; (g) [bacteria]f = final number f staphylccci/ml. These terms refer t the K strain f Staphylcccus aureus and t the hmlgus K phage. The experimental prcedures emplyed were as fllws: 1. [Ttal bacteria] was determined with a Klett-Summersn phtelectric clrimeter which previusly had been calibrated with bacterial suspensins f knwn density. 2. [Viable bacteria] was ascertained by plating prper dilutins f the suspensins in tryptse agar; cunts were made after 24 hurs incubatin at 36 C. 3. Activity titratins fr [phage] were carried ut by: (a) remving 4.0 ml. aliquts f the experimental samples and chilling immediately in an ice water bath fr 30 minutes; (b) adding ml. f a phage slutin cntaining activity units/ml. t each f the 4.0 ml. aliquts; (c) chilling the bacterium-phage mixtures fr 10 minutes in an ice water bath; (d) preparing dilutins in tryptse phsphate brth fr activity assay accrding t Krueger's methd (4). 4. Plaque cunts were made by Gratia's methd (5). 0.5 ml. f the experimental sample, prperly diluted, was mixed with 3.5 ml. f bacterial suspensin cntaining 16.0 X 107 bacteria/ml.; ml. f melted agar was then added and the whle suspensin was thrughly mixed. ml. f the latter was remved and spread ut n the surface f an agar plate. All plates were read after 18 t 24 hurs f incubatin at 36 C. 5. [Extracellular phage] in a mixture f phage and bacteria was determined by first passing the mixture thrugh a supercel filter (6). This prcedure has been shwn t remve all the staphylecci and the assciated phage withut reducing the cncentratin f phage free in slutin (6). Plaque cunts were then dne n the filtrate. 6. The grwth medium fr all experiments was tryptse phsphate brth. EXeERIm~N~nL I~SlIT.XS 1. The Effect f 1 M Sdium Chlride upn Bacterial Viability at 42 (]. - T determine the effect f 1 M NaC1 upn bacterial viability at 42 C., staphylccci frm an 18 hur agar culture were suspended in either plain brth r in 1 M NaC1 brth and incubated at this temperature. Samples were remved at the beginning, after 1.5 hurs, and again after 3.0 hurs f incubatin fr determinatin f ttal cell cunts and viable cunts. Table I summarizes the results btained. It shws that while cellular reprductin des ccur, a cnsiderable number f the staphylccci die. Only sme 55 per cent f the cells grwn in plain brth are still viable after 3.0 hurs f incubatin at 42 C. The crrespnding viable cunt fr staphylccci suspended in 1 ~ NaC1 is apprximately 33 per cent f the ttal cunt. It is evident that 1 ~ NaC1 des nt exert any measurable prtective effect n staphylccci grwing at 42 C.

3 ~. FONG AND A. P. KRUEGER The Effect f I M Sdium Chlride upn Prductin f the Phage-Augmenting Substance at 42 C.--Suspensins cntaining 108 bacteria/ml, in either plain brth r in 1 ~ NaC1 brth were prepared frm an 18 hur culture f staphylccci. These cell suspensins were then "activated" at 42 C. fr a perid f 3.5 hurs accrding t the prcedure described in earlier reprts. At the end f this incubatin perid ttal bacterial cunts were made and the preparatins were tested fr capacity t increase the activity titre. Table II shws that thse cells which have undergne incubatin at 42 C. fr 3.5 hurs in plain brth r in brth cntaining 1 ~ NaC1 retain the capacity t TABLE I Effect f 1 at NaCl upn Bacterial Viability at 4~C. Average values fr 2 experiments. Sample Staphylccci suspended in plain brth Staphylccci suspended in 1 NaC1 brth Incubatin hrs [Ttal bacteria]f [Viable bacteria]f X 10s X enhance the phage activity titre. As a matter f fact, certain ther experiments in the curse f these investigatins demnstrated that cells suspended in either plain brth r in 1 M NaC1 brth and grwn at 45 C. are still capable f prducing this effect. 3. The Effect f i M Sdium Chlride upn Bacterial Grwth and Phage Frmatin at 42 C.--T demnstrate the effect f 1 ~ NaC1 upn bacterial grwth and phage prductin at a temperature f 42 C., suspensins cntaining 1 X 10 s bacteria/ml, in either plain tryptse phsphate brth r in 1 ~ NaC1- tryptse phsphate brth were prepared frm an 18 hur agar culture f Staphylcccus aureus. T these suspensins the hmlgus phage was added s as t btain a [phage]0 f 1 X 108 units/ml. The mixtures were shaken fr a perid f 3 t 4 hurs in a water bath kept at a temperature f 42 C. and samples were then remved fr activity assay and ttal bacterial cunts. Table III shws that staphylccci mixed with phage are able t grw equally well in the presence r absence f 1 M NaC1, at least as far as we are able t determine after 3 t 4 hurs' incubatin at this temperature. Hwever, the presence f 1 ~ NaC1 definitely affects the [phage]v A lss f apprximately 60 per cent f the [phage]0 ccurs in plain brth mixtures, while thse cntaining 1 M NaC1 exhibit neither lss nr increase in [phage].

4 282 EFI~ECT OIF SODIUM CHLORIDE ON PHAGE FOliATION 4. The Effect f 1 x, Sdium Chlride n Srptin f Phage by Susceptible Bacteria.--Since a culture f staphylccci grwn at 42 C. cntains "activated" cells amng the survivrs, it might reasnably be predicted that a mixture f phage and bacteria kept at 42 C. wuld exhibit sme prductin f phage. Hwever, as nted in the previus sectin, the ppsite appears t be true fr [phage]f is actually lwer than [phage]0. TABLE II Effea f I u NaCl upn Capaci y f Cells Grwn at 42 C. t Increase Phage Activity Titre Average values fr 3 experiments. Activity titratins cnducted n mixtures f cells and phage kept at 5c. Sample [Ttal bacteria]0 X10a [Ttal bacteria]f X 10 D Activity titer [Phage]0 X10 a Activity titre [Phage]f X IO~ Staphylccci "activated" in plain brth at 42 C Staphylccci "activated" in 1 M htacl brtl at 42 C TABLE III Effect f I lr NaCl upn Bacterial Grwth and Phage Frmatin at 4Z C. Average values f 3 experiments. Sample Incubatin [Ttal bacteria] X 10s [Ttal bacterialf X 10s Activity titre [Phage] X los Activity titre [Phagelf X 10s Bacterium-phage plain brth mixture in ]tt$ Bacterium-phage 1 x~ NaC1 brth mixture in One pssible explanatin f these experimental results is that the cnsiderable number f dead cells accumulating in a staphylcccal suspensin grwn at 42 C. remves phage frm the system thrugh irreversible adsrptin, a weu recgnized prperty f dead ceils. If this is the case, then sme change ccurs in the presence f 1 M NaC1 fr the salt mixture shws neither frmatin nr lss f phage. In rder t get sme idea f the differences in adsrptive capacity attributable t 1 ~t NaC1, suspensins cntaining 5 10 s nrmal, nn-activated staphylccci were prepared in either plain brth r in 1 u NaCI brth. The sus-

5 j. :FONG AND A. P. KRUEGER 283 pensins were then mixed with phage s as t btain an apprximate initial cncentratin f 1 X 10 s plaque units/m/. The mixtures were maintained at 36 C. and samples were remved after 0, 0.5, 1, and 2 hurs fr determinatin f [ttal phage] and [extracellular phage] by the plaque cunt methd. The results f three experiments have been averaged in Fig. 1. It is clear that the presence f 1 ~ NaC1 entirely upsets the nrmal prcess by which phage is adsrbed t the bacterial cell. Very little, if any, phage is taken up; the cells remain uninfected and n significant amunt f new phage is frmed. At first glance these results seem t cntradict thse recrded in sectin 2, fr in the latter case bacteria grwn at 42 C. in 1 ~ NaC1 brth give, n the average, a 15-fld iincrement in phage when tested at 5 C. by the usual prcedure. The explanatin f this discrepancy may be derived frm a separate experiment designed t evaluate the influence f temperature in testing fr "activatin" f cells. Fr this purpse staphylccci were grwn in plain brth and in brth cntaining 1 ~ NaC1 fr 2.5 hurs at 42 C. One sample was held fr 0.5 hur at 5 C.; phage was then added and the mixture was maintained fr 10 minutes at 5 C. The ceils were then subjected t the usual test fr "activatin;" in additin, plaque determinatins were made n the whle suspensin and n supercel filtrates. Anther sample f the staphylcccal culture was kept at 42 C., phage was added, and the mixture was held an additinal 10 minutes at 42 C. All the measurements mentined abve were made n this sample als. (Table IV.) The results demnstrate that the temperature impsed in testing the cell suspensin fr "activatin" markedly affects the results. When staphylccci are grwn in 1 ~ NaC1 brth at 42 C. fr 2.5 hurs and are tested at this same temperature fr capacity t raise [phage], nearly 85 per cent f the phage added remains extracelluiar. In cntrast, when the test is cnducted at 5 C., nly 16 per cent f the phage is left free in slutin. Much the same situatin is bserved in grwing mixtures f bacteria and phage maintained at 42 C. When 1 ~ NaC1 is present during the incubatin perid f 2.5 hurs, the final plaque titre is essentially identical with the [phagep~]0 and 85 per cent f the phage is extracellular. In plain brth withut NaC1, the titre is < [phagepl]0 and nly 2 per cent f the phage is extracellular. The plaque determinatins cited in Table V incidentally cnfirm the activity data f Table III. An imprtant pint remains unanswered s far; namely, are the drps in activity titre and plaque cunt nted in plain brth mixtures f bacteria and phage kept at 42 C. fr 2.5 hurs due t lss f phage thrugh adsrptin n heat-kilied staphylccci? If this prves t be the case, ne wuld like t knw als why the same r greater lss des nt take place in suspensins cn-

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7 ~. ~FONG AND A. P. KRUEGER 285 taining 1 ~r NaC1 despite the fact that smewhat mre dead cells accumulate here than in plain brth. Additinal experiments were perfrmed t determine hw 1 ~ NaC1 affects the srptin f phage by heat-killed staphylccci. Staphylccci grwn n nutrient agar were suspended in 1 ~ NaC1 brth and in plain brth. Bth TABLE IV Effect f Temperature n Results Obtained in Test fr "Activatin" Cnducted withstaphyiecci Crwn at 42 C. Sample Treatment f sample.i Staphylccci grwn Held 5 C. ½ hr. 2.5 hrs. at 42 C. Phage added and in 1 ~ NaC1 brth kept 10 min. at 5 C. ] Staphylccci grwn I Kept at 42 C. 2.5 hrs. at 42 C. ] Addphage, hld in 1 ~ NaC1 brth I / 10 rain. at 42 C.! r I ~x ";.~ < 50.( ~x Plaque titre [phage]f Ttal Filtrate X l0 s X 10s J 3.2~ Effect f I ~ NaCl n the Distributin TABLE V f Phage between Staphylccci and Medium at 42 C. Sample Plaque titre [phagepl]o X 10 7 Ttal X10 7 Plaque titre [phagelf Filtrate X 10 7 Staphylccci and phage in 1 NaC1 brth at 42 C. fr 2.5 hrs Staphylccci and phage in plain tryptse PO4 brth at 42 C. fr 2.5 hrs, preparatins were kept fr 2 hurs at 80 C. t kill the rganisms. Equal prtins f the heat-killed staphylcccal suspensins were mixed with phage and kept either at 5 C. r at 42 C. fr 20 minutes. Plaque determinatins were carried ut n samples remved frm the whle suspensins and n filtrates (supercel). The results f this experiment (Table VI) may be evaluated by cmparing the initial titres with plaque cunts at the end f the test perid; the drp in plaque cunt is a measure f the phage taken up by dead bacteria. It is evident that 1 ~ NaC1 vastly alters the adsrptive capacity f heat-killed staphy-

8 286 E]~FECT 0]~ SODIUM CHLORIDE ON PHAGE FORMATION lccci. In the tests cnducted at 42 C. fr 20 minutes, 65 per cent f the phage in the NaC1 mixture is left unadsrbed, while in the absence f NaCI nly sme 0.3 per cent f phage remains free in slutin. When the experiment is perfrmed at 5 C. the salt effect is smewhat less prnunced. In plain brth 3.5 per cent f the phage is unattached t ccci and when NaCI is present the extracellular fractin is 30 per cent f the ttal phage added. Since the grwth f staphylccci at 42 C. in tryptse phsphate brth is attended by a cnsiderable accumulatin f dead cells in the medium (Table I) and since such cells irreversibly remve hmlgus phage frm slutin, an explanatin is available fr the bserved drp in titre (60 per cent lss in Table III and 56 per cent lss in Table V). TABLE VI Effect f 1 M NaCI n Srptin f Pkage by Heat-Killed Staphylccci at 42~C. and 5 C. Heat-killed Plaque titre Plaque titre staphylccci [Phagepl] X lo t Medium Temperature Time [Phagevllf X loa per ~ul 5X10 s 5 X l0 s 5X l0 s 5 X ~ NaCI brth 1 ~ NaCI brth Plain brth Plain brth When 1 ~ NaC1 is present in the brth (Table I), even mre cells die but they present a'less efficient adsrbing surface and mst f the phage remains active; i.e., free in slutin. DISCUSSION Earlier wrk has shwn that the prductin f phage is inhibited in a brth mixture f staphylccci and phage maintained at apprximately 42 C. althugh the bacteria multiply at a fairly rapid rate (2). Mre recently Fng (3) has experimented with the thermal destructin f "activated" cells; i.e., staphylccci which have been allwed t grw rapidly in a favrable envirnment. Nrmally such rganisms when reduced t a resting state at 5 C. pssess the ability t prduce a six- t tenfld increase in activity titre when added t phage (1). Hwever, Fng fund this characteristic t be rapidly ablished by expsure t 44 C. in distilled water alng with the capacity f the yung cells t survive at this temperature. He nted als that the presence f 1 NaC1 permitted the cells t resist expsure t 44 C. and t retain their phageaugmenting prperty. We began the experiments described in the present paper with the idea that the prtective functin f 1 ~ NaC1 cnceivably culd be brught t bear n

9 J. ~FONG AND A. P. KRUEGER 287 brth suspensins f staphylccci and phage incubated at 42 C. In this case, we hped t eliminate the inhibitin f phage frmatin reprted by Krueger and Pucheu (2) and ascribed by them t repressin f the cellular mechanism respnsible fr synthesis f phage precursr. The current experiments were cnducted in tryptse phsphate brth and revealed several pertinent facts: 1. Staphylcccal grwth at 42 C. in plain brth r in brth cntaining 1 ~d NaC1 prceeds at a mderate rate. It is attended by measurable rates f death, with the result that at the end f 3 hurs in plain brth nly 55 per cent f the ttal cell cunt represents viable cells; the crrespnding figure fr the salt-brth preparatins is 33 per cent. These results d nt appear t be due t the frmatin f clumps. 2. Cells grwn at 42 C. in plain brth r in salt-brth are "activated;" i.e., when brught t 5 C. t stp grwth and tested with phage they very rapidly raise the activity titre 15- t 16-fld. 3. Mixtures f staphylccci and phage maintained at 42 C. in plait, brth exhibit cellular reprductin but fail t increase either the activity titre r plaque cunt; in fact, the activity titre falls sme 50 per cent and the plaque cunt abut 56 per cent. When 1 ~ NaC1 is present the final titre is precisely equal t the initial titre x~ NaC1 in brth suspensins f staphylccci and phage kept at 42 C. cmpletely alters the distributin f phage thrughut the cell ppulatin. Nrmally, phage is taken up very rapidly by the cells but virtually all f it remains extracellular when this cncentratin f salt is in the medium. 5. A similar result is bserved when dead staphylccci are tested fr adsrbing capacity. Phage is readily remved frm slutin by dead ccci and the rate increases with temperature. With 1 ~ NaCI in the brth, relatively little phage is adsrbed at 5 C. and even less at 42c. 6. The temperature at which the test fr "activatin" f rganisms grwn at 42 C. in NaCl-brth is cnducted greatly influences the activity titre btained. At 42 C. much f the phage remains free in slutin; this is reflected in an insignificant rise in phage fitre. At 5 C. under the cnditins f the test, mst f the phage is picked up by the cells with the result that a large increment in activity titre fllws. These experimental data furnish a basis fr certain generalizatins with reference t the effect f an elevated temperature (42 C.) and increased electrlyte cncentratin upn the relatins btaining between phage and hst cells. In the first place, the increased temperature seems t favr "activatin" f staphylccci. The immediate rise in activity titre when phage is added t cells grwn at 42 C. and subsequently held at 5 C. t inhibit reprductin, is 15- t 16-fld as cntrasted with the 5- t 10-fld increment cmmnly bserved when the grwth temperature is 36 C. The additin f 1 ~ NaC1 t the grwth medium has n appreciable influence n this phenmenn.

10 288 EFFECT OF SODIUM CHLORIDE ON PHAGE FORMATION Despite this evidence that staphylccci grwing and metablizing ver a perid f time at 42 C. efficiently synthesize the substrate required fr phage frmatin, a mixture f phage and ccci maintained at 42 C. nt nly fails t develp an increase in [phage] but actually registers a cnsiderable lss. Hwever, if 1 M NaC1 is present in the suspensin the lss is prevented and [phage]0 = [phage]v At least tw factrs appear t participate in this seemingly anmalus result: (a) If living cells infected at 42 C. behave as they are knwn t d at 36 C., 20 t 30 phage particles are prduced in each bacterium which then underges lysis releasing the newly frmed phage t infect ther rganisms. Hwever, the rate f bacterial death at this temperature is cnsiderable and a sufficient number f dead cells accumulates in the medium t prvide an adsrbing surface fr the uptake f phage. Phage remved frm slutin in this fashin is irretrievably lst and cannt later participate in the phage-frming reactin which nrmally ensues when living "activated" ccci and phage are brught int cntact. This, then, might be cited as the reasn fr the bserved drp in [phage], in the mixture f phage and bacteria kept at 42 C. It is pssible, als, that the same end result may be attained thrugh the peratin f anther mechanism. If the bacterium-phage cmplex were quite thermlabile, [phage] wuld be expected t fall ff in similar fashin even withut the added factr f lss by adsrptin n dead cells. Experiments which will be reprted in a separate paper indicate that, in fact, such thermal sensitivity n the part f phage attached t susceptible rganisms may exist. The data presented here affrd n prf that it is an imprtant cnsideratin in the experiments discussed but in certain respects it prvides a mre lgical explanatin f the experimental bservatins than des lss f phage thrugh srptin n dead bacteria. Fr this mechanism t functin there is n necessity t pstulate the liberatin f newly frmed phage under the cnditins btaining in these experiments; even if lysis did nt ccur, thermal inactivatin f phage-ceu cmplexes wuld prduce the bserved drp in titre. If remval f phage thrugh adsrptin n dead cells is invked in the case f the grwing mixture, it shuld perate equally well in the curse f testing ccci "activated" by grwth at 42 C. in the absence f phage. Viable and ttal bacterial cunts have shwn that 45 per cent f the cells present after 3.0 hurs are dead. Yet this relatively large fractin smehw fails t cmpete successfully with the adsrbing surface presented by living" activated" rganisms. As a cnsequence, infectin f the "activated" ceils ccurs and the activity titre exhibits a 15- t 16-fld rise. It is true that the efficiency f adsrptin f phage by dead staphylccci is greater at 42 C. than at 5 C. s that ne might be tempted t intrduce this difference as a factr in accunting fr a psitive "activatin" test at 5 C. and a negative ne at 42 C. Hwever, the degree f difference is minr in crn-

11 j[. FONG AND A. P. KRITEGER 289 parisn with the large quantity f phage which culd be adsrbed during the perids f expsure f test suspensins t phage: (b) 1 ~ NaC1 has a prnunced effect n the uptake f phage by living and dead cells. When staphylccci are grwn in salt-brth at 42 C. and subsequently are tested fr "activatin" at 5 C., they adsrb 85 per cent f the phage added and prduce a 14-fld increase in activity titre. Hwever, if the test is perfrmed at 42 C. (actually nt a satisfactry temperature since it permits cntinued cellular reprductin) little phage is adsrbed and the increment in activity titre is negligible. CONCLUSIONS In experiments with the K strain f Staphylcccus aureus and the K race f bacteriphage suspended in tryptse phsphate brth and maintained at 42 C. it was fund that the presence f 1 ~ NaC1 prduced certain drastic changes in the relatinship between the hst cells and the infecting virus: 1. Staphylccci grwn at 42 C. in plain brth r in NaCl-brth are "activated," i.e. when grwth is stpped by lwering the temperature t 5 C. and phage is added, the activity titre immediately displays a rise f 15- t 16- fld ~r NaC1 tends t prevent the srptin f phage by ccci and this effect is mre prnunced at 42 C. than af 5 C. When the activatin test is cnducted at 5 C. (the usual temperature) mst f the phage is picked up by the cells and the described increase in activity titre fllws. If the test takes place at 42 C, there is little srptin and crrespndingly little rise in phage titre. 3. Mixtures f staphylccci and phage incubated at 42 C. in NaCl-brth fail t prduce phage; the final plaque and activity titres are identical with the initial titres. Here, als, the influence f 1 ~ NaC1 in preventing cntact f phage with ccci appears t accunt fr the results. 4. Similar mixtures held at 42 C. in plain brth exhibit a drp f abut 60 per cent in activity and plaque titres. The lss f phage may be due t adsrptin n dead cells accumulating in the suspensin r t the thermlability f the bacterinm-phage cmplex, r t bth. BIBLIOGRAPHY 1. Krueger, A. P., and MundeU, J. H., Science, 1938, 88, Krueger, A. P., and Pucheu, H., Prc. Sc. Exp. Bil. and Med., 1941, 46, Fng, J., J. Gen. Physil., 1948, 31, Krueger, A. P., J. Gen. Physil., 1930, 13, Gratia, A., Ann. Inst. Pasteur, 1936, 57, Krueger, A. P., Scribner, E. J., and Brwn, B. B., J. Gen. Physil., 1946, 30, 25.