THE INFLUENCE OF TEMPERATURE ON THE RATE OF GROWTH OF SPOROTRICHUM CARNIS, FROM -io C. TO +3o C.

Transcription

1 379 THE INFLUENCE F TEMPERATURE N THE RATE F GRWTH F SPRTRICHUM CARNIS, FRM -i C. T +3 C. BY R. B. HAINES. (Fm the Fd Investigatin Bad f the Depatment f Scientific and Industial Reseach, Lw Tempeatue Statin, Cambidge.) (Received ist Mach, 93.) (With Six Text-figues.) THIS muld is f fequent ccuence n sted cacases and in cld stes. The lean ptins f cacases which have been kept f eight ten weeks belw ze centigade but abve i C. ae fequently cveed with muld gwth. N such gwth has been s fa bseved n cacases sted at C. Bks and Hansfd, in eviewing muld gwth n cld-ste meat, fund that sme stains f Cladspium hebaum develped and gew at 6 C. (Bks and Hansfd, 923). They als suggested that the mulds including Sptichum, wuld pbably gw between C. and 6 C. It is theefe f inteest t attempt t detemine the pecise gwth limits f this muld. Tw cases have t be investigated : the influence f tempeatue upn gwth pe se, and als the effect f physical changes in the medium cnsequent upn feezing. The pesent cmmunicatin is cncened with the fist f these nly, all measuements belw ze having been made n supecled media: i.e. media cled belw thei feezing-pints but fm which ice cystals had nt sepaated. EXPERIMENTAL. The paticula cultue used was islated fm a cld ste. Seveal platings, n a mdified Czapek's aga, yielded a pue cultue. This aga, which was used thughut in the gwth expeiments, had the fllwing cmpsitin (Waksman and Cutis, 96): NaN 8 2 gm. Sucse 30 gm. K,HP 4 gm. Distilled wate lite. MgS 4 7H gm. ph 6-4 appximately KC 0-5 gm. measued climetically. FeS 4 7H 2 00 gm. The stck cultue was kept n aga slants f this medium. In geneal the spes wee taken fm a cultue at least a week ld. 26-2

2 380 R- B. HAINES A small quantity f steile melted Czapek's aga was allwed t slidify n the cente f a steile cve-slip in a steile cveed Peti dish. Cae had t be execised nt t make the dp f aga f t geat a thickness, it wuld have been impssible t fcus n its suface with the f in. bjective used. A little f the cultue t be studied was then tansfeed with a steile platinum wie t the aga and spead as evenly as pssible. Cve-slip and aga wee next pessed-dwn n the tp f a small glass ing f suitable size, cemented t a micscpe slide with Canada balsam. The tp gund suface f this ing had peviusly been cated with a stiff ubbe gease which did nt "un" at the tempeatue f incubatin. A single lage dp f wate was placed in the cente f the fl f the chambe s fmed t maintain a satuated atmsphee, Tmkins having shwn the imptance f humidity in muld gwth (Tmkins, 929) (see Fig. ). In many cases the special facilities f the Lw Tempeatue Statin pemitted the caying ut f incubatin and measuement in the same chambe, maintained at the desied cnstant tempeatue, s that nq es wee intduced by the fluctuatin f tempeatue caused by tanspt fm incubat t micscpe..vp. Klip x / / ^ agcu~ -f spes ^f / wate Fig.. With the highe tempeatues, hweve, the dinay labaty incubat was used and bsevatin caied ut as quickly as pssible n emval t the micscpe stage. Stictly speaking, measuement f the ate f gwth shuld be a measuement f inceased vlume. If, hweve, bsevatins ae esticted t the ealy phases f gwth and nt made at the exteme tempeatues at which the ganism will gw, the diamete f the gem-tube is appximately cnstant. N e is thus intduced in substituting length f vlume. This hlds tue nly duing the lgaithmic phase f gwth. bsevatins n lde cultues shwed that the theads wee ften seveal times as thick as in the ealie phase. Duing nmal gwth the spes at fist thicken t abut twice thei pevius diamete, then elngate in ne diectin, a gem-tube being put ut. In sme cases anthe gem-tube makes its appeaance in the same staight line as the fist but in the ppsite diectin. Me than tw such gem-tubes have neve been bseved. Banching begins t take place sme hus afte geminatin at the highe tempeatues. At 30 0 C. spes wee seen t geminate and the theads t incease in length f a time, but the theads wee thicke than thse gwn at me favuable tempeatues. Afte sme few hus a geat deal f thickening and cnttin f

3 Influence f Tempeatue n Rate f Gwth f Sptichum canis 38 the theads tk place, s that mst bizae shapes wee ften pduced (see Fig. 6). In this case the substitutin f length f vlume des nt hld, and the gwth at 30 0 C. cannt be estimated accuately. At 5 0 C. the theads wee slightly thicke than at me favuable tempeatues, but slw extended gwth withut futhe thickening tk place f abut 00 hus. Afte this, extensin f the theads tk place but cmpaatively slwly, me thickening and banching C Ci Time in hus Fig. 2. ccuing. Pvided that the measuements ae cnfined t this ealie peid, the e intduced in egading inceased length as an index f gwth at 5 0 C. is small. F tempeatues abve ze abut half a dzen pepaatins wee made f each expeiment and incubated tgethe. Measuements wee then made at andm fm ne f the slides, fllwed by at least tw thes. In this way es f sampling wee educed t a minimum. Althugh nly a factin f the ttal spes n

4 382 R. B. HAINES any ne slide was measued at any ne time, that factin was cmpsed f diffeent individuals at each bsevatin. By using a lage numbe f slides than was necessay, epeated measuements n ne slide wee avided. In all cases at least tw sepaate batches f slides have been used in de t shw that the esults btained wee epeatable. With thse t be incubated belw ze it was necessay t make me slides and t select thse which had nt fzen. At 5 0 C. it was cmpaatively easy t supecl the aga, abut 50 pe cent, f the slides put dwn nt cystallising. At 7 C, hweve, nly tw f all the slides pepaed did nt cystallise initially, and emval f these t the micscpe stage f measuement caused thei subsequent cystallisatin. Gwth was measued by means f a Ramsden micmete eye-piece and a I in. il-immesin bjective. ve ealy peids f incubatin the esulting theads wee faily staight and measuement was nt difficult. Afte lnge times the seveal bends and banches in the theads wee measued sepaately as accuately as pssible, and ttalled. In all cases thity measuements wee made at andm and each pint in the cuve is the mean f thity such measuements, except in a few f the last measuements taken at the highe tempeatues. Thee the theads wee s inticately banched that measuement became difficult and labius, and the mean f ten lengths had t be. taken as epesentative. Refeence t the lgaithmic s> gwth lines (Fig. 3) will shw in geneal a falling 3 ff at the highe pints. At the extemes f tempeatue makedly abve 25 0 C. and t sme extent at 5 0 C. thee seems t be definite depatue fm nmal lgaithmic gwth duing the peids f the expeiments. At the the tempeatues, this falling ff must be ascibed t the difficulty f measuement f the lnge theads, such lengths being unde-estimates. The mean esults btained ae tabulated in Table I and shwn gaphically in Figs. 2 and 2 A. They ae expessed as divisins f the micmete scale. Calibatin shwed that ne divisin f the Ramsden scale was appximately equivalent t 0-3/x. In Table II is dawn up a epesentative set f values btained in ne expeiment at 0 C, in de t shw the individual vaiatin which ccued fm thead t thead in a given selected gup. It will be seen (Figs. 2 and 2 A) that thee is an initial lag phase, fllwed by a peid f lgaithmic gwth, that is, a peid f apid gwth duing which the lgaithms f the length pltted against time yield a staight line. F pesent pupses thee ae included in the tem "lag phase" bth the "initial statinay phase" and the "lag phase "f Buchanan's nmenclatue (Buchanan, 98). If the length f "lag phase" a 'at = JI * 80 Time in days Fig. 2 A. 00

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6 384 R. B. HAINES be pltted against tempeatue, a smth cuve esults, shwing that thee is a definite thugh nt a simple elatinship between tempeatue and length f lag. Fig. 3 shws the lgaithms f the lengths duing the lgaithmic gwth phase pltted g f c ep c 3-0,5 C 'ice 2-0 & We Time in hus Fig. 3- against time. The gaph indicates that the gadients f the staight lines s fmed decease at fist slwly, then me apidly. Using the expessin n lg Z», lg L, tan0 = ^=? 7j -l, whee 8 = gadient, L t = length at time T t, L^ length at time T lt the mean value f the angle f slpe f each line f any given tempeatue can be calculated, in tems f the abitay units f time and length used. mitting values depating fm the mean value, and multiplying by ten f cnvenience, the mean values s btained ae given in Table III and gaphically in Fig. 5. The pints lie in a smth

7 Influence f Tempeatue n Rate f Gwth f Sptichum canis 385 cuve appaching the tempeatue axis asympttically, but becming vey clse t this axis at a tempeatue f 0 C. In the wds gwth n supecled Czapek's aga becmes infinitely slw at i C, which is in accd with the vey slw ate f gwth bseved expeimentally n the tw slides which it was pssible t supecl t 7 0 C. Table II. A epesentative set f values btained in ne expeiment at 0 C. Scale divisins Time in hus Mean ' ig ' ? i in I i Table III. tan0 = The value f tan 8 was calculated f each pint n the staight line f lgaithmic gwth, and the mean value f tan 8 x I is given belw. Tempeatue 25 0 C. 2 C. 5 C. i C. + 5 C. C. - 5 C. tan 8 x i (5) 00 (2)

8 386 R. B. HAINES APPLICATIN F THE ARRHENIUS EQUATIN. The expessin used abve f the calculatin f tan 9 is in effect a measue f the velcity cnstant f gwth (k). Thus fm the equatin dl dt = kl, 0 / / 2-00 \ M X / / " X a: \ \ \ / , Tempeatue C. Fig- 4- Fig. 5- whee L = length f thead, t = time, f the ate f gwth duing the lgaithmic phase, it fllws that, _ i j k = gl0 ^~ x 2-303, whee Lj = length f thead at time T lt L* length f thead at time T t, that is, k = tan 8 x The fm f the Ahenius-van't Hff equatin geneally used in bilgical wk,,. d\g,k_ -n ^. = "citical themal incement," indicates that lg k pltted against the ecipcal f abslute tempeatue shuld yield a staight line. Values f k btained fm the expeiments with Sptichum ae tabulated in Table IV, and ae shwn gaphically

9 Influence f Tempeatue n Rate f Gwth f Sptichum canis 387 pltted against =, in Fig. 5. It will be bseved that they lie nt n a staight line but n a cntinuus cuve. Table IV. Temp. C. j abslute T x 0* lg k fm expeimental esults X 0' lg k fm smthed cuve x 0' u calculated fm expeimental values f k / calculated fm smthed values f k fm Fig '4i 3H 'II I-02 i ,495 7,670 3,290 8,050 26,640 53,57 4,830 0,0 4,280 23,930 30,090 48,560 Gwth at C. x hus G wth at 30 C. x hus Nte thickened unusual shapes Gwth fm a single spe at C. x400 Fig. 6.

10 388 R. B. HAINES Integatin f equatin (i) gives T x and T 2 being abslute tempeatues. Values f p can be calculated fm this equatin. These als ae given in Table IV. Hee again n is nt cnstant but vaies cntinuusly with the tempeatue: a finding in accd with the views f Fulme and Buchanan as ppsed t thse f Czie (Fulme and Buchanan, 929, and Czie, 926). SUMMARY. Measuements f the ate f gwth f Sptichum canis n Czapek's aga indicate that:. The ptimum tempeatue f gwth is at 25 C. 2. Gwth can take place at 30 0 C, but is esticted and unusual shapes ae pduced by the thickening f the gem tubes. 3. Faily gd, thugh slw, gwth was btained at 5 0 C. n supecled aga, and gwth als tk place at 7 C. n supecled aga. Thee ae indicatins, hweve, that gwth is smewhat estained at 5 0 C.; the gem-tubes becme thicke and cul me eadily than at highe tempeatues. 4. In n case was gwth bseved n fzen aga duing the peids f incubatin f the slides up t tw mnths. 5. A cuve is given shwing the elatin between tempeatue and ate f gwth duing the lgaithmic phase. The fm f the cuve suggests that gwth n supecled aga becmes infinitely slw at i C. 6. Applicatin f the Ahenius-van't Hff equatin t the esults btained is cnsideed. The auth wishes t expess his thanks t D G. S. Gaham-Smith, F.R.S., f his inteest in this wk. REFERENCES. CRznt, W. J. {926). "n cuves f gwth, especially in elatin t tempeatue." jfun. Gen. Pkysil. 0, 53. BUCHANAN, R. E. (98). "Life phases in a bacteial cultue." jfun. Inf. Dis. 23, 09. FULMBR and BUCHANAN (929). "The themal incements and citical tempeatues f bilgical eactins." Pc. Sc. Exp. Bil. Med. 24, 446. TMKINS, R. G. (929). "Studies f the gwth f mulds." Pc. Ry. Sc. B, 05, 375. WAKSMAN and CURTIS (96). "Sil Actinmyces." Sil. Set., 99.