Itano, Arao

Tsuji, Yasuhiko

Okayama University Scientific Achievement Repository

Investigation on Agar. 1. Physico・chemicalProperties of Agar and their Influence on the Growth of Microorganisms. By Arao Itano and Yasuhiko Tsuji. [Feb'luary 10， 1935.) The previous repor切り.2)，S) dea.lt with the genera.l review on agar倒 tothe seaweed自 fromwhich agar is manuf郎 t.uredand t.he iodine contents a.nd a1日Othe history together with genera.l chemical composition. In thi日 inve圃tigation，the physico・chemi叫 1proper“es of agar of different gra.des 1， II and III on the markeも， were de旬rminedand their subsequent infl.uenc自 on出egrow也 ofAzoωb蹴旬rchroococcum，. Bacillus subtilis and Sacch町'Omycescerevisia.e was tried. The quality of agar is di貸erentby the locality where it is produced， the kind of seaweed目usedand the process of manufl郎 turing. Consequently the ag町should be carefully cho自由na.ccording 旬 its u自eand examined minutely before iもi自国ed. Especially for the use in也emicrobiologica.l investigation， it is neces-sary加 considerthe quality of ag町制 toi旬 physico・chemicalproperties since a minute quantity of foreign cOllstituent infl.uenceもheirphysiological activiもie由.With the recent increased dema.nd for aga.r in the scient.ific and industria.l ueld自， the fund回 lentalinve自tiga.tionshave been undertaken， a.nd a日 theresu1丸the grading of ag町 ismade on日omephysico・chemicalba.ses instead of出esu perftcial method自which had been em ployed in the past. To obtain some defini旬 informat.iona.s to也einfluence of a.gar of di貸erentgrade upon the microbiologica.l activiti由民 the following investiga.tion wa.s under-旬ken.E玄perimental: Three di貸ere叫 gradeof a.gar， na.mely 1， II and III， manufa.ctured in HyらgoPrefecture were ground up in powder o.nd subjected to the following tes旬:Parlよ Delerminalion0/ Moislure Co腕前.I. Moislure conlenls: Two grams of agar was dried at 105-11伊C.until the weight became con-i8tant. The results are given in Table 1. 576 A.ITANO and Y. TSUJI: Table 1. Moisture Contents of Agar. Grade of agar. l¥Ioistme . • ???????????% 20.37 23.24 22.20 Table 1 indicates that出esecond gmde agar showed the highesもmoisturecontents. 2. Ni'frogen c01Ilenls : Five to ten grams of agar was taken and analysed for the tota.l nitrogen by K.TEr.DAHL method as usua.l and obta.ined the following results : To.ble I. Nitro!len Con旬ntsof Agar. ???? ?????I ~町附i比加t廿叩r agar. (m宮)u.371 0.840 1.862 Nitrogen in 1 g.oven-<lried ag8r. (m芦.)0.466 1.064 2.398 Grade of agar. As shown o.bove， better th自quo.lit.yof o.go.r is， conta.ined less nitrogen. The second grade o.go.r conta.ined more tho.n twice of nitrogen tho.n the first， a.nd the third gra.de was more tho.n twice that of the second. 3・Ashconl，伽 Is:Three gra.ms of ago.r wa.s burnt in 0. crucible a.nd th自residuew倒 weigheda.nd obto.ined the following results : Table III. Ash Contents of Agar. ????? ? ??????Mh in 1 g.air-<lr山 ar. I Ash in 19叩…町(g.) (g.) 0.02112 u.0354 Grade of agar. ???? ???? ?? ? ?? ????????Ta.ble III indica.防自， worse the gra.de contained more a.sh which suggests tho.t some of the soluble inorgo.nic constituent ha.ve been wo.shed down o.nd reta.ined in the third grade. Investigation on Agar. I. 577 4. VelocilyザCoagulalion: A fixed quo.nもiも，yof o.go.r of differenもgrade'0.日 notedinもheto.ble. was t.oken .and mixed with 100 c. distilled wa.ter and boiled for o.n hour with a. return condenser a.nd白ha.kingocc闘 iona.ly. While the solution i白 hot，10 c. of it were poured into a.旬日t-tubeof a. definite日ize，pre-cooled in 0. consta.nt temperature bo.th of 4500. for 20 minutes a.nd tra.nsferred to a.noもherba.th of 3000. a.nd wo.s -exa.mined every hour for the coagula.bility. The日tagefrom the liquid to coagu-la.tion wa.自 notedby A， B， 0 o.nd D， a.nd the degree of coagulation de日ignatedby (+) or (ー)in ea.ch自ta.ge. The re自ult自a.renoted in出efollowing to.ble日:Table IV. Velocity of Coa.gula.tion of 1.5 g. Aga.r in 100 cc. HrO. Time in Grade ofおで竺| 1.5 2 2.5 3 3.5 1. A B C C(+) D H. A B C C(+) D II. A A R C D(ー)Ta.ble IV indicate自tha.tno o.pparent di宜erencewas not，ed beもweenGro.de 1 .and II while Gro.de III wo.自slow.Ta.ble V. Velocity of Coa.gula.tion of 0.7 g. Aga.r in 100 cc. HIO. 弘法王竺 3 4 5 8 7 1. B C C(+) D D I. B C C C(+) D II. A B C C D(一)In this concenもra.tion，a.日lightdifference wa日ob自ervedbetween Grade 1阻 d:n while Grade III wa白muchslower tha.n the other自To.ble VI. v'elocity of Coa.gula.tio岨 of0.5 g. Aga.r in 100 cc. HJO. Time in minutes. Grade of agar了-------、|5 7 9 11 13 • ????????『????C C C(一)C(+) C(+) C(ー )D D C(+) 578 A. ITANo and Y. TI自l;.TI: Table VI shows thaももhecoa伊Ilationra旬 becamemu巴hslower in al grades. and no complete∞agulaもionwas obtained in Grad自III.Table VII. Velocity of Coagulation of 0.3 g. Agar in 100 cc. H20. 示恐竺 20 30 40 50 60 I. B C C C C 1. B(ー) C(ー) C C C III. A B C(一) C(一) C(ー }In this case， no complete coa伊1叫iontook place il any grade， and the di宜erenceamong thr自egrades be叫 memore distilct. Table VIII. Veloci色yof Coagula位onof 0.1 g. Agar in 100 cc. H20. 30 50 70 90 120 A A A A A A A A A A A A A A A In出eabove concelltration， al.grades remained a呂田olution.5. Delermi'na#慨。1/Sol必，1，;砂:Placed 0.5 g. agar in 100 c. H20， and immersed in a hot water bath at 9000. and sbaken at di笠erentint自rvalsas below， and the自olubilit.yw乱日observed.Table IX. Solubili色yof Agar of Differen色Grade.コ吉正 5 10 15 20 25 30 35 40 一 + + + + + 1. 一 一 + + + + + + + + + 1. 一 一 一 + + + 一 一 一 + + + 11. 一 + + + Note日: (ー)indicate日noもdi回目olved;(+) comple飴lydi目白olverl.In¥'e同igationon Agsr， 1I. 579 A日 Ta.bleIX indicates tha.t Gra.de 1 wa.s di鮒olvedfirst which was followed by Grade II a.nd m il the order. Parl Lよ Delerminall・'on0/ Speci_戸'cGr.n砂，Elecll1'cal Conduch:叫y，."伝cosily，Suiface Tension and勾砂ogenIon Concenlralion ザ 0・.1%Agar SoIu的n.Dissolved 0.1 g. a.ga.r in 1∞c. H20 by sha.king one hour阻 dboiling for 10 minutes with a. return condenser a.nd left sta.nding a.t roomもempぽature.Then the following determina.tiolls were ma.de a.nd obta.ined the results noted in Ta.ble X: Table X. De色ermina.tionof Physioa.l Properties of Aga.r. 1. I. lI. Sp. gr. . . 1.0∞12 1.以J014 1.似)()19Electrics) conductivity. . (a) 3.536x 10-d 3.8i3x 10-6 4.284xl0-S ORmotic prω目ure. ・・・ (h) 0.1815 0.4235 0.4719 Vi舵ωity. ・・・・・・・ (c) 1.697 1.576 1.529 Sllrfaceもension.・・・・(d) 7向.0 73.2 70.5 PH' • • • • • • .・・・ (e) 5.98 6.43 6.32 NoぬI!: a. Determined aも30・C.and expre園田din mhoR. b. " hy freezing戸intme仙剖1.c. " at 45'・C.hy U8TWALD apparatllf!. d. " by DU Nlluy me仙吋， in dynel!/aq. cm. 8・，. hy qlllnhydrone meもhod.As the results given in Table X indica.te，自pecificgrn vity，自lectrica.lconduc-tivity and osmotic pres白urewere la.rger in the order of 1， II and III gmde while the visco圃ityand surfa.ceぬtssionwer自由er自verse. Oonsequently it may be stated that more impuriti伺 especiallyelectrolytes wel'e cont.ailled in也eorder of 1， II a.nd III grade. Grade 1 was highes七inhydrogen ion concentmtion while gra.de II alld III were jUl!t about the same， and叫1of也emwere slightly acidic. Parl Lよl DelerminaHon 0/ .Spec折'cGrav砂.Eleclrical COnductl叫)1， OsmoJ必Pres抑 re，Viscosity， SuゲaceTension and勾IdrogenIon Concentralion 0/ Agar Exlracls. S凶cethe results obta.ined in Pl1rt II illdica.ted tho.t出edi住erenceof propeトもiesi日 mainlydue to也epre目enceof water白olublemat旬r，it wa目experimentedwith the wa.もerextracもofo.go.r to SUbsta.lltiate the foregoing re自ults.δ80 A. !TANO and Y. TSUJI: The extrac七wa日preparedby自haking10 gram日ofagar in 300 cc. water for 2 hours and filtered through出epaper and the determination自 weremade a自inPart I. The re自ul旬 are自hownin Table XI. Table XI. De旬rmina色ionof Physioal Properties of Agar E訪問cts.1. I. II. Sp. gr. ・ 1.000152 1.ぽlO291 1.∞0434 Electrical condnct.ivity. . ( a) 1.401 X 10-5 2.687x 10-5 2.885x 10-5 Osmotic pressnre. ・・・(b) 0.56A7 0.6776 0.8470 Viao日ity. ・・・・・・・ (c) 1.140 1.268 1.402 Snrface ten自ion. ・・・・ (d) 70.4 65.2 66.7 PH' • • • • • • .・・・ (e) 6.52 6.86 6.81 PH ・・・・・・・・・・・(f ) 6.67 6.88 6.79 Note日: (a)， (b)， (c)and (d) were determined in the日amemanner as in ca円e01 agar; (e) was deもerminedin the extract which was prepared by shaking 1 g.agar in 20 c. H10 for 1 hOllr， and (f) was tested on a広島γsllspensioninstead of the extracts. The resul旬 givena.bov自， as a whole， indicate the 自泊lilartendency as in もheprevious determination excep七vi自cositywhich may be due toもhecolloidal nature. In case ofもhehydrog自nion concentration， both出eextract and emulsion were aboutthe自am自， Grade 1 b自ingthe high自前followedby II a.nd III inもheorder. Part IV. Injluence 0/ Different Gradeザ Agaron Baclerial Growlh. By th自 resul旬 obtainedin Part 1， II and III， itis apparent thatもhereis a considerable di貸erenceamong the different grade of agar as加出自 physico・chemical prop自rtie自・Since th自agaris used in a larg自quanもiもyinもhepreparation of solid culture medium， itis natural1y expected加 infl.uencedi宜erently出egrowth and 0出eractiviもiesof bacteria，迂differentgrade of agar i自usedinもheircultivation. However日ofor as the authors are aware， no inforrnation i自 availableon出e自ubject. Consequently出einfluence of di館erentgrade of agar on Bacillu自白ubもilis，Azoもobacterchroococcum and Saccharomyc自由 cerevi自iaewas undertaken， and the r自由ul旬 arereported below. Experimental. Grade 1 and III ag町 wereused in order to bring ouもthedi貸erenceplainly if there were any di貸eren伺 inthe influenc自， and出eforemenもionedorgani自msinour自tockwere used. Im'estigation on Agar. l. I. Injlu伺 ceon Bacillus subHlis : The following solid cultur自medio.were u自ed:0.) Albumin agar. Glucose Dipoも朗自iumphoBpha.te (K2HP04) Ma.gneBium自ulfo.t白 (MgS04.7H20)Fer吋C 自ulfat，e(F~(S04)3. 9H20) Egg albumin H20 Agar (Grade 1 or 111) 1.0g. 0.5g. 0.2g. t.ra.ce O.~お g.1，似ゆ.Occ.15.0g. 581 DiJf erence i" Gr側 '11zザ B.sublilis : - By using Grade 1 a.nd 11 agl¥r in the o.bove media a.nd made pl叫eculture of B.日ubtili円 andincu bat.ed 叫 300C.The ex阻 lInationw剖 madeeveryda.y forもhenumber of colonie自a.ndtheir size together wiもhthe ammonia produced. The a.mmonia Wo.R det.ermined by separa.t-ingもhemedium from Petri di!lh and diRsolved after恥 idifyingwit.h sulfuric acid a.nd di自tilledwith Pn回El.'smicro-KJELDAl!L app町atuBo.nd fino.lly by NESSLEB'S colorimetric me出odw帥 used.The results are given in Table x.n， XIn and XIV. Days. 2 3 4 5 8 7 Table xn. Numb.er a.nd Size of Colonies of B. subtilis on Albumin Aga.r Medium. 1. Nllmber Si:-.e of colonies. of coloni酬.51.0 ( 1/泊)mn.) 0.34 77.0 1.26 74.0 1.74 1.98 2.10 2.30 To.ble XIII. Nnml同rof colonie札76.0 78.0 78.0 II. おizeor colonies. 寸市出訂アU.62 1.72 2.10 2.26 2.56 ~.74 Numb.er and Size of Colo'凶esof B. subtilis on Nutrient Aga.r Medium. Gr副le 1. 11. of agar. Days・~ Number Aize Nllmher Size of ('.olon ies. of ('.Qlnnies. of colonieA. ofωlonies. 77.7 ( 1/己oern.) 2 2.45 76.5 ( 1/20 cm.) 3.32 3 76.2 3.10 77.5 4.62 4 73.7 3.52 74.5 5.07 .582 A.ITANO and Y. TSUJI: Tab1e XIV. Ammonia Product1on of B.自ubtilisin Albumin and NutrienもAgarMedia. Ammonia produced in 100 c. medium. Grade of agar. L 111. AIhl1min agar. (mg. N.) (mg. N.) 1.55ヨ1.4~61.667 1.587 Nutrienもag晶r.(m日.N.) I (mg. N.) 12.505 I 9.845 14.196 11.046 To.b1e xrr o.nd Xlr indico.t白 tho.tth白 numb白rof co10nies d白ve10pedwo.s about the Flo.me in 0.1 medio. but the size of co10ni朗 in出自 medio.mo.de of Grade III o.go.r， wer白 1o.rger飽 pecio.llyit wo.s very mo.rked on the nutrient o.go.r medium. Also， o.s To.ble XIV shows， more o.mmonio. wo.s produced in both medio. Qf Gro.de III o.go.r， and o.go.in the di宜白rencewas very mo.rked in t，he nutrient o.go.r m白dium.2. Injluence on Azotobacter chroococcum : The insuence on Azotob郎防rchroococcum was determined in A.SHBY'S medium Qf the following composiもion:Mo.nni加1(OsHs(OH)6J Mo.gnesium Flulfo.te (MgSO.. 7H20) Monopota.ssium' phospho.te (KH2P04) Sodium chloride (No.Ol) Oo.1cium sulfo.te (Oo.SO. 2H20) Ca1cium co.rbono.te (Co.008) H20 Ago.r (Grade 1 or III) 10.0g. 0.2g. 0.2 g. 0.2g. 0.1 g. 15.0 g・1，∞O.Occ. 15.0g. A日祖国時 ofB. subtilis， the size of co10nies o.nd the nitrogen fixed were <1eωrmined， o.nd the results o.re noted in Tab1e XV o.nd XVI. To.b1e XV. Number and Size of Colonies of Azotobacter chroococcum in Ashby's Medium. ¥ポfL 1. III. Nl1mber Size Nl1mher Size of colonie目. 。fcolonies. of colonieR. of colonie目.( 1/乙UCIl.) (1/iO ern.) 2 36 一3 179.8 0.99 1i5.0 1.73 4 183.8 2.02 185.3 2.57 5 191.3 2.53 187.5 3.15 8 一 3.45 4.05 7 3.67 一 4.62 Inv舗もigaもionon Agar. I. 583 The results o.re 0.1自ogiven in photogro.ph (Plo.te XXX). To.ble XVI. Qua.ntity of Nitrogen fixed by Azotoba.cもerohrooooccum in Ashby's Medium. Grade of agar. Total nitrOllen in 100 CC. ι .. 町】cum町1.L II. ????、?????? ??????Nitrゅgen台xedin 100 CC. medillm if 10 day払(rng.) 11.348 14.230 The foregoing results indico.te t，ho.t th自numberof colonies were o.pproximo.tely tho RRme but their白izewo.白 muchlo.rger in Grade III o.go.r medium than in co.se <>f Grad自1. Al日omuch 1ぽ gel'quo.ntity of nitrogen wo.s fi.xed in Gro.de III o.go.r. 3. Injluence on Saccharomyces cerevi，叩e:Th白followingmedio. were u岨00:A. LAUR聞 T'白 medium:Ammonium自ulfo.ω((NH.hSO，lMo.gne自iumBulfo.te (MgSO，. iH20) Pot闘白iumo.cid pho白pho.t自 (KH2P04~Mo.ltose (CI2H220U) H20 Ago.r (Gro.de 1 or III) B. Kodi (mo.lt) extro.ct medium事:Kらdi(mo.lt) extract Agar 4.71 g. 0.10g. 0.75g. 50.∞g. 1，αX>.α)c. 15.α)g. 1，αX>.O c. 15.0 g. * 300 g. Kddi extracもedin 1，000.0 c. of waもer.The number o.nd白izeof coloni倒 wered白色erm泊ed回 inth白previousexperi・men旬， and the quo.ntity of sugo.r cODRumed WRS determined o.lso in this c卸 e.The reRults o.re shown in To.ble XVII， XVIII o.nd XIX. To.ble XVII. Number a.nd Size of Colonies of Sa.ccha.romyces cerevisi随on Lo.urent's Medium. ¥三て 1 m. Nllmher Si:r.e Number Size 。fcolonies. 。fωlonie岡. of coloni帥 . of colonies. 2 一(l/:!.りcm.)29.3 (l:!.O cm.) 3 134.3 0.4L 178.8 1.14 4 17.0 0.83 185.5 1.79 5 177.2 1.22 183.5 2.31 8 一 1.38 一 2.43 7 一 1.62 一 2.76 584 A.ITANO and Y. TSUJI: To.ble XVITI. Number and Size of Colonies of Saccharomyces cerevisiae on Kodi Agar. G!'sde 1. of agar. Nl1mher 向i7.eDay凡 of colonie同. 。fcolonie自.(l/;!'U~m.) 2 224.0 2.26 3 222.5 6.64 4 221.0 8.32 II. Numbe!' of colonies. 225.7 223.5 25.。Si7.e of colonies. 市芦同市ナ2.66 7.45 9.70 To.ble XIX. Quantity of Sugar consumed in Laurent's and Kodi Media. LAURENT'S. Kodi. G!'ade of aga!'. Resicll1al 8l1gar'. R回iclnal 8ugar* 同I1gar.- consumed. slga... 巴onsumed.1. 1l 6g.4j 14 1(4只.0)87 1(3g.2.) 46 4(0広.9・j19 III. 12.888 18.687 11.411 43.148 -8uga!' in 100 c. medium expressed aR malも0枇In 0.1 出e自eco.seR， beもerresults were obtD.ined in Grade III o.go.r medium tho.n Gro.de 1 which seems 加 indico.te もho.tthere is 出8 自晶me自ubsto.ncewhich stimulo.ted the physiologico.l o.ctiviもiωoforgo.nisID. The reRul旬 obto.inedthu日fo.rmo.y b自由ummo.rizedo.s follow自:a.) Size of colonies grown 01 di宜erentmedium is given by the ratio. dividing the meo.surement obto.ined on Grad自HIago.r medium by tho.t of Gro.de 1 /Size of ColonieR on Gmde III o.g肌¥ag町¥. Size of ColoD同 ODGra.de 1 a酔r:-)Bacillus subもili自:Days. Albllmin aga!'. Day凡 Nutrient agar. 。 ? ?3.325 1.419 1.836 2.0fl6 Azotobacter chroococcum : Days. A自HRY'Saga!'. 2 3.050 1.584 7 Invesもigaもionon Agar. l. Saccharomyces cereviRiae: I>l¥yS. 2 7 LAURBNT昭島gar. Dl¥ys. 3.406 2.902 KlXli exもrl¥ct.1.385 1.359 585 The自edat.a indicate plainly that the coloni倒 produωdon Gro.de III agar medium were much larger than tho目。 onGrade 1 agar， especially it was marked :at an e町 ly白tageof growth in albumin， As.ElBY's and LAURENT'S media although in the nutrient and Kodi media， the ratio remained由。 same.、(Grade III agar.¥ b.) The similar ratio， ( ，-，~:-;_...;..._:~:.. ) is given in regard旬 ammonia， ~~V D'~"~' .~U.~， ¥ Grade 1 agar. Jproduct.ion， niもrogenfixation乱.ndsugar consumption: Bacillus subtilis: ammonia production in 10 d~戸・Albumin agar 1.112 Nutrient agar 1.121 Azotobacter chroococcum: Nitrogen fixed in 10 days. A阻町、 agar 1.253 Saccharomyces cerevifliae: LAURENTj自 agar ].326 Eらdiextract agar 1.054 As shown abov自， in geneml， the白出ilartendency 1.8 in c朗自 ofcolony was <lbtained. From th倒 eresul匂， it is evident. that the quality of agar influences the growth and physiological activitie自ofmicroorganisms. Summary: This inv飽 tigationw阻 undertakento制 certainthe qualiti倒 ofui宜ere凶grade of晶g紅 andtheir probable differenc白intheir influence upon出ephysiologi-<la.l act.ivi tie自ofmicroorganisIDs， and obtaineu the following result日.1.) B白tterthe qua.lity of a.gar contained less句ta.lnitrogen a.nd while the velocity of coagulation a.nd solubility is gre乱ter.2.) Both the agar 801ution and extr由叫， 油owed自mallerBP伺 ificgmvity， d舵凶ca1∞nductivity，ORmotic pre銅 ureaud surface tension a.自由equa.lity w朗better， but th白concentrationof hydrogen ions w嗣 greaぬRtin Gra.de 1 a.ga.r. 3.) The bett.er growth of B.白ubtili自， Azotobacter chroococcum a.nd S叫 ch町 0・myce8 cerevIRiae w嗣 obta.inedin Gr8.Ue III a.gar than in Gra.de 1.FrOID th剖 ere自ults，it is clear tha.t aga.r di宜erswidely in its properti伺 amongthe di宣erentgrade and i旬 U自由 insuence自t，he phy自iologicala.cti vitie日of micro-586 A.ITANO and Y. Tst:JI: organisms. Consequently iもisnecessary句 purifythe agar obtained on th~ market before iもisused in delicate microbiological investigaもion.Literature : 1.) ITANo， A.， Repo巾 onGenerl¥l Snrvey and Inv側 igationon Agar. Berich色ed. 。hara.Ins色.f. landw. Forsch.， VI: 59-72， 1933. 2.) ーー一一一， Inve挑igationon Agar 聞もoits Iodine Content. Proc. Imperial Academy. Japan， IX: 398-401， 1933. 3.) ITANo， A.and T8U.TI， Y.， Investigation on the Iodine in Agar. (Japane鈎 pnhliω，tion).Ndgakll Kenkyd， xxn: 168ー 193，1~34. PLATE XXX. Colonies on Differen色 AgarMedia. 3. Bacillus snbtili円: (4 day日old.)1. Nutricnt agar (meat-exもract-peptone)Grade III. 4. Ibid. Grade I. Azotohac飴rchroococcnm: (4 days 01<1.) 2. ASHHY'目 agar，G rade III. 5. Il>id. Grade 1. Saccharomyces cerevi自iae: (1cla刊 old.)3. LAt:RENT'自agar，Grade 11. 6. Ibid. Grade 1. 5. 4. PLATE XXXI. Colonies on Different Aga.r Media. 2. 5. sacillu日円ubtilis: (7 days old.) 1. Albnmin-agar;乙GradeIII. 4. Ibid. GrR.de 1. Azoもobacterchroococcum: (7_days-old.) 2. ASHBY'自Ill!'ar，Grade III. 5. Ibid. Grade 1. Saccharomyce目cerevi日iae: (7 day目old.)3. LAURENT'日agar，Grade III. 6. Ibid. Grade 1. ~. 6. PLATE XXXII. Slan色 Culture on Differen色 AgarMedia. 2. 3. 4. Azotobacter chroococcllm: (5 dayll old.) 1. AIlHRY'S agar， Grade 1. 2. Ibid. Grade III. BaCillS sllbtiliH: (3 day同 old.)3. Nn色rielも乱gar，Grade 1. 4. Ibid. Grade III. 8accharomyce日cere¥'Isiae: (4 days 01<1.) 5. J_AURENT'S agar， Grade 1. 6. Ibid. Grade III 5. 6. 

Investigation on Agar. 11. Physico-chemical Properties of Agar and their Influence on the Growth of Microorganisms.

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