July, 1923 Research Bulletin No. 81 
 
 STUDIES ON STREPTOCOCCUS 
 PARACITROVORUS GROUP 
 
 By B. W. HAMMER AND M. P. BAKER 
 
 AGRICULTURAL EXPERIMENT STATION 
 IOWA STATE COLLEGE OF AGRICULTURE 
 AND MECHANIC ARTS 
 
 CE) CURTISS, Director 
 
 DAIRY SECTION 
 
 THE LIBRARY OF The 
 
 NIV ERSITY OF iLLINOIS: 
 
 AMES, IOWA 
 
 
 
SUMMARY 
 
 A study of 124 cultures of 8. paracitrovorus from milk, cream 
 and butter confirms the characterization of the group as those 
 streptococci that produce a definite but variable increase in the 
 acidity of milk and a high volatile acidity, which is greatly in- 
 creased by the adding of citric acid to the milk to be fermented. 
 
 Organisms having the same general appearance in ditmus milk 
 as S. paracitrovorus but which fail to give a high volatile acidity 
 or to show an increased volatile acidity when citric acid is added 
 to the milk are readily isolated from dairy products. 
 
 Cultures of 8. paracitrovorus destroyed the citric acid in the 
 milk in which they had grown, while the organisms failing to 
 produce a high volatile acidity, did not. 
 
 In general S. paracitrovorus produced considerable CO, but 
 there was no clear cut difference in the CO, production of these 
 organisms and of other types so that it seems a determination of 
 the CO, produced will not afford a means of identifying S. para- 
 citrovorus cultures. 
 
 Certain of the S. paracitrovorus cultures show considerable 
 resistance to heat and are undoubtedly capable of withstanding 
 the usual pasteurization exposures. 
 
 Ropiness was observed in one culture of S. paracitrovorus out 
 of the 124 studied. 
 
 No satisfactory basis was found for the division of the S. 
 paracitrovorus cultures into types; further work may, however, 
 provide such a basis. 
 
 S. paracitrovorus is rather widely distributed in milk and 
 cream, It seems to be more common than SN. citrovorus. 
 

 
 Studies on the 
 
 Streptococcus Paracitrovorus Group 
 
 By B. W. HAMMER AND M. P. BAKER 
 
 For a number of years the dairy section of the Iowa Agricul- 
 tural Experiment Station has been interested in the organisms 
 that are capable of producing considerable volatile acid in milk. 
 These organisms have been repeatedly isolated from starters, in 
 which they are evidently of importance from the standpoint of 
 flavor and odor development, and largely on the basis of a study 
 of these starter-cultures they have been divided into two species 
 —Streptococceus citrovorus and Streptococcus paracitrovorus. 
 The present paper deals with S. paracitrovorus from the stand- 
 point of its characters when isolated from milk, cream and but- 
 ter, special attention being given to whether or not the charac- 
 ters suggest a division into types. The distribution of the or- 
 ganisms in these dairy products is also briefly considered. 
 
 CHARACTERIZATION OF THE 8. PARACITROVORUS GROUP 
 
 The S. paracitrovorus group is considered to include the 
 streptococci that produce a definite but variable increase in the 
 acid of milk and a high volatile acidity ; the volatile acidity pro- 
 duced is, moreover, greatly increased in amount if sterile citric 
 acid solution is added to the milk to be fermented. 
 
 The appearance of the S. paracitrovorus cultures in litmus 
 milk, which is of importance because it is the medium into which 
 colonies from dairy products are most often picked, is quite 
 variable, largely because of differences in the rate and extent of 
 acid production. Occasionally a culture is secured which shows 
 considerable reddening of the litmus in a day or so and when 
 a heated loop is plunged into it gas bubbles may form and rap- 
 idly come to the surface; such cultures are likely to produce 
 a gassy curd in a comparatively short time. With other cul- 
 tures the acid development is slower and when coagulation 
 occurs the curd formed may be without gas holes due apparently 
 to the fact that the gas development had largely ceased before 
 coagulation occurred.t Some S. paracitrovorus cultures never 
 
 “seem to show coagulation and gas development is difficult to 
 
 observe at any time by the usual methods. 
 
 1. The importance of coagulation during the time of active gas de- 
 velopment in the production of a gassy curd has been dealt with by the 
 Iowa station in a study of the formation of gas in the making of cot- 
 tage cheese. Studies on the Formation of Gas in Milk, Hammer, B. W, 
 iets <ptiasta. kes, Bulw 27, Jan. LOG, 
 
 742755 
 
20 
 
 There seems to be a gradation from the S. paracitrovorus 
 organisms which show a high total acidity in milk down thru 
 those which show a low total acidity to a type which does not 
 cause a reddening of the litmus because of the very small acid 
 production and which has been ealled S. citrovorus. This or- 
 ganism, like S. paracitrovorus, produces considerable volatile 
 acid in milk and an increased amount of volatile acid on the 
 addition of sterile citric acid solution to the milk. 
 
 ISOLATION AND IDENTIFICATION OF 8S. PARACITROVORUS 
 
 The isolation of S. paracitrovorus from such materials as sour 
 milk, sour cream, ete., can usually be readily accomplished by 
 plating out on whey agar and, after incubating at room temper- 
 ature, picking colonies into litmus milk. The usual type of SV. 
 lactis cultures can be easily eliminated by the coagulation and 
 reduction that they cause. Cultures of S. paracitrovorus that 
 coagulate rather quickly are occasionally encountered but can 
 usually be detected by a few gas bubbles that appear along the 
 side of the tube and in such cultures there is often a copious 
 evolution of gas when a heated needle is plunged into it. The 
 more common behavior of the S. paracitrovorus organisms that 
 are picked into litmus milk is a slow acid development that is 
 usually evident in two or three days at room temperature by 
 a change in the color of the litmus. Cultures producing a slow 
 reddening of the litmus that are not 8S. paracitrovorus are also 
 commonly found however and certain of these which have been 
 studied are streptococci and cannot be distinguished from S. 
 paracitrovorus by any rapid tests such as morphological or eul- 
 tural. The odor of S. paracitrovorus cultures is quite charac- 
 teristic and after experience with the group it is possible to make 
 a reasonably accurate separation of the acid producing strepto- 
 cocci that are not typical S. lactis into 8. paracitrovorus and 
 those yielding only a low volatile acidity. A determination of 
 the volatile acid production is the final test in this differentiation 
 and a second determination on milk to which sterile citric acid 
 was added before inoculating supplies added proof. 
 
 Some of the differentiating media such as casein agar made 
 with peptone and lactose or whey agar to which transparent 
 milk? is added at the time of pouring plates seem to be helpful 
 in the isolation of S. paracitrovorus since by their use many 
 of the S. lactis colonies can be detected without picking into 
 litmus milk. The cultures that are picked, however, must be 
 studied as are those secured from whey agar plates. 
 
 2. Transparent Milk as a Bacteriological Medium. Brown, J. How- 
 ard, and Howe, Paul E. Jr. Bact. 7, S. 1922, p. 511. 
 
21 
 
 METHODS USED 
 
 The S. paracitrovorus cultures studied were secured by plat- 
 ing the various materials on whey agar and picking a consider- 
 able number of colonies into litmus milk. The cultures giving 
 the changes expected in litmus milk were plated to insure purity 
 and then tried out for volatile acid production before being 
 definitely considered as 8. paracitrovorus. 
 
 In studying the cultures isolated particular attention was 
 given to the characters that past experience suggests as being 
 of importance with the typical milk organisms. Certain char- 
 acters which are extremely valuable with some groups are be- 
 heved to be of little value with those that are primarily milk 
 types and their study has largely been omitted. 
 
 The volatile acid production in milk or milk to which citric 
 acid had been added was determined by the method previously 
 used by the Iowa Agricultural Experiment Station? which gives 
 the results as the ec. of N/10 alkali required to neutralize the 
 first liter of distillate when a 250 gram portion was distilled with 
 steam after the addition of 15 ec. of approximately N/1 H.SO,. 
 The total acidity was determined by titrating 20 grams of milk 
 with N/10 NaOH, using phenolphthalein as an indicator, and 
 calculating as lactic acid. 
 
 When citric acid was added to milk it was sterilized as an 
 aqueous solution and added to the sterile milk just before imocu- 
 lating. The method outlined by Supplee and Bellis,* for the 
 estimation of citric acid in milk and milk products was used for 
 determining the presence or absence of citric acid in milk fer- 
 mented by various organisms. 
 
 SOURCES OF CULTURES STUDIED 
 
 The S. paracitrovorus cultures studied were isolated from 
 milk, cream and butter, most of them being secured when these 
 products were plated and colonies picked for the purpose of 
 classifying the organisms contained. A number of cultures of 
 another type that were looked upon as possible 8. paracitrovorus 
 when the original cultures were first observed because of the 
 change produced in litmus milk were studied but failed to show 
 a high volatile acidity in milk. Some of the results obtained with 
 these organisms are given in order to bring out clearly the im- 
 portant characters of S. paracitrovorus. 
 
 A total of 124 cultures of S. paracitrovorus were isolated and 
 studied but not all of them were studied from the standpoint 
 of each character dealt with; when it seemed that a certain 
 determination was of no importance for either characterization 
 or classification it was investigated with only a part of the cul- 
 tures. 
 
 meres tulis: la.Agr. Exp. Stas 55,63, and’ 66. 
 4. Citric Acid Content of Milk and Milk Products. Supplee, G. C., 
 and Bellis, B. Jour. Biol. Chem. 48, O. 1921. p. 453. 
 
22 
 
 RESULTS SECURED 
 OBSERVATIONS ON MORPHOLOGY 
 
 The description of the morphology given by the Iowa station” 
 for the 8S. paracitrovorus cultures isolated from starters apples 
 in general to those secured from milk, cream and butter. The 
 cells were almost spherical, altho usually slightly longer than 
 broad. A paired arrangement was very common but short 
 chains were frequent in preparations made from agar slopes and 
 also from milk. Occasionally chains somewhat longer than those 
 usually found were seen but these were not correlated with any 
 other variation observed and were finally considered to be of no 
 special significance. The organisms giving the same general ap- 
 pearance in litmus milk as S. paracitrovorus but which failed to 
 produce a high volatile acidity showed the same general morph- 
 ology as the organisms classed as typical S. paracitrovorus. Both 
 eroups showed some variations in the size of cells but they were 
 quite alike and the size of each agreed with the dimensions re- 
 ported by the Iowa station for the S. paracitrovorus cultures 
 from starters. 
 
 The data obtained on the morphology of the 8S. paracitrovorus 
 organisms indicate that it is of no value in characterizing or 
 classifying the group. Organisms producing only a low volatile 
 acidity showed essentially the same morphology as did the high 
 volatile acid producers and morphology was entirely valueless 
 in attempting to select organisms of the latter group. 
 
 ROPINESS WITH S. PARACITROVORUS 
 
 Of the 124 8. paracitrovorus cultures studied one—no. 27 of 
 table I—was regularly ropy when grown in milk. The mor- 
 phology of this organism with the ordinary stains showed nothing 
 unusual and it was impossible to find any evidence of capsules. 
 
 The formation of a ropy condition in milk by organisms of 
 the S. paracitrovorus group is rather to be expected since ropi- 
 ness occasionally occurs in other groups of organisms common in 
 milk. It is not lhkely, however, that the production of ropiness 
 will be of value from the standpoint of the classification of the 
 S. paracitrovorus organisms since it is of no importance with 
 the other groups on account of its variability. Data secured at 
 the lowa® station showed that certain pure cultures of S. lactis 
 showing ropiness yielded non-ropy cultures in small numbers 
 on plating rather regularly and that these non-ropy cultures 
 showed a morphology essentially like the ropy ones. 
 
 5. Volatile Acid Production of S. lacticus and the Organisms Asso- 
 
 ciated with it in Starters, Hammer, B. W., Res. Bul. la, Agr bx ptesca: 
 a (Oly IEP Oe 
 
 6: Studies on Ropiness in Cultures of S. lactis, Hammer, B: WW.) Res: 
 Bulls la eA Sr Bip tee stein 4.0 degen 
 
23 
 
 TOTAL AND VOLATILE ACID PRODUCTION 
 
 The total and volatile acidities in milk and the volatile acidity 
 in milk to which 0.4 percent citric acid had been added are given 
 in table I for the 124 cultures of 8. paracitrovorus; the source 
 of each organism is also included. 
 
 The total acidities produced in milk varied from 0.27 to 0.99 
 percent, calculated as lactic acid, with an average of 0.46 per- 
 eent. The distribution of the cultures in the different acid 
 ranges is Shown in table II; the minimum, maximum and aver- 
 age volatile acidity for each range is also shown and these values 
 will be referred to under the discussion of the volatile acid pro- 
 duction. From this table it will be seen that 33.1 percent of 
 the cultures studied fell in the acid range from 0.41 to 0.5 per- 
 cent while 24.2 percent fell in the acid range from 0.31 to 0.4 
 percent; accordingly over one half (actually 57.3 percent) of 
 the cultures studied produced acidities ranging from .31 to .5 
 percent. Outside of these limits the acidities produced by the 
 cultures showed wide variations. 
 
 The total acidity produced in milk by a certain culture of VS. 
 paracitrovorus would be expected to show variations in different 
 tests, as do other organisms producing acid in milk, In a con- 
 siderable number of trials repeated determinations of the total 
 acidity produced by a certain culture of S. paracitrovorus 
 showed wide differences. It was a rather common experience to 
 plate out a pure culture of one of these organisms and after 
 picking colonies into litmus milk find that some coagulated and 
 some did not. In one instance a culture that had quickly formed 
 a gassy curd at room temperature after being inoculated was 
 plated out and 20 eolonies picked into litmus milk; none of the 
 cultures developing showed coagulation after one month at room 
 temperature and after two months only three were coagulated 
 and these showed no gas holes. The variation in the acid pro- 
 duction may be due to variations in the composition of the milk, 
 in the temperature at which the milk is held and probably in 
 other factors; it indicates that the presence or absence of coagu- 
 lation cannot be considered of importance in identifying cul- 
 tures of S. paracitrovorus, since the usual differences in acid 
 production may result in one organism causing coagulation in 
 milk and another not. 
 
 The volatile acidity produced in milk by the S. paracitrovorus 
 cultures studied (table I) varied from 12.5 to 40.0 and 
 averaged 28.6.* There seemed to be no very definite rela- 
 tionship between a high total acidity and a high volatile 
 
 *As pointed out under “Methods Used” these values represent the ce. 
 of N/10 alkali required to neutralize the first liter of distillate when 
 a 250 gram portion was distilled with steam after the addition of 15 
 ec. of approximately N/1 HeSO.. 
 
24 
 
 TABLE I—THE SOURCE, AND TOTAL AND VOLATILE ACID PRODUCTION OF 
 THE S. PARACITROVORUS CULTURES STUDIED 
 
 Volatile acidity in 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Culture number Source Total acidity | ———————_—_ a 
 in milk Milk plus 
 Milk 0.4% citric acid 
 LC ee eee ees Souracreainiy sos se ee .5D 18.5 70.0 
 PO 8 ee oe SOUrpCrea ines ee “BH 31.0 5120 
 PASO t ee SOURPCICAIi =e eee .36 32.0 72.4 
 Para ea SOuUreGre a eae eee 39 21.0 88.5 
 Parayee one ee Source sins. == aes .40 23.8 76.0 
 NeW ATS | Apex eta Ee Sour crea ing aes 43 32.0 78.5 
 JET Wiel tos | Yee ee ee Sour creania.--=soeee .30 26.2 66.9 
 Pare eres SOULACrEa = eee .62 30.9 82.5 
 Pala dee ae Sour Creaiieesse eee 59 SHEA) 76.8 
 joecass § Se 20 SOULNCreAli =a eee ee 259 32.4 80.0 
 (etwas ahh eee & SOUETCredn == eee .38 29.6 Slay, 
 IPB AAs ee) Sa SOUrCreaIn sae see 60 Bites 81.8 
 Palen 3 oe Sourscresnp eee 42 33.4 82.8 
 OTe Pan ee eens SOurECrealnis = eee -40 24.0 With 
 PAT. eb 2. eee Sourvcreanies ee ape 22.8 (Glee 
 PAs ae lO eee Sour cream 2-2 .58 31.6 Cond 
 detianguly GW ep ce ees SOUr Clea ee 22a .42 29.4 72.4 
 PaTrasveels sees Soureerea m= .29 19.1 66.5 
 AL eee LO pete ees SOUT CLea ni n= === ae .45 26.8 86.9 
 Para eae Sour Cre nin ee ae .39 31.8 74.4 
 Pa hinge Lee ee SOutsCclea l= ae 52 31-0 77.8 
 atl see eee SOUrecre ale. === .36 Done 87.0 
 ieee 2a eres oa SOUteCre ain seas 639 26.0 63.2 
 PATO ade eee SOUT. Crean =e .48 31.0 toU 
 Pata eee oo Sour (cream si) 31.0 73.0 
 PAT ote oO aa SOULECTOAT === === ena .39 30.4 70.9 
 1G PAP See cee Sourecte@arnp e222. =a 259 36.6 Hleoe 
 Pata nu28 2. eee = Sourecreatim eee .48 25.5 76.6 
 Petia =e Sounpecrenniy sae eee .48 36.1 83.9 
 Para eo eae Sourecren eee 31 95.6 87.0 
 Palisa Sourscereame eon 253 28.9 74.7 
 (Be Ehe BPA Se a ye SO cre sin) eee 30 21.9 68.0 
 Para wos eee eee SS OUT Cre alae eee PA 20.3 80.4 
 Pata agote ee Sourscreali eee ee .46 23.0 72.3 
 Paraneoo eae os Soutseres ae ae £50 31.9 12.2 
 Pi ee oO eee SOUTECTGs Titman ae .48 31.6 81.0 
 Para me (eee SOUie Che aimee 45 PA ses 61.0 
 Para aeose ee Sourschen ae eee RET 25.1 12.8 
 Para aoe = Sour cream 22-222. 2 254 36.5 77.9 
 ieee, Ci Seurscream 2 soso. 54 33.4 73.0 
 Para SoOurscre 21a 43 26.4 74.5 
 Parad eee SO uliew Cie cri) eee 56 29.1 80.5 
 Para) 243 Sea SOUEECTCAI a= =e an nae 61 19.4 74.0 
 1etige, CR ne Sourere alia eee eee .50 82.5 44.4 
 Patiie4) eee Sournmencn lm aaeoaee ae ae BD, Son 85.2 
 Seed Oe eee Sour creams eee .40 23.6 61.4 
 POT Oe ee ee Sour creamy 2 oe .40 26a tose 
 (Pala oe see ae Butter woe ee ee .28 HDA T 50.0 
 Para, 0490.2. Butter eee 44 37.3 77.9 
 POT 0 ae ee Butter ee eee oe ee .46 20.0 75.8 
 Pavarsia Butter 22 ee ee .b4 o2ee R23 
 Ralarolge oer Butter eee eee 30 33.0 75.0 
 Pardee DS poe ee Bitter ee ee .74 32.0 85.0 
 PAT Oe 4 eee Butter ee eee Set Zou. 70.0 
 AEH ATT aoe Soe cres is eee .55 32.8 83.7 
 IDAalhes pO ee Butters eee ee TG 88.4 80.2 
 Pas AO (ee oe eee Butter sence .99 81.4 75.4 
 Paraeeoo es Buttereeeee eee pe ha Se Ped Sag 41 38.0 89.0 
 Para ae Ope Buttere oe ose ee eA Bien 59.5 
 Para Oe eee SOU Cr Co, 1) eee ea .78 34.0 85.0 
 IPA a6) eee SOUT sere din nee [83 28.0 76.6 
 PAT Rem O pee eee Sourrercanis see .86 Bie O 69.0 
 Lee We hee Sie, Sees oe SoOuracredin a= see ees .39 bears 66.2 
 NetWarahe ave ee cot as oe SOMPMecredi =e eee 48 25.9 83.5 
 PAT hee One eee SOUnSCrCAln oo .45 B20 96.6 
 PAT ae OO. eso oe SOUT CrGA Ty == eee .60 40.0 83.0 
 
 
 
25 
 
 TABLE I—Continued 
 
 Volatile acidity in 
 
 Culture number Source Total acidity |-——-——____-_———_ 
 
 
 
 
 
 
 
 in milk Milk plus 
 Milk 0.4% citrie acid 
 
 ay MeeeeOd eo = an SOUrs Creag o- = = eee ADT 28.6 62.5 
 TEE, SOUP Cre Alig ae ee -Sy/ 28.8 61.4 
 LEONE 0 SOUT ChCA IIs! oa ears 44 28.9 73.4 
 rat, ae SOUtmere plies ae 41 17.6 80.4 
 IEC G7 a SOUDECrea Ii g= === aaa mae 41 30.0 57.5 
 lesniGhy 212i SOUPECTCAIN = eee ee .39 95.3 67.7 
 PA ae ee fois. soak el SOU CLe hill a eee .40 30.0 v0 e4 
 Tei "7c eee arr SOUDeCrCAlI Maes eens .46 82.0 (ALW 
 Ded ih AT re SOUmCcreaniee see 52 39.0 81.0 
 Pie, aie SOUPECreC al === = ee 43 26.5 69.9 
 IEC, “Wie SOUDMCrCA lI) areas eee 525 24.3 (200 
 Do. 1 ——— SoOurmerealne= =e eee .36 B2eo 77.9 
 Tet hoi (0) ne ISOM Cre niin 30 19.3 76.9 
 Se i ‘Sour eran 222.02 22. .28 26.2 Wei 
 LES Wetley US ee eee SOUrecrea nin == eee .49 33.5 89.5 
 Lets ty ars SOUMMCLE A lee ees .38 84.9 78.9 
 1 gy ‘Soursereain 252.2262. 40 34.7 86.0 
 JER. et SOULS Crea Ie eee ae 50 34.0 85.0 
 Ue Wee SO cee = Sour cream = 22. == .36 32.0 76.8 
 Wea RA OG oe SOUMECCCAT eee ee eee .48 34.8 (Del 
 12h Beli SGOUmuCreanive eos = 44 33.5 78.7 
 eae Sooo eo SOUrserenins seo eee ee 28 20.0 70.1 
 WANA OO 2a ken. SOURPCIGAI ae 2 ee .29 SEO) 77.0 
 Peano O0o2 2 oo: Sour Creamy ss ee 45 26.0 75.8 
 Jeph to) ier SOULSCl6A Meese eee nO 35.1 78.2 
 IB oa Ctolibe Gee tem 2 re ae 45 30.7 65.4 
 IPG he Sours creainmesss oe .45 26.8 68.5 
 i2ariis 4) Sour crea ee 100 30.0 64.0 
 lenin, 955) Sourecreaiies. ==. =e .29 32.0 78.0 
 wanraenc96. 2. VC cere Se ane ee 47 26.5 76.9 
 edhe, (ore aa Nea teeSualte lee =e .36 29.0 83.5 
 ICR Wish eee Metallic buttermilk --_- 54 28.9 64.8 
 apa 09,22 Metallic buttermilk —__- .70 aoe2 80.3 
 earaes LOO pes ee. Metallic buttermilk —_-- 20D 834.0 83.0 
 Parade elles oe =: Metallic DUttermes == ons .56 31.0 68.0 
 1s et oa kl ee Bitters ce eee ee 4c 15.8 48.5 
 arame Os co) 5 2s Butterer eae .86 28.0 50.0 
 PAT LOs oe Sweet cream butter___- AUS 36.0 85.0 
 Iara \Obs2=. 22 2.- Bitterman. Ss eee .46 97.5 74.0 
 Para L0Gm.=-—- = Mil eee oe ee 47 29.9 68.5 
 Para lO, sc. 25.2 Mil kgeeee ee ee > eee ee 53 26.7 77.8 
 Inara use. oo VE i) ee i en eee 45 32.8 80.0 
 1247075 | 3) a ir Mil knee ees cee a .48 TO) 72.0 
 PAT vee lO Milk-geeeeresa ewes 2 ees 54 31.8 78.2 
 Wardell o. Milkaeeeece = tae Fe .65 31.0 87.0 
 ae lo ee SS VET) ee ae Se eS ee 29 Py atl 58.4 
 eid, Mbp CU GUW ES - 2 Ct Say oe heer Be ea .38 eo 66.0 
 arnt 2s 2 Mil ieee ae ee ee ee .56 26.4 75.5 
 
 ions The Butter having ‘burnt’’ 
 TlaVOlEe es eee n= .49 30.0 73.0 
 
 ae NOs ss es Butter having “burnt” 
 flavOt meee eee eee .58 ilenes 73.0 
 
 Leite a Butter having “burnt” 
 EON. ae ee ee Boe .58 26.2 75.6 
 
 iia 10 ae Butter having ‘‘burnt”’ 
 i aiViO tae eee 47 24.0 82.0 
 
 SE Weta a Co ee Butter having “burnt” 
 {laviorwee= wees eee .49 26.4 64.5 
 Ae Lae sees can |SYU ELT, See Sew ee .28 76: 57.3 
 IEW, BD SOlliss Cre At eee at ron 12.9 59.4 
 TEC Se. BAZ Aelia ee moter Buble es eee ee .36 125 56.6 
 Paine teom ean .S SoOumeecreant 2226-22 -22- ear 13:55 60.0 
 AT G4 2S ee Sourecrealtees. 2s. ee AF 1lS}.ah 80.3 
 Wihtobbaa\wags) 2 All 1220 44.4 
 Witp-quan\eped, oS ae .99 40.0 96.6 
 AVeLai Gmc mee ree 46 28.6 73.9 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
26 
 
 TABLE II—DISTRIBUTION OF S. PARACITROVORUS CULTURES IN DIFFERENT 
 ACID RANGES 
 
 
 
 
 
 
 
 
 
 
 
 No. of |% of total] Minimum | Maximum] Average 
 eultures cultures | vol. acid. | vol. acid. | vol. acid. 
 Cultures giving total acidities 
 UPigbO” 231 p sete nee wee ae 17 13.7 13.1 34.0 23.4 
 Cultures giving total acidities 
 from 0.31 to 0.4 inclusive 30 24D: eA 9) 34.9) 26.8 
 Cultures giving total acidities 
 from 0.41 to 0.5 inelusive A 33.5 15.8 88.0 29.0 
 Cultures giving total acidities 
 from 0.51 to 0.6 inclusive 23 18.5 18.5 40.0 31.8 
 Cultures giving total acidities i 
 from 0.61 to 0.7 inelusive 4 ee 19.4 36.2 29.4 
 Cultures giving total acidities 
 ADO Vel RT a ieee ee owe 9 hae) 28.0 38.4 33.4 
 
 
 
 acidity since some of the cultures giving a rather high total 
 acidity gave a comparatively low volatile acidity while other 
 cultures giving a low total acidity yielded a comparatively high 
 volatile acidity. From table If it will be seen that while there 
 was a tendency toward a slightly lower average volatile acidity 
 among the cultures in the lower acid ranges than among those 
 in the higher acid ranges, the differences are of very little if any 
 significance. 
 
 From table I it is evident that all of the S. paracitrovorus 
 cultures showed a pronounced increase in the volatile acidity 
 produced when 0.4 percent citric acid was added to the muk 
 before inoculation. The volatile acidities produced in this modi- 
 fied milk by the 124 cultures studied varied from 44.4 to 96.6 
 and averaged 73.9. The increase in the volatile acidity caused 
 by the citric acid added was quite variable either on the 
 actual or percentage basis as would be expected from the 
 variations that occur in the volatile acidity produced in normal 
 milk. The inerease in the volatile acid production with all the 
 cultures of S. paracitrovorus when citric acid was added to the 
 milk is further proof that the citric acid normally present in 
 milk is one of the important sources of the volatile acid pro- 
 duced. These findings with the 124 cultures studied greatly 
 strenethen the evidence of the increase previously reported’ for 
 a limited number of 8. paracitrovorus cultures isolated from 
 starters, 
 
 Table III gives data similar to that of table I for 27 cultures 
 that were tentatively selected as S. paracitrovorus but which, 
 when the volatile acidities were determined, proved to be another 
 type. These cultures gave total acidities quite like those pro- 
 duced by the S. paracitrovorus cultures but only low volatile 
 
 7. See ref. 5. 
 
27 
 
 acidities and it was these latter values that first indicated the 
 organisms could not be classed as 8. paracitrovorus. None of 
 the cultures showed a significant increase in the volatile acidity 
 when citric acid was added to the milk fermented and this abso- 
 lute correlation between the low volatile acidity in milk and 
 the failure to get an increase in the volatile acidity when citric 
 acid was added before inoculating is further proof that the 
 organisms were not 8. paracitrovorus. The failure to get an 
 increase in volatile acid by the addition of citric acid with these 
 organisms that produce only a low volatile acid is an additional 
 indication that the citric acid normally present in milk must be 
 a source of volatile acid in the case of the organisms producing 
 a high volatile acidity. 
 CITRIC ACID FERMENTATION 
 
 The results obtained on the increase in the volatile acid pro- 
 
 duction in milk by S. paracitrovorus as a result of the addition 
 
 TABLE III—THE SOURCE, AND TOTAL AND VOLATILE ACID PRODUCTION OF 
 CULTURES NOT S. PARACITROVORUS BUT APPEARING LIKE 
 IT IN LITMUS MILK 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Volatile acidity in 
 Culture Source Total acidity - oo 
 number in milk Milk plus 
 Milk 0.4% citric acid 
 ‘Cini! Sie SOU CLealiesss= eee 7, Dial 6.1 
 Cilio ts SOUre Clea leet= ee ee .38 Eyer 6.8 
 Cia eee See SOULHCreAI pe ee eee 39 Gan 7.8 
 Sine Lt ee SOUTPCECAMIN ee =. = ee ea aE sey 5.4 4.7 
 Oil Sa aes VE eee ee eee. Se Eee 742, 4.6 5.4 
 Se Oe VET Kae eee rr ee 41 Tok 10.7 
 COLE” ee SOUrECrCa iinet = ee .40 6.4 5.5 
 (Ck) a er Bitter yee eee oe ee 42 4.0 5.0 
 (O10 hoe Glue (Mrs hail Se ke .43 6.6 6.7 
 Obs Kt ae eee BiGteTeeee sees. ee .49 4.9 5.4 
 SC gleelieos BUG te tgeee™ ee ee ee ree 2 a Sail 2.8 
 CORDINE,~ iby ti ce IBiGGerc gee a .46 5.8 5.8 
 ‘CCUG SABE SOunwere ale eee eee 63 4.6 6.5 
 Chaat SOUDSCLE QTM ese eee 51 6.5 10.7 
 Cielo Butter from sweet. cream__ .25 3.8 2.9 
 GulemelG.22= Butter ee re .45 6.7 6.0 
 CRA fanaa Se BUC Crae eee eee ee .48 3.6 3.9 
 Coulee Sas UGLC eee are ee is as .49 6.6 6.0 
 (Oiotiy Pak eA ee PES UETRG GT. ere ee =e ee 47 8.3 8.7 
 C0 S OU Cha Te a eee ee 54 5.4 5.5 
 Citi, Die =e SOUMRECCO AI eee eee eee E “49 6.2 6.5 
 (Obi) Oe SOUrseCreaMimese 2esee = yi 4.5 4.3 
 (Cibile VS IV eae ee eae ee aval 3.4 4.1 
 GE, i ee Mice eet OT ee SA 47 4.5 4.3 
 CChbT hs yd soy oe Milica ee eS Pe ee .5T 6.7 8.0 
 Oul.26-__..___|Butter having “burnt’’ 
 TLV OTe eee oe .63 38 G52 
 Cals 275. 5.2: SGRTigt eset 56 5.1 5.2 
 Woeapeoai vine, = 22. eye 21 Spill 2.38 
 NL AUEMELEN ee eee .63 8.3 10.7 
 IAVOTACE: poe oe ee tos ok 45 9.4 6.0 
 
28 
 
 of a sterile citric acid solution to the milk before inoculation 
 suggested tests for citric acid* on milk in which these organ- 
 isms had grown. The milk was sterilized in the autoclave and 
 then the organisms allowed to grow for from 9 to 11 days at 
 room temperature before the tests for citric acid were made. 
 
 Forty-seven cultures of 8. paracitrovorus, taken without any 
 attempt at selection, were tried and all of them completely de- 
 stroyed the citric acid present in the milk as determined by the 
 method employed (see ‘‘ Methods Used’’) while the uninoculated 
 checks showed the usual amounts of citric acid. Five organisms 
 of the group whose original milk cultures suggested S. paracit- 
 rovorus but which failed to yield a high volatile acidity or a 
 significant increase in volatile acidity when citric acid was added 
 to the milk before inoculation were tried out for their effect on 
 the citric acid of milk and with all five organisms there was no 
 evidence of any decrease. These findings with the two groups 
 of organisms are what would be expected when their action on 
 citric acid added to milk in which they are grown is taken into 
 account. 
 
 Bosworth and Prucha*® found that the citrie acid in milk was 
 changed to acetic acid and carbon dioxide during the souring 
 and also that starter and buttermilk contained some agent eap- 
 able of bringing about this change. Kunz® observed that the 
 citric acid gradually decreased with the aging of milk, particu- 
 larly when soured and Supplee and Bellis'® concluded that, 
 ‘‘the eitrie acid content of milk decreases during aging in the 
 presence of high developed acidity, and is more rapid in raw 
 milk than in pasteurized milk.’’ From the work of these inves- 
 tigators it is evident that there is one or possibly more agents 
 in milk, starter and buttermilk that are capable of destroying the 
 citric acid present in milk. While the increased volatile acid 
 production of S. paracitrovorus in milk, when citric acid is 
 added, suggests that this organism is capable of destroying eitric¢ 
 acid, determinations showing the disappearance of citrie acid in 
 milk in which this organism has grown is undoubtedly more 
 convineing proof. 
 
 CARBON DIOXIDE PRODUCTION 
 
 The evolution of gas by S. paracitrovorus in milk and milk 
 plus peptone was reported by the Iowa station™ in the descrip- 
 ticn of this organism. The production of gas with S. paracitro- 
 
 
 
 
 
 *The authors are indebted to F. F. Sherwood for assistance in mak- 
 ing these tests. 
 
 8. The Fermentation of Citric Acid in Milk, Bosworth, Alfred W., 
 and) Prucha, M. Je ech iBbuly NY. ASE xpt. state Nee Lomas 
 
 9. The Determination of Citric Acid in Milk. Abstracted in Exp. Sta. 
 Rec. 36. 1917. p. 415. 
 
 10. See ref. 4. 
 
 Le See rela: 
 
29 
 
 vorus 18- however very different than the gas production with 
 such typical gas-formers of milk as Bact. aerogenes since many 
 cultures of S. paracitrovorus can be studied in a routine way 
 without the formation of gas bubbles being noted. In the usual 
 cultures, the gas production with this organism is apparently 
 so slow that the diffusion of the gas into the air seems to remove 
 it and gas bubbles ordinarily do not form. The production of 
 gas should however offer a suitable method for the preliminary 
 selection of organisms that are likely to be S. paracitrovorus, if 
 the gas formation could be readily determined. Attempts were 
 made to detect gas formation by sealing freshly inoculated milk 
 cultures with such materials as paraffine, bees wax, vaseline and 
 various mixtures of these with each other or with paraffine oil, 
 the sealing material being poured on in a heated condition, and 
 the cultures then watched for a forcing up of the seal. Some 
 of the results were very satisfactory and the plugs were readily 
 shoved up but in other trials it was evident that the seals were 
 not tight and that gas could escape between the seal and the 
 wall of the tube. It seemed to be impossible to secure a material 
 that uniformly gave satisfactory seals. This was apparently in 
 part due to the coefficient of expansion being so high that the 
 contraction of the material on cooling was likely to draw it away 
 from the wall of the tube but it seemed that getting milk along 
 the wall above the surface of the milk during the inoculating 
 and handling of the tubes so that the seals could not be gotten 
 tight, was also a factor contributing to the unsatisfactory results. 
 
 The tubes used by Eldredge and Rogers'* in determining the 
 CO, production of organisms isolated from Emmental cheese 
 were next tried out as a means of detecting gas formation. Ten 
 ee. of milk were added to one arm of each tube and after stop- 
 pering with cotton, sterilizing and inoculating, 10 ce. of 
 N/10 Ba(OH), solution were added to the other arm. The cot- 
 ton stoppers were then cut off level with the glass, shoved down 
 and rubber stoppers very firmly inserted. The usual incubation 
 was 10 days at room temperature, after which the excess 
 Ba(OH), was titrated. 
 
 The effect on the CO, production of adding citric or lactic acid 
 to the milk before inoculating was also determined. A sterile 
 lactic acid solution was prepared so that 1 ¢c., the amount to be 
 added to the 10 ce. of sterile milk in each tube, contained 1/30 
 ec. of Mercks lactic acid U. S. P. VIII. The citric acid was 
 prepared so that 1 ee. of solution contained 1/30 gram of crystal- 
 lized citric acid (1 mol. H,O of crystallization). The acid solu- 
 tions were added just before inoculating; the citric acid in the 
 amounts used caused a rapid coagulation of the milk while the 
 lactic acid did not. 
 
 12. The Bacteriology of Cheese of the Emmental Type. Eldredge, 
 ia and Rogers, L. A. Centbl.’ f. Bakt. etc. 40. 1914," p, 5. 
 
30 
 
 Dupheate determinations of the CO, production were made on 
 milk alone and on milk plus lactic or citric acid and while the 
 results were not encouraging from the standpoint of agreement, 
 a series of organisms were run both with and without adding 
 acid to the milk. 
 
 Sixty-four S. paracitrovorus cultures were studied for CO, 
 production in milk, in milk plus lactic acid, and in milk plus 
 citric acid. In the milk alone the cc. of N/10 Ba(OH), equiva- 
 lent to the CO, produced varied from 2.5 to 8.8 with the values 
 for most of the cultures ranging from 4.0 to 6.5 ce. The effect 
 of adding the acids—lactie or citric—was quite variable. With 
 some organisms the CO, produced was increased by adding the 
 acids, as might be expected in the case of citric acid from its 
 effect on the volatile acid production, while with others no such 
 increase was observed. The failure to get an increase may have 
 been due to the acid limiting the growth of the organisms in 
 certain cases altho this does not seem lkely because some of 
 the organisms that failed to show an increase, produced quite 
 large amounts of acid in milk and accordingly would be assumed 
 to tolerate considerable acid. 
 
 A number of cultures that gave the same gross appearance in 
 litmus milk as S. paracitrovorus but which did not produce a 
 high volatile acidity were tried for CO, production. In general 
 the results suggest a lower value for these organisms than for 
 S. paracitrovorous but there was no definite difference and the 
 figures for one group shade over into those for the other so that 
 the CO, production does not seem to offer a means of separation 
 upon which any considerable reliance can be placed. The CO, 
 production of S. lactis was also studied and a great deal of varia- 
 tion in cultures, all of which produced the usual low volatile 
 acidity, was found; in some instances the values ran as high as 
 the typical S. paracitrovorus cultures. 
 
 ACID FORMATION FROM VARIOUS MATERIALS 
 
 In the description of S. paracitrovorus the Iowa station™ re- 
 ported on acid production as follows: ‘‘ Beef extract bouillon 
 containing fructose, galactose, glucose, lactose, maltose, or, with 
 certain organisms, sucrose usually showed a sleht increase in 
 acidity at either 37° C. or room temperature but with glycerol, 
 mannitol, salicin, raffinose, inulin or starch no such inerease was 
 observed.’’ 
 
 Additional fermentation tests were run at room temperature 
 with a number of S. paracitrovorus cultures and these were in 
 entire agreement with the above statement except that some of 
 the cultures fermented salicin. Accordingly salicin should be 
 classed with sucrose as being sometimes fermented and some- 
 times not. 
 
 
 
 18. See ref. 5. 
 
31 
 
 Considerable variation was observed in the acid production 
 with different cultures in the same bouillon. There was a gen- 
 eral tendency, modified, however, by a number of exceptions, 
 for the cultures producing the higher acidities in milk to yield 
 high acidities in at least some of the bouillons. 
 
 The results obtamed up ‘to the present time do not suggest 
 that the fermentation of various materials will afford a method 
 of dividing up the S. paracitrovorus group. 
 
 RESULTS WITH JANUS GREEN 
 
 Altho the S. paracitrovorus group has very little power of 
 reduction, sterile milk to which 1 ce. of a sterile aqueous solution 
 of janus green (0.5 grams per liter) had been added per 10 ce. 
 was tried out with some of these organisms since janus green 
 has been found to be of value in showing variations in the reduc- 
 ing power of the S. lactis group. The reducing power of the 
 S. paracitrovorus cultures was so low that certain of the cultures 
 showed only a shght pink color in the bottom of the tube even 
 after periods of growth such as 20 days at 21° C.; some few 
 showed a pink color thruout the tube and occasionally this ap- 
 pearance was presented in as short a time as 4 days. From work 
 done with S. lactis it seems that Janus green has a restraining 
 action on bacterial-growth and while conditions may be different 
 with the S. paracitrovorus group the time required with many 
 cultures before there was any change in the milk to which janus 
 ereen had been added also suggests a restraining action due to 
 the indicator. A few cultures of the organisms appearing lke 
 S. paracitrovorus in litmus milk but giving only a low volatile 
 acid were also tried in janus green milk and the changes pro- 
 duced were quite like those produced by S. paracitrovorus. It 
 seems evident from the small amount of work done that janus 
 ereen is valueless either as a means of identifying the S. paracit- 
 rovorus organisms or dividing the group up into types. 
 
 GOMPARKISON OF THE GROWTH AT 37° C. and 21° C. 
 
 With organisms producing visible changes in milk only slowly, 
 a comparison of the growth at different temperatures is rather 
 difficult without elaborate methods, such as counting, and these 
 cannot be applied to a large number of cultures without a great 
 deal of work. The method followed in comparing 37° C. and 
 21° C. for the growth of the 8S. paracitrovorus type was to inocu- 
 late two tubes of Ltmus milk from the same culture with the 
 same loop and hold one at each temperature. After a short time 
 the cultures were observed for the extent of the changes in the 
 appearance of the litmus, since this can be assumed to be due to 
 the amount of growth, and a record made as to whether one 
 temperature seemed to favor these changes more than the other. 
 
32 
 
 TABLE IV—INFLUENCE OF 87° C. AND 21° C. ON THE GROWTH OF S. PARA- 
 CITROVORUS AND LOW VOLATILE ACID PRODUCERS APPEARING 
 LIKE IT IN LITMUS MILK 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Growth best Growth best No difference 
 Athomeees at 2ie8@= in growth 
 Total |—————— —— 
 cultures 
 No. | Percent | No. | Percent | No. | Percent 
 S7 Daracitrovorus eae e aaa 116 20 Ip) 56 48.3 40 34.5 
 Low volatile acid producers 
 appearing like S. para- 
 citrovorus in litmus milk__ 23 6 26.1 9 39.1 8 34.8 
 
 Table IV gives the results obtained on S. paracitrovorus and 
 also on the organisms appearing like S. paracitrovorus in litmus 
 milk but giving only a low volatile acid production. With the 
 116 S. paracitrovorus cultures tried 17.2 percent seemed to grow 
 better at 387° C., 48.3 percent at 21° C. and with 34.5 percent 
 there was no observable difference, while with the 23 organisms 
 of the other group 26.1 percent seemed to grow better at 37° 
 C., 39.1 percent at 21° C. and with 34.8 percent the two tempera- 
 tures seemed to be equally good. These data show that with both 
 eroups of organisms there is a certain variation in the way 
 different cultures respond to different temperatures. With 
 neither, however, is there a clear cut dividing line with some or- 
 gvanisms growing well at a certain temperature and others grow- 
 ing poorly or not at all, but rather a gradual variation with all 
 degrees of response on the part of the organisms. 
 
 RESISTANCE TO HEAT 
 
 The isolation of S. paracitrovorus from pasteurized materials 
 such as sweet cream suggests that at least certain of the organ- 
 isms of this group are rather resistant to heat. Preliminary 
 trials were made on a number of the cultures. The method used 
 was to stand tubes of litmus milk in a large volume of water to 
 a depth considerably greater than the depth of the milk in the 
 tube; after the milk had taken on the temperature of the water it 
 was inoculated with the organism to be studied and then held the 
 desired time, care being taken that the water remained at a con- 
 stant temperature. The tubes were then placed in cold water 
 for rapid cooling. After holding, the tubes were examined for 
 gsrowth by noting the appearance of the ltmus milk, staining, 
 and often by culturing on a whey agar slope. While this method 
 may not be as accurate as the use of Sternberg bulbs or capillary 
 tubes, it seemed that it would supply conditions the most like 
 those existing in commercial pasteurization, and for that reason 
 the method was followed. There was no evidence of scum for- 
 mation on the surface of the heated milk; this would not be ex- 
 
33 
 
 pected since the cotton stopper and column of air above the milk 
 would largely prevent evaporation. 
 
 The exposures used were, for the most part, 30 minutes at 
 60, 65, 70 and 75° C. Some cultures failed to resist 60° C. for 
 30 minutes but the majority did, while 65° C. for 30 minutes 
 destroyed a considerable number. Only a very few cultures 
 resisted 70° C. for 30 minutes. Certain cultures were run a 
 number of times at the different exposures and there was in 
 general a very good agreement between the results obtained on 
 different days; in a few instances discrepancies occurred but 
 these are to be expected since such factors as the age of the cul- 
 tures, the acidity, etc., probably influence the resistance of an 
 organism to heat and in addition there is a variation in the 
 resistance of individual cells in the same culture. 
 
 Table V gives the final results obtained on cultures which were 
 run by the method above outlined, all the tests on a culture being 
 earried out with the same inoculating material. These data con- 
 firm the preliminary tests in all respects. 
 
 The results given show considerable variation in the heat re- 
 sistance of different cultures of S. paracitrovorus. This is to 
 be expected from the data reported for S. lactis as well as for 
 other organisms and there seems to be little reason to expect that 
 organisms whose morphological, cultural and bio-chemical fea- 
 
 TABLE V—HEAT RESISTANCE OF S. PARACITROVORUS 
 + EQUALS GROWTH — EQUALS NO GROWTH 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Heated for 30 minutes at Heated for 30 minutes at 
 
 Culture eS ao ay Culture |— ee 
 
 (BOP AC Boll Ose 165 4 CO rat LO. (GOP Ok HGS TO Th (Ok 1 4GP LO; 
 ae eS 2S — “= — — [240 eee e. — - oe — 
 Gh 1 5 se ee ++ -- — — AS a oe — — — — 
 Ap el a — — — — 40.0 eee de — = 2s 
 eee. + + — — 50 were eee — — == — 
 (ile oo ee — — — — Sige ee ae — — — 
 i 233 + — — — 5 aeeeweie. = — — — — 
 ct ae — — — 54> ee — — — — 
 No ae “ft — -= ~- DO pe eee te — — — — 
 ik Ape _ — — — LOO Rees eee + — — — 
 G3, 2-year + —_ — — PST Se eR Ae rote =F — — — 
 1h 3 —- — os -— 26 eee + -f- — — 
 itl = =e -- — — ~- DAE | Bie Se apes ++ + = + 
 100) ee ak os — = As Raph Nae Oe ee as =f = = 
 ili aie ae = — SORE een aia == == = 
 Co a + — — — Soh ae ae aie — —— — 
 CCl oer — _— — — Bi heer es - _- oo — 
 Of ee = AO SS — — — — Sige ee — + — — 
 Os pees eS — — — — Bip eee ee + — — — 
 TSS ot ele a -— — —- — Bio) patio nka eee ae — — - — 
 ICON 0 pe eh -= —_ — —- 4 es bie =f. ++ -- 
 LG are eee => te — — (Oe eee =f == i = 
 TRIN ie in eee 4- -+- -- -= Abyss ae — —- — — 
 JV 5 + =e + — 4 Opens see fe ce — —- 
 liigmessasse et ob -- = -—— 6 ee -- — — — 
 ih = Si a. a — _— wpe eS -f + — —_ 
 Ml Gierece + + Fa —_ Ae ren EE =e = a= =s 
 ile == ee + ar _— —_— Shite ee sta — = — 
 
 
 
 
 
 
 
 ——— 
 
34 
 
 tures are essentially alike would have exactly the same resistance 
 to heat. Moreover variations in this resistance, unless extraor- 
 dinary, cannot be considered of significance from the standpoint 
 of classification except where they are correlated with other dif- 
 ferences. 
 
 The tests on heat resistance that have been reported show that 
 S. paracitrovorus may be expected to frequently resist pasteuri- 
 zation exposures and this explains the rather common presence 
 of these organisms in pasteurized products. 
 
 Tests of the resistance to heat were also run on a number of 
 the cultures having an appearance in litmus milk suggestive of 
 S. paracitrovorus but which failed to yield a high volatile acid- 
 ity. In general these organisms responded much as did the S. 
 paracitrovorus group and there seemed to be no possibility of 
 separating the two types of organisms on a basis of heat resist- 
 ance. 
 
 VARIATIONS IN STARTERS PREPARED WITH DIFFERENT S. 
 PARACITROVORUS CULTURES 
 
 The preparation of satisfactory starters by mixing cultures of 
 S. citrovorus or S. paracitrovorus and S. lactis has already been 
 reported by the Iowa station.’* Many starters have been pre- 
 pared in this way using S. paracitrovorus cultures with S. lactis 
 and it seems certain that excellent starters can be secured from 
 such mixtures. Satisfactory results are not always obtained, 
 however, and there is undoubtedly a difference in S. paracitro- 
 vorus cultures from the standpoint of the character of the 
 starters they will yield. From certain observations it seems 
 that S. paracitrovorus cultures are very consistent in the quality 
 of starter than can be secured with them. One culture that was 
 studied over a considerable period, quite regularly gave the best 
 starter In any series in which it was used; it came originally 
 from a sample of off-flavored cream, and accordingly its source 
 did not suggest its value as an organism to be used in starters. 
 
 Altho there are variations in the quality of the starters 
 vielded by different cultures of S. paracitrovorus and these 
 represent important differences, they cannot at present be con- 
 sidered as a satisfactory basis on which to divide the organisms 
 into types because they are not known to be correlated with any- 
 thing else. Moreover differences in the flavor and aroma of the 
 products formed by the organisms are probably due to extremely 
 small differences in metabolism which cannot readily or accur- 
 ately be detected in cultures so that these differences in metabol- 
 ism are not likely to be recognized as related to the variations in 
 flavors and odors produced. 
 
 14. See ref. 5. 
 
3) 
 
 RELATIONSHIP BETWEEN S. PARACITROVORUS AND ORGAN- 
 ISMS PREVIOUSLY DESCRIBED 
 
 The relationship of the organisms here referred to as S. para- 
 citrovorus to organisms previously described and named is a 
 matter of importance from the standpoint of proper nomencla- 
 ture. Freudenreich’ isolated an organism ‘‘ Streptococcus b’’ 
 which Migula'® later named S. kefir. Evans" studied organisms 
 which she considered to be S. kefir and also reported some of 
 the characters of another type which was referred to as Strep- 
 toccus X. Evans found that both these organisms produce con- 
 siderable volatile acid and it seems that these two types are more 
 closely related to S. paracitrovorus than any of the other organ- 
 isms reported in the literature. 
 
 S. kefir is reported as producing gas in milk but the S. paracit- 
 rovorus cultures as observed in milk are usually not recognized 
 as gas formers, except when coagulation occurs early. As already 
 pointed out, the gas produced is readily lost to the air without 
 the formation of bubbles in the medium unless a curd which 
 can hold the gas is formed. Evans reported that S. kefir rapidly 
 acidifies peptone milk, with curdling in from three to six days, 
 and that the curd is rent with escaping gas. The cultures of 
 S. paracitrovorous studied sometimes did and sometimes did not 
 curdle peptone milk and escaping gas was not regularly ob- 
 served. Moreover the description of the lactose agar plate colo- 
 nies of 8. kefir given by Migula is not a description of the colony 
 of S. paracitrovorus. . 
 
 S. X. is reported as curdling milk in five days with partial 
 reduction. The organisms studied as S. paracitrovorus certainly 
 do not do this and while, as already pointed out, the question 
 of coagulation is not considered of importance because it may 
 depend on a very slight increase in acid development, if the 
 results obtained with S. Y give this character importance, then 
 S. X can hardly be considered to be the same as the S. paracit- 
 rovorus cultures studied. 
 
 One of the important characters of S. paracitrovorus is the 
 fermentation of citric acid. Data on the action of S. kefir and 
 S. X on this material are not known to be available, altho the 
 high volatile acid production of these organisms together with 
 the correlation usually found between a high volatile acid pro- 
 duction and the fermentation of citric acid suggests that they 
 ferment it. From data presented by the Iowa station in a series 
 of reports it seems that the fermentation of citric acid with the 
 production of a high volatile acidity together with the reddening 
 15. Bakteriologische Untersuchungen uber den Kefir. Freudenreich, 
 HWmaVOnlemmOentbl. Bakt. 3) 1897s p. 92. 
 
 16. System der Bakterien, Migula, W. 2. 1900. p. 44. 
 
 17. A Study of the Streptococci Concerned in Cheese Ripening, 
 Mvanseewlice CC. sty, Ager Res. 13. Apr. 22, 1918) p. 235. 
 
2 042501814 
 
 3011 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 of litmus milk is unusual and important enough to justify § 
 being considered as a characterization of a group of organisr 
 If such a group is justified, the data presented herein show t 
 certain variations such as the acid produced, the resistance 4 
 heat, ete. exist and suggest that, if the organisms are at presege 
 divided up on the basis of minor variations, only confusion ci 
 result as so frequently occurs when descriptions of organisigg 
 are written from a study of only a small number of cultures. j 
 is entirely possible that some of the organisms such as S. keggaae 
 and S. X represent certain combinations of characters that mim 
 be found in the group that has been referred to as S. paracitig 
 vorus and that further studies will yield evidence justifyi# 
 some sort of a separation into types. It will be necessary, hog 
 ever, to guard against a division on the basis of such differen 
 as the coagulation and non-coagulation of milk which are depengg 
 ent on neghgible differences in acid production, instead of § 
 differences that are of some real significance. 
 
 DISTRIBUTION OF THE S. PARACITROVORUS TYPE 
 
 The distribution of the 8S. paracitrovorus group was not 
 vestigated systematically but the data secured in connectif 
 with a study of considerable numbers of samples of dairy prog 
 ucts allow of certain conelusions. Sour milk and cream veg 
 commonly show these organisms and frequently in consideralge 
 numbers. They have a been secured from sweet milk ai 
 eream and have been found rather commonly in sweet cream wig 
 a high bacterial count but it seems that sour milk or cream off 
 more suitable materials for isolation than do these products § 
 a sweet condition. This is to be expected since S. paracitrovor@m 
 like S. lactis, grows well in milk even after acid developme§g 
 
 milk organism. Butter samples have frequently yielded S. pay 
 citrovorus; this is in all probability due to the presence of the 
 organisms in starters but their resistance to pasteurization & 
 posures suggests that some may have come from the cream. 
 
 From the observations made it seems that S. paracitrovorus® 
 much more frequently encountered in dairy products than 
 S. citrovorus. This is particularly true of sour milk and crea@ 
 from which organisms producing no observable change in litm® 
 milk (which inelude S. citrovorus) are only very rarely encow 
 tered. : 
 
 The wide distribution of the 8. paracitrovorus group in daig 
 products indicates that there must be some common source § 
 contamination that supplies these organisms. Altho no attemg 
 has been made to locate this, the fact that these organisms gr@ 
 so well in combination with S. lactis suggests that the usu 
 sources of this organism such as utensils and the ERE GLau 
 
 cow may also be important sources {Of §.\pRadterdb 
 sep 24 1930 
 
 UNIVERSITY OF LLLINOIS.