IDENTIFICATION AND PHYSIOLOGICAL ACTIVITIES OF PSYCHROPHILIC MICROORGANISMS IN MILK ‘ Thesis far file 033:» of Ph. D. MICHIGAN STATE UNIVERSITY Frederick Weber, Jr. 1956 L"- This is to certify that the thesis entitled The Identification and Physiological Activities of Psychrophilic Microorganisms in Milk. presented by Fmderick Weber, Jr. has been accepted towards fulfillment of the requirements for Doctor of Philosopgydegree in Microbiology 8: Public Healm Major professor Date July 13, 1956 0-169 JR..«-I..I...,Iefl.u.. 11.x 4.1..“35 r. I l p.’ . T i "‘\'T1"‘TT'1T‘:11'“1 NrfiT‘f‘t“ .LA- .o-'~'¢ pence no m.:v eflflnm0hzobmm pasoo no mmu Hapoe meadow msaep hAflee mohfiv GoaMIwomwdwpe XHHE emu mweam mo wmamwww 3H mo mvnsoo Hewawpodp mo m.4qwoflafl£moa:ohmm was A0 mmuladvop ewaaepd N mumde do 000.m omda0b¢ omfluofi Hm ooo.m 0H own a oamuom we oom.: on sea a QJH-OH :5 oom.m on :mH a omaaoa om oom.H ea mH a omfluoa mo oos.m 0H NH a on-OH gm oom.m ea mOH a macaw mwdae>< meagfidm pnsoo owawsm0hxommm padoo Hepoe no means: 300 we newsdnmwmem vb chap aw whee Hfim Seam mmacfiem Mafia QC %o mandoo Au m.£v afiflwmmo mohwm was A0 mmv Have? aweaob< m mqm3,000,000 milk B 22,000 26,000 10 50 C 2.500 7,600 2 1,1 D 53.000 19,000 6 1,0 g 150.000 190,000 170 50 G >3.020.8gg >3,000,000 25 >3,000.000 : J50,“ 52,000 0 200 If 8.400 >3,000,000 O 19 30,000 79,000 125 12 J 95:000 61,000 20 15,000 K 100,000 119,000 8 < 10 L <15.000 1 000 0 H <3 000 0"700 800 11 18,000 7' ‘, - O <10 0' : , 9,000 >900 13 P {@3338 6, 900 26 20 Q. (5030 11900 O 90 ’ $9500 5 l 300 a <3,000 15,000 0 ’ 20 akifiui abl‘fi . i _ _ ... Hind. \m...’l..lrnei.... .433 TABLE 12 (continued) Bacterial populations of milk and cream from several Tichigan milk plants 39- Type of Dairy Plate counts sample 55 C 20 C coliform h.5 C Homogenized S(bottle) <3,000 11,000 72 5, 300 milk S(carton) (5,000 1,700 O (10 '1‘ 11,000 8,200 1 1,000 v 28,000 35,000 5 13,000 w >3,000,000 >3,000,000 o >3,000,000 x 10,000 230, 300 L13 63,000 Y 111,000 90,000 0 £13 2 380,000 1,300 2 60 AA. 38,000 100,000 2 < 10 AA(frozen) 25,000 590000 IL 4110 Skim milk R 3, 300 111, 000 0 3,300 5 43,000 10,000 3 9,700 1' 43,000 111,000 1 < 10 U < 3,000 11, 900 6 311 v 10,000 5,200 12 20 x >3,000,000 >3,000,000 >300 > 3,000,000 M 18,000 61,000 0 10 AM frozen) 18, 000 26, 000 0 111 AB < 3,000 6,000 0 20 Chocolate R 200,000 710,000 0 530,000 milk T 3,000 5,1100 8 2, 500 U 70,000 270,000 260 160,000 AA < 3,000 6,1100 0 114 Coffee G 17,000 11,000 0 11 cream J £110,000 >3,000,000 >300 > 3,000,000 K 170,000 116,000 6b, 3,700 L 5,000 9,000 111 2,000 1: 4 3,000 2,500 110 12 N 220,000 >3,000,000 >300 > 3,000,000 0 5,000 700 35 20 P (3, 000 311, 000 18 3 , 500 R 60,000 320,000 15 190,000 U 4 3,000 >3,000,000 L1 700 _ v >3,000,000 >3,000,000 0 >3,000,000 . O o u o a o v 0.. a a . I... r\4l.'I4J.||I Jil‘ assent}! .. ‘01..- -....r. L nikev. . J... TABLE 12 (concluded) Bacterial populations of milk and cream from several fichigan ~ 1 milk plants Type of Dairy Plate counts sample 55 C 20 C coliform h.5 C Whipping A. >3,000,000 >3,000,000 >300 >3,000,000 cream B 29,000 85,000 lho 25,000 D 21,000 53,000 51; 19,000 E >3,000,000 >3,000,000 0 >3,000,000 F 13,000 >3,000,000 21+ >3,000,000 Q >3,000,000 >3,000,000 >30 )3.000,000 v 500,000 >3,000,000 0 >3,000,000 =4 >3,000,000 >3,000,000 0 >3,000,000 111. The results of counts obtained from plates incubated at 20 C are generally higher than comparable results from 55 C incubated plates. Eighteen samples had 50,000 or more bacteria per ml as indicated in counts from plates incubated at both temperatures. Only four samples gave counts of 50,000 or more at 55 C incuba- tion but not at 20 C, while the remaining 16 samples were shown to have 50,000 or more bacteria per ml at 20 C incubation but not at 55 C. Since the dilutions made to determine plate counts from 55 C incubation were 1:100, 1:1,000 and 1:10,000, the results from 55 C incubation include counts between 5,000 and 5,000,000. 'Uhen_ less than 50 colonies were counted on plates made from the 1:100 dilution, the counts were recorded as less than 5,000 bacteria per ml of sample. Similarly, when more than 500 colonies were counted on plates prepared from the 1:10,000 dilution, the counts were estimated as greater than 5,000,000. However, 68 samples counted from plates incubated at 55 C had counts between 5,000 and 5,000,000, and these samples, when compared to similar counts from 20 C incu- bation, were generally lower than the latter. Only in 1h cases, about 20 per cent, were the counts from 55 C incubation higher than the comparable 20 C counts. The psychrophilic microorganismS'were present in 75 of the El milk samples while coliform bacteria were found in LB samples. h. Isolation of pure cultures a. Fresh raw milk The types of microorganisms most commonly found in the pour plates from the 20 fresh raw milk samples are shown in tables 5, In table 15 are recorded the results obtained from the 55 C incubated plates. The predominant organisms were gram positive spore-forming rods and Actinomycetes. Three gram positive rod , could not be identified; tney may have been members of th genus Tacillus, but no spores were detected. The five cocci which could also not be positively identified were gram positive tetrads. The isolates from the 55 C incubated plates are classified 0 ' -3 o o o o in table 1;. JaClllUS and Actinomycetes were still predominant, but no Streptococcus was found. Contrary to the 55 C isolates, 15 gram negative rods were isolated as well as 11 cultures be- longing to the genus Ticrococcus. From the 20 C plates (table 15) predominantly gram negative rods and 3ran.positive cocci were isolated. These types were also isolated from the 55 C plates but less freluently. Actinonycetes and Bacillus cultures were evident, bu all of the types isolated from the 55 C plates were also found in the 20 u plates. Of the 106 cultures isolated from the nsvchronhilic plates (table 16), 65 or about 61 per cent belonged to the genera Alcaligenes, Achronohacter, Flavohacterium and Pseudomonas. AlcaligeneS‘was most frequently found in the mi h from barn C. The cultures obtained from the milk of barns A and B were too few to indicate the predominant genus, but members of the genus Flavo- bacterium appeared to be found less frequently the the other genera. The largest number of gram positive organisms belonging to 11,5 . m m mm m cm om Hapoa m o NH m a m o o H m m w a m m m J m m m « woooo .Omwu weep \‘Edaw mepoohfioswpod mneoooopmoapm mSHHHowm moamndw msmwsdQMOOHOME mo momma mo penafiz chem Mia .38." Swepm flopm even mopadm .Hsom 0 mm Seam #009302.“ mfimflgupoouoflg ma mamae m H J m m HH Hm om om H309 m H m H m m OH m m o o o H H o J 0 HH a m m o H H m 1H m 4 m .« mecca Esflpepodp hepodp mmpoe meoH lowsewm Iobwfim I0E0h£od mosomdeOHd m500000howzelrfio:wpo¢ mSHHHoam meamndm mEmHwapoopoHE mo momma mo popasz chem MHHE awn Among Soak mesa mopwam hsom 0 mm ammw wePwHOmH wEmflQdmpOOAOHA 4H mafia h5- m m J m m m mm m mH om H38 m m N N H J m o m w o H H H m m m w m w a m o m H H m m m w .4 m 4 upon memos Edwuowoam Imowowp momma anoooe wepoo \ Edam mmHoHH lovemmm 10.53 3080.33. 5.3603. touofi; 130er3. @3393 meHmme msqudquOAOHE mo momma mo hepapz spam MHHE any Swoam 50am mesa mwpdflm know 0 ON anm UmpeHOmw mEmwcdprOhOHz mH Em: mm wH mH NH mm m om Hapoe mm 0 0H m PH m w o o m H m o m a m m N H J N N m d mnozpo mesoaowzomm.EsHaepoanobmHm hepoepoth:o< mosomHHwOH< muooooouowz meHmem «duesow exp op wsHNGOHen 209552 .3 .8252 Pam MHHE 39H ch95 Goa.“ evens mopdam .30m 0 mjd Scum wopdaomw mamwgwaoonofim «CH ”,3de 1+7. a single genus were found to be members of genus Kicrococcus. fl Organisms listed as others" in table 16 were as follows: barn A- two Aerobacter, one mold; barn C- 20 yeasts and nine molds. The 20 fresh raw milk samples from individual cows in barn - 1 "Big Four' C gave similar results (table 17). Kembers of the genera were again most frequently found while the yeasts and molds were not uncommon. Of all the genera listed in table 17, Achromobacter and Pseudomonas were found in the fewest samples. b. Stored raw milk The 20 samples of stored raw milk from the three barn gave the results shown in table 18. As would be eXpected, the same organisms appeared after 10 days as were originally present. However, it is interesting to note that, the gram negative rods, particularly Pseudomonas, re- mained prominent. The yeasts, molds and Aerobacter strains were not as prevalent after 10 days storage as they were in the fresh milk, while members of the genus TicrococcuS‘were commonly found ‘ throu hout the storaee eriod. 'Witnin the erouns listed as Hiero- g L) P x.) ; coccus and Pacterium'were several strains of gram negative or gram variable organisms. On subsequent transfers at 20 C all of the cultures listed as Yicrococcus became more gram positive than gram negative. Some of the strains listed as Bacterium were de- cidedly gram negative, but could not accurately be placed in any of the gram negative genera defined by Breed et.al. (lghfl). The gram positive or gram variable strains failed to produce endospores. o o H o o H H 8H a N O O > I o c c o H HHm a N H O H o o m HOH a o o o u o a H H mom a H H o o m o m o m moH a o H o ) m H c o H mmH a O O O o o s H o m omH a m o m H o m m HEH a H m o o m 02 a o m H m pH a m m o m . m o m m HRH a o H o o H o H m oH a mHHOOOo meOE EHHwhOPOGm hmpodo. ! mcaom mpmdow Ichowm Ioooemm Ioboam IoEoygod mememfiaeoad moamsdm see me nonemow an» op mmflwsoaon muonssz no 909852 qofipdqwflmoa 0 seem ma mace Hosofl>fivefi anm moxoe xHHE amp Smopm Boga wopeHOmH mEmstmhoouowa OHHflsmohsommm NH 33.9 )'0 NH mHoap none spam * NH Nm N MN 4. mm mm mm Ho mHapoa H H m n o NH m o 0 OH o H N N 0 0H m N 0H m N a N J H NH o HH HH a 1H o o J H N N N o N OH ON 0 o N «H NH NH N .o mooooo peWoom mosofi .ESNQOWoom uewoen momow Amhaov moaou mpwoow.Edfiaopoem onowm Iopo< loosomm Ioboam IofioaSod Iwfieoa< MHHE ”daemon exp op wqwmmoaop mucosa” AJ. a ,8 Fa MHHE awn come no onmfiom 0N Eoum oopwaomw ma mqmda wfimfimeupooaofla oHHHsmoamohmm 50. B. Taxonomy 1. Ticrococcus Sixty-one micrococci were isolated from 55, 20 and h.5 C plates. Forty-seven of these cultures came from fresh raw milk mile the remaining lh'were from stored raw mi h. All of the 1h cultures from stored raw milk'were picked from h.5 C incubated plates. Fourteen other colonies were picked from h.5 C incubated plates prepared from fresh raw milk. Eleven and 22 cultures came from 55 and 20 C incubated plates respectively. Thirty-six of these isolates were readily classified accord- ing to the description given by Breed et.al. (lghfi) while 25 were not. The 56 cultures which had the characteristics to match the descriptions given by Breed et.al. (lQhS) belonged to 10 species, while the remaining 25 cultures were placed into 11 groups. The distribution of the 61 cultures is sh ‘.tn in table 19. hr. he predominant types of micrococci were hierococcus conglom- eratus and: ' icrococcus epider m. M13 the former found in plates incubated at 20 and h.5 C, the latter found in the 55 and 20 C in- cubated plates. Iicrococci belonging to roups D and K'were also 3 frequently encountered, but never in 55 C incubated pla tea. The cul tures identified as Hicrococcus freudenreichii, Iicrococcus caseolyticus and those in groups B, D, E, F, H and I did not initially grow at 55 C but after several transfers at 20 C, all cultures except one strain of Ticrcccccus freudenreichii and three strains of licrococcus caseolyticus grew at 55 v. TABLE 19 Type of micrococci isolated from pour plates incubated at various t-mperatures Name of organism number of Source cultures Plates incubated at (C) 35 2o 1:.5 hicrococcus luteus 2 0 Kicrococcus ureae 2 0 2 0 Iicrococcus fFEEHen- 2 0 l 1 reichii 113.3175??qu flavus l l 0 O Ticrococcus‘conglom- 9 O h 5 eratus .— Kicrococcus varians l l 0 O Ticrococcus cascoly- 6 O l 5 ticus Hicrococcus aurantia- 2 l O l cus Kigrgcoccus epidcrni- 10 h 6 0 dis _— Iicrgcoccus roseus l 1 O 0 Group A 5 1 O 2 Group B 2 0 O 2 Group C l 0 1 0 Group D 6 O O 6 Group E 2 0 O 2 Group F l O O 1 Group G l l O 0 Group H 1 0 O 1 Group I l O O 1 Group J 5 l 2 0 Gr up K h 0 h 0 * For a description of each group, see appendix B. Strains of icrococcus epidermidis, groups G, J and K could not be detected after 1h days incubation at h.5 C. 2. Alcaligenes Eighty-h vo cultures of ‘lcali enes were obtain ed, five from ight from 20 C and 69 from h.5 C incubated plates. All of KM 5 C. (D the cultures were readily characterized as shown in table 20. Alcaligenes faecalis‘vas only pick (ed from 55 C plates; the 3 Alcali- .4 .. O \J ( *3 H {D (1' (D U) 0 remaining five species were found on the genes bookeri was isolated most frequent y. ~ 3. Achromobacter he 50 Achromobacter cultures were classified as shown in table 21. All of the cul ures except one group were comparable to the descriptions given by Breed et.al. (lQLB). The descrip- ‘ O oth this unidenti- "'1 tion of this group is given in appendix B. fied group and Achromobacter super ficiale*rere the predominant * Achromobacter. h. Flavobacterimn The descriptions which are given by Breed et.al. (lghs) to distinguish members of the genus ilav011eterium are not com- plete enough to allow for adequate separation as is indicated in table 22. The 5h cultures did fit into 10 groups; six of which are comparable to the descrip ions given by Breed et.al. (lth). he remaining four groups are probably also given in Bergey's Vanual, but too incompletely described to*w'arrant conclus ve identification. Types of Alcali genes TABLE 20 ate d from pour plates incubated at various Iame of org anism t1nnneratures Humber of Source cultures Plates incubat 'ed at (C) 35 20 11.5 Alcaligenes faecalis 5 5 0 O Alcaligenes viscosus l5 0 2 ll Alcaligenes metalcaligene§_ l9 1 5 l5 Alcaligenes bookeri 5h 1 1 52 Alcaligenes recti 9 O O 9 Alcaligenes marshallii L. O O h ¥1 Types of n,hromobacter isolated fromgpour_plates incubated at various temperatures flame of organism number of Source cultures Plates incubated at (C) 35 20 11.5 fichromobaqtgr liquefacien 5 O O 5 Achromobacter iophagum 1 1 0 O Achromobacter delicatulum 2 0 2 O Achromobacter cycloclastes h l O 5 Achromobacter_superficiale 15 O O 15 Achromobacter butyri 6 O 2 h Achromobacter eurydice 5 O O 5 Achromobacter delmarvae 5 l 0 2 Group A * l5 0 2 11 * - . .. . . For a description of this group, see appendix B 55. TABLE 22 iypes of Flavobacterium isolated from pour plates incubated at various temperatures fame of organism number of Source cultures Plates incubated at(C) 35 20 11.5 Flavobacterium devorans 2 l O 1 Flavobacterium suaveolens 5 O l 2 Elavobacterium invisibile 6 l 0 5 Flavobacter'um lactis 7 O l 6 Flavobacterium esteroaroma- 2 0 0 2 ticum Flavobacterium ferrugineum. l 1 0 0 Group A * 9 O O 9 Group B 2 O O 2 Group C 5 O O 5 Group D 2 0 2 0 Group E-l 7 0 l 6 Group E-2 5 0 5 2 Group E-5 5 l 0 2 For a description of each group, see appendix B K” O'\ o 5. Pseudomonas Since 75 out of 78 Pseudomonas cultures were Obtained from plates incubated at L.5 C, the results of classifying this group of organisms applies particularly to psychrophilic Pseudomonas. ma ine findings recorded in table 25 indicated that two groups, Pseudomonas pavonacea and Pseudomonas ovalis accounted for near- n—‘o 1y 75 per cent of all Pseudomonas cultures. C. Temperature studies The representative psychrophile cultures subjected to 62.8 C in skim milk failed to show any growth after the heat treatment. host of the 25 cultures (table 2h) grew better at 20 C than at either 55 or L.5 C. Whereas the h.5 C counts were generally comparable to the 20 C plate counts, the 55 C plate counts in seven cases fell far below the counts from the 20 and h.5 C incubated plates. Although the cultures may have grown at 55 C, the colonies on the pour plates were often too small to count with any degree of accuracy. D. Influence of psychrophilic microorganisms on the keeping quality of milk The plate counts of four raw milk samples stored at h.5 C are recorded in table 25. In a similar run previously described, the 55 and 20 C plate counts vere initially higher than the h.5 counts, but within a short storage period, the psychrophilic count equaled the other counts. The population determined in 55 C Types of P8»udomonas TABLE 23 57. isolated from pour plates incubated at various temperatures Name of organisms Number of Source cultures Plates incubated at (C) 35 20 h.5 Pseudomonas chlororaphis 2 O 2 O Pseuaononas synxantha 5 O O 5 Pseudononas fluorescens 5 l l 5 Pseudomonas-oavonacea 57 O O 57 Pseudomonas gericulata h 0 O h Pseudononas syncyanea l O O l Pseudomoqis iodina, 2 O O 2 Psculon5333:s;jnqriiafii Efpudonengg_g:a££i 22 O 1 21 Pseudomonas SEEEEEEi l O O l Pseudomonas immobilis l O O l [‘5 /v 000.00m 000.0 4 000.000 undo» 00308.2... 000.08.0mm 80.08.08 2 . . - t 5... . _. q L .0 _)_ _)\. .) \ . . - .. LLLL- mnnqn00.0cw. 000.0r0HcC\ 000.0r0.0ma dHohfioo udcofiopnOmm 00 000 owe H 000 000 000 000.000.H maqdbo mesouo.som 000.000.0 m 0 . . . A . .r... e a . 00 000 OCH 000 00% doomncbdm macoaovzomm 0. sq Q . a. 000.000.m 000.000Hm 000.000.; mfiwoea EsflaepodnobfiHh 000 000 OH 000 com . OM14 J H5 \) .3. ; 4 OOOQOOOQ04.J OOVOQOAJQQC Q \J G) O'MH #HfHQ.fiUF-Cr0ktd.flrg 000.000.0xm 00 . . .. -. 000.0(0.c Hum Esflpopownobdam . or; . 0 000 0Qv H 000 000 0H0 4 Edwpepoapobwam 0. q 0. _ .\ \ .. 000.000.03H 000.000Hmcm 000.000.0NH .WwwflofimpoCSm hoped:ofioamcd 000 00> 4 000 00> N mm mdoaoe-mdwa nepodocfioudcw . . . ux. . e .H . . .a 000 00a m 000 ocu m 0H d hopompoE0h£e¢ 000H0w0. 000H000H00m 000u000H0HH “poop manoeuflmefim 000q0cogomm 000.000.0mm 000.000.00w whofloon menomflawoad 000.000.00 .\ 000.000.0@-. 000.000 OHH mecomwwfleadpofi.moqoufladowm 000 000 000 n 000 000 000 m 000 000.000.H msmoomflp monomfiHaOHe 0. _qa 000.00m.m 000H00wum 000H00>Hm noflumHommdo mSOOQOOLQflA 000.0mn ; 000.00; 4 000 00m : mzpdaeaoamnoo adeoooopoflu 000.0-0 000 00H 0H d.mmmmmmmmmm. 08 05 000.02 2 .. ....m....8..fi.m..w.. 000.000.0 000.000.0 00H.m m osommuy--m¢ .) 4 , \ up. -mu;.: 000 00w.0 000.000.c 000.H 0 msoo.oono-p 000.000.m 000.000.90 . x. .; . . c 000 00a H 4 mneOOOOAOHL 0 mo: 0 Qm 55.. 0 mm mfimwfi0umm « pd wopaQSOCfl mopafim Seam mp:500 Hawpopoam Ho cowpaumwmem .Il'n. II I t..- 'n- ' III. . \O.+. 4 ..I u )0 .3. J...) \ . ... .... l4 .. .. .1 «in... .1 J 1 0 r ; no rowPSrrcrfl wrap >4: henna yHH: Yqfim r0 mfmw_c ac OHHMEQOHdome mo Pdsoo adflaopodn - C .- OI..'-I‘.I.I.I‘.I. 0'... v ‘5 ‘l TABLE 25 Bacterial count of stored raw milk usine 86V ver temperatures Days stored at al incubation Bacterial count :l‘l //. 2.5 C Incubation temperature (C) 35 20 55.5 l. Iilks mple A 1 1L,000 15,000 2 ,L00 2 33,000 30,000 20, 000 5, ’1 f‘, a 1, 500,000 2,200, 000 1,100,000 6 2,700,000 28,000, 000 15,000,000 8 L3,000,000 65,000, 000 50,000,000 9 100,000,000 200,000, 000 210 ,000,000 13 970,000,000 2 ,000, 000, 000 2,300, 000,000 2. Iilk sample B 0 8,L00 5,600 170 2 35,000 30,000 8,700 l. LLO, 000 L00 ,000 360,000 6 2,L00, 000 9,500, 000 13,000,000 8 b:,000,000 210, 000, 000 57,000,000 12 .0. 630,03 0, 000 060,000,000 720,003,000_ 5. Kilk sample C 0 200,000 310, 000 10,000 2 190,000 9L0, 000 870,000 L 360,000 8 ,200, 000 7,600,000 6 1,700,000 35,000,000 L2,000,000 8 2,L00,000 190,000,000 110,000,000 10 12,000,000 1,L00,000,000 7900,000,000 h. filk sample D 0 85,000 07,000 37 2 22,100 L9, 000 110 L 10 ,L00 150, 000 22,000 6 13,000 L70,000 , L00, 000 8 100,000 5,L00,000 0,000,000 10 2,900,000 31,000,000 27,000,000 12 25,000,000 L50,000,000 L 0,000,000 / 00. plates, as Opposed to the 20 and h.5 C plates, failed to increase. From the data in table 25, a slightly differen trend can be noted. The initial 55 and 20 C plate counts were well above the psychro- phile count, but within two to four days, the psychrophile count was as high as the counts obtained from the other plates. With milk samples A and B, except for an occasional variation, all plates gave similar counts. The 55 C plate counts from milk samples C and D indicate a definite lag, especially in the latter case, but the increase in the psychrOphile count is event- ually reflected in the counts from the 55 C plates. A portion of milk sample C, which'was pasteurized immedi- ately, was divided into five parts. One part was stored'without inoculation while the other four parts were inoculated with an char- 0 actively growing pure culture. The plate counts and test acteristics of these milk samples are recorded in table 25. The uninoculated milk did not deteriorate after 16 days, although the psychrophile count increased slightly. The inoculated milk samples had off-flavors when the psychrOphile count was 10 to 100 million, vhich occurred at varying times of storage depend- ing on the amount of inoculum and the rate of growth of the particular organism. The milk samples A and B were pasteurized in two day in- tervals as shown in table 25 and inoculated with 0.2 ml of a raw milk sample containing 853,000 psychr philes. The subse- quent growth of psychrOphiles and the deterioration of milk 22:12 26 Plate counts and taSte of a pasteurized raw milk sample from ‘Tresh raw mileESample C; table 252 Days stored at Plates incubated at (C) Off-taste 55.5 C 35 505 l. Uninoculated 0 1,300 10 o 2 1,100 10 0 1. 1,200 10 0 6 2,L00 10 0 8 5,000 10 0 10 h,300 ll 0 12 5,900 65 0 1L 6,100 95 0 16 6,700 700 0 2. Inoculated with Flavobacterium.(E—2) 0 1,300 970 0 L 1,700 1,000 0 8 3,800 9,000 0 12 6,h00 270,000 0 16 16,000 2,500,000 0 5. Inoculated with Pseudomonas ovalis 0 1,700 550,000 0 L 1,L00 2,800,000 0 e 1,L00 L,500,000 0 12 950 L0,000,000 ,1 16 1,300 L70,000,000 ,AAA/ L. Inoculated with Alcaligene§_viscosus 0 1,L00 , 3,000,000 0 b, 2,000 12,000,000 / 8 L,000 L9,000,000 12 L0,000 28,000,000,000 ,4! 16 370,000 61. (A .' 0 TATZE 26 (continued) Plate counts and taste of a pasteurized raw milk sample from fresh raw milk (Sample C; table 25) Days stored at Plates incubated at (C) Off-taste L.5 C .55 h-S i_ 5. Inoculated with Flavobacterium (A) 0 2,L00 1,700,000 0 A 2,000 2,900,000 ,/ 8 30,000 150,000,000 // 1 12 300,000 200,000,000 11 16 L6,000,000 <__ 370,000,000 curd A Agg 6. Inoculated with.¥icrococcus conglomeratus 0 1,900 26,000 0 L 3,600 370,000 0 3 10,000 16,000,000 0 12 59,000 59,000,000 ,1 16 1,100,000 #59,000,000 _1411 proceeded as indicated in table 27. Both raw milk samples, h ving a psychroplile count above 50 million, produced pasteurized milk 'which was unfit for consumption. Likewise, the pasteurized milk produced from raw milk with a low psychrOphile count, was able to withstand more psychrophilic growth before the milk developed an off-flavor. apmbm mu w a O o . o o o . . Mme dmwo ow aowowwoepwwos omiwpmdozepmom HHHN.wHthH00 fines and DHHW mdowou 90 3. deMOSm wwnmm 9:0 wsoocwpamg Eva: m prom mowsprwo: ow mmuoxfiomwwwom “‘ i‘ wmmwoCdMNom BHHW wmwowdowwwwo >00 0% H05 Swww pd wwao ow wpmdmzewemawob mfiOflQa. Aflpwdv 006.5%. ... O H N f. 0 m 5 Hm H. HMHW mwfiwwm P \ 0 000.000 --H om 0 0 0 x 0 KM“ -- xxxx H H. 000. 000 .i 0 0 0 \H\ .. x .. .. m 0.000.000 .. 0 0 0 \mx xx .. -- .. m 3.000.000 .. 0 0 “a x .. -.. .. .. L 00 000 000 I- 0 u- -- u- u- u- m 50000.08 .i rmxxavm xxx .i u- .i .. .. m. 190% mmnwwo w 0 000.000 0 .. 0 0 0 x .. xxxx .. H 2000.000 0 .. 0 0 x x .. .. .. m 5.000.000 0 .. 0 0 \k xx .. ..- .. w 0:08.000 0 .. 0 0 xxxx .. .. .. r 00.000.000 0 .. 0 x \Mx .. -- -- .. 0. 00.08.08 x -u x xx I- -- -- -- 0 258000.00 txxxx -- xmx xxx -.. .i .. .. .. are wmwoueowowwm 00550 wm p5 m10,000. They conclude, quite logically, that Pseudomonas probably inhibits the q development of Flavooacteriun strains. The differences in the types of psychrophilic organisms given in this paper and those reported in the literature can be partly explained by the individual techniques employed. Some ators have used special media and methods (e.g.,'3agenaar and Jezeski, 1952) to find a particular organism suspected as being the causative agent of spoilage. This is particularly true in the Po papers dealing T h Pseudoronas fragi and Pseudononas putrifaciens. Furthermore, the incubating temper ture is extremely important in determining the types of organisnS‘which will predominate in stored milk. The terperature of storage at h.5 C was selected becruse it approximates the temperature recommended by the American Public Zealth Association (ITEB) and, it corresponds to the temperature at which milk should be stored. Deviation, however, has been made from the incubation time recommended by the American Public --' 11" A r‘ 4 . 1C"? '11. 1’1 6 «p. r‘ ‘C‘ fi'w ~r . -~ “ea en Association ( ,3J)‘wn1c. suggests lire dads. In uSan a ' O O I O V V. Q 1 dav incubation eriod onlv slicntlv ninner countS'were oo- 14 v p . . a . s tained, but the colonies were lar er nazin 1 count. Other incubation periods anr temperatures which have been {7 " for 13 days, h.§ c for seven dars (Erdnan and used are: 10.5 o ‘ ' h a r— Thornton, 1:51a); h-7 C for 2 days (Iogich and burgwald, 1932); 5 C for 10 days (i‘iatrous, Doan and Josephso or, 1952); 0.7 C for 10 days ( D ahl J Tlri 1 l 011 S 11132; , 1951);, I.LI_U berg, Adans and held, 1:5 Thomas and Iforris, 135 " for 20 days (Boyd, 19 C7 gm )1 7 )3 53). 17 ('1 U ('3 for 10 days (Olson, for five days (Proutjf, VI. VOICLJSIOIS A series of experiments has been conducted'uhich indicate the following: 1. Only bacterial counts fron plates incubated at h.5 C gave a true nixture on “We ~cvchrochi"’c “c““ "*’c" in nil” ‘ 4 ‘, v ‘.I a... J. U.LJ llfi)" J.~- .- L24. -..---.} L. Ml- »’..~~.-. )- .‘a .1“ nt Lb. raw milk multiplied, C When the psychrophilic population of it imparted a characteristic to the rarr mi k‘wb Mic} decreases the keeping quality of the pasteurized milk. 7. PS"chrop11les were killed by pasteurization. 11... ed milk developed an off- flavor when the psychro- ! - 3 4. Pa teuriz (I) phile pepulation reached a partic.iar evel of gronth. 5. Certain speciest nded to predominate in mi : stored at h.5 C for sev eral davs. VII. SI.I;C. From th discusssion on he relationship betreen plate counts incubated at 33 and 1.5 C, counts from plates incubated at 55 C did not reflect the n""cnr0fn1l1c population of the sa ple. The in it al count from the 35 C incubated plates 13 higher than the psychrOphilic population. 'Hith cold storage, th counts from plates incubated at 35 C may decrease or increase 8115‘ The psycl 1roph1 ilic population increases rapidly'with storage , and after four days of cold storage, t1 p“"c"ro,n111c pol1ul ation is :1 greater than ’ndicated by counts 1rom lates incubated at 35 C. *3 Some commercially pasteurized milk samples, which'would be juig ed good or satisfactory by the count obtained iron pl tes incubated at would not be considered satisfactory on the basis of the psy- chrophi1ic population. An insignificant number of the organisms which are predominantly found on plates incubated at L.§ C grew better at 35 C; most grew practically aS'well at both temperatures. 3 N ('1 One—third of the cultures studie‘ aid poorly at 33 u as compared to 1 n V ' '— o a s "W o _ o the counts outained fr m tne 4.; C incusateu pl tes. ”nen KIIL lS ' f" a 1 o u o o 1 stored at 4.3 C and no psycnroph1l1c ~ra..t h is ev1dert from plates (D 0‘) 5:1 '1.) (I) O s: f 1 o H H incubated at L.5 C, the counts from 35 C incubated plat not Show sijns of bacterial ‘ro'tl durinj storage. In short, the counts from pls tes incubated at 3; C can be used to evaluate careless liandling of milk or inprOper refrigeration, but should not b the criterimafor judging the general bacterial quality Of mi1:. ha I1 An examination of counts obtained from the plates incrbated at 20 C and those at 55 0 indicate that the latter temperature is more nearly optimum for growth of bacteria found in milk. The slate counts obtained upon incubation at 20 C are influenced both by the organisms found on plates incuoated at 33 C and h.5 C, which explains the higher counts obtained at 20 C incubation. This fact invalidates he use of 20 C incubation for determining the psychro— philic pepulation in milk even though 20 C more closely approx'mates the Optimum temperature for the group of organisms. However, the fa t that at 20 C incubation higher counts are obtainable than at es th- use of incubation temperatures below 35 C. Raw milk generally contains a small pepulation of psychro- philic microorganisms. ihis population increases to about iht days. If this miln is pasteurized, f" t.) 10,000,000 in four to ci the pasteu ized product will probably have an off-flavor. Past- eurized milk does not contain psychrOphilic microorganisms unless post-pasteurization contamination occurs. Iost comnercial milk contains a low psychrOphilic population. Pasteurized milk ob- tained from fresh raw milk will generally not have any off- flavors due to psychrophilic multiplication until the psychrophilic population approaches 10,000,000 bacteria per ml. If the raw milk, from which the pasteurized product is obtained, is stored for a period of time, the pasteurized product will deteriorate before the psychrophilic pepulation reaches 10,000,000 per m . A series of standards is proposed, based on the psychrophilic population of raw and pasteurized milk which will safeguard the dairy and the consumer from selling and receiving poor quality milk respectively. The maximan psychrOphilic count recommended for raw milk is 100,000 C SD 0 cf‘ (0 H P. a per m1 and 100 for pasteurized milk; the latter being probably too conservative a figure. From the discussion on the relationsnip between psychrophilic coliform plate counts as indices of post-pasteurization, data {is 8.31 ’ have been cited indicating that the psrchrOphilic pepulation is probably a more sensitive measure. Rather than neasure the extent of contamination, the psychrophilic population is suggested to be used as an evaluation of'whether or not post-pasteurization con- tamination has occurred an the relative holdiig time of the milk. The following characteristics are preposed to designate psy- chrophilic microorganisms in milk: 1. They generally have an optimum temperature above L.3 C and below 35 C; some may grow at 35 C. 2. Their growth rate approximates a 10; increase every two days of storage at h.5 C in mil:. They impart an off-flavor to milk when their population \.‘1 0 has develOped to 1,000,000- 100,000,000 bacteria per ml. A. They are feebly saccharolytic and predominantly gram nega- d- tive, consisting of the genera Alcaligenes, Achrom bacter, Flavo- bacterium and Pseudomonas. 5. hembers of the genus Ticrocoecus are the most common gram positive group. 0. Yeasts and molds may be present in large numbers depending on the particular barn. 7. Psychrophiles are killed by pasteurization. 8. Certain species of the genera.fiicrococcus, iseudomonas, Alcalijenes and Achrcmobacter tend to predominate. A mechanical key has been prepared to aid in classifying psychrOphilic bacteria associated'with milk. V') 10 ' Ft'fivfia >‘ «v & aL‘ .11-; 4J5 . Iechanical key for the idcnt 9 :3 E3 H. }.J W I‘D v hey to the genus alealijenes \3—4 a [I‘d r. Key to the genus Aehromobacter Key to ,he genus Flavobacterium U] o C\ 0 Key to the genus Pseudomonas l. \u I _. 100 11. nopendix A .L- ric key to the gram negative rods* Iotile Hon-motile Polar fla3ella Peritrichate flagella Litmus milk alkaline Litmus: nill: not alkaline Yell W to orange pi'ment Kc yell‘." to orange pigment Acid from glucose 30 acid from glucose Yello" to orange pigment Ho yellOW to orange pigment Crecn fluorescent water soluble pigment No green fluorescent water soluble pigment Litmus milk alkaline Litmus milk not alkaline YellO"I to orange pigment “0 yellow to orange pigment YelloW to orange pigment Io yellow to orange pigment Co lifo orm group not included Pseudomonas \N ext? \n A1c ligenes Jh—C lavonaCte i m :glcali * "ones *w‘ ’U C) 9 ;§ ? 5 59 m 1% ‘-b 0393 6:. ~ s.’ w - . , _ . :iaxooactcrium AT‘ 1:. a Flavobacterium jbhromobacter 2. Key to the genus Kicrococcus l. 7. C) o ... J. Pigment formed Io pigment formed Gelatin iquefied Gelatin not iquefied Litrate reduced to nitrite Kitrate not reduced to nitrite Litmus milk acid Litmus milk alkaline Litmus milk ccasulated k.) Litmus milk not coagulated Pigment yellow Pigment red to orange Litmus milk peptonized Litmus milk not peptonized Pigment yellow Pigment red to orange Litmus reduced Litmus not reduced Nitrate reduced to nitrite nitrate not reduced to nitrite Pigment yellow Pigment red to orange Litmus milk coagulated Litmus milk not coagulated i nus milk acid “ilk alkaline Litmus milk acid Litmus milk alkaline Ho reaction in litmus milk Litmus milk coagulated Litmus milk not coagulated ON Com \OJT‘ 0w Iicrococcus conglomeratus 7 Kicrococcus pyogenes var. aureus Iicrococcus caseolyticus Iicrococcus citreus Group A Ticrocoecus roseus Vicrococcus flavus [icrococcus freudenreichii ll 18 tJo C3 is U} Ticrococcus var, fiicrococcus aurantiacus Group’B 15 Iicrococcus cinnabareus- rhodochrous ,_ Licrccoccus cinnabareus 16 17 17. 13. O /o 21. [o 3 DJ \21 o R) Kl Acid from lactose Io acid from lactose Iotile Hon-motile Pigment "ellow Pi 131ent red to orange Litmus milk acid Lit r1 3 milk alka ZIo reaction in l Gelatin liquefied Gelatin not liquefied Yitrate reduced to nitrite fiitrate not reduced to nitrite eptonized IJitrills dilkp 8 0t peptonized milk ’3 Litmus milk acid Litmus milk alkaline Ho reaction in litmus milk Litmus milk acid Litmus milk alkaline So reaction in litmus milk Iitrate reduced to nitrite Sit ate not reduced to nitrite L rus T“il? ac1d L ttus milk alkaline to li ..us mi k Litmus milk acid Litmus milk alkaline Kc reaction to litmus milk ' i-icrococcus 07 O Q C T.Ill".&09.l‘0‘.‘-.S [icrococcus fl“ fl I'..lu€-;o Group C -icrococ cus rkodochrous up D Ticrecoccus cascol"ticus fl .7 r"-v L9 Ticrcccccvs enidermilis Group E Group F 1i rococcu freudenrei cilii Tic receccus llTUCf aci ens Crone G . .L ' CCII‘BI‘. ul $017.3 licroccccus -v .4 A‘ Group Srcup Ticrococcus 77 .‘L candidus ' ’ av o~\J 4 U .L .1." U0 U-L genus Alcali~enes .32 Gelatin liquefie Gelatin not liqrefied Iitrate reduced to n ‘9. i e nitrate not reduced to nitrite QP __o C produced in milk not produced in milk Alcaligenes \‘3 (A o O ‘ L. Aloaligenes recui . ”a ., » ~ ~13» . Alcai genes UkLh'fl —- fi '— ' j / LL. g.- D J I --I l. ‘4 Ci) 0 v It to the genus Achromohacter Iotile Non-motile Gelatin liquefied Gelatin not liquefied Fitrate reduced to nitrite Kitrate not reduced to nitrite Some reaction (, ction and refill in litmus milk 30 reac 1.. tion in litmus milk Litmus milk acid Io reaction in litmus .ill: 1w. nitrate Litrate (D :val H. tra id. lb J. aU ‘i- ra u (D Gelatin Gelatin Gelatin Gelatin reduced to nitrite not reduced to nitrite nitrite to nitrite reduced not reduced liquefied not liquefied d lique— Mi t Mg Mfi d no $7- *JID CNKN Pcnromo to cter delicatulum Achrcmobacter ionhigum AA. L, 0% ‘ .L . O b Achro tacter ""jerfiiciale Aczromobacter liduefaciens Achrcmobacter c"cloclastes uclroucoacuVr sugerfic1ale 8 O / Gresn A dc rouobacter delnarvae r 1 key to tne 1.. 5. O\ O 7. \O o 10. ll. 12. enus Flavobacteriii Iotile Hon-motile Gelatin liqu efiedn1 Gelatinr not liq ed Titrate reduced to nitrite Xitrate not reduced to nitrite formed not formed Indole Indole elflzali;.e Litrlus milk I re in litmus .o eaction allcal ine in litmus tmus milk To reaction N ..J H. H ...J I" k acid k alkaline in litmus milk Gelatin liquefied Gelatin not liqL ed Iitrate reduced nitrite Uitrate not reduced to nitrite Litmus milk acid Litmus milk alkaline Ho reacti n in litmus milk Litmus reduced Litmus not reduced Litr.us milk peptonized Litmus milk not peptonized Acid from lactose Io acid from lactose CDKH \D R) 0‘17 'L 9'7".) ‘1‘.” U3; ....... -lavooac win” at *96‘4 bacterium rnenzmus rlavobacterium difiusun or ‘ri n~— ‘eet MrLun r1" -..“: Ljv‘VJ Flavohacterium ‘a"““"onii -1j er\j*4\,j - )v.‘ , W I ~ ‘ .11 U sax/(v UL- 11.. L marlnun. Fla 'oricter"H lactis Group A Flavobacterium invi 4. ble 0 U) l 12 15 13 .L. J.‘ esueroaromaoicum 11; Flay obacterium ferrugineum Tlavoaacterium Dalustinum — J. 17. 18. mu milk hen tonized c; we .1. U Litmus mil; n tor ized Acid from glucose 30 acid from glucose Iitrate reduced to nitrite Hitrate not reduced onitrite Acid from glucose 30 acid from glucose llOW'pigment Orange pigment litmus milk pepton ized Litmus milk not peptonized Acid from glucose Io acid from glucose Litmus reduced uced Litmus not red itrate reduced to nitrite . itrate not reduced to nitrite p.01: Acid and gas from glucose Only acid from glucose t... *4 t a s: C) E?! F. H h {‘3 0 P. Q; Litmus milk alkaline Ho reaction 1n litmus milk 0C xx. Group B TanFAhnhi‘fiWI‘fis‘q 1“ & ax" -4 HJV-‘ SL5 --LJ s ?lavonacterium ferrugineum) h V ‘- -L‘ .- n gla:0uacucr1um lucatum 3 avobacteriam ostcroar mat cum 10 / '1 -. 1 ' 1. ,. ' :la ooncterium es e-oaromaticum “filo w!‘::,r1L:1 es {nunirCV“atIC‘tl ~ "V ‘ C uroup arborescens or oacterium Lorrugineum) acteriun azyatile or avobacterium ferrugineum 211- 25 (Flavobacteriun 1 ave “1 ‘ 1~v,r\ ,_ a: .‘L".~. x) k.‘ I‘lavohacterium roteus or :lavoba cterium hrevi 'ul‘O Lip D (Flavohacterium brevi or Flavopacteri n flavotenue) Group E-l (“laVObac crium brevi or glawmiacterMX1solare) Grozzp‘E-E avo .3:1c terium nrevi or Fla ooac terium solare) _‘ w: UFOUP “13-; ’11 ,J ( (Flarobacterium br-vi or lavoaacterium solare) ll. It Key to the genus Pseudononas l. Gelatin liquefied 2 Gelatin not liquefied 16 30 growth on gelatin Psesdcnonas erythra 2. Yotile 3 Hon-motile 15 3. Grows better at 35 C than A at 200 Grows better at 20 C than 5 at 35 C h. Litmus milk acid Pseudomonas caviae Litmus milk alkaline Pteudoxonas effusa 5. Litmus milk acid 6 Litmus mil? alkaline 8 Ho reaction in litmus milk Pseudomonas syncyanea o. Iitrate reduced to nitrite Pseudomonas perolens Titrate not reduced to nitrite 7 7. ndole formed PseuComonas airmountensis Indole not formed Pteudcxonas fragi 8. Indole formed 9 Indole not formed 10 9. Iitrate reduced to nitrite Pseudononas myxogenes Iitrate not reduced to nitrite tseud0monas schuyIkiIliensis lo. Distinct "ellow to orance sol- Pseudononas svnxantha J uble pigment produced in cream No distinct yellow to orange 11 pigment in cream Hitrate reduced to nitrite 12 Iitrate not reduced to nitrite 1h The terminology and descriptions of the genus Pseudomonas are based largely on the recommendation of Haynes (I?§l3:~lgjlb). According to annes, this is " essentially as it will appear in the next edition of Ber:ey'si“anualr,i.e., the seventh edition. 12. 13. 11L. 15. C‘ L. Eitrate reduced milk coagulated milk not coagulated reduced not reduced ized to nitrite fiitrate not reduced to nitrite otile Hon-motile Crows better at 35 C than 23 C Crows better at C thai 33 U Ti trat reduced to nitrite Zitrat reduced to nitrogen Uitrct not reduced Acid from glucose Io acid nrom glucose Shuth :r“&olrzed Starch not hgdrolyzed Inlole formed Indole not formed Litrns milk alkaline Litmus milk not alkaline Litmus mill coagulated Litmus Aittn .xl milk not coagulated for? ed not formed milk alkaline mi k not alkaline 1 Pee cu doronas cnlor ran 4 J 0:13.53 Pd cI‘i C O .ZQGI‘CllS flmx rescens . Pscvaononas naTIonacea geniculata O Pseuc r . d0“0nas v» .3 snare tn_1 ha 0 s o o P"c“dc"onas 1"*O”1115 ..... h 4- ' rsendononas ecu zcri Pseidononas striata m ‘ 'Q 0 \.'»s01. .O.:vL-S rau1L‘311lS .Zj '3 " 4—K ' :seuaornonas ral,oa:c, '.«-‘~n“-"f\1-qwnfi fi-~‘nj\1r\‘:(“,n ‘ u\.~ \LVJ.‘\_--\.~) u; ;~__-;,u_~4ufi Psenaomcnas Ed”lda A;- «:2 ",f L—U ..(r .1....‘n,.. J. 04. 1 L40». .0 O.av\.u Svr._\A-Ua fihn' in r‘wwr‘ ‘00 7'4" .5 '—“]‘—§4 .U .\.4.\) C.-u; 0.5.] 10a 102. C38. 1 Z desno Pseudononas acid lk not acid )4: 1~+ pa I‘ll Li tn: 8 .ST"O ‘ .’.f‘ ‘7' A“ no . i\_, -.1 .u) xx ‘ L4 Cr m u n a L 9 a." n... L. J \“ q. 2 t H .‘L W». «L A,“ at, P: S C. S S a... u W ‘ “A 0 O m. N . O n. fin... sm. n e a... a.” ....Y. W: .0 e Z un'u l n. O .hl. ‘w t :3 ~fl my C .1 r a a .3 .3 n0 Cu 0 n‘ n... J- .. :1 EJ O. I -. I L ya (_‘. (- H _ -.Lv.kJ D 'Jr ‘ fl - 'A ”'trnzs Jqfi u. L1 S T ‘.;~ an inc - lital mil“ not ni Va “—— acid 1 2C il 5 . Litmus n itmus no reac P) a; .m w 01 ‘0‘." ”I" (1020:1618 oval; S , I bua' Dr: an .0. ~‘. alkeline in l n _ 1... .l:_ i T ..J 4-” UM i ion t ‘v \J A; 7) 7-1 uiCC‘. L 0' \--'.‘ \ ‘uce Iitrate re: ra TJ 7/ .1. v _. I- ..i—dh \J --o u ..E. n, W. 3 am w; W. o- m ._1* .. S no 1. _. h m, mu 0 C .1 o o. w. mi. rd nu P hi 9 - A ...... l d a .1 a, _. C ul~ a a f; — h 1.; ‘ a .1; .I‘ .1 :4 3 no u 1.“ na-n :L ..L v.“ T. no .II .I 2/ on. aline u 1‘— .L n J} S .1- m -a. V a ) v L vexa V‘ .L CC C“ n.) (T7071? 1 (I MA 3011 Ll 103, n‘l A... U. . nxq «w; 13 ‘l TGOOCC -o 1 n -.. \J . AC In‘orrg‘: :13 1301- ”1 .‘ ctcri 7 av 01'} C. -l . “\ ’vx flrj. '_ Appendix o n I hi :4.‘ ' ‘ ,. ' .‘ 3. , ., .1. group n. rar POSlolVQ cecc1 occurring in pa rs and clnsoers. "Oi-n tile 'nnecr" to be 'dentical W’L“ “‘n a”. men n v e t - - -.. U..._ . . il'va A.) L; .. l... /.I. V..- ( ... Ill. U-L --.1quCOC‘-/~.uo rv~J~.L]Q e...c up ° J. ,1. ,. . '1 s ..- - ,._,,, -. o -.. ° , , .n r- .A .n -, v. - , - lu produce- a :e_iCn piwnenc. dice 1.ueiae.ien oi elasin. Colonies Q -. V ‘1 ‘2': t L, a- ‘P \ ~0- ‘ n agar are c re: lsr, encire, s- c..1, centre“ a: d dollar. n ‘wrctn, ' -14- .L... ‘ -' PL. ., ..' . .. “.... J- a . " ' . . . m. slignc caraicic; and :OllOJ seaihenc cereleas. LlCfiLS mill tirns ' .«Ta4— l‘r ' - I " J- . ' ,. J-‘ J. - . sliuncly inali.e and sea “laces. no neoconiaacion or leutecion J o " J. J- F - W . . ,.. .. ‘k ..x- .1- ' .1- OJ. ' . dececced. ACld proouced from Ulucose he not in lac ose. uerCh is .. .'_. l .. ~— T ... J— D - “n. -u .' a ' ,3 1 M .1- ‘ not ngurolpsed. indele is not lorn d. an; niun aCiu pnospnace lS - .1.’ 'v : _ .. ‘2' .' J - fl ,1 . ,1 .L. . r .2- used as one sole nitre on source e is reduced so nitriuc. 3* ,I_ l. r- n r'fq (a w “r:- H 1 '“roils av l1..3 V, L—‘v V OJ’er // kl; 'm u a v" A : -!-. . us . ... ‘ 3. I group n. dram p sicive COCol occurring s‘c' y err 31 pairs. - '1‘- V ' 1. u .9" . ‘ J- . ‘ ~ ‘ . a Mon-me..le. herpsoiccicall; Slullar . icrococcus rarians. 9' ° . J- .1 . .1, ~ 1 ' ° - an. n 1‘- z , . 'ellca pi n. proarceu. Leia in net lja.eiicd. solo“. a on afar .- T 1- C K. ““5 PjrCUlnv‘ paa4-%w_ VQ v 4 . . you. ’ \J.. U-‘ ., ‘ an s x o turbidity * yello: sediwent develops. Litnus rclk turns alta- line; the litmus is reduced. Io eca :ulati n or peptonization ‘fas detected. Acid produced fr n c wmco e and lactose. Starch is not hydrolyzed. lndole is not formed. Arronium acid phosphate is used as he sole Litro;en sor.rc Iitrate is reduced to nitrite. Grows at h.3 C and 20 C; leerly at 933 C. Group C. ”ram posit ive cocci eccllrrin* singly and in clusters. Yon-motile n " ‘ except 1 produces an e Similar to .ierecoccus cancidus t .nt. Gelatin not lieu efied. Col 3 on afar are cir- e, snoeth, convex and era; 0. In broth 51 -ht pellicle, i o J- r . '01. '1‘ V 1 " -* and SllUJb LClLCJ se< itmus .milk e \ .eid; no reduction of litmus o o; tne milk detected. field prodiced Starcn is no hydrolyzed. Indole H. U) pho Sphate is -ot us d as she sole nitr :,n scrrce. trct is not reduced to nitrite. '"o's a. 4.5 C, 20 C and '3 C° cost a 23 C. Group D. Gram positive cocci occurring in clusters. Ion-nctile. Appears to be similar to froup 3 re orted by 21* (1:; L) exe ept it produces a pink pigment. Gelatinn not liquefied. Colonies on agar are cir ular entire, smooth, convex and pink. In ‘roth roderat olruld qr anZl pink sediment develops. Litmus mill '3 inchanged. Lo acid produced in g-ucose or lactose. Starch is not 1ylrol‘r'mu. Indole is not formed. in: nium acid u gate is used as the sole source of nitrogen. Litr- to i e. Grows at h.§ C and 23 C; poorly at 35 C. Group 3. Gram positive cocci occurring 1; pairs and clusters. Yon-motile. Appears to be sinila to group 1 reported by Ely (1953) except no pigment was observe'. Gelatin liquefied. Colonies on agar are punctiform, entire, smoot 21, raised and transluc cent. In broth, moderate turbidity and granular sediment develops. Litmus mil}: t~rns alkaline. Io coagula tion, peptonizat ion or reductien'was Lletected. Acid is produced from "lrcose bu.t not from lactose. Starch is not hvdro "zed. Inlole is not formed. Ammonium acid ohosphate is used 3 the sole nit r0; ;en source Nitrate is re- dueed to nitrite. Crows at L. C and 23 C; n t at 35 C. Group F. Gram positive cocci occurring singly and in pairs. Hon-motile. Gelatin liq1€lled. Coloni- s on agar are punctifor en tire, smooth, con ex and opaque. In broth, moderate turei I‘~- O . T Q :oroer J.A. leS naeterial Cnen1sor' and Pn s1olocv uonn ‘v - Wiley and Sons, 14-. 1. IO'mell, 3.3., 1533; Flavor and anterial changes occurring dur- in; storage of svcet cream'wnioh has been flash pasteurized at various I‘e*“r‘r£3.“”‘c-3s, J. Dair Sc 1.; 21; 215-223. Prescott, 8.3., Dates, P.I. and Icedlc, 3.7., 1331; The affect of disc ontinvous refrigeration Abst.), J. East. 21; 25. Prouty, 8.3., 1933; Observations on the presence of faculte tive psvchrophilic bacteria in milk produced unéer tre farm tank ~ ":—-«—'-:‘ -.r‘O" system, J. h1lL 1d cod Tech.; 12, 220-159. Ravenal, I.P., Has tings, B.G. and Zammer, 3.7}, lClO; The bac- terial flora of milk held at 10? temperatures, J. Inf pis.; ....3 Elk/‘43. . w ,1 1 .1 -~ Roaahousc, C.L. and Henderson, J.L., l"1; Tne rarkct hilk In- ,v 1' dustry, IcGraw Hill Book Co. Inc., “.1. on psyc- 10:31.1 111 10 Rogick, F.A. and Burgwald, L.I., 10 50; A stud 01.; 33: ’05. VD bacteria in market milk, Aost.), J. Dair r; S O 1 Rogick, 1.A. and Burgwald, L.Z., 1952, Some factors 121cb con- tribute to the psychrophilic oactcrial cowl t in market milk, J. 71‘ :and Food Tech.; 13; 131-135. Salle, A.J., lCMZ; Fundamental Prir if 10 s of 3acteriolo;;, '1- ~' tr 3rd ed., -cCra' Iill Book Co. 1nc., n.1. . 1pc. A Schm1dt-Lielsen, S., 1902; Ueber eini~e psychrOphile “1:roor an- ismen and ihr workormcn, Ccntr, Baht. II Abt.; 25 laS-lL7. Sekhar, C.V.C. and talker, 3., 1d 7; Preliminary 03s erva' ti ons on various temperature charac tori s ics of some facu tative ps ycl1ro- J- U philic bacteria, Proc. Soc. Appl. 3a C't. ,21-27. ‘ ‘. ' ‘ 7 "‘ o ,n' 1.! _ a Snerm.n, J..., Cameron, G.M. and unite, J.J., 1,11; The bacteriol c U1 cal s:oi1agc of milk held near the freezing point, J. Dairy oci.; .21. 1.3.2.7 7E»... “( _ a _ ||i . .Kal .Vm‘fi-tl’ffi‘lirhnx TgJ Shemuan, J.I. and Start, P., 1931; Strep tococci .Ihic-1 grow at 11133 I, ; .:. 0'1 0 i‘ no terreratares, a. sacc.; 11; 173-co5. —_ 0‘ an .:. 1 9" .‘I ‘1 ‘ A “r‘.‘ — -vr onenen, J... ., Ssar1-, o.-.. 1.:1 {.L‘l‘lSS..-‘ s, I.-o., 1;”; L hrioio '1, fl F H“ u 1 o. of R111, Lop. cornell A r. 11s.. Sea. gall. 137. 07 a T " 1 ~'- .. '7 7 ,7 m1 1 .n . ‘ 4. ‘ 0 - - - '1 1, ,. _- .:-1 0 n n“ , «- DQCuGlla 11 .111, Jlofl spoolal reierence to stasis ds, u. airy - 04 {F 2 801.; so; log-17,. - -‘ c‘ ' . q o o o o o Sherman, V.o.4., 1949; A.nechanical key for tne generic 1-ent1f1- cation of bacteria, Zact. 2evs.; 13; 75-133. Sulzbacker,'Y.L., 19:3; Su vital of microorganisms in frozen meat, Food Tech.; E; 330—390.? Sulzbackor, 2.1. cud TcLean, 1.1., 1:51; The bacterial flora 01 fresh pork sausage, Food Tech.; 5; 7-3. — Tanaha, 22., liozalzi, IE. and foshida, . ., lg 3138 tuaies on the pork ‘ in storage. III. Bacte riolo ical studies on t:1e porL in storag 1‘, V ' ’ ‘ 111111. -:El'il. IHS‘G. A::Z‘.r U04... (10118» 05., 93.13311) SCI‘Q G;23h5-L1'.9. 3101. Abst. 1320 1953. ///’ Tanner, T.U., 1938; Bacceriology, 3rd ed., John Wiley and Sons Inc. ~1- «‘7' «LI.-. 1i,e1 C.C., 1913a; The inflience of various factor on the fer- rezitation end-products of the letero-Ierleucasive strep ococci, J. 93.3-1.3. --CS.; 11; 51—Jl. . ,1! ~ . . . el, 3.3., 1‘” Do; TAG influence of various factors on the for - meat tion end -pro'uucts of the hetero-ferzm cntative lactooacilli, J . pair; .583.. , ll; lBJ-U.Lr. Thomas, S. 33. and Seldiar, (3.173., 1:3;3; Ps sv'.;c irophilic bacteria in raw and commercially pasteurized milk. Proc. Soc. 3331. East. l 47.50. Thoma , 8.3. and Thomas, 3.3., 1947; Some observations on the bac- terial flora of farm'water supglics, Proc. Soc. Appl. Zact.; 65- b9. sq 7 ~\ 0 A, a fi‘o 0 Thomas, S.g., Tnomas, 2.7. and Ellison, D., 194;; .11k 1 ctcria § 0 - I ‘ 'which grow at rcfr1gcrator te poratrre, J. sa1ry Ind.; 1;; n? -mOT !/ ;"-l ‘1 “-J .0 94-00 ~ ~- {‘1 1" fl 0 .:t. Ell/Id .x . J1 S-»C:,r, U .‘.> . l T 4- “, J- an crao meat, (AJSuo) C T ' T? P", ., at. uOUlS, L0. 1910. I . . 1941; The baccerial Tobin ,L., Alford flo aaer A 20, ;orty-sccond , J era of ' ced, frc uCIl. :‘-GC451.L-S S oil‘LQD 117. ”a enaar, 4.0., 1f52; The bacter 1010"? of surfae taint butter: T U O ' ha Q . “F: I. _ . I A leviea, . eaird Sci.; 3,, 133-112. .D l - tne 1*“ "r,. n . » ”no .70 m, - ”a -naar, 1.C. anu Jezeo.1, J .J., lgyl; 118 in flaence 01 ed Wee‘(n"enfis nutriiae uer’ on “e QrOIJh and surV1i"al of - , ry Sci.; :5; 738-754. Whtrous, C. 2., Jr., 1931; Ha eceriologieal studies 01 some refrve- erafied dairy products, Ph. D. Thesis, The Pennsylvagia State College, S'ate College. ‘Yatr us, :.Z., Jr., Joan, T.u. andu To se17h son, D.T., lj52; gore bacteri ologieal studies n refrigerated milk and cream, Penn.“ doabe Coll..13r. 329%. Sta. 3ull. 551. .n . YT ' O O Q. C .nite, A.1., lfl'; A bactei1ad1scolora“1on of print butter H o . ’wn /‘ r- 001. Agric.; 20; oyJ-a49. 203011, 0.3. ahd Conn, J,E., 1T503 SLudi ' ‘q of marine bac er v . ram +0 at ...!