a thesis submitted to the graduate committee in partial master … · 2020. 9. 28. · a um or...
TRANSCRIPT
E
A STUDY OF BACILLUSALBOLACTIS\V
. byz
I; LEO SIEGELr\(
A Thesis Submitted to the Graduate Committee in Partialfulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
CinBACTERIOLOGY
l Approved:In
Charge of Major Work Head of Department
Dean of Agriculture Deio?~Applied Science
Virginia Polytechnic Institute
l9l+l
7 / 5 7 57/
Thasid am advios of U¥*„•jF!‘•f! S. Ormät this work ;>o¤¤1§!.o•
A
um or coznmms APART L A S'1‘L¥DY GF §sGILI1J’ä$ AI„HJ1„AüTIS IN 4%. LOGAL HIM t*$"».}2.'é·I.I
mmmmm or am momm 2
OFHomo}.fozmmtatim of milk 5
Abncwml t*•x·m•¤t¤t1¤¤• 4
Sowas ot cozxtaminatioxx 5
importance cf Strcüosggous ggg; 6
Impox·ts.¤¤• ot @1111:; albolaotu *2
KITHW OF PHOGEDURS _ 9
RWILTS 11
DI3G'US$3IOH O! RESUISS 23
MID €„‘0NCL1I5:1If§‘lö 26
PART IL A ÜTUDY GI" HE CH2d€AC“&ZE€I5T”It25 AND (ü1!I‘UiäAL
Wilißffifäää UF °L LAC‘I‘IS•
O? ZEW
AEID GEIEEML Aä\l*EC'l”§5 OF Üiß l‘ROBLBä
History 29
'i*yp•m of mriatiozs 51
Cultural variutxoxx 51
lwlatioat of ~3«.5,.l€•ul’a.il. Zw tgyyms ui v¤1·$.¤·L5.o:+.x 34
Factors iacitiug vmrimicm 35
AA
e
Taßm oz?Cct‘3THN*°i‘;=3·~c¤&«?ä‘I;b¥tfzé:Dcmpariscu
ct S and R fame BB [E=¤!"Ph°l°fLB' co 9
Phyeiologyculturalcmzmcteriatice 59
Emtic cf S to R, und R to S, cclomee 45
The effect cf the medium 43
The effect of heut isrezztmexzt 59
The effect of lcmqthczmd iucubatiorx 63
The •ff•ct cf grcwth in milk 62
e The effect cf pv. 64
Bläcvaälüßä mh cmäcwälüüss 6669
CI'Z‘é§D 70
1 I
ILlst of Tables 1
l.···6. Study of raw and pasteurlzed milk
frwxsourcesA and B.‘
1.. Souroa A. ms mllk mllk Ct1118• 1.2 I:2. Source A. im mllk from holdlng vats. 15 I3. Source A. Fasteurlzed mllk fromthe~
cooler. 144.. Source A. Pssteurlzsd mllk from bottles. l5·l6
1 5. Source E. äv milk. _ 176. source B. Pasteurlmed milk. I 1.84
9. Gcmmrlsou ot mllk la the raw and pesteurloed
states from source A. 19-808. Gmparlsou ot milk ln the row und pasteurlsed 1
states from source E. 23. 1 X
9. Bletrlbutlou of llquefsstlou ln relation tothepercexxtcf Q; albolactla of the total count. 82
Part 11.1. Gcmperleon of S und R ratlos ou tryptcme·k;lucose•
extract arm. 4S
B. Gomparlsom of :3 and R ratlos on uutrlent aggar. 49 I
3. Gomparleon of S und R ratlos 0:1 etarchautrlemtA* nger. 50
III1 I
List ci Tahlcs•«uo¤tiaued ,Page
+4. Effect of various media on the S and R ratlos. 5l·-52 15, Effect ot heat treatment or; the S nac! I: ratio;. öO·-611
6. Effect of growth im milk om the 8 ami R ratlos. 63
7. Rtfeot of gi ami oarbohyérates om 5 ami R growth. 65
List uf PlatoaÄ.’&;r§€
4 Plmse I• ;2.ZeJ<J’l'£?£ <:<„zm?m;2 522 ;~,2<?*§* ;„z‘;.2*:ä. -21
ume 711. mmm-; üülßäiiisäé 442 2:U*§?«:I2:t4·:2* 42
Flma 111. 144 HGU;2 G—2.„»1„2>2·iI;;:s 2* 222, QÄA;.{—”%.§{• 56
Flmza IV. 144 55
Iziata V. 144 54N 21446 VI. 57
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3„x;C.·w.."’ EEIEJZ 1Z!,!;iZ"LT£•
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2
I During a period of~obse1~vat1ou over several years, milk from
a creamery in the xricintty of Blacksburg, Virginia, uns frequently IAshown an abuomnl fermcntation in the tom of rapid acid11quefac··tion
of the card. Previous vertraege indioated äcillua albolactiu gas the cause (21,22).
The following investigation was ande of th.em$.l1z supply in anattempt tc determine the prevalence am action of §_,_ albolactia,as sell aa factors influeaciag its appearance and increase.
V r!
E
XX
X · XHISTORY Arm XGMERAL ASEHCTS O! THE PR¤H.H«£ X
Iilk, the normal eeoretion of the manual? ttlanda, ia one of Xmost complete premrationa elaborated in nature. among
1
its more important conetituoute are laotoae, glyooridee of
oleio,pelmitio,nyrietio, and hutyric aoida, oaaein, laotalbumln
andlaotoglohulin,und traces ot other orgenio end inormnicconpounda,auch
an oitratea und phosphatea (19). It is nmphoterio innatureend
nearly neutral, with a normal p E range of 6.5 to 7..*3. mus,
aa milk leaves the ¤o¤*e udder it is a very eatietectory medium X
tor the growth ot many haoterinl apeoieu, and in ooneequenoe
itwillundergo many changes. 'lhe prooeeaea produoiug thesechangesare
temed milkfermentutione, und theee may be ocneidered ae
normal or abnormal dependiag aainly on the regularity ot their {oeeureuoe (ll) . X
X Xgornel 1·•:·a•¤t¤•1E• og nilk. u X
The couuoneet change ooourriug in milk is thedecompoeitionot
lactose to laotio acid, with e oonaequent coegulation ot the
cesein. Bale eourinn ot milk ls regularly round under usual
conditions, und le called the normal termentation or milk(ll).Otherchanges auch as the production ot unusual flavore
undodora,roplnees, sweet uurdling, liquetnotion ot the euro, gas1
production, etc., conetitute the ebnormal termuntstioaa (11,3.5,25). E
X
4
ttOriginally believed te bu due to something im the casaiu,
~eourizu; of mil}; was proved to be cau.sed up saicroorgeuiwwe by
the work: ot iaateur in 1857, when ha de:aoaetm:·ted theexietezzceot
crgaaimes capable ot procluciag lactic acid, ofLister,who
ieolatml cue ct theee teams in 1?37¥£=,. (1}.).\
The 6:2:112: orgauiera involved im the oorzcexl fe1·:1e¤bct ion of
milk io ätreptococoue luctgm The type cpeciaa rcpidlz; ‘:;¢r*od¤¢@ß L
only lactic acid from lactose and, since this Emo :20 flavor cr
oder, gives sheet is called a '*clouu e<:·u1·” mit ia the dairy ia-
dustry. It jproduoee up to cue pcrceat aci<äit3„·, or were, iz:
milk, but its growth ic sczzcohati inizxibitod above this and
thelzxctobucillusgroup bogi:1 to up, ami to groduecoaciditlescf
over two amd ooe half pcrooam altäaoumh their ggroasth ie a
eccurresce, am desirahle 111 many cheeecs, it ie cc·m··
uidered uudeeimble in UUÜSIT mshufactux·e• The vaio.crgaxzismsef
this group are Lectooacillue g3._e__g_;_, ,g„_,_ ‘€}L2£,‘i£°l1‘1CU$• smc _;_,_,_
~eegg¤ghi__Zg_q_§_„ They proctuce pz·1acipc12.3,> lectie acid Lac- 4
toue• (11,19}. N
Ahaorcml £?em»utatioae•
There are many types of aämormal 1‘e:*me11tatio:1s, aaa these
may bo caused. bg: a eiaugie species, or groups
ofEechnrichga_qg_g,_i_ cad aeretacter a¤ro:·re:1c__a_ are tec of
thectomszaoaercauses of gesay fermehtat icme sad oft'··£‘lavo1·s amd odora.l
t _ ja J
5
They are almost always found 111 raw mllk, and come from duet and
, !‘ecee• Gertein members of the genue Olcstridtom, the aaeerobiceporuformere, end sone non•eporeform1ng yeaete, (Torulae) , eleo
produce this ebncrul type of temaentaticm (l1,l£)„
— iThe genue Peeudcucnee, members of which producee a water-
° eoluble blue piment, ie reepoaeible for blue milk, while membera
cf the geaue grratle produee ••bloody ¤1lk*'„ (11,1.2), Ropy er o
ellw milk may be caueß by viecoeug, or highly cap-i
culated etraine of gbccteraegi
nee, or gt lagig; _y_a__:g_. _h_g_l_·among others. (1.1,19). Sweet eurdling, 1.e. coegula·
tion of nilk by the productiea cf the ename rennet by baeterla,
y rather than by acid production, ie encouatered minly ln wemo vveather und when milk mzet be held for ooaeldereble perlode after
iapeeteurizetioa, (ll). The oeueetive ageate are gene:-ally aembere
of the aerobic, eporedoraiag youp, •,g. __!;,,_ g,_§_,_ eubtilie, _ etc, (15); although eembere of the
y geaue gggteue (ll), and varioue yeaete and melde my cause tblei
trouble. {23),
@g__eg_„a cf cont-eM.net;on•i
Milk, ee ÖPGUSI freu the udder, aormlly contain: Materie.
cconted by the plate method the average in about 500crgaaiemeper
cc., though ccuate have been reported ef over 1.00,000 per ¤c• T
ötephylooct end mtcroeocci are the cmoaeet orgeniene found. (11,25}.
6
(From then on everything the milk comes into contact with ll eerves ss a further source of contamiuatiouz air, theanimalcost,the milker, and probably the most important source of
bacteria in milk, all the dair utensils such as milkingmachines,coolers,
milk sans, etc. (ll).
_ Impgrtance of Streptococcus lustig; —Btreptococcus lactis, previously mentioned as one of the
commonest milk orgenisme, is undoubtedly one of the most im-
portant for two reasons, lt produces only lactic acid in milk,
und so gives a *cl•un sour* milk that my be used in all types
of deiry manufscture; end it is a very rapid growing organism,
and the acid it producss, though at first ineufficient to teste,
will inhibit the growth of other orgsnisme. (11,25).
It is this latter factor ehich ie of the utmost importance
in keeping down the growth of abnormsl fermenters, particularly
_when their relative numbers are low. Organisms ceusing elimy
·"¤1lk, for example, or those auch ae Alcaliggnec fecalis, and
certain.¥ierococe1 ceusing bitter milk, are relatively acid-
intelerant und will be held down by the acid production of,§L
lggg;g• (20). Hembers of the coli-aerogenes group, if not pres-
ent in too great a proportion, will also be inhihited by lactic
acid production. (ll).‘ ~
However, in certain abnormsl fermentations, the rapid growth
l and acid production of §g_lggtig will not be inhibitory (5); or
a__ e lggo___
, 1certain unecmnd deu'? prccteloes auch an ovezurosteurinntion myso lower their numbers, or increase the relative numbers of
other hnrdier crganiwae that the action ot all). bepraetioally worthlssau (l2,Z+3£>)•
The Iegtaaoe or gillus elbolnctgmThe type ot ebnormel rermenteticn with which thin investi-
gation has been ooncerned ia described es acid liquofoction ofthe eure. In this, there ia a rapid production or acid from
leotose resultihm in ooe;-äulatioh of the cnscin, 1.6, card i’ox·mn···tion. this ie immediately folloned by preteolysla ox- 11qce£‘hc··tion, in whlch oasein, or its insoleble derivative, inbrokendown
to water eoluble compoundm This ls gäonerelly eooompahied 1
by bitter flovor developing; in the milk, an woll os hadodors,due
to com breeadmm prodnctc of the cnceih, ami possibly from 1leotose nc well. (l1,äß)• ätgggtocoooue lignefhcieno, Bociliuslnteroew Qogllue gyoolaotte, end certain oembcro ofthehennaGloetridiom ere the ormnlms oc„::osly responsible for this 1
elmormhl fementetioo, (11,16), ee cell es certain amd
in essooiatlve (11,17,10). lrrevious horiz (äl,;:i::)has indlocted Bnogllns albogtgs es the cause of this particular
ebaortml condition in the milk supply being; stndied here, LIn 1904, Flugrze, Ford, and rryor pointed out that milk heatod
to tenperetnres above GOG C., ehoued exoessive develoment ofscore-benrina;
beoterin, ordinarily inhibitod by the ls¤tio•eoid·pr0duoin.;: I
baoteria present in milk. (SSL I11
1
_ -_ 8
4 Loetfler ln lßö? tlret ncted an organlm ln bolled milk whlch
asldlfled, coagulated, and peptonlned it, sad he proposed theneue lllas far lt, This eas changed by Laurence
and Ford ln 1916 tc Äclllus albolactus Iälgulau (15), although thefollowing year dann clulmed that thle oraenlm was merely e lac··
toeuteruentlng variety ot glllus 9_Q__1"_&Q_• (4), In the first
tour edltlone of "ßezmeyw Hanna}. cf Detemlnatlve mcterlolog"s this orynlm me listed as a eeparate speeles, Ä elbolactis
Hlgula, (1), In the 1'ltth sectlon, however, thla hat been changed1
to lllu: £_e__;_·•_____u_e_ _y__a;_,_ albclactls Hlgula. (2), Borlano also lists
the organlua es a varlety ot _Ä__g__•1·___gg__1_g_, (24),
A8 synonym: tor Ä albolactls Laurence end Ford llst mclllus_l_ggg_a__l_b_:;_g, Loeftlera (as dose Bergey (.1)), Ä _gg_r_gg Helde, Ä
¤lgnln, Ä _1__g_g;_t_l_g_§g,__;_;_ Flagge, und Ä bernennleLehman and Heumann,(15),they
were the first to lndlcate the lmportance ot Baclllus
ggglactlg in the llquetectlon or pasteurlzed milk, They also
nentlcn lt es a contrlbutary factor to hltter mllk occaslonelly6 obtalned when paeteurlzatlon ls narrled on between 60° and 65° G, (15),
The organlem hu also been wldely reported ln soll (4,24|, which
¤·g•y•e manual liste ae lte habltet, (2),
1
1
‘
NIGTHOD ÜF PROMDURE :
Fresh samples ot both raw and pesteurleed mllk were collectedfrom the dalry over a period of two end a half months, Milk easbrought to the ereeaery true two different souroee, and theee are
treeted dltterently, end ee were kept separate in the aaalyeee
mde, iülk from source (A) le paeteurleed nad bottled, while thatfwß/eouroe (B) ie paeteurlaed and put into milk eau: tor use in ·e large eating eetahllehment, ·
ln both eaees the mllk le brozmt in from the ferne in large
milk oane, poured into a eelmlng vet and then eenveyed to a hold-ing vet, lt le peeaed throzmh e preheater into the
paeteurlzerg it ie heetedie 1440 E (62,20 G) und held for0
thlrty to thirty five nlnutee, then held tor another ten min-atee et 1550 (57,20 G) before being passed through the cooler,Bilk (A) ie then pat late a bottllng machine, end bottled in0quert, pint, and plnt guentltlee, while milk (B) lepaeeeddirectly
from the oooleriato milk cane,Jus fer ae poeelble eemplee from either eource were taken both
before und after peeteuriaetien, and cn the average er once e weekthe samples were taten from various points along the line, Samp-ling wee done in eeoordenoe with the mthode outlined in "ätendard
ltethode tor the-Eoenlnetloh ot Dalry Prcducte", (26), amplee were0
held at l0° G in no eaee tor lßßßf than two hours, and all due-
tion: were made at leeet ln duplleate,
4 10
Nils aus plataä ln 2 routine wanna: using standard trypsan¢•‘g1u¢oss·axtrant ugs: {id) at s p E sf ö„d ta 7,ö, there dilutiusgreater than 1:10 wenn made sktm silk, und not bhs pnder, uns
¤eod•Total neunte er the orgnnlss sera made, und after a study ot
the nolenlnl aherastarlaties ot ggoillue albcggeggs had haas made,the numbers er this nrgeniam were eettmated from the eolonxes an
Tthe ylatee, pereestaga et the total tiere ¤a1eulated• ‘One hundred
eusxe eentimeter portions of nach sample were ineubated st raumtemperature (Elo to 24° G,) and at 3?° G. amd exemind in t¤enty~ter und totyeexght hours, und later it neeesnry, to determinethe type cf fermsatatlom the milk uoud u¤d¤:g¤•
u t Repneaentative culamles were ieolsted trum time to time, und
the action an litmue milk nated, to serve am a eheeh on the p•r·
eentage counts, und also to determine the types 0: ergsnims
pre•nt•
‘ ’ ll T V 9 A
RESUDT8 g
Plates with less than eeventy eolonies were not included inthese results because it was felt that in such cases the approxi·mate count of_§L elbolactie would be too inacourate, Ga the otherhand plate: with over three hundred und fifty colonies were not
used because of possible ineccurseies of the count.Tables l through 6 are the results of samples taken from
both sources, A and B, both raw and pssteurized, and from variouspoints in the line. Gounts were made at least in duplicato, and
the average recorded. Deshes in the observations on the tye of
fermentation indioste no change from the preceding obscrvation.· Slight liquefaoticm mens that less than a tenth of the one hun»
' o dred ee. sample was whey,‘ In Table V a companies is made between raw and paeteurizedenples of milk frc source A, types of fermentatione and the per~
cent increeses of Besillus slbolactis notes, es well es the de-oresse in total numbers of orgaaieme. Table 8 does the same
thing with milk from source B.
Table 9 shows the relation of type of fermentation to thepereatsge of Beoillus slbolactie in the sample. Gases showingelight liquefsction were omitted, while those samples that gave
no curd ( and no liquefeotion ) were included under ”no lique~
faction". The pereentages of §&_slbolactis were rounded out tothe ueareet whole number,
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23ITable 9—-Distribution ot }.1*4;1.l6t:£1ß‘Gl0ü in 1616111611 totheparcemt
of QL albolectis ot the 11:16141 count.Q
3'7° G. Iäoum '1‘6m ·e1·a1u1~eQ
______w_____,__‘__L_ 24 14.0uxe ___Q 43 hhurs :24 mum 443 uouraä
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I 50 1: x 1: 1: Ql = Fastourized milk QI 3 Raw milk
I Q
25lDISCESSIGN OF PESULT3
IThe samples cf milk fall into four rain classes, those that
l
coagulated (norsally or with gas formation), those that rapidly
liqueficd, those that shared a slight amount of Whey, and those
in which there aaa no visible change• I
4 There were very fee cases of really normal cards, most cases
of ooagulation being attenésd by gas production. This was part1cu·
larly true cf the raw milk samples. The fact that this eas more
common in raw than in pastearized milk, end from several trial
runs on violet red bile nger, eosin methylene blue agar, and Endo*s
agar, that gave a rather high co1l·serogenes counts (over 1,500 per
co, in some cases), this group is probably the cause of this gas
,production•
When less than one·tenth of the sample was whey it uns report-
ed as slight liquefaction, There are several possible explanations
of this pheneenon. It might be due to partial inhibition ofBacillus slbolactls so that maximum growth, und maximum activity,
was not obtslndg or orgsnisms that coagulate caseln by production
of the enzyme rennin give a tight card that squeezed out whsy in
some cases (•,g. g&_gggggg was found in several cases); or frequent-
ly gas-produeing crganisms may grow vigorously enough to break the
card and result in the expression of some whey.Samples of milk seeing no change, two even after forty·eight
hours of incubation at 57° C•, were fond, although only rare1y„
24
The peroemtegee of B, elbolsotls ln these cases ranged trom 5.31%,V
ln Ihloh oase there ess so change in tesnty-four hours at eitheru u
room temperature, or 3'I° c, thoumx both showed e normal surd
infortywlghthours, to 55,3%, which ess liegzefisd in twentpfouru
hours st :5'?° ¤, hat shoreed so enorme after fortyweimht hours at
rosa temperature. In three eesee of this type s predomimnoe er
Stggßgooous uns found, but outslde of
these samples no unususl types or numbers ot mioroormnisms could
be found.4
The comparison cf rem end pasteurized milk shows that the
numbers of orgmnlems killed vsrled from 663% to 99•ö$’¤• Althoußx
there was no sorrelation between this end the psroent increase of h
B• elbolastls, there ess s elmlftoent increase in the rslstlve
k numbers of this ormenim after psstsurlzsttom The ormnlse in theu
vegetstive stehe ls more resistent than many mxcroorhsnlsms
(ll}, and in the epere form ls able to wlthstsod partioulsrly high
temperstures. Qms, although their actual number desresees, since
Btggooogs }_._a!_t_;_s_, the nein orgenlsm in milk ls not s spare form-h Ä ler,
the relative numbers ot gz Qbclsotls inoresses In the cases
listed there were inoressee from laß to 1,820%. This im-
ereese ts important when the milk is to be used in any sort of dairy
manufeoture, for, although a fee samples of rse milk shoeed a
sllmhtllquefaotlon,for the meet part they gave a normal curd, while
*pss··teurinedssmplss showed s signlficantly high nusber of ossee of liq• {
p u•!'ectlon• fnk
I 25V
VAlthough there were eeverel samples that ehovred no lique!'ac•
tion in twe¤ty···foox• hours at 3?° Q, when the percentage of §_,_
elboleetie reuged 11% to Bü, all the eamples that did liqaefy
in this period had 11% or more}, alholactig present, Below thisVpereentege undoubtedlyVßtmVgwdg; lactle, or §_,_ __lg__c__gg_g_ variety
terdus, büth er which have been iscloted from these samples, pro·
dose enough eeid to be scmewhet becteriostetio, It ls also later-I eating to note that liqaetaotion occurred more rarely when samples
ure incubated at room ttur•, and then only when the peroent-·
age ot gt albolectie ms et leeet 16%,
II.. I [
II
26
md} 00NCLU:3Iül8
1. The type or rermentatlon at room temperature and 3*/° G,
total becterial count using the nger plate method, and relatlve
numbers or gclllae albolactgs were determined in a number 01*
samples obtclned from a local milk supply,
2. In almost all samples or both raw end pasteurlsed milk,
from the time lt entered the cneamsry until it lert, gg albolsctis
was sound to be present, Its percentage or the total count gener--·
ally rauged from 2% to 21%.
:5. The rev milk rormed • gassy card in most cases, a normal
_ card in several cases, a slight amount or shey in a ren, no change
et all in a tea, und llqueraetlon er the ourd in two cases,
4, The pasteurisod milk gave rementetlone similar to those
er the row milk, although about one third or the samples zxqosuea.
5. A 16% to a 1,880% increase or the percentnge ct _!;,__a_;_„_lg_•_;_·
lactls or the tülitl count occurred in pasteurlsatloc.6. Llqueraction or the mllk can correleted with the numbers
1
or gt glbolectgs in relation to the total count, proteolyals ¢>¤¤¤I'1‘*·•
lng when its peroentage ot all the lorganiaue present was over 11%.
xx S
xa. Smm or im mmmmmcsMm mum. vmmmous or
28
8'!A%4‘f‘ OF THE PROBLm
In the tu-et part et this theais s study see made thnt
entalled xnmerene ebeervstiens et ooleniee et Beeillus _g_l_)___e-
ggg. It was notes that when this ergenim eas leoleted trem
· Milz, endelse when plsted ent 1n pure oulture, two mein types
4 et eeleniee teemedt
The purpose ot this investigation ms te make e detailed
etndy et these two verietiee et _p_,_ elbelaetgs te determine any
dittereneee between then treu the etendpeint ot norphelew and
~ bioehenieel estivitlee, and to sttenpt to find n possible caueel
taeter, er teetere, tor this Variation.
GFHä7
The queetloa ot the eouetaaey or yerlahillty ot hacteria
huplayedan important part in the etudy ot these a1¤ro¤a•gaa1ms•
At dlftereat tlmee ia different countries, am or the other“ doatrlae has held been ot la oheervatloae
mda, interpretation draw, procedure: used., (30},
Early Iaotloae ot varlabllity, am traamatebllity, ot bee-·
teria were eryatelllaed 111 their extreme poealbllltlea by Nagel!
in 187*7, and egia ia 1882, la hie theory ot pleaaorphim, He
maintained that ae far ae baeterla were coaeeraed, there eas e
single cell type ot extreme veriahlllty that aus able to change
tte morpholom, bioahealeal reactioae, termeatativa abllitiea,
and eo torth. ‘¤xae he alelaed that a alaeelticatloa et bacterlal5
Y gehera am! epeclee oa the heels or aerphelogy md hioahmical7
chereeterietloe eas autmmded. (9), Hueppe, Kruee, Lamcestar,
Bllbroth, am others, ameed with Heegelvu work oa variahility,
although they dldzüt carry the idea or pleomorphlaa to the leugthe
that he had gem.(23},Aboutthis time Cohn (18*72,1876) presented a classification 2
ot batterie, based aa etehlllty ot the aorphology amdphyaiolowof
hacterlal speelem (25). Eoeh ia 1878, ölld may tiues after5
55 55 55 t 5
50
that, insisted on the dogma of nonomorphisn-—a constuncy er form( and unifornity of action. His followers in Germany, haglund, and
this country added rurthcr support to his theory. (hu). This con-(
(copt led to a sauer view of bacterial constancy, und rapid pro-. grass cached in the entire field, particularly in classification. ”
Migula, in lö97,·brcugnt out a rather ooprehensive classification,ed this work ess follosed by-0rla•Jensen (1909), Buohanan (1917,1918), Gastsllani and Ghalmars (1919), end Hergey (1920,1923), andothers. The tendency has been towards a more conplicated o1assi•ficatlon, with the diffcrencea between speciee being split more andaore finely, (9,10Q, ° ’_ ( “
All this tim, hoscver, evidence contrary to the theory cfmonaorphism had been ecennulating. Lehnanu und Neumann offered aclassification cf bacteria in their Wdtlaa and Grundriss der ak- (t•rio1og1e* (1896), but er the tina they pcinted out that many so-
V called specica were suite often only varieties of one apecies,Heisser (1906), Hnssini (1907), Kowalenko (1910), Baerthlain andEisenberg (1912), and others, werked cn cultural variatione of g,
99,},;,, (9). nexmxr (1921), Webster (1925), ana Hutt (isst), r•-ported on cultural and norphologic variations of the Pasteurellagroup, (8); Griffith (1923), and Reimann (1925) on pneumococci;Eagles (1928), Todd (1928) and others on the hemolytic streptoeoccig
T and many workere on other speciee, particularly ou the cc1cn·typhoid-
dyeentery group. (27).”Top1•y
and Wilson (27), Hadley (10), Lewis(16), and others mainteia that cultural varieticne may probably he
A ,31 y y Ä
‘ _ see in all baoterial atreins lf they are suultted to a prolonged [
y lllßlhßßiüh under suitshls conditions.A übul, today, in epite of various clasalfioatlous in general
use aaaed es the idea ot constsasy of bacterlal apecles, the ques•
tion, are the bscteris elaselfiahle, is still a dehetable one.
Hhdley (10), and ßinseer and Bayneeäenes (S0) clals that theyare!
A hot, nur will be until bactelologiste know what conprlses a boc-d
tortol speclee, The trad of the species concept cf bacteria ls
away free e reetricted uns ef chsreoteristice towards a concept
cf etatletieal distribution about a mean. And in the msantloe, it
ls evident that the varlsbillty of bacteria le still unestlseted in
tte wxtent and elgnlflcanee. (30).
l Qzggs of varlstlg. hSelle (23), in e list of bsctarlsl varlstlons taken from · A
Thomsen (1955), offers sixteen hsctariel characters and the veri-
etiose of these that have been obsarved. These characters inelde
callular oerpholoa;y—·s1se, fern, , stalnlng properties, spare fernse-
tiou, flagellatlon, and cnpsulatlon: growth on solid und liquid
mania; vlrulence, antlgen. und toxln prnduatlog prcteolytic and
ternentatlve power, etc., with varlatleos in all of thom•
¤ul verlassen.Gulturel vertatiens may take several forest The progeny of
a single cell, or e few calls, taken from one source may give
Iidely different oolony forms. Ä One of tho most common of then · Eta the change from smooth rough oolonies. (30). The smooth or ;8-type, colony io usually smooth., glistaning, meist, soft, homo-geneous, round, und entire. Tito R•type, or rough oolony is gamer- 1ally eaewhat Larger, urinkled, irregular, granulsr, brokenorfimtriateat the edge, flattened, tronelucent, Two or moreofp
‘ these dlfferenaea may show ap, indloatiug a transition from one t
form to other, though the most etrlklag of these are eine,face, und dessity. (9).
· The amount change ia from mooth to rough, and this may bea grsdual change our a period aa long ae several months, or a ä
„ sharp change lith so internediatem Intermediate forms are de-eig-· unotes aa *1** hy xoser and styron (13,14), aa *0* by malay(26),(or, varying frw true uooth, 8, to true rough, R, they are Sr,
I
7, SB, und Q according to ikdley (9) end Dienst. (7). The change
from the smooth eoloay to the rough from through ihteraedxates. been report•d.h1—Xo•er and ßtyron (13,14), Dawson (6), Dienst(7), Edwards (8), end many others. Gases of direct paseage from
lS to R with so intemedlctes have also been reportad by Iowls (lo),am van neakel (ss), rar example. Hudley (9), however,nahtlosethe
fact that tntermadlate forms are all highly unstable, and aug-gaats the pooolbillty, because of this fact, and the greatnumberof
ormnlmas a how to have intermediatea, that all 3 to R ohauges Fpass through translttaaalforms.Ä
V ‘ . _*
‘ 53 l
Until the beginning of this century it was believed that
thechangefrom S to R was permanent. Pirtsch, Dyar, corbett, Phillips,
Zupnik, and others, in the lust century, and White, Schutze,
Cronell, und others in this century, working on many different
species maintained that the oonvcrsion from the rough form.back to
the smooth uns Impossible. However, the verk of Bordet and Slees-
eynk In 1910, Baerthlein ln 1918, Wreechoner In 1981, Lewis in 1934,V
Koser and Styron in 1930, Hndley In 1932, and·many others, ldlcated
that with eufficiently long peride of observation rough forms could
be found going to smooth (in many, just es easily as the reverse)
(10,23,28). hsdloy Indicates that this reverslon does not seem to
pass through Intermediate forms, and raises the question es to
whether this ie really a reverßion or further *prcgreeeion*. (10).
huooid oclonies, generally formen from vedgee, or eeetors,
_( (
cf smooth eolonies, are a third form that seem tc be more comon
then previouely thought. (10). 0erta1n_worhers (8) have attempted
to show that there uns a direct ocrrelaticn between M und R solch- ·les, though most evidence seems to point to this ae a distinct
colcniel type. Transition from R tc M is particularly rare, the
change generally being from month to mucoid, cr muooid to sooth•
The fourth main eclonial type is the gonidial, er G type.
This type ie claimed tc have been separaten from parent cultures by
filtration, and are at first not visible in the subcnltures. In t
continued cultivatlon tiny *d•«·drop* coloniea form, with the celle
34‘
es mlnute oocaue forms, uaranlly wenn-nogu’tive regardleae of the9
original source, These oolonies revcrt, on further ehculture,
hack to the original S form (23,1G;, Hot very auch ie han of
these, however, ann reports on then are rare, (30},9
There are several other types er oelonial variatione possi-
ble, The production of aaughter ooloniee very different fra the t
original type in an old or aying culture, er within or o the Vmether eolony le a rather common phnomenen, This has been re·
ported on es fer tech ae 1695 by Gunther• And, ot eouree, the
V appearanoe, eonettnee aelayea, ot eeconary eoloniea tollowlog
partial er oonplete lyele of a mother aoleny le not at all unueual,(9).
gglatiog ot Gglggggg to otheg tgggg gt verietlgg,
the question ot eorreletion of cultural verietione with varie·
v tione in other oherecterlstlcs ot nloroergenlse has long ben
apointof conteatlon among beoterlologlste, end is still unsnsered,
Healey (10) in the sone discussion er mlerohec dtsociatien olalms
· that ach culture phase, when existing in a pure state, 1a associ-
ated with a def1nlt•~grcup ot characters, and that when one phase
beeee traneforned oonpletely to another, some ot these oharaotere3 are lolt end othere seinen; and again, that each attribute of a
epeolee may be traneeltted to aub¤•qhent generetlone, inaepenaently,
theraoters host trequently oorrelatlng with oolonlal type in- V1
olue sell nrphology, aotllity, peeseseion of certain
carhohy-dretee,antlgnio etrueture, end v1ru1eno•• Such things es chrm-
ogeaeele, heolyels, ternentetlena, aa other physiologlcel rec-1
P
35 e E
seem to be unrelated to oolony phase, (23}.I
Virulenoe has been oorrelaten with eolony phaee at least aeIfar
beck as 1911 when Freies omonetreted that what ie now called
the rough form of Qgggugg @@3 was virulent, und thesmoothphase
ie evirulenh Beiémxif in 1921 oorrelated virulenoe of Q,
with smoothners, and also 4 diftused growth in
broth,whilethe avirulent form was room, and granular in brett:.Smooth·neu
has .o.£;'ten been correlated with virulanosg Griffith in 1923,
and Moss in 1.925, and others, with the pneumooocci, and Topley
and Ayrton in 192t, with for example. ßuthough1
here again, Todd in 1928 ahowed that hemolytie etrepto-oecci were
virulent in me rough phase, and not the smooth, (23), gEdwards (8) nn the other hand, etudied rough and smooth I
variante of §h§,5g_]3 gg,i,gg]„g|_ and found no aerological or bi1··1
ehemioal ditferencee, and equal virulense among the rough mueoid, _
smooth mueoid-, ana non-mueoid phuea,
hithtggj,uSonne, in 1930 Einser ann Styron reported no difference,
other than eolony tom, with the 8, I, ann Et etrsinm (11,).Thun, although many eorrelatione een be made of other beo-¤
ter.141 eharasterietios with cultural Variation, it seeas that each I
charsoterietic of a spooies ie subject to independent ltran•¤1•si··
(23), 'mmman hmm- I
an "ineiting'° suhstanee, or condition, is one that eets
upsomephyeiologmsl mechaniem by whose activity ultimate changes I
I
I
. a Ü - ·
Üresult, (9). Tnpley and nilsun (27}, Edwards (3), Hadley (9),
end others aintain that when the ”normal“ form is the southÜ
phase, the change to the rough phase is induced by unfavorablaÜe¤dition•„ Besson puts this in a eomewnat different lightwhenhe
oleins that the chemical constitution of baoteria can he
changed hy changing the media end, with this, auch things aa an- Ü
nyne production or agglntlnation may be changed. (16}, Lewis (6)
y in his work on the coliturn group, states that in sons cases at
least, variation san ecour spontaneuuely and without regerd to
•avironmant•
However, many things will bring about variation in becteriat
Growth et n temperature above the option; special food auch es
isodulcite, UPQQ, lithinm ohloride, various engere, and even a
lach of road, 1.e. ue or a minimum amount or nutrients; aatiseptice;Ü
auch es phenol, potasnium diohromate, dyes, e„g„ methyl violet,Ü
malaohite green, gentian violet, ete,; animal aussage, month
in normal sorua, aacitic fluid, blood, etc.; and use of the pro·
ducts of growth, are some of the more important means of pro-
duoing veriatiune among haoteriet (9). Selle, however, reines Üthe question an to whether these changes 1n environment merely
‘ lead to tempurary changes in the organiam, er whether they de
lead to permanent inheritable differensee, (23), Ü
Various explanations of the phenoenon ot vmriation have been
propoad, und they include auch things as tertniteua Variation,
Ü
nÜ Ü
37
he, Duaiaian; 'vnwiazias. an reapanaa an nnvirn:;:a:·.·n·tal nozzdie
ttaaa, er Lanarnriaaisa; natetian, in the nenne af Eatriza;
_ aetaagaaaaia vnrlazina; er aynlagnny, pragranaiva antngenatie
übungen in aaa iara ai a lila cyela, {lé},
Great cnafualan han reaaltud in beta ayataatia and applied
baaterinlny frau trying tc aampare calturen in different phaaaa,
wur Lean af a *nanan1“ bncterial type, ann variazieaa {son this
seen an ba uatennrle alann the tern er aba eryanzan may very nat
only from ana median.te aba next but even n uns anna aediwa. (9,10),
Altnengh.aa¤h‘aorr has bnen anna, there in vtill a great dealto
he dene Untere enaugh ie rauen te really eaplain variaticn, ap·
pawßiatm ltr exten? und determine enmotly what
ccmpriaee a bactarial eeniaa•
Ku
38
EXFERIEKETAL Y
‘ Congggleen of 8 und 3 forms
South and rough forms of Becillus albolactl; were ieolated
·. from stock oulturee of the Virginia Polyteohnlc Institute and
fra platee of the milk from s local creanery, These were traue·
ferrcd to tube: of litmue milk, plated from this on standard
„ tryrtene-g1uoooe~•xtraot agar (26), and ieolatlos from these
plates were replsted und relsolstd four more times, Isolstions
from the fifth plate were grown on ordinsry nutrient agar slants
and all the phyeiologieal tests mentioned in Berg•y•s Manual (1,2), _
und several others were run, Cellular morpology, including size,
fer, spores, capsulation, and mtlllty uns also atud1•d• YA great number of workers in the fied of mlcroblo dissoci— Yation, including Dedrutf (1921), Hellon (1919), Webster (1925),
Amos: (1925), und Jordan (1928), used single cell technique and
indicated that their results were the same as those obtained using
colouy isolatioe, (9), In vie of this, and because colony leo- ‘l
letions ts very auch simpler, it nes used in this work, In all
this werk coleslee were used only after a minimum of five isola-
tlons to insure purlty of the straln, ~
Helther physiologiosl er·¤orpholog1ca1 differenses could be I
found between these tee forms, The following description, there- Yfore, eerves for both the smooth und rough strains,
\
3
3Msmnalaa .
Size. 0.5 to 0.75 to 2.A microns.
4 Sporen. Central to subterminal. Ouoid.7 Ends rounded; occur singly, in pairs, chart chains, and occasion—
ally long chains.3 Gapculatod. °
Actively*mctile at 30° C. and non—motile at 370 C. in tsentyefaurhours.
&xa‘L@Starck not hydrolyned.
Litmus milk acid, reduced, and peptoniaed.H: S not produced. _’ 7
;Indol not formcd.
U
Hitrited not produced free; nitratea. 4Gelatin. Crateriform liquefaction.Re gas produced fra any carbohydratee. y
Acid fcm dcxtrin, dextrcae, galactose,4glycerol, lactose,levulase, maltose, aucroae,itrehalcse.
Ho acid from adonital, aeaculin, arabinose, dulcitol, ince-Vital, inulin, mannitol, mannoae, melezitoae, raffinose,
‘ rhamose, aorbitol, xyloae.
Optimum teperature 30°C.y
The cultural characterietics of these two forma are different,
3
¢·° $
uf eouree, und it in cm this haelt, täzet they am :sepnx*e;ted•InmztrientbI‘0·t•h bett Tome gave eimiler growth, e tufoldity,a. at the tmp, and a eltght emomxt ef ee=:2§.m•m·:;t.
The ezeneth type in a mxtrient. nge.? eteh ,g§.v»•:·a £'i.}..i.i'c>ze2. gr·¤vrth•0:1 mxtrient enger- the eoleay ie eixeuter, white, entire, raieed tceemmec, ezseoth, ehiny, with the center genemlly atmen, und
sTeecaeienally mxeleetem (? 1, Plate I}, käicrueeepically the
appeezmeee ie free eoereely grenuler in the center ta xtiszelyQwenglwut the met ca: the oe3„er„;··. (Figmxe 2, „?late I).
The edge ie eemetimee eleer, genere„1.ly entire, abhheugh occasion-elly netched, On continued incmtbetien the intemel appearezzee
became e¤x·led• On e matrient engem elant the c¤.al.tere ia white,rslud, ekxiny, month, lohwzte, with. a very etight eäepreeeiuu demthe Güßtbäfee
. y The mugh type in e nutrient nger stab in a vlllaua to
e rhieold fashion. Ga nutrient the eeleny ie elightly spread-ing, flat, grey-··wh1te, eeebeid te rhlaeid, rough we ahingm (Figure1, Plate II), älicroeeepically, pertleularly when. the aol-
, any is finely tilementeum (Eigen 2, Flete Ill}, Spreadlng ia
in the tom ct milzoid-1ike g1··eeth atraight aut, er in ecloekvrisedix·eetim·x„Of ‘ several theuxmedn eelanles ehaereed ea}.; tm wereeur seen getting in e eoueterelßekeise äireetiem 2'äeplatmg ofthese failed te give! this type ot gwowt:.1 again, The grwth an a
mxtrient nger elaat de reagh, rhieoid, undT
ehi¤y• e ,
41 'I
@Side View Surface View
Fig. l. Natural Size, 96 hours old
Fig. 2. 100 x, 10 hours
Plate I - Smooth Colohies ou Nutrieut AgarII
III
I4F)VI
Fig. 1. Natural Size, 96 hours old
V
/ n' 14 A
„ •"·’$‘x'I•~7~'°*~'¢\;~,‘ "
Ü3([avV;Q\‘~\f" #.3I‘\Fig.
2. 100x, 10 hours
Plate II · Rough Colonies 011 Nutrient Agar V
I
T U1 1 1+3 ,
Z_§_ggtg,_g_,_g_§___;ef% go lg, gd R 1
SPlatonmade from either type of oolony almost always ahowed 1several eolonieo of the other form, Gontinued repleting and re-iaolation of typioal ooloniee failed to yield a ntraih that wouldresult in ooloniee of only one type oa replatiag; in one case arough ntrnln een replated seventeeo times, but smooth oolonienstill ehoved ap,
1To determine whether there any oonstehoy in the ratio of
mouth to roußx, or rough to month, atraias, several plate: weremade oa txqptohe-·g1ueos•-extreet nger, autrient nger, and nutrientnger with etnrelu The results obtataed in twatptour hournarelisted
in Tables l, 2, 3, Gombinihg these results with thoseofthesuse media listed in Table &,, gives an average of 3,.%eaootheoleaieefrom the rough etreina in a range from 0% to 6,5%,Haug;eoloniesavereged 7•25$ ef the month etrain in e range from 1,6%to 12,9%,, This fairly nerrow range seems to iadioate aeonetenayin
these ratiom l
Usim different neunte of the colonies were made |
es that previously abeervel, 1 ¢JThe following ie a list er the media used, and their compani-
44 11
Nutriet ogg; e 4 [Beef sxtmot 3.,6EZPepfßul
5.,0
15.0 ECDistllled water-—l litnri
1 gggggggg gguoo¤•4•xtr¤ct ggg; 1 5ke! extract ELO 5Tryytone
45,0 54
Glueoee _ ‘A 1.,0 3
Agar * 15.,0 3Dlstilld q¤ter-1 litsri 3
,44_
4
Bee; Lootoae ggg;B 1
Infusion from 40 3 baer heart.
Pr0t•o¤••popt¤ne 5 5Bacto (B.) lactose 10 54B. ewr 15 3 “ 445 mu. per liter ot dtatilled water.4
Blood gg.·e1• hu
Infusion from 500 3 fresh beef heart.B., peptone 10 3Ha 0}. 5 5B., amt 3 15 g
45 me. per liter of dutillsd water.
V 45(
wein Vu; ggrInfusionfrom *250 5 «c&.1.i‘ brain.
In1'¤¤1oafrom500gvpq1NaHg; 1*04
1 Y 11.25 6
Ba G1 1 _· 11
3.75 6;B. p•pt0n• · 1.0 5
53 ms. Pl! l1$l1° of distillsd watsn. 1
Intu•10a from 50 5 com mal A1 B. dsxtrom 2 5 5
B. nger 15 519 ms. par 11t•x• ot distxllnawatsr.l
B. plptms 10 g4
fB. lactou 10 5
° - V Kg HP04 BJ} EBasta fushsin 0.5 5Sodium sulphits 2.5 5B. man 15 g
Dintillsd mt•x·•·1 litu-[.._qV1n•'| Moin HstggggnoBlgB.
poptozu 10 51
B. lnotou 10 g 61
6[
46 5
4E2 HE04 2B, ecslu Y (D$··2} 0,4 5;B, methylene blue (DA···2) 0,0655B, nger 15 5Dietillea wat•r··é·l liter 5
Gtlectoue who; mar
y Whey frau 1 liter orlmilh°· B. peptcme ZS 5,
d gelaotoae, mrm 10 5;1 B, nger 10 g
65 gmx. per liter of dietilled warenLee! Aoetete gg
B« tryptoxxe4
20 5B, doxtroae 1 5;L•¤¤\ Aeetate CL2 5B• ¤£;&1‘ 15 5Distllled ••at•1•···-1 liter
Belt
htrect'ßechnicel meltoee 12,7*5 gBextrlu, Ditco 2.*75 5;Glycerol 2.35 5B• pepteuo OJ?} 5
Ä B, h 5 q 1 - l 1 l 15.A: u
Ulstllhled ¤at•1··¥4-1 liter xB. » w
_ _;_
47
ßltm~B. naar armer. 3 6B. Feptom 5 g
zam:} (cr.}1B•&f€«Z\1‘ 1.2 g
Dietllled water-·-3. lite:( The results cbtained am right of these media are listed in
Table L, and a- comparison of the teelve meüa in made in Table 7.
to those ebtained an nutrtlent nger, but in may caeee they M!' net. Decedptime nl the ve.r·lat1.¤nc follow.
Por description ot eeloniee see above. (Aleo
mm 1 anc 11)(Plate IV.) Smooth colcmiee were never
lergerthan 5er6¤ee• indi•m!—er• Tlxeedgeevrere cndulate
to euriculatn
Rough coloniea were large, yey-ehite, epreeding, edth '*hesy“
engen. 'the eolnnien were irregulerly opeque te trenelucenu(Plate V} Coloniee eere the ems ee
onautrientnger, though the color ct the eedim made them ebend
eutevenmore clearly. Theedgeeet theemethcelenieewere
dark. The rough celenlee typically ehoeed growth in e _
eleekwiee direction. After incubetlcn for L8 to 72 hours Ä
met ct the mooth eoleelee changed over to rough.
48
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' /Fig. 1. Rough Form, Natural Size
WIINSide View Surface View
Fig. 2. Smooth Form, Natural Size
II
Plate IV - 144 Hour Colonies ou Beef Lactose Agar IIII
I
I- *
‘
54 „I
I
Fig. l. Rough Form, Natural Size
@ @Fig. 2. Smooth Forms, Natural Size
W?}
2Fig. 5. Smooth Form, Showing Sectoriug,Natural Size
Plate V — 144 Hour Colouies on Blood Agar Base
55
§gaig‘ygal.agg;. (Plate III) Distinction between the two
types wasn*t clearcut until after incubation for forty-
eight hours.
Smooth colonies were up to seven or eight mms. in diameter.The edge was raised, and there were frequently two or three
raised, concentric rings.I l ·“
Rough colonies were quite large, up to forty-five and fifty
g mms. in diameter in many cases.: The edge was either lacer-i
ate, and finely filamentous for about three millimeters
around the colony, or was undulate with a clear zone of
Vfrom one to three millimeters around the colony. Micro-scopically the centers of the colonies were coarsely gran-
_ ular, and the rest amorphous.)· Corn meal agar. In 72 hours no colonies were over four
V
millüneters in diameter. All the colonies were myeeloid,
(and thepgh this isn't typically rough, there certainly is
_ no resemblance to the smooth type.
Endo's agar. In 72 hours the colonies were still pinpoint
in size, and the rough form indistinguishable from the
smooth. After a week most of the colonies seemed to be
either rough or intermediates. LLevine's eosin methylene blue agar. (Figure l, Plate VI) l
Rough and smooth couldn't be distinguished on this medium, ;
„ i.e. the colonies all look rough, Colonies on plates
Z6 ,I
//«_IÄ‘
r
‘¢ ‘ {I •;‘ '
fÄl‘*:='· ;€’a “
„ I ll glI
x
Fig. 1. Two rough forms, Natural Size
,
IFig. 2. Smooth Colony, Natural Size ,
Plate III — 144 Hour Colonies on Brain Veal Agar lI
I
I
III
I
I57 I
I
II
2% gg
‘SmoothStrain Rough Strain
Fig. 1. 144 Hour Colonies on Eosin Methylene Blue Agar. 5x.
’»\ ‘? 5
l-
Fig. 2. 144 Hour Colony of Smooth Strain on Galactose WheyAgar, Showing Sectoring. Natural Size.
II‘
Fig. 3. 144 Hour Colony of Smooth Strain Subjected to97.5OC. for 10 Minutes Showing Partial Lysis. On NutrientAgar. Natural Size.
Plate VI — Unusual Colonial Types of_BL albolactis
I
58 {
made from the smooth etrein, however, were not es finely 5woeolid, end were more deeply oolored than coloniesofthe
rough strsins.
ggg ggg;. Smooth colonies grew eoccoedingly wellen this medium; some colonlea were ae large es forty mi1li·—
meters in diemeter at the end of s week. They were consen-
trioelly ruised, lobate, with very fine, short myoelisl··like
projeotione surrounding the colony. Sectoring was noted on
several of these colonies. (Figure 2, Plate VI).
gg Agggggg ggg;. Smooth colonies were the same es on nutri·—ent nger, though they were eomewhut coarsely grenular around
theedge:.Rough
colonies had a large wrinkled nucleus, finely granular
around this, to irregular course granulation at the edge
(auriculate). They were flat coloniee and up to eighteen
millimtere. Some regular rough forms were alsopresent.gl;
gägggt gg (jh 1;.7). No growth.
I4;tggte _z;ga_1;. ~Both forms show up clearly, colonies appear-
ing the same SI tm namens ego!. _
Qzgtom gluooae extract Goloniue évero typical and thedifferenoes between the two foms show up very clearly.
As shown in Tables L, und 7, the ratio of smooth to rough
oolonies, from either etrecln, will very dependihg on the med-
ium used. Beef lactose agar comes the closset of any medium to
59
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of Mw S tum 6:: tm: R tom- Table: 5 mdiaataa aha mault of
1.«ng‘l‘-hsnsd imubation- Tlatas made ot um mhh-·tma¢„m atxaims
ot ä- ware eometad in hwonty-tmw md ewmtywm
hours, and a raarkad dacmaw in the mzwam of Zehe aeamuv oolmniu
uu hawa-Saweral pur·t2iaI.}.y lgmed uoluniaa mm obwwod an platoa
af the www st„z·a§..¤ that had bum huhad at 9'¤° G- und 97-5° C-
(1'igura 3, Plaw V1),
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oexlhvua, after fiva td swzdla miilk,
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ama weeh und Taäzüä plutcd {Jill- uuirxg nger-
ääaragal geurouhbsagma af smooth ami raaggee aaloniaa frau the
oppoßitn atrzzäm ewru hbbaizzed; 5.-eu. abuut Bü? aaa»Tat„2*5 ‘c»:·le>n;.2.aa 4
oa plateo asia frac: the: aaußa atruhua, had ax little wur A55
rough oolmias troa. am mouth ukraine. raaulta am raaurdud
in Table 6-Growüz in mliz uvädusxtly Lade am effoch üéfä uaszwraiaex uf
either aaooth ta: um mugh tum, mr vice mama. 'ku
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63
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t64[hgegfect of g H IThe various media used in the first part of this experiment
en the ratio of smooth to rough coloniee, had a range in p H [A values from l+•7 to 7•5S• Table 7; indicating the p H values of “these media, es well es the carbohydratee present, shcme thatthere seems to be no eerrelation between either of these tac-tors, and the favering ef the growth of either the rough er —
smooth form. H A
65 {|
Tabls ?······!äI‘£a¤t ot pi! and cvmbohydratas{ . { cm 6 22 .6 . { {' ?:rp• tm;··vm·¤d„ Garbohydram !
G? { aodium { 0·····m3.6thm• f¤·m~:3 , „pedia}!.7käalt axtract wo prowttm ääaltczaa,
dsxtrizzGora Com mal iafuaicm,
claxtmaß6.6 Galactoaa whey xi milkwkmy,_
{ _ _ _ _ «.;ala2ct·::>s0 {*.:3 Lead acamta H Baztrosa
·· .6 mzmw R -—·-·
‘=•7Hutriaat sugar ··-··~·
6,8 Boaf loctxoßa Laxßtom
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1)epreedixxg,csssbeid to :•hiso·id im shepe, with lecemteeé er ciliatededges, }§1’£3§:’•“vä“}‘iit»ö, rsugyh, ;;;listehire,a3, lesxgger them the e:·e:>eth
eelenies (neteEil}essozpimlegicsl or physieleegiceß. uiffereesews eseuea he fuuexd
between these two fame, s.L9t2*eoz;gh re.ethe:· escterxsive utueäieaää were
?i'1äÖÜ•
Fure cultures ef either Yum, when plste:«:i out am uutr·£..mt sgsr,
or tx·;7;t>teme glueoee sseteeect sgger, elw¤;m geäezieä ee certwih ;>ex•··
Gßiltßäjfü ef the eppoeite t;,rpe„ These ;;:>s:·eszat4—1;:e:; see fe£.z·3. cemsteht
psrtiezxler meezäie, ttzougtx they very free; one mee$3.xs~e te s:m·thez·•ÜGPÜHÄII :;eclL.s ·e29.l'l. fever she fem over the but. there
seems te be es .§.h.eli¤st„§.M0h of why this shock! be so, Zähe
cesitlommei the ph seem te hsventThezeeräexu will slss have en effect oh the type of c::>E„::»u;lsc [ferhxed, äeiycoloiei calcimies WigmF9ersbee:teri,estzetic sezbstaezces pres»eh·t, such es im [·sc>eé.n e:··„e·t„hyis¤e blue sgsr, for eeesse;,>le; er ;>:>ssih3.;; gs? vs; ses st tmNN
N
67 PPlover limits of the orgmiaaw tnloranca, as in com anal nger at
;a ph ot 6•0• g
Feportad quite comnonly with army cvmganiwzs (23,30) ,andr¤lly
roculting: in variant fame, aeotoringg was notea! am mv-9
4
oral oocaaions (Fßwrpxc 3, Plato X3; and Figtwc 2, Flntc VE} . P
Icolattona, however, ware never made of thoae foren. Partial lggraia
cf coloniee, due to man atraim et bactariopäzago, has ciao been
roported (9; 1.0; Bü) • This wan owervec cmly an platcn
ef the smooth etmin that had been haatcc at 909 und 97•5° C. for
ton a:·.ic1ut¤a(Fi;;‘¤1r¤P 3; hlntc 91),
nltzuoaxggtz interewiate fame were cbcervec, nm; a vaz·;;ä.ng: pur-
centage af mouth fame will ochenge over to the rtnxgéz t;,»·;>e of
wlona sm continued incubation, lt dose hot seen to bcnecessarytor
gmnamgo to be thmugh an intarnewiiata form in changgirzgg fm.; monthI
to rough, or rough to üzzootm ho! is there ang; chamggc of physio-I
legical or morpholovggical charaoterieticc going; tion waooth to
magen raw; hack again tc ¤«·w>oth• }hoth fame vom acpeulated, and their coloniea gglictmirzg,
which would anke than nucoid, an described with otherorganisaaby
slciwarce (3}, Saale (2*5}; mc othe·x·c• In no caec werecalonicacom
which nigghgt tm dcacribecl an non-wmcoid• $‘·ha.atnm colcnios, Pau notcd bj; xßoule (25) with clescly related ferne {c•;;• §• mgggg)
were not men; ner were genidial colrmica ever obacx·vnd„I
The question es to which ot these two main fame might beI
considered "no1•·zna}.", und which the variant, accrms to be unanswornblc•
P
„ ’ I68
I um emu zum hey be ie•:s.¤•a es um am en nem.-einl. media, hat veviea net enly Imm erm medium te metbo,
II y‘ but avm en the ume medium The normal. tere might b• defined ee e
mat easily ieelated fame, bet this veries {mm milk um- I
I · · ph te the mut, smooth type, heewer, scene to be eemeuhet6 I une in mim. am the num head, hw the etmdpeiater‘·I etebility, the magix femleexweewe lese eeeily than deee the ueeth
lhenneitherfemmeyheeomeideredmeremmalthautbeethev,I
im my eymm er memxeeum, ee Mswmuen, mt take um ee- II omimt the wicie peesible within thieeingle •p•¤.i••• I
II I
69
l. Sacillus albolactis exists in two main cultural formson solid media, one described ae a smooth form, the other esa rough type. 6
2. hmooth, rough, and intermedisto forms are morphologi—
cally und physiologically ideotioal.
3. The rough form is eoeewhat more stable than the smooth,
but pletiog out pure straius of either the rough or ehooth typewill yield some colonies of the other type. On outrieht agsrund standard tryptone gluoose extract agar the rough type
yields about 3% smooth type colonies, while the smooth Fone
yielde abut 7h of the rough form.
A. Various media, although yielding both oolohial types,may fever the growth of one or the other form, There seems
to be no oorreletion between this and either the composition
of the medium, or its ph.
V
«
70II
I., Buggy, I3. H,. atalEäergegßeot metemimtive älocwriologyäilliama md äülkina, Baltißsora, Ath od. 1931,
2. Borgay, I3. H., et el.
8or·.gey*¤ mnxml ot Uetcnuinative Be„ctev1¤3.og;;Eäilliama md 2>éi11d.n•, Ba1..t.1m¤z·a, ßth od. 1.939
3, Cheastar, F. D, Vübsarmtiona on an hqportant e: up ot mil b., ctoria
<>=•@¤¤i¤=¤¤ Nlßßßd ¤=> mmm ¤m1.L#.¤·6V_ Del. Coll. Ayia. ééäxp. Sta. 15th import, 1,903 9 II,. cam, sa.
J.Sparetoming b¤.et•1·1.s in the wu. I
2*:, Y. Sta. Tach. Bull. 58, 19175. Bavie, J. G., md C. S. Thiel
The effect af p!§ um gzvwth am gas pmduation of I
IJour, Zmiry Ems., 10, l»55··k63, 19396. Damon, éä.
I,Zhctarialvariutiona induoed by ctmngos im thec.¤mpoai··tionaf cultwo media,
Jour. Beat. L, 2, 133-lU•9, 193.9 I
I
v1I
7. mama., 6.6.Aatm; cf soma of tim factors disaociationofJour.
ßaot. ä: 5. l.89·-50i.,3.9336.Eädmrda,ätucüaa
cm cnc! smooth variantaofJour.
Bam. 2f., 1., 263-$@9. 19329. iämilay, P.
zäicmbac diaaociation.Jou-. 1»·«.£¤¤t. ma. 1.0, 1, 1-312, 1927
I
Fwttxar Advamaa in tha study of 16ic1•ob;i.c diaaociatioa.
änsact. Dia. 60, 2, l2‘)···J.92,1937ll.äiarzmar, B.1;.*.Üairyßactoriolomr. I
Jcxm Üiluy am Sana, 11. Y., 2:16 cd., W32 pp,193812.Jansen., 0.
Fßaotewiology.lllalzastomSons, 1931116. . läü pp, 192*1 I
3.3. 1.0661*, :3. A., am?. 11. C. astyrou I
Üflvhfiü; (E5I
by variatlons 1.:1 culture msciiwzzI
Jour. Izxfact. ma. 1.7. 6: l:53ᢥ77. 1930I
II II II
172I
aI1A. Heuer, S. A., and M. G. Styron
The production of emoth from roch forms of Qgggggggg
Sonne.Jour. Iafect. Dis. A7, 6, AA3-A52, 1930·
,15. Lawrence, J. S., ad h. W. Ford4* A Studien on aerobic, sporeebcaräag, non—pathogcnic bacteria
Jour. Haut. 1, 13, 273-320, 191616. Lewis, I. H.
Bacterial Variation, with special reference to behavior of
some mutable strains of colon bacteria in synthetio
medla.
Jour. Haut. 28, 6, 61.9-638, 193A17. Marshall, C. E.
Extended studies of the aaoeiative action of bacteria is
the of mm.Mich. Sta. agric. Coll. Exp. Sta., Spec. Bul. 33, 1905
18. Marshall, C. E., and B. Farrand6
Bacterial asociatiene in the aouring of milk ‘Mich. äta. Agric, Coll. Exp. Sta., Spec. Bol. A2, 1908
196 Olßang To EQ
Elements of UairyingMacmilla Co., H. Y., 570 pp 1939
20. äogers, L. A.
The inhibiting effect of on ggggggaggllgg
äHlE£Zl§§£•Jour. Bast. 16, 321-325, 1928
7:6
21. Rubin, B.A.A
Study of the cuuses of periedic abnormalities of a highgrade pasteurizd milk supply.
Thesis, Ysrginia Polytechnic Institute, 1558S22. Rubin, B. A., and F. S. ürcutt
Study of the causes or periodie abaormalitles of a highgrade pssteurlzed milk supply.
Virginia Academy of Science, löth Annual meeting, lüßö23. Selle, A. 3.
Fundamental Principles ot Bacterialogy.nccrau 12111 Boek Go., 1:.*2,*., lat ed. :*3*79 pp., 1959
24. Sorianc, A. M. de
Estudlo slstemstieo de slgunas nacteriss aspurulndos aerobios‘
Rev. Inst. Haut. D. M. H. 6, 5G7·ö%2, 195nSaale,21.Diseociation in the spore—£ormlng, aerobic group.Jour. Beet, 23, 1, S0-52, 19:6:2 . 4
aa.d5+—————————————————é—~ B T
ßStandard Methods for the Exemination of Dairy kroducta.American Fublie Health Asaeeiatlon, H. Y., 7th ed. 190 pp.,1959Z?.Toplsy, W, W. C. und G. S. Wilson
4
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