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A CI I'A'IiJI’ai} 07f! ‘s‘f'sfi TILTKAT‘ILIT‘Y bi‘ I‘J‘LGI’ZLIA

23103123

Lutritmd tn the Faculty of E‘Iioleignn Ltato 00110530
of Agriculture and Apzliod heinnco in partial
fulfillment. of the requirements for 1;}:

6051300 of Photos.“ of 30101100

Ogden L‘razollo 132-.an19

Juli-.10 ’ 19:13 0

1' .;' '
7/2.! 1* ‘0/

Que writer desires to express his grateful
uuknuwledgwunt of tho ablo nonletunco of Easter
w. L. hullnunn, 333001339 firoiesaor of Laozoriology,
under whom the work waa carried out, and of ozhor
members of the Faculty of the chtxtnont of anteric

elegy and iygiene.

199250

'1 3,... .I ‘b‘?‘ .‘v v'.’.'f I-‘O_,
Ath-‘AA 3}; tr 311-44.! 'L‘J

Title

[3012110311663’ 19:;

1. Introduction
II. diatoricul
III. :-..:;xr1m:~z;?.u

L. nttempta to produce 6 :orua of bacteria
by physical methods

8. fittompts to demonstrate the presence of
G ;0rm8 in the bacteriophufo filbrutoa
of Salnonelln rullorum and StegflgJocnucna

5.3.0.1’ it ”B

C. the production of microcolonics of
bacteria by physical influences

D. Studies on the filtration of bacteria
IV. Summary urd discuaaion

V. Literature Cited

A critique on the Filtrebility of bacteria
Introduction

In the commercial world a propoeel meet have an
iqmediuae inherent materiel value, whereas in pure
science ufly scientific fact irreegoctive of its
practical value is ingortent. Fragmently observations
that eeperently have no material worth, later become
eoeontiul stepping stones to objectives of positive
economic importance. the proof of the existence of
filbreble stages in bacterial life cycles may be too
basis 0; ingorbezt economic dieeovoriea in all phases
of bacteriology.

In 1983 Lbhnia and Smith presented a ruthcr
intricate lire cycle for the Azotubeutor which included
a filzroble snags. They goesuloted the ioruuLion within
the bacterial cells of gonidie of ouch also that they
would page through a herkefeld filter candle. Lore
recently Honduroy, Hadley, and Kendall have presented
data on other organisms that appear to veliflete the
findinge of Lohnis and Smith. Further observations are
necessary toth 35 to proof of the exiazonce of filtreble
forms and of their relation to the non-filtrablo
beeserie. as the data at the present are exnremely

fragmentary and highly unorganized ae to continuity.

-2.

Historical

nscseriologists ere civiacd into two schools of
thought concerning the existence of life cycles in
bacteria. The first consists of the followers of
plconorphisu, the chility of the bacterial cell to
prssont mun: varied feciercs of structure and sis ;
the second composed of those who believe in mono-
uorphisu, the fixicy of the bacterial cell in only
one form.

£33911 (55) in 1877 presented the View that hccterim
are extremely ploomorphic. us believed Lth all bacteria
belocvsd to c sinfilc type of fission fungi, highly
Variable, greezlg adaptable morphologically. fermentciive-
13, end biochcmicullj. 4n the other Mend. Gone (11)
in 1&75 presented the sheer: of meromorphism V th a clan!-
ificetion of fission fungi bcacd on the clove character-
istics. Koch agreed wits cohn and was able to present
onou;h proof 30 swim: the ponculum of thought deiinitely
to unis View. Koch's teachings have dominated the trend
of bcctcriolef: until recently. Luring this ycrlod
manifestations of u 330019110n were attributed to con-
taminc.ion, involution forms. and even to poor technic.

About 1911 the tide changed and from this time the
concensus of opinion has gradually turned towards the
Opposite vicw;oint. Jones (33) 1930 summarized his
former super of 1913; He noticed that cone Acotoluctor
possessed a complex life cycle. one obserVution was

that the cells of those organises formed two types of

.3-

granules. a s oihoble m d u x on—otoinuble. 153m former
were apparently regrodnotive bodies or junidiu. .men
the mother cell disintegrated thooo granules were
liberated, whereupon they grew into normal cells. ro-
produoed indefinitely b3 binary fission, then each cell
ogoin forced gonidio one disintegrated thus completion
the o3ole. jooover, he stated that they were not
filtrotle and need no mention of the size of the colonies
:Todueod t3 the "oxidial earns.

Lofinis uLd Smith in a grolimin-ry tote (4%) 1916
showed this the life history of the szototootor axe
other hectorie was very complex. In a liter commonicution
(41) 19:3 tho3 :03: ted out that the Anotobootor goo eased
seven distinct cell types. Shay reported that all types
of bacterial reproductive oriens were found in those
orgunlena, gonidiu and gonidanglu, 33goeporos, arthro-
spores, mierooyets, sodosooros and ozosporoo. ”hiya stated
that when the nuclear material was oooomoonied *3 econt
amounts of other cell elevents, gonidie were formed.
”ho3 also stated that these gonidiu were in port filtrable
or had ultronioroeooo io portiolcs associated tith them
which were ougoble of reproducin: Azotobootor colonies
after filtration. rhis pay or is o 013 @310 on the
dissociation of bacteria and is t;o basis of Lroutioolly
all “orL cu- disoooiztivo yfionononu.

Load and :orren (5.) in 9L3 regorted the isolation
of a roodil; cult ivoble vi%;rio. he3 ettrioutod the

filtruhility of this organism in port at least to its

«.4-
motility.
flouduroy (be) in lio? presented a technio for the
cultivation and ,roouotion of visible orguoisas from
the :iltretes of filtroble forms of bacteria. Ihis
process consisted essentially in placing one or two so.

3

of the filtrate on u lootose gelatin plate, inoutotin

C”

the seeded plate for 24 hours, wasting the surface of
the medium with sterile broth, and transferring the wash-
ing to a sterile lactose gelatin plate. Skis roe repeat-
ed five or six times until fine, pin-gain: 0010;509
appeared. fie stated that at this stage it was possible
to grow the filtroblo zeros in broth. this was the onl3
method by which he was able to rrow these forms.

Jadloy et 01. (35) in 1831 wore perhaps the iirot
to renerote artificially and subsequently to eeltiVuto

p.

in pure culture the :iltrutle virus-like staée o- on3
bacterial egooioo. Skis Ziltrutlo or 3 form was yroduoed
durin~ dissuoiotivo chunjes b3 reyid transfer ELG aging.
Que G 10339 were grown as pure do torus; their filtra-
Lility woe demonstrated and b3 means of special tooonio,
the ruyid serial plote transfer of douduroy, tho3 were
recovered from the filtrate. the different reactions of
the G Lorne were found to be different from the normal
R or S t3pe, but after suitable munionlotion they reverted
to the normal form. 0 forms for other organiens were
3130 demonstrated.

Kendall (36) in 1931 rogorted e oulturo medium waioh

was looking in protein digestion products. This medium

-5-
was unpublu of changin; ordiniry Laotoriu to primitive
forms which woru able to page filters. flheoo filtruble
organisms were unuultivuble in ordinary media but grow
well in ; medium.

Vurnoy and ironfenhrunner (06) in 932 extended
the observations of {unfilll and d monetr-.Ld that the
cause of tho iilgruhilitg might be attrihutcd to the
suspended material TPOSGht in E medium and adoortcd
by two iilter. :do: noted fifiut the organiuqo in the

filtrate were equally cultiVuble in K or other media.

p.

-0-
prerimentul
(A)
Attempts to ?roduee 5 forms of Luateria

by Physic&l finthoda

l. The effect of rapid transferring and akin;
bacteria in different media

There exista in the literaiure much confusion as
to tue exact import curried by the nyrn G flora.

Hadley at al. {33) in 1931 introauocd this designation
applying it to colonies, micronOPio in size, groduccd
by ooccoid forms which were :iltrable throu h Various
types 0f filne‘sa. ; uecéumr and ;.:hz.3r:r~.:;n ('7) in 191.23
ieoluted or :uia 9 which the; called grimitive bacteria
uycultivgrla *3 0?dih r; mcihods. Ubher Lanes further
cload fine situation. In this paper the Iollouint
terminolOéy will he used.

1. 3 forms which consist of any organiaw in a
form.di£forent from the normal uLd which will pusa
filters and produce colonies microauopio in size upon
tha application of rapid aerial plate transfer or aging.

2. Filtrublo forms which oonaiat oi any orgnnian
in the normal or chwzeod form which will pugs filtera,
and mill produce OOIOfllGS and have rcuotioua nurmul for
that organism upon the urcliUution of suitable teuhLio.

3. m forms which oonaiat of any organism which will
not pass filtere, bufi will produce colonies microaoopio

in size upon the applicgtion of auitublo tachnic.

-7.

who nature of thia study required the use of
several controls in order to evaluate correctly the
data ottzaixaed.

1. Control and method of filtration. the iorkoield
filters were oleonéd before ouch filtration by passing
through them under negative pressure 25o cc. of a solution
of potassium permanganate (1 gram KHnoi + 6.5 gram
concentrated moi o 1 liter of distilled water), zoo co.
of l per cent oxalic acid solution, 500 cc. of hot dia-
tilled water, and 5J0 00. of oold distilled water. the
filter was then connected to the ace mponyinx flook, tho
outloto plugged with cotton, and toe u;yurotua sterilized
in tho autoclave at 15 pounds stow; pronoure for 30 min-
utes. who Soitz filter pads were renewed after ouch
filtration afld the apparatus sterilized as above. Loch
Iiltor was tested ior its utility to rotuin Serrotio
moroeoooug after evory 13 filtrationa.

Tho proosuro and time of filtration were adjusted
to permit the filtrate to pass at the rate of one or tvo
drops per second. for 20 00.. the amount generally
filterod, tho preosure was 103 to Booxwo. of Hg, and
tho time woo three to five minutes. loose methods were

polled to all filtration studios.

2. Control of sterility of media. The glassware
was sterilized twioo ot loo’o. for In minutes or longer.
Boo media were prepored in the usual manner and sterilized
oooording to tho standard proooouro. Toot tubes contain-

in; media were ylooed at 37°3. for 4“ hOHFB. then at

-a-
room temperature for one wecz. n11 tubes chewing growth
were discarded. :he agar pletee were poured cecptically,
ncubeted ct aV'o. for 43 houre and at room temperature
24 houra before oeing; those showing contamination were
discarded.

3. Control of test meteriel. The E medium tubes
seeded with the filtretee were controlled t3 duplicate
filtrates placed in etorile cotton etoygered teet tubec,
in plain nutrient beef extract broth, or in differential
media for the particular organism studied.

Rapid truneforring and aging of bacteria under
various conditions have been studied by eevorel authors.
'Jedley (p3) 1931 obeerved that rapid transfer or
Shicelln gggenteriee in media which had alight inhibitory
effects on this orgeniem ccuecd a change from the normal
colony else to one which could only be econ with the aid
of the mieroecoce. He reyorted the S rcrxe of gfilg.
Qgeextcrieg arose lo Limos dcrin: Spontaneous or enzorcec
diseociu:ion of the S or the R forms of this orchiem.
Incec cultures orieineted from both B one 3 types uncor-
going ragid transfer and aring in broth, with one without
lithium chloride. in pancreatin broth, from bacteriOphege.
and by animal injection.

inc former methods will he considered in detail.
Plain beef infusion broth. p] 6.8 to pH 7.8, was used
for spontaneous dieeccietion. reenty-five hundredtha
to 0.5 per cent lithium chlorine was addeJ to broth.

pH 7.5 to pH 7.8, and 5 per cent pancreatin (Squibbe)

-9-
was likewise placed in broth, pH 6.8 to pH 7.8,for study-
ing enforced dissocie*1on. Dots H and 3 type cultures
underwent rapid transfer end skin: in these media.

Que technio of rapid transfer consisted essentially
in transferring from one loop to several drags from tube
to tube at 34 hour intervals. After 24 to 48 hours'
iuouLatlon ct 57's. the culture was yluoed on u sterile
ugur glaze and secured ovUr the surface "inn a sterile
glass L-shuyed rod. These seeded plstce were incubated
from £4 to £3 hours and differertisl colony plate counts
undo. She studies in sting consisted in part in placing
the organises to be studied in he Vurious media and
mskixg subculture plates at intervals for differential
colony counts. dcdley found that on about the seventh
to tenth transfer microcOIOhies uygeured in large numbers,
sometimes preceded by a sterile plate. These afpcercnooe
were very regulur in recurrence. u 33 repeated trsnsfor
the colonies disupyeured. returning again when urther
plantin;s were made. The cultural, morpholo~icsl, and
chemical reactions of the 3 colonies were obtained. These
diffiered from the normal Shir. gggenterlue reactions.

The cultures were found to be filtrable and the filtrate
under proper manipulation produced mierocol nice or were
true G {eniso

Euros (54) 1931 obtained results with Salmonella
enrtrTekc which confirmed Hadley's work. She technic
used closely followed that of the letter. At the

termination of the experiment six cultures of the 0 forms

~13.

of 331. aortr~oit were seeded on veal agar platea, sealed
with adhesive tape, and placed in the flue box for iivo
months. at the beginning of this period the culturue
were microscopic in 8139, but after two prriod of china
only yellow, oguqne colonies 2 to 5 mm. in diameter could
be discovered. an oxglanatiofi of this phenomenon will
be offered later in this pager.

a. T?e 6;? act 0:: ru id Lrnnqjurr1v~ uld u in; Ski

filmewteriue in different media. {a an at:em_t 30 ohttin

 

G forms he influefice ofr fid Lr"*”err1n aufi u 1:3 8
strain of Shig, #399338? ac was tested in different mefiiu.

 

2he culburu wua obtained iron Hadley of the University
of: ich113n. ‘his strain was the one used by Hauley to
obtain the G farms that he has re; norted {83) 193 . in
order to alaulato the conditions under think his work
was conducted the following media were eelootcd and pre.
pared according to his.recommendutinna: (1) Plain beef
infusion broth. pi 7.8, (2) 0.25 per cent lithium
chloride broth, pd 7.6, (3) 5 per cent p: ncr<zatin broth,
p! 6.8 . (4)5 psr cent puncrxutin hrot n, pi 7. u, (3) plain
beef infusion a3xr, alld (G) litmus lactose thP (biEBU).
A anificient amount 0f ouch medium was made at one nine
for the entire e.<pp eziwunt. before using each take or
plase of medium WfiG incubated for sterility a8 outlined
previously.

Daily transfers were mafia in each of the fowr truth
media. All tuiea warn incubated at 57°d. After 45 houra'

incubation each culture waa streaked on litmus lactose

-11.
and plain beef infusion agar plates. Qhe gletea were
incubated 43 hours at 37‘6. for 5 to 10 days be room
temperature and examined for colon: f rantion at a
magnification of 1)) diameters.
The initial tube of each eeriee was placed at room

tougoreture after the incubation varied at 57's. to
o 1’ I

-\

determine the offiect of aging. fiver: Bi huure each
culture was streaked on litmus luetuee and plain beef
infusion agar. rho plates were incuheted and examined

ea outlined chore. Although Shifia‘gigrnneriee was aged
for 53 Gaye, end in 33130 of 5J reeid transfers in four
different broth media, no effect of dissociation was
observed. $he colonies present on each plate in the
eeriee were rough and Opaque throughout the ooureo of the
experinent. ‘

B. Syontnnoeue and enforced dioeociotion of bacteria
by rapid truneferring and ugin3. Although no G forms
could be obtained from Shigs'gieenteriee. tne method of
rapid transferring and aging wee extended to abhor
bacteria and to several other media. SeVen or3nniene of
different genera, both pathojenio end nonpetho chic,
motile and nonmotile, p finent and nonpigment forneru
were selected: (1) §n13e11n_Eerad:eentoviee, (2) Ccrrntie
indicn, (3) chrofiobecterinn violnceum, (4) Escherichia
.2233, (5)_§nlmonellniggllorwm, (6)_§orohccter ucrogenea.
end (7) Shieelle.g;penterieo.

The medie were prayered as outlined below: all

media were sterilized in the autoclave at 18 pounds

-12-

steam pressure for 15 to 20 minutes and :ueched for
sterility by incubating ue described previously. ?hc
seven oréwnieee were planted into ouch o: the truth
media: (1) Klein bee! extract broth, ;3 7.8, (2) ;luin
veal infusion broth. 31 7.2, (3) 0.5 per ocnt lithium
chloride broth, (4) 1.3 per cent lithium chloride broth,
(5) 2.0 per cent lithium chloride broth (6) E medium
{B1foo}. and (7) 0.007 per cent trilliunt green broth.

in addition a tube of ouch was need as a control on the
eheence of 3 rorae from the media. The culture tutee
toeether with the centrole were ineuteted et 5 ’J. ior
24 hours. Lolly runefere tore mode from ouch tuie in
each eeriee. after 43 houre‘ incubation the cultures

and OJDSTOIS were streaked on voul infusion fixer plates
that had been previously checked ior sterility. th
seeded gluten L620 incubated at 27’0. for 45 hours,
followed by 5 to l) duye at room Lcnjeruture, and examined
for colony foruntion at a magnification of 1;Q diametere.
Colonies reeeHEliLg 3 :orma were subcultzred to reel
infusion broth, incubated 43 euro at 37's., and filtered
through berkefeld N filterc according to the toohnio
previously outlined. the filtrates were utudied ier the
presence of G f0 we.

20 determine the effect of axing the initial tubes
of eucu series were pieced at room temperature after the
incubation yoriod ut 37’s. Lt the intervals riven in
3uLlec 2, 4, 6, 8, 1}, mad 13 the material in ouch tuic

wee streaked on veal irfueien user elutee previously

-13-
checkcd for aterility, and after incubation at c7’£.,
followed by room tcnycrnzure, were examined for colony
formation. :he control antes remained sterile for the
entire czgcriaont. The plates producing microcoloniea
were incubated one weak to observe any uhahxe in colony
size. Licrocolonios which reuuihed cuhstcnt in six
were carefully transferred under a M';niiicution of 190
diameters, to veal infusion broth, pH 7.0, and incubated
at 37°d. for 43 hours. Broth cultures which chuwed faint,
opaleaoaus growth were filnorcd according to the mezucd
previously daecribed. ihe filtrate was clear and yolor-
1033. vne co. amounts of the filtrate were placed in:
(1) ioal infueion broth, pH 7.2, (B z’meuium (piioo),
and (3) a aterile cotton stappcrod that tute. Iheee
tubes were incubated at L7“&. for ?2 hours, followed by
one week at room temfiercture, when they were atreuked
on veal infusion uga platen. thse were incubated at
b ’3. {or 4a hnurs, followed Ly one week at rcum temper-
ature, uud than eXuminod at a mmgnifioution of 100
diawetcra fur colony forzntion. fio colonies were dia-
covercd on cry of the cubculLu39 pluses; thq media contain-
ing filtrates from tie cultures forming EIUrDOGIOhififl
remained sterile uncroacnpically and microscopically.
this donunsnrutcd that these organiana were not filzrable,
aid for fihis reason the term 3 form could no& he applied
to any culture icoluted ty rapid Srunsfcrring or u lug.
According to the Serninology outlined previoue y, tho

microoolonica formed were labeled K ccloniea.

-14-

1. Liaaociution in plain boot extract broth,
pH 7.8. ?lain beef extract broth was made in the usual
munn.r, adjusted to pi 7.8 wink P/l Laid, tnlod in 10
cc. amounts, sterilized and seeded with the toot
organisms‘

a. Rapid flranofer. Soto in fickle 1 Show thvt
only three organiSdc produce M oolcnioa, Shin. ;ߣ;9
giggntorioe, Tech.‘gg;;. ond‘ggl; Ezllorum. they
apyourod when 5 colonies were the predominating tyoe.

Show 353% only three

{‘13

b. fifinf. Fhia in table

organisuo produced 5 calories. Goran. violmocnn, loch.

 

coll, cull”: '. filth the acception of chrom.

 

viol~ccug_1n which the apgocranco of the u Lypos woe
:reccdcd by three aserilc plates, they arose when 3
colonies were the groonminatin; tyyo.

2. Spontaheous diaonciotion in veal infusion
broth, ?K 7.2. veal infusion broth was prepared in the
canal manner. checkod for ezcrllity by incurution, and
Seeded with tho_tcat organiama.

a. Hapifl transfer. Hutu in Qable 3 show that
four organioaa, Barr: indica, Shiv. gzgfiQBGVIQfi. carom.
violoceno, and 23;; pullorun. produced H type colonies,
the first two when R colonies were the predominoting
$336, the o;hcro when S colonies were in echao.

b. Aginr. iota in Table 4 show that the production

of u colonies in dhrom.lgicloooufi and Sol: pullorum

 

occurred «hon R colonies were predominant in the former,

and when S colonies were in excess in the latter.

~16.

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mafiaaaaompu ”seasonoflm I I
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muumgficoohu«: I fl

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moaaoaoocuoax I 3

ammo ham :u wwamoHoo fiuoofim I m
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1 : Acosnwaaov.
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epsuwuwumh: hflazzrhw I so

mnuvaoao muccaan0§ofi I %
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unwo has a“ uuuaoaoo nmcox I m
mamfihfiwsc: «hawara npcnm I m

 

 

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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-20.

 

manna
«nonaoooam
cuedddom

II'
(145;!)

ouauunuoonq hdaunmue I :0

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unaccuooouoau

«coo how ma unano~oo :aOQEm
unoo ham nu nounoaoo naeox
nonwhaoama uncanno gnosm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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--21-

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waacnono aaoocmmofiox I fl
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ammo hon a“ mvuzonou :uoasm I m
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ri b b b

 

..cos:«pacm.
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~25-

3. Enforced dissociation in 0.6 per oent lithium
chloride broth. 20 Veal infusion broth, preyurod in
the usual manner, was added L 31 q.a. to make a concou-
tration of 0.5 per cent. rho medium one eteril sod in
10 co. amounts and seeded with the teat orfuniamao

a. Rapid trunofor. Data in Euble 5 Show thut fl
tyre colonies were produced when 3 types were in excess
in Sorr. indioa, and when H ypee prodofiinutod in Qhrom.
violaceum and Shiq: QXSentoriae.

b. Aging. Data in Table 6 ahow that 3 types and
R 3963 were equal in amount when H oolonioa urooe in
Barr. indiou. and that 3 $3398 predominated vhen u tyyos

egg ared in 390%. coil and fial. golloron.

 

4. Luiorood diooooiotion in 1.9 ger cont lithium

chloride broth. the medium Wu. prepared as in yurt 5,
zoop: that the conoonzrution of 1131 wag made 1.0 yer

cent.

a. Kufiid transfer. buta in fable 7 Show that E
types were obtained when 3 ooloniea predominated in fierr.
indico und‘§§;‘_§ollornnt .

b. Huging. Eotu in Table 8 show that R afid 3 types
wvro equal in i235. indion, and thoz 3 types predominated
in $21; Eullorom.when H colonies appeared.

5. Enforced dissociation in 2.0 yer cent lithimn
chloride broth. Che concentration of L161 wus'mooo 2.0
per cent in veal infusion broth.

a. Rapid transfer. Iota in Ioble 9 ahow Bhut h

colonies were produced in fierr. inaioo and Tech. 0011.

-24-

nomao

unmasoooam

mnflcsoao msoonomoEom
mofiaoaooonoaé

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ammo hon no mmfinoaoo swoon
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‘ .26.

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b. figinx. Data in Sable 19 chow L t"p68 apgeur-
ed when N 83963 yrudominuted in Egng indiau.

6. fiisaociation 1n 3 medium (Difoo). A 5.0 per
cent snaponalon o; pondered K m~dlwm (rifuo) was menu in
distilled water, Bturilized, checked for sterility, unfl
seeded with the teat organisms.

a. Rafiid tvynefer. Buta 1n futle 11 Show that}:

£3363 appeared when R types predominufiud in gargn.

violucann, u:d “hen 3 t3

4

was prcdoniuated in ail: rullorum.

b. .Afiing. £0 3 typea were prcducod.

7. Fiesouictinn in brillianfi g?cun broth. lrilliant
green broth was prepared by adding pepzono, 13 grams and
geut entrucfi. 30 grumeta 1 liter of distilled water.
this euaponeion was heated until dififiOIVfld, udJusted $0
p1 7.0, autoclaved for one-half hour, filtered, and to it
was added 7 00. Of a 1.) per cent solution of brilliant
green. ghe medium was tuned and sterilized.

a. Ragid transfer. Batu in table 12 show that a
$3303 were obtained when S Lyfiea were preuogingnt in
122.4 13!} 1 10!" mil.

b. aging. laau 1n Buble 13 show that a Lgyea were

obtained when S colonies wove preuuminunt 1h Vsch. c011

 

and :31}; Enlla'n'vii
Biéauaeion:
Seven different species of bacteria were transferred
Gully infio BQVQH different media and the EngAPMNUe,

persiszonce, and disupgcuruncc of 3 00101103 mused.

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It was oheorveu thin in most uuaoa a colonies
appeared on the plates fiudfl from the fourth to Bi£3h
transfer into, pureiczea shroujh five to eo?cn transfer
and than diauppe red. :hey uleo agyeured uELar 1» days
axing and diauppuared fry] the fiLth to figth day of aging.

fho regularity with wxich the G fonia arose in
Experiments by 136163 on spontaneous and eniorced
dieaouiution of fiki;& disentrrian by rapid transfer and
ugin;, and coniirfled by {uro& with figlg aertrguxe, was
not oteervad either with fihivz ggggngcriaa or other
orgauiafia. Home of the media produced a types in all of
the cultures; vedl infusion broth itself agonuuneously
gave rise to more 6&803 of H cOIOLleB fihun any of the
ayecial enforcing mudiu. Enema ouloniea occurred hap-
hazardlx, and no relatianahip between the yrosenco of H
or 3 colonies. the tyge of media, the variations in the
churunteriatica of the urgunigxs, or the taohnio unfl the
appearance of S galaxies would be diauovared.

icprcaenustivo coloniea 3510439d at random and
filtered us Outlined previously did not yuan Lcrkciold
R filters. For this reason Ede writer did not feel
justified in npglgin: the term G form to say of the
microcolonioa obtained by rapid trgnefcr or ujing.

2. Attempts to obzuin pure culturea of J types by
serial dilution.

filthouzh rapid transfer and aging of pure oulbures,
an indicated previously, in Various media produced micro-

ooloniea which rcaembled G 33303 very closely they were

~44-
not filtrehle. Several authors, Hadley et el. (23
1931, turney end hronfonbrenner (66) 1982, end Linoeer
(70) 1833 stated that a true G culture meet to filtreble.
fine data obtained in the experiments on euro cultures
indicated that these forme were not present or associated
with the orfoniame studied and could not be demonetreted
by rapid tron.fcr or aging. Goneeouently e different
method, ecrial dilution, was employed on material known to
contain eeVorel different tygee of organic a in naturelly
occurring aubetunooe, milk, sewage. ote.

:he method or procedure was similar to that employed
by Erueokner and Sherman (7) 1932 who roeorted a high
incidence of primitive forms of bacteria in many naturally
occurring eubatenoee. milk, soil, sewage, etc. they were
able to obtain these forms in pure culture by eeriel
dilutions. 1 x 13-13 on. and higher. obtaining growth by
special methods as regid aerial plate transfer. They did
not conclude thet these forms w re filtrable, tut they did
state that they would not grow under ordinary oelturel
methods. In a study of milk from.normel cows they demon-
etruted that primitive types of bacteria ooourred in larger
numbers than the ordinary type of organised. This was
ehown by obtaining growth of primitive forms from high
dilutione of the meteriel studied, 1 3 10’8 co. and higher,
although the usual forms of bacteria could not be isolated

Six eoeelee of sewage and four Bangles of skim milk
were used for the test material. Lextroae beef infusion

broth was tnbed in 9.0 cc. umounte, sterilized in the

-45-
autoclave at 15 pounds eteem prceeure for BJ minutes,
and checked for sterility by incubating before using.
one cc. of the sample one placed in tube 1, this tube was
carefully shaken, end 1.0 cc. of the mixture trcneferred
to tube 2 with e eterile pipette and continued until the
20th transfer had been mode giving a eeriee of dilutions
from 0.1 cc. to l x l) ‘3occ. of the original sample.
Duplicate dilution eeriee were run on each eomyle end on
a control tube for each hatch of media. :ne eet of dilutions
wee placed at 25’s.. the other at 37°c. for 72 hours. all
tubes showing growth at the orpirotiou of this period were
discarded; the remainder were incubated at 25’0. for three
weeks, eftcr which they were streaked on dextrose beef inn
fusion eger. In each eeriee the lowest dilution tube that
had not ehown growth at the end of the prelixinury incubation
period was examined by the rapid eeriel plate trenefer method
of fiedley (23) 1931. At the time the eemplee were diluted
they were also filtered froctionclly through c herxefeld F
candle. to difference could be shown between the couples
incubated at 26°). for the entire period and those receiving
preliminary treatment ct 37'3. The material treated with
rapid aerial ylete trcnefer did not show growth macroscopic-
ally or microscopically. tete in Sable 14 show that no
growth was obtained on eubculture plates at the end of
three weeks at 25’6. from tubes which did not chow growth
after 72 hours et’S ’3. This did not disprove the presence
of G types in the materials studied, but merely chewed that

they were not present in the samples in greater numbers

-4 .3.

Gable 14. Growth uLtulned on Subculture Plates from

Serial rilutioh tubes of Eewuge and Rhim_L11k.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fl. _' T
Latex-m1 ' x on .- x --x .
T I U T

' ; ' 2 ' a ' .4 ' 5 ' c. n , '

T T I I LT r —:‘T

39:1{3fe ' . O O I § 0 O I O t - .
r ' T 1 T 1 r

SB‘HLX‘Q . . ' . l 9 O 5 U ¢ 0 - g
T '— I f 1 r T

SBWEEG ' ‘U’ 0 O I O t O I c | - .
r f r V 1— ' f

Set-73:86 . ’ . O 3 ‘. ' . . - ' O .
:egytéizo ' . U . ' . I .’ I 0 fl - '
vw 1 ' ‘ I T j T
Sewage ' ’ ' * ' *" fi v o 0 ~ .
r ‘ ' ' 1 j ‘v‘

aglm hilt ' ’ ' ’ ' ‘ ' * ' ‘ ' - o
' ' I v r I 1

53:1” 1, 11K I § 0 4- a O o o c o g - .
T T I I I 1 T

Skim 1:11}: 0 O O O I O 1 9 u o t -_ g
T T I I 1 T

Skin i113 ’ ‘ ' ’ ' ’ ' ‘ ' - ' - I
.: 1 r T r T I j
' 1- I o t o c c. g - . - .

Control

9 - Growth oltuinod in fillution tubes after preliminary
incubation at 2533. or 3 °C. for 72 hours

. a £0 growth obtained by rapid aerial plane transfer
or on atreak anboqlture plates of the material
after final incubation at 25°c. for 3 weeha.

-47.

than the (1:31:91 11:00:;01'113. '21::0 000111.30 to (702-:01'mtrato
the g::1(1(:(.11109 (:3 (.1: tygma 1:: (my emneontnw£35,011 is: the
BDszlo 11:7 thc ((10.20 s of fleetional 1“11‘:°z.(t10n Lave not :11: ivc
results (:9 (3111.11.23: 1:: 1:01-23 1:25. 11.9 CPL (1: 1.131718 pm 00:1: in
the {3"123910 (11C: (mt. 3:17:09. 1:219 filth? (2.111311 the Few-.11. 21 to
eighth fraction, and (70.1.10 the usual typos fmmd in 171.113:
0.1.1 301.10.;0. (23:11:11.0? filtmfi‘fln 10:11:11.9 o: G $011229 11.101.13.62 have
passed in the first 0:1 0009230. f‘mction (3m: according; to
71.11.103132921 (and: 311101122371 (7') 11-33" (1.21:?! i'tzdloy 9t (:1. (“3.- 13:31
(101210 havn 1.1m}. lif‘f‘ororzt (abnmtgctcmistica from the 0:1Lm:1m2:3
(1.9102131?! ,7 (fits. fined.

£1001: 30:10:: 0.1.“ (.2091: 10:: n.

1. (100110133 1:20:10 13:06.0 (:0 (303701100 1531.0 arcxiuction of

1:"

G fox-3:123 from 91.(:1u‘(;(((:0(_:1.19 (1.11. (3 (1910:1006. ((13:00 1(- tin :3 113-
(1932313 ’l’flilsf'fil‘ (1:15. (:LinL; 012‘ 001mm (iifi'orom: (11011:):113. 1. (3:13
1:1 Vurioz-m (1.06.213. A. 1101.13": (313:2.1015: of 31:17.; '1. (“11:00:11.11‘01m was
aged for £21.“) days and 0911:1106. 1:19:01.th Ex) (31913.3; trcrmfora.
{:0 31110023003 0.? (4131:0013 nttrm 1 (1:10 (10:10:2- in an}; of 1:310 17013.:-
r:ec‘:ia used. 1.1220 I10;;1.:1::1:11t:; mated (1:; 1.01210}; with which

(3 $021.35 0001011190. is“: 571-14". grcmggcricg (1:03 nut 01"(3011ved in
any (sf 11310 (3011011 m-Lflzflmn stucfiiod in (my of‘t, :.:(.—.:(.'z;in.

0 “(:Int1(11:(1“1:>0 could 1m onw‘rlia} 00 101:: (0cm 1:: :0 1-0..-
1192931521033 03:" 13.229 (gm-1:00:131100 0f (frievcmolcuioa and 1.1.0 type
of“ madly, t1 0 92': (111111101111 .193 of tin: CPL (1:113:11, 011 trio

8

state 0: (i211 (..zcci(1t‘.i(:n. 191101111}; ti (:30 re: 3:111:13 002:. 1:171:0(1
those 0181:1300. 1.1;; 3":165103; of; 0.1. (1'35?! 11"):131 and (70110.1( (#1:)
19531; but (1.171200 I10:31:1o::0::ta.tiv0‘(11011000103103 (rm-0 trans-

i‘orrod to 111.731.], 1:13:51011 h" 1111': cm}. filteroé, they (kid not

-43-

mmuduficonsm a“ 0:0 newnoakm camuufiwm aw

mozaaumo nuEOhw on I I

aokaua30000 an 033 acuuodpm unmanawm :« ceausupo

£9 Eek"... I ...

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. . + . + . + . I . + o I . I a I a 0 . + . o . I . I . I . Md“; Ewan

.Dr» P Dr D D. L F (P p D P P D D: (D. D

. . + . o . +, . I . o . ¢ . I o 4 . 4 . I . 9 . I . I . I . Mae; Eaxw

P D D P D b} D bi D D (Dar D D P (D ID:

. . + . + . 0 o I . I . I . + . I . 0 . I u I . I . I o I o xauz flaxw

hr - P b p b L.“ - pll1nP b L p b: (n: b

o . +, . o p 4 . 0 . 0 a + . 0 - o o + n I . I . I . I . I . Mfldnnfiwm.

h D h {P (P L P P bDl Dr b D»: (D r (P (2’1‘

. .¢.¢.+.+.+.+.I.I.I.I.I.I.I.I. 01.036fl

- b .- 11%: p L: n b r by L P p p L p 1

. . o . + . + o + . + . 0 . o o 4 o 0 u I . I . I a I . I . muarom

b b P, b L .- h n p L hlP P brib - . HI.

. . ¢ . 0 . o . 4 . + . + . + . I . I . I . I . I a I . I . Ornfiom

P .1 b (- P p by F 1P 2» p .p p b L .p (1

. . o . + u + . Q . + . I o I o o . I . I - I o I . I . I . etfikom

b (D P D P D} D D D D (L! D D D P D

. . + .1f - + p I . + . é . 4 . I . I . I.. I . I a I . I . OMfiBQm

.P P b P p P LP b (P b b b P PL in .

(P b ... P6 p 4 r4 -+ P+L+b OPIbEILIL IbrI pIi. 09.90....
. r n- O a . a . o I 4 .

. aficcfis a“ .20 O acauOQhw cwahuflua . daaumumm

Pl D

 

.Maua_s«xm o:0 endamm no meumuuaam «mucauowam :« suzouw .mH edema

~49-

paoa Eorke?01d U filtovs. Tocnuso those microcolouios
warn not filtratla, tho term : COlOfiIOB rather than G
form was aptliod.

3. 950 tachnic 0? sayinl dilution rovertud by
rruookzer and LbflPfinfl (V) 103? was up lied to 513 samples
of mara¢o and four annmlao of skim.rilk. :0 9 forms or
u typos could he demonstrated in thong unnnlos which were
also filtornd fractionally. fho Cygnniama Pocovmrmd were
tho u<ua1 typos 9? seat in sewage or in Skim milk.

m forum of bacteria rather than G tygeo were Conan-
atrntod 1n Ofigfifi 3P3 unco?tcin; éisnociation and in two

natural substances, sewage and skim milk.

-501.
( 13)
Attortgfits t0 3.3011011311111130 1.31.0 P30001100 0.13 {310111.10 1.11 {3’10

1301101110 311133 11113133131111 0f

 

1.111 ’ .1. 0111.111 and

fire-u}? Xivl 000(‘Q? 1:2 p11nn11uc3

...oti'or 01‘ not G I'm a try 110 01: to mod from 11110

 

1mct03103h050 filtrates 0f Vn310113 or1rnisma 19 still a
mmtfior of controvoray. Varnoy and 1r0nfenbvonn0r (GU)
in 1932 wore unable to ohtsin 6 $0300 from 23 strains of
1’111ot0111051‘110L0 filtrates 1’1"1:1<.10d. 210610;; of. 0.1. ($2.33) in
1U 1 r300rt0d the nreaonce and 1001M10n oi a G 1'0133

131011 £3.11!" 1.71-1.1'00111131'fis0 W103"‘1'103i'ifi1--0 i‘iltrato. in the

l

literatwra may 1:0 I'mmd other 1101011011000 0111121033 1‘1'11’211-
1.111;; 01'- 10113133 the 13101301100 05’ 1.33000 farms-:1 in 13110 1-12.01101110
{£1150 flit: 11.1303 01’ various 01101113118. 1.011'01102', it”. 0017:1110
unnecessary t0 cite ‘11:. :30 1*0‘0'0'1009 1.11: 1:114 ..0 1:111 :0. in
V1011 01' 1131000 01.1111?) 101.1013. rcvpovts it 11110 t?'*01.1.__}:t: 01131017310111:
to 11131: 111111.110? 0111131100 011 he 1-001131'1011110170 1111111311100
1:11:10 for __0_.;_._

1. fttompta t0 (101101131. 11111110 G for-1110 11:; Panic”: 1101:1101

111.11, “10131.1": 0116. 52110—111. 01111011.-

         

3110.10 1111111101701? of‘ 1.7110 17111011013101.11111L,0 filtrate 01? 53h
3.1111111111'1 egg-ed 1.11 1-: 11011111111
'1’3310 11133011111113 used in 11:10:10 013-11.31.00 consisted of
orinl filtvutos 01 thiflfld fzmn :31) trans .1003 0f 51 moto‘rio-

11111 own. 2110 filtrate 111 011031 011110 was

    

plunge 01. 111;.
flflfled 1n 01H01nts of 6.1 00. and 1.0 co. in duplicate. to
1.3111111 1113191011: 13001: 1-: 111.130 broth, p‘i! 7.7, to which 1.0;.

“011133 111.161 m‘(_}V10’113 1:; E10011 31313011. $117130? 91% 11011391?

 

incubation at *“C., 1:310 1.111951 1.13 101-35111; 1.3.10 Lwroto .1; 1:31:10

.91.

action were filtered through Emits filters. This transfer
was repeated 62113 using the freshly ohtninod bacterio-
3fiugn filtrflfio. At the time each of the eLovo transfers
W090 mafia, 1.0 cc. pOPtions of the filtrate were Almcod

in fi.m0dium and plated a liver infusion nLuP plates.

fine K medium was prepared from fresh hog intestines
according to tho motzod rec0""0 mod t3 "onLLll (UL) 1931.
A 3.0 pm? cont sunpension was mfifie in 'yrouo so 810:: and
bubod in 10 cc. amounts. k’ho tLutoa voro autoclo vod at 15
saunas atoan.pressuro for EU minutes and incubated to
cheek ntnrility. inc K rzedium tuho and the liver infusion
agar plate were used an ccmirols to check the sterility

of the bactfiriaphngo filtrate at the time of its preparatica.

‘imo brotn culture contfiinirxgé n3. 4 5*; .& £310 bacterio-

 

phage filtpmta was filtered by diluting it with 30 cc. of
sterile fihgsiolofiicnl salt solution and fi' torin; under
a negative pressuro of 130 to 1:9 mm. of L3. Tie time of
filtration was 1033 than three rinufios.

it tVe tine tLe'Iact =1104hh'n i'iltrato x.raa planted
in K nodLan L to 13 cc. of the filtrate was also placed
in a sterile cotton atonnored test tube. These U0 tuIea
cunstituted the material fer Btndyina the effect of rapid
aerial plate transfer on hnctnPiOpfingn filtrates aged in
K. modirm o

laundin 013 a?tor pragnving, these ti I03 \voro ifxcub:1t 0d
at 57°C. for ?3 hours, then at room temperature until the
cmvpletion of the expoviwont. In tie studies fade on
thos a f‘ilt1ntos, $5.0 onos aged in K.xo@1un'x.‘ill b0 Coa-

1‘nut0a 33 series n end the others as movies I. All tales

.53.

more otorilo mooreoooyicnlly at tio time too rnyid oorlal
plate tooaofor nntkofi was nufiliod to moon tube of both
903109, which rouvmsontod poP10€3 of fi;ifi¢ from 1 to 30
day-. T30 procedure consisted in placing 1.9 08. of the
untoviol in each tqu of movies a Qua b on a mterilo veal
infusion agar plate which had boon chocfiod for storility.
Tlo filtrate was sprood ovop tho surface of the ago? with

a sterile Lmohnpod glass rod. After incubating the aoodod

{I

4

plates at o7°C. for “4 hours they wore examinofl at a
vagflifiontion of 100 Q.nmotora for the prooonco of col flies;
the surface of tho agar was then was ad with Q.U cc. of
Antorile veal infusion broth. one co. of this wanting was
transforyod to a sterile veal infusion agar wlato. ibis
procedure nus repootod until the colonies nUDOQPOd. those
were fished ona planted la storilo wool infusion broth for
Further study and ifiontifioation. whore no growth could
be discovered tho sorioa was continued for at least 16
transfers.

Data in Traffic: 13 film 1: Iii-mt groom‘s could to attained
rvom only 15 of 6a macroscoyicolly storilo filt°atos to
which rapid serial plate transfers moo boon apgliod. If
K.moaium.onhnnooo the (Tooth of G forms, colooioo ohoumd
be obtained from filtrates ogod in h.modiun.o shortor
period :f time than from the corrosooading filtvstoa
aged alone. T313, however, was not tho cnoo, indeed no
regularity of envenrrnco was noted in either series a or
b, thus the occurwonco of colonies on the ramio soriol

plates fvom filtrates aged alone and in K medium must be

Tahlo 16. Growth Obtained by Rapid Serial Plato
Trannfar of 33;, gallornm.ngod in.K.Modinm.

 

 

Mu fir

Filtraio'number' Orgwth obtained b2 eerie; plate tranggor'
number ' of ’

' day ' K medium 0 filtrate ' Filtruto alon-
' we lieriog a orio b

 

 

 

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I u

r - I
T - r - ——T
I - T - r
4' 2 w' o T O w :r
WK T . r - T
...} r - T . i
r , T - 1 - 7
T1 c F c ?
o r .\ T - 1 . T
-.(o—‘J 1 . ' - '
,1IIIIHIIEO ' - ‘— - ‘T
2-.» T - ' - '
2—..1: ' - ' - 1
(IIIIHIIP " § T . “r
IAIIIIIIIHE “Ti - '7 o ‘V
GIIIIIIII+ ' - ' 0 it
lMIIIIHIIIE ' - ‘7‘ o '7
.EIIIIIIIIIIk ' 9 r' o ‘fifi'
VIIIIIIIIE " - ' o 'w'
Jfllllllllfl! Vi + ' - "
.QIIIIHII!‘ " - T o ’r
g! = '* . #7: u T
2? " K V o ' o :7
. , I 7: T . T O T
T T - T - T
v T . r . f
i j T O T 9 :7
V . ' O '
i I I . 7 - if
r E 'I ‘ v - *‘V

 

o - Growth ohtuinod by ragid aerial plat. transfer

. o no growth obtained by rapid oorial plate transfer

~54-

Js". a . I -' 1 ‘ . . ‘~ ' (1. . .1‘!’ -;< ,v- ‘7 .0 ,w 4‘. -~ ‘91 .‘0
0.313.331.1330100 to 01.1.0.0 31.001093 1.11011 C3.L3CLIL,. 3.3. is 1.0000030

Q

not (30273031110311; 0371 {3'20 31.06.133.113 13.00:}. 131.1330 2111117310 .17 :3.- 07

11:11: 1331000 “@110 3223100 {1:11.53 1317-3053 of 0010:1103 130 c "rum...
$7330 1 11:29 a. 31331'11'101110011t, 00'10011300 00 133233;; 1.302131331115

£11.30th C3130 1.111. 10 (2101:1133 1:301 , 0.1.3qiLwL1L 031‘ 0.3.0131; r0630 which

0130 {313031 11“.; native. 31'3030 170300 3110113131; 33.13.21.013 :3 33013119

 

0.11:2 [3130:3001 the filter 1:1 (31:21:31? 1.2.0
nonzml for-1'3 or 3.1-3. (.3310 11211011 (211102213; revertcad I to 33230 1123111011,
{30001100 13330:] 17000 21000301300 (‘31) L20 1‘11“”; 10: 11 :1“ 31’ 113011
3.1130 13113300 of 0011100 0., £11306 {3' 11:10. 11 (3.1130, and from the
011303 01“? 001110913, 0.; 0:1 1:3 011:1 1.1}: days.

Typo :3 . The majority 03? 0111111113011 11000310110321 formed
translucont, 0:3103'H3'31' 007-023;: :3, r100." .. 10133 1; 33 C3: 34.3131 [373.
to 0.1 17:31. in (unmotnr at tho and CF 13:0 11301.93: 83- .011 33:11.00
at: {37°C.'1.:0 Cumin 010.513 1.090 Pi 32.1t0 {I '01:: p0 .it-ivo 00001.

3:30.00 00.30 13' 1'1: "133111 I' to 1330 G t." 300 (213000113de 1:;

000103 at a] .(9d) 105.1 and fucox ( -£) 1JC1 b0 12 (“tor
0350310215144 000 01° two wool-'3 at 901.11 t:01::300:1 1.1.3.330 ti 0 001031103
boa-1:39 110‘1013 011C? 0 "300110, 11100131301115 2 to 4; 311. in (31:31:03. or,
Consisting; of 00001, 111 the 0331011: ,03-"0311'. of tetra-"1'33 which
3.10300 C130": ponitivo. '13'12000 0.3.3.: 0111:3053 130130123110C1 3131:0530 of
11110 £003.10 ‘3"“120433 and were 311103033311] introduced £13011 the
air d‘~~-31“i-I"~{£ 1,22. rapid 13030101. plating; toolinic.

Type c3 3'03.": pd 0:331:310 (1'01 .L';, 1:3; to, 0.1111 Cam-«1.30 003.013.1013.
310 0.; 133331913 :3 0031;131:100 0001.1 11 nwuw 30.003. 37.0130 0115111113110
11039::- 1101111100 0.313.? 3.30110 9111130137137 (31311:? {3.113 cmtmflzmnta
or were pros-cant on ti‘m 0401' 3310.110 ‘01“01'0 it 3:0 0 3006.031
with the filtrate, 1:113; 1.25.110 to $2105.? 010W [1101.151 €11.13. not

03373001? until after 133:0 37:31:11.0 hat“; 130011 1210111 03300. 1303101301

.3 320...... no 03.3» o» .333 .3000:

30.33.“ on 0.3mm“ gangs: ... 0025.: 00:305.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

bihvfimom caoawtmb 0103 +.b. mugs; b co, 3 b m .0 00m
FIheipnn 000mm.mp..ltmb00 +,p 000:: b3 edm . .m3t . dauJI
. Humbwflmmfiufiw HP 35.“ .. r mmomemoo 0 mg. F H . 0......“31
b h...!....ho.., 0km... mu CAMP may?” ... - mi 41.... P M b 90m
#3! ............H......«.. .. m p H0330. + P 304me ... ._ H: r... p a» p 3.3.3.“
bl 2.3.0.43. sub «3333 A. . ”......OgmHOCH ht! pom” P of P L b QM...“
0, 00000;. W. .1mvooo 4 b tcHHaH p com .5 H.“ .b, r. n .1 00m

b 3MM000 . Acocoi¥ . roflweh. b. o.u .a.0 p 13 .H p nHm.l
ranshOHHw C. .Hu. Hr mask .... » mwodhomoc 1...... L . .. .0. .3 *1 L E... m.
b3. 0.wuuwa m. «0000 4.b Bofiflmp p o.m _ H241 3L .JCN ..1 pm“
030000~H00.H00 b . mung I b 000H00403 » Hflr » aha
bihmfiucw,wm0me. 003; + . ausfin . 3.!!W r fimflii
b35000HH00.H2w a. 0:00 I p mmwahoaoo b .b _ 90H

. égfioz;w m p «3030 + . aoéfioh. b . m . no 11
b; uflfivmg. m . .euweec + » roaaaw P, . n W » .WWWII
. amumamm m . «0000 o . aofiachl . ..n . . N . mcflli
C p b » LIN - b b

. . . enmeshemfi$v .Momfi H . mummz mv . . a

. . Emaazmuo. Esau . mzu . hazaun 000 .

. . Ho . «a M003 H . Iadpuonu heuma .OmmSHu aufiam .

. can 000 . 000x» 000 .hwamm h:oacu . oeaflcu knoaoo . Hflauom . 0090:;
. 0:0 @039 .nuaum 00mm . M0 hoaoo . Ho heuommam . as £01000 . omsaamz
b b D b a b

 

omanno«uouoaq mo nommnzhu on0ma aswmwn cums; hp canaaaun 000mm oucuazu

3000000 3 :«

 

can: firps-mfl..~0m Ho uaazuaa«m

.ba mandm

Gays lonLor than tic po?1od used for checking sterility.
with the exception 0? the fcur culgurns in type 1,
the Opt-{13113118 Prrocwm‘oci (1216, mm r-nVoPt to ___I_}_,_ 1:31.11‘Ie__03"’=u‘9.
Eho aged filtrates tboxsolvon were lrtic for ggga,
gallownfi, wfille filtrntos of tie ovinniszn rGOUVOrod Ly
renid sapial pinto t?cnmfor we?o not. All of two colonies
tvgo 9 and 8 oltainod closely rosovblod & colonies
upon troir f1?st an on?cnco 9nd during ouhsoqncnt rapid
aorinl plate trnnsfor, but uhsn stauc'ng at Poem tempo; aturo
for Ono to two weeks 1101? nnponrnnco van (roatly chnnbod
and 0réinnry typos of colonies arose from £10 rlcrocolonios.
Whom those “ colony revue of 1rd1unry bacteria arose it
was d1f”ic:fl.t toui11wcntiflto'thom from the G colonies
doocpirod by Inaloy ($3) in 1931. fire P".;11d sorir.l pinto
transfer 1c w1n10 was 913,0020d of having been tho source
of the colonies oTtninodi yon tio tilt: ates. 10? this
ronnon a clock expuriwnut uno plannofl 131ch olininntod
this fhctor.
2. fitiompta to Gononatvuto 3 forms vy subculture
plate technio in fi.noa1 1.1nctov101ha 0 filtrates of 5&1;

1. 0?“ W1

   

In part 1.tho preaonco of colonies resembling G
Forms, as coyozstrvtod by re p 1a cerisl plate transfer
in 1:310 lmctorioflgrunt) filtrates m" __;_._ “~11 1mm"; :1 ed in
K medium, was studied. xnrt amonsurutoo £210 atseneo
01‘ G fort-"3 in t’ 0 ii r-nflimtw. Ir-ctor’ionhrzto 111 r0130 01‘ _"_;;_L,

mufigorum.thon tro influence of 1&0 rapid serial plate

-57-

transfer method wet; eliminated. iii-:0 preparation of the
filtrvtes was similar to that outlined prQVieuely. To
(Zupllcete series 01‘ 1'1 rmdixm mad plain nutrient brief
:ztrect broth, pi? '7."?, tubes, prekuely seeded with
$13.11. 1311130111131. was ed: fed bacteriegfinge filtrate in
amounts of 0.1 0.1161 1.0 00. 113.30 1.!) co. 01' the filtrate
of each of tie 4 series wee edfled to atrrile plain nutrient
beef extract; broth, 1)?! 7.7, sterile 1’. nodium (Lii‘co), a
sterile cotton eteppercd test Lure, a glues sealed ampule,
cad a veal infusion nLer plate checked for 110511113 by
incubutien. ?hese culturoa carved as a control of tre
sterility ef‘ the l‘ecteriezi‘m;_;o filtrete at the time of
filtration. The first three n ntiened also furnished
the rmterinl for tile cxyxrriz‘mnt. fifter 2:2: l'xem's’
incubation at zit/“C. the E: medium and plain broth tu‘: ea
containing; {33.710 bacteriedmge filtrate and: ;_;§,_,_ imilegfiup
zero filtered t?>1»cn.1LEz I‘erl-zefeld 1‘5 cenfilee and 19011391
filters previously'checked for their utility to retain
Elgrr. run.” racers. A hotel of 10 transfers were rmde.
{El-.0 i“: medizm and centre]. tubes cmtaining; 110th lerlzofeld
end Leitz filtrntea remained free fren.mror0300pic changes
for six.menthe; thus we may assume that neither erainnry

center: ientien nor 1'27; I .1111 err->1 was present in t3 .0

        

filtrate at the time of“ filtration or mfieeqnently.

As rentienod above, the results of part 1 txfiiceted
that the remid serif-11 plate transfer untied raidzt be
susceptible to OOntCfl“.ifl (123.1011. Fer this reason a variation

in treatment; 01‘ the filtretoe was 1:13:30. In place 01‘ using;

the rapid eerie]. plate transfer notice, subculture plates
were neeo at intoxwola of four weeks Wet-1 each of the
tubes of 1:120 four series of the meteriedzege filtrates
aged in '5‘: medium and 0*" Use controls. T110230 plates were
m-zru-zined for Lrovth ei‘ter lacing; incuheted for 445 hours
at 37°C. one. again after etenriing, two weele at 2:3"6. 33o
growth was obtained on any of tine subculture plates Wide
either frcm tine lame-12m or the controls. Although in
part 1 cultures egecl for a abniler pericxl chewed growth
when the netted of rapid aerial plate transfer was agroliod.
1120230 (late indicrtto that coloniea roeezifl‘slim: G tg'mo my
appear from the extraneous sources of cootemizmtion in-
herent in the rneid eeriel plate transfer tecknic.

5. fattm’:‘.pt8 to dOEfizOI’H’Jti‘flilo G femur; in the bacteria-
13110430 filtrates of gtmfiw. morons

til-no I".flt01“ifi1 for this experiz'zent was olteinod from
14'.) daily transfers of the t‘mctorioflmge of gig-inn :3, gcamgug.
{motoriogfimgco filtrate in 033101315313 01‘ 0.1 and 1.0 00. was
ad ed to glein nutrient bee? extract broth, p3 ?.7,

previously seeded with $3" on. One co. ..ztzoamta of

 

the filtrate were placed in sterile 1'; neclimn (3315300),
sterile cotton steppered toot tubes, and on sterile veal
infusion radar plates. After 1731 heurc' incubation at 37°C.
the tubes containing: 1_.,ecteric~1im;o and :te‘h. gm’eug
showing the {Teotoet ly'tio action were filtered and the
filtrate treated at clove. 23:19 was rOpoetod 10 times.

‘l’hc filtrate in sterile K medium and in the cotton

atopxn‘od teat tul‘zee (control) was ulcerated. at C2'2'°C.

for '72 lawyers Lima at; Pom tmwpmmturo mam}. the cmplotion
of the oxmrkzcnt. 33:0 visible vowni: hac‘i npgoaz‘od in
either series (”2211* mg an 1::1cu1mtion put-10d of 91:»: months.
Lt intervals of four woolza mlbculturo plates: wore 1:331:10,
which were 1110:1123. tod for 4-43 hours at 237°C. follcmod by
no I'm-:21: a * {253“0. T30 growth was o‘lvtaizmd frat: the sub-
ulturo plates of 03.1th sermo-

:mnnry of Emotion 2'3.

"l- 3%
I {Hath-x;

1. l-‘ncttwiofimgjo filtrates of .‘._ and

     

$51 2311. Win aged in f: r;.:0dim1 togathor with controls in

   

plain nutrient beef ext act tram, pi: "33/, and in cotton
stepfmrod test tubes were 8t:.1c2.10d for tie presence of“ C.-
forms.

2. Organlmza fanning:nut-000103103 (3f: tat-.308)
resemblim; G forms were ohm-aimed by using; Pa pit-:1 Box-'18].
plate transfer tad-mic on the tmctmfiozi‘mgo of ‘53...

T... v.11, 392w aged in K 172961111221. {L310 so upon (IL-4111;; were shown
to be £110?! Erna-115:1; air contaminants.

n

a. 23::9 z'fi‘csonco of G f‘omta in tho Tmctoriazfimgo

filtrates of 35:11. an; 071.133 or

dmonstmtod 17;; subculturing; portions; of the filtrate,

'2‘“ 0.13%; could not be

- 0.1135011 .

       

aged in K medium or in ‘cmtrols, on sterile van]. infusion
agar plates at intoz'vals of four 1.700123.

4. It was alzotm that rapid aerial plate tmmsfor
must; he apgliod 2:11;; nation and! b0 carefully controlled

for accurate results.

~60-
to)
The Production of’aioroooloniea of fiaoteria by Ehysicol
Influences

1. The effect of touporohure on colony aize.

Hodlo' (23) in 1931 advised shot plates containing
inooulwm of the G tyyoa of organiomo ohoold be incubated
for 1 to 13 days at 37°c. before examination for the
formation of ; oolonios. iho oolturoa with which he
worked pooooea on optimum temperature of 57°c. as do the
cultures in Sections A and B of Lhia paper. Lruocknor
and shormon (7) 1932 reported tho isolation of G or
prixitive forms of bacteria by a method of dilution.

They udvieod ihut the tubes be incubated for 72 hours
at 37°). and then placed at 25°G. for two weeks before
they were plated.

To fietorqine the effect of temperature on $39 size
and structure of'bootorio grown at other than their
Optimum tomoorotoro, 16 cultures were selected for study
that were 0 toinod from eXporimente on the bacteriOphuye
0£ §gl;pu11orufi. Five cultures of the 6 forms 0f.§&l;
oortrzoko obtained iron Lurox (64) 1951 unfl 1 culture of
Acre: oero;onos diosooiuted on 0.25 per cent lithium
chloride veal infusion brozh. Shoes cultures Vere planted
into vool iniaoion broth. incubated 24 hours at E “3., and
streaked on veal iniuaion and litmus lactose agar plates.
rhoso plates were incubated for 72 houra at 3 ’c., folloo-

ed by two weeks at 25’s. Qho average diameter of the

~61-

colonios of each cultuv'o was rmoaourod at me (and of '{3
hours and at tho and of the two woolza' moulmtion poriod.
The data in 3:11:10 Iii: 31.3.ch that there 13 a Izumimd élif'or'onco
in the colony 91m I-aofoz‘o and after the maturation period
at; roan tompornttmo. Lovrwn‘: of tho org/anions Very close-
1y roemnblo {farcfino Baton. 12.11 of the colonies at the
0nd of 13.310 1.2'2c:,1t--:.2tion at 33'}'°C. resembled " £03323 in also
and structure 12211: were greatly changed at; 122:9 and. of room
temperature incubation. 111331105139 plates incubated only
at room temperature showed XIQI‘Eml 8120 colonies at tho and
of 772 hours. 21:18 suitciootod the use of a pure culture of
M. 23320:: in an effort to éotozmino the effect. of tomporntlmo
on colony for-ration, because it 13 (.1 0102.7 crowing, 0123212191.:
with en Optimum temooratnre of amt-025.1 12190137 530°C. 'i‘wo
plates woro streaked rim: 1! 24 hour Culture of this: orumim.
(mo was incubated at 2:2“:’°C. for 73 hours and at room
tmzpemturo for 14} (Faye; the other was inculmtod at room
tonmraturo for 1'7 days. ‘1‘!"20 roaulta are Livnn 1n ’i‘ol'rlo 19.
It can be 530022 tat-rat. similar results woro obtained 21th a
pure muturo of 4;. lutoa :23 with Davao-(.21 of.“ the suspect-
ed G forms from various sources. Itctorin Lmdor adverse
tompomturo ooméitioam may produce nicro- or If: colonies.
This oxoorrtzont offers an explanation of the phoncn‘zorm
observed with. G cultures: of Euros: 9:230 10.

2. The influence of rapid 3019:2131 plating on colony

£3130. T270 cultures were selected, to: y . :2 having an

 

Optimm tmnpnrnturo of {33°C. {2310.11 1:22.030 orgmalm'fs 0:330

{tram under $2: 1? optimm tempers: are roouirorzonto they

Table 18. The Effect of Temperature on the Structure

062.

and Size of colonies.

 

 

 

 

 

 

 

 

 

 

 

 

 

r U a? W”?
Culture'Broth ' Golani1 diameter ' Gram etain' Organiln. '
fiumber :84 hourl' ' ' '

I D
""'"""""‘f W r r *T
' or '0 .0 06 ' 2 ma» ’ + on 1 ' '
e o a -0 2 mm. ' e 000 I ' ‘7
a! Q ‘ e o - ‘ ‘7
' 0 - r 3 o
a: 'T D e
' - r run
0 qga_. e ‘
" . r orun
0 ' e o
r, 0 e *‘ ‘1
V 0 e o o “ ‘V
e ‘ e r '—‘ V
n ' - r are:
o e
I r {'3 O m . r T
a *To -' mm ' e "V ‘V
. a: f.) o f r t
d ' -o o o o' ' ‘7
our ‘ - e *1
U 0 u e j
o O - ink» e “
z o 0 -' Ega'V' o o '7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sable 19. She ttfeot of Somporuturo on the colony 8130

of tar. lutea.

 

I1

 

j
j 1 21:11.1 : FEW 2 .

T
24 hour' Colon" diameter after'24 hour’ Colony diameter aftor'

  
   

 

broth ' 9 hours days ' broth 'vgfhoura VT 14 35:9
gulnure' 3 *6. ' 32’s. ’cultnro' 22°3. ' 2233.
... T V I

 

clear 'leaa than ' 3 m1.
: 0.01 mm.

5 mm.

T
i
1
clear : 2.5 mm.- :
I

....
«ecu-q

 

O04.

{'03. :‘2 a 3 2232.312'ntnlg tho 10.2.0 81223 00103210... about 1 to 3 rm.
122 (31321212313012. '2': o 133.301.0001 consisted 1232 2322212212222; 2223221221
231:1;0 tz'232221"o1'~s on 022072 (32211322220 at 3"“C. 01.0 at 252° C.

'I.’: o (:22 Luroo 23232 0 planted 1321109.-..{212'2 hoof 0:2" tract "moth.
(22-20 00. of 22210 latter 222:5 placed on 12. 312022110 vonl

122925212222 12., .222 31:2 to t2“tor 2'3. 122321223! 132022‘2'2t2tion (22223 23230212er
with a otorilo L-ohng'md £119.35 rod. The plates 2322230
inculfltod for ”.42 hours (2226. 922272122021 for 0010223} fom'mtion.
“12052 22131322 t1: on wanlod 22"1th<:2.0 on. of atorilo voal 12211151021
t3r2t2th (2132.1 the 2222523513133 were (2111113061 1 to 10,000. 213220 00.
of the (I 2122212222 was 22:23..ch on a sterile veal infusion agar
plate, and moored 2-15.32 on L-al:2e.;30d glass rod. 211.13 2722.3
repeated 1?". times. 2;; utoo for-2222361 2.2123220001022109 at S?°C.,

formed

 

and normal 133-303 at room temperature. mm“ .
12232332221 23130 color-212.23 ct PDQ“! tm'22g2om'L22ro and at 37°C., but
after tho 2222003251 13132222 for tho (3610:2103 at 255°C. lost their
pig-22.3221; fo 22212.2, ' preportios. into. 1'2 Table 22) 3122327 t3~22t
rapid 3322,1221 t2.2.22:z3"2'-1‘ 2222.823022t1y has 11’5th effect on
colony 231322 except 22212022 temperature is also a factor. The
important np-flioatior; 1103 in the foot that: 122 the 220131321
plate transfer of Laudupoy (2 G) 1927 R 2d nauloy (“3) 1931
“31:21:00 are 151210111‘11321'1 (at 37°C. .132... Eaton] in particular
(2222.3. 02.122322 22.2122 02323.2 8.221221312133 in {oz-3012121 having. cm Optinmn
tow 23092222322130 of 122°C. 12222302" Lit-o 02"2n231tiona of this tootnio
two 113213-122 to Tom r102mcolouioo which {any to 022311;;

confused with tho two (‘3 33:39.

Table 23. The Influence of Rup1d Serial Plato transfer

on Colony Size.

 

 

 

 

 

 

' T w“ '
bay of ' Serr. indioa ' Eur. lutea '
transfer' 37’2;1 an 9: ' 37°Q. EB’C.‘

‘7 F T r T

1 '2 mm.(rodljz mn.[;p¢l ' m (3.31 naJ' 3 am» '

T 1“ U 1 l

2 '2 mm (redlfa mm {colorlcqglf H (O 81 mn}' 8 mm '
__._ T . r- T . T but ' t :H
3 '2 2321423021 I‘B mulcolorloeal' 2.2 (21.22132221' 5 m'uL '
1 W ' I V" i w"r

 

 

 

‘1‘ . It) . , I pt I n 3. U a '
g _‘d 22211311262315w mm.(00101le§§2 m (3,1; unit 0 mg,
' 'g mm,lrcd)'2 mm.£co;gr;es§l' H>Lo.fil mml' 2 mm.

.1

 

5

6 '2 my. [red ['2 2m,_(c3010rle£23 '
I T

7

B

 

 

 

 

.66.

3. Z‘Em effect of selective pltzfiing on colony (112.0.
A 01.13.451.110 of tiqrg'. 13372951 was; plantecl in plain has)?
extract Inf-032.1 and in 0.93 pa:- omit lithizm chloride veal
ini‘asion broth. 231030 tubes were incubated 2'1- hcmrs at-
57°C. and streaked an sterile litzi-me lactose and. veal
infusion agar plates with a satmiLI-wt needle. The plates
2022's inmzbated {‘24 hours at 3WD. and 0:: mined at a
xmgxii‘ication of 100 characters) the largest mud £2.29 srm11-_
oat 001011103 tryore transferred to 31:13.21 beef ozzract 12:90:11 '
anti to 0.9:?» per- cont lithium chloride veal infusion broth.
Those tut-res wax-o incu?~ated 2:1 hmu‘s and restmalmd. {Li-31:3
process was: Formatoa 1:": tin-03. Observations were manic on
the 0010113? 5120 and structure througfimut flue eximrizwont.
late in 'i‘ablo £31 slam? timt the only characteristic tend-
(may 19 toward. a madinn 912.0 of colony. Cultm‘es vii-wish
produced a greater murmur of cclonioa of 0.1 1:23. when
picked to broth. and streaked on agar plates may deem a
majority of 3.0 1:371. colmioa. The 881110 eff‘octhulds tvue
if a colony of {-3.0 mm. 15 1:101:06. from a plate containing;
a mmir-rz'm of this size} tE'se'ro the tendency is taxman-1:18 tho
smaller colony also. were was no &§)1_")£'.P031t permanent
effect an coleny Biz. produced by aoloctiVO plating.

4. Coytmrison of serial platis'xg trmmfor and aerial
test tube transfer. 3310 filtrates of the ‘mctoriOphage
~1110ru2~

of 1:2 mm: and f-‘azird transfers of ‘3}; Lrom 1n

   

bacteriomaco were planted in 1.0 cc. amounts in 1'; medium
ml in vonl inmaion brain. Tog,ot3'-.or with the filtrates,

they were incutmtad for '2’?! Loewe at 3’?°C. At; $310 and of

 

 

.F 03,."

:3
"J
O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

 

 

 

 

b N h a u . b .3 b 3 a b
P fioH L m {P of... - «mum _ Dwain flan {P
P ......w . Hr . "mom P D. b DonLIL. o? b
Li: fiaH . w P “Him... L 31.). P ”H b J ...D n
P. fi. 3 m on fun .3 . C w
P HoH. b N H Mud H H35” h W3.” L A... L-
FDofi b H p113-H n 39H b 3.4 hi3...” -
b 30H L H p 30H P Gov” P 00%.. P 00w.“ p
P H 3.... P m... - H103 . 3&3 L m cm... . 3 TH p
blfioH P H . Ofn p Do P 6.3:! PHaJ .
P1 1...... P N b Nam” - O , Nd? Om . .
. fig: . m . Hqc . Own a H O o o m a .,C a O. a Nmmfi mH o
P L p _ p P h p b .
Li ¢wfl~2d m.u:Hm :3 Guam HQOHOu .
bIHHusm . owaxH,p Haxrc . w,mrw u «H.1d . owth,b Hamfim b wwHQhIP
h . .. . . - all...
+ fiinrgfl . 52; 5:3 _. .....PS 3...... m. Er
h Scam mzzficnwu
ht Mina :awm:m:w H10» n:.n omegaaw mafiMMMWIIIA. wanna
. Go:- wcué N M0 mafia
p .

 

oeuum hzcaou no m:«»3Hm abduoodwm mo oonecAch any cam Danna

III-L38.

this time they showed no growth visible to the naked eye.
One cc. of the material in each tube, K.medium, veal
infusion broth, and the filtrcto, were placed on a sterile
veal infusion san plate and smeared a th a sterile lpchaped
glass rod. After 2% hours’ incubation the surface of the
agar plate was covered with 2.0 cc. of tee sterile Veal
infusion hrcth. This was rotated carefully to mix thorough-
ly, one cc. of the wasting being transferred to smother
sterile veal infusion char plate and the process repeated
until growth seesarod.aruutil 11 transfers had been made.
At the some time 1.0 cc. of’K medium, of veal infusion
broth, and of tLe filtrate was placed on sterile voel
infusion chP slants. These coco rotated carefully and
incubated cs h are at 3V06. ?ue cc. of sterile veal
infusion broth was coded to each tube. One cc. was trans-
forced to a sterile Veal infusion agar plate and 1.0 cc.
to a sterile veal infusion scar slant. The tube and plate
were incubated 94 hours at 5'°C. fit tVe end of this time
the broth was coded to toe tube and the plate inspected
for colonies. Each flag a new series was also started.
Any colonies discovered on tfo plates were planted on K
medium sné into veal infesion broth. Part of the tales
were placed at reen.temposcture and tie renninfler at 37°C.
At tre end ci‘thrcc Geys'in ubetion they woos streaked
on sterile veal in¢usion cps? plates and incuhnted at the
respective temperatures. At the end of an hours micro-
colonios were found on the plates incubated at 5?°C.,

while duplicate plates at Penn tenpcveturo showed yellow,

-09-

fl 4.

piizlonted colonies 9 to 4 mm. in die t1eter. ibeso colonies
were composed of Eran positive cocci occurring in tetroas,
.1113 wees o”: wi We.) only from the re 1111 seri: 1 9 ate teens-
fers end set from.t?e test tube series chic: remained
steriae ts 'OUVHOJb the experim mt. “he result s are given
in Table 27. 11:13 ehner'vr 1t does not prove that true G
forts can not to ebtsi: 10d Ly test tube t ansfers, but that
tfiis motfiod is not so susceptible to extraneous contamination
as th rapid serial plate transfer method.
eumrery of Section C.

1.' savorse temyoretiro confiitions were shown to {ever
the eroduotion of microcolonies. I

2. Likflid serial p3.:1ti11;1";1e<1 little e- "(h ..t on colony
size wkon ccnsidered alone, let eten t mpereture one also
a factor, the predestien or nieneeelenies res favored.

3. selective plating use shown to have little effect
on colony size.

4. {e h~l test tuoe transfer was slows to be free
from.msny of the difficulties inherent in tte rapid serial

plate trans fer re ’6‘; 10d.

Table 22.

-7 p-

Compurleon of the fiuyld Serial Plots

Transfer and Jest Cube transfer Ketaeds.

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

  

 

 

 

 

V T ~"T

Qrsnsfsr ' Rapid Serial Plato ' Rapid 2set Bubs '
ouster ' transfer ' trensfer ’
' ' series 1 to 10 '

T I _T

1 o - I - u
"""""'L r A” r r
2 ' - ' . !
rw 1"w * “1"

3 u - u . o
”t r T r
0 ~ 0 I

4 'T‘ £11m r ¢ '1

5 ' Film ' - .1

T T Y

6 ' Afilq ' o '

W I I 1
'7 ' z: ' - .

fl T Um T
8 ' a ' - '

r T T

9 'trggsfer discontinued - ’

m T ____ T
10 O i - O
"""""t t I j
11 ' ' . v
""""' 1 1r 1F
4:23 . ' . '
T I T

13 ’ ' - a
“w T 1 T
;4 n u . v

 

. - no growth

u - microc

olenioa

-71.

(D)
studies on the biltrstien of Bacteria

The removal of bacteria by passing the suspending
liquid tlil’CJLle‘E'l s mattress or structure hemis'zeeble to
the ordinary ferries of rticroorLsnisms is of value in
preparing water and other liquifis for human consuxption,
in sterilizing thermolstilo substances, purifying filtrshle
viruses, and in prepsrisg vsccines, toxins, etc. Lowover,
the literature is replete wits references which indicete
hat in many instances there may be failure of numerous
types of filters to prevent the passage of‘micreergesisms.
easyghe difficulties which are encountered in their use.
Reese (31) 1&56 confirmed by Please (Go) lShG stated thnt
cley end asbestos filters retained microorganisms, prevent~
leg direct transmission, but trst they allowed the
organisms to tree t%rougi tie pores permitting indirect
transmission. v. ssmsrch (is) less presented ghotomicro-
traphs of stained sections of filters which chewed the
course of the bacteria between the pore wells.

An excellent discussion of the history and technic
of filtration is given by Feuduroy (27) 1929. He mentions
some of the following filters.

The Pasteur-Chemberlsnd filter composed of dense
biscuit percelsin was the first one erepsred which was
more or less Depornesble to esoteric. Bullock end Crew

(3) 1908 stated that these filters retained berr.

 

during five days continuous filtration, but that they sllow

T
-3-

inc1rect transmission of bacteria.
Yorkefeld filters made of Liesclkuhr were produced
in three graded pore sizes, N (normal), V (coerce), and
(fine). Helicon et el. (9) lOuc stated that they per-

mit bacteria, Eberthelle tqfihi and {*1f, figmenferieq, to

pose 11 £0 per cent of t1;e instances 3 tueiod.

 

m1 ler filters, sL’ ziler in structul e to Forkofolds,

were produced in the Salted States fluying the Eerld her
because of the flirtice ty of eLtsining tLo letter.
iilt ers have also beln.msce fPOTI inorganic substances
other the n siliceous rusterill, 9.5., plaster of Paris, etc.
Collodien secs, pnrd‘ment raexifiefiezleo, and 0011016111 3011};
filters have 11x0 :ise been cent’suctee. Bic chief disc
ecvsntege e? the Leitz filter, fitted with ecLectes
61303, is the difiiculty of preventing contamination Que
to air leeks.

where have been many theories nevenced as to the
reason tLy tee erie ere retained y filters or eermitted
to pose. Leee Loy Le clnssii 'ied in {our LPGUpS!
1. Frejertiec of the filter, 9. Properties of the
organism, 3. Preeer tiec of tLe recline of suspension,
and 4. She technic of £1 t ration.

1. Preportioe of the filter.

a. ILetelect ricsl czyurg e oi‘the filter. :Lero
exists between the filter and the surrounding liquid an
electrical field, tLe Iu1.heltz-Lphn 1r"er. Tqu ald Eudd

(CB) 192$ a, Llievc and SusPes (11) 1937 stated that

00110631011 norflmanea pomjesaod a positive electric 031211130
when an noid suhstnnco was filtornd and a negative
notentinl with on nlkpline mixture. Kramer (37) 10??
found that filters of siliceoua earth were flabutivoly
charged and did pass certain organisms. By making filtara
with plaétop of Prria, a calcium aulfnte 9nd calcium
carbonate mixture, the charge became positive and the
filter retained the organisma filtrable through the
siliceous onvth riltnrs. A neutral filtor did not retain
any of tho substances filtered. Kramer (52) 1993 reported
the construction of an nmphotorio filter, that is one
capable 0? removing both positively and negatively charged
00110163 nnfi pnrticlos. this was mofla by ndéinu a water
inaoluklo, thermostuhlo basic material, E50, carrying a
nositivo charfio to the siliceous material in tho filter.
F0 stated that this would retain tho virus of tobacco
toggle, rhetoriOphago, and filtrable bacteria.

b. Tie method of testing the filtar. Lrobiahor (21)
1925 found that testing filters by air pressure was in-
adequate. Eiltora tested by this unthod ullOwod organisms
to pass.

a. Aésorption. Eliavn and tucrez (1a) 1927 reported
that oéaorption pinyod nn imuortnnt pnrt in the ultra-
filtrntion of bacteriophage thrauth collodion membranes.
Eng first 50 co. containad no bacteriophage; later fractions
contained thn lytio agent with the naflo titer as the reeiéuo.

They rooortod hora ndsorptioa in a positive suspension than

-7 4.

in a negative. Lqu (53) 1997 reverted thrt eCeerpt uion
played an ineertent part in filtvntion. ”rPHL: and {Games
(39) 1980 reported that beeteriefiheLe was adsorbed to the
pyetein frnctien.

d. Fechnnieel retention. Equ (‘1) 19”) reeerted
thet mechanical retention due to crannier;e Lei g ceu;ht
amen; the pores of the filter neeisted in their removal
from t}? e Pieeenfiien.

a. Fave size. Lucid (5.31) 19,..3 reported that J1? 1‘10
gepeelree did not filter through ierkefeld a filter
candles unCer a pnnza ure ef 0.0 to 76.9 on. of Li§bl1t did
threuih.fierkefeld V filter candlee.

2. Properties of tLe organism. Sued (53) 1997
feund size, netility, n23 flexiLility to he very important
in tPe filt rebiliW er retentien ef cranniere

l3. Prepertioe of thy rr'2m11un of suepnneion.

e. Cyanogen 1021 00110021 trs: tien. gimme em {err-sea (52))
1“}0 rover ted tiaet hectorle:ere wee rerzoved 1y M'rltler

Hid Leitz fiIters fees seepenaiona et p2 4.5 to 5.0 end
pH 9.0 to 10.0, by plaster of Perle filters at pfi 7.0,
hit ensued at {ii 41.!) to {3.0.

b. Kimediun.ienég11 (38) 1331, Verney end Prenfen-
evenner ((6) 193 P PO)0Pted that cultures in E.nedium pose-
ed mere 0 -1013}; and in lflt‘é e? emecmta ti an in 021?.wa
cult ure nelie. A aimiler ef?ect was obtained by nrrely
suspendint the ergenimma in K medium. They claimed that
this was net Cue to fut, Lut to may epbed prof eine.

4. Yfie teehnic e? filtration. TLe tir 0, pressure,

-75.

volume filtered, and manipulation of the process of
filtration is very important. I‘irobisher (21) 19:28
attributes ultrsbcstcris or filtrsblc forms to poor
filters. technical error, or imperfect autoclaving.
Elisvs and bum-cs (14) 1928, lieronfonbrcnncr and ruckonfuss
(6) 1927, Ludd (63) 1927, and other authors reported
retention of bacteria followed by passage as volume filter-
ad was increased. owners have reported passage of bacteria
with increased pressure.
tinny authors did not [.170 sufficient data concerning

the method and conditions of filtration used in filtering
s particular organism to permit a complete interpretation
of their results. Thus we discover in the literature con-
flicting: reports on the i‘iltrsbility of various organisms
Visible through the microscope in their classical morpholog-
ical state. In many instances we may assume that due to
one or more of the reasons previously outlined that passage
of viable bacteria was accomplished. Among the organisms
reported as being; filtra‘olo or having filtrsblc forms

associated with them arcs

Es. _ L Andcrvont and Simon (2) 1924
JUN...» tolion 46) 1921

% mm Kellen 47) 1926

3 E! 3 ' 5. n : a -: . 30’1“ Rm Valtis (49) 1986

E um mg. Laurens (42mm) 1907

y... u.- Burnet (10) 1926

L3 my -; - fonts: (19) 1910, Valtis (65)

199.4 amend (13) 1924, Dal-cm!
and fiendromsr (12) 1924 Arloinc
:36. Dufort (a) 1925, Vote:- (68)
26
31‘ m Almeuist (1) 1911 Wisdborgcr
and Iicissner (20 1925 licudm-ay
(slings) 1924, arm-e1 c and

Fnudtroy (99) 1929, rojfiin (17)
1135.3.)

hort (92) 1917

Eaguohi (So) 1919

Envy and 1.1mm (:39) 1996., "i-reinl
and ilnghorn (5) 1909, hicollo
and flood (57) 1914, ‘-.';d':11m011
(69) 1215

   

 

  
  

 
 
 

 
 
 
 

1‘ T3" um 311'" '
vii-o .1 \,-¢ .

   

   

" ElEEE.and uenlbecn (so) 1215
‘ ' ’ *Pm 308mm (en 131., 2;;me (16)
0"“‘0‘193-“1923

“1299 Di 0’ “‘- ° Pollen ( 1-5) 1372..) ‘

" Innr (34) 1994 ,diForelle (29)
19253, f’nucmroy (53.1) (2L5) 1021,
Tonnsolli (GE) 1925

d'i orollc (2 ) 191-32, Iov.dnroy
(21)(22) 1924, d'forello and
iIEuduroy (29) 1929, Lndloy (23)
193

211222.19. d'iorel'ie (2’ O) 19.13
:i;‘%~.€ ‘ d'Lerello and hnuduroy (29) 1925

 

 

Benin (4) 1923, Eellon and Jest
(GS) 1994 ”01113 (59) 1929 6
1922, Veeiliu end Irxinoiu (57)
1926

1:1 preterm £19 10.35111 (17) 1924

fine filtratiene renertod in this paper may be div1dod
into tJO 018.celficntiono, those in which stall amounts of
materiel were need and those involving larger amounts.

1. filtrations of amounts involving less than 40 cc.
of'flntorlnl. The conditions under which tPo materials
were filt cred is very 1r portent. Two types of filters
were used: Forkefold V (Genroo), fl (nernnl), end 2 (fine)
and Seitz. The Perkefeld filters (2 1/2 inches by 3/4

inch) were tested for cracgs end leeks under air pressure.

Filters ontiefectery by this test were cleaned as previously

.77-

outlinea. The fittings were eyeliod to the filter flask
and the entire enoeretue tested again. It was then
sterilized and teetea as outlined on page 7.

All cultures filtered more fiiluted three to five
times with etevile ghyniolotieol Belt eoluoien, placed in
the filter aseptically, and filtered under conaitions of
temperature and pressure, such that one to two drops of
filtrefio penned per SOCOnfl. The filtrate was collected
in sterile test tubes, inserted in the filter flask (Plate
1), which were renovcfi and incubated at 37°C. for $5 to
V2 heave, at 25°C. for one week or longer, and the rapid
serial plate tvumofor or fire streak eureulture plate method
was apolied. The series of filtratione reported extended
over a pooled of nparoxinctely 12 tenths, ceneieting in
teats on a nufiher of different organim e from a variety
of sources, and under different methods of peoporatien.

A. Filtration of 39;. .11 even and

   

2411*; gag
crown in the presence of bacteriojhate
in.grewn in the

Leily filtrationo of 53;. eullo 1

   

presence of bacteriophage were made tireugh aeitz filters.
Ehe filteetee were placed in sterile fl medium and sterile
cotton atop oped tent tubes. After suitable periods of
aging the rapid serial plate teanefer methods were used;

on another series of filtratione of $91. eullo.um and

   

§t§g%. ;n?eu4 bacterioehage streak plate subculturea were
used.

Data in Tehle 23 show that in 40 filtratione where

the method of rapid serial plate transfer was ap lied a

.-78-

 

 

 

( ]--cotton
plus
---glass

(“W nmntle

h-n-filtering
‘ cylinder

 

 

TV

 

 

   
 

--rubber
stepper

 

 

vacuum ....
4—.

 

 

insert

 

 

 

 

 

 

Plate 1. Apparatus for collecting small

amounts of filtrate. (1/2)

~79-

 

- p o a u p a o -

 

 

  

 

 

 

 

. O . o o a . N . k a m o N . N o . 0v

r b - Sr P 1
.ssuoea M.eaansuwm.nsmmos u.owaumHHM.E=amos.M.e»enuflwm.es no: . .

hiccupszwtsusoo p Echoawrw .me b neausamasscce » EsHOHHo .Hem .

. mousam ehdmflso£3m . hommzdnp oafia Hoanom .uflouucth«N
. so eonspooea mehsanso . canon an eene>oooa nonsense . no possum
.P u st! lift»! .P

 

it.

.nsnefifisa .Hsm no mcasAnaun emaxmo«ueaeow on» seam cohohceom museum .mm capou

-8 C-

.AIIGVHV

filtssble fort-=1 of Sn}. m... was recovered in 4

       

instances and contamination in 13 instances, also that
where streak plaza subcultures were used no contaminating
organism were recovered. In a 815111.181? series of fig. :33.

e filtrations like results were obtained. A

”“071

    

dis ussion of the fonts obtained was presented in section
B . Attozrzpts to demonstrate the I.)roso:'1ce of 6 forms in

the Yflctoriofimgo filtrates of Us}. £R11.10P?._L?‘.

and Sta 1".

   

sarong.
B. Filtration or organisms recovered by rapid serial

plate transfer- from filtrates of ital.

 

bacteri 0; 11113.50

Qhe 16 organisms obtained in port 1 (including
organisms shown to be contaninations), together with 5
of ate mod frost-1

cult mos of the G forms of 3113'. martIWcF-f .

   

Iturox, and 4 I3- cults “es of 5.9.29.3. .osorfono dissociated on

 

0.25 per cent lithium chloride Veal infusion broth, were
planted into veal infusion broth, and incubated so hours
at 37°C. ?he cultures were oilutod with 24 so. of sterile
physiological salt solution and filtered thsough Soitz
filters. Ehe filtrates were placed in sterile K.modium,
veal infusion broth, and sterile cotton stepporod test
tubes. after suitable periofls of incubation rapid serial
plate transfers were spelled. Data obtained showed that
Aof 25 cultures filtered onr HO per cent of the filtrates
remained sterile. after the nethods of rapid serial plate
transfer were applied, cultures WOPO recovered from each

filtrate after the first to fifth transfers. The Game

-131.

stein and tie so; or reactions of tie cu tures recovered
were different from these filtered in each cese. After
a period of 16 months suing st reom.temeesstupe, these
organisms hove not yet recovered tic Gram stein or sugar
reactioxzs of the original organisms filtered. It.is
probable tlv wt 21sec Grip axioms represented.ecntsminstiens
end were the we suit of tie repid serie.1 pleto technio
because they zero not obtained on the streak plate snow
on tuse plates.

0. Sue effect 0; K podium on :ho filtrsbility of

F3" 76‘ @3231 use pleated into 2.0 cc. of 1’. medians and
slain beef mzterot teeth; these tebes were incubsted 2«
hours at 37°C. its some filters, Yorke: 03d L, V, and
and Soits, were used for cool] iiltsctiox1 series. VLoy
‘sese else nod, test ed for sir leeks, and sterilized, so
given voices, after ouch filtration. The following series
of filt Potions were mac? 0:

a. A tube of sterile K medium.felloeed hy a plain

beef extract broth culture

b. A K medias celturo

c. A tLJbG of sterile beef extract Irroth followed by

e K.medi um.cul cuss

d. A plain hoof extract loot” c lture

o. A suspension in rh"siele ice] salt solution

1'. A suspension in it medimn

Ono cc. of the filtrates was placed in K medium,

plain beef extract broth, lactose beef extract broth, a

n5 .1.-3-

scaled ampulo, and a sterile cotton staggered toot tube.

no culturea of chh. gel; were recovered from any of tho

six filtpnsion sorion. hpgarontly K moaium.éooa nct enhance
the filtrahility 0f Hsgh. coli_1n firnunta loss than 40 00.

D. Filtration of camhineé oulta‘os of Each. 2011

 

g2;;,and Envy. gnfiicn were planted
to separaze tubes of’moéia. hftor 94 hourat incubatian

at 3‘?‘°G. “ti-1030 were 00311112106, diluted to 4%.) cc. with
sterile physioIOCical salt solution, and filtered through

a Borkofold fl candle. The filtrate was placed in Gupliuate
1n lactose hoof extract broth, plain beef extract brotb,
sterile cotton staggered test tubes, and on dextrose hoof
extract agar. .

Data in ?uhlo 34 slow that gggn‘,13§1gg,wcs retained
when both crannismn were grown in plain hoof extract broth,
was filtrablo alga icb. g9;§.was grown in K mofllun, and
when both were grown in a medium. fisgh. 07 was retained
in all three @3908.

B. Filtration of miscellaneous cultures

Cultures were selected at random tram the collection
of stock cultures usod fer clasa work in elementary
lactoriology. whose were planted into E medium (uifco)
and plain hoof extract broth, incubated at 57°C. for 24
hours, fillutod with 20 cc. of sterile pfiysiolo;1cal salt
salutian, and filtered through Zorkofeld fl and Soitz

filtora according t0 the procedura prcv3omaly autlinad.

«£325-

   

Tablo 24. k’ilfiration of combinod Gulmrol of flog
Eli and Sign, 1116103

 

 

 

 

 

 

 

Volume 21130:“ 40 on.

064-

Z‘I:e filtrates were placed in plain beef extract broth,

E: medium (Difce), lactose beef extrect teeth, sealed

011211.103. sterile cot-ten stegatwred test tubes and dextrose
bear extract smr pistes. fiasco were insulated fer 43 to'72
hours ct 3‘?"3., for two weeks at 25°C. and streak plate sub-
cul'ifimes more rude. Growth clue to cex'xtenizmticm was present
in 3.0 per- com; of the 3:120 filtratiens media on stock cultures.
The per cent gran-'15}: resulting; from filtraticms in this ex;_\eri-
men: was considerably less mam that reported by .‘t-ro’eisher
(31) 10213. 'ilze letter was able to obtain as his; as 4.0 per
cent: growth fret”: filtrates of beetel'iogflmgje of sterile broth
and enmexirmtely 20 per cent from filtrates passed tire-0134b
air tested filters.

Swmznry of filtrations using small amounts of suspend-
1:13 rrzedilm.

1. A total of 403 filtratiens were triadic using; less
than 40 cc. of summndiug: nedixm. Growth was obtained by
the technic of rapid aerial plate transfer or streak plate
subculture from spgs’oxk‘etely 3.0 per cent or the filtrates.
The cultures recovered from tE‘rose filtrates censisted of
either filtmble fonts or I colonies.

2. True G fems were net recovered from the filtrates
.of cultures eras._u"':x.'160d in less than 40 cc. of amterisl.

a. K medium was mac-rm to enhance the filtrebility

of m zrlrlm‘igc 1n the presence of

4. Refiltrstien of the forms nwcevered gave only

T?" 1"}
. \ .I .

ee 1 .

   

sterile filtrates.

-135.

2. Fractional filtration of amounts involving m“re
than 40 cc. oi'mcteriel

Bulloch et el. (9) 1908, huad (53) 1387, firenfenbrenner
and tuckenfuce (6) 193?, cue Hadley et el. (93) lDSl'ueed
frectieeel filtration in investigatienc of the filtratility
of microorganisms. Ac this method of filtering offers
slightly'mcre difficulties in.menipuletien it should have
advantages which the ordinary method does not poeeecs.
fractional filtration makes it possible to compare the
filtrebility of identical or different micreorgeniemc
uneer the came or varied conditions, especially with
reference to a determination of the moment of passage of
the organism.

The generates used for making the filtratienc was c0;-
ctructed from.uleee water ctillc, Lietig‘c cond0n30?8,
(Plate 2). A ring of glass was removed free the middle
of the outer Jacket, the central core was cut transversely,
and made one inch cgertor than the Jacket. The filter was
placed in c eneéhele Putter stepper and fitted to the out-
er jacket 30 that the outlet projected into the central
core. The smell end of the jacket e:c inserted into ane
and of e 531:: inch section of heavy vccwzm rubber mung,
to the other end wee fastened a sterile medic tubing shield,
Eellmenn (44) 1951. A clamp was placed in the middle of
the tubing. The inner jacket wee celibccted to coliver
10 cc. and the cpynretue cleaned and sterilized. lo choc
that tho epinrctuc excluded all sources of extreneeuc

centcmienticn, 490 cc. of cterile plain beef ext ect broth

 

 

 

 

@"etgn

+--glass
mantle

h--filtering
cylinder

 

 

--rubber
stepper

n---central core

vacuum “ ‘
K/

---Vacuum rubber
tubing

--Liebig’s
condenser

 

 

 

 

 

Ifi---shield

 

 

 

 

Plate 2. Apparatus used for fractional
filtration. (1/4)

WDB filtered in 10 ‘3. {motmta (bnrmnetrie nroesuro 10a) mm.
11%;, time of filtration of each fraction 1 to 2 minutes).

'3‘ ese frr‘actions were incubated at 3'1"”0. for three days ant}
at room tmt'mrnture for two t-aentl'zs. lac grer-zth annenrod

in any of the fractions.

A. Effect of sutculturin" filtrate imitations
L

iwmf‘onl‘eronner and t‘nclmnflmc (i3) 192’? rmwrtnd that
so few bacteria mused the filter pores that the ez-‘xtire
fraction had to be iz'lcubntod in order to Ger—mmtrnte viable
OI’fl"fiif3!ifS. A filtration was rat-.2630 to (intertiine \zhetl‘mr this
phenomenon wuld be observed using 10 cc. filtrate fractions
of a plain beef extract broth 0111?..731'0 of Finch. 8312;. She
culture was planted into 26$ cc. of plein beef extract
broth, pii 7.0. Tide ws-s incubated 2.”: bums at 57°C. and
filtered f‘nctlezmlly through a Iterkofelcl 1'! filter. the
10 cc. fi‘actions were incubated £1 tote, and at we on
of no 17991: 1:329 factorial in each tube was streaked on
oesln-methylone blue agar plates, $110121 which typical
colonies were transferred to lactose beef extract broth.
7330 19.013030 ,utuea Lilia-3121;; {gas farmzrztien were planted into
heaer's 50:31am citrate radium and checimd for Eisnb. £2.10
Table 2'35 shows that III-iinch: e]. was recovered From the
eighth to twelfth fraction inclusive.

5310 effect of sulmulturing the filtrate to other
1.106151 at the time of filtration was c?m;em-xtrntod (as follows.
{339%. SEA was transferred to $300 cc. of plain I"O'~‘.‘f extract
broth, 1acu1:atod {-24 hours at 3’?°C., mm filtered throng-1

the some filter cs Wm used in the previous OIE“)()P1J".O:?-.t.

Qublo 250

Growth Obtained from Fractions

Ineubated.$§ Toto.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I
Fraction ' cc '
of 1!) CL . M recovered from plain '
r* broth igltr-te. ‘
_T
'“T
A 2 : . '
3 ' 1
F - '
flT
a ; «- '
j
5 'r . '
T
6 : - 1
7 ' T
r - '
“T
8 ‘__ .r o '
T
9 : fl 0 4
r
___4 10 : Q I
T
11 j. § 0
1
13 ' O ’

 

« , n v . - ~
3 .19 e; filtration 01 canon fraction . 3 minu

Filtration pressure a .20.) um. Kw

(.3

tea

.. \
«II-U9-

cne cc. 0? each 19 cc. {rection was transferred.irnedicte-
1y after filtering to plain beef extract broth, K.neoiun,
lactose beef extract froth, is sealed engulo, to plain lixoef
extract agar plates, and the remaining portion of the
filtrate to a sterile cotton steppcrod test tale. Siege
were incubated 43 hours at 37°C. and for two weeks at 25°C.
beta in Table 26 show that inch. col was recovered from
the eighth to twelfth fraction inclusive. Thus little or
no difference in the growth spwecring from the filtrates
could be shes-m whether the Fraction was incubated in its
entirety Or by subcultures in various media.

There was no apparent macroccepic difference in the
appearance or tfie fraction at the moment of collection nor
in tte mmnner_in which it come through.the filter. It was

apesoible to tell wLich frect1on weulfi rennin sterile and
which would later she's: growth.

B. Effect of ii. medium on 13-29 filtrability of Eris-ac}:-
922-1....

Kendall (36) 1951 reported tint K.modium enhanced the
filtrsbility of nicroertcnisms. Fronfenbro;ner and
Puckenfuss (6) 1927 reported that organisms grown in K
medium.pcseod the filter more quickly and in larger amounts
than those grown in plain beef extract broth, also that
subcultures grew more quickly in K Hediun,then in plain
beef ox rcct broth. we have already diown that this is
not necessarily true when small amounts ef‘matorisl are
filtered. The follow1ng series of exocrinents were planned

to demonstrate the presence or absence of this effect when

-909

Table 26. Growth Obtained from Subculturcd Eiltretc

Fractions.

 

I" T
Fraction ' TGGOV0r(m after 1 week ireubetion in '

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

of 10 00. p t ‘ cetoee eoelod'filtruueTtolohica on '
'broth'mediem' broth 'o.’“tulo' alone 'Qlein agar '

“T I 1 I r

a I - I - I - I - I - I o I

T r 1" r Y I V

2 I - I - I - I . I - I 0 I

A I r I I I I r
3 I _ I ‘ I . I 6 I . I 0 I

T I I I 1 I *r

4 I . I - I - I . I . I O I

W I I I T A I I TA
5 . o ' o ' ~ Q ’ o ' I. . 0 '

r j V I r r T

6 O I ‘5 I -- I . I ‘ I . I 0 I

I I r r T 'T r

7 I - I . I . I - I . I O I

I I T T U T T T

8 I - I - I - I . I + I o I

T I I I I f r

9 I , I , I . I - I , I 3 I

T T ‘ 1 I r V T

1 I , I , I , I , I . I 8 I
'4‘ T ' fiT ' r A V T
11 ' I ' I ' I ' + ' + ' 20 '

r V ' I r I T

: l2 ' I ' I ' I ' I ' I ' 45 '

 

-91-

1.110: or 111.11.12.13 of I atorisl 11:01:10 filmmed :‘r{'1.ctio:m1;i_y.
To el 01: 11.13'10tinr or not 1;. 11.106.11.117: has an ef1‘oct on the
{MD

filtrehility of a ricroorganiem, cell was 7.1tod

   
 

in 2J0 co. each of? K. {1101;711:111 {1.111.131 plain beef extract '6'1-roth.
filmse were incohetmd h?o1.:re {1t 37°C. and filtered t3 1:100:11
s Iserizefem If: fut-111. 'fl1e fractions were collected.
osc-wticnlly in 5.1;) cc. amounts and 11101111121301] at "1"" °C. for
'72 hears. i1t~c1:.re:"1e (ii.i‘f'l.01'1lty we 0:5311'1rione1i1d 11.1 f’ilteriz'lg,
the 31‘. medium. 15719 time for each fraction increased rapid-
ly as did also the air {110001311110 necessary. In both cases
the re {.6 of filtration did not excord one {11210:} per second
nor fell lose then one eroD in 10 seconds. Ho air hubfilee
"rere cert-rest. The {10 )00'11 nee of the filtration was ncrmsl.
T1110 point at 1'131ich the 0:11:30. x'lie111s unarmed 0013.113 not 1'10 feare-
toléi at 1:370 1:53:10 the filtration was 0 "1310131363. ’.1.’."1e (late
nresentod in Trble R? 8107 tlnt hoe“. g£;é_L< Tow.:1 in a
medium passed the filter after 25 cc. of tutorial had 300m
filtered, however, growth was Goleyed for one week.
Urgznisrm 1:':!'rie:1_'1 moved after 1333 00. had. T1011] Filtered
(1-1121? 01.11:. in 113 11011113. 021,1 {00102110 {gr-rem in 131151.13 leef
11.3mm; 1:10.11 (.1131 not pass-.1 11171:: 11 1:51;: cc. had tern nfilterod.
It is thus comrent that K modim: Cece have a m rlced
effect on the filtrshility 0.? 1.3713 011:1.1111311'1. In order
to cmmoxntrate 17110121110111 this ei‘tect is one to a of {111:0
produced in the organism itself or to 1:11 6 no re ml": ysical
presence 01" 1".‘2'10 i1; moditm, groom-'1 was transferred from
03.111.110.01" 0:5: tr{.0 0t sijor slants to ii nedi'em until a sus-

pension of the {31.1.1720 t11:rl:§.dit:1 as in t*'- 0 nrrvioue e: :periment

.92.

cohohooou eel I a
ozoPQOOh I ...
538. a I .5 .H
gangs gauge a .m.m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P P lbw; o . o
r » 111.1!5 I P C P
b L” f- 0 u I b
r O b 0 MW . b I. F
P 0 LI 4 kn O [P I
I? + 1?! ¢ L C P O p
rt 0 pt .I p I. p I
b . bl - 1b” - b -
.F O r l W O b I .
hi .9 iP O hi I p I. b . . ... ..
r + p .. b .. Hr .. . . 4.3.0:“...
Ln. . p, - . 1 . c .. .mu-I-saznuuu...-nu-uaunna
P O r I k 8 P t b..1i.NiuIi
*1 ' I - b - D! - + h.
{P} C p U h U ? 0 11-
b! - # - ‘1'! i n 3 b
b lo
. b . .I. .. u I a c f
o .00 and . swam-Andra . HO
. nun-00km . no . nauuoauh
. by 081%th .

 

 

.8453.“ H a: and ducks

 

aoguaxn «on: adage a“ usage «sou .go-m no unauunauunam opuaanangoo .pu canny

mm

was ob‘. ainod. 11113 8115001101011 1703 filtered fracticnnlly
and the filtrate 11113006. 113 12130 various 1310121111 1371 1.0 cc.
0:11011z1ta. Data 133103011t0d 1231 1‘01le 531's area that .33; 001
paaaod the filter when 55 00. of." 13111150111511 had 13330171 filth?-
od, this was 111201.11“. half way between tE-io 011031111: for plain
1301f extract 13110111 culture and K medium culture. T730
effect was not so 1312-1111-3011 when K mcfiizm war e :133110157 present
130-3132,.0 . 22;; was actually L110 n 111 the material.
C. 11tt0333pizs to 1301;313:1100 1.23.10 1'1: CLOI‘B POP 130.183.1119
for 1.130 afoot of 11' medium on the filtratvility of “139321.. 991;.
1110 130:1; 110112131113 1.10110 (001331111 to (3.01:3:2111ttix1o 11"
3100311210 11133311: factor 111101333231: 111 K 12.011111131 1.03.116. have the
effect 0?." 11101100311111: the filtrnbilitz; 01" 01133.11 .1.-1113113
0890010113] With 1101'01103'160 to F330“. &. In (1111?:ch to
0113131330 the £111.33» oloLLiini-j 11110 to (3.11053 pfirticulato
r111 11311111 51 48 1101111 c .11t11z10 1.3" :1 filtered 13311ouL.1 sterile
absor130nt cotton, t10n f‘mctio 3011;? 13110 .131 0 5011120130161 N
filter. 1110 filtrate 131113 incubated in 5.0 cc. fractions.
Data 3111093311tr'1d 1n T511310 91') show tits-1t {33.0 01111130 present
111 F. 113.06.111.33 13011111011 11101100530 nor (3330110000 1113.113 .edly the
i'ilt11r3.‘.1111ty of 3700.11. coli. ‘10 01111.1'1r1tn 1:310 possibility
...... ...—......
of 1:130 physical 13110001135105 of 1.110 K medium and: to 051311111311
the fact that the 301111.119 011 filtra‘lt-le povtions 01‘ this
31.1! 31301100 119.0 cm of’oct on 121.0 filt Minty of tizo organism

two armpanion 03:1301'1m011t3 1210119 £1tt0r3ptod.

.94.

Qablo 28. Fractional Filtration of Each: 00;;

‘K

supondod 1n 1: mam.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T ' H“ - t': ' « ,_ Hoover“ '
traction ’ 1 ' 8 . V' m [rte o ’
Gt 5 “a ' -' ' ’

' 13.3. Boo - n rot 13 median

' ' 0 ' ' . . g 0 \, . .n. .\. '

. , . .' - 9
'._ ’ . o ——r - r - WT
» M“' T o ' . “T
*f a u- r .. V - ‘f
’ ' ( § ”.3 T o T . A 'T
« ”mun-J. ' - ' - 1
. m1..- 1 - T - W
3 “filing ‘ - T - WT
. m - f - 1
' t «13".. " T - 1 - VT
1—11.10 ‘ * V «- ' «I» T
v ' Mo ' - 1 o T
k 1 3 v r - r W
VTT—'2%*1 I T - r v
. » "V T f' - T _—T
, 1 1 1 . I T

 

:- Time in minute!
".P. - Baromcudo prcaam in comment.

'0-3

t.

~05-

fuble 29. Lifect of Clamps 0’ K Leaiun on Filtrubillty

of gash, 92;},

\

 

 

 
   

 

 

 

 

 

 

 

 

 

 

 

 

'* I r 3? ‘TTBch. 0611 recoveredi “7
Fraction V k - cured wit‘ pot cloure

Of ' T . BOPO. T . Dupe. QOtth . Wit"! cotton '

5 00. I I " . I 48 I 1 I

' ' ' ' 'houra ’ week ' houra ' week '

I I I 1 I W F I I

a I 1 I 5 I 2 I 5 I _ I . I - I .' I

I I I I I I" I "T

I I 7 I I I - I - I - I - I

2 I"; I 1 5 r l; I "I . I r 1

I I ,A I I I - I - I - I - I

3 Ilhfl'“;J I 8146 I 1* r I I

4 I 2 I 29 I13 I 34 I - I - I . I - I

1 I It 1 I 1 I I I

5 I 3 I 20 I14 I 49 I . I - I - I . I

I I 1’” I I I 1I I 1

6 I 3 I 30 I12 I 50 I - I - I - I - I

“I I I 1" I f F I I I

I7 I 5 I 46) I27 I 4Q I - I . I - I - I

I I "E I r I I I I I

8 '2‘) ' 49 '5Q ' 4Q ' - ' I ' - ' I '

I 1 I I r I I I 11

9 I39 I 49? I25 I 40 I . I :L I - I . I

 

Q a Time in minutes

b.P. I barometric pressure in cenLimotora

Tee'hunered cc. of sterile n meflium was filtered
through e sterile Ferkefeld n filter. This was followed
f3 9 9; hour eiltuze of 3eeh. gglg,1n plain beef’ex-rect
broth. ’fi'le data 3.1 ’i’ehle 30 $31” that (mammal the organism:
pansedumere quickly than when plain.beof extract broth
culturee are filtered alene, It did not eenpere with the
filtration 01" a culture grown in K medim'n. ‘me K meciiium
filtrete free tie above experiment was collected in a
sterile centniner and seeded with Each. 091;, incubeted
fer e4 hours and filtered frectienelly. If any difference
in netefl in the filtrehility of the orgnnieme under these
conditions, it must he flue to a einLle factor, that is,

e 021an e prequeed in the OnL eniem itself 1y soluble, or
at least filtreble, portions of E medium. Date in Table
31 shew’thet t .1e alumna of insoluble he teriel have no
effect on increasing the filtrnhility of Hggfi.‘£g;‘,
Indeed, the oruanieme grown in the sterile filtrate of

K modium.peeeed the filter more quickly than those grown
in mmlenred K Bloc-21mm.

D. Erectionel filtration of combined cultures of
fieefi. 222$.“3d Eeyg‘ indigg.

’re“ g__;,end Ser 2. neiee ’ere select ed to shoe
t3 9 ef ect of t" :e presence of eaotnn“ er h“iea on e
filtrebility of bacteria. One hunfired cc. of plain beef

extree t ITete mg-e no God with Mia; and a like em.unt

 

nfllco. fifter being iaeubeted at 3?°C. for

 

with SQPr.
24 hours the cultures were combined and filtered fraction'lly.

Table 80; Effect of Cloxsing Filter with Sterile K

medium on Filtration of Each. cell.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I T T
Fraction'
of '
5600. . .

I I I

I I I I

‘7' V r

IaIZJIgIbI-I.I_-_I.I
é'z'o'z'ar-r-T-I-1
_‘ 13 _) 07-1-T-I.‘W
‘I 'r O ’ l7.'-'-1—-:—T

JEIIIIHIIIES ‘7 eriT' - ' ~ ‘ - '

W1.T.I-I-T
:' ml. -'-I-T~I
I I U f. 1.I-T-f-T
-EIIIIHIFIIIEIIIIIEIIIlw " -' ' ¢ ' - ' -::'

'-'-I-'.'
2.14m;- ~o1.r-I¢‘I'
EMQ . oI-'-I I 1
1-19 aorT '.'-T I1
17. -’. '0 ILI-1 1 1
2.1]: II 'O'g' ' T

 

! - Tim. in.ninntoe

£3.33. .- Barometric pronoun in centimeter.

giltered

1h

eeli Great

. ‘.
5.90 ”.0

I

.2"? 11hr.::.tien of
Freetien‘ 2 ' B.P.'

'fiblfl 310
terilo K nenium.
I

)5

'3
I.)

 

r.

free

cell recovered

K

T
'brefih'eediun'lfi

‘7
‘eeh

I
J...

 

1.1

of
3 ca; '

etoeo'emeule

,4.

..

lQ'
e

2
' 2
I 4 I

T

 

 

1’

 

I

 

mu

5-")

584 '
' 645 '
' 7:56 '
' 692

.p
'I'
...

 

 

 

'3

:g‘

I5I
I

7

 

TI

904

. 101

 

 

 

 

(

'I'

4.

74¢

IgoI

 

 

B.P. - Euronehrie preaeure in centimeters

T - Time in minutes

...—19

-99-

The following; 0021?“:12'122t10ne we'o filtered. (1) 330311 Crown
in plain beef exerect broth, (2) git-(9‘22. m in if. medium,
33302;? . men in Main beef extract breath, (35) Pet}: in I:
mediw-I.

Date in Table 3.52 mew that in com! inetien (1) Each.
93;; passed in the ninth fraction, 530122;, HUI-3.9; wee retain-
ed; (f3) gel}, egg; was retained, :39?“ meg; passed in

the fifth weeeien; (:3) Path organism passed in the third

fwectien.
22.6.1". was probably more filtrehle than '27" cc! .
co 3,2,. K rze ”1mm wee ehevm to ezfimnce tin e filtrebility 01'

both errU- nieme, h wever. its mere presence was sufficient

    

to cause the peeeege of git-erg. 216.1022 22272110 21:21 e."

 

had to to {Tom therein, in order to effect ”:2 e peeeeUe.

”Z20 peeeere of? rem. £93; in t2» 9 nintu fraction (1) wee
probe‘ely not significant for the feet 1:}: at. it wee leyend
the range of the other combinetimie.

E. 'flao effect of 04121215 £23302}. 29335 in 1’; medizm.

A culture of Eire“, m (yarn in if. 111061114311 at room
12021901222122-1116 for 43 deya GEEQWHG no (37:31.2?! vent clrenUe in
filtrehility from a :34 hour culture. The (late in fable 3:5
show that the eswnniems penned the filters in direct ratio
to the (period were size of 1c gmrticuler' filter need.
waltz filters were 3 mm to remix; 1;} e organisms for the
longest period of tine.

In Table 3.22} is (:iven a euzermtien of the various
exmrinente en the effect of K medium on the f‘iltretility

of 22.229722. 9271;, 21-2050 date shew that a soluble er filtrel'lo

-101}-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. m . w. . .8 ow . 827 . om . E.-,.L....33.._w1. 5,23% a
o b b IJLIL - 2p - 221 p

. m . o . .00 OH . nova . be . gnome nemam . maee9e_m
- h b b P b b 2

. a . m . goo on . (coma . mo . sweep nflaem2. recap nenfie
- p P P * j by if p

. . . a . QLmeen. .

. a . o . dun:- .

. downed .Lumm . «moo irvm@.:eauoanw .. memo .ac . cemmeao. . .
. . goes mo . a“ . nowanfia. an naonm . em gnome
..0m acewoehu :« weee>ceoe . cgbao> . casewohm . a“ mafia. mOchu ukuom . «doc .mowe
F D 211 pl b P D

.eeaaca .uuem ewe

 

 

«Hon crowfi Ho mehzafifio memdcfioc Ho Gadaehafidk H5fl0wu0flha

.mm macaw

moxmw 0m... 2.... ._
p «(Fee :a mndwmoem ode» Tone: I .4.e
.0. n”. .V.-
mwéfiflwfi in QwHA I 4
rt. C

Sateen 0g I o

 

 

0 \o . q. 2!]
v habOOUL .mr D offlmna I #

F
2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 
 

 

 

 

 

 

 

 

4 3
P 0 pc. P am p OH p .9
2" bake. P M!) b .3. ‘. 2P .FIH. ‘.
- .- . -
b O PfiH b M23 .2 u b b2 A. ? mHP Gav - d1
. I 2 v.2 » >2 ? ... p o . b e 0 an
1 r h»... D so.) 3 b a . LHH. ...... P a
0 LI; 0 2 7. p 2 b i b o b + . me. .33...
1 p, H 5 M33 b nu - P #2 .ibafl .... b my
- (Pll - Le? b .23 w.» b - P b - 3 L b A“; h fit“ 2P
:2 p . e 22 m.
bl ... .3." F 2. w». p I p . O 1 p L! m p . m b
bl - 4 .2‘ D an.“ LP ! 2P - L I-L' ‘ 2P (‘0 mused...“
.3 . £2 1. e 2 . . 22
- J 1 L b N P - b - ... Pr - J G1
. .u . on 2 ,br p 3 b o . .H .
(P P 2P O H O - O D - LP D .. . WW 2. - II N b
H- m9 .3 a ,b lb 6 - I 2. '
bkw ORV Hr - 0‘: Cwehfiowa OCH ,4 . b n P o a I. ep‘H h
P N 54 F» f. .2 P J 5; 00”.; 4. O .4 I ...,o 2-2 . I H
Duo; {rt P : .bZOHafljhe . L w #2} c
. V pl? 5. .1. w 2. w. IRWJ “much‘Iu 4 . [v . .11 . 2. -
I? mH02ewee: 2b,: L2; rpneuaeena : . e p
r V r h 1 .1 I... R IN? fluke-”"2“? .‘ . ,.. On." 1.:
...~.202..F.HOMM r. . ». Lszfideoflh
I! D .5... Cumuwumkmfiw

 

 

.omam meow ancheaa a 3 .
22a: anaemommom ououaun

 

. .. n o I: GO .H.Dmuw
e A. 3r ace 3 a mo 303333.» ofiifie
. V .0 u .... rfiwoo .\O.\d o
“a; .Hnuwm“

K

-103-

 

 

 

 

 

 

 

 

Table 34. Fractional Filtration of {3022. c011.

V T .. —T
weblo ' Description of the oonditionn' Izomont of passage '
nnwber' of filt -r tien 1n co. '

I V T

25 ' Plain Lreth filtrate traction' '

' irnn‘utnd in tote BO ‘

V r I

26 ' Plain brozh filtrate frautlon' '
' 5'2_bc"1t—21rnd ' 80 '
I T T
27 ' E medium (grqwn inl; ' 25 '
T V T
28 ' K,tediufl (aenfiended inl, ' 55 '
I ‘; “flF* T
29 ' 3.50diun (cleared through ' ’
' coaten);‘ ' 40 '
r U I
80 'flr1lzor 0102306 with atcrllo ' '

' K moeizlq Lellored b”a ' '

' URL in Lreth QHj tnro ' 55 '

I I T

31 ‘ Grown 1n.K medium pravlona- ' '

: 1y filzored tuxoufh a : '

I

aerkcfeld P filter

uliifio

substance present in the medium affects the filtrubility
of the organism. Prom.an examination of this table it
is aawnront that there is prosoflt in K.noéium a soluble,
or filtrnblo, substance which has a marked effect an the
filtrubility of ysgh. gqli, Table 31. iho more presence
of K.modium will cause this effect to a lessor oxfiont as
shown by Tables 23 and 50. The Cross particles pfiosont
in the suspension inhibit the filtrability, TaPles 33,
89 and 30. Tho effect on the organiamlhny be Bufiiciont
to overcame this inhibition, Table 27 or 23.
{Jury-13.12:] of Section D.

1. In a series of apnroximatoly bug filtrations
involving nm.nnts less th n 40 cc. {rewth wvs obtained
in three per cent of all cases.

2. Fofiltration gavo sterile filtrates.

5. K medium.enhnncod the filtrnhility of Sony.
gaging in ths.prosoncs of Each. £931,

4. The effect of K.modium on the filtrability of
yficg..£g;;,was shown by a series of fractional filtrations.
5. A soluble or {filtrmbls portion of E medium was

shown: to 0313135169 the filtrmtdlity of £5393“. m.

6. The solid, gross, particulats Esterial inhibited
this ofioct.

7. Aging of gscb‘ 22;1_in K.modium.boforc filtration

had no effect on fho filtrnbility of tho organism.

Summary and Discussion
(A)

1. Attempts \2.-'e'z.2e race to produce G ferns by
soontcnecus and enforced Cissocietien of vsrieos bacteria
undergoing; reoid transfer and. siding: is a series of media.
flesh organisms snd.meois were selected so that teere use
presozrzted a wide variation of factors; p11 of xztedium,
concentration of lithium.chlorise, dye, characteristics
of organisms, etc. ih many cases colonies which here a
striking resomfilsnce to the G colonies reported by Lsdley
were discovered on pistes seeded with cultures in the

crises stsges of rsoid transfer. These colonies Lenorslly
obscured after the fourth or sixth transfer. rescues the
orgeeishs forming the microcolonies could not be fiemonstreted
as being filtrnble, they were labelled s colonies according
to the terminology introduced in this paper. ?he 3 colonies
forced during tfie etvLcs of dissociation of the various
organisms did not so oer with the reculerity noted by
Hadley or Purox. 1L0 sets did not show any relationshiy
between the production of K colonies and any of the various
fscsors considered. There seeks to he iséicntiens that
the soccereace of U colonies in dissociative phenomena
may be due to an inherent characteristic of the organism,
retEor than to external influences which may not Le strong
enough to hreok down the sturdy mechanism VhiCh controls
the dissociation of the bacterial cell.

2. The_tochnic of serieléfilutiou and fractional

filtration was swelled to a series of six samples 0; sowoLo

~135-

and four samples of‘s:im.nilk. The resort of breechner
and Sherman that primitive forms were present in milk
in vastly greater numbers than were ordinary bacteria
was not confirmed. no H colonies or filtrable forms
were discovered. The number of samples studied was not
la'ce enough to justify conclusions as to the occurrence
of pr mitive forms of bacteria in milk,lh0mover, there
is a suggestion that many of the organisms recovered say
he contamination resulting from the very delicate technic
mployed, that is, raoid serial plate transfer.
(E3)
:iorisl bscterieshagc filtrates lytic for Lie a 51103333:

and fits 7h ,

"T1n'1
. ‘1'»: s K».

were studied by rapid serial plate trans-

    

fers and by streak slate sahculturcs for tie presence of

6 f' ms. The filtrates were aged in sterile ce'ton stop r-
ed test tubes and in K medium, E colonies resenhlinu G
colonies were ortsined by the rapid serial plate transfer
notice from 15 of Lie 60 filtrstes, There was no regularity
as to their sewearance. Three rein types were found, two

of which were air contaminants. tron streak plate sub-
cultures were used no colonies were recevored. The data
suatest that K nodium.in this particular instance had no
effect on the prodn tion of G colonies from the filtrates

of bacterienhnge. ifiere is also an indication that rapid
serial plots transfer is reasonsiblo in many cases for

the production of G and similsr forms of Lsctcris.

~106-

(C)

2’30 {mahzcmon of 23 colonies, which obviate the
mngmarancg of G 001011100, by various physical influences,
toa'“1.-:1()rz_1t‘1:*0, rapid nor-1:11 plating, and 00100111er plating
12-03 stz.1fl10d. (1’1":0 (Into. nit-ow that Lt-ncteria having; Lin
optimum tonpormure of n31;.yrouz'imntoly £3{;:°C. 11-1113; fem 12
colonies 1f arm-'01 at 111910? torvgopaturoa. £21113 1:) of
090cm}. irz'aortunce because $3.1“- toc?~nio for Gorge-natmtinig
G tonne P00111003 the uncutwtimg of 9101.03 at 227°C.

33501123101? rapid aerial plato- transfor- noz- acaloctiiro plating;

were 01101011 to have Marl-:06} of acts on the: production of

6 13012713 01" 1”: forms of hecteria. Data obtained when rapid

0011101 010110 and test tube transfers were mm ooincidontly

ale-wed that t:10 format- was very ailmentnflca to centnmination.

It 1:23 suggested that the oer-3.01 test; tube transfer be sub-

stitutod 110001100 of its cormzzrativa froodem from contamination.
(D)

A review of 00:10 0:? the filters 11-11122; their (31113-
ndvantag'oa and faults which 1011:: been used for: removing
brtctnrin from suspensions and a discussion of tho theories
of: filtration are prosontod. 11;: 00311111213011; {BO-I.) filtrations
wore 1:10:30 using; 1023:: tff-mn 40 cc. of sunponsicn. ‘1‘}.10 revert
of i-‘ruE'r-ishor 1103 not cdafizmmd. 0:11;: times per cent {fr-ova:
was 0111;021:1061 from those filtratiozm consist-inc; of :3};
and £53012“.

bacterflorfimgo, orginnlsrm grown in 1: 12106111271, amt-binned cultures

411(1)::th 0:112:01: grofi'n 1n the 331' 6001100 053

         

~107~

11:". ion mfl niecolleneeue cultures.

 

Of g::ne’1, m mud finer.
reotisber has reported as high as £0 per cent growth in
filtrates of bacteriophage or of sterile beetb. The eete
indicate that uneer preger coneitions and controls, en‘
where the capacity of the filter is not exceeded, sterile
filtrates may be exeeetee unprecticnlly all one s.
hefiltretieno of the erecnisms recovered (three per cent
of’the total filtered) gave sterile filtrates.

A seedy of fractional filtration woe mode with special
reference to dotermining;tho effect that K.meéium exerts

on tee filteebility of :ech. cell. It was demonstrated

3‘

\I

J
-

 

that there is present in K medium.e solukle or filtreble
fraction eiich increases the filtrehility of peek. §@§1_
to a marked extent, while the gross particulate matter
hes a slight 1ehih1tery effect. Eim11er results were
shown whether the filtrates were 1ncuheted.£3_tg§g,er in
subcultures. No particular medium was demonstrated as
possessing the ability to eecreoce tbs period of time
necessary for tke opeoerence of growth. in many cases
one to three weeks elapsed before macroscopic growth
cooeered 1n filtrates or tefcre 1t ceula be demonstrated
by streak plfite suhceltere methods. It_1e well known
that in many cones single cells obtained by the verious
single cell technics require periods of one or more weeks
for growth. 1'30 suggest that, as was cleirseneti‘eted by the
eta presented in Section D, n13 one or two organises

may pose the filter. There would tion to present in the

-108-

filtrote coucMi ions, one cent Ior the e; :cess of sos3eecing
medium, eeeeetielly the some as found in single cell
technic end the extended time period would be expected.
This 13 very like} y the cone ition in t} -e earlier frt'ctions
where growth is delayed, while in later fractions whore
greater numfiers peso through the filter pores growth is
obtained is 24 to 41 hours. {his is co'eeetro'ed by the
dots of many of tie fractional filtratiens. s 45 Gay old

culture of £2.11. and e 9311- heor cllturo possessed

 

identical filtrehility properties.

Constantly but he. Licencrdly t33r0uL33m.t the es-zporinents
presented there have bees observed microcolonies. Those
were similar in size, aoeeercnce, and behavior to the G
colonies reeorted by ”noisy endcfinors. There was also
a striking similarity in t: 3e 33303353301033‘ of the orLe3-3isns.
Lohnia end Smith mentioned that gonidis were microscopic
granules formed by 333303131 cells in one stzu e of t3eir
reproauctive cycle. The gonidisngio'upon disintegration
released the gonidia which then grew i.nto normnl sir 0 cells.
other authors postulate thst from so gonidie were produced
colonies microsOOpic in size, G colonies, which after Stilt-
oble technic would form normal colonies coincident with
the formation of too normal cells. In the literature may
he Toned scottered references stetiev that the gonldie
are filt rs11e but 30 mention is made as to the size of
colonies formed from them. Thus microscopic oemenstrstion

of the granules, proof'of their filtrebility, and ability

~109-

to £01111: 1103117111 00130 11:11:} or t 011 01:: 0 0110010115: 1:3 1:10
crib-rim 1‘01: G 13:73:10.0. 1101113031 1'0poz't.0d 1’11 1.0310 for:
but did not {1'10 01111101100 01' 1:110 13210003100 01‘ £01110 10.

1311.10 1:: litorm: are mach 001112001011 :10 1.0 1:110 term-311010;}
exists, and 1:30 difficulty of 6.001111111112115 311.101.1101 0113. with
17110.1: mrticuler 11310.00 0:: 33110110110non 1.23:0 01.11::01113 {moornod

10 011310103113. 172090 01:11:11: (3.000111313102133 0:101 ("011011310110 01"
1:120 1:012:10 0013,1061 would clarify 1:110 0115;101:101 and 11.. 1:11
t: :13 1111100111011 1:10 (13,12,111 of: or a tel-1:: 3..1101r.:g;y which it is
11090:: will aid. rather than 00113 to 1:210 001151101011.

1. 3 0010:1100 fozmd 110:1: 0111111101: 1:: 1.1 1:110
50211131111111.1171 1111:1110, 1303:: 3:11:11; {11014110 01:11:10.1. which
1111011 1:110 00"111012131011 cf." 01:11:11.3 10 1:00- 21:10 11113. 1.1.1; 01.11100
0010:1100 11101111000010 111 0130. 1‘70 111111110110 1100013101113 of
1:110 c::-.L.tu2-30 11:30 (311‘2'01'0013 £33011 1:3":0 1101.201 form.

2. Tiltmblo £011.10 - or 0:111:10 00110330 01 pas 01111;;

'1

1‘13zsez‘0 111.1011 01110911113 controlled 0011131111033 but 1.: 1021
1101:0211: 11-0 111.101.1131 130 1:3-:0 1101121101 fe'mu 1.71:1: 0010:1100 10:10:10-
0003110 111 3130 1100 not 31:10:31.:0011.

3. 3' 0010:1100 - 0795;21:110113 normal 1:: £01m "1.101; produce
00100100 microscOpio in 0130 due to the influences of

01231101300- 1:11:10 101:». 1 0011012101111.

1.

2.

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5.

6.

7.

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9.

10.

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~110-

Literatnre Cited

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Bronfenbrenner, Jo'and P. fiuokenfuaa. Mn the filtra-
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fl
V.'Lonuroh. Uhor Kleinsto Eaktcrlon and dug
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lytiquo anti-3.;19 at lour rap)ort oveo lea aouohea

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:riedborger. E. and .;ortrud Loiasnor. zur rothonenoso
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Frobiahor, Kartin Jr. 0n the action of bacteriophage
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dadley P. microkio disoooi'tion - ‘ho instability
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Kudloy, 9., Ldnu Lolvea, and John Klimuk. Lhe
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28.

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'2
Cl '.

~113-

.‘ .. . . \
flauduroy, 2. Lou onlzgres\eccan alrea aprca
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, \
fiauduroy, F. L08 culturea\aeconu¢1res uprea
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cough. 10nd. Reba. 300. de £101. 91, 1b25, (1934).

flandnruy, ?. :cohniques d9 cultures dea forges
filtranzca inviaiblos dos microbes visiblea.
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fluuduroy, F. Lee ultravirua e: 163 forges filtruntea
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\
d'Eerclle, ?. and 2.,Jnufluroy”, Bur lee oaraotcrea
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52.

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61.

~114¢

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I
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68.

-1153-

Tamaaelli, 3. contribute 5110 studio del
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Valtis, J. Szr 13 iiltrubilita du ik 01110
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Varnoy, P. 1. 33d J. Lronfonbrenner. Lfiffecta of
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"easiliu,”1tu, uLd Oh. Irlminoiu. Sur 13
filtruhilité dea Mioillea tuberozlcux den
produita 1,,1- who *“Lnulau $0113. LU‘lpt. Bond.
.161; '6. 5300. (:6 1.410109." ”11. ‘19‘46 ).

Veber, T. Lur la i’iltration du 32101116 tnbnrculeux
du liquids de pneunothorax artii 10131 aur rougio

ChamberL .Ld LL. conga. Rand. fiebd. 303. 66 L101.
94, a, (1936).

Iofilbaoh, S. 1:. in the filtrubili Ly and biolojj

of the spiroowx ates. LL. Jr. Erup. 113. 2 49¢,
(1915 ’-

“imager. aura. in 3108tu13t08 of proof in problems
of L19 bvoteriul life cycle. Science 73, 237,
(1932 .

A Critique on the filtratility of Lecturia.

(Abatraflt)

The term t colony was introduced to dietingcieh
between colonies closely reecuhlin; the 3 Lygce, reported
by nedley, except For filtrchilitg, which were erodnced
hephezerdly b; regid transier end u’in: 7 epeciee of
micrccrwnnieee in 7 cifierent media, aid the true G type.
Lo relationship could he established tetucon the uoyeurancc
of the * calories and the p] of the necie, concentration
of chemical (L131), dye, characteristics oi the organism,
or the etute of diceocieticu.

She serial dilution methoé of Pruecknor and nhcroun
dic not demonstrate the ~reecnce of prititivc forms or G
tyeee in sewage or ekim milk.

Ihe appearance of m colonies from the bacteriophage
iiltrutea of Salmonella Bollorom and teghglOJnocne enrooe
upon the upg‘xlioabiufi 0:; the I'LL;X&I Geri—ail isze tl'uheie-I‘
method of ieuaurcy wee not dependent Upon the fecter of
u~iug but opiurently noon the metnod nf teahnic itself.
Similar series of bacteriOphare filtrates to which etrcek
subculture plates were applied r.mained sterile.

Adverse temperature conaitione were scout to fever
the production of u types. Bupid serial plating, selective
platin3, had no effect upcn the procc'tion of fi_typee.

An historical review of filtration end the factors
influencin; filtration were preeonted.

It was shown that when unconte lees than 40 cc. were
filtered throuxh Lcrxefeld filtere (2 1/2 x 5/4 inch)
sterile filtrates were obtained in 97 per cent of too
filtratione.

ihc method of fractional filtration Wee up lied to
the stud: of the efiect of x medium on the filtrubility
of'Lch. 0011. A filtruble or soluble portion of K
meaiun Wes eucwu to enhance the filtrebility of this
ergonien.

Ihe following terminolo'y woe need to differehtiate
the three forte of orwudeme comet-3141;" encountcmed in
filtraticne. l. G colonies - formed free or enigma in
the fonidiungie phase, poeeceein: iiltruble gonidic.
which upon the apglicution of e“ table technic will
produce colonies microscopic in size. the Various re-
actions of the culture ore different from the normal

form. 2. Hiltruble icrce - crfiunieme capable of passing
filters under carefully controlled conditions but «nick
revert eo quickly to the normal iorm that colonies
microscopic in size are not groduced. 3. m colonies -
organises torMol in form which produce colonies rioroecopd
10 in size due to the influences of udvcreo physical

com! 1 t iota.

1993550

(18

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