STUDIES ON THE NUTRITION 0F
CLOSTRIDIUM TETANI IN RELATION
TO TETANUS TOXIN PRODUCTION

“1.3!; for the chrcd OI pI'I. D.
MICHIGAN SUITE UNIVERSITY
James R. Howenstine

196.2

 

THESIS

IIIIIIIII III I III I IIIIIII

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

’ i fli'éSis entitled

Studies on4the‘Nutrition of Clostridium
tetani in Réiation to Tetanus TOxin
fiPfoduction

presented by
James R. Howenstlie
has been accepted towards fulfillment

of the requirements for

Ph.D. dapfiein Microbiology and
Public-Health

Char] es Mtjf’r Spamfifignente

Date 27 February 1962

0-169

__ ._._._____._-

LIBRARY

Michigan State
University

 

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STUDIES OF THE EUTRITION OF CLOSTRIDIUM TETAEI

IE RELATION TO TETANUS TOXIN PRODUCTION

\
a
James Rf Howenstine

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCT R OF PHILOSOPHY

I

Department 01 Hierobiology and Public Health

A/
-n
lSUc

ACIQIOWLEDGI-LENTS

The author wishes to express sincere appreciation to
Dr. Charles L. San Clemente for his patience, encouragement, interest
and guidance during the course of this study.

Furthermore, the author is indebted to his many associates at the
Michigan Departnent of Health for their technical assistance and
interest in the study. The author wishes to acknowledge particularly
the helpful criticism and guidance rendered him by Dr. Russell Y. Gottshall
for many stimulating discussions durinc the course of the study.

The author also wishes to acknowledge the support of both the Michigan
Department of Health and the National Institutes of Health (Grant E-2088)
for the funds and facilities which were made available for carrying out the

study.

II.

III.

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temperatures by controlling p3, ionic strc or“_, and protein

~.

concentration. ThC crystalline toxin contained 15.7fi nitrogen, 0.06%

phosphorus and 1.0Mfl sulfu. t had an isoclectric pOint of 5.1 and a
sedilv ntation constant of M.5 bvedoers units. It cod. a purity of 66 A106
mouse MLD/mc N and its amino acid composition, dotC‘WWNed uic1'obiolocally,

0 I n ’W 0_ '_ J- . ' ' q . -- ' , ,_ 'I /
con81Sted or 13 amino acids. Its Mlfllhum molecular weight was aoout 08,000.

since been oLfirmed in two other

C0

The protein nature of “he toxin ha

laboratories (Turpin and Raytaud, 19 9; Largier, 7056) and no difference

U

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was found betVCcn the t xins p1 oduce C‘ from two strains f ClostriC ium tetani

 

which differed both morpholo mi all and physiologically.

PrOQJess in the study of the nutrition 0“ C. tetani in relation to

 

he product101'tetanus toxin has been hawperel by the failure to produce

' I ‘I ‘ .\ i- ", .. ‘-, . . ‘ -- "‘ . '1 —'- ~- ‘ - -n . - -
it except on a cachicallr uacciined medium. also tce bOLlD is lEldblV91“

' O r- 1 *1"' .—~-n'. _ r“~r ..r_‘ ‘ '0 - - mart
uBStable (P 1.] encr ct al., lTMo) and Jeiy difllcilt to assay. LhLJlflCthelo

. u - M J m ,n n: - L — :71 JV 1
of tetanus LOXin tust talze special piecautlons to av: c ootu tne loss of

. J "-\ ‘ ~-.- J— c" -L- >. ‘ \-'O '1“ ‘ \ ‘Ic . - J“ a -‘ -,-1 J- \1r-. - _ ‘ . ‘-.4 1—, . ~
the strain as well as cue mutation Ci tie ciganism to a hOn-DCAlb‘th stlaino

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nost of the Sivuifjcant cont-ibution 18 which ‘»vc been Lrje du lug the
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last 22 vears With rcfercnce to b;@ nutlition O; the or3anisu and toXin

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production were made by the late Jio J. n. uelle and co-molneis at

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m
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Thou Vovld 33: Il was aoout to st: t tho; e Haw in intensilicd demand loi

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The veneral nutrition of C. tetani without special consid—~rc:liorl

 

for toxin roduction Las been studied by various workeys. Clifton (l9h2)
concentrated on the f"i1izsction of amino acids and rela‘ed compounds by
the organism. Lerner and EU 3l1e: (l9b9) were especiallv interested in

the utilization of filU‘emine. Lerner and Pickett (l9h5), Prevot and Boorsms

(1£1hl) , W‘Ssribn zinfil Ekmidxuzn (11911-) aund “"‘ti‘v‘z Enid.iliirterfl3exqg (1f95gfl

reported on glucose utilization by c. tetani. Kaufman :nd Hump

,
J

(1958)

flE3

U)

 

d_evised a chemically defined helium unich they founo would i--consisth1y

SUp mo tgrowth of C. totani.

 

III. 'leujs LID flimfiilgLB

‘1‘ .1 .y __.~_._ —_ _ -.‘
., I A“) 5'” - -.x '1,C:_:i_ _"~~‘_
A. - o ‘g v.___ L} L'... U, L Lpgiil LIE-4L k}

 

 

’- '1 "1'.“ r11 '7‘ UL.” 7 -~‘- .1- " I. —. - 1—1 ‘ ‘ 1 ' ‘ ..1. ’

13 1924. lne Uhuc streig mes Uoseiue1 111m Une Lastaceus ett Uolic Lea
("\

DIE IO“ ‘ '“1 T :10; - Q‘W‘eqd -' 111W“ M‘ C ' n“ m - ‘ , - ‘1 q -_ p ._

.1.) --U, _1C,.l ' 1 L .L _. L ll]. / /':, o 41.1— _1_U_-‘ ) U... C 11‘ 171“.) L-‘~_.-'_ lV-C l 1.01.]. Mile

tettnus toxir in e neatlfiss HUbLUMo 6p01e IO Jebior if either straiU hU
not been demonstretel.

Both st aies were maintained in the frozen state and transferred 3
thawing at weekly r SCLiLOTUDIJ interv'ls. “Le 33—8 strain was transferU“
1ailv into thioglyeo1lcte seel gediu . 11- Lelle“ r““111“ va" subcultu:
into veil instiLq seed Lecium UHC iHUUJUted ”1ewo“icell; (Roscnthel, l9

 

Dy PWO“OUQOPIC ext IT‘L1OW o1 —UUU strUiUed excess.
I‘ N- '- J—‘w ‘ ' ‘ I — I 1—‘
i) o 1C: -L‘O -;rU.!. - .‘J ..‘,Sll_'€' -3: u
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which were chromatographcd at the same time under identical conditions.

 

 

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Iv . EXPERDEI‘ITAL

A. Variability of Toxin Yields with a Medium
Formulated by Mueller and Miller

In the large scale production of tetanus toxoid, the medium used
at the Michigan Department of Health and widely throughout the world
for the cultivation of the tetanus organism is the one proposed in l95fi
by Mueller and Miller. The formula for this medium is shown in Table l.

The problem of variability in yields of toxin from this medium is
well known to commercial producers of tetanus toxeid. The data given in
Table 2 illustrate this variation in the potency of eight consecutive
large scale lots of tetanus toxin.

In the production of these lots, six, thirty—five liter stainless
steel kettles were used for each. - Such results are typical of lots

produced either in stainles. steel ke

U)

ttles or in pyrex animal jars.

B Standardization of the Medium

ormulated by Mueller and Miller

 

Flj o

 

 

Mueller and Killer‘s formula contained two components of unknown and
variable composition which could cause significant variations in the yield

of toxin. Since it was desired to study the metabolism of Clostridium tetani

 

in relation to tetanus toxin production, the first goal of the study was to
develop a standard method of cultivation.
The relatively recent innovation of growing the organism in a cellophane

bag (Figure l) was found to be undesirable because of easy contamination and

1

volume Changes.

 

 

 

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. . ; I n 9 n a
u n a u n a - a
a l’ n 1 n I n -
p . . n n . n o
n 1 ~ p a 1
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o s n I 1 a e .
n n d 1 n r - A
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u , . . v

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n. ' .-

v. n It.

 

 

 

 

Table 2. POtEQClGS of eight large scale lots of tetanus toxin
produced in the medium of Lueller and Miller (195M)
Lot MLD*/m_ T.D.*/ml Lf*/ml ml harvested
1 300,000 250 an 145,800
2 450,000 300 60 123,000
3 150,000 200 M1 1h5,000
u 150,000 300 M5 1M8,000
5 350,000 250 17 123,100
6 200,000 350 #7 127,500
7 300,000 300 17 150,300
8 300,000 300 #5 119,900

 

 

 

 

 

*ELD = minimal lethal dose; T.D. = test dose; Lf = floccu-
lating unit of toxin.

 

 

 

 

 

 

-—t—-'”*

 

W\r~\\\\\\“ - Cotton between two

layers of gauze

T\
Cellophane bag

600 ml physiological
saline plus inoculum

__fl_,,__—————- 5&00 ml medium made up
as per six liters

“__—————-——'Glass jar

 

 

)5

Fig. 1. Dialysis apparatus
using a cellophane bag.

 

1 1!-

2. Use of cellophane tubing
lnother method (Fi wire 2) using cellophane tubing instead of a sheet
of cellopha1 1e gave hi 3T1 MLD values without contamination but the volume
a

of the toxin inside the tubinn decreased from 680 ml to 156 ml during a

12-day incubation period. The difficulty of Ieesurinr volume changes

(9:

in such an ariangc nt witiout disturbing the culture remained a problem.

3. Use of predialyzed medium
Since the organism could grow and produce good yields of toxin using
only dialyzable me- mrials, the possibility of growing the ovoonism in

This would eliminate the errors of

Q:

predialyzed medium was inves tig ate
volume changes durinO the growth of the organism. When the medium (complete
except for sugar and vitamins) was dialyzed at 0-5 0 at a sixfold concen-

{51,1

tration against an equal volume of physiological saline thi cL1 was ey chan.C ged

ysis resulted unless

I_J
1' 3
Ho
(D
a-
Q
[—1
[._J

for fresh saline the following day, inelif
the ban as rota ted at a slow speed. house KLD values of M00,000 to

s when the organism

r‘b
H
{.1
c;—
H
5:)
(7‘
(D

600,000 per ml were obtained on the culture

.‘

was grown in predialy eL iodiww out oecs us the rotating cellophane bags
ruptured easily, it was decided to dialyze only tl1e two complex components in
the medium-—NZ case and bee: heart infusion.
H. otandard17ation anc amino acid composition of NZ case
ting of iris ions

"7‘- 4—‘ . ‘.- -- " . - " J—
a. greetional Li 1ySis and tes

Craig and King (19:5) were able to fractionatc complex mixtures of

. . .1 ’j . "\ ""' . I‘j r'\ 1 A“: - — . --. -'\r1- — -:
amino ac ids and peptides oy ulmlySlS- ncShltS o1 pLeliuinary rperlnerts
.1. I
. ' ““7 .1 '1 Dan 'I- ~ '. r14- ? “.3“ ‘ J'I . "V"‘ . ‘ i -‘-7\,_«.L.
indicated thcfi r2 case could oe liact1o1ated using bAlS p1 nciple an: that
‘1 n1 ' -0 q /’ LI? m j (no Ll w c fix "on ((30 0.: me" "er3"; "WQ'ICS
tne dialy81s 01 o; o sc \ oi _ UL c c L_cac .0 l H u” vi l
1 o o ’r_ ‘ -” _r“>"_‘1 , \fl 1 .- 37 fi_(j_{_'\' w “j -—1'_\_1IVI .1
Pancreatic dieest of casein o ouuoeu o; odcllieio 39; 5 co.,
“ ‘“ " ‘ g) ‘ .:_

 

 

15

f Cotton between two
layers 91 gauze

#flflfflfldfll..——-Cellophane tubing

H”

 

 

 

 

 

 

 

///////////r—-hedium for six liters
’/
,/////////”F‘ 680 m1 physio?o;iea3
% saline plus inoculum
/ C lass 3a:

 

 

 

 

 

J)

Fig. 2. Dialysis apparatus using
cellophane tubing.

 

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Amara

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at

 

 

 

 

 

 

 

 

 

DJ KO VI 00
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[2.21.4] 'l

 

 

NZ case Diolyzed (‘70)

 

 

80—

60-—

40L-

20—
4 I 1 1 J
IO 20 30 4o 50

Dialysis Time (hours)

F133. 3. The rate of dialysis 01'“ a 6%
NZ case solution in a 0.1 N acetic acid system.

 

O
O
'3
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9
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7 -0 -- ‘7 -. an « ' — .7- —- 4— 5' - < ' ~ -~- -.-. -’ , n —' - —- .
none oi the L- case flictions JOUlC suostitute eifectively foi UJLTLCtLOpaIQQ
HZ case in the me it: for toxin production.

a ' edudzrd in gredient 10? future

0
,‘3
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i...)
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C'
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Axperimeuts as a result of the knowledge
from exocrimeuts in UllCd

_ _p __ V ... {"1 / '_.'
producoiou Oi toxin. lable h ShOMS

 

stinula“e greeiser yields of toxin than some other loot (lot OOF3).
Lot 70D13 was the lot “hoseu as a staudevd US case source.
b. 4mino wcid comgositioe of F2 C906
Tiuce NZ case is a povle"ed Cryp'ic digest of caseiu, it was expected

b0 CODtFleEI‘COWDlex Fixture Ci TAV1JJzicixs and peptides. fmme manufacturer

has stated that o thicel lot or L2 case wnull have a total M_i ;ogeu value
I I
‘0 I ' * <8 /j w-. ‘ 4 "L r ° ~ IN 1 ' ’\
Oi wbtut li.T\ adv aJ flwlflO nitfio,er vwi_yo o How q 1.,

o ' ‘ Tr.

“ " -. “* 1 “' J3 . ~. vv-A N J— . I' ‘r "'- ' i 1' (“v V -‘ *' r w ”- “(1-...“ ‘1" ‘ r~ —‘ .’ r" ‘0 - -
Ouljxcmv: bioup oi JAbujbleJbOmg HHS tsie. LA) -.:cuion-te i“ case io-
- - -.,. -1, -.. 1-, --: _'_! -, .._..r. _L. -,'...\ rrr ‘ u, . .1 - +7. ,.. _ 7 ‘
studios 1A tnc LOiLCbiOi o; Cbu us to“: . leis gioup, unue: Jae leedecsnio

(‘ 'n~'r - x: ”T ‘ . ' -.. . J-—~- '7 IN .‘ 1 - J- —:‘--. J- . ~ ~'- '—. .
u_va:u quJGuSluJ, Mes eolo b0 iiflCblOQEbe lL case

into neutral, ociCic, cud oesic portions WJlCl were fGULl to be very complex

by auxan05rephic studies. Tot GVGL a qualitative eua_ysis of the material
was 0. clic ed.

After muck consult: tiou and a review of the literature for 3 pr cedure which
could be used queu‘it JtiVC 13 for the determination of exino acids end snail
ucleculer weight péptf.1es i1 t“~ -Qdiflm, 1L Vuo c:cide€ to ckoose tte pIOCLCUTC
of Loors .gue dteiu (lTil, lffiL) oud Loore et el. (l;§9) using i0: exchange 3951p

 

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R3

 

 

Table A. Veriability in the yield of tetanus toxin
from two lots of NZ ese
NZ case Liters of
Experiment lot toxin harvested MLD*/ml T.D.*/ml
] 70013 20.2 350,000 300
l 00F3 20.8 100,000 <<100
2 70Dl3 8.7 400,000 300
2 00F3 1.5 150,000 150

 

 

 

 

 

 

I‘D

}-._.‘

State- that their procedlres coull letermiue
J..°,. ‘ .L --. ' :.. .-
end peptides u; to te: units it siso. Aese

These workers have

amino acids

P orodwrn based

rinciple oi adsorptiOu and contltuous sequwntiel elution of compounds
'“om the resin with apprOpVIHB; suffers sud rigid ZOUtTOl of pH, ionic

-_ . '1 .‘ . _ , 5'17, - '— -.. - “' . .. J- ‘l '
strenflt and torn iiU’“. in“ clULed Latirleis were 00118008‘ in
9‘4 ~ '1‘ . ‘ --A~'~ . ' '- . \- 0 —.- :1 1 r- 1 —- - -O .. - J--, ~ 1‘ . .
llactlous and cummtltu luely usssyeu 03 a eesolully coatgolled reaction
*T ‘ "W t‘ u ’11} rd' ‘ ”'C. ’3 “ t t "‘i "'3“. ‘l C‘ 1J—-~1 1y“ '6 r ' ‘ 1r 1 ‘n t O r 1 '.
l -1 :'. I’lJJJ; . 11 a. 1' O é... LI .4 .. ”DBL Lu. L.-.-.L O_L - Llnlld 0.1]. 1308.1 1V COLPO {"111 (LS
. 1 _T a 4.7 .... -. -° 1 — . 1 ‘ . -. - —, . J-'
Tte details 01 MG. ploceduyes whiCfl were used nose were es ltislly the
r 1 '1. ~ .1 , - ‘1 , -1 ,— -—'-‘- .. - . -I- . °-- " ' .1-
ssuw is tnose puollsned J tue lutuogs ercept is indicated.
rJ11A . — I 4" --J W w . (V J" . "‘ " '. . ‘ -'- fl ‘ ..- 1
inc fllst tee pyozeutyes to be lLVmbtlUSbCQ was he ch (Loove '-u
7"L ." - H. "‘ ‘ 1‘ .1 r/ -‘/- 1" fl . r I‘ l " fl - " C’\ F‘ ". ‘f ‘ ‘ '~ *6
Obblp, 195M) usi.é . J,J A 0.8 :1 col1 w, WJOI " ,o-x4 cesiu, and uu eluent oi
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3c:“1uuou~lv ~1””QW*¢ p; Lid LOLlE st_fr: _ht.. ‘lL OU’ll seiosll not as vese
- ‘ - -. " . --_ . 1- _ , M -_ ,, ”_'_- n - 1
Spért uSlv' tuls Diocrnuvl to “-«l .o iniwn S hPleS, wdc 01* t0 coitrol OI go
1 . ~- . - -. -- J— . /\ 1» w - —— A -'- . '. ‘f "‘- . 1 " “."W'Y‘ 1 ' J' .. -0 ‘1 ’ " 1" - 'K ' ‘
.illr; o” Lin:l>wf;e-s I» 5 two gixmLLs.t élutlor tu.'tsi coupoul<k3tL.s got
:iQE:iT¥Vg€J earl ’tfljgw rx‘cp;jr' ‘"3 T" s aliiu:ct”ti:nfleé? iL‘ £1.1ci- cf? ‘ttx: (tie If‘;ic}1 fires
_. . - -,._(\\
'I o A. - _ fi“_t’_. _ ‘ -l_ ‘ '1" . _‘
‘01»??? SFW’J 10.“\ ‘:.:;.r . (-(JO ‘- 3‘ ~-—-; ~-./L} .
...... ‘ l , _. .._ ' .1- ‘73 e an ,3 - .... - _‘. _r _ «. J. . .«J- - 1 -° .I-
It if is lTiYNV‘l,;ip -.. be- JJA,_Li-;2v, -}_;3 il_;m_S L‘Cn-ulOUuAA;L lute
‘-
fi . ' ' 1 " ~ fl -. , fi 1 “"3 'fi "‘4 r J“ r f" T I“ ‘-. —‘ JI‘ . fl '\f_(" 1" ‘ ‘1
live psfits at 3:9 IV’QLLRLJ . nesloul lo es20v o- i.-l_sou l:1w . L
L U
' ' ‘ ‘ ‘ — " .~—- -'—- _ "K . - .. 2‘ -"‘ . -'—‘.
*fiictlon U (o““tl1lc Jl' tx‘ :7 (Pp”> l to?“ ,- a) W”" uSCu lo: tge
- '. .. .xf. , ~ :1 ., .‘ a- 7' ,:*: --
7737 2“ :oflrfi: Io '{§1"O’f“UO‘VKJ3;_Z - ..1. ~7 0. gr. '01-;. c EmlLJLC (7‘sou.1f .
-- "F‘ ' '1‘; ‘v‘ 1 -' ._J_.' "-1 ‘ ‘1': ~ ~".' ‘ _ fin- ’i15’\-——\ ..~ -'- w If] 1" T_v' C‘ 1~g‘ ‘\ 1 .q '-\“ "‘\_/'\ :fl d)“
_J . :‘ #0, J T) , L1] J;7J.'_) , _ -., - u , ' .L' Q. \J- | I {JV J“ “I‘v '-.-- . -.|.) ~- LL. L‘.‘ :.n .‘V 1' - ,-
:oluw to? Tl”- "*o '"c‘ii m.'7y“ls of *fe Josie 'ex90t16s. Tte upl““
. r ' - -. .. .. .J. , -.- -. o- 4 .. -.
_‘r'n V‘f‘ qflj.1 n 1 1-: ‘40:? \j .J- i J, J j 'J' ° J). go ' ) k :1 VOL (13.x: _'_/‘0. - JJ..L.O'- ‘O 1.“. 'J0
- . 1 \ '1--.“. .--~. .4 x --: '- "o'x’ 1m: --3 W
F‘ “‘:_-013 " ..U.‘. .' .... -5 _' ‘ k‘ u. .L- .. I'-..:\‘ O'. _‘_- k. _‘_JJO .).L..~. -- -.. AJ. .p--. 0' k... V
—~ ~ . ~ --.?.-‘ ..‘40 . . .fi '_1. , :. -1- - 'yfl“,\ .‘ 1
“e.ls:t;‘ 1‘ T” rovoletsi it- ;lu e. o- t - gsi -olg---. i_c “3.

n.
P

 

 

.. _ .
. r
. . .
f h
.
.
t I t L
—
f
w
A f r\
....
.. L
»
.
. \. .u
.\ .
u .
.1 .
.\ . 4 v
A

m 0 .
n
.| l 4
_ .
I.
F. .—
~l
n ._
. . r
.. a
—
. r.
1 .. f
F. r; u
IL

PO
PO

1 1

was pipetted into a matc‘eu see i: pnetcmeter tubes and

the UiUHVdUiU color develOped. accorxiug to the procedure of Loore and

eniho acids was Studied with the modified hihhydriu reagent um? e: the

sane conditions as bhOS‘ escablisheo w;th

eat wss p1epvred. iresh ea"h KQV. iae rdrihdantin which

was US+L as an aLtioxideut 'u Lee reagent was purchased from Nutritional
'I

Biochemicals CorpOtJulL '. ’ 'erdaid curves Wsre pvepared for each of the

o_ r, 0’: ‘ —\~ .‘~ _.,-. q u _ -_/_" 1 , ._..,., ,1 ---,—. .._ _'._ fi. _00 --.,
swieo aCluS ioueu in pieiimigex; coluL. -uh- lu tie iltia

J
o

. _ ..L «I? r fr‘ .'._ r. 1 Pi: J‘“ r ‘ 1 - ‘ 1," 1 "1’ r \ ‘ . ... 1
5, 10, o; lw #1 Ci a 3J2 0 -rdte.—~,p EVQWMOL QlAbULd as e diluer_t sue

/

J- —. .A ‘. -' . —. - - . —.. J- - - - . l- rc J" '--J/- r] “.1 ‘1'. .-‘~ - 's‘ r- ' 1 . . -(~' >~ ~-. _'-
plot Lug optical LGQSLUJ un be at he eppiop.iete Waveleubth &6mlh8u
‘=-°cn~w3 t wadQVVFJFi“. wa‘ c »~~ n~r31 *vw -« tdehm ‘Lfi 1o7~“ q ~u n -L
CLJ. L.-- 91” DO; . LL. 3... LL. C.L u.J_OL..._I OJ. punin -0 W'V—hk-L O .J. _'_ C)J.L iipDL. S LIOJHC C4- 0. LL11. f0 0 ) 1 LI

ves possible to COUvert directly the total number of uiu-ydrin color units

*\

OthlJOQ 1U ail the sc p-es coilcc ed LSOm de sihqic Pfihfl (a sex Curreg lop
‘0 t‘ 3 "" ] a(~ " -1 4—“(3 ("V") . If \ “\Ixfl’fi -J , TV"; 1f) ': “ . 1A7 (‘10 1 ,- -D c 11.--', r- \.(-“
o. .L v4_L,o io_ be- :x---c- b-0c *L'e , 4) L .1 _o-.-cs a; s-1 0 4-1-.
D'Kr '1" 1"“ 1*'fl""" . "‘1 " ' ‘ "3.1"" ‘ -/‘~. _I . "‘ ,- "7'“ " T'W- " “ )1 ('1; 'l" r" "-r" (4.1“ r.‘ 1'? ”WA 61/3 . r1 f‘ 1 -. *ICA "3':—
UV LJ_.,C f. . -_L ‘.’Q_,_ 5.1107..- L .J- L‘-_i_\,.J_ L A L, -!_J4, s.) \Ja. p».-....L-O V J. ..'—\. L14 J. '44 4 .._ 45.1: .._._-'\_‘ L,u\_,l\,J. ) (.J. O._LJ. v V
"‘ 1""I' ". "IC‘JW'; 1' "‘T h '\ 'r 'J'j. ofilzj ’3 ”V: I" . "“"‘ “ “'1‘ ’tr' ’1‘ '1 J "J" ‘n "' :3. "1)“ 'J‘ " ‘11—. ‘l“'](_) "‘ "t ‘x’ (‘ '7} '3’. j
.L 9. -.ch bLOL...JL_...D De LH‘JC 41-. 03 a“ .4 “'41....01 b“) .Lli_. o 4.L,LL- oilC ”6.1.ka _U -.,-_ LI--«‘, ».'_.1;._L...) 2,-.\_,,J_LL

A- .1.
, '1 J _o ’ ‘1\ v . .] ‘1_\"| .‘N .——
11:0 on \JL—‘J—l—LJQLQ LES 0;- J‘. 1' J.“ i-IJJ__\. ,"
- , fl . 1 .1-“ -L J 1 - : - ..... ‘- _ . - 1
It 1.33 ”X “_C‘- ' (:LJ J _4‘ TM; ‘1‘ L--i-03. _.L ~J4L}4‘_- _LL--.u..c)LJS T11"" “CL“..JL UL:
1 o _ "f“ I _:_1 w _ ‘ “‘_\1-' ,. --.“ 'v01‘~ -

“a“ no” j.~ 11 0-: ohc‘ 'd ;J, 3; -ce 9 -1?“ och._-u possiole ~lunl“
"3 " ‘11 ‘ “n 7' " “\- -.-, 1‘~/\““ 4-7,: . 'IF‘J "D" 'T‘W . 7:11 ‘1"J7rP.-J r.‘ {fin—u. 11/“.1 4‘wa) T'd" """io‘fl
CAJ... “0-1; a J L1‘. 1? vii? 941.14. Law"; 4... .-(4'... r. .‘..l‘ -- '4 V 4-01).) \J-;.' OLLQ._ u_i4 .- 44‘. , L1 ..

. . ' I. . 1 ‘ " .4 \ :- n r‘ O n C ”x a 1. .

- .n . _ .....t .\ v - . v . ,\ A a u . , ‘ _ ~

OI -Ll-11J1« L.J ".J.V 174'- 'LJ‘J. ”u- 4'. 1.]. "-' (-4 :-J- J"«,’ (J. )4 '4‘ )l—J. ‘ u.) l .-4-J. LI‘L‘ 10k 2 pp; 10 LL 0

1‘ \ 1 'J' . 0 fi“ " . ~"' “ r. w" 1 ' r“ un—J .““ ' ‘ ( “fl. ‘ , C‘) -‘ r) ,"\/') (W!)
--L-J Sou-tOL (.J ulC D194, 'x/ :1. ';4. J. x J _L ‘4. .‘ L2H.) 4,49. lU-. '4; abv. LIQ LLLLJlj- ..U\ 0 \JJ

.1.
' . —'- .. J-.." ~ 7 '37-? ‘ 4 J -°-‘ - -.-‘ :J- ‘ ~—°‘.‘ -~ r - -,.. f-..‘- ~-‘ n
iljfLJCLib DC-’ wit)? SELL p‘,,_.Li.r .. -. _C.J..L '4‘ ..L.,L'.C .1. 'u pOLLJi4-JJ, JC .10 -.;O_'C UL-L"4’S (1.} ..
" _1 t .1 . o ‘7 F‘ “ W ~_ I‘ ”‘11 _ fl ‘V’fi__ ‘5 r. :_.:.
"ouiu cewc o>~- -cse -‘ L ul snfi ‘wv‘— a. ;-44- --- -L.U Dies
1" .1. ' . ' —\ ' ‘ ‘ j ,1“ s W * 4
’—' ~\> J . . . ' ‘rlr\ ~-— '\—f" . . ‘ ,. '.
~ub itionel sioczsnic- 3 Jo: po _epioc, si-ye-u-u, Odio

 

O. rV

.LJ

Table 5. Reference table expressing optical
equivalent to 0.01 ” " dcid iu thr—e

(7 fl:
(D
{3
\ CD
[41.
d-
«A.

 

 

 

J
O
H
CO
CO

lispal1‘tic aCido o o o o o I 0 3C.

a
o
o
0
LL)
[\3
CO
}__J
‘ Q
L‘J

Lhrecnine . . . . .

Serine . . . . . . . . . . . hgo 252

Glutamic acid . . . . . . . 31M 187
-. o 9/ Q C
‘fall‘fle o o o e I o o o o o 0 J03 .._.O/
MetLionine . . . o . . . . . 322 195

Lo

\Jl

\O
[\3
O
O\

Isoleucine . . . . . . . . .

U)
\n
"1
m
C)
(D

Leucine . . . . . . . . . .

O

O

O

O

0

r J
U}
4*:
H
..r
O\

Tyrosine . . . . .
Phenylalanine . . . . . . . 267 15o
Glycine . . . . . . . . . . 607 352
Llanine . . . . . . . . . . /l #05

‘1
L\
...

P. (311118 0 o o o c 0 o 0 o 9 log 65

: _3
LL)
-t

Argginine‘)é . . . . . . . . . 271+

oi
[D
i...‘
09

[11311.1]? i118 o ‘0 0 9 O ‘ ' ° ’ ’ 3 7

- O I I ‘I r!—
; lStldll'Jxew o o o o o o o o o 292 1.0 (

.LJ.

0
0
LL)
C
r-'
[\3
|_J
O

Lysine* . . .. . . .

 

 

 

 

*Calculuted as

 

paotometer tubes 150 313 mm were 'al ibrated according to hoore and

ayl red solut 10 in 0.03 N HCl so that a readinx
of 0.655 t 0.005 was obtained on the optical
525 mu against vats-r set at zero. The sa mple tubes were read on t
Coleman Junior spectropao tou1eter, model 6., at 570 mu. e::cept for proline
and hydroxyproline whicn were read at HMO mu. The olanks were read agaiL.t

a “eierence tube of use 6ilueut. The blaLk tube with the middle value was

l7.-- the ouelirs s 6’ hi. 68.88 for ...1 ho acid outert, the 4:4. case xas
Lylrolyzed by the prscedure “ocom:.e%1de by Dr oelua L. Baniemer. in

"$4.1 A.,] ,w _r " ‘9‘] ‘1 n7’ (7" M: ‘ HT: "‘0‘ ‘i3 1 13"". J“ \ 3'.“ 7* r3"-""-'\r11 " ‘1'“, 0 1'“ r‘ {‘1'} rm m -'- ‘| C5, ’1 T-
..11. .'_L;'__'.-C;. e; -_ longlLo _-;k., :.-4,L1 _‘_ 1 .14to Ui;(.,:. it,L.L14L- J41- C11..- (“11.-quw44 ‘fe gnu _/- _|__:J
- “/w‘xhn‘T‘"; * (1'1 “1 1r" rub!“ '37-'13 1 r} n ("v-L ."1 -~r*uu T1 0" r-\/'-‘ 01’“ "’VD. "L r “T," >NC‘C‘ «“
p 4'70L4.\, -144 SL4- lLUL- '3. t 1.1- km.) -44. L L 41p _L- UL»- [JO v _UwL 4411.) 4,1-

,_ .1 1‘." n I‘. _ : ,‘T A - _ __'_>“(_ _‘ r‘ _f_'\ ». _ ,. j _1_ 1" Op ..- ”I 7.. - - ‘ 4.1 O ...
r\ I ‘rj r_-\ 1 C‘ 1y , ‘ (‘i - . ., ' fl.._\ \ ‘ .. L r\ a . ‘ .,
a s eem seen. lie mouluuc “as e audio -cu to a l u ml case; lefl 5 ml
3 . _yfl o 1 7 _'_ ‘1_ _‘ -,«. fl 1_ _‘ 1 4 .. 1‘- ..\ j _ v.4. ,, ... 1- _‘ _J- , n...“ 1 r _:__1_ ifl'l- A -1“ n: “I 4 __ _n .
ols L113. 1-630. .8 L103? 8,. C'. 7 C ‘ .,( ~ L‘ U L On ..1 0.. Cl. L. been .. D.-. on. .._L‘c‘ i gulche 1148
- _ -. -u w .. . rd 4.“ n: ...le-11, .1 -4-.J- _ . '1 :.- -I- . 4 3- 4...
‘4 ." _\ *. _—‘ . .~ ‘1 j .. 9 i u , y \ ._. _ '\ 1 . .\ n (x . 1: _y r ‘ .n. {\‘C C
4‘. e 8119: i L). (I 9?. _'1} 3,: .. 0-1-5: _'_. 1 , 1. ..- {42.1.3 ‘J__-.__L_'4U. ‘.. -4, LA. 4 a .'.1'«.' L.\_)41._‘5 [IE—4:]. -CL. [JG (4--- J .L'... L) L)

-, -v- - - .. - -m r‘v. , -.. 7. -° ’* V. L" .. ' " 4 m -. -74 h . 4N4- .-. - .“I 4 D 4' -..'- .' - :1

'w r- H . .3 .3 , _ WA [/1 W111 w . . ’ r v_ . / .‘. ,_ r‘ 5
CHL'. '4}, L) UTJLJZL {341L1'.-. 4-114 _ 4L.'_L\~|—‘. 4 ‘J.---'.'-3 (.1313: 1‘."- 14C) .14. _1 ’J4--.L-LJ.L\;4..- 4'4. 11.1.U1'. Kin—L ULl].L‘-‘.

— C‘ — '1 “-3~~»»3 'n'lr' 7' n C '«3-1‘ J's ‘1 1'1 "‘1 r- 7 F7 r‘ r1 1' ' w
Tfl" LC " 'J(- ; N3 '1'] V C LU. 4 ~ - ..L‘: .L - £131.- '4_L 14.44 -1. U ‘U ox: Ln”: \ C1 1... _‘_- C) K_»O]_‘--T110-

.— — - ‘ -- ' .1 —'—~-‘ :— - ~ , '-—‘~-r~1 -- (I. ,‘- Trrfi '1 \ '1 ‘2 l ‘ ‘wfV

_L‘LEI -1"SJI\l.’ /:\ U,L" I}, _ - (.._ \J. ...1 IUL‘ OI ‘3 . O- I 4.! v LtL. .03-- $-00 1-; O V‘_1_.:LO~..LJ
.1. 'J a.) J.

‘ 1 (\er nmr‘ -’ ~ "‘ * j " . ' ’° 4‘1\': I‘ C‘ ’ .1 ‘J‘: q A ‘ ‘1 5 4 " Yor r 1 fi 7 r‘ r' -'-7‘ u" 'I' —“1

DHOC1‘.L_J;‘_ ‘ F" ' '4 7 VL 4_Jl EQ U_-—LKJ UL)-.L_L1-4O-- Us. _ __c ..LU 1U LL44... L. L1-'.v-.1b L1I
. r‘ ,—

.. ,— 3 » — _r‘: ‘ ' —-’ 4 "t ~ "‘ . .1 1 "' ~ "‘11‘_‘\ Ijl “I I“, I A! 1’.) ‘ r (\ “ll ,1" 1.1 ’ ‘-
p‘oc--rx> 0. 3-01;- 1 u b4 uliue u. n‘ p_e 1.- .ul one 9.5 l o 2., “J
”fit. A» 1 .3. \I‘l w--—< - -'— ”I - - " I“ -~ ‘ '2" ”“1 . _1_ ~ -\- 't ‘ I ‘w" f “'I r ‘*1“ ~"(‘

l-. cc K; LVLJRL_; silo?) :trfiale: to ego...uiie Igloo .esilu azoli. u e- .Lu. L ifls.
l. U _-

"\-n 1 " — a , '-n r_: 13 E: .1 vvh rN h t3 I 1" fi s « ~ - *‘ v .1 ’1’“ 4““ r'\

L-.. (:Q'J‘ _._ or) {1- LIV L" _I...’ ‘/"-‘l_: :LI: LL) 0 -__ ‘.’ - CI 4 --4.1_' I { 14.5.1.1- :[Or7 _. OJ. u--\,

- -:1\‘~‘1P1-flfi‘.q Tamr) f:\‘fi‘:‘

— 4.4-4”. --_ --. -. -4 _\V~ \J.

   

1 I‘
1 L . . .
. l __ .
. \ r: - I ~ ‘
r ‘ . . K '\ 1 I - r- | rd
‘ n l I.
‘ ‘ _ ' _ _
. ; . . . ‘
v Q - “I --
... I I I ‘
. r* i'
. t - i-
. . f 1 . ' r
1 ‘ Ir
. | ‘ . .
‘ . f
_ . .
1' ' - . '
- _ _ , r ‘ r'
. t 1 L" (—

 

Table 6 stow: t

‘ _ -. 7 - .- — ’ _ "l '1 “r‘! I
Or". 71,0/1 «5‘ ’1’... ~ ~. .7 1- ‘ ‘v A: ' --:— - -.- - ' , -1- ~ 1 -
0e obs-_1el *« u t.v a T .m. Jaeu«0-¢ 01 vi? Lu case covnoveuus.
.L.

Such recoveries are tTpiflll for ofoteifi lvurolysat s siucc 0*m voleeule
., L b - - V _l_ a“ u _ -
'0 v: -- ,— ‘ . J- . r'» ' ‘ ‘ "1’" ",' "‘1 7 " " fl . . J" _. ’j ‘ "I ““ '~ 1 ['1‘ \ '1‘ 1 A ‘ -
ol tater is true- .9 it» -MCJ3 flllcpbiu; ale wee. lg? b&Olu also snows

'1'

.1 ' 4‘ V r"; r‘ -: :‘Q i" ’. ~ ~1". ‘r‘ '5 1 . \ ~ 1‘ .1 .~ 4-1 '/ -.. P. J- , .'__ —L-. -- ' n‘ - _f‘ 'I'.
tiat tne analysls aoeouits lOl a value oi 10.1w Ol use UObdl xelbht oi up

I“: f‘ S I} ‘*. 1“ 3'3 . ~'-— 3 r .f'\‘ \ iAJ—Ir -. 1n 1 r. _J 7~ - H- (o ‘1 /)1’ r" {x ‘ ‘v r H- '1 x c '- {—1 1 —. -r‘r" - -. f‘ J-1, '\ _ 'r] "I
.. ,_ _‘ ‘, , 1 . _. - . , . ‘ g'_
‘4—' V tau ..‘lU- O p‘-"‘. _.lle-J J'-‘.-\—v ..uw —.\I~-U\/L“-o~)_‘..v L41 ~-'. u-.L...L .LsJ J-...VL-. Unit. .-' .._uc
0 --~' - I
“‘l‘\r’_‘5“-', j\‘_~ ':_1‘\~" , IIV‘ Vfil—r“ “"“1 n | n *j "1 v. " t-r KI)? 11-", —‘- t! | l‘..‘ "0.-\ I ,’ “' ij‘r r‘\ 1 -~ I“ )1"
i ’ 1_, . . ‘ y , 4' A.-‘ _. . . t,. J- '. ‘2 ‘ __ w ' ... . J, V ' ' . “"
g) . pv g--~ ,__ J 1 a U .L , :-. .L .J .a' “_Jk, ..\_ u’ L}. “d J. '1 o
— 1" a 7 _o _ - n ‘ 1 4 . . . 'T . q '1 j
. '13 ; \ fr‘ ‘1. u; \ f; ‘ ,, ‘ ‘." ' m \f - 1 1- ,—. . ‘1
:I L—-4.- _, ), _Il _,_- ,1 .LI ... ,L UK’kT— __ ‘_)A . 'J _ . l 7‘] l_' .) '\- .1. _IL‘.’ ... (C .1. , _ 1 .L; D 8.}. Iph' _Z: ’

n ‘7 - ‘1 "I 0’ . r' _ " . .. . .. 7' -

" ' f1 r‘. / r- fi ‘L‘ . J4 ‘ 'I - l ‘ 1 y 4 r‘ (a e (- ‘ -? ' —‘ _: ’ 1 ,_ l
"\ "‘x I ‘ I" ' ' ‘ I. \ — -.’ ' ’ "' \ \t"'|
V_ ‘_, '_ 1' u.._ “"'"|“‘k_J U__V _ 7. ..-. p (' L M.) Jr.) J- ...x Jk’. ....-L .1-_' .1 UV _, .LO" Ol‘
_ 1“ fl - .- ... _I_ - —1 _ . -l_~ .' '\ v o ,‘3 n . "I Q I . Q fl '
r 3 j 13“, T" . . , ‘ » . . . _ ~ ' ‘ ' ‘ ' ~ v , 1 1 _ ‘ ' " ’ ‘j ,‘4 - ‘. ~ '~ -‘r -‘- ‘- ' 3‘ ‘ - < ‘*~: ‘
.3- ‘J.,'\J‘Lt.‘. _.__‘_._’ -Lvul .l ‘Jx; . u p ,-,.,- .18. .1 u.l_. ._-.'.J JLLlVL‘x \. 8;',1J..1L.u, v-'_. Jk.‘_‘_u ,3 u.:C‘-'
, 9
.1 '. \ (‘c *7 l’ H “’1 ( 1 ‘ ‘ 1‘ - n m ‘1 , - . \ -' 1 -‘ , e. -. ’\ ’1 " ~ ' r“. "I V - I I
‘ .~.-~--'. f . x . ‘ . - n x’ . ~ - ‘ x _ ‘ | .1 ‘- ‘. —. - - - .; - . ‘f ' - 5‘ . ‘ .. .
ll. - all . .L‘fi“--- "" _L (*J,)\_4_, _: .‘- L A. UOC) ' .‘J: Liu‘ I-“‘J iD—L‘ -_O‘ 3"" U k‘ |-.'\_.,, J-Ijr-(' . J-J.L«. .: ,J_'_ In: 1.1.jdl-l ‘JJ—Oi—L
.4—

or —zfl;&g_fll ;hl pgxxr: s; 4:) ;¢rtu¢y;0’_ugx;f, Ol‘fi. two—

C1

.- ._ :..,- .--.-~—. .. ,. . - - .. 7" -- ‘ 1.-.'..'. ...,I - :3 _. ,A. .1-.. _--__-,
)— L:.IL- KILL. Jr: -'\J_I_.O ~IK' ._’.. 1, l..~. 033—1— '_ .~‘-—L.‘_. 0-,. Lille L‘CJ..—t \JJ— ‘Jrk: Pf) _ J— b—l—Oi. S O-‘-- U-..Iw 1:. ~---AO.-"L_C'

, n q o - u 1 | o ”I I

H -,-.- _«__- - , __ , -: ...,-.-fi . .... .. .. ..-.-.” ., . . x. a _. _- - .
‘-. PI-‘.‘1_._L;3L=.L1.LO L . '.1.“ (a .. '-'..'_1.'J--O.2 L.O:Lz.DO-:.LU_O’.. 0.1. 1395.1. Lib--...‘U 3.1..-- .-.~'l(.)L.

I. . . ’fi q .I-. - n . - o _ ‘ - ‘0 .
1 ‘ ‘ . . _ . .. '

v 1 o , q . _ . _. , .. _ _ _. _'_ -. _ _ _ 7 _ ‘._ 3 - _—-, _.
1" ..-,-s r. ,u - —. . ~ . ‘ ‘ ‘ I , I 3 I \ I s I ' > n j .r “ ‘\ 1 3
. (J U_I:\_)] Ls": E: .‘. ..L .1 x), kl ) 3.-- -Li I \IIJ L3» . .1. 0-. x E) V .J—\ ‘- V3 ..L. vu ' K- -1..A. ._ ,-4 L’J—L - . _1.-.l UL .p g, '
o I r1 - _‘ r—- o I -\ j w - - ~ " I u n n ‘ 1 ~ g -\ —7 v I
- »- - ‘v .5. \1‘-'.‘ ' " . V _‘r «1" . “ \ *-"1"" . ‘ ‘,\ " ' "0 “ ~‘1‘“. ’ "'7‘ "1 ‘ " ' —‘ "‘
1’\ 4 ‘ ' ~__- { db ‘1 ' T I -._ K} .14.V.14Q . . _5. [.J 'v I) U_u‘ _'_--'-tJ - x, U .L: \J. ...) 'J ... “a. .._\ C. UO- -L-—
_ - . , V ,_ . 4 ~ _ . .... .. _ ., . y

"r; Q q I _I_1 # _t .‘ _ _ . .‘.. _'_ ‘_l' 4)-? J- _ ‘. .‘ '1 ~‘ ‘ . ‘ .L.‘ -7 1‘,
‘ ' ‘ ' ‘ ‘i l .
+lL—tLg1Lv’ 841'... u“ '\ .l. "- .1-ka uO-.-LV-_ 9‘ c-1fi--. . \_ :4 U ..-.Je .
~ w

Table 6.

1').{\

(i.

Amino acid composition of NZ ca

Chromatographic method of Moore et a-.

 

Amino acid

Amino aCLd
in N9 case

Nitrodeo in
NZ case

 

fl ‘ _[_ ' ‘
hSparblC asid

Lhreonine
Serine

Glutanio acid

Phe my] 8133.].“8
\‘nrr fl ' ,
r q] __]_I r—5

 

LI ( 12
K‘. ,/
7,.oh

3 J28
5.81
i.56
3.35
8.66

._1

f)
O
o\
3*

 

 

mg/lOO mg
10.52

11.72

13-33
0.52

}_J
to
{—1
~1

 

mg/lOO mg

0.590

(3

0
L2.)
\ 1 1

O\

3.h20
1.530

1.772

 

 

 

I“
_

 

 

 

 

 

 

 

 

 

 

 

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VA‘ . ‘ -n, | . --. ("fl-H "- "‘ *4 -\ r ‘ 1 . -,-An"- ‘1 - . x- z-r -r ’1‘

since a iacui;emeus 101 ’081 16? U luiUSth udfl bCCu )3eblisnom
J“ 01 mihqti ‘ ‘ 7mm: 1- 111‘?“ 173i '3‘" '1 .1--«~ - *1 a -.eJ« MW“!
LII/l“, \4_1.L'.-_.. _L.‘ --Ofl OJ. ...=_k.-U_L UK, L..O-.CC&- (’34- ..Lflf‘ ”NU C0111 01-x-..1 LID O_L U111, Lugu gel. 1:1,“

throueh the use of some Ciel
W

Water. r11he 1i1t3ute 0° the eoo1ed eed do uted b1mt was Tivi1e1 egually
into tweity-iive ee1_ono%o tubes, each 0; \I11ieh was immersed in 3,000 m1
of disti11e1 water within a 923 K 102 mu p; ex tub . 1Le love? end of the

(—1
...J
J
.1
'11
,.J
«.1
C.)

_ 11 1... .--1- .A ,.. -_ . .
e: sends and St; us end the upper end
I“)

-- .0 , -1 A -,,_°.. _0 _. , . 4-- -1-. . _- --7 M ‘4- r;
was JfiDLdCd cnwn: the ~._.31 o- LAMB operx<3fld ao_‘tee ritess :Jibe vuxaae lL;1fl;S

s then covered with e 1e

(‘1
Q

J
A
{J

secured by SEViLj. ihe g1ess

so t’éd to the tug: vita str‘13. 13e tabes were

.._)

" winutes prior to ixeu tion for 15 ours

zit M 0. {Eve iiPJQ“HXCQS vexm1:poole1.:wx1Iiispetjxil irto si r'jr 11 sexing
bott1es so that eaeh oot’31 ” voulfi eortmir material having a dry weight of
62.2 mg or the d‘y weigtt of etude beef heart whieb is tequirefi for 5 m,,
tfie usua“ amount of euiture medium in one tabe. This standardized uelium
ingreTicpu Jns ”“iefi ivo the froze‘ SEC" in bott_es. It 7:” “owed t3 be
a satisfactor“ s1hstitvse o“ a 1:3 veidht basis fo; the erude beef beert
inxusi01 used in th; 00 ' pm1 odvetiov V1e1UW (fleo1es « ””fl 0).

Q

The beef Kemrt infusion difliyzete before drying from the feozen state

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Tflfleg

 

 

 

V. Compa‘ison of crude t0 dialvz ed beef heart
L E'Llsion in toxL1 p_,010tlou
. I ,1 I
Jahfiyml L1*/ 1
Crude bee“ hea;t 1L“1sion** 1,100,000 45.1
Dia1vzed. 0e Qef heart 10ft01on** 1, 600, 000 h3.75
Crude beef heart b:0tn*** 5,000,000
Dialyzed beef

00“fi;h"“cfi**

2,500,000
*ML ‘

 

51.9.
— LlLiflal letha1 0050; Li — f1000u18t103 uni of toxin.
- n r‘ /
**Ha:veste d 81te1 o davs at 30 C-
L" I ‘v '/ /
* "Tn‘VCSBeu afte? 6 days at 30 C plus 0 @335 at h C’

n production

heart preparations

...

 

MLD*

 

Beef heart brott dialyzate
Non-dialyzed beef heart broth

.1.

Crude beef heart broth

500,000
700,000

500,000

 

avg at Oak C.

 

.w 1‘ K. n
v f' I
‘ .
' .
l. A p" ' . u : I
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;

 

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1
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Ido:

itified axino asids con"ributed by purified

iniusion to the toxin production medium

 

" Y1
a. ..A.;

no acia

Ho

v- ,H. -f-’ i ‘1 -,. .L' ‘.
LOnCGLLi¢ulOfl lu VODCGAtIEUlOL 1a
puiificd beef hPart bOX E medium
'1

 

Taurine

 

 

 

12.012 “.995

 

 

 

 

. ,_‘

J J

Table 11. Yield of toxin from he same cul
by two filtration methods

 

 

 

of filter: Mouse Mime/ml
Seitz (asbestos) 100,000
beitz (asbestos) < 100,000
itz (asbestos) S 100,000
'qtere- glass 1,000,000
fimceted glass 1,000,000
tered glass 1,000,000

 

2 minimal lethal dose.

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T ab 1 e

T1-o-c1 -L.
12 I .._II ‘ 18C L1
.1;

at veriou

J/

of different anounts of penicillin added
intervals upon toxin production

 

 

 

Incubation Optical Average KLD**/Kl value with units
time density* penicillin G/ml culture
(530 mu) 0 unit? 1 unit 10 unit; 100 unit: 1000 unit
hr
2% 0.23 10 80,000 80,000 70,000 10,000
#8 0.hh <<100 300,000 300,000 100,000 100,000
72 0.M7 50,000 300,000 300,000 300,000 200,000
96 0.36 200,000 200,000 200,000 200,000 200,000

 

 

 

 

 

 

*Optical density of the culture dete:

addition of penicillin G.

**FID = minimal lethal dose.

 

mined just prior to

 

 

36

Table 13. Effect of an optimal amount of penicillin
at various intervals upon toxin yield

 

 

 

 

Average MLD**/ml
Incubation Optical Penicillin G per ml culture
time density* 0 units 10 units
(530 mu)

hr
0 0.135
17.5 0.239 100 50,000
h1.5 0.h3 10,000 200,000
65.5 0.538 600,000 700,000
89.5 0.368 700,000 h00,000

 

 

 

 

*Optical density of the culture determined just prior to
addition of penicillin G.
**MLD = minimal lethal dose.

ds
1110' ue

ac}.

nino

 

 

 

 

 

 

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m 11. one V. as.“
A m}. Tl mi Di

 

 

 

 

An estimate of the percent of amino acid in peptide form in the
medium was obtained (Table 15) by subtracting the sum of the individual

ree amino acids detertuincd in the {medium f1om tr ne sum of the corresponding

P.)

amino acids found in the NZ case acid mrdrolyzate and the free amino acids
added to the medium. Such an estimate ignored the contributions made to
the medium by the beef heart broth dialyzate which contributed only 3.7%
as much weight to the medium as NZ case.

The changes that occurred in the free amino acid composition of the

metaoolized by the org anism for various periodsare shown

U)

medium as it wa

A study of the data in Tables 13 and lo eveals that threonine, serine,
and clutamic acid disappeared from the medium at the time that toxin was

‘I

and

0)

being roduc ed rapidly. The basic amino acids, lysin nis tiline,

increased in concentration whereas the other amino acids were decreasing
in concentrati01_ at this time. A number of other ninhydrin positive peaks

0 c 0 n r“. ‘ u
appeared in tLe various spectra particularly alter -5~ nours when the cells

q
C.

(A

were autolyzing. These 11inihyd1in positive mate rials were not identified.

 

j. affect of hodificetiois of the Stande1dizec Medium
on Toxin Pr roduction

 

ractions pi' eviousl _y described

1--:,

A comparison of the NZ case dialyzate
to unfractionated NZ case in the stonda1d1sed medium for toxin production
was made using the pencillin treatment 3L0 sintered glass :ilters in the
procedure. The results given in Table 17 reaffirmed the fact that none of
the NZ case fractions could substitute e1 101tchl" f0; Infrzctionated NZ

case in the medium on the basis of the amount of to: {in produce'.

 

. . .
1 1. ._ .. .
. N . .
. . i
.
r t .
. . 1 1
. . n
u I
_I . 1|
. f v .
. .
.~ I _
. .\ ..
._ . L .
r . . .
. . . f
. . 1
JI f1 .
.L . . .
1 . . .
1 . . n . .
.. .n r I u
. .. _
ls . . ..
. .3 u .
. . n r . 1 . .
. .. _
, 1 . . .

 

Table 15. Estimate of combined amino acids
the standard medium

 

Total
amino acid amino acid Portion 0"

added in medium Ffee Combined amino aci
directly from amino aciu amino acid combined
to Helium NZ case in CGCiUfl in medium in medium

 

 

:1 01 F7”:
1 1.1 Jo 1, 1 11 .o
1 V
1:12 2C“ s13 E“ 3 - 5
ll: / .r _/‘\
)T -‘_ )‘fl ‘1' lax/‘00

 

 

 

 

 

Ale '70 ”W
7 a} 1f 1‘ I\ I
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11 JL“ ‘ / I .J
7 Ft. '1 {3) f" ’1 n
_‘_, , _'_q'i' Q?! 310.:
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'0 U, vb _’_}. .._Joql
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1’1: _’o 0:1 o
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5 ha (WC —‘ Fir—I. f4: _
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a _1 “ 1“_? "‘

 

‘11

-I\'-

I
.\

 

x - -a-r-.a.t. ‘

to ai 'ua ic culture filtrates

”vowth of C. tetani
x—J

 

 

I l . I
.oncen'ra'ion (MQZlOO ml

 

c c J
:r 65.5 hr 89.5 hr ll .5 hr

U)

 

L)

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>;

 

 

 

‘l r. j '1- -'— ”a 1‘- - ‘ - “— -'- r ".-11 :‘\ > ‘ - -. \-l l - .. , ‘ -T‘ _ ‘
n SGLCHQ abbeflpb tc SUbSb uue -lccilons oi is case -or tee

‘. -C'-, '1— . * -:- 1 7‘1". .fi . 4‘ r-x -1-r\ f. ~u j . - - j sz- -. < - 4 ,1 ‘l.
ualractionaued “a case 1U his ste1daldizcu Leliuu was Jade Usi1¢g tuc

"" . — .—. Tl ‘ 1,..‘,- ‘7 pan“ -'7‘--.l,.-L‘ .... . _ . _ 4-1 - 1
aCiLic, neutral, due «as c is case il'ctions prepared b1 sue Down; and

fl... ° . .-- - i1 -,, “3‘. -, ,. ..3 'M- - ._ 33/ ,~,—.. _ -_
lufimarlite resin pEOCGthzjfiileU€1lei cqm111illc. (1550). zLLUhough these

workers had ZOIDd tha iree iractiocs o: a combiia ion of tae acidic

[_.l

J'W

fraction, neutre.l fractior and ac“ yl-L-hisoiainc c0U1d subs titu.te for
unfractionated HZ case in thcir mediu_m for toxic production, Table l8
shows tbat this r-eport was flOt 01f :med .

Based on the amino acid avelysis of the standardized toxin p: oduction

:uuleted in which no NZ case was added.

'63
p.
[:-
C
{3
I..i.
I
U)
(J
:3.
C:
3
1.-
(3
-
o
c
E
g:
to
O

The formula for the conpositior of ‘his mediuu is given in Teal.c 19. The
only itgrediect in this tulle which hgs a ;he”i:ally, undefined ccnpcsition
is 'bhc -3eel \e”n'b tv“e:iu.

Although this mediuc Uppc; de tbc grcv'h a” unwashed cells of the

.—
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. , . -

 

 

 

,4

[‘3

L

. nifect of substitutes for HZ case in the
taidard medium upon toxin production

 

 

- . ., /
Variable MLD*/ml
KZ case 100,000 and 300,000
Neutral + seidic + baSic 10,000 and 50,000
Enactions of NZ casc*‘
NeLtral + acidic fractiOis 1,000 and 10,000
of NZ case + ucctyl-L-
Listidire***

 

*MLD 2 minimal lethal
**Frdctions according

Miller (1956).

***21 m3 scet“l-L-hist

l;
a

dose. Two tests veue done.
to procedure of Euellsr and

iline used per 75 ml medium.

A
-7-
l

for 100 m1 of a
f]

h of C. tetani

 

 

Ingredient

 

Ref-“311000. .130? o o o o o o o o o o
Il’t11ri3 C’I] ill-18 0 a O O o o o o o 9

L-VE‘.liL’le o o o o o 0 O o o o o o
L

T (W rh‘j \j‘ "r‘ TT’NW

..1" .i. L'.__f --u o o o o o o o o o
-'——.

1:-03’8 b--1-e o o o o o o o o o a o

0
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l".‘l‘- ° . .' .
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'n - --C- --: .
Lil]. x/L 1.84‘JLL’1 o I o a O o o 0 o o o o

' '\ Q
nyinoxine . . . . . . . . . . . .

‘n ° .1. 0.
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0
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cncess (:Nd.c a; oi T-UKUuU‘ij 3010 was Lsec Co a supplcuent ic hue set;-

A I ~, a

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30““, \i _O ‘ _ J" to, go _ O _L U-k.\-'w l._L\ _ , _ o _ Ac: 0

 

Eodified
replaced my

st9n

ro Le€ium

(q {'7' «OI-ll)
..‘l b —-

OI

 

Inorsdicnts

T ‘l ' ~ ,u "a
1180-10.sz _--L

Control Ledium

 

If)? 30L, 0LT T20

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tacil
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Wyndies.1ede :9 G’o"“’ and Toxin Production

\ - w

in €I\Jo'1flf7 09731 WJ:_1Eea.7 ediu

 

1 . J».- . ,n «4- . -. A'- J—‘I . 1 . “-'i ‘9 —-«1-1 . 1 _.T‘ T;- ~ ...... r“ -A t 7.". fl v .
7. IHVeStifesion e1 oflC mAQlUw ioiuu a 01 ngflindu dLQ Humphries
1r, “a. . _ - r— .. _., - ~ (7 -._ .1: - -.. -3, -. _ _ _
AdhiMdn and duwp711es (7950) weie the 0213 VO;Q€:S who had reported
1 a l u f“-

tne compo31tion 01 s SJH+hGELC meélwiu:1 1o: t_te cultivation of

_r medium.

A...—

— )7». -. - ‘ - ~'—-7 ~ 1 "'h - ‘f‘ _‘ ‘ '7‘ “. ‘ . ‘I- “1 *‘ ,~-~-1 1r v-‘L- , . - \
o Qaptsm and peel OwaluGQ on73 inCOQSlSUChL 31 ”be 1m

 

l?urth1“0”o they dil Lot deterniue wflether their pertieulsr strains

_. 1 - 1 ..z. _ _'_ '-, .1.‘ '.. 1' 7‘ 43' - -, “ . ‘v‘- . _I_ -'.1, ' -. _
procueed toxin la oflGl; _eoiux. deoium prepared aeooroing to tgeir formula
was “ested f,r its ability to support the riowth of the Mueller strain. Fo
growth Wes obtdiuel and a further work was done with it.
2. Harshall and Kelsey's medium17u1d modified for study

Iarshell and Kelsey (1960) have made stuiies of various culgure Hedi

ir order to est? .b7ish 3 abbrflflvd mediuw for general bacteriological usage.
In @?C of d7eir Wedie which appeared to be guite satisfactorv for their

purposes there were two coupeneets, e ein digest and yeas t aito7rvate

the COWposit ion of which was determ1ned. U51ng this analysis and their

semi-synthetic medium for nle as a basis, a medium was developed which
‘ a r ~‘ 0 Ai- _’_‘V '1 1" -'- -' .'\ . m1 .'\ C . - _f'l "-1“ . V "'1 '1'“ -— . v, a . . -
sappoited tee o~.wtw o1 s. tetini. lee nfimpositioe o- bulS SJLDLCblC QCQldd,

 

“I.. “:1 '0.“4('"!‘ fi‘r r3“';“‘~'\“"~"' I; ‘,,"\,W .“Y‘ -Tric‘ “'11—:— f‘] le-.-‘(S f *"I 90 V ‘.~l-‘—L. ‘r‘ r\--
_11__1Q __ :L' Q t, "‘ -p‘; _'_ .L' "I' ‘J .'..A.'\;U-lw_'..- \.;;-J L’ --UOL.-Q.‘/C\L 0.1- - 1.1.1.1-L; be”; it ‘u

K’v ‘.'.\_, ..1. U) _l

A
'7 . _ . ,- .2. 1 .:. -_ - ‘ 4 , - .1_~ _ ‘
97 ” Fooled and ivocrieted with 1.0» of Dewe513u 33118 01 use in-o

 

 

'7
'- ' I) "1 .' ..'. .' -: 7.. .2. .WI-L a . .. - . .
C‘ 545:; v‘ . I rm (1 ‘r" ' ’ ‘W‘T. ‘ '- i x '-‘/\_o- - 1 {'1 4—\ -~‘ 7. —~ W. '2 4-11 --. '~. ‘ a ' J- 1
elm «~15» ,1 v ' v‘v Uw-w’v u“ V~‘-‘— ‘LJ ~- -- -~ '.A’;\.=.1.‘I..7~l_ - 1-77; :.=GL.-.LI‘L‘.: TTLNS L/ZJC‘--. ll..CL1QL’.' :CL
“
- ‘1 _ ’7 ‘ 1 1 « 5‘ ‘w‘: J , .3 ‘ 4— -~1 '3; 1 4—41 -'—‘ -'—‘~-' 1
“wee7or7‘717v ”u 3: C r~~ Lu. 1101s mos a; owe UULQlu Let11 U18 tiiid
_- -0 '. 1 n4. ~~ -'—‘ -'-—‘ \ ~ ~- N - n \ r~ - 2 ~'- 1 -~. «‘4-
'V‘ o: 1r'nn3 tioe; ‘71 sols xbux‘:oalg o. x/7rw‘7 ceils kENTEJfGL- wo 7; piemmn.s
_ A
-_ f‘ _1_ - ,., _. -L.‘ , , "J. - ‘ - .f-' ., .i- "J.. _ .
1: 1 :ren nufi'UCd ”es” C" t;7 onitujo. 1 ,OEolOU o lee ‘Uib“3 USS
151 J-7 - -_ 1 “ x S, ' 1
1 : m‘Lofij"JO]I“Ax3 Se3,17 o 111‘.

 

Table 21.

synthetic medium (5

Composition of first successful

9’.-
l)

 

Quantity

Ingredient

Quantity

 

L—glutamic ac30
L—glycine

L-threoninc
L-tyrosine
L-valine
313301". 71120
unsou.hu20
F8501!) 71120

135ng msqk
##,0 mg N&2HPOM
72.0 mg Ca012

K01

Glucose

Thiamin.HCl

Pit-r} idoxil’le . HC 1

82. mg Biotin
32.; mg Calcium pantothenate

Riboflavin

hicotinic acid

Inositol
11-900 1'9 312
15.5 mg Choline chloride
60.2 mg Folic acid
”J 9 We 0"amicobenvoic acid
‘ AOJ “-0 Lam. \, .l _J. D V..-..J~,_. . L-

.- i. .,.-L. - q * -
Lila.‘ 4010. 0101:. _L-Yr’X

L7
2.0 mg Water, distille
2.0 mg

 

C!

.A

0.151
0.515 2
0.002 2
0.204 g
0.200 g
1.0 g

0.015
0.015 mg
0-75 us
0.30 mg
0.015 mg
0.3 mg

0.85
0.00 4’ 11g

/

3’1 0‘
LU

1.0 mg

0.01 mg

 

*The

medium was ad

solution.
**Solution I is

water and 2.7M g

sci of 5 ml 850 lactic acid,

A
{‘\

V

7.0 with diluted sodium

.05

hyd roxide
ml listilled

 

 

 

\, _-

.
. .
. c
)
..\
I.
u n u n
. .
‘
.. L
n u -

 

 

.
VIJ \J U. ......L JV _L...,_ ‘J._'. LIV: . V .; c ‘77- . U1,- ., ‘77), “’..-.J .1.- _7U VLLV _. v..-\--.__.'.~u\.,__
# . . \ (W .-..
'er‘: . F" ... 7.1-0,. ‘rfi‘j 17 :-.; 1 '. ,7- ,.J. _ 7 .._7- it 1 . . y 7 _,‘..I_,(, 1‘11‘1
. — ' tr ' -._ _ '4 ‘- 7.; ‘r .. . U_-- .4. \JV... aJ U‘v...‘ Q - - U A \J _L,\7__ '.’ ...-K- . _‘_ ._) . __77_'V
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_- ~—«~J ...— ' ... -- U .... .4 (J .J . \4 .' ..‘A U-_ .. \J-I\ ...—— U” .4 U7. £J . _ .4 RJ -.. _'_L__ _L_ L)_7_'\, L77 .
r- u . 'V n I H r‘ , / r'j -‘ o 0 a - A
7 "777'“7'” —. 7~cl .77 7.7 7.. . 7 ...
-‘hf—LV v-__. L.) ...--- .-_..~ ~ v‘ -_ -” -' ’ ’ 7\’_ .«_-_‘. ‘4 -~.)»\, 1kg. \xe,.,, LILV \J «/\,,_ "_ 777:7 7‘." 77 _7
.‘ i 4.1 _ ' _ ° . '1 3 . 7 '1.
7 . a ’- .40 I ”(x or . 7 w
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7 ~ . ‘\C 1 " ~,.)r~ ~. “C‘ ‘l I] no r‘. . .u v.?.: C‘ . 1 . ~ . *1. ,— >¢
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11"} 'q C‘fiW -; o 777 7 —] ‘ ..- ff: .._1 7‘ ".3 (\- r---'-‘ .,,A _. ..- _. _'1 -.2 ..l_K§ - .2 ‘W _; 7 1‘1 '1 1... w 7‘ I 7, r .
-.- «~ 7‘ ~ ' ' - 7 ~ .~ 7- ’ -. -7 r ,. 7. ..fi .77’.‘7
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—
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y- ..
. 77 7 ‘ 7. _ 7 7 7 7t_ -. _ 7 7 _7 v 7 7 7 J _1‘ _\ _ 1 7 77‘ 77,7 7 7 7 7‘ j w _. 7: \ 7! 7. 77‘ ’77 (77 v7 7 Q
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.._J .0.... _' - .- - J... '.0. .2 U v v -4... UV -.-.'..1~_,\ . a --/ _ 'J U\. ..i’)U7' O _ 0-]
J. U _‘L 7.
v w n a . '1 v__ 7 u _l7 _7 7 _’1 _' J_‘ 71 y 7n _ o o y '
'7- -‘ f\" r~ - - v-«\ - f a .H ‘ ~ " 1 ‘ , / . . I/ ~ ) /-\ \ (7*- - f . , - - -»-,
U- 7- ' - .L'_- Tl 11_‘.‘~l_.“_'. U .- ..MCFM :1- UK) - - ./ ~JI-.Lx, .-.. U_‘.L,'(~--..,- 1 J __ _:3.__.‘_L'Jv ,L;;‘
777 -1 1 _ . 7y— 1 _n _ 7:7 7* 7- 7r:_ . 7 _7 _1_ 7 _: 7 .7 _f _I_ _ _’_7 1 o n _ 7:7777‘7 7 77,7 J— J— 77 . .1 . 7
» n. a r7 1.-. - - . =_ , 3 ‘ -'7 (7" . ~w.- C‘ c‘ ,. w’
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.L'. . ‘1 _- . . 1 :7: ’1 -7- .t-’ a". J-‘ . ‘7 ‘ .,. —. .' 7 -‘ .. -' .- .'- . - J- J..-" -
,7) . r .. ~.- . 1 . A , . . 7 . - 7 ‘\ \ . -4 '
LI ‘. ‘.fl‘ - _ .17 .... .1 uum - _, .1 xi . 7'7 _. u 1' v '\ _'. - ._u u~vflpfl -U—'U>LO‘.,
F1 , ‘ 7’ , 7. .l_ 7". 7'7-! 77 7 77 -‘. 7. 7- I 7 7'7 7'7 77 7 J7 . 7-
f _| .2- \ ’..-fl’ ‘Y_ '2 ‘. - . -‘ l , —‘ . r. . ‘ l _ . w I“ ‘0 I u - . | ’. .A. \'.. C‘ 1:;
7“... 0.1.,- ‘~.7 .—. . ‘1 0 ~' . - -—-7.‘ j ...-:1. db. “U7 -' .. 7,- -- .J
-r—fls-m - m .-.,7 7 7 7 7 —: ' \ " .7. '3‘ . 7 a, c ,. -7. (J-r) w-tgea 'trr‘
~-- ‘4 ‘ -— ‘ 4. I ‘ I. 4 . . ..- ‘ . A
d.- J. ._ 77/ . \_ J . J -- L -~ . J 7’ 77 ) J U_- 777 .7
_I_m flj‘ - ‘l‘q' V“ 15"“ J-" " ‘ ' A .' 7‘ 7 7. 1-7 ‘ ‘ ’"Mfi ’ ’ A j . r37 . '_‘ f'v- "‘1;’\‘I’ ‘. \ '\ "j 1].
(J7 - -. ._, _ , ‘1 ,- ‘J .. - ‘1 2 -- -4 , - ‘7 ‘ . J - 4’

Table 22. Toxicity of culture filtrates
before and after storage at h C

 

Mouse symptoms*

 

d-

Dilutions of the filtrate Days after mouse injec i n

 

o
l 2 3 A

 

Before storage at k C

 

a , bot”
lOO 2+,2+ 2+,2+ 022:

1,000 _,_ -,- -,- _,_
10,000 _,- -,_ _,- _,_
100,000 -,- -.,- _,- _,_

 

 

 

 

 

 

 

After storage at M C
'1 I1
‘1 '3 ’_)77 1301311
100 J+,JI 7 .
0930
500 l+,l+ both
dead
:L ) (3130 -' J "" 2'1“ ) 24" b04011
dead
C‘ . '1
))OOO -)- "j- “1" l'T‘,_+
*Two mice in‘ected oer lilution of the filtrate.

"r11 ' "N j . V" ‘41 ' ""_ . I n 'l‘ I“ "' . ‘.W " 1’ . N x H. I“ m
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— -—--4~ u -~« ~~-» v 4.4.- »—- —~4 7- , w :~.. - .: - ) _...-, ,_~-) .0 .,L_IJ
“DCHWtG were obtainéa iv 31LKic t; +ub33 of 'edlu~ or in difife?eWt n‘nofil-
melts. If fho MQFLMV v;n Usoufi“u t0 w wwil By Tucva *rifl" LO inoculation,
we ifiprcvehent cf jffiwth ever LE9 chtroln c<uld be absurved. If tho
medium was Overluil witU ‘iWCVul Ciig growtk fiis f1: 1033 t“fifi in the
”CTiTOTS. InconsiSLEfit Jésults wave 3130 poCiieC WES" 3” ml Ff 6C nwdnua

'1 _ _‘_ 'V _ ~‘ I_‘ ‘ 3. "‘f" “ C} _ I a o p o 'v A ,\
'rrfi‘nrh _‘.‘1_~ r 1.1 ‘ ,1 - L .m- Cr’x ”.r ~~ fii’ffl" T ,w .-‘-< .- 7- v “ (\f‘u. - .
.. V _- -’ 81113733 31-;0 - USO. .‘ -._-Lu, .‘ '._; - .LJ ‘.. ,_ .4". ) \‘ . I'U , ,3 u”. j '-,I__‘""_1__--) U]_1_:_ w?) :1: U . \.~‘(_ -1\ 5

. ‘ .1 n "I '1 - . , , ~. 77 H u 72-. . -. ~s . . '1 (:1 A . .H ., - ." _I_”,
1 (\vver, . V (a ...v \ w _ V I A.’ g r \ 11 1) ‘.. , J ... | ,\ 4' _,_
‘1..' :‘ir‘..o WC“ r“ ) ' _.’_I k.‘ I L Tu .) ‘ 1 v .L. La) .: ‘--__L[ _ .- c .1. . -L "U ~:OLILUOL_-G-;+\;S 0-4. V. .' .3

l
1‘. it? C. 11TH

) ~«

 

_ o .- . v_ _ ‘ A 'l _2 , _J,_ ‘ _ ’ ' _ _I_ _ r _!_'f ‘ _I_ , ~ _ '3 o ., ' ‘ >‘ _ a _I_ ‘ J-
O” CtluulaLOPy; I'—3 L“ 'r(w;?t lin o;“ JS'IJ Lc‘lflu MloLx slyh uho c0113.

I” +1» ...w- (Ma -:' cw: --i « me «qn \ «O- 1w
[1. ':-__‘ l ‘ ’ L, L)“. A 0.. '.,'..-)7‘.. .L'm'v '.Ln‘i’ DJ.“ fl'aogwuh‘v. "‘~,.-_’) L-,_,..JC D,_p0(.-1_e'_l ,J'b.

‘1- '1 (a " '— ‘ 1-« w. '“ ~~. vvr V '- x V fi 7 " ’. (H r1 . «1‘ 1 ' ~\ ' ‘ ‘3. ‘3 .
" ‘.“ ”W . " ‘\ 1 _‘_ " ' ’ i ‘i _‘ ... ‘ ) I ‘ “' I " "" "“ .t‘ “‘4‘ " “ ‘
...I]. ---._, m J L ' _A 1.1L 1 . ..'_ I » _ _ K J # .-.J -L, /l,. -K ,' '-- ., (“3 .. Q LJ ..u \JL) Ln 4.3.“. K. _
/ ~..
/
o '1 c j I u w ~ . ‘ "I v - u , —‘ .1 :1“ -‘ _ ..- w -‘- - ‘1
~‘\ '- (' 1 ‘ ' * 3" n '7 V - rm” » nrx ' a‘ ‘ ' r“ . ~ ~ "‘ I! ' ‘~’ "\’.' ‘- f) " "7
,L l lJ:’]j_Q : C\:_L ,. n J. l! : .'.' ' ‘1 _. A. _ L’ O .\ x,‘,4_ .uJ x'__].l' “K ‘_ ‘0’) L\ v 7 :L, 1
\
‘ ° )4 _ ‘ " V . A —' ‘ " ‘— ‘ ‘ ‘ ‘ 1 A. -‘ ..- . . - J-‘
“ r r I 4 ‘ -I" fl f" j - ‘ ~) ‘ ‘ 1 \ ' \ “ A V/\ \i‘ .‘l \ ‘ ‘ I l ‘ . “ 3 ” ' ‘
1 .L1‘ .j.1\114 .... UHIH - ~ I. ! , . :er ' ~ 4 .( r. U ,x - _‘ “ ._- LI . './ ‘.-V _ .. ‘ —-» ULA\- \_/ O l.
n -‘ 1 q _7 P o y _ ‘ 4‘! n ‘ ‘ . ‘ .-L 1 _3_V _ ‘ IV _0 1 _Q ~’-..- J. J- ' ‘- H F.
lg-q , L ‘ r , (a "v n ‘ w 1 w 1 I (‘.‘I \F . \r \ < C“! \. - L m ’\.
K11. "T'IEIEL'V' '3'“; J .1] i] 1 LL-‘ ' ,1 , 'g‘)."'\ ‘3‘ \-¥"" JVW LL'V i) )..)n.).:~ll ”1-1.1. ‘.J‘_'--LJ ‘L-L’J ' »
1 a ' ‘I F‘ ‘ 7 \ 3 - . ~ 1 fl ’ ‘ -! 1 J (- 'V ‘4
7—: --, r. - --.\\\rwp --, -- fix 1* ".' "\ ’. " ‘ 3' “~ —“ a J " “ .‘V' Trrx-v'vr) '-\* 'Qr
t‘ie C t F) ‘\."(-V|)\} LI.‘\.' ,4 . m -er. , 7," -._ v- J“ L _' \Id VJ. , , O"\,\.Lo

 

 

 

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n .-
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The cells Vere moi sigfiificantly 11‘0”“u in the Les_ :_iuw; procedure

SiLCG a serial tube dilution of Josheo c;lls ’n T nediuw indica.ted the
l 6 - .

sum of cells to be at least 10 per 1.0 ml. Unwashed cell inocule

fl
containel less than l0“ per 1.0 ml as determiree by the sanle nethod at the

.1.

same tim

‘\ . _. ‘ . _‘
An examiletion

the presence oI at leeSt owe
\ ‘ -, “I“ 7 ‘ ’1'. ,‘x . I -1 -"- '
These c0wponeu s we": reset c
n -._.1 ..‘JJ u -) 4.- _.
supplementeo wlbu tuesc two 3

not support growt” o: a wesne
As a cont:ol 0.l *l of T LQQl
obtained.

“9’0" 7 and "romfl 07" r“'.7:'\.'~T"L‘,h 7““th
-. (.41 L a‘- \— _._ '.—,‘;—.)x.__ 1,- L). .7. ., _ L ‘ L
til): A. 10 '09:“ti'71 e €«3II.r?G r1
740 larval ~. ‘37} ”- (N'IF ' " O n"
-,pi,ccs LLCCL liti is t,
.C‘ —' ‘PA 1“" f\ -/I 1Q ‘ '\ ‘ J‘ . ‘.‘ ‘ "\ I C‘ ‘7"
.1. 19C] __C (J \J\_. L ' I \_,>T)t.’—l. U ]_‘_. ' ~ 1L_| U
__ _'_s _ .. c _ s ‘ . u ‘ _I_-I -;-
0.. Lnie wegfigvoligcl l-‘ L,_e 1-,__s
sodium bi7etbouate "is ntoyil
sfl;tile fil+.eto fie” .d"o to
b (-1 :f‘ 1 Q h Ill: \f‘: "" '2'“ ”a "N j '.
lCc- DO] -:- Ca, -;.< (,0. LA » o 4 _L

. f’ _ ».
Fls‘tcw7l T7fe l. IDCV7~7CD C‘ L>() ' " H7 1f
1 ’1‘ ‘ " . "’1’ 1". 7‘. r\ r“ J' ""‘1 ‘ “.1” 1‘ -
‘.‘C‘ bo-~l.LU‘_L;.U g. 3"- .uo bull
- J- ‘n ‘ ._ 1 _u v‘ D I .3 ., _
to he e m)QLUL C; 0.; .1 0?

L’—
- -'1 ("x

nr‘x" 1. S T‘Iji: q 7’237‘:‘ .... 1(vav ,L ,. _‘ ..Lll'

composition

of 0.1 ml of T medium (Table 23) revealed

components which could provide unkuowu nutrients.

a

xtrect and Bacto-casitone. medium was

 

‘\ -. ‘ J.— "‘y r " 'I“ - 'l-‘I ' a "‘1 “ ‘A
" DOUCI US c._L.OI .'3 Q" [1008 0118i , I18 'BQlLJIL. U 10.
7 "v" w; J . 7‘1 rm Lil ‘wd 1" '7 ‘l'L 7 7 1"" '1 ‘ ' "1
111C k111.-[210-.£. . *- pl l J sue _LiOT'. O l'J‘uOlL‘A.
‘ _ _ r 1 ’5 :i |_ _I_“ 'w ‘ '1 o 1‘ _fi ‘0 P-"n __I-‘y
up was deCo so tie so gteU‘ ale Q-owbn was
a
“1 ,w ‘."L- J " ('\ J— .rj - \-x (1'. ~23 1,3 T (-1 [XI-‘0 fi'l‘ .n O -q
l 1,90: LICLJ. 7 '..r. new 00- u. 1 (“‘--iCk. 7.8.0 oboe; 15.]. lo;
- . . ‘ '1 . -- 5‘ '1‘".- I 1 . '. 1"“ "- -
.mal 1100119 of qTOSULlQlHU pa U )OuullNUl
V _o r j .21. J_ , _ n _q 1 _o _'_ I 1 , . f! a o _‘ *-
QHla; cue gist QQIDOu elolioe coulu oe p? :‘i ll;
~.--1 . "‘ r‘ I.’* ‘.' T J v . 7‘ A ‘1 "I" . '\ 1 r\ - I" -.- J- .
st.7t= is 00A QlOLlUu oeplet Cu or an unidenti—
)\ ‘ r.-"- r y“ '4 --‘ 1-- '. 1:\r I“ ‘3!“ f‘" (V: ‘C, ‘0 Y‘
E“ _ u. ’l ,. 13010 13 'CV J.(‘-L‘-- .. polo _.O.:_ 0-,;-
0‘ zv4429. - Ta i‘vie 3‘”x i e':"7 "olut’crn F
.._.\-L, ~_,. J- V b. _‘_ ' -! 5.} L_,__- .._1 ’-l L) ,__ _kJ_. ‘... C;
-. . x x v r —-. A '? -‘- 7 -)'fi . I \ a ‘ ' . N 5‘ r (N “-n -
#24:, L l, sJ’-‘l J; J. ll U [JlO' - 1: . 3*. J o 1 .11]- 0‘? {SEE
.0 w ,L _ -L .vn .. a ._ J- _o _ .° ..,
lflL" Ol Ln .rxprn so the sze C'ffll.‘ O; sodiumt
/
1P7 TKLS ,— FM“. I?‘ Lf'e ;7u.w\ FifoPV'i°¥7 't lCl It“ (3?
" ' L "‘k-) ‘ "’ V " ..L ‘" ‘ 'V ‘ ' "\.J '-
‘ ‘ '.. "a" . J3 , -.-.-" ' ( . r, .m' —‘V,
_hw_ to ;0 ml oi u «cuium *s c LOQlele
uh
‘ .. “—‘q .0 . J-1‘-. fl .’ . ‘> \ ‘J. (‘ _L‘fi,‘ ‘ '1 fl - _'_.t _>
_‘J- ale 4.. ta. -,’,;-. :3 LDL: - ‘ C" b 3"“ o u 'L' «Krill L;...O.:
" ”_I_! _‘_. W , .r“ c _‘- 4 I“ -7. ’ .. I - - _- v 'W-u u ‘ r . '
. V I':,'_ ,2,_‘_3 Llj_ " bk) “1.0. “03$; 20D-.. 1L1”.-- D 1;»).14 k};—
F:7fs i‘ j ngiVW.

51L

”able 23. Composition of 0.1 ml
of thioglycollate seed iedium

 

Amount

 

C1"

m n .. .
c o c I 0 Q o o o o o o o o UOUE 1:18

. . . . . . . . . . . . . . 0.075 mg

. . . . . . . . . . . . . 0.50 mg
0 o o o o o o o o o o o o :1]- 0 SO lug
.‘odium thiOSlYC01let€ - ~ - . ° ° ° ° ° ° 0°05 mg

. . . . . . . . . . . . . . 0.0001 mg

distilled, us . . . . . . . . . . 0.10 ml

 

 

 

 

- L
.
. H c . . .
a .
v ' .
a
u n O . n h -\
.
i l I I l n I
. . u . q - u .
l u . . . ._ u . . u n
1 K I O n l l I n
. u a . . . . u .
. . . . . . .. a a .
- I a n I 1 a F U
y . n . u . . > u r y
c . u r . . . . .
. s o - a . . . .
u . . n p . n . .
u . 1 ~ . - . . .
l
u n . a . . n v -

 

In” 83/ n:-
iLle _‘_» iLf‘

‘

Q

1

t

0

se 81*

e.;te:1t

 

plemeuts

d cell
77C

would

”I 4‘

uppo-

"—1 -
Lve

-1-

L;

I1

#—

r‘ro*.I'I:.h of wasned cells to the sa
L times tbe nun ' oi “GllS (tub
I 1

- ‘.. ,. - — ' . 1-". 4—7 7 '1- . — -.,- _, . 1 ”-0 .--I. -I-
In otqe" CLLGflMGnuS Wlufl l medIum, agar, asa oi (”ClHU} 3east Egbfac U
- - ‘9' -I w. .0 7. _ :.- -'J . . _ --_ -_ w 1 .
or puriiied a -i gesgt iciuSi'c as ”Hoolere ts to 00 “20 um, no L"OW h could
C.
1 - l 1.. '1 .: _'_.1' .... ‘1 -~ , ~. w I-~I -w _I_w__ ‘ _ 1 “TIA r) 4.- _ -_ ... '--
0e reported vi a uablcl cells. nit iou : t.e unfasnod sx—O strain brew lu
J-1 I“ .- — . ‘ ‘ 'L-I - t w 1. - -'—-—- . . fl . .‘J - ,_\.'_
use 00 ubdldfl, tne unwashed -uelle: Sufalfl Lie use.
C.
["1 '.- 4.. /- - .. 7. .-. ...c" ,. ‘. - .J- ..-1. _ "7.7.1 0 4.. ' -3
'aus, lu uh» pye Ious so 7cs oi CipeJiAclts, Lne uA-C st:sia 0
r1 -1. - . . ° - -.- , ’-. "7 . “ -. .--, ‘ ’—‘ .— - r» --." . f?‘ . " — - ' 1
o. be uni Has Qrowg in l Lediu- sud tue same .uMuc oi cells fiere e1 0L8
“
_‘- C-‘WO: w»: -. - {-3111 (‘N"’/\ :1 .1". 11/‘\. fl I‘l"? '7 “I I'— .' F} T ’i 4"...“ 4,4 77". J“. j V1311 C‘ 1
./';.uI:_\,.i OJ. -LLlOpLALc-LICL- ~.__.Ll vv 0. U; - -u .22 '1ek-ll..:. 7... tn... 0;- n b110lb V0.4. 10118 gIlpp~ €—
r ‘ o 1 - .11-: -'.1. r. - , _"I _ _‘ 'fi _‘_3.’ 1 _'_ _I_? _ ._ 1” 'u ‘ Fj . ,.
metts. ltflOJw -Lsopuion tne unuss. ed cells 5-ew out Lie wzsdei cell< did
I
-z-\' -- (-..-E. TNT -- (‘{\—- n ~n1 \r
1‘0 U U-~ O- 0.; p40-—
7.2- -I-. .. ,1“ .- ‘..-7 a... ...~—'— ~27 .-.. — ‘I ' . 4.-. '17.-..‘1
Slcec the T delUd nod i smdll suOuut Oi ugas ”fllch was ceflbfllLbeu
- - :.m. 1-1 - “I. 'n — 1 - .1- -.- . . :1 ‘- - -1 1 L1
‘..LO‘.‘.L' 7.1L ml 0:-C cells 0031': L0.“ L8 “i1.“ 3005.)”- ::_Li.-.L-. ’.‘Tlllc'il COuipc- L LAG
-,~ _ o ‘ v, _‘ _f _I_ ~7- 4 I.‘ . , ,: J- '71 _. A.<—-\ '_ J- fl- , -I , _- T - '.- '1
cells '1 tie inocull. i. is qdite aukus_r~ou so lewyu tIut tic wells
7
- . -‘I '- ‘ - —'- .- ?-,-~ ' FT"‘-— .3, -' . ‘. - -1..-'-- . 7 . . .. 11-. J-‘ -
could be cuiuivsted i: -u nei7u coutalsed to dbwi. 13¢", tje cells coulc
-I - 1 , n--- . -0 , ._ — ~- s-- -'--, .—7-'- -. , -, r11- ' ‘-- - ' — -
so massed lugs Cl .11- ii -li~2:Is lugluwlyu _ch. luis 2C: Jos7ou ass
-I- ,- ..- - . -' , J.-
' :olocc d in the we- c: eglmg:u.
I \
7 , ‘ ,I‘ | ..‘3 _'-‘ - ,. ' ---. , I I.‘ _'_. | " \fi .1 A K‘\:' ' "‘1. ‘n‘ h“, _I_ -? 4‘1fi-
T. 3. "if {.1- b '..... u-_-’..u L-_J\__).‘.’ H1 ‘3- v \ ...; U‘nx. (_’_ ) .._- ! L110. 1.11-; U: (_I_) 0.1;
-' I
. :.. :0 .7 7 _ ~ l. . - " _- , .'1 - _o. _, .. . .7
IJJEIQ C:"'5W "25 its -uis: . ; - .- cu~ cI illigu-tl ___1i T. IHC“77‘ ‘.ds .us,..
_I_ A q “ I '\“-w‘ 'l‘.’\ VIA17‘ fl ‘ «1: _l "I 3“. [J ..‘_' 1:! C 7—». J_‘! 1‘ -V“_'; H. ,l 3;“ j ‘l. n (f -7 \ . _‘Ijvj y... “'7' 730.7 f-"W r; V 2 A
L}; ‘.-. L ‘J. ....k LIL, U ‘.. _ ‘ ..Llp-i): 5... Av! u \ LID _. IJ» V——-\_/ 4 -_ ) ,___,/ 9&1»; k.) V. -3 ulgI ~...__»J .,
otiiged fbey .7 E7V slof i;.71l:- iqc JD: “'3 ::h b- l‘CQVlflb; t‘h XLSNJQlt-
. - . t- .: ' .._ J-‘ . I “ -: .g . 7 7—.) / 71.17?
gut-”3'31 {3"117'5337 “."."F 1.18 3 (..--.-) 1;. .5». ..~ .‘ I. '2- -4.--- so": -._: ._ ~. 3’ .mn \sll)
A I-I " ’ ‘ “ -'- __ V ‘ - , _—‘ 3.,“ Wm ~~.- ‘.. ~
;; 79:;23 '“W7 7:?“ ; :sfii“: J;';.-- 1' u7j. *x.8.312 .7; ..cs, ;:ll- . .-i.
. l. L» .- i--s: -14.. usa~ - -. -.. -~ ..... -- -r: e. (:e) . o 7 i
\Jw'.jw.~ -L, L} C I" - LJ-_ ,1» K1 .1}: _ __ ' 7.1. u. 2. U.... .1.) _ H'LD‘V 39;\, ~—- ~"“ -*-' -’ * J \ ‘ ‘ . —‘ a
'1
'/ .— fi'- 41:!”— '-
‘ ,4 _'_,“ :: t.) -7 j . -L-‘ -‘— LJ J» C ‘x-' I.) - U--.

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

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seex medium

 

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to

 

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Thiaglyoollate -,— Goud,
filLrate, 0.1 Good
Lfl

Thioglycollatc —,- Gooo,-
filtrate, 0.1

‘17
:.--L

Darco G60, —,— -,3parso
l0 n~

k.‘

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.duguo GoO, -,— -,bparse
70 “:.N‘

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'Jpq_~\,

 

 

 

."
{'1 r n lflF’ ' i _I_. ‘ 'r - ,- W \ . var-\~ .‘l' r‘u J“, '..I -r . 3 . ’7‘-
Iebl4 :5. oluscv 0 '7'.S oupplsuonUQ 0: 0, hodiuu UpGu
,,,. , l -_'_-I '1 '11,, n _ ‘I —. _‘ ,7an _- .- _'_. 3:1"- . 1 0 ~. --
lemufl 0; 00139 l--o) . o. ullley-nb seeu neoium

 

iwentHLnt O.D.** ut 533 uu

 

“God Incubation time

 

o
' ‘1 I r w , r~' 1 \
iho ulum QQQ'ULfi uupplo out lo a? 20.3 or 39 hr

 

Unwasned 3TB 0.17% 0.19h 0.300

393 J-_ i :l éu.tu:e 0.139 0.135 0.101

- 7 W ‘-’ ‘ ' / ' ‘ ’ A
l1 we boo JED Agar Dlus 0.1 fil 0.1MO 0.140 0.25?

CIIlbeliwz ifll_-

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—“"1h _ 4—.— ' -'.I—.- '— " 4-" . —. -:,_..--_ ,- 'I .3. . -.
*olo — sbevillu 0030 0. on; . — LIlVQ ~iollaue seep meoium.

- - 1 .r't 1 . 4-2... .0 _;: . : __,_.:-,_ . _._ _, a
**0.D. : avoiaqa oi nglifll fleQSlUQ O; uupll-hoc Vuluugca.

 

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.. ._ J- 7 q — .._ - ., _ -. _ 1 y_ __ - . ..- _1 _‘ _‘ 1
(j jcwxw’x A. {73/ § 1 7‘. . .fi ‘»‘ I (11' 73 j x a 1“ . _ 1‘ \v- \ u r l—q ."‘V_, -. f“ I '7‘
(... _ --.-U \_,“_ ... ..V'..--7__... _.J 7. 1.4., \JV‘-_- 0.1 .Ll. .._. ..7-U _L\1 7 ‘J.- . .J \. v: ‘4‘- _z... .LILJ ... ~-..,-__‘-7J
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3 " "." O‘T'V" ';,’3"l/‘- ' “/1“. "vv 1‘,“ ‘. ' "-'0 gm] r" '.‘ O"- O r. - -. (1.47.,1' (1.75. .—I_7 " ”r" 1:.‘.._'-"\ .‘
.. Um... UV .-U_-\., -- V . . 7....-77 V.-t._. - . -1 ..-./ - ~ . U-.. __ ._ . Q ._ A, Us..._/, 74...; v ....'_ U-‘_[
fl": ‘n r“-" -'~r-'—‘\ -- '111‘ r t -\ -~'-‘ f I“\ .'~-‘r--‘«—'-‘ 1n~'—-- 3 _7 - 11 . — r' r4 0““ “I “1"\ 7‘.' \ _l .‘ J-‘ O “I
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(_,._ '.. O Q" ‘..' U_J O .,‘ .-_ .. . :..- .._ U'...L‘.7 "u V'J- ~ _L‘... \T 7-72 u L i)”, ,'.,..-‘.,- ‘uLu. _L ULI. \ 9 _~'_ '1].
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—— U44.) .4.’ -— ~J.’ .- t."._-. i. _‘_ d--\4'7. kl O
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‘ a-l 141.173 ~/-\ ‘1 . , g 1" r. 73hr} 19“.. ., H“: L 'D‘\ ‘.. _._ 'I — M‘ ‘. .:’\.\___'_.x.l __, _k ... VC . .. _JC} .
-V ‘ " - ‘ .- _’- . .- ‘ - -' \_/
- W l ‘ I '\ ‘1
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‘ ._ ‘ . 1}— (fi)-,z ; {‘1 . H" ‘ j “.7' \r- ‘ {I} ' ~ _ ’ ’ L \I. . . 1 .-D _ ‘ o 7 , El I '1) ‘j_._‘.: - A. J 0.. ‘71-»)771-
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q C n
-'.':~ 1 7.: .._ .. '.' .~- ”,7 A -.
jj. _‘O I '\..'/ if] 1‘ .'l ' .‘..v\ ' J ...) . I.l . -- J 5‘) -'- . 0

.r

 

/ -. _ -. __ .12-. ' _ 4.
2o. Importance of 1gesh thioglyeollmte
in the preparation of media

H3
3.)
G’
I__1
( D

 

Treatment Type 0.D.**

 

 

Tubes of Seed sodium supplement Incubation time
inoculum nedium* tliogly- 16.5 hr 19.5 hr 2h hr £0 hr
col1ate

 

O
f 0
\n
O
O
l\)
(“D
\n

1,2 Unwashed T Pres. 0.297 0.320

C)
'92

“ h ersshed son yresh 0.162 0.200 0.198

{I £44

/ 'r" w ("1‘ 1|_' w ... l—- , I
5,0 wasned 0TB r1esn 0.1 m1 3 0.1L5 0.170 0.207 0.2H5
r-* -v w '1'" . ‘.‘_. ‘ ,..,- f . . n ,
(,8 massed USE fixesn géac 0.080 0.000 0.093 0.102
9,10 Washed sea Fresh Agar + '.1 0.130 0.105 0.155 0.196
11 5TB

11,12 Unwashed T Old 0.030 0.030

13,1M UnwgsLed 3TB 01d 0°030 O°OBO

 

 

 

 

 

 

 

 

 

bid AI—'_‘!P‘
... '..,- LIL_. 0

Ci'

*T = thioglveoligte soefl refliun; 31b 2 steriiity tes

-— ~| ‘ 4—: , ffi ... r‘ .J— v‘ -'1 r 1 . J—I_ " '1‘ -n.~
D. : eve as: optical 00.010» o1 neplieate ChlUULQS-

 

 

.x. .*

 

[*1 ‘fl "‘ ‘ _'1 r» ", ’1 ,“ "‘ro .' '.‘ .3 ,3" "L‘ ' ‘ "'5 J". ._./ a. -q / .-" -W , -/' . . I, , ,V‘ ,' _I_ .,
1he 1esults 01 11 :Lp81inen b0 COuPafit 01: 000 L1 1 medium ant Lbu

component pnrts as wopWe“ins to 30 .ediun sic chordcd in Table 27.

Again, unwashed ce113 :eqnirefl no supplement for L? nth in the 52 medium

and t ere was a difference in the growthi e sponse C ependinn on the
Aposition of the scefl ncfiium. Then wasned cells were use; the best
mppl ment was 0.1 ml of T mnd’nm. dince this supplement varied from

STE 1 in =ts ce‘t;nt of agar anfl rzsazarin it souln be expected that

1“ ”:61. "E “f‘ '.‘I 1‘.’3 \‘fi ('1 (q f; "1:“ 'Y‘ "““v' . 'P'i‘n‘ ",' ‘. ‘ ' 1 ’23'1’)“ " "" “t17‘5\’) (* ‘" r1 ." \ 3 "x
;.5_C/.\. : “ C. k4 -' '.- \.;\.L~z-lv| _ UO UHLk’ 5—)...__1 l.) .-pp-x_v'-.L\J:L LJ DO b-J\;L_) .2. ‘,- «L\«. __L__’ L)_ AOL. U;_ iii

/
.1- ‘ , _'_ a . 1 .. 1 J- .1- 1. _ 3 .._ f .
. ' , x 1 . _ 0 \
tnesc pages would 1 .5 0:1» w0o1t we sine as 1- buous / aim o. It was
1 V‘ 1 '- "' r- ‘ ' J' I“ I j ' \ J“ "1 J ‘ "7 -‘ H I J (F—‘D v“ - ~ "‘ "‘
8!.1O'I'LI J :10‘ '9 \/e _ ) _LL‘. U -1, _, / ' nA‘ ». _'_ J -lL-I CI :1,Qsil__1 L~( U. 4‘.. LI _) v\).l _‘_) {is 14 {JWL‘LL I ]_e: LELAL

di1 not inc1ea.e the unongt cf L,.o"t;‘:- obs93701. R rl1apzt these apparently
contradictovv yesvlt; ev'id be ,1p1.iccd by the fast +51" if T ueTinm tbe
corporefts V330 st23*17“" .3 V*::I:“’“U a“: the ‘“”poLe1ts togctfler,
whereas tbc suppIc‘cnts 11 311 .n; ”5;. wood 1- 0‘“ S 0‘0 1'1 We?“ Dt<c10ved
separabuiy a16 ‘Hde 33p“ica:1; to tbc 3? zcjinm 0 ‘”pp1cicrts.
.. . --.-... " '~ mm ‘ 71‘ “ 11: "‘r\ _. -. n-\~ T ,-

-- 1,0 13-4;1Qx . .1- J L, x) LI - .1.-- Uv"n-) _.J _'u. _ ‘ L) E- cqb .....J LII .44.-) I “.11
In] i.Ciun 313 "U “0 kn ; :ic Iwa‘flL13 *1 3T; (rti *fingtcn ¥,Kr*0n sin:3e '11NTL1
was not as 330i when the cs1 of QT; #33 substitute: to: 03:. ;““”“““1s
ifiorgtnic in catzic in 5T1 stifinl cu 'O”t: sizzc if 02L :0; was onitten
littlc, if 3-3, gtoxtb occnxrhm, is Seek in tukss 7 “_Q C. T‘bc; 7, 3, 5
:4'3 :13 :1:C"ecfl*35 'tltxt .\J 4: Lilo.'~ r it- “oi; 0"};pcx1t kgsoirbi.

To sunnngize, ; suppl‘fie“t o; 3.1 31 o: E McCiu; pe" 13 n1 oi u: gciinn
figs :;qui?ei for 900” ;:0"tv o; “asflefl coll. ;n s; -ciini; U“: L ‘;if:-
p ocCLT; go t;;;03 - 1. _i-3. CL] :ss,:ti-3_:;‘3:cfi:jtz.

m.

 

 

Table

27. The failure of certain components of T medium to maintain

61

growth equal to T medium itself added to 82 medium

 

 

 

Treatment O.D.**
Tubes of Seed Incubation time

inoculum medium* Supplement 15 hr 2 hr
1,2 Unwashed T 0.280 0.310
3,4 Unwashed STB 0.192 0.220
5,6 Washed 8TB 0.1 ml T medium 0.195 0.260
7,8 Washed 8TB Agar 0.105 0.090
9,10 Washed STB Agar + 0.1 ml STB 0.135 0.202
11,12 Washed STB 0.1 ml STB 0.1u0 0.210
13,1h Washed STB Agar + ash of STB 0.126 0.09M
15,16 Washed STB lO-fold agar 0.105 0.091

 

 

 

 

 

 

*T = thioglycollate seed medium; STB = sterility test broth.
D. = average optical density of two cultures.

HO.

 

Since progress in identixyinfi tnese essential insrelients by
individ1al components 0 medium was rather disa wppo tilg,

it was leciled to determine if appropriate supple me11ts added to the S

[‘0

T?

medium in addition to T medium would stimukme grea te1 growth. nxperiments
were carried out in wnicn variables sucL as the size of inoculum, various
vitamins, EZ ease, acetyl—L-histidine, and various purines and pyrimidines
were tested but inconsisoent results in duplicate culthres were 01
obtained. Then uracil 1as added as a supplement, however, there was a
greater growth response.

a .

‘0 the synthet is on medium supplehented
C.

J.

with the five purinesa nd pyrirm1 ines would support good growth of the

1 -! ,0 _1 T1 " Q 1,.”— .° -. f .4. .._, .A ' _- _I_ ' n _ ' ._ ,.
wasned ceils 01 tne nA-o st1a1n 01 . bebcul. If the 11ed1um lso contained

 

the T medium supplement etter grow‘h was Obtainel. Growth in the 52

adenine and sood grewth was cute ainel by supplementing the 52 mod ium witn

7 '-,.
uracil and hypoxen hide.

' r . " r. - a Li. a .. . ' .— ' -2 '1 " 1" . 7* .-.-c‘ n -..-~-' 1. 1. -
Finally, Table 29 ohOHb t e COmgoblLth o1 10* ml 0 a 531508013

" . ‘ . -‘ I” d " .. 1" “ ’ "j "1 r 1 ‘1'“! '-

mediuh neizh SLPPOIUCL no' only the . LTtb 01 tne “b‘fll ULQCHlS. out

! - . -— . — - - «u. «w: A, ‘ ' “1 ‘- " .'I" 0* ., ~" ~- ' 7“ . *3 h “1:! - - '15 (“j

alSo the product1en 01 3231“ QHOJQ s of tetanus tonia. leis 1ornula
_ .A I- 1.: , ., . -. ' ,. J." —'-1. .,.. , 2 - ‘ w _. - . -2". . <7» —,.-.- . — —. _

rcpcese LS 018 compos101oh 01 0H8 dediun 1n 1ts inal igiu as 1 ras

filo 8130.03”.

Q1
( D
6
I"
Q
"d
1"!)
Q;
}-—‘l a
d

w
T
(

 

63

Table 28. arrest oi purine and
edium* on growth of w

 

Tub

1 -X-~x-+<- ,- :w 1.

0.1). gab LJ‘) 11:1).
Incubation time

L 23 j L+ 7 (rib: V15‘73'hr 337-5iT37 EH-YI" Efiy-S'In: ‘fizi1xr

 

e

 

 

_ _ - - _ 0.03 0.03 0.03 0.03 0.03 0.03

 

 

 

 

 

 

 

 

 

 

 

 

- L. . 1 . m r"‘--/\ r‘ ' ‘ . -,-\ ,-~ ~r- . ya -- . r“ r‘ _'—
%3n medium — Svnkhetlc uedium Ciutalfllub SOdlUm thOélyCCllwbeo
v- - . _1-. - O_T_’—\.—.‘—~. . _ - . _
**1 : gyacll, 2 _ needine, 3 — appoianta1ne, h — Cyt081ne, 5 — Thymine.

***O.J. = averace optical density of duplicate cultures.

'31- 6(-

‘I-'l"-(-

 

Table 29.

adeouat~

(a
\d

Jinal
for

of a synthetic
_ some toxin production

edium

 

 

 

 

Ingrediert ua-t1t" Ingredient luentitv
L—alanine 13.5 mg K01 0.20M g
L- ar nine hh.0 mg Glucose 0.200 g
L-aspartic acid 72.0 mt Tbiamin 0.015 -5
L-cystine 5.6 ng Pyriloxine.HCl 0.015 mg
L—glutamic acid 22H.0 mg Biotin 0.75 ug
L-glvcine 26.0 mg lc1um pantothenate 0.30 mg
L-histidine (fiee base) 29.? mt 312 0.009 mg
L-isoleu. iIe 66.0 m’ CLolinc cbloiide 1.0 mg
L-lCUCil’le 98.3 [15 F011? 8C..C10.0l Ilu
L-lysies (:ree base) 82.7 15 Para inobanzoic acid 0.003 mg
L-ietnioeine 30.9 L; niboflavin 0.015 mg
L-pbon"lala_ine 57.2 n; Ricotinic acid 0.3 m;
L_pyclwl> 108.6 gr Ieosltol 0.55 mg
L~sorinc S&.5 ‘5 Uracil 0.75 mg
L-tb‘eonide #3.0 A; Ade1:lc 0.250 mg
L-tr“ptophan 15.5 n; ngpoxauthiue 0.250 mg
L-tyrosine 60.2 a; Cytosine 0.250 45
L-Vallne 79.: u; Tb;;iLc 0.250 m;
3‘51 ‘1‘ N—jL 0p ‘08 ”.3303, 1 .3 L; '"Oi‘qfiliOZ‘. 1* Q .5: ‘..—l

disodiuu I’11t Idolution 111* 1.0 11
Sodium tbilclvcollauc 0.070 3 solution III*** 1.0 C1
EafiflPOL 0.151 Q Tater, distilled 23, 00.0 11
.)ng J\ . JD; L.)

W 01 uticn I is composed of 0.M g gQQCL.{Ip , 0.0% 5 “100p.4120,
0.3h D FCSOL.T:00, and 10 xi 11stilleJ E30.

1' C. C.
M“deletion II is couposod of 0.0151 g 0100; cud 100 m1 distillef E9
***Tcld‘ion III is ceppose? of 5 _ 85p lacti: acid, 8.25 41

 

 

L; \

$N1e "cd_wwm :is IXJSEKI"‘G.{ECCCw'llWéJ'CO iflde :follrriinr':1tepm3:

(i) All itens in the :irst column of Table 29 except eystine and
tyrosine are Weighed into a 20c ml beater.

(ii) Cystinc and tyrosine are dissolved in 1.25 E EC? and coded to

(iii) 1 100 m1 donut uf a
tLe solution is varied to 35 C a

(iv) n 0.5r0_emeuwt.fi730
. * ‘ a . A ’9 f ‘ .‘ M‘ " F“-/-.I 7.? I
is emuLoued cl 3.¢ ; LQWOMoYHdC,
10 rfl_’iis+ilfrgl1vate:.

(V) f 1.0 ul n.0unt o: “01
is c1wposed of 0.0151 g Cu?0b an
(Vi) /; 1.0 V1 ’“Cmflit cf 9C)

2 1

. . n w
a III is coppeseu el 5 a

0

end 2.7M q of K03.

(vii) i 0.01 r1_orxyu:: 1f
tbcr'bezfl333. TEHe rrtoeli solittion.
distilled water.

(viii) A 0.01 11 anount of
1dc:d to tbe beaker. The stock

(it) A 0.t5 ml a2ount of a
beaker. The stock solution cont

(3) 0.05 n1 abount of s
coded to tbe beaker. The stock
31d 50 11 of distilled water.

(1
k)

i

18

C) [— "1‘ ‘ _I_ I F .
U)g gaCth @010,
.I, .. .4- : -,
a stock SOlUULCU

... _I_, '-. r71:- ,1 -71
{jOibleljr‘ {/s [1". O
.J— -1.- . ....L. ‘ -
steel SCflALblON

solution

stoc‘ sol: ion 01

Pt I
O‘.‘ 'L‘I‘j‘If? ‘..

1
v
I

v _ fl 3‘ L _I_ 1 ’_ a _‘ ‘
atmleca 0:) L01? )etcier‘zin

W ‘l ‘
oeacer.

, - . ‘7 p - w r- u

3 a 9L!” C, _'. -1“.:)Oll- o LLi‘LCiO) K.) 0 0M1 C) 1393.301!) ('LIEO) 3.1 11.
utfon II is aédcc to the beater. SOlUtiOn II
.a 100 L1 distille: 1&tCT.

8.05 @l distilled water

J

1131

:4

C
L)

Extridcrzine.ifijl

‘ _'_ '. ‘ . .'1 '3 J- .l-
-blOUlL is eu1u21 to see

v.“ '— f‘.': " 21.11-- \ w
CDC 300 1;]. :"/’}‘ .5 D' c-1101.

L4

calciuu pat

 

(xi) A 0.05 ml amount of a stock solution of B12 is added to the

beaker. To prepare this solution, 0.7 ml of a solution of 15 ug of 312

per liter of 95p EtOH is added to 104.3 ml of 25% ethanol.
(xii) A 0.1 ml amount of a stock solution of choline chloride is added

to the beaker. The stock solution contains 25 mg of choline chloride and

.9 ml distilled water.

/

f0

(xiii) A 0.1 ml amount of a Stock solution of folic acid is added
to the beaker. The stock solution contains 1 ml of a solution of 9 mg
folic acid per 10 ml distilled water and 9 ml of distilled water.

(xiv) n 0.1 ml amount of a 1:10 dilution of a solution of 3 mg
p-aminobenzoic acid per 10 m1 diStilled water is added to the beaker.

(xv) A 0.01 ml amount of a solution of 3 mg of riboflavin per 2.0 ml
of water is added to th: beaker.

(xvi) A 0.1 ml amount of a solution of 3 m' of nicotinic acid per
1.0 ml distilled water is added to the beaker.

O

(xvii) .A 0.1 ml amount of a solution of 0.5 mg inositol per 1.0 ml

distilled water is added to the beaker.

(.3

(xviii) A 1.0 ml amount of a solution of 7.5 mg uracil per 10 ml of
distilled water is added to the beaker.

.5 mg of adenine per 10 n1

A
k

I \
'..}

L. A

r ‘1

V
lo
0
i
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__)
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to
R)

distilled water is aided to the beeker.

(xx) A 1.0 m1 amount of a solution of 2.§ mg hypoxantblne per 10 n1

/

*. I -. -- r -vn '-‘ "~ '. 1 (JP ':- J‘ ‘1 ‘1 .'K'. -,'n
if QLSEllleu haLef is added to tee beaLel.

' ." 1 w t“ 1“ J“ -.-l.-‘ r‘ .1 J" ' \‘q m'u’n "n ‘4 r A m 3 ‘ -\ 1 ’f *1
(3X1) 5 1.0 ml alount ti o SOLUleu ml 2.5 iv egtoslue psi 11 L1
“w _ _1_ ‘ 1 _~_ '1,“ '1 —'- L1. '1 . ‘.-. ~,.
elstilleo later l3 coded to tie segsel.
‘ _L fl_{jz . 1. 1 1'1 _‘_: -. _V 3 __ ,
(3’::_Ll) ;‘ .‘_.O I] C Idll’xb | ': UCLL‘ElDLi O. 2. / [ t_1 l3: pr *1 1\) ‘ |7_

(xxiii) The solution in the beaker is now adjusted ‘0 pi 7.0
vith _ * sodiu; hydroxiie. The C. R. of the neliun is 0.0 volts.

(1"17) 3h? contents 0: tne beaker E“? filtc:ed tlrtur: a clear and
sterile sintered glsss filter.

This medium when prepared in 100 ml or larger volumes supported the
QT vth of wasnel cells of either th Bf—S ea hueller strains 01 the tetanus
:rvanisn. Both strains produced as much cs 10 PVT/n1 of toxin in the
uedium after an incubation period of 23 hours and both cultures contained
107 to 108 cells per M” at this time as measured by the serfal tube dilution
Wetbol. The highest tox‘r titer which was obtained in this medium was
obtained “8143 the 53-8 utT41fl ml the organism grown in 75 ml of synthetic

medlun Tor 2U hours at 35 0 followed by another 2% hours at 0—4 0 p310“ to
the sterile filtration 01 the culture using a sintered glass filter.

Fift; ul of the filtrati Were harvestel and tie mouse MLD value was lOO/rl.
Torin symptoms occurred in the mic; at a dilution of 1 to 500.

i- so, the otlain was succersfull; subcultured in this svnth tic medium
on tro su~,essive davs uriur 0.1 ii of inoculum per 10 ml of medium. Tro-
Jirerseoudl paper chr vatOjraphT gave the saws spectre of ninhydrin positive
;stevials cite trout“ i? the synthetic flediuu as it did before inoculation.

 

V. DISCUSSIOH

Ii the product_on OI tetanus toxin certain factors influenced the

yields obtained in a goon growth medium. lirst, it was necessary to

keep easily exidizable ingredieLts, such as sodium thioglycollate, sealed

and protected from inactivatine ageits. Second, . it was necessary to
prepale sufficisntly large batches f media to avoid significant
adsorltion of trace uptevials during sterile filtration.

fl third :actor rhich affected the yields of toxin was the growth pease

.3 -- - .v- _ _v- - -v-.: - :v 1. n... _ .._ .1 .'- -._..-.- . - _, _- ,-—1
ol tie culture at the time oi calveso as teoernlusd microscopicallV. lhe
... U

n u I

fir‘" r ~ . 1 'V - . f. "'.1‘ ,1 r“ 'l‘ 'r " WIT ' J'" " J‘“ . 1 "‘ "-
c:.telyo oi eultolfiwsls in -cs.lltetr vel ”'Wel] oi oi uClCfllIJLOELKEbLO

l/ . . .. .- . ‘1 .... _

scio precipitcble Asteria and KID values n

a 0 u n o o ' L ’1 . '_ ' - ‘ "' " 1
- . —- A —\ ~(m‘ ’1 — ““r ”‘0‘;— ' N 3 « .123 w u I "" j '- ’ 1 1(. r _ ,— _‘ ..., ’_
i' luS uCu-OL ugcl')u ‘uoscpuiolc bao' "la ugch tn‘ cello Jcl‘ lap at

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“Li ‘1 L '. " ,.. * 1 N "' -'-‘. w ' .NJ‘ '3“ - .u~—- -" .3 .1 \ "‘ i - ’1 —‘ a“ - x a» ’7 1 "‘1 \
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r‘ ‘— r - ~‘ ~-‘ ‘J— ‘<‘ o‘- t”- w‘ 1.3...- N“ ~'— 3 — \ -§~‘11‘s .. “J-_’
10 pL.uS\.. ‘ 0. 01.2.0 U13 c...t.'_l'C.i_OLi Li _'_elb'cl .. be- 0-. “_‘_ '9‘” _313___j__h _I_C’LJLLlL; t“

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formula for a synthetic: medjurl which

“oxin. These approaches cons 's ed of
complex medium or to devise a svrthetic
the organism anl toxin production. In
for peptides iL a complex ~redir' and th

would supp rt produc

8

study to preper a succe ul_

ion of tetanus

either to simplif“ a good

1

suppo;ti115 ooth growth of

spite of the reported requirements

/

'7

e unavailaoility of an adequate

synthetic nediuu it was decidel first to undertake the difficult taSko of
simpliIVing the forWHle for the oomple medium of MLloller and Miller (1956).

'..—1‘ V“ 1' o .1- '1') _.“l _ 1 n '. _ 7 o _ l. a _ _- I V ‘ -- _'_.
lie co pleuity of AG Heciun ;as indicated by the 1151 per ce nt oi
J- . '1 . J—': o -r ('71 3" .1 v 1x"... 1; .- - ”1.!- . * 'r' . c\‘~
Deptiuc iitrogen (iaol: 32) WLLCQ mas presc;t in BA case c”d by tne oeef
w -. - q - : mm. ’7 —. u "'4 _ -0 . 3:- J—‘!\
aeert “eeuivel n iaole l) in nueller and nillec'" loimula. Since the
q f - .,_ _,- - . 3 . - 1‘ .1- _,,--. -, . _’_ _5 . . ‘_ .
or5aaisu caull grow and produce tenin in a cellophaleb 35 (Figure l) Khlch
- 7 v ,. - - 1, ,1 1‘ .- r. . .- -- 4 5., e-" a 4.1 -
WOUIU enclude *e‘ ;ls oi larbe molecular \ei5ht llOU one cells, it seemed
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logical to Simpllié one NSOLUU DJ I:&Cbicflu] ulleSLb o tne tqo lanGdlCDtS
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and so _nentilw caeuicalig nei; coupCSit_en. apparentlJ th JEUAOU did
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the amino acid acaL ses anion were Laue 0L uni SbmnfluidlaeL L;Qth
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VI. SITEEEXY

 

A complex wediML which supported a good yiell Cf tetanus
the Ihellzg strain Cf C Ostridiur tetani was standaxdizefi an&
by :;plac1ug bee? Besrt infusion with a beef hefigt fliclyzate.
~ntire S”0Wtf p;ri03 ggcrges it the J0wu~~trati018 of free amino dcid

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BIBLIOGRAPHY

Block, R.J., E. L. Durrum, and G. Zweig. lTSS. Amino ac'ds _
proteins. Section 1: .Amino acids and peptides, p . 77-113.
s

\n
9
s.
D
(D

J..-

2
A manual of paper chromatography an paper electrophoresi-
Press, Inc., New York, N.Y.

Clifton, 0.3. 19u2. The uti

.1

1 tion of amino acids and related compounds
0y Clostridium tetani. J. Bac

teriol. Eg:179-183.

 

Craig, L. 0., J. D. Gregory, and W. Hausman. 1950. Versatile laboratory
concentration device. Anal. Chem. 22:1M62.

, aLd T. P. {ing. 1953. Some dialvsis experiments with
polyp ptiles. J..Am. Chem. Soc. 77:6620-662h.

Descombey P 1924. L'anatoxine tetanique. C. R. ‘oc. Biol. Paris.

- . IQLQ. The amin
n. Arch. Biochem. 22:37h-37

)
composition of "etanal tox'

jaton, L. h., E. H. Laebert, N. A. Chris’ensen, D. L. Thurber, 3. D.
Henderson, and H. H. Young. 1957. Symposium on tetanus. Proc.
Staff Meetings of Mayo Clinic. 32:141—167.

Faber, K. 1890. Die pathogenese les tetanus. Berl. Klin. Usehr.
”72717-720-

7 I C

zre. 1958. The role of tissue spaces an“
ad of tetanus toxin in the rat. Univ. Kansas

 

-.., ' “6,. fl‘h ' ,.. -0 (11
.1. 901111 gallon-CD OJ. v.1.
F1

--. -'-1. , - - ‘ —
OJ. 12-1C OJ. bCLO.lSiL'.o

Feeney, R. 3., J. H. Ewelle' :nd P. 3. killer. 19M3a. Growth
0 etani II. Factors exhausted by growth
1

. h6z559-362.

 

 

 

Feeney, R. 3., J. H. Lueller, and P. A. Miller. 19k3b. Growth
rceuireuents of Siestridiur teteai. III. A "synthetic" mediui.
Jo :38.C'C€1“101- )l“ :f63-57’lo

“ildes, P., T. Bullock, R. A. O'Brien, and n. 1. Glenng. 192$. Bacillus
TcJani, p. 298-372. .32'3 svstea of bagter'olog; 1L ~'clatioh to neticine.
7. 3. His Majesty's dtat’onery Office, London.

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