27
Doc No 55388B
MExperimentation with Typhoid Bacillus andTyphoid Vi Bacillus, tt hy Epidemiology Laboratory
of Army Medical College, 31 Mar 42
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salting doeroooo I* tonics and Uoma* la oatlgaal* strength. 2ow-oror, tho influence exerted on thorn tectorial cell* Igr eupereeaicwmoo of ftiTlAC frt|iiuifi hut of tho omm energy lerel ham notboon reported.
Hr ala «m to f»Dwr iaprovo Ihi gaalitgr of vaMtoathrough a study of tho deetruetiro strength of saporsoalo wares ofvaqrtoi frofo—oloo oa tot mho oatlgen.
Chapter 1, Outline ef papertout
allowing the procedure in the cholera bacteria experiment lafart 1 ef thin series, the survival, bacterial count, eerpheleglcolchanges sad turbidity ef typhoid bacilli (Shedebata strain) aadtyphoid Y1 bacilli (ho. 17) were exaalaed.
Chapter II. hxperlaeatal prosedare
A. Bacterial flvtUi Hu typhoid bacilli were obtained fro«the strain preserved ter this laboratory; the Tl bacilli (do. 27} werecontributed by technician bOJltti of the Infectious diseases tesearchInstitute. loth strata# indicated uaifem turbidity in Tallinn?gas furcation In lactose and dextrose vert leal e&r sac negative;whey culture and milk oiafvlttkB y«r« MfaUu; lead acetate «f8rculture sen positives indol react ion was negative* and the a«slut inn-tie® titer of the typhoid lasunt i«r« was positive at 6,400 SU»s,
6. bacterial suspension; ffuaoreus celtarea of fi tec 1111 werenode since each 6 per coat agar cultare (ft 7.4} produced snip ft caallquantity ef colonies at ST* 6 ever a 20-hoar period. After weiring,a 10 as per o« solution *es prepared with a physiological saline so*lufcica; this was diluted to 1.0 as per sc aad 0.1 as per cc.
Bach ef the procured bacterial eolations wac cultured in bouillonaad asari toots for siseellanesas bacteria were eoadsetod after pre-!>nr .ag sneer spec Incas. Bacterial count# were performs* before eachexperiment since the sane beeter ini suspensions were act employedwhenever the supersonic wave frequency was changed*
0. >«pereoiJ ic wave treetaaati As already related la Part 1 ofthin series, the plate current was regulated to produce a teet tabstasrporstunt (internal} rlee of 10* 0 per alautc at each frequency inorder to maintain a eeastaat energy input into a teet tube containinga specific volume ef transformer ell* She following ourreato wereneed to provide a naifera energy levels
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1, 8, 3, 4, 6, 6, 7, 6, §, 10, U, 80, 30, 40, »0, 60, 79, 90, 106md 180 ainatas.
0. ittalitatir# taata ©a esllallsldal atraagtht *ws platiaaal#sp psrtisas saah af tha aaparaoala wara-traatad baatar 1*1 saspaa*aiaaa war# alaat-#altor#d with agar and plata-aaltarad with bouillonand agar. Aftar incubating at %7* 0 far 84 hoar# th# raaulta wars•valaatsd Mttt, -rr, a and - aaeardiag ta thstr grawth.
S. Matarlal count* fh* mparsant* wara-traatad taatarisl aa-latia&a war# dilutai progrsatiwaly %## tlaaa cash with a physiologicalaalias solatia*. Oaa a# af #a#h was alxad wall with agar (46° G)and allawad to solidity. Agar was addad again la aa aaDaat sufficientta aerar tha aarfaoa. hastsrlal count was takaa aftar incubating at37® 0 far 34 hours.
laamaaa th* paxpaoa af this last vtt i# dataraia# it# aaaher ofdaatrsjrad fcaatarla, count® vara taka* #a th# aaatral mad mlao duringtha lataraadiata ©tag# mad tha ©tag# ia&adiatsiy prior to da#tractiaa.T*m rata af tla# dsaraass aaaparad to tha hactarial count #f th# con-trol warn #aiiaad. la fcrlaf, th# par*#*tag# af daralapad aalooisspar ailllgraa ia asaparlaaa t# th# #aatral was dataralaad.
7. Qliimlio #f iiylali|lwl chang##* I—#61Italy followingto# war# tr#*taa*t9 a aaaar apaaiaa* was prsparad with 9a#platina* laop parti## of th# i##t aalutioa. Staining with *#thBrlsa*ala# was aecaapllsfcad 4«n 6ii vu triad mad ftxad. aiianpi#oftedsalldAf van mad# la urtar to iMr 6« aataara of tiw daatrsyadiMlaria.
0. Ma&aarsaaat #f turbidity* 71m bastarial aaapsaslsaa war#according I# Iht nliH afcow* fcslaw villi * phjralslsgieta
•alia# solatia* Md »M#irtd 46 a iulfrisk's ?hato*#t#r. Thaafcsolafca larHdlly **# «o*p*A#d fm Hit rHallTt IwUiitQr.
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fhs rM«U« art «bm la fahlss 1-6. At# hatlirU iwlnlUmm ara show* la figuras 1-6, tha aarpholsglsal ohaagso la Tahlao7 aad • sad tarbidiUaa im tMm 1-6.
Doc No 55388B
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Me Ko 553861
v 4
Xahl* 3. results on ecUulicifial strenstiij survival and turbidity «f hftiMA bacilli treetod mi.it-, 280 ka uus»r««i4. •:•*»».
Flat* voltet.'* 3300 v» fOat* currant *00 as) grid currant 190 oe. tct* of «peri*«t 3 dtO. 41. -cob t*c«.<araUire J|* ti.
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40 — 0.23433
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Timfateu}
Survival ofbacteria
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Doc Ho 553888Table 5« Test results m cellulieicial strength$ survival and turbidity of typhoi Vi bacilli treated with 560 kc supersonic
Plate voltage 3000 vj plate current 550 na; grid current 130 raa. Date of eacperiBMRit 2-5 41* *00tt teisperatuxe 26° t.!
•
0.1 mg 1.0 m 10.0 xag
TimGain.)
Survival ofbacteria
Turbidity Bacterial count -
0.1 mg(x)
Survivalcompared
tocontrol<»
Log 2 T*0*C»&il3. • /
Survival ofbacteria
Turbidity Bacterial count -
1.0 m&
Survivalcolored
tocontrol(«
Leg *£ Hob' (jain.J
Survival ofbacteria
Turbidity Bacterial count -
10.0 mg(2)
Survivalcompared
tocontrol<«
Log Z
Bouillon Agarslant
-jouillon >Agarslant
Bouillon Agarslant
K -Hi- -H4~ 0*01174 72.447 x 106 7.8599 % , iilII I 444* 0.08929 724.499 x 106 8.8599 I +H- £ 1 1 iI TT 0.83655 7244.79 X 106 9.8599
1 -+44- ■"Ml" 0.01X26 l 4H- s 11rit 0.08700 1 1 t 1TTr j 1 iTTT 0.S0309 -
2 4+4 -H+- 0.00226 2 44-4* H4+ 0.07919 2 +++ I +++ 0.73759 I
3 4f 4~ 0.01312 3 11 1J ? ! 4+ 0.07752 3 1 1 >TTT .illTTT 0.59116
4 *4+ -H- 0.01360 k | | | , tit■ ITT 0.07585 4 +++ | | | 0.635785 -4+ 4 0.01362 39.357 x ID6 54.326 7.5949 5 1 l |r T r 444 0.07562 17.147 x ID6 29.973 8.3367 5 -H+ 4+4 0.646936 -44 4 0.03630 6 4+ 444 0.06748 6 -Hi- l j >TTT 0.680397 -44 44 0.01532 7 t 1 1
[ 1 T 4+ 0*07362 7 1 1 .
TTT illITT 0.680398 4- 4- 0.01582 6.683 x 106 9.225 6.8250 8 44- 44- 0.07473 117.526 x 106 16.226 8.0m 8 -H4 | 1 1 i1 1 { 0.602329 4 4~ 0.01582 9 44* 44- 0.07429 9 -H4 I 4-j*4 0.57/^43
10 + 4- 0.01560 0.115 x ItJ6 0.160 5.0607 : 10 44* 4* 0.06525 4V. '76 * 10* 6.384 7.6778 10 4+ 44 0.58001
15 4- 4 0.01266 0.33 x 1C6 0.046 4.5185 15 44* 4“ 0.06915 39.471 * lfi'' 5.448 7.5963 • 20 ■++• 44 0.47962 274.83 x 106 3.793 8.4391
20— — 0.012675 20 44* t 0.1X692 33.929 x JIT 4.683 7.5305 30 i 1
TT 44 0.41828 15.00 x lfi6 0.200 7.1761
30 — — 0*07027 40 44“ 44 0.44951 11.46 x 10^ 0.162 7.070540 — — 0.061347 50 4+ 44 0.86251 6.03 x 106 0.083 6.7784
60 44- 44 0.32347 2.55 x 106 0.035 6.406575 4“ + 0.35693 0.13 x 106 0.002 5.U39
90 4” 4- 0.29558 0.09 x Ifr 0.001 4.9542
105 + + 0.25456 0.012 X 10^ 0.0001 4.0792120 — — 0.25096
0oc 140 553ags
Table 6, Teat results m cellulieidal strength* survival and turbidity tf typhoid ¥1 bacilli treated *&h 28© lee wine
Plate voltage 33©© vj plate current 400 m$ grid current 190 aa» Date of escpejrlisent 5 *>el 41* oon tenperaturc 31° li.
*
0.1 S?g.
1*0 Eg.
io.o mftae(®in*)
survival ofbacteria
Turbidity Bacterial count -
0*1 mi<*>
-survivalcor-feared
tocentre!(»
log 2 He*ioSjo.)
urvival ofbacteria
Ttsrbldit//
Bacterial eount -
1.0 eg(4
survivalcellared
toeuntraltfi)
40g 2 tto(ala*)
Iv&l ofKorin
turbidity u+ctorlal count -
1CW mg(.)
survivalconp&red
to
w
log £
Bouillon Agarslant
ifcmillon Agwrslant
BauOj [on %erslant
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1 -H{ 444- 0.01467 65.935 x 106 90.10? 7.8191 i f-f 1 i i L441 <h<mr? 79.736 x 1S+ 10.^93 7.9016 1 4+f- 0*8120
2 444- 444- 0.0253 2 444 444 0.07/AO 2 1 1 17 rf 0.64693 i ♦ iT rr 444- 0.010x5 50*441 at Itfi 66.959 7.6993 3 444 4+4- 0.05120 78.690 * 1# 10*771 7.3967 3 4Hir»-
111MT 0.5577
4 4+4- 4+4 0.01048 4 4+4- 41 iTti 0.063Q4 4 44- - Tf 1 0.52965 4+4- 444- 0.00959 45.126 x 106 63.656 7.6544 5 444 4+4 0.0603 50.681 X 106 .924 7.6995 5 4+ *» tilTfT 0.55206> l t iTfT 444- 0.0004 6 +4 1 • 44 1 0.05166 6 4+ 1(1m 0.46737 44+- 444 c.mm 7 4+4 i i *
TTT 0.COL35 7 4+ j 1f r 0*4450
8 44 44 0.00766 11.539 x 3UB6 13.769 7.061*' 8 44+- i 1 1IT T 0.05800 l. *18 x It*6 1*666 6.0856 8 -4- u 44 0.4450
9 — — 0.00761 9 +44“ 4+ 0.05855 9 4+ 0.433O
10 —* — 0.00988 10 44- 44 0,06079 0* -24 * 1JT 0*004 5.0156 10 *4 » 4 0.3825• 15 + 4 0.Q&906 15 4 4- 0.2900 3345.039 * 106 42.990 6.4962
20 H 4 0.271® 1&>3*655 x M>6 25.469 9*270230 4 4 0*223© 2.341 * 0.0J2 6.369440 • 4- 00617 0.15S x IQ6 O.0GC 5*1937
50 — 0.1520
i 60 —- . — 0.3427b
see Ho 563888
A, Collulioldal tlao.
Th# results shov aa aooolor&tloa la osllul lethal tlao (la thoardor sf 280 ko sad 1120 ko) with higher baotorlal solatia* dilatloaoat tho saao suporseaie ««r« aaiflr lml. Dostraction, tevmr, 1*retarded at 560 ko, Tho oollulioldal tlaos for tho typhoid bacilliat 280 ko are tlx aiautes for 0,1 ag, 15 atautoo for 1,0 ac *h& 40ainutes for 10,0 at* At 1120 ko tho tlao for 0,1 as i* four alautesor a somewhat faotor rato than that at 280 ko; tho othor tlaoo aroaloyor, tho tlaoo for tho T1 has 1111 at 280 ko or# alao alaotoo for0,1 mg 9 30 aiau&oo (15 aiautoo at 1120 ko) for 1,0 at mad 50 Miaulosfor 10,0 fit* Consoqooatly, tho dootruetlvo strength at 220 ko Isdirectly proportioaato to tho froquoaoy and inversely proporttenetsto ware length oad lo suhjsot to ooao asaaat of floatoatloa.
2, tarrival toots (too films 1 to 4) Tho graphs show thata ooporooaio warm troataoat produoos a legarlthato dooroaoo la bac-torlal eouat, ir tafctag tho bacterial oouato la tho truphs aolacorlthado oaalos (vortical aaoo) aad tho troataoat tlaoo ao arlth-ootiaal soaloo ihorlaoatal area), opprextoately straight llaoo aroforood, A step-like dooroaao io ladloatad hr tho 10.0-at oeaooatra-tloa. igr troatlag tho bacterial oouat ao r aad tho troataoat tiaoao jLp tho relationship
log y « o*# or
r sin(e • negative oonstoat)
la obtained.
As illustrated hr tho baotorlal oouato for tho 10,0 ag/1.0 soconcentration la fifara 5 aad d, tho dooroaoo la lira baotorlaoouat, at tho saao saotfr level, boooaoo aoro proaouaood as thofrufusaar doortooos or, la othor eerde, ao tho wave length laoroaoos.
Tho tlao required for tho loo of tho lira baotorla oouat of tho10,0 ag/1.0 oo eoaooalration to booooo halved lo shoya belay.
1120 ko 90 ala,560 ko n ala,
' . * - II tffciv
1120 ko 40 atm.mo km 90 aim.
k 280 ko 20 ala.Typhoid U taollll
0-1 ao/l-Q an 1.0 a*(l-Q oo 10-Q ag/1-0 aa
1120 ko 4 ftl&. 16 ala. 50 ala.tea tin 560 ke 15 ala. 00 aia. 106 ala.
260 ko 6 ala. 16 ala. 40 ala.typhoid ri uao ko 9 aia. 16 ala. 60 ala.teUU 640 ko 20 ala. 30 ala. 120 ala.
200 ko 9 ala. 30 ala. 50 ala.
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Doc »o 5536BB
Table 8. Microscopic observations of morphological chan ■;o4r .in typhoid Vi bacilli at various supersonic mve frequencies1
Frequency 1120 kc 560 kc 280 kc
1 Microscopic Cellular Cellular
—
Cellularr
iotoplasm Inability -ellular Cellular Cellular J Inability Cellular Jellular 1 ellularr
1 iliabilityLGranular Cloudy Dust-like formal to Granular Cloudy Dust-like Normal to Granular Cloudy lust-like Normal to
stain1 observations spelling defoma- dostrue- hCBaogeni- stain celling deforaa- destruc- homigerii-* stain swelling iefoma- < estrue- hocaogeni—tion tion nation) tion tion smion) tion tion aation).
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3 4 — 4 — — — 4 4 — 4 —• 4 — — — 4 4 — 4 — — — 4 4 T4 '"I""* —— 4* — — —- 4 4 •— — 4 — 4 -pi 4 4 44 — —M 4 4 4
ime
5 — 4 — — 4 4 — 4 — 4 4 —• — 4 M' 4 —. 4* —— — 46 —f— — 4* — 4 — 4 —— OttMHi — 4 — — — 4 4 4- -f- — 4 — —. — 4 4 4
5 7 4 4 4 — 4 —— 4 -H- — 4 — 4 — — — 4 -4- — 4— — 4 — —* 4 4 —
a ■4“ 4 44- — 4 4 4 4 — -4 — i# 4 — — 4 "4* — 4 — *4 — —> — 4 —
9 —j-~ 4 4 — 4 4 — — 4 — •wt W — — 4 — — — # — — — 4 —
10 4 — 444 — 4 4* 4 — — 4* —* 4 — —» 4 4 — — — 4 — — — 4 — —
15 — — 4 <— — — 4 — — —-— 4 — — — 4 — —
20
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X — — .— — — — — 4 —* 4 — 4 — — — — 4 4 4 — 4 — 4 — 4 4 —
2 — — — — — —— — 4 — 4 — 4 •— — — — 4 4 4- — 4 — 4 — 4 4 —
3 — — — — — — —* 4 — 4 —— — — 4 4 4 — 4 — 4 — 4* 4 4
O4 — — -H- — — — 4 4 —- 4 — 4 •*—. — — 4 4 4 4 — 4 — ")" — 4 4 x
1.0 5 — — 4 — — — 4 4 — 4 — 4 — — 4 4 4 4 —« 44
— 4 — 4 4 4•P
- & flag 6 — — -H- — —- —— 4- *4- — 4 4 n—. — — 4 ■i —- *— mmt - — 4 iJlP 1*1* .
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t ft ,X-t t 1 1 r 4~ TT TTts
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1 ° 10 4- 4 4 4 4 — +• -+- — 4 — 4 4 — 4 4 4 «m|» — 4 4 — 4 4 —
1 g 15 himIi 4’ 4 + 4 — —. — 4 4 — 4 4 4 4* — 4 4* i. 4 — 4 4 —
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4— 4 — — — 4 4 4
5 — — 4 — — 4 4 4 4 — 4 — — — 4 4* 4 — 4 — — • — 4 4 46 4 — «4* — — 4 4 4 4 ' 4 *— —> — 4 4 4 4 — 4 — 4 — 4 4 —■
s 7 4* — 4 ... — — 4 JLm 4 —• 4 4 *— —• «m|w» 4 4 4 — 4 — 4 —* 4 4 “4“10.0 - 8 • + «4* — 4 — 4 4 4 4 — -j- —. — 4 4 4 4 4 4 4 4 — 4 4 —
m 9 4 4 4 4 4 — 4 4 4 4 — 4 4 -— — 4 4 4 4 4 4 4 4 — 4 4 —
10 4 4 4-’. 4 4 4 4 4 4 4- — 4 t —, — 4 4 4 -|n« 4 4 4 4 — 4 4 —•
20 4 4 + 4 4 4 4* 4 4 — 4 — — 4 4 4 4 4 4 — 4 — 4 4 —
1 30 4 4 4 + + 4 4 4 — — 4 4 — — 4 4 4 4 — 4 — 4 — 4 4 —
40 4 4 4 4 4 4 4 4 -4— *— 4 4 — — 4 —4— 4 — — 4 — 4 — 4 4 —
50 — 4 4 4 4 4 —- 4 — +H- 4 — •— 4 4 4 — — 4 — 4 — 4 —* —
60 — — — 4 4 4 — — — — 4 <*|ii 4 — 4 4 — — — 4 — 4 — 4* — —
75 — — 4 — "j- 4 4 '— — — — 4 4 4 4 4 4 —
90 — — 4 — 4 4 4- —
105 — — 4 4 4 4 4 4* —
120 ■— — 4 4 4 4 ——.
—
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It mi lonroad fwa the foregoing rooulta that the optima foreollular deetruetioa to at 280 lea far both trpw of bacilli.
9. Iloaeuroaoct of taikldllyi da ohora la Sablae 1 to I, trans-parency o*e act eboerred aneag euoyeeeleaa of typhoid bacilli *ithiatba eupereoaic acre fnqmgr rang* aerated by this experfeMat, *&
extressaly alight turbidity decrease wee dotectod la the 10.0 ag ftbacilli bat change* ia tba other* eooaad to be non-existent.Chapter IV, Sniaqr aad eeaclosleiM.
?ha following conclusion* vara derived upon observing the colls*llcldal tine, bacterid count aad turbidity and upon caking aioroooo-pic atudiaa on oollular destruction of typtaid bacilli aad fi bacilli,tba susps&aloaa of thick vara treated oa tba aaaa caargr laral with«og»er*e&ie varaa at fraguaaeiaa af 1190, SCO aad 880 too.
4. At tba aaaa auparaeaia rave aaargy laral the aaat effectiveecliuUcldel tiaa ia iadloatad at 880 ka aad 1180 kc, ia that order.Collulieido at 580 ka la alar, fhle actioa la diraatly prepertleaateto tbo froquaacy (inversely proportionate to tba rara length) in cor*oaatrated baatarial eolations aad appears to bo adbjeat to ooao aaeu&tof fluctuation,
3. A slightly greater aaouat of difflenity lo eacoustared iadootreylag tba VI baailli coopened to tba typheld baailll.
0. ffea oallulteldal tiaa decrease* aa tba baatarial aaaaaatra**tioa is lesacaed.
L\ The ebaagaa produaad in lira bacilli, rboa baccd aa treat*sect tiaa, caa bo ayresacd legarlthalcally aad pcaacaa roughly tba
lag y • ex.y * lira baatarla countx 3 troataent tiaaa * negative eaaetaat
s. A nlorooeople oxoalnutloa af tba opaalaaao raraalo cellulardo*tract ion occurring at 380 ka, 1130 ka aad 580 ke, ia that order.
1. i-ranapavaaer doao cat moult# rbaa physiological salineoolutloa suspensions af typhoid baoilH and VI boaUU (only a alighttraao aaa bo aatlaad la tbo aaaa of 10.0 at Vi bacilli) era traatedtfttfc supersonic ravaa. It aotlaaabla chcngcc ia turbidity ara pro-
ktlTulor destruction cannot bo gained by changes ia turbl-dity.
Bibliography
!• Mari, and o thorn, Biological aad laanBolegloalStudio Baaad mi iuperaeatc «*res, fOtTO IJI iAddfil (Tokyo KadlaalJournal), JM>. 3011, februasy 194*.
*. 014X1, Shlgekl, Cellulicldo, Turbidity Amm, TtMrUOhangoo and internal tmpwmtvrm af f*«l Solutieaa fmlod vith
Mm, naei roiumu nwuoAKo rai u*pan dauraai ofMiarobtpiogy aad rathaiagy), v#i. », *>. 10. otioiw n«.
3m v* 5ssta$
S, A. C. K, TW m*k 3IK uat Pro®. ttxp. BUI. «a& a«tMA. fol. 31, Bo. 9, 125&0 (19*4).
4. rUBAZO, J«, v«»truetiT« Aotioa on 3a*f*rla witA 3qp«rse&l«Bbtm, &«ooad loport - £xp*ri»mt* vith Bacillu* soil, MoUlutrohl, SmUIm pmijraat a*d £toeUlu» 4jr*«nt«ria#, IXSOABlfcaimo AAS981, fol. 38, Bo. ft# Jum 1939.
| j UbCLASSIS ISD j f.QttMTRv Jft.pa.ri
REPORT NO. .
rr, '.,..' ../-... 1 WpGS- INTELLIGENCE REPORT fc D. NO {subject: with Typhoid .Bacillus .and Typhoid ¥1 BacillusFROM; «P/T . __&s2
. ftVQ. __h?EQ—REFERENCES:
J EVALUATION: DATE OF INFORMATION: l$kZ. DATE OF REPORT: ,l6 _AUg_ 1ftfiQ .. ..
incl. one, prepared 3Y:—A3!I54-JE“£*JSE£iJ-ESC„ -«^4Sfl4CaLj*0JLL.0^S**«-
SUMMARY OR SID REPORT:
Por-./arded herewith four (4) copies Allied Translator and Interpreter SectionDocument Uo e with Typhoid Bacillus and Typhoid ?i Bacillus5 ’by Epidemiology Laboratory of Army Medical College. 31 March 19U2C
This report concerns experimental tests, which ware made in the laboratories ofthe former Japanese Army Medical College in 1342. to improve the quality of typhoidvaccines. The study outlines the nature of the experiments, discusses experimentalprocedures and results and gives a summary and conclusion*
For the Assistant Chief of Staff, G~2s i
/AtJ. E. POLK ( 2 J-/Lt Col s GSC >' *’
1 Inclosure (4 cys) Executiveas mentioned above
NOTE: This document contains information affecting tho ns- j DECLASSIFIED tenia in any manner to an unauthorized person is prohibitedtionaf defense of tfio United States vitiiin the meaning cf the f *'
by law.Espionage Act, CO U. S. C.*3l and 32, as amended. It3 trans- $
| CCS Form 17A mission qr the revelation of its con- j |.•■•»NurACTa«5 it oio toKK msao" a o.:w>on coww.nr. pnoccirx. w.y. april s. ism 1 ' 1 " *
