Doc No 55388B 
MExperimentation with Typhoid Bacillus and 
Typhoid Vi Bacillus, tt hy Epidemiology Laboratory 
of Army Medical College, 31 Mar 42 bm a* aMW» 
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I, KUMWOMfti of Iwlliitf. 
ite.ptor HI* Jtooulto of oxparfcaoat. 
A. CollaliOidol tiftO. 
1. 3arrival toot, 
C, of aorpfcoiofloal ortacao. 
». »oft»oroftOftt of tarlidltp. 
CMptor If. saiiwiy and ooaalaolofto. 
Mil lag—ply B»e M se»o 
Maanl 
Ttm iam» Miti at WUUUUH, Tltullll US2. SAfcUA®, 
cawi* Ml r«W«fi coat*la (Mtciti of ifao aocfaulesl dsotroetioa of 
ImUU Igr aeoas of m supersonic war* trmtmnt and tho ft- 
salting doeroooo I* tonics and Uoma* la oatlgaal* strength. 2ow- 
oror, tho influence exerted on thorn tectorial cell* Igr eupereeaic 
wmoo of ftiTlAC frt|iiuifi hut of tho omm energy lerel ham not 
boon reported. 
Hr ala «m to f»Dwr iaprovo Ihi gaalitgr of vaMtoa 
through a study of tho deetruetiro strength of saporsoalo wares of 
vaqrtoi frofo—oloo oa tot mho oatlgen. 
Chapter 1, Outline ef paper tout 
allowing the procedure in the cholera bacteria experiment la 
fart 1 ef thin series, the survival, bacterial count, eerpheleglcol 
changes sad turbidity ef typhoid bacilli (Shedebata strain) aad 
typhoid 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} were 
contributed by technician bOJltti of the Infectious diseases tesearch 
Institute. 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 «f8r 
culture 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 celt area of fi tec 1111 were 
node since each 6 per coat agar cult are (ft 7.4} produced snip ft caall 
quantity 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 bouillon 
aad asari toots for siseellanesas bacteria were eoadsetod after pre- 
!>nr .ag sneer spec Incas. Bacterial count# were performs* before each 
experiment since the sane bee ter ini suspensions were act employed 
whenever the supersonic wave frequency was changed* 
0. >«pereoiJ ic wave treetaaati As already related la Part 1 of 
thin series, the plate current was regulated to produce a teet tabs 
tasrporstunt (internal} rlee of 10* 0 per alautc at each frequency in 
order to maintain a eeastaat energy input into a teet tube containing 
a specific volume ef transformer ell* She following ourreato were 
need to provide a naif era energy levels 
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1, 8, 3, 4, 6, 6, 7, 6, §, 10, U, 80, 30, 40, »0, 60, 79, 90, 106 
md 180 a in at as. 
0. ittalitatir# taata ©a esllallsldal atraagtht *ws platiaaa 
l#sp psrtisas saah af tha aaparaoala wara-traatad baatar 1*1 saspaa* 
aiaaa war# alaat-#altor#d with agar and plata-aaltarad with bouillon 
and 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 physiological 
aalias solatia*. Oaa a# af #a#h was alxad wall with agar (46° G) 
and allawad to solidity. Agar was addad again la aa aaDaat sufficient 
ta aerar tha aarfaoa. hastsrlal count was takaa aftar incubating at 
37® 0 far 34 hours. 
laamaaa th* paxpaoa af this last vtt i# dataraia# it# aaaher of 
daatrsjrad fcaatarla, count® vara taka* #a th# aaatral mad mlao during 
tha 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 aalooiss 
par ailllgraa ia asaparlaaa t# th# #aatral was dataralaad. 
7. Qliimlio #f iiylali|lwl chang##* I—#61 Italy following 
to# 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 daatrsyad 
iMlaria. 
0. Ma&aarsaaat #f turbidity* 71m bastarial aaapsaslsaa war# 
according I# Iht nliH afcow* fcslaw villi * phjralslsgieta 
•alia# solatia* Md »M#irtd 46 a iulf risk's ?hato*#t#r. Tha 
afcsolafca larHdlly **# «o*p*A#d fm Hit rHallTt IwUiitQr. 
0.1 ng/1.0 «e &taak solatia* 
1.0 #j/l.Q a# 10 IlM 
10.0 ag/1.0 ec 100 tlaaa 
Qhaptsr 111. Basalts of ttsparlasat 
fhs rM«U« art «bm la fahlss 1-6. At# hatlirU iwlnl 
Umm ara show* la figuras 1-6, tha aarpholsglsal ohaagso la Tahlao 
7 aad • sad tarbidiUaa im tMm 1-6. Doc No 55388B 
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Table 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. 
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5.0607 
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4.683 
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7.1761 
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0.162 
7.0705 
40 
— 
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50 
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44 
0.86251 
6.03 x 106 
0.083 
6.7784 
60 
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0.32347 
2.55 x 106 
0.035 
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75 
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0.002 
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90 
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0.001 
4.9542 
105 
+ 
+ 
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0.0001 
4.0792 
120 
— 
— 
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. 
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60 
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b see Ho 563888 
A, Collulioldal tlao. 
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. 
Th# results shov aa aooolor&tloa la osllul lethal tlao (la tho 
ardor sf 280 ko sad 1120 ko) with higher baotorlal solatia* dilatloao 
at tho saao suporseaie ««r« aaiflr lml. Dos traction, tevmr, 1* 
retarded at 560 ko, Tho oollulioldal tlaos for tho typhoid bacilli 
at 280 ko are tlx aiautes for 0,1 ag, 15 atautoo for 1,0 ac *h& 40 
ainutes for 10,0 at* At 1120 ko tho tlao for 0,1 as i* four alautes 
or a somewhat faotor rato than that at 280 ko; tho othor tlaoo aro 
aloyor, tho tlaoo for tho T1 has 1111 at 280 ko or# alao alaotoo for 
0,1 mg9 30 aiau&oo (15 aiautoo at 1120 ko) for 1,0 at mad 50 Miaul os 
for 10,0 fit* Consoqooatly, tho dootruetlvo strength at 220 ko Is 
directly proportioaato to tho froquoaoy and inversely proporttenets 
to ware length oad lo suhjsot to ooao asaaat of float oat loa. 
2, tarrival toots (too films 1 to 4) Tho graphs show that 
a ooporooaio warm troataoat produo os a legarlthato dooroaoo la bac- 
torlal eouat, ir tafctag tho bacterial oouato la tho truphs ao 
lacorlthado oaalos (vortical aaoo) aad tho troataoat tlaoo ao arlth- 
ootiaal soaloo ihorlaoatal area), opprextoately straight llaoo aro 
forood, A step-like dooroaao io ladloatad hr tho 10.0-at oeaooatra- 
tloa. igr troatlag tho bacterial oouat ao r aad tho troataoat tiao 
ao 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 so 
concentration la fifara 5 aad d, tho dooroaoo la lira baotorla 
oouat, at tho saao saotfr level, boooaoo aoro proaouaood as tho 
frufusaar door too os or, la othor eerde, ao tho wave length laoroaoos. 
Tho tlao required for tho loo of tho lira baotorla oouat of tho 
10,0 ag/1.0 oo eoaooalration to booooo halved lo shoya belay. 
1120 ko 90 ala, 
560 ko n ala, 
' . * - II tffci 
v 
1120 ko 40 atm. 
mo km 90 aim. 
k 280 ko 20 ala. 
Typhoid U taollll a* b# mam 
n|tar« l# ml vaflilliai 
%t Oml ag/l«Q m 
(typfaald, mull) Minia* 
mm 
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(«/piu»U teailll) tolutloa 
mm 
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UK 
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S fiat (tinl««). a*t Bo 959MB 
figm 4. Bofttftrtal roust wkthRi 
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QUII 
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1 Bootoriol wort. 
A fttoo* solution. 
3 ¥1m (aimlM), 
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Table 8. Microscopic observations of morphological chan ■;o4r .in typhoid Vi bacilli at 
various supersonic mve frequencies 
1 
Frequency 
1120 kc 
560 kc 
280 kc 
1 Microscopic 
Cellular 
Cellular 
— 
Cellular 
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It mi lonroad fwa the foregoing rooulta that the optima for 
eollular 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 *ithia 
tba eupereoaic acre fnqmgr rang* aerated by this experfeMat, *& 
extressaly alight turbidity decrease wee dotectod la the 10.0 ag ft 
bacilli 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 effective 
ecliuUcldel tiaa ia iadloatad at 880 ka aad 1180 kc, ia that order. 
Collulieido at 580 ka la alar, fhle actioa la diraatly prepertleaate 
to tbo froquaacy (inversely proportionate to tba rara length) in cor* 
oaatrated baatarial eolations aad appears to bo adbjeat to ooao aaeu&t 
of fluctuation, 
3. A slightly greater aaouat of difflenity lo eacoustared ia 
dootreylag tba VI baailli co opened 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 count 
x 3 troataent tiaa 
a * negative eaaetaat 
s. A nlorooeople oxoalnutloa af tba opaalaaao raraalo cellular 
do* tract ion occurring at 380 ka, 1130 ka aad 580 ke, ia that order. 
1. i-ranapavaaer doao cat moult# rbaa physiological saline 
oolutloa suspensions af typhoid baoilH and VI boaUU (only a alight 
traao aaa bo aatlaad la tbo aaaa of 10.0 at Vi bacilli) era traated 
tfttfc 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 laanBolegloal 
Studio Baaad mi iuperaeatc «*res, fOtTO IJI iAddfil (Tokyo Kadlaal 
Journal), JM>. 3011, februasy 194*. 
*. 014X1, Shlgekl, Cellulicldo, Turbidity Amm, TtMrU 
Ohangoo and internal tmpwmtvrm af f*«l Solutieaa fmlod vith 
Mm, naei roiumu nwuoAKo rai u*pan dauraai of 
Miarobtpiogy aad rathaiagy), v#i. », *>. 10. otioiw n«. 3m v* 5ssta$ 
S, A. C. K, TW m*k 3IK uat Pro®. ttxp. BUI. «a& a«tM 
A. 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, MoUlu 
trohl, SmUIm pmijraat a*d £toeUlu» 4jr*«nt«ria#, IXSOA 
Blfcaimo 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 Bacillus 
FROM; «P/ T . __&s2 . ftVQ. __h?EQ—REFERENCES: 
J EVALUATION: DATE OF INFORMATION: l$kZ. DATE OF REPORT: ,l6 _AUg_ 1ft fiQ .. .. 
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 Section 
Document Uoe 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 of 
the former Japanese Army Medical College in 1342. to improve the quality of typhoid 
vaccines. The study outlines the nature of the experiments, discusses experimental 
procedures and results and gives a summary and conclusion* 
For the Assistant Chief of Staff, G~2s i 
/At 
J. E. POLK ( 2 J-/ 
Lt Cols GSC >' *’ 
1 Inclosure (4 cys) Executive 
as mentioned above 
NOTE: This document contains information affecting tho ns- j DECLASSIFIED tenia in any manner to an unauthorized person is prohibited 
tionaf 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 " *