3o« So 58389 A 
om 
o«rm 
5 /f r . in *AST CQKHTO 
mmax nmo&xamret ascvxor, wm wnn 
ALL TO TBAS3LATOH TO mmmfm. SECT 10! 
Tra&ilatlofi Hoqaootod by ?X8, 90 
Dftt* K*c»A A3PI3 U fob 50 
Dooeriptlosi of Ckmtontoi full troaolmtloa of *Supor*onU Waro 
Sonorator; Sxporlwontatlon with OJiolom 
by Away Mo<Ue*l 3ollo#o 
Hpldoololocgr Laboratory, 31 Mar 42. DOC SO 5538$ k 
Amy Nodical Collofo Sptdomiological 
loooareh Baport 
Section 2 Sunbcr 324 
Bolmtloaohip of Superaonic Ware Fraquancy 
and Oollulicidal Action 
Part 1* iferc fcracxmtor; 
Iferrarlaoatation vith Oho1arm Baetorim 
Army Kedieal College %id*aiology Laboratory 
(Maj. Qob. I SHI I, coanaanding) 
3WLO, Takaahi Won-official otaff 
Action S 
Original Copy 
Olaoti floation 
STS 
;|76-SS 
43S-1 
•feooirod 
31 Mur 43 me To SS398 A 
fablo of Content# 
Shrtrfcar I* mvm caaaratar; application af auparaa'ni© 
mcwmt 
A. Svxparaaaia *»▼» ganarator 
B. ApplloatioA af auparaanio waaaa 
Chaptar II* 9baarvatl<ma 
Chantar US* Sxpariaaatal nraaadura 
A. Baotarial atrain 
9. Preparation af tusatarial auspanaiaaa 
C. 3up«raoaia vara traataant 
D. sjialitatlaa taata a» eallullaidal atraaetti 
1. Saatarlal count 
?. Obaarratiaa af Borpholafiaal chanya* 
9. Maaturwaanb af turbidity 
rttiaptar IT. of axparlaant 
A. CaUmlieldal tia# 
8. Survival taat 
C* Saaolta on aarpfcalacteal ehaagaa 
D* Baaulta as i«n«artiMOl of turbidity 
Chap tar ?. SaMaary sad conclusion 
libliagaaply 
II Doc So 58368 A 
denevttl 
Humorous studies bars been performed on the effects of super- 
softie waves against different types of bacteria. In the study of 
cholera bacteria alone (1) £49akaBE sad his fellow rasearehsrs 
discovered that antigens produced by destroying cholera bacteria 
with supersonic wavs* were la compartton to ordinary bacterial 
solutions weaker la toxicity, and that their agglutinin-complement 
fixation properties, antibodies sad immnl fact eat powers contributed 
favorably toward producing immunity; (2) QXITStr observed in his 
complement fixation reaction tests that a bacterial solution treated 
for 30 to .10 vinutos with supersonic waves was most sat 1 *f nets ly 
end produced results superior to those ef past antigens; and 
(3) 02AXI reported that the cellulicldnl action of supersonic waves 
io governed to a con tide ruble degree by temperature, that tempera- 
ture rises destroy bacteria, but produce a decrease in trans- 
parency and that turbidity is not pronounced at SO0'! or below. 
Xaeh of the above studios was performed on a single frequency. 
Tho effects produced by varying the frequency have not been 
Investigated. The purpose of this experiment was to stake compara- 
tive studios on the effect of different frequencies (1120 kc, 
560 kc mA 280 kc) on bacterial cells end to clarify the relation- 
ship between frequency and eellulicidal action. 
1 
Chapter !• Supersonic wave generator; application of supersonic 
waves » 
A. top* ramie wave ssnerator? The rape ramie ware generator 
(Model Humber 3) employed by Ui« labor*to 17 for this experiment 
was designed $a October 1994 upon the request of tho lot# 
Or* VAfiiili Qatari. Construction mo completed last year* 
(Army Hediaal College Epidemiology Laboratory import Bomber 11-96*) 
The principal features of tho generator t,rt do tori bod below. 
1* tho range covert five frequencies (140 be, 380 be, 860 be* 
1180 be and 3340 be). 
2. Two resonators hnTO boon provided; both curn bo 
ooelllatod eimultaneeusly* 
3* Increased dioloetrie strength around tho crystal re- 
sonators i« obtained by the automatic temoe return control and the 
oil purifier which io provided for tho insulating oil. 
4. Tho plate Inpaot of tho 0telllater tube (one per 
crystal resonator) is fixed at 1*8 tines or 1.6 kv. 
8. Compensators allow for volt*$40 fluctuations la tho 
power supply. Doc 9o 55388 4 
Sapor tonic war© gonorator 
Front rlev (Part X) 
FHQfO 
Sapor*®*!® fnwrfttfir 
front riow (Pari ?) Doc Ho 65389 A 
the bacterial solution containers were redesigned by this 
laboratoiy. Ordinarily it has been the practice la supersonic 
ware treat sente to place the bacterial solution la a thin-bottoned 
tsst tube which in turn wae iaaersed la the ell froth formed oa the 
oil surface above the crystal resonator. Under such conditions the 
bacterial solution is sprayed upward by the supersonic wares. 
Because the spray droplets adhere to the test-tube wall a consider** 
bis length of tine si apses before those droplets descend to the 
surface of the bacterial solution. Though eons of the force from 
the supersonic waves passes through the test-tube wall and acts on 
the clinging droplets the Intensity le considerably less than that 
acting on the solution lying at the bottom. When the supersonic 
wave treatment le discontinued after a fined period tite portion of 
the bacterial solution which received a smaller amount ef the treat- 
ment nixes with the portion at the bottom. This leads ts In- 
accuracies in dote mining the tine required to destroy bacteria. 
A metal container built according to specifications des- 
cribed below was used la tbs expert meat la older to reduce such 
Inaccuracies. 
&rj«<3lAl eon%*in#r octploym! 
la *x#mrtamm% 
mofo Hoc Ho 55388 A 
Hard glass was used in making the special container. Based 
on past experience a 0.5 mi thickness at the bottom section was 
determined te be sufficient. The bacterial solution was placed 
in part A at the lower end ef the email inside tube* the length ef 
which was set according to the relume ef bacterial solution to be 
handled• 
An opening approximately 80 ms in diameter to he used In in- 
jeotlng or extracting the bacterial solution was provided on the 
hemispherical surface at the top of this tube. fMe enables the 
majority ef the spray droplets to defleet downward from the opening 
toward the tube wall ant drop rapidly, the droplets which pass 
through the opening and strike the wall of the larger tube descend 
to part 9 and cannot six with the solution at the bottom. 
This container was tested at our laboratory last spring. Tbs 
tests rsToslsd a slight shortoning sf the ties required to destroy 
haetorla. < 
B. Application of supersonic wares i As stated previously 
comparative studies on the effects of supersonic wares on Bacterial 
solutions were performed at three frequencies (1140 ko# 860 kc 
mA 280 Vc). In order te ashlers# a constant supersonic were output 
50 oo of transformer ell was placed la the abera-sientloned special 
container and the oscillator was tuned to produce at each frequency 
a 10°C per minute rise in oil temperature, (figure 2 illustrate# 
m example produced when tuning wan accompli shed in eueh a aannsr.) 
Other experimental conditions are listed Below. 
ncy 
'Inn 
length 
H*U 
Flat# 
current 
Orld 
mmnttti 
0*) 
(•) 
<*> 
(**) 
(*0 
nao 
266 
3000 
460 
166 
560 
535 
3000 
560 
160 
260 
1070 
3300 
400 
160 
Crystal resonator position* 8 m below oil surface 
Busts rial solution 
containmr oositlou* Container Cotton 3 cm *feor* 
•11 surface 
Oil tatfc tespemture 
(internal)* 30®S 
factorial solution 
(constant)t 48°C (-f- 2*0) 
Bacterial solution rolunsi 100 so DO# BO $5888 A 
tiadar condition# irhtrt th# initial liquid (nil) t#*p#r*tTir# 
elot#ly approach## th# **fei«nt tonporoturo, th# r*«ult *hchm In 
Figvtn> 1 1« obtained by riottin* th# troatraont tin# *nd th# 
tanpcrnturo of th# tmmmformer #11 oo&t*ln#& in th# opneinl t#«t 
tub# vh#x> «up#r*onle war#* not again#! th# bottom of th# tub#. 
Figure 1 
ORAjrH 
(top) 
(1) f««t tutu (intorn&l) 
(3) ?r**t*#at tla* (»l*ttt#«) 
Tha straight taamormtura rlsa Auria* tha initial stag# 
lad & eat • a that tha hast #anarated threap the aboorptlon of 
nuoeroiic va»«« la considerably greater than tha thamal eonduatlrlty 
vi thin and without tha tuba. Tha rat# of teapemturs rlaa in thla 
pa rtion la proportioaat# to tha supersonic oar* input Inside of tha 
tuba. DOO 10 36388 A 
2. Baetorial eoaat v&rlfttiosu of 
1 oo Notorial (eholora) oolutloa 
<am 
(lower 
ion) 
Ik 
(1) Sectorial count 
(2) Stock solution 
(3) nm (miautoo) BOO BO 05383 A 
Hrure 3. Bacterial eomt variations of 
10 mg/1 ee bacterial (cholera) solution 
mam 
(lever 
right) 
.&ET' 
(1) Bacterial $*aat 
(2) Stock aalutiaa 
(S) Tia* (alout**} 
Ohaptar XI. Observation* 
Hi* iie*» ll*t*d k*lov v*r* okitrred on tact*rial *u*t>«a«loa* 
containing 0*1 mg, 1.0 mg and 10 mg *f cholera bacteria par 1.0 ee. 
Thaaa cb serration* ver* aade at different fr*<pum©lee. 
A* SellaHciAal t#*t 
B. Bacterial aoiaat 
0. Turbidity aeaeureoeat 
0. flffl* change la a*rph*Xegy Boo Bo 55388 A 
Chapter XIX. Experimental procedure 
A* Bacterial at«da: The bacteria used 1ft this experiment 
were of the Kitaai strain preserved by this laboratory. The strain 
was always subjected to animal pas sags before use. Virulence against 
mice was 0.1 mg. Uni fora turbidity and "bacterial film" resulted 
with a bouillon culture (FK 7.4). * Bacterial film," unifora 
turbidity and excellent growth were observed in peptone, las 
formation with a vertical dextrose or lactose culture was negative. 
Cholera-red reaction was positive. The agglutination titer of the 
original seres was positive at 12,$00 tines. 
B. Preparation of bacterial suspensions* The product separat- 
ing after 24 hours in 3 per cent agar (PH 7.4) was cultured at 37°o 
for 20 hours and weighed. Bacterial suspensions were prepared at 
a retie of 10 mg per cc of 0.B5 per cent physiological saline 
solution. The suspensions were employed after completing tests 
for miscellaneous bacteria and bacterial counts. 
C. Supersonic wave treatmentt The 0.1 mg bacterial suspension 
was treated with supersonic waves for 1-10 aiaute periods, the 1.0 
mg suspension for 1-10, 15 sad 20 minute periods and the 10.0 mg 
suspension for 1-10, 30, 30, 40, SO, SO and 70 minute period*. 
P. Qualitative tests on ee Hull cl dal strength* Immediately 
following the supersonic wave treatment bacterial suspensions from 
the respective treatment periods were slant-cultured in 3 per cent 
agar and plate-cultured in peptone sad agar media with large 
plat!nun-wire loops. The cultures were observed after 20 hours 
(4$ hour# if growth was insufficient) at S7°0 and their dswelopfceat 
was graded with symbols Ht, t + and — 
8. Bacterial count* The super tonic wave-treated bacterial 
solutions were diluted progressively tea times each. One cc of 
each dilution wee placed In a JPetrl dish Into which 10 cc of 45°C 
agar was later added. Upon solidification, agar was again added 
in a proportion sufficient to cover the surface with a thin film. 
Inch was examined after a 24 hour, 37°q culture; colony counts were 
made after a 4$-hour period if growth was inadequate at that time. 
donate on the masher ef destroyed bacteria were taken on ths 
control and. also during the intermediate stage and the stage prior 
to destruction. The rate of time decrease compared to the control as 
well as their logarithmic values were obtained following the 
bacterial count. These included ths number of developed colonies 
per milligram, the percentage of developed colonies In 
to the control and the logarithmic values of the bacterial count. 
f. Observation of morphological changes* Simple stains with 
Pfeiffer* • solution were made following the supersonic wove treat- 
ment (after one platinum leop portion of the test solution placed 
on the object plate had been dried and fixed). Microscopic 
observations on the destruction of bacterial cells were performed. 
0. Measurement of turbidity* The bacterial suspensions were 
diluted with a physiological saline solution, according to the 
ratios shown below, and were measured with Fulfrieh’s photometer. 
Vpon obtaining the relative turbidities ths absolute turbidities 
were computed. Doe So SB5S8 k 
.Bhttn esassMiiaila* 
auirtiaa 
0.1 mg/10 e« 
3toek «olutlo» 
1.0 **/l.O <w 
10 tlM« 
10.0 af/1.0 ec 
100 %i**M 
Qhmp%*r XT. Sosulto of •xporlBont 
(3#0 ?rVU« 1, 31 *a4 .*?.) ooe m 553aa a 
Ml• 1. t'MROtc of n»\W1*fih1 t**&a$ and of i oUr« bacteria treated with 1120 kc aoporaoaic ism*. 
Flat* voltage 300G vj pinto currant m; grid cormrtfc X65 m, Otto of a*j)orL««t ZL Apr 41* Sooa toqporaturo 22®C. " «mfch«r d«*dy. 
o.i m 
,, „. - — 
1*0 ag 
4 
lo.o ag 
Tim 
(min) 
Survival 
at 
baetaria 
Turbidity 
ii&efcariaX 
aovrit - 
0.1 &g 
z 
3ur*2*ftJL 
«s^Mapa3pv»l 
to 
COIitfOl 
00 
So*; Z 
\sfai) 
of 
b&etari* 
-kv1' 
* 
lt$r 
B^u&urivtl 
oouni - 
l.o m 
lz) 
Survival 
e glared 
to 
OWUWWM* 
<« 
las. 2 
Tf/yaft 
(®ln) 
Survival 
of 
lit aria 
t^rteidi^r 
final m\ il • 
const - 
10.0 m 
m 
Survival 
compared 
to 
©ootrol 
«> 
log Z 
Pup- 
icivt 
•gar 
P^>- 
tcm 
*C«r - 
elaat 
P9/P"! 
torso 
■g-ir 
•Imsi 
X 
-t- 
-+- 
0*0105 
68.81 * 336 
K 
: . f - 
T TT 
tH* 
o.iyr 
53(00 x ID6 
3.7694 
X 
-Hf 
4H> 
0,6246 
5381 x IQ6 
5.7695 
1 
-K 
0.0333 
X 
t“H" 
tH- 
O.IF 
i 
M 
/* 
1 
fH- 
444 
0.6023 
2 
“4* 
-t- 
0.010, 
t 
4+1 
fH* 
0.JJ® 
2 
t+4 
144 
.5688 
3 
-t- 
0.0117 
0.06 x 106 
OJ02 
3.77*2 
3 
t++ 
T]t 
9.4f* 
3 
ttf 
HI- 
0.5464 
4 
— 
— 
0.012? 
k 
Hi* 
iLi. 
fP 
o. M 
4 
H+~ 
4+4- 
0,5353 
5 
—— 
0.0150 
5 
44- 
"H 
o.f 
5& 
54.29 x .to6 
9.239 
7.7326 
5 
i (i 
|TT 
iii 
!TT 
0.5130 
6 
— 
— 
0.01% 
6 
-H- 
TT 
7.^3 
4 
All 
TTT 
H+~- 
.4796 
7 
— 
— 
C.OU3 
7 
H- 
25 ! 
7 
1 44— 
44- 
0.4796 
* 
— 
— 
0.0149 
a 
-+- 
[ ; 
o. n 
67 
9.16 x HJ6 
1.553 
6.9542 
a 
44- 
44— 
0.5130 
9 
— 
— 
0,0146 
9 
—f~ 
o.| 
OD 
7 
+4- 
44— 
0.5353 
10 
— 
— 
0.0126 
10 
•4— 
0. « 
67 
t .OOOOU 
1.3261 
10 
•f4- 
44- 
0.5&X) 
15 
— 
i — 
0.$ 
:30 
1“ 
-4— 
0.59116 
5.42 x 106 
0.092 
6.7340 
30 
4D 
t“ 
"4~ 
0.75847 
3.39 x Hi6 
0.057 
6.5302 
50 
■+- 
+- 
70333 
0.6336 x 106 
0.00142 
1-9191 
60 
—— 
—— 
0.61347 Doe m i 
T&bjfc 2* Mmm&Ms of otiivXlo&AaX otonMstftfe t—t»| toifoi&it# ani at cho&tr*,/4f*&tod mih .540 ke —pagnoli w#»#« 
**&&<#» JM90D V} pinto mvtmii S5Q m$ grid mmvml M3@ m. D*&m of 1 aqporjjMat JBL lagr &« Im %«np<r»itmnB 26°c. 
0*1 
,1,0 tag ‘ 
30*0 iqf 
Osin) 
Surriir&l 
of 
&svciari& 
Aor^iJiiw- 
-tttAarlal 
-wwaft -» 
0*1 4% 
<z) 
?fcir*4ral 
r. 
s^itrol 
c«) 
!■• z 
t$IK 
(lain) 
IwM 
Tarb&dl* 
BaetarlT 
aannt ~ 
1.0 
U) 
iarviml 
ee^aapwi 
1 a §1 
«> 
TOf; Z 
’©bosi- 
iMdn) 
rsurriswX 
of 
TtirbM.it 3 
Bwctterlal 
- 
10.0 m 
z) 
SaarribraX 
eoaparad 
ft 
6«M 
m 
' z 
Pap-1 
UM 
Agar 
Slftttt 
Pap- 
A0MT 
alaai 
Pasp”- 
tasna 
S 
+f 
.TT.-r ..,...,r(r 
0.0881 
-7.35C x 10* 
7M315 
I 
i. i ..< 
m 
j ft* 1 
' s 
0.100*1 
671*585 x Vf 
M9X$ 
I 
413 
>.. -A-,. 
Ttt 
0.62462 
'785.85 x itf 
9.8313 
X 
ft 
-+- 
o.oom 
1 
fTT 
ft* 
0.074T5 
X 
tt+ 
,-i, 
ITT 
4MM 
; a 
-— 
0.0062:1 
a 
fTT 
t++ 
0.0604 
a 
11 1 
lit 
TTT 
04W 
* 
3 
j 
| 
ox&m 
3 
fff* 
1 i l 
■fTT 
0.09095 
; 3 
1 4 1 
TTT 
*H 
0.45731 
4 
— 
— 
0.00636 
4 
TTT 
t > i 
TTT 
0.04853 
4 
In 
fTT 
141 
TTT 
0,45174 
5 
; 
— 
0.00636 
5 
Ht 
Ttt 
0.05131 
0.271 x is)6 
0.0396 
5.4330 
5 
kit 
TTT 
ff+ 
0.4405® 
j 6 
— 
0.0069? 
6 
tlT 
HT 
0.05666 
6 
44+ 
44+ 
0.41501 
7 
— 
- 
0.00692 
? 
H+ 
H+ 
C.C4796 
7 
m 
il 1 
TTT 
0.40154 
$ 
■ 
0.00747 
a 
»11 
fTT 
i “ftf 
0.05465 
a 
+tt 
+H 
0.40154 
9 
_ 
— 
C.00814 
9 
i. i_ t 
rrt 
i LX 
TTT 
0.05800 
9 
ft" 
t++ 
0.53434 
10 
— 
0.00781 
10 
11- 
JT 
*T 
0.060% 
10 
i 
Tf 
111 
ffr 
0.40156 
15 
— 
0.00848 
u 
i i-i- 
T? 
fT 
0.06583 
0.314 x M* 
0.0026 
4*1401 
» 
ft 
ttt 
0.W 
0.348 x 10* 
5*3145 
ao 
— 
— 
0.0)903 
30 
~+~ 
0.0577 | 
652 
0.0009 
2,6453 
30 
, 1 
Jf 
in 
ffr 
0.:34577 
• 
30 
-—! 
— 
0*04999 
|j 
40 
ii 
T» 
H*~ 
0*37734 
0.023 x 10* 
0.0036 
4.3617 
40 
— 
-— 
0.05^6*1 
50 
■■■ III. <^Wl» 
+4- 
0.37934 
0.007 x JO* 
0.0013 
3.8451 
• 
fji 
60 
T 
-f— 
0.2.5731 
O.OOOS xlO* 
0*00®? 
2.3010 
* 
75 
' 
: 
-.35693 
, 
90 
— 
j.39006 Doc Ho 5538? A 
m2* 3* iittn&ts of OoUaUfttfeL strwftgtii topktdity m*I *t*rvfcr&l of ohoX*H5 b**t*rU troatod «lth 390 ke *up«snml« war®*. 
Flat* volta*$e 3300 vj p2*t* mrrmt WO »? g**4 oormit 190 at*. oC «aqp«*4wafc ? ML 41. &Mt taqpmlavtt 31a0. ttoa&MNr oloar. 
s.i m 
t.0 fflg 
10.0 jg 
Tint 
(*&ra) 
9ii«M 
toetari* 
Tarhldity 
SsjImIaL 
{S' 
3arvival 
m 
COTitjPOl 
j&g Z 
Ha» 
(lllfl) ; 
aurfftpul 
baetori* 
1 S&afcarlal 
temty Ottflfe - 
1 1.0 em 
<7.5 
‘urtftvaa. 
ccHpaiml 
CfiTitrol 
'AS z 
Tim 
(&£&} 
Wvival 
b-ict^srla 
3a«fc«ifjj4 
eaust - . 
ap* 
Jurv&raX 
#q^P&e*gc| 
E 
centred 
w 
■ z 
tai» 
Agar 
•Iwst 
5) 
t*r« 
Sgar 
slant 
—,—...—* 
m 
rifK 
Ajpy 
ilaflt 
S 
t~ 
4— 
0. 0998 
58.410 * 10^ 
7.76H 
I 
~+J 
0.07361 j 
154.100 * 1C6 
8.7664 
X 
-H 
4~ 
0*1*2530 
—— 
ej*i 1 M .#.6 
X W 
— — 
9.7651 
1 
I ~4~ 
4~ 
0.01174 
4.523 * 10* 
7*743 
6.6551 
1 
H— 
0.06556 
1 
0.59136 
3. 
— 
I — 
Q.01126 
2 
4“ 
: 
0.06525 
2 
>*52424 
3 
— 
— 
0.01253 
3 
~4—j 
■4— 
0.06134 ! 
4.768 x XO6 
4*210 
8.3927 
3 
| -+* 
-H 
0-55770 
4 
| — 
— 
■.01267 
4 
4~ 
0.06033 
3.833 x 10f 
4 
Hh“ 
0.'-9635 
5 
— 
— 
0.SU99 
5 
~+~ 
-H 
C.O606C 1 
2.710 
7.1995 
5 
-t- 
0*4 W7 
— 
— 
0.01239 
6 
— j 
— 
0.06470 
6 
-H 
4~* 
0.45731 
7 
— 
— 
0.01025 
7 
; ~ 
j 
0.96692 
7 
-+~ 
4*~ 
0*41270 
a 
— 
» 
0.00960 
a i 
— 
— 
0.07361 
1 
a 
*m#nnA» 
0.42301 
7 
-— 
0.90942 
9 
— 
— 
0.06660 
9 
-f- 
-H- 
0.41827 
& 
— 
— 
0.00760 
10 
— 
— 
0.0713^ 
10 
■+* 
4- 
0.41270 
30.719 * 106 
3.995 
8.361? 
15 
; 
fc.06581 
15 
”4- | 
■4— 
0.48960 
53.740 x 106 
0.930 
8.7243 
20 
■'4— 
«—■W^i 
0.39597 
0.246 x 106 
0.004 
5*7303 
30 
— : 
— 
>;*39®09 
40 
——- 
I —— 
C.36908 
— J 
50 
— 
— 
0.339* Doe Bo 55388 A 
the eellulioidal time Is shown in figure 1, the morphological 
changes in Table 4 end turbidities in fables 1, 3 end 3. 
A. Oellulleldal tlae 
(Summarised from fables 1, 3 end 3.) 
*T* 08 
9 
«l« 8 
•* ose 
8 
*T* 84 
*X« 08 
«T» 8 
088 
8 
«T* 06 
«T« BX 
*T* * 
031 0811 
X 
oe x/W crot 
oa'i/Wt 
Wx/itf i*"’6 
At the earn# supersonic uare energy, cellulicide w« accelerated 
aa the bacterial dilution was increased and ae the frequency mat 
decreased. At 380 ke cellulicide occurred In the 0.1 mg solution 
in tve minutes, the 1.0 mg colutien in six minute# and the 10.0 mg 
solution in 30 minutes. Higher destruction occurred at 1130 kc 
than at 560 ko. Oonsequently, the eellulioidal time (directly 
proportionate to vats length? inversely proportionate to frequency) 
fluctuated according to differences in save length. 
3. Survival test; Ae illustrated in figures 3 aad 8 the 
decrease in bacterial count produced by treatment with supersonic 
waves is logarithm!cal. By treating the bacterial count in the 
graphs as a logarithmic scale (vertical axis) aad the treatment time 
as aa arithmetical scale (horlsontal axle) It can be said that tha 
relationship 
leg T s cx T » 
( e s negative constant) 
exists (when I represents the tact*rial coast aad X represent® the 
treatment time) elnoe approximately straight line# are shown when 
viewed in tome ef logarithmic values for the number of surviving 
bacteria. 
In the aaee of the 10.0 eg/1.0 cc eolation graphed In 
figure 3 a marked reduction in bacterial count occur* at the ease 
energy level them the frequency decreaaee and tha wave length 
increase#. 9hle reductitn ie net ;>renounced in figure 3. 
The tlae required for the legarlthm of the lire bacteria 
count in the suspensions of each concentration to become haired, 
or their fractional eurriral time, le ehoua be lorn. 
0*1 MC 
1*0. Mk 
lo.o « 
1 
1190 Ice 
% *i* 
8 ala 
40 ala 
3 
660 kc 
1 Mia 
18 aim 
30 aU 
2 
880 ke 
1 aim 
6 aim 
30 ala nee Ho SS3BS A 
0. Haaulta Oft »omhologle&i ehaagaat (Saa Tabla 4.) 
Tha iaflu*n«a§ axartad bjr f ra<nMmciaa oa lha bnctarlal 
call*, whoa axpraaaad la lam of Uaa raquirad for ealluliclda* 
har* boon aaaaarload la lha following labia. itoe X» 583*8 A 
Freq(ke) 
1120 
560 
200 
Bacterial if*g) 
0.1 
1.0 
10.0 
0.1 
1.0 
10.0 
0.1 
1.0 
10.0 
(1) Stared endurance period of e*al« 
3 
(atin) 
9 
(min) 
50 
(min) 
1 
(slu) 
70 
(Min) 
50 
(i&tn) 
1 
(astn) 
5 
Oak* 
20 
1 
(2) InsteUity to stain 
m 
- 
4 
- 
2 
2 
2 
5 
*i 
1 
(3) OeiluX&r swelling 
- 
3 
4 
- 
l 
1 
1 
1 
1 
• 
• 
(4) Celia3U*r degeneratior 
• 
3 
4 
► - 
- 
• 
- 
[ 2 
10 
8 
4* 
M 
(5) < rotoc (feaciof eriiaa tioii) 
* 
4 
i 
- 
3 
3 
- 
1 
10 
(6) (?rssmlar precipitate 
1 
3 
5 
9 
4 
1 
- 
* 
9 
Cloud’- precipitate 
+ 
<*** 
« 
15 
f 
3 
9 
Up 
$ 
Oast-Ufei precipitate 
1 
2 
6 
7 
1 
1 
1 
X 
2 Dec 80 53388 A 
The steps tAteen in ths destruction of bacterial cells by 
mean* of vibrations Included cellular swelling followed either by 
changes in the cell morphology or by immediate destruction nxtd 
homogenisation upon the release of protoplasm. The destroyed matter 
from the bacterial cells displayed dust-like, granular or cloudy 
forms* A* a peculiarity of supersonic wave treatment* certain parts 
were unable to take stains. The above "steps* were taken In rapid 
succession at 350 tec. 
3* Be cults on measurement of turbidity t (See Tables 1 to 3*) 
Within the range of supersonic wavs treatments covered by 
this experiment, transparency was not dot acted among any of the 
suspensions of cholera bacteria. In every case extreme differences 
in turbidity were not present* 
Chapter T. Summaiy and conclusion. 
The following conclusions were derived from observing the 
oellulicidal tine at well at chance* in the turbidity end the 
morphology of bacterial solution* after subjecting cholera bacteria 
suspended la a physiological saline solution to the action of 
supersonic wares possessing the sane energy level but of a different 
frequency (1130 tee, 560 tec and 350 tee). 
A* At the sane energy level the cellulleldal tine is accelerated 
as the frequency Is decreased and is Inversely proportionate to the 
frequency (somewhat directly proportionate to wave length), 
3, Oellulleldal tine decreaees as the concentration of the 
bacterial suspension decreaees. 
C. The changes occurring in 11 vs bacteria during the treatment 
time are logarithm!eal end poseoes the following relationships 
x*» r - « 
y * lire bacteria count 
z * treatment tine 
c a negative oonetaat 
D. Hardly any e»M of traneparency 4\» to the effects of 
supersonic waves on cholera bacteria and physiological saline solution 
can bo observed. Turbidity appears unchanged. 
I* The atop* taken In cellular destruction by sup* ramie vstve 
treatment Include cellular swelling end deformation or immediate 
deetruction and homogenisation upon the releaee of pretoplaam. the 
destroyed natter* dloplay duet-like, granular or cloudy forms. 
F, The degree of cellular deetruction eennot be learned through 
changes in turbidity. 
16 Doc So 55383 A 
Bibliography 
X. Vatarlj 0SAXI, Shield{ SHIMm, J««9; amt 
0KXT3U, Biaayo; X®«uuoiogical Hotoarch vith 3uportonic tfawo~Yrented 
Tholere Antigone, SUE® BXSBIIOTSG SYTmXGAXU 2AS3HI (Japan Journal 
of Microbiology and Pathology), fol. 2, So. 10, October 1938. 
2. QXXTSU, Hiaayo, Heeearch os Oonplenent Fixation Beaetion 
with Supersonic torc-Trented Ant leant, 3# riot 1, Vel. 82, Bo. 11, 
lOTeober 1985. 
3. WAfASABS, marl i UK**!*, Akio; BAHAI, ft'onto; and 
IAJiaUUHX, Toyoki j 7 art out Symptom and Serological React l oat 
Following Inoculation of Humana with Supersonic Vawe-Treated 
Too tint, Amy Modi cal College Reeeareh Report 
So. XI-36. 
4. OZAFX, 3hif eki, Jollulicldo, Turbidity Ohaagte, Volumetric 
Change# and Into real Teoroeraturaa of Toot Solutions Treated with 
Sopereenic SUvet, HIFO* 1ISBIBUTSU BVBtlGAltf BASSHI, VoX. 32, 
Mo. 10, October 1938. 
3, VAXTO, dyoiohl. Turbidity Mot.au ratio at s of Vaccina*, H*»ry,a 
Antigen and Piagnoetlc Solution* with Fulfrieh1 a Photometer, Amy 
Nodical College Bpidealologioal tteaearmh Report So. 11-29. Doc No 55338 A 
Table 4* Microscopic observations of morphological changes in cholera bacteria at various supersonic wave frequencies. 
Frequency 
1120 kc 
560 kc 
280 kc 
Microscopic 
Cellular 
Morpho- 
Cellular 
Presto - 
Granular 
Cloudy 
Dust- 
Normal 
Inability 
Cellular 
Morpho- 
Cellular 
® 
Prot>- 
Granular 
Cloudy 
Dust- 
Norraal 
Inability 
Cellular 
Morpho- 
Cellular 
Proto- 
Granular 
Cloudy 
IX»t- 
Normal 
Inability 
observations 
swelling 
logical 
dostrue- 
plasm 
like 
to 
av/elling 
logical 
destruc- 
pl^urt 
like 
to 
swelling 
logical 
destruc- 
plf»em 
like 
to 
deforma- 
tion 
(homogeni 
stain 
defonaa- 
tion 
(hom^rjenlr 
stain 
deforms- 
tion 
(hoKOgeai 
stain 
tion 
zation) 
tion 
zatidn) 
tion 
zat ion) 
T l rpn 
... .4 
■ 
K 
4H- 
- 
:-jj-» 
“ft 
- 
+4 
• 
+4+ 
1 
— 
■ HIM 
■ ♦ 
— 
-4— 
-H- 
— 
——- 
—4* 
4" 
■■■■■ 
■■■ - 
*4- 
4“ 
_+— 
1 1 111 
“rr 
■'* " 
4— 
r 
2 ' 
— 
— 
-4— 
— 
—t— 
— 
-4~ 
4-4- 
— 
. 
— 
-f- 
— 
— 
— 
— 
— 
-4— 
—— 
—— 
4— 
—— 
—1 ■ 
3 
— 
— 
7 " 
—— 
—,— 
■4— 
-4— 
— 
—— 
— 
— 
j 
— 
— 
— 
— 
4-4- 
— 
— 
— 
-4— 
— 
— 
4 
— 
— 
44" 
— 
—1— 
— 
—f** 
— 
— 
— 
— 
—h W 
— 
— 
—— 
— 
— 
44- 
—— 
*+— 
— 
'r'iri1 
5 
-— i 
-.I 
44* 
—1— 
Ml 
—j— 
—... 
— 
—1— 
— 
— 
— 
— 
—— 
—— 
-4 
—— 
"- " 
4— 
1,1 
0*1 IQ 
; 6 
—— 
44 
■ 
—p- 
*4— 
— 
— 
4- 
— 
— 
—— 
"■ ■— 
— 
——— 
4-4— 
s 
1"” 
-4— 
Tr 
n 
. 
1 
| | 
- - , - 
— 
, 
—p- 
—— 
—1— 
— 
— 
— 
44 
—— 
—* 
*4— 
■ 
( 
a 
9 
10 
— 
— 
44* 
44* 
44" 
— 
-4— 
— 
— 
— 
— 
44 
H4 
J 
ILI - ,n 
1 T--I 
44 
44 
— 
4— 
f4"*' 
4 4 — 
-— 
— 
15 
— 
4'H 
— * 
4— 
. -4— 
—l— 
' 1 
' T1 r"r ' 
20 
* 
ill 
TtT 
4-4 
44 
I 
$ 
is 
8 
§ 
K 
1 '■" 
—— 
—— 
—— 
—— 
44 
— 
— 
—— 
—— - 
— 
— 
— 
4++ 
— 
—— 
— 
—— 
—— 
+44 
— 
g 
1 
— 
— 
*H-t~ 
— 
4— 
— 
—»-► 
— 
— 
HI lU^IWMl 
4- 
— 
4- 
— 
H+ 
—1— 
— 
— 
-4— 
1 4"' 
—— 
cj 
2 
—-■ 
—1— 
-*— 
— 
— 
—f~ 
■H4* 
— 
•— 
. 
— 
— 
—4- 
4- 
| 
4- 
—4— 
ili 
TT“ 
•H— 
— 
- 
-4— 
44 
3. 
j.. sn&sSs 
,. T~ 
-jr. 
' . 
■ .. 
t4 
' 
w T'liUjBP'IT1'1* 
>14 
•*3'’ 
•mil a 
*4" 
— 
—— 
■ +4 
-4 
4- 
-p. 
. 
■f H* 
L • 
—— 
"■ 
44“ 
“T7_, . 
; & • : 
• 
- Sg 
■■ v 
+4 
1 
E 
444- 
t 
■ 
t± 
1 
44“ 
"1 pr - 
"J" 
4 
44— 
' 1 L 1,1 J 
"I™ 
41" 
" ’’ 
"'+1"n 
, 
' * 
+r 
111 
1 
5 
-4- 
-4- 
44- 
r— 
-4— 
— 
-4— 
44— 
— 
p- 
— 
tft* 
1 
—j— 
— 
tt4 
—1 
—1— ' 
4- 
*144 
— 
—■— 
44“ 
—1— 
w 
. 
6 
■■ 
—H 
44 
-T- 
4“ 
——— 
-4— 
44“ 
— 
4“ 
—— 
. i . t 
4— 
-H4 
—t 
■ . 
—— 
+++ 
— 
—— 
-4— 
M 
1.0 mg 
7 
—f— 
—i— 
44“ 
-4- 
-4— 
— 
« 
4+— 
— 
4“ 
— 
4f4 
4-L 
4H- 
4- 
— 
— 
— 
> i A 
TTT 
— 
— 
——— 
-*+— 
—— 
— 
•S 
a 
1 4 
—P 
j 44- 
-4— 
-4— 
— ■■ 
—t— 
44- 
—— 
— 
44 
-ft 
-4— 
*4*4*" — 
4H- 
—|— 
—— 
—— 
—— 
tft 
——* 
—— 
— 
9 
—j— 
_ 
44* ! 
—p 
44“ 
— 
—i— 
-4— 
— 
—4 
< 
44 
4- 
4- 
4*4* 
-H4 
4- 
—— 
— 
ft4 
— 
— 
— 
-4- 
—— 
*8 
20 
—f— 
— 
44- 
-4— 
4-4- 
— 
—j— 
-4— 
— 
—4 
— 
44 
4" 
4- 
44 
t'4 
—4* 
— 
—— 
++4 
— 
■■■■■ 
• — 
-4— 
— 
—— 
& 
15 
—— 
—— 
m 
—— 
4*4* 
—i— 
—— 
— 
— 
44 
-4- 
ift 
44 
4 
—1— 
— 
20 
— 
— 
144 
— 
44- 
— 
-4- 
— 
— 
— 
— 
44 
— • 
44 
*44 
44* 
4~ 
30 
44 
— 
44 
4+" 
44 
■ 
" 
40 
44 
J 
t*4" 
44“ 
44 
K 
— 
— 
— 
— 
— 
—. 
trt* 
— 
— 
— 
— 
*—j.. 
.. 
— 
— 
44 
— 
— 
— 
— 
—■ 
— 
— 
— 
t+t 
— 
1 
~ —1 
■ 
" 
— 
—- 
444 
—— 
—p. 
—* 
■' i|—■■ 
—1— 
— 
4- 
| +■! 
— 
—i— 
— 
—j— 
— 
——- 
—- 
+44 
—— 
2 
— — 
- 
— 
— 
— 
— 
44 
— 
—4 
— 
-f-4 
— 
— 
4- 
Ill 
TTT 
-4— 
-4- 
— 
4- 
— 
— 
-4* 
■4t 
——- 
3 
— 
— 
— 
— 
— 
— 
44- 
— 
—4 
— 
4- 
L —i.. 
4r 
, 
-+•+■ 
—+— 
4- 
— 
4- 
— 
_ 
— 
-4— 
44 
— 
4 
.■■■■» . 
— 
—p- 
_ 
— 
— 
— 
44 
—f 
—1— 
— 
44 
-4 
44 
4- 
44 
4*4 
-4— 
-4— 
— 
—1— 
—— 
— 
-4— 
44 
— 
5 
—j— 
r- 
—— 
— 
——- 
441* 
—j— 
—t— 
— 
a A l . 
' r t "T 
»i i^m 
-44 
-++* 
4- 
—j— 
*4— 
4'"4". 
—— 
—— 
—— 
-4— 
44"1 
4— 
6 
—1— 
—p. 
—4“ 
—{— 
— 
—4“ 
444- 
—p- 
—p. 
— 
44 
4 
*44* 
—1— 
4 
—1— 
—1— 
—1— 
— 
*+*4 
— 
— 
—4— 
44— 
*-+—* 
7 
— 
—p- 
— 
-4- 
— 
—r 
44 
“4“ 
—4 
— 
4+4 
4 
44 
—f- 
4H* 
-4- 
—j— 
4- 
— 
44- 
— 
— 
—— 
-4— 
4.4". 
-4" 
a 
p. 
—t— 
p- 
—P_ 
—*—. 
—r* 
—r 
44 
—(— 
—p 
44 
—p,. 
"14 
-4- 
4H* 
4- 
—1— 
H— 
44 
— 
— 
' 
—4— 
—4— 
9 
. —j— 
~fl~ 
“4“ 
—j— 
"44" 
—p- 
—4“ 
44 
f 
—j— 
+4t 
4- 
"44 
H— 
4H* 
—1— 
—1— 
.* 
44 
—— 
— 
H— 
44 
4* 
10 
—p» 
4+“ 
44" 
—h* 
—rt" 
—p- 
—h 
-4 
—4 
—-— 
+4t 
4- 
1-1" 
-44 
4H- 
4- 
—1— 
4“ 
4- 
4+4 
-■ " """ 
—1 
-44 
—H- 
10.0 
15 
—+— 
TT 
44- 
-4- 
111 
—p. 
—p 
,44 
— 
—4 
—— 
44 
w 
44 
4* 
4ft 
4- 
~i— 
H— 
4* 
+44 
“+— 
——* 
— 
4" 4" 
-4- 
”+— 
me 
20 
—1— 
44- 
44" 
44- 
44“ 
—P 
-44 
——-— 
—j— 
' 
444 
4^ 
4-4 
44 
44 
H— 
—j— 
4- 
-4— 
+44 
* 
j 
—1" 4 * 
”+ 
25 
30 
40 
IfI 
44 
44- 
44— 
414 
444 
444 
Ip 
~r- 
-rr 
-4-+- 
44- 
~~p 
44 
14 
44 
— 
3 
44 
4+ 
. .. 
TTT 
hh- 
1 | 
■44 
—44 
4*4 
4+* 
4+4 
1 i .1 
TTT 
+*4t 
EH 
— 
— 
Ttf - 
44— 
444 
— 
— 
k 1 
f 1 
44— 
44— 
-44 
-44 
44- 
- 
— 
50 
—r— 
4 JL 
1 * 
444- 
1 "t 
4-f 
44*- 
—r- 
-4- 
— 
—4 
—~ 
■4+1" 
-44 
44+ 
* 
-H 
60 
—— 
— 
• *. t. 
t 'V 
— 
444 
44- 
44 
— 
. 
■ 
44 
1 mm 1 
-4 
1 j1 '1" 
+4t 
—ir iir" ~ 
75 
120 
—— 
— 
44 
—4 
-4-4 
44 
t+4 
_i 
'lrj ' - 
44 
44" 
4*4 
++4