Airborne ultrasonic sound in combination with propylene oxide on spores to achieve sterilizatio

Pisano, M. A.

English

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An Invest.1gat1cm or a SonO<o-Chem1cal 
Approach in sterilizat.ion Problems 
Fourth =Annual Progress Report 
1 January 1966 to )0 June 1966 
Michael i f> Pisano» PhoD" 
Grant N ., NsG-684 
Submitted to a 
National Aeronautics and Space Administration 
Wasbingtoo, DoC., 20S46 
Department ot Biology 
Sto John's Un1~sity 
J ca~ New York 114)2 
1805 
https://ntrs.nasa.gov/search.jsp?R=19660022845 2020-03-16T21:11:06+00:00Z
1 .. 
10 Purpose of Investigation. 
The posslble use of air""borne sound in sterilization problema 
confronting NASAls continuingo studies on the effect of sOnic d ultrasonic 
trequ ncies, propagated in air, on spores of !o subtilis var" nig r ha 
alre~ been reportedo In addit1on~ the combined efteets ot sound and 
ethylene oxide have been noted in past reports 0 
The pre8ent port cootaine data on the antimicrobial effects or 
ultrasonic vaYes at 60 C and at )0 C,) The use of propylene oxide in combination 
with ultrasonic waftS i8 also included .. 
20 Alterations in Procedures Employed .. 
Th teat apparatus itself 115 dentical to that described in the Third 
Semi-Annual Report (1 Julr 196, - 31 December 1965) (I The change 1n8tl tuted 
presently invol.a the use or propyle oxide in place of ethyl oxide., This 
change vas made becawse or the ditficul ties encountered with the use of ethylene 
oxide in our test apparatus 0 Th higher bOiling point of propylene oxide as 
compared to th t of ethylene oxide el1m1natas the problem of vol tll1zat101l 
experienced with the latter compoundo 
30 Effect ot Ultrasonic Irradiations on the Spores of 80 subtills Yare niger 
at 60 C and 30 Co 
In the Third 'Sem1=Annual Progress Report (1 July 196> ... 31 December 
1965) data were presented for the irradiation o~ "pores at a distance of 1 inch 
f the t.ransducer at 60 Co Since the last report, .additional experiments have 
been run and the results are included in the tinal figures" A8 & result, aUght 
d1.tferences in per cent Idll-will be noted" Table 1, therf'ore, contains a 
repetit.1on of data trQlll the preTious report ( 1 inch irrad atlon) and it also 
presents DeW data compiled from experiments em the irradiation of spores at 
other distances at 60 Co 
Table 10 Average cou.nts (X 106) and % reduction in "i ble counts (in parentheses) 
or Bo subtilis var~ tser spores exposed to ultrasonic irradiation 
(3476 Irc/sec) at 60 0 
Hours Distance fram Transducer a inches 
90~ ~o~ ~o~ !o~ 
1 1050 (0%) 1042 (O%) 104S (O%) 1<>30 (8%) 
2 1036 (4%) 10)2 (6%) 1040 (1%) 10 24* (l2%) 
4 1 .. )6 (4%) 1 .. 27 (10%) 1027 (10%) 0096it (32%) 
8 10 26 (11%) 1.14 (19%) 1019 (16%) 0 ,,91* (36%) 
* Denotes etatisticallT significant value at 99% 1eYe1 of certaint,. 0 
In order to ore tully eruu :te th role of temperature on t he 
antimicrobial effects or ultrasound on B.. ubtllis Taro ni or, th experimental 
-
design vas expanded to include irradiations at )0 Co Table 2 81IEnrurtaes th 
results obtained: 
Table 20 .yerege counts (X 106) and % reduction in viable counts ( in p enthese&) 
. ot Bo subtil1s val"o rger spores exposed to ultrasonic irradiation (lh78 lie/sec) at)O ... 
Hours Distance tram Transducer & inches 
'oS So~ ~o> 100 
1 103S (S%) 1047 (0%) 1029 (9%) 1026 (11%) 
2 1037 (3%) 1025 (n%) 1040 (1$) 00 94* (33%) 
4 1020 (15%) 1016 (18%) 007S* (47%) 0092* ()S%) 
8 00 72* (49%) Oo76it (46%) 0,,83*(41%) 00 67* (,2$> 
* Denotes statistically significant value at 99% level ot certaint7" 
Comparing th results obtained at 60 and )0 C presented here vi th 
those obtained at 50 C (Third Sem!.,Annual Report, 1 July 1965 go 31 December 1965) 
it appears that temperature has a direct effect on the properties of ultrasonic 
waves" Irradiations at 50 C and 60 C yielded 4 and 3 statistically significant 
values with respect to redu tions in viable countso At)O C, on the other hand" 
a total of 7 statistically significant values vas ·obtained. These results support 
the concept that t.he antimicrobial. activity of air=borne ult.rasonic waves i6 
emanced by lower temperatures" 
lene Oxide and Ultrasonic Irradiation on S ores of 
gezoo 
In the experiments being reported, propy'lene oxide was used in place 
of ethylene oxideo Initial efforts were directed toward finding an optimal 
cambinat.:ion of ultrasonic irradiation time and propylen oxide concentration for 
most effect1'f8 and r liable sterilizationo Accordingly, spores of Bo subtiUs 
-
var .. niger were exposed to arious levels of propylene oxide alone and propylene 
oxide plus ultrasonic irradiations for different periods of time o Numerous 
trials were made and the data given represent average values" 
Based on our experience with ethylene oxide, a concentration of 
250 mg/liter of propylene oxide vas selected for our initial experimentso 
Table 3 summarizes the data obtained with proP71e~ oxide (250 mg/llte ) alo 
and. with ultrasound at 60 c: 
· . 
Table 30 Average counts o£ spores at Bo subtills var. niger osed to 
propylene oxide (2$0 mg/U.ter) aIone, and in combination with 
ultrasound ()4,,8 kc/sec) at 60 C for varying periods of time 
Expo Noo 
1 
2 
Period of Exposure, 
minutes 
20 
40 
Irradiation distance Mil 1 inch 
Without Sound With Sound 
41,43S 
59 
To determine the role of temperature in the pro as of sterilisation 
nth propylen oxlde, experiments similar to those shown in Table 3 were 
planned using a temperature of 40 C instead of 60 C I) The reaul ts are shown in 
Table 40 
Table 40 Average counts of spores ot Bo subtil1s var .. niger exposed to 
propylene oxide (250 mg/lltW) &lone, and in COmbination with 
ultr aund (3408 kc/sec) t 40 C tor varying periods of timeo 
Expo Noo Period or Exposure, Without Sound With Sound 
minutes 
1 40 729,000 79»000 
2 60 312,000 319000 
3 80 268 S1 OOO 44,000 
Irradiat.ion d1stancct .. 1 inch 
C ng the dat.a in Tables 3 and 4 for the 40 minute exposure 
period, it appears that the lethal act.ion of propylene oxide is faywed by 
higher t peratUNs 0 This tentati,", conclusion also applies to the combination 
of prOWleD& o.rlde plus sound" 
To determine it an increase in propylene oxide concentration would 
8ignif'lcantly &tfect the data obtained thus far. the level of this gas was 
increased to Soo mg/l1ter and t.be temperature vas maintained at 40 Co Table 5 
illustrates the reaults obtainedo 
Table So Average counts of spores of Bo subtil1s var 0 nigsr exposed to 
propylene oxide (500 mg/litei=) alone» and in com illation with 
ultrasaund ()408 ke/see) at. 40 C tor varying periods or timeo 
1 
2 
3 
Period of Exposure, 
Jd.nutes 
40 
60 
80 
Without Sound 
833g 000 
4019500 
378 g 4OO 
With Sound 
Irradiation distance • 1 inch 
Ccapar1.ng the data in Tables 4 and 5, 1t is possible to gain SOlDO 
insight em the effect of increasing the propylene oxide concentration while 
maintaining all other variables constant., It may be seen that the effect of 
propylene oxide alone at e1 tiler 250 or 500 mg/l1 ter, respectively J did not 
result in appreciably lover countso Tbe addition of sound» however, reduced 
the viable count Moro effectively when coupled with a higher concentration of 
propylene oxideo An observation worthy of note here, vas the tact that much 
les8 fluctuation resulted between ind1Yidual determinat1onso 
To fmother reduce the degree of nuctuation noted betveen indiddual 
counto, a final. series of experiments were run in which the exposure tius 
were increased to a fun two hours 0 Propylem oxide levels of 2S0 and SOQag per 
liter, respectiftlyJ) vere plo,.ed and the temperature selected vas 40 Co 
, . 
r 
Where sound was employed.s the irradiaticm dist.ances vere varied betnen one 
and 9 oS inches.. Table 6 shows the results obtained using propylene axide in 
concent.ratioc or 2$0 mg/l1tera 
T bIe 6 .. Awrage counts of spores or B., subtilis val"o ~ exposed to 
propylene oxide (2$0 mg/ll ter) alOone, and 1n com nation with 
ultrasound (34 .. 8 lee/sec) at 40 C tor 120 r:d.nutes at Yarlous 
1.rradlation distances .. 
Without Sound With Sound, 
Distance tr Transducer (inches) 
1 .. 0 
82,296 2,82S 462 
Table 7 Illustrates the results obtained when the prOW1ene oxide 
left1 vas inal"eased to $00 mg/llter .. 
Table 70 Awrage counts of spores of Bo subtil1s Yar. ~ exposed to 
propyle:oa oxide (SOO 1IIg/lltei=') titone, and in COiiiD1natioD with 
ultrasound ()4 .. 8 Ire/sec) at 40 C tor 120 minutes at Yarioua 
irradiation distances 0 
Without Sound Wlth Sound, 
Distance from Transducer (inches) 
2, 200 o 
The data in Tables 6 and 7 show that doubling the 1eft1 of p!'OpTlane 
oxide frca 250 to $00 mg/li ter reduced t.he viable count by ore than S~ 0 The 
d1 t.iOD of sound to 81 ther 1ew1 ot propylene oxide brought about ignit1cant 
deereaa in th count 0 Further, ultrasound coupled with the higher propylene 
oxide coneentratioD prOftd to be the ost &ttact! ft means ot sterilization 
accomplished thus taro The importance of the irradiation distance i8 well 
illustrated in both Tables 6 and 70 As vas the ease in previous work reported, 
r , 
• 
the closer the sample to the transducerJ) the greater the killo Total 
sterilization vas achieved. at a distan of 1 inch using propylene oxide 
(500 mg/liter) coupled with ultrasmmd <34 0 8 kc/sec) tor period of 120 minutes o 
IndiYid fiuctuations in the latter experiJllent were great17 reduced and it 
now appears that reliable conditions for achieving sterilization using propylene 
oxide and ultrasound in combination can be described. 
50 D1SCU8aiODo 
The work reported. here indicates the 1.Jllportance of considering 
ai.rc->borne ultrasonic 1rrad1ations as a potential '~001 for spec1.t1c sterilisation 
probl " The use ot ultrasound alone signiticantly reduces nable counts or 
!o 8ubti118 Yaro niger" Ot 1Dtere8t is the tact that the antibacterial. ettects 
of sound alone most evident at lover temperatures o In add1t101l$ the closer 
the specimen to the sound source, the greater Vi.ll be the killo 
The use of propylene oxide in our present experiments has permitted 
us to establish certain base lines tor achieving total sterUi tiona C ns1derat1oll 
must be gi ftm to the concentration ot propylene oxide employed, the period ot 
exposure~ and the irradiation distance when ultrasound is add 0 It has baeD shown 
that the cOIIlbiDatiao of propylene oxide and ultrasound resul.t.8 in more eftectift 
ldll than when 81 ther agent is ueed alODe" or importance in our present ettort 
1s the tact that reproducible results can DOlI be aeh1eved ore readilY'o 
60 Future Ertorto 
kper1ments employing propylene oxide in combination with ultrasound 
wUl conUnueo It 18 planned to further vary the propylene oxide levels employed 
as well as the exposure times 0 Similaril.y'. the effect ot temperature and the 
distance at which irradiations are made will also be evaluatedo 
As the above experiments are progressing, it is also planned to initiate 
a series of studies using the thermophilic orgauism ~il1us stearotharmophUua. 
It 1s expected that the use or a thermophile will be or nlue in ass.ping th 
role or temperature in the propylene oxide-w.t1'"asound exper1mentso The 1188 of 
!o atearotbel!1OPhilus w:Ul make it possible to compare the beha"fior or two 
different species in our sterilizing systemo 
70 Publication., 
A paper entitled. "Sterilizing effects or high intensit;r al.r=borne 
sonic aDd ultrasonic vawsn bas been acoapted far publicationo Fi preprints 
are enclosed vi th this report. as well as a cOP,1 of the letter of ceptance 
for publlcatlC1lo 


An Investigation of a Sono-chemical Approach in Sterilization Problems Fourth Semiannual Progress Report, 1 Jan. - 30 Jun. 1966

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