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eryland 21701 se Branch/

fEPARTMENT OF THE ARMYFort DetrickFrederick, Maryland

TEXT NOT REPRODUCIBLE

INVESTIGATION ON ARY'A i).,vmINFECTION WITH BETA-PROPRIOLACTCTT IN AEROSOL FORM

(These investigations were condLucted with the support of theGerman Research Association.)

Zent. Biol. Aerosol Forschung9: 1960, pp 179-194 by K.-H. Husmann

From the Hygiene InstituGr o* ",ainz University (Prof. Dr.H. Kliewe, Director).

The effectiveness and appl'[cab~lity of disinfection agents in aero-sol form are limited not only by "he multiplicity of ways in which patho-genic germs can be transmitted but tiso by the physical-chemical behaviorof the disinfecting vapors and aerosols (1, 7, 56). Disinfectants used inthe form of aerosols are therefor, generally used exclusively for area airdisinfection which, however constf.t-2.;s a valuable measure supplementingexposure prophylaxis (1, 2, 3, 7, 2E. 27, 3h-4o, 54, 57, 58). Aerosolsused nowadays can give us a more or 1. 'ss adequate disinfection of area(room) air but unfortunately it c;.n cL achieve an adequat infectioneffect along surfaces 5hree-dne,.csic¢nal as against two-dimensional dis-infection effecg. Only formalin aerosol does not follow this rule becauseboth the air and objects and surfaces can be disinfected as a result ofthe formaldehyde gas which is rel,!ase- when formalin is atomized (8, 59).This property is responsible for :_xtensive use fo formalin in the pastand in the present, that is, for pur:,oss of room spraying as part of athorough final disinfection of sick rooms in hospitals and elsewhere. Ofall of the other substances tested for their suitability in room or areaair disinfection (triethylene glycol, triethylene glycol-containing pre-parations, propylene glycol, preparations based on hexylresorcin, etc.)none offered the advantages inherent in formalin, that is, good solubilityin water, easy volatility, relati- .-.. rmlessness, and a good germ-killingeffect both in the air and along .tr. aces. Ethylene oxide gas, which hascome into use recently and which z -eployera primarily in other fields(41, 42) is likewise unsuitable f, r" - om or area spraying for a number ofreasons (50). Final decontaminat_:- .3ing formalin vapor or aerosol istoday prevented by a number of wev*-,' Ut; -reasons. The chief disadvantagesof the formalin method reside in 'h[ transportation of the equipment, inthe long period of time, that i s . ..ral hours, required to have theagents take effect, and in the su,.scjent neutralization with ammoniawbich again takes time. Final dkinL'ec-:ion with formalin vapor is considereda necessary evil particularly because o the overcrowding of hospitals andthe shortage of personnel. We thr-rcCorc do not need to explain any furtherwhy it is of the utmost impotanc: for us to discover more convenient methods

- - Best Available Copy

for reliable room disinfection.

The discovery of the germ-.i * .ng effect of beta-propiolactone (BPL)during the vapor phase by Hoffman anu Warshowsky (31) might therefore possi-bly constitute a valuable contrioution, to disinfection and sterilization in

general and to room disinfection in particular.

Beta-propiolactone (G3k02) hnas the structural formula GH2--CH2

0--c - Of

in concentrated form it is a colorlass stable liquid with the following py-sical properties:

Specific weight 1.149Boiling point 1550 QMalting point -33.40 CVapor pressure (250 C) 3.4 ,mm HgWater solubility (25o C) 37 Vol. %

In watery solutions BPL is unstable and very quickly is hydrolyzedinto nontoxic decomposition products; here the hydrolysis, measured in half-

life periods, is a function of 'the temperature. In concentrated gorm, BPLremains unchanged for several years If kept at a temperature of 4 C; at

higher temperatures (for instance, $9o C) it is polymerized within a few weeks.

It reacts quickly with hydroxyl-,amino-, carboxyl-, sulfhydryl-groups and

g phenols. Inorganic salts, acids, and bases catalyze the polymerization of

the liquid BPL or form ne reaction products with it. Details on the chemi-

cal properties of BPL have been publAhed by Gresham and associates, as well

as by Bartlett and associates (9, 10, 15-25). The virus-killing, bacteria-

killing, spore-killing, and fungicidal properties of BPL in watery solutions(4, 14, 28, 29, 30, 32, 43-48, 51, 52, 53, 55) make possible the successful

disinfection of plasma, transplint tissues, vaccines and other biological

material (Hartman, Kelly, Lo Grippo and associates). A report on the anti-

microbial effectiveness of a german 3?L preparation in vitro was made else-

where (Husmann).

So far, BPL has been used for disinfection only in watery solutions;

Hoffman and Warshowsky, however, were able to show -- in experiments on germ

carriers to which adhered pathogenic staphylococoi, respectively, spores of

Bacillus subtilis -- that the suhsta:ce is more effective than formaldehyde

after aerolization and that it tzakes affect much more quickly and has less

harmful accompanying effects. Enplo- ing similar experimental procedures,

Dawson and associates (11, 12) achieved corresponding results in VEE-, small-

pox, yellow fever, and psittacosis viruses and Rickettsia burneti.

In view of the importance of the prevention of the spread of micro-

organims through the air ("airborne infection") I conducted the first in-

vestigation of the antimicrobial property of BPL in aerosol form on micro-

organims in room air and I also tested its disinfectant effects along sur-

faces*

12 -

f&Cell.

I 1ethod T EXT NOT REPRODUCTBLEA. The investigation on sre <3sn 'ectint effect of BPL in room air

was conducted on the basis of a r,.t.r swges'ed by Albrecht (5) and reportedon elsewhere so that we need not ;:o :to a detailed description here. I willdiscuss only those points where dr':.: -tcI' roni this method. The disinfectanteffect of BPL aerosol was tested :t:a>-st artificial germ aerosols consistingof staphylococci (micrococcus pyo;er.:' vs var. albus) and Coliphagen-(T 3 ). Thegerm suspension (18 hours with pNy logical iaCl solution in a ratio of 1:10diluted bouillon culture, respect'vcly, its filtrate) and the disinfectant(304 aqueous solution) (the beta-i)ro.iolactone was obtained from the Dr. Th.Schuchardt Company in Munich) were atomiz~od by means of the centrifugal ato-mizer (8) and the quantity of the ger.s in the air was determined with thehelp of the so-called modified Moulto:. unit (6). In addition I determined thenumber of germs in the air also according to the sedimentation method usingsettling plates. During the experiment the temperature changed very lttleand the relative humidity in the test room, which had a volume of 35 m, waskept relatively constant by means of a Defensor air humidifier with hydrostat;the fluctuations were very small. The experiment took place in the followingmanner i

Minutes After Start of Experimrent Measures Taken

0 - 3 Atomization of germ suspension9 - 12 Settling plate A9 - 14 Moulton Unit A

after 15 Atomization of water (empty ex-periment) respectively BPL sol.ution

29 - 32 Settling plate B29 - 34 Moulton unit B44 - 47 Settling place C44 - 49 Moulton unit C59 - 62 Settling plate D59 - 64 Moulton unit D74 - 77 Settling plate E74 - 79 Moulton unit D

A quantity of 0.5 ml of the wanshin liquid (sterile tap water) was --concentrated and diluted in a rat.o o.1 1:10 and 1:100 -- inoculated in adouble deposit on nutrient agar plates and the germs (viruses) contained in1 liter of air was calculated on -e zasis of the colonies which grew after24-hour incubation. Of the phage-conLaining washing liquid I also spread0.1 ml together with 0.1 ml E.-coli-2ouillon in a similar manner on agarplates on which the phage holes could be counted on the next day. To preventany possible bactericidal aftereffect of BL in the washing water for thegerm determination equipment I adrded 0.5.. sodium thiosulfate in a parallelexperiment in order to deactivate the afrent. The evaluation of the experi-ment however did not reveal any difference with rospect to the deposits notinvolving NA-thiosulfate; this appears to have been caused by the extraordi-

)

TEXT NOT REPRODUCIBLEnarily rapid hydrolysis of BPL in acucous solutions. This is why I did nothave to conduct any further expeci-.c:-ts involving the addition of NA-thio-sulfate.

B. To deterine the disinfccting effect of BPL aerosol on surfaces,I applied, to the top of a 6 X 6 cm large unwaxed sterile linoleum plate,using sterile swabs, suspensions of 18-hour bouillon cultures of micrococcuspyogenes var. albus, 1.1icrococcus pyogenes var. aureus (SG 511), Escherichiacoli, as well as a culture filtrate with coliphagen-T3 and I exposed the in-fected substances for 15, 30, 45, 63, and 120 minutes to the action of a BPLaerosol (0.5 mg pure substance/l air). Untreated controls remained in sterilePetri dishes for the corresponding periods of time until further use was madeof them. To control the diginfectant effect, I made moist swab skimmings ofthe bacteria-infected treated an& untreated surfaces, after the periods oftime mentioned earlier; I transferrd these skimmings to agar plates and tu-bules containing nutrient bouillon; Lhcse skimmings were then checked forgerm growth after 24-hour incubation at a temperature of 370 C. The swabskimmings of the surfaces infected with T3 -phages were spread on the agarplates which were covered with 0.1 7r1 of a bouillon culture from the corres-ponding E. - coli strain.

Remults and Discussion

A. I. In experiment groups iI to IV and VI, VII (Table 1) I testedthe effect of PBL aerosols in vairjin,; agent concentrations for artificialgerm aerosols of staphylococci and coliphagens. During the pertinent emptyexperiments, I atomized sterile tao ater instead of the BPL solution. Therelative humidity in all experiments was rather constant and fluctuated onlyvery little around an average value of 70,", respectively, 50i* (experimentalgroup VII). Table I shows the data -or the series of experiments conductedon the problem of room air dis'.rfection, arranged by experimental groups Ito VII; every group of experiments consists of five, respectively, two indi-vidual experiments. The measurer~ent results Na, N , N , Nd, N as well asthe computed survival rates (Q Qc, Qd, Qe)* and the disintegration rates(Kb, Kc, Kd, Ke)** are shown in Tabl1 2.

N [b..,, " 100

Q~b 1b. c. d." Q~,.,,.] =N.

log N, - log N [b, . .]K b. d. 01

CI

The formation of the survival and disintegration ratios made it pos-sible to compare the measurement data also when the initial values were notidentical. I did not evaluate or reproduce the germ numbers determined withthe help of the sedimentation method because this method does not give uscomparable results and therefore doez. not seem suitable for a quantitativedetermination. The average survIval ratios from the empty experiments werecompared with those obtained in the disinfection experiments and the signi-

4i

I

TEXT NOT REPRODUCIBLEficance of their difference was Similarly, I compared two disinfec-tior experiments, each time, wit:: jing BPL concentration (Table 3). The

statistical analysis was perforn.d on the basis of equation (13)

(x') - iS(x) + S(x")- i'S(x') (Zommon dispersion of samples),

N + N'-2

1X, X' SIX, X'

Sn - +- (Standarc deviation of mean value difference),N N'

deviation T- ' * he poirt.: rent probability p was taken from thet -

probability table (13).

SERIS OF EXC. SCONDUC TED

Al (3) (4) 0 (4)Gruppc Versuche Tekcir Mafinahmi Feuchte 'Is 0C

St. Albus Vernhlung von sterilem Wasser 70 T 5 22 T 2(Lee(6) 1111 Ling ab .,,,,,. (7)

I S St. albus Vrerntblung von BPL (30'/.i;) 70T 5 22T 25 rin Iang (= ca. 0,44 mgR .in t .ibtanz/i Luft)(8)

III 5 St. albus VcrneI~ung von 11PL (30I/,ig) 70T 5 22 T 2I ,'h ' In ; ( " ca. 0,21 mgteit'u.bsTanzAl Lui,) (9)

IV 5 St. albus Verncilun von BPL (301ig) 70 T 5 22 T 215 w'c lang (- ca. 0,11 mgRcinsiib.anz I Luft)(.O)

V 2 Coliphagen Vcrnc'ung, von sierilem Wasser 70 T 5 22T 2

(er. (6) -,) 5 m i.ing (7)vetode) .

VI 2 Coliphagcn Vrn.bilung von DI1L (30hig) 70T 5 22 T 2(T,) 5 min ling (- ca. 0.5 mg

Remnu,,b-tanz/I Luft) (11)

VII 2 Coliphagen Vcrnmhlung von BPL (30'/.ig) 50 T 5 22 T 5(T,) 5 min lang (= ca. 0,5 mg

Reinwub~anz/1 Luft) (11) ____

Legend: 1--Group of experiments; 2-- Number of experiments; 3--Test germ;4-- Measures taken; 5--Relative hur di ty '; 6--(Empty experiments); 7--Atomi-zation of sterile water for 5 minutc:;; 8--Atomization of BPL (30%) for 5 min-utes (--about 0.44 mg pure substance/l air); 9--Atomization of BPL (30) for

S) I minute (--about 0.21 mg pure sabstince/l air); 10--Atomization of BPL (305)- for 15 seconds (--approximately 0.11 m pure substance/1 air); 11--Atomiza-

tion of BPL (30) for 5 minutes (--about 0.5 mg pure substance/i air).

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SVM"uu N. Nb Nj N4 N. Q, Q. Qd Q9 Kb K, K4 K,

i 2580 757 549 229 104 29..4 21,3 8,9 4,0 0,0266 0,0192 0,0210- 0,0215

2270 1063 1072 758 572 46,. 47,0 33,4 25,2 0,0165 0,0093 0,0095 0,0092

* 1485 587 344 263 160 39,5 23,2 17,7 10,8 0,0202 0,0181 0,0150 0,0149

1109 569 586 266 178 51,3 52,0 24,0 16,0 0,0145 0,0079 0,0124 0,0122

' 756 361 215 175 183 47, 33,5 23,1 24,2 0,0161 0,0156 0,0127 0,0095

I 1640 667 553 338 239 43,C 36,4 21,4 16,0 0,0188 0,0140 0,0141 0,0135

- 4258 25 16 32 0,53 0,37 0,75 0,1116 0,0692 0,0424

3556 13 12 27 0,38 0,35 0,76 0,1218 0,0762 0,0424

6219 45 33 36 0,72 0,53 0,57 0,1069 0,0650 0,0247

847 1 2 2 0.12 0,24 0,24 0,1464 0,0751 0,0525

3419 6 9 4 0,17 0,26 0,12 0,1377 0,0737 0,0586

t 3660 1 14 20 0.39 0,35 0,49 0,1249 0,0718 0,0441

III 1957 13 2 1 0,92 0,10 ,0,05 0,1018 0,0854 0,0658

240 2 2 4 0,07 0,07 0,14 0,1576 0,0901 0,0570

2456 31 1 4 1,26 0,04 0,16 0,0949 0,0969 0,0558

2205 114 25 22 5,17 1,13 1,0 * 0,0643 0,0556 0,0400

1839 63 IS 14 3,42" 0,82 0,76 0,0732 0,0598 0,0424

2 2259 46 9 9 2,17 0.43 0,42 0,0984 0,0776 0,0522

SYbols: N -- number of germs(viruzcs) per liter of air; ab,c,d,e - first,respectively, second, third, fout -, fifth measurement; Q - survival rate;

K - disintegration rate; x - mez rtlue.

Legerds I-Ezprimental group.

O)-

4 TEXT NOT REPRODUCIBLE

TABI.-, - ('cuntd)

• uen. N, Nb Ne Nd No Q1, Q Ql QS

IV 5740 1144 15 5 19,93 0,27 0,08 0,0350 0,0738 0,0612

3064 42 3 6 1,37 0,10 0,19 0,0932 0,0159 0,0542

695 47 12 4 6,75 2,02 0,57 0,0583 0,0504 0,0448

3655 41) 59 46 11,3 1,62 1,73 0,0473 0,0512 0,03#0

2320 244 92 is 8,67 3,26 0,63 00531 0,0425 0,0439

1 3196 371 36 16 9,6 1,45 0,64 0,0574 0,0401 0,0414

V 1220 400 354 310 194 32,s 29.0 25,4 15,9

1966 710 422 404 319 37,6 21,5 20.5 16,2

" 1593 565 38 357 257 35.2 25,3 23,0 16,1

VI 731 0 0 0 0 Oi 0,0 0,0 0,01928 0 0 0 0 0.0 0,0 0.0 0.0

t 2329 0 0 0 0 0,0 0,0 0,0 0,0

VII 2366 19 27 0 - 0,3 ,1 0,0 -

1010 34 12 2 0 3,1 I,1 0. 0

172I 20 1 1,95 ,1. 0.1

Iap n:(1) &xperimental oup.

SyIolsa N.-umbr of eras (vna:r o ) per liter of airl a. b, o, d, 9-. first,

disint tlcr, rato t - man alue.

FZSULTS OF STAT-T2 : A,.ALYSIS (t-test)Vtr ,,i,cnc 0, f ,VernudQ-ruppen S11 t P Sp, t p 51) t "

1- 11 3,842 11,0905 <0,001 6,175 5,83R0 <0,001 4,037 5,1795 <0,001I-ll 3,952 10,3314 <0,001 6,170 5,9298 <0,001 4,031 5,2046 <0,001I-IV 4,231 7,8940 <0,001 6,201 5,6361 <0,001 4,046 5,1309 <0001

1l-ll 0,2968 -,9973 <0,00t 0,2329 -0,3434 >0,05 0,2324 0,3012 >0,05II-IV 1,7374 -5,3010 <0,001 0,587,3 -1,8713 >0,05 0,3201 -0,4686 >0,05

III-IV 1,697 -4,3783 <0,01 0,6279 -1,6244 >0,05 ._0,3498 -0,6289 >0,05Legend: Groups-of experiments cor .;cd.Symbols: Qb, Qc, Qd-- survival rac3 in of initial value (20 respectively35 and 50 minutes after 1st meazri-"nt)

SD--standard deviation of mean value differencet --deviationp -- probability of ceviation.

2. In the evaluation of tho exporiments we must keep in mind thatthe number of living viruses of an artLft1ial virus aerosol will decreaseeven without treatment. In the eAity Wpriment, the average survival ratesfor Staph. albus 20, 35, 50, recpoctively, 65 minutes after the first measure-ment was 43,, 36.4, 21.4, respectivoly 165; the rate for Coliphagerswas 35.2,25.3, 23.0, respectively, 16.1,.

3. After atomization of O.4 - =G; BPL/l of room air the survival per-centages for the staphylococci were below 0.5,. Wen the room concentrationof BPL was about 0.21 mg/l, there wore 2.17? surviving after 20 minutes,and less than 0.5; after 35 and 50 ninutes. The corresponding survival ratesat 0.11 mg BPL/l of air amoumted to 9.6, 1.45, and 0.64%. The probability(p) that the difference in the oian ialues was accidental is always less than0.001 (Table 3). In this connecton: We rmst consider that the time of actionof the agent at the moment of th- 2nd measurement is not 20 minutes but 15minutes - actually it is less t'an 15 rzinutes -- because the effective con-centration of the disinfectant in te air could not have been achieved atthe start of atomization. It was i .,ossible v'o kill all of the staphylococcibeyond 99.% with BPL in the concont:ations used. In this connection wemight point out that the method ,sod for determining the concentration of BPLin room air -- through conversion from the determination of the volume of theBPL solution sprayed -- undoubtedi-.- .nvolves a by no means inconsiderablespread of error. Experiments aincd at a continuing photometric concentrationdetermination for BL in room air ha-e so fz.r not led to any satisfactory re-sults for methodological reasons so that I did not deem it advisable toreproduce these results here.

4. If we compare the disinfoction experiments with each other, we dofind a reliable difference ( p = <0.01 respectively< 0.001) of the survi-val rates at the time of the ist measurement after the atomization of BPL,although not during the further measurements.

(-.' -- 8 -

RESULTS OF STATP; .. : AIXALYSIS (t-test)Verplj,dbene Qhg,, ,Versu -i.truppen Sl) t P t P s) t P

1- H 3,842 11,0905 <0,001 6,175 5,8390 < 0,001 4,037 5,1795 <0,0011-ii 3,952 10,3314 <0,001 6.170 5,8298 <0,001 4,031 5,2046 <0,001I-IV 4,231 7,8940 <0001 6,201 5,6361 <0,001 4,046 5,1309 <0,00t

I1-I11 0,2968 -5,9973 <0,001 0,2329 -0,3434 >0,05 0,2324 0,3012 >0,05-IV 1,7374 -5,3010 <0,001 0,R7 -1,8713 >0.05 0,3201 -0,466 >0,05

|1t -V 1,697 -4,3783 <0,01 0,27' -1,6244 >0,05 0,3498 -0,6289 >0,05Legend: Gk'ups-df experiments co;rtred.Symbols: Qb, Qc, Qd-- survival ral..s in ', of initial value (20 respectively.35 and 50 minutes after 1st mca~ir.-ent)

SD-standard dviation of mean value difference

t --deviationp -- probability o deviation.

2. In the evaluation of the experiments we must keep in mind thatthe number of living viruses of an "zrtificial virus aerosol will decreaseeven without treatment. In the e-'ty experiment, the average survival ratesfor Staph. albus 20, 35, 50, rez-pec'ively, 65 minutes after the first measure-ment was 43, 36.4, 21.4, respectvo.:.y 16 ; the rate for Coliphagerswas 35.2,25.3, 23.0, respectively, 16.1-'.

3. After atomization of 0.41 ng 1PL/I of room air the survival per-centages for the staphylococci wero below 0.53. When the room concentrationof BPL was about 0.21 Mg/, there wore 2.1?i. surviving after 20 minutes,and less than 0.5 after 35 and 50 ninutes. The corresponding survival ratesat 0.11 mg BPL/l of air amounted to 9.6, 1.45, and 0.6l4. The probability(p) that the difference in the roan values was accidental is always less than0.001 (Table 3). In this connec-oa we ramst consider that the time of actionof the agent at the moment of the 2nd measurenent is not 20 minutes but 15minutes -- actually it is less than 15 minutes -- because the effective con.centration of the disinfectant in t;,, air could not have been achieved atthe start of atomization. It wao -.-,possible to kill all of the staphylococcibeyond 99.5 with BPL in the conentrations used. In this connection wemight point out that the method used for determining the concentration of BPLin room air -- through conversion :'from the determination of the volume of theBPL solution sprayed -- undoubtlr i nvolves a by no means inconsiderablespread of error. Experiments ai.:ed at a continuing photometric concentrationdetermination for BPL in room air :.-v so far not led to any satisfactory re-sults for methodological reasons so that I did not deem it advisable toreproduce these results here.

4. If we compare the disinfection experiments with each other, we dofind a reliable difference ( p = <.01 respectively< 0.001 ) of the survi-val rates at the time of the I st ne.-aurement after the atomization of BPL,although not during the further meaaairements.

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TEXT NOT REPRODUCIBLEtg0VALES AS EX"ESSION OF . - :LL::G ,FFECGT OF BPL A,,OSOL

((2)

t, . Ct, k K1, Signifikan hbnung ffir k-K bE 1/Ik) 5

11 0,44 9,0 3,96 O,11,11 0,1116

0.43 8,5 3,66 O.1176; 0.1218

0,45 9,5 4,28 0, 1 ; 0,1069

0,45 6,75 3.04 0,1.-t2 C,1464

0,44 7,0 3,08 0.1I,' 0,1377

1 0,44 8,15 3,60 0,i2;o 0,12490,01148 0,00871 > 0,90

111 0,24 9,75 2,34 0,12v 0,1018

0,23 6,5 1,50 0,15314 3,1576

0,17 10,5 1,79 0,052 0,0949

0,39 15,5 2,95 0,0645 0,0643

0,21 13,5 2,84 0,0741 0,0732

1 0,21 11115 o2,28 0,' 0 0,09840,02254 - 0,0177 > 0,90

IV 0,15 19,0 2,85 0. .,26 0,0350

0,14 11,5 1,61 0,CS,4') 0,0932

0.12 20,5 2,46 0,04S 0,0535

0,10 20,5 2,05 0,0484 0,0473

0.06 21,5 1.29 0.0465 0,0531

2 0,11 18,6 2,05 0,0567 0,0574 ,0,01240 - 0,05645 > 0,90

Legends l--Group of experiments; 2--BL concentration, mg pure substance/l air;3--Computation of significance wher k - .Symbols: t~o -- Time in minutes required to kill 90- of the germs (viruses);

C O -- Product of BL concentration in mg/liter of air and valuefor t

k -- Value for decay ratc obtained through conversion from equa-tion t9 0 = 1/::;

Kb -- Disintegration ratio at time of 2nd measurement, calculatedfrom

65 N.,-log Nbt

The fact that the survival r~tios become increasingly siniilar as theduration of the action of the agent increases could not very well be an indi.cation of the greater significance cV the tim e factor for the bactericidaleffect of the BPL aerosol (apart Vro.-: the -'act that this development is cer-tainly dependent on the concentraticn). instead, this appears to be ex-plained by the below-threshhold do.. n of the disinfectant, especially sinceit was impossible to kill all of the viruses 1001 (experimental groups II,IIZ, and IV). In future experimonts it will therefore be necessary to increasethe concentration.

5. In experimental groups VI and VII we checked the disinfectant

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TEXT NOT REPRODUCIBLE

effect of a BL aerosol, con.ta..-. .- gent/1 of air, for an artificialvirus aerosol of Colipha-ens u: : .:. Ccon used as model viruses in place ofpathogenic aninal viruses. As . - scu 'n Table 2, it was impossible toprove the presence of phages af'x..' _ first measurements following disinfec-tion, when the relative humidit: - hc air was 70'. At 50% relative humi-dity there wore very few viruses ch survived, that is, we never had morethan 3f surviving. The dependence c' the disinfectant effect on the humidityin the air will have to be subjcct. to further detailed study. There willnot be any need for a statistica. :.=iycis in this case and at this point.

(b) veme 6an von BPL

100' .t

, 40 -J .. ? ,U I

..

a'

0,01 20 5 50 65 rnn

(d) noch der t.Messung(a) Graphische Ermitdung cines t"-Wcrtes

Legend: a--Graphic determinatio. . a t9 o value; b-- atomization of BPL;c--Aof surviving viruses; d--after 3rst nasurement.

6. We can also comp4are the coffectiveness of disinfectant aerosolsif we determine the t9 values (Taolo -), that Ls to say, the time requiredto kill 90 of the given viruses. he value for o9 according to Phillips(49), corresponds to the reciproca. value of the aecay rate = -... This

can be determined very easily in 7:',:hic form (see illustration); we simplyplot the percentage of survival : . ainst the time in a coordinate sys-tem on sei-logarithmic paper; .n -u, cal cases the points plotted will liealong a straight line whose gradc-.t-' will be equal to k. in general we getsufficiently precise to values if, in addition to the initial virus count(survival rate--i 00,), we also na::o a further virus number determination af-ter disinfection. To the extent t*.: the points in the coordinate systemare not located along a straight 7 proceeded as described in order todetermine the values given in Table 4. The fact that this does not give usany major errors is also borne out by a comparison of the graphically de-

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A'ET NOT R

terined t0 valus ou.. co:....... o: dccay rates obtained from theequation to with thec di::'.. ratcs K computed according to

log N-log Nh (Table 4) w!:j .. >n vale:c differences are far outside

tthe range of significance (p = > C.!Z). Zf we want to be able to comparethe relative activities of various (. .sinfoction aerosols to each other, itis a good idea to dete..no -.,he &to values (Ct 9 0 -- concentration of disin-fecting aerosol in ,.g per lir -o: ... r.ult ioalie by the time in minut3s re-quired to kill 90.' of the viruso/. The product formed from C and t 9 0 inthis case (with the exception of c;;:;rirental group Ii) appears to be quiteconstant so that we can assume t'> -. ,re is a direct proportionality be-tween the concentration and the -.i:;'..::'ection effect. It will, however,take further experiments before we :.n make a final statement on this.

B. The results summarizo-. ,. Table 5 shows that the BPL aerosol wasable to kill staphylococci and J &a-es, adhering to the linoleum platelets,after 30 minutes while it was ahL t.o kill 3 . coli after 15 minutes. A simi-larly successful disinfection ws :x.. :orted by Hoffman and associates and byDawson and associates with regar 'X the effect of the BPL aerosol (partlyhowever involving higher conccntrat'.!ons) on ger- carriers consisting of linenand paper which had been infected th pathogenic staphylococci, Subtilisspores, VM yellow fever, smallpox, )sittacosis viruses, respectively, Rick-ettsiae.

According to results availabo to date it appears that BPL, in aerosolr form, would be a useful agent in t'.< decontamination of rooms and perhaps evenof entire buildings -- because of i- s good antinictobial effect even at lowtemperatures. It is of course not z substitute for ethylene oxide gas whichis known for its good germ-killing properties and penetrability even at lowair humidity and whose applicabil'-t is found primarily in other areas.

S5DISN FECTING EFFECT 07 ZPL AEROSOL ON SURFACES

Keirar() (2) min-Einwirkiet15 30 45 60 120

Staph. albus (V)evrsuc (+) 0 0 0 0(4) Kontrolle + + + + +

SG S,1 Versuc (+) 0 0 0 0Kontrolle + + + + +

E. coi" Versud 0 0 0 ,Kontrolle + + + +

Coliphagen (Ts) Versuch (+) 0 0 0 0Kontrolle + + + + +

Legend: 1--Germ species; 2--Action ir. minutes; 3--Experiment; 4--Control.Symbols: + Heavy growth (bacteria patch (turf) respectively countless

numbers of phage holes);

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TEXT NOT RELJJUCIBLE

S(r) }oeak row:th (a :: e- counted bacteria colonies, respectively,phage holes) ;

.. o growth.

Si4 ;e -LL achieves the . z o. effectiveness of formaldehyde gas afteraerclizat -n -- and since it excct *.:-dis [as in many ways -- we might havesome new ozibilities here for :.c-. cc-Lc e and more convenient room dis-infection. A comparison of the o-e: -ro-crties of formaldehyde with those of:e 3ML z. ,rosol also clearly brir;-.,z -; the superiority of the latter: the

vei-y annoyL.n:- persistence, the coni -ation along surfaces, the polymerizationinto para:orr-aldehyde and the requ-:: long period of ventilation after theanplication of formaldehyde are isa'vantages which we need not anticipate whenwe use BP' aerosol. Here it is er; Lnportant to note that BPL aerosol doesno- lc.ad to the corrosion of me tals and, according to past experience, doesnot a.tack other materials which arn aoually found in a room. We might fur-thermore mention that the agent is noL cobustible in concentrations whichmigh'. exist under normal atmspher'c conditions. We do not get a combusiblemixt.re until we have 2 vol in t-c a5.r. However, the vapor pressure of BPLis z;ch that, at 300 C, the concentricfon in the air along the saturationboundary is only about 0.6 vole so t.-.at there is no danger of fire at all.In addition this concentration i. ccn- derably higher than the one requiredfor disinfection.

So far we do not have much in:fo-ation on the toxicity of BPL aerosol.It becomes perceptible to the sense _" snell at about 0.05 mg/1l. A room con-centration of more than 0.1 mg/1 can:oz be tolcrated by man longer than 5 min-

Yi utes because of the irritation to tne tear ducts. Another disadvantage is thefact that best results with 3PL can be achioved only at a relatively high hu-midi-y. 3ut this shortcoming can be somewhat reduced since, in the productionof the BPL aerosol from an aqueous solutLo,, the humidity in the air can alsobe increased simultaneously.

It will take many investiation on the properties of BPL aerosol beforewe can arrive at a final judgement. According to past experience, however, weare justified in hoping that we can find a fully equivalent and even bettersubstitute for formalin in the form ofE Beta-propiolactone.

Sum mary

Thi--i-the first, reporton '.voeti 2ations de .ling with the disinfec-tant action of a Beta-propiolactone erosol on artificial germ aerosols ofstaphylococci and Coliphagens used a- nodcl viruses. in addition we-4eted-the disinfectant effect of the aeroscl alon; surfaces- 7"he* article discussesthe advantages and disadvantages of to aerolized substance, particularly incomparison to fornalin, as well as I suitability in room disinfection.

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(2) Albrecht, j.: Contr.outL._. c: --he D-i.n'ection of influenza VirusAerosols,i Zschr. Aerosol-" -. . (Journal of Aerosol Research), 7, 3:274; - 278 (1956).

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TEXT NOT REPRODUCIBLE

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(Author's address: Dr. .nod. Zarl-Heinz Husmann, nygiene Institute,University of Mainz, Langenbeckst.asse 1.)

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