Propylene oxide sterilization of media containing soil bacterial populations occurred after 11 hours exposure in a closed system. Population reaction to the sterilant was characterized by inhibition intervals in the sterilization process. These stages represent minimal survival rates of 70%, 40%, and 7% respectively. A sterilization threshold (less than 1% survivors) occurred between 8-10 hours. Homogenous populations of Bacillus subtilis showed survival values in the first inhibition interval until the 4th hour. Lethality was noted at the 5th hour of exposure and other stages observed in heterogeneous populations did not occur

Goss, Robert C.

Naylon, Micheal J.

English

University of Northern Iowa

Proceedings of the Iowa Academy of ScienceVolume 74 | Annual Issue Article 71967Propylene Oxide Sterilization of Media ContainingBacterial PopulationsMicheal J. NaylonRobert C. GossState College of IowaCopyright © Copyright 1967 by the Iowa Academy of Science, Inc.Follow this and additional works at: http://scholarworks.uni.edu/piasThis Research is brought to you for free and open access by UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academyof Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu.Recommended CitationNaylon, Micheal J. and Goss, Robert C. (1967) "Propylene Oxide Sterilization of Media Containing Bacterial Populations," Proceedingsof the Iowa Academy of Science: Vol. 74: No. 1 , Article 7.Available at: http://scholarworks.uni.edu/pias/vol74/iss1/720 IOWA ACADEMY OF SCIENCE [Vol. 74 Propylene Oxide Sterilization of Media Co·ntaining Bacterial Populations1 M1cHAEL J. NAYLON 2 AND RoBERT C. Goss 3 Abstract._ Propylene oxide s.teriliZation of media contain-ing soil bacterial populations occurred after 11 hours expo-sure in a closed system. Population ·reaction to the sterilant was characterized by inhibition· intervals in the steriliza-tion process. These stages represent minimal survival ral;es of 70%, 40%, and 7% respectively. A sterilization threshold (less than 1% survivors) occurred between 8-10 hours. Homogenous populations of Bacillus subtilis showed survival values in the first inhibition interval until the 4th hour. Lethality was noted at the 5th hour of exposure and other stages observed in he-terogeneous populations did not occur. Previous work has demonstrated that sterilization of microbi-ological media by exposure to propylene oxide occurs within 24 hours at ambient conditions and that the nutritive value of the media is not altered (1,4,6,7,8). The present study was under-taken to obtain information on population characteristics within a heterogeneous population versus a homogenous population (Bacillus subtilis) when exposed to propylene oxide in a closed system. PROCEDURE Heterogeneous populations of microorganisms were obtained by serial dilution ( 105 ) of soil samples. Nutrient agar was poured into sterile petri plates which had been previously inoculated with one ml aliquots. B. subtilis was used in parallel for a study of a homogeneous population of spore-forming bacteria. Twenty-four hour nutrient broth stock cultures were serially diluted 104 and 1 ml was used as the inoculum. After inoculation, the plates were placed in a closed system consisting of an inverted four-liter battery jar with 6 ml of pro-pylene oxide in a 25 x 70 mm glass vial. Glass plates were used for bases and the system made air-tight by coating the jar lip with petroleum jelly. All test series were conducted at ambient conditions. The in-oculated plates were exposed to propylene oxide for 1-6 hours ( B. subtilis) and 1-11 hours (soil population). Twenty plates were used for each treatment and colony counts made at 24, 48, and 72 hour intervals. Chamber controls consisted of a series of twenty plates each. Controls were terminated with the corres-ponding treatment interval. 1 Abstracted from . a thesis submitted iu partial fulfillment of the requirements for the MA degree, State College of Iowa, 1967. • Present address: Director of Research and Experimetation, Biological Services Cen-ter, Minneapolis, Minnesota. 3 Associate Professor of Biology, State College of Iowa, Cedar Falls, Iowa. 1Naylon and Goss: Propylene Oxide Sterilization of Media Containing Bacterial PopulPublished by UNI ScholarWorks, 19671967] PROPYLENE OXIDE STERILIZATION 21 DISCUSSION Analysis of surviving fractions in the heterogenous population slt6ws a straight line correlation (slope: 0.345) until the fourth hour. A straight line relationship (slope: 2.864) is reestablished between the fifth and ninth hours, with sterilization occurring at the eleven hour interval (Figure 1). This data is in opposition with that of Goss and Marr ( 1) which indicates a straight line relation from the second to the sixth hours on plates exposed to air contaminants. Figure I. 10 • z 0 6 u 4 a: u._ Cl z > > • a: 4 :'.) V> ."\ ~ ......... • ........ ......... 0 PO SURE TIME - HOURS --' 10 11 Surviving fractions of heterogeneous populations of soil bacteria exposed to propylene oxide in dosed systems. 2Proceedings of the Iowa Academy of Science, Vol. 74 [1967], No. 1, Art. 7http://scholarworks.uni.edu/pias/vol74/iss1/722 IOWA ACADEMY OF SCIENCE [Vol. 74 Step phenomena implies the existance of an underlying pop-ulation survival threshold. Plotting the treatment as a function Qf the control was used to compensate for population variables. Intervals which appear to represent stages where new population factors become operative may be delineated on an argument whose value remains constant under the conditions of the ex-periment (Figure 2). 30 INHIBITION I INHIBITION 2 ~ z "' l: ~ 20 4 .., "' ~~ z ::i 0 .3 u >-z 10 0 _, 0 INHIBITION 3 u z <! .., l: 10 30 50 70 MEAN COLONY COUNT - CONTROL Figure 2. Step phenomena occurring in the sterilization process when populations of soil bacteria are exposed to propylene oxide in closed systems. Data from the 72-hour counts can be interpreted as demonstra-ting several distinct chemical reactions of a lethal nature within the propylene oxide systems. These reaction stages may be sep-arated into inhibition 1, inhibition 2, inhibition 3, and steriliza-tion. An intermediate interval designated as the sterilization threshold occurred between hours 8 and 10. Conversion of the surviving fraction values to precentage sur-vival showed that Inhibition 1 was characterized by a minimum survival rate of 70% of the population. Data from the 3-hour inter-val indicated that about 40% of the population had recovered and was designated as Inhibition 2. Survival values of 7-20% were noted at Inhibition 3 (hours 4-T). The exposure intervals from 3Naylon and Goss: Propylene Oxide Sterilization of Media Containing Bacterial PopulPublished by UNI ScholarWorks, 19671967] PROPYLENE OXIDE STERILIZATION 23 8-10 hours exhibit surviving fractions of less than 1% and have been designated as representative of the sterilization threshold. Sterilization was interpreted as ocurring when no colonies de-veloped after 72 hours of incubation at ambient conditions fol-lowing removal from the propylene oxide system. Under the con-ditions of the experiment, this stage was reached between 10 and 11 hours in the chambers. Populations of soil bacteria ex-posed for 11 hours showed no growth when subcultured to nu-trient broth. This was interpreted as indicating that the propy-lene ocxide system was lethal to the bacteria and that the petri plates and their contents were sterile. 10 • • • .8 z ~ 6 >-u 4 Q'. u.. (.!) z -> -> Q'. .4 ::::> (/) 2 .3 5 6 EXPOS0RE TJME - HOURS ' Figure S. Surviving fractions of a homogenoous population of B·, robtilis exposed to prQpy]ene oxide in a closed system. 4Proceedings of the Iowa Academy of Science, Vol. 74 [1967], No. 1, Art. 7http://scholarworks.uni.edu/pias/vol74/iss1/724 IOWA ACADEMY OF SCIENCE [Vol. 74 The progressive lethality shows that the mortality reflects a lag phase near the end point. Jordan and Jacobs ( 2,3) described this lag phenomenon as a function of environmental conditions rela-tive to simultaneous multiplication-rapid death population dy-namics. Increased colony counts at 48 and 72 hours in the heterogen-eous population may prove to be a characteristic of species acti-vation which is reflected by the surviving fraction. Other research ( 5) demonstrates ( i) metabolic retardation at sublethal expo-sures to bactericides with recovery occurring after removal from the sterilant system, (ii) adaptation by induction may result in modified metabolic activities which show increased numbers of organisms, and (iii) cells may demonstrate .an increased. sensiti-vity at certain stages in their growth cycle. There was no detectable sensitivity of B. subtilis to propylene oxide for hours 1-4 (Figure 3). Sterilization occurred between the fourth and fifth hours. Nutrient broth subcultures incubated for 72 hours at 37°C and petri plates incubated 15 days after 5 and 6 hours of exposure showed no growth. This is interpreted as indicating that the mortality threshold lies between 4 and 5 hours and that the 5th hour interval represented a lethal exposure for this organism ( Figure 4) . ~ z ::> 0 u >-z 30 ~ 10 0 u z <! w :>: INHIBITION I 0---- •s-:.6-------------STER I LI ZAT ION 10 30 50 70 MEA~ COLONY COU~T • CONTROL Figure 4. Comparison of the survival of B. subtilis exposed to propylene oxide in c)Qsed systems to the interval phenomena found in heterogeneous popula-tions of soil bacteria. 5Naylon and Goss: Propylene Oxide Sterilization of Media Containing Bacterial PopulPublished by UNI ScholarWorks, 19671967] PROPYLENE OXIDE STERILIZATION 25 Comparison of the homogenous population to the heterogen-eous population data showed ( i) that coloni(:)s of B. subtilis can be expected to appear within 24 hours and will make no further contribution to the overall colony population trends, and (ii) the surviving fraction and threshold phenomena observed in figures 1 and 2 is a species characteristic. The sterilization lag phenomena noted in the heterogeneous studies was not demonstrated in the 4-5 hour interval for B. sub-tilis. It is speculated that the organism's sensitivity is greatest at this point and that the lag occurs over a matter of minutes. Gaseous sterilants and disinfectants exert their influence by first dissolving in the moisture surrounding he cell ( 5). Organ-isms exposed to propylene oxide may be protected from its le-thal effects by (i) reaction of the O'Kide with the chemical'envi-ronment, (ii) adsorption of the sterilant to organic material in the system, and (iii) formation of protective films around the cell. Literature Cited 1. Goss, R. C., and Marr, J. L. 1963. Media sterilization with propylene oxide. Proc. Iowa Acad. Sci. 70:125-129. 2. Jordan, R. C., and Jacobs, S. E. 1944. Studies in the dynamics of disin-fection. Jour. Hyg., Camb. 43: 275. 3. Jordan, R. C., and Jacobs, S. E. 1945. Studies in the dynamics of disin-fection. Jour. Hyg., Camb. 44:210. 4. Klarman, W. L., and Craig, J. 1960. Sterilization of agar media with propylene oxide. Phytopath. 50 ( 11 ) : 868. 5. Sykes, G. 1963. The phenomenon of bacterial survival. Jour. Appl. Bact. 26(3):287-294. 6. Thompson, H. C., and Gerdemann, J. W. 1962. An improved method for sterilization of agar agar media with propylene oxide. Phytopath. 52 ( 2) : 167-168. 7. Tymer, L. E. 1958. The effect of water on the partial setrilization of barley seed by propylene oxide and by heat. Phytopath. 48( 3): 177-168. 8. Watson, R. D., Carley, H. E., and Huber, D. M. 1966. Storage of cul-ture media for laboratory and field use. Phytopath. 56( 3) :352. 6Proceedings of the Iowa Academy of Science, Vol. 74 [1967], No. 1, Art. 7http://scholarworks.uni.edu/pias/vol74/iss1/7

Propylene Oxide Sterilization of Media Containing Bacterial Populations

Name not available

https://scholarworks.uni.edu/cgi/viewcontent.cgi?article=1994&amp;context=pias

Propylene Oxide Sterilization of Media Containing Bacterial Populations

Abstract

Similar works

Full text

Available Versions

University of Northern Iowa

Name not available