Evaluation of a New Bactericidal Spray Effective Against Staphylococcus aureus ARIEL A. ANDERSEN Andersen Samplers & Consulting Service, Provo, Utah Received for publication 8 January 1963 ABSTRACT ANDERSEN, ARIEL A. (Andersen Samplers & Consulting Service, Provo, Utah). Evaluation of a new bactericidal spray effective against Staphylococcus aureus. Appl. Microbiol. 11:239-243. 1963.-A new germicidal spray consisting of an active ingredient, 2-chloro-4-phenylphenol, solvent, and propellant was evaluated against aerosols of Staphylococcus aureus and spores of Bacillus subtilis. The method of evaluation consisted of determining the decay rate of the bacterial aerosols in a small chamber, with and without the aerial germicide present. The method is unique in that the effect of very short exposures of airborne bacteria to aerial germicides can be measured accurately. By use of the method outlined, extremely potent aerial germicides may be evaluated against highly sensitive organisms. The 2-chloro-4-phenylphenol spray was found to be extremely effective against S. aureus but only moderately effective against spores of B. subtilis. Hospital-acquired communicable staphylococcal disease is recognized as a problem of increasing importance. The fact that patients discharged from the hospital may serve as potential spreaders (healthy carriers) of antibioticresistant strains to the community at large is of real concern to hospital and public health authorities (Communicable Disease Center, 1958). Greene (1961) has shown that antibiotic-resistant micrococci, as well as other microbes which are airborne, might easily and quickly move from room to room and floor to floor in hospitals, thereby contributing to the cross infection. Obviously, an effective aerial germicide, one that can be used in hospitals, is very much needed to combat the spread of Staphylococcus aureus and also other microbes. The basic problem in the evaluation of proposed aerial disinfectants is the absence of a common, acceptable testing procedure and method of expressing results (Kethley, Fincher, and Cown, 1956). Because of the many variables, the many types of microorganisms, and the lack of adequate controls, the evaluation of germicidal sprays under hospital conditions would be fraught with difficulties and, at best, the results would most likely be unreliable and confusing. Thus, it seemed necessary to develop a laboratory procedure for the evaluation of aerial germicides where adequate controls and precise measurement would be possible. The purpose of this paper is to report the evaluation of a new germicidal spray for airborne S. aureus, and to describe the method and apparatus used in the evaluation. MATERIALS AND METHODS The germicidal spray evaluated in this study consisted of an active ingredient, 2-chloro-4-phenylphenol, incorporated with solvent and propellant in a pressurized spray container. Preparations of the germicidal spray were supplied in which the 2-chloro-4-phenylphenol occurred in concentrations of 3.,.3, and.3 %. The culture of S. aureus used in this work was obtained 239 from a local hospital and was carried on nutrient agar. The effectiveness of the aerial germicide against airborne spores of Bacillus subtilis var. niger (globigii) was also determined by the procedure outlined below. The laboratory test method developed involves the use of a small chamber and other necessary accessory equipment. The method consists of generating an aerosol of bacteria into the small chamber and periodically sampling the air in the chamber to determine the initial concentration of bacteria and the subsequent normal decay rate; the procedure is then repeated with a measured amount of the germicide dispersed in the chamber. The difference between the normal decay rate and the decay rate in the presence of the germicide is a measure of germicidal activity. The chamber and sampling equipment used in these experiments were basically as shown in Fig. 1. The chamber consisted of a horizontal stainless-steel cylinder with hemispherical ends (over-all dimensions: approximately SYMBOLS RUBBER TUBE a PINCH CLAMP FILTER FIG. 1. SYRINGE _a![. CHAMBER CLA SPECIAL SYRINGE NEEDLE ATOMIZER VACUUM PUMP VACUUM NO. NO 2 Apparatus for the evaluation of aerial germicides. li ANDERSEN SAMPLER Downloaded from http://aem.asm.org/ on August 16, 218 by guest
24 ANDERSEN APPL. MICROBIOL. 12 by 24 in.), having a smooth inside surface and a volume of 35 liters. It perhaps approaches optimal shape and size for such studies, because ideal circulation of air seems to be achieved by a slight temperature difference on the two sides of the chamber, and complete dispersion of aerosols within it is rapid. With no obstruction to interfere with circulation and no fans to remove particles by impaction, the physical decay rate is reduced to a minimum. In one end of the chamber, a syringe atomizer was located which was used both to generate the bacterial aerosol and to disperse the germicide undergoing the test. In the other end of the chamber, a sampling tube was mounted which consisted of a stainless-steel tube (inner diameter: %.2 in.) protruding 8 in. into the interior of the chamber. A double T in this end of the chamber made it also possible to connect to a vacuum gauge and an outlet through which the chamber could be evacuated or air permitted to enter. The syringe atomizer mentioned above permitted measured quantities of the bacterial suspension and the germicide, one after the other, to be very finely dispersed directly into the center of the chamber. It consisted of two syringe needles (3 and 18 gauge), one soldered inside the other with an air inlet tube inserted in the side of the larger needle. Aerosols of different particle sizes may be produced by using needles of different gauge, or by varying the air pressure or bacterial suspension feed rate. The germicidal spray liquid was cooled in a freezer, sprayed into a cold vial, and drawn into a syringe from which the material was aerosolized into the chamber. The sampling apparatus consisted of an Andersen (1958) sampler and petri plates, a vacuum pump, and a 1-ml syringe. The sampler was used as described by Andersen (1958), except as noted below. Because of its flow rate (1 ft3 per min), the sampler could not be used to sample directly from the small chamber. With the syringe, 1-ml samples were withdrawn from the chamber and immediately inserted into a special intake tube on the sampler; this intake tube permitted air to be drawn through the sampler at a rate of 1 ft3 per min while the 1-ml sample was fed into it. The syringe had been modified by cutting the tip off flush with the end of the syringe, enlarging the hole to.5 in., and cementing a one-hole no. 7 rubber stopper on the end of the syringe. Three 1.5-in. lengths of stainless-steel tubing (inner diameter: 5%2 in.) were inserted into the stopper flush with the end of the syringe: one for drawing the sample from the chamber, one for carrying the sample into the sampler, and one for airwashing the syringe and connections. The vacuum pump designed for operating the Andersen sampler at a rate of 1 ft3 per min was used to evacuate the chamber, and to operate the atomizer and the sampler. The procedure used in evaluating the aerial germicide in general was to determine the bacterial aerosol decay rate for the test culture under certain conditions, with and without the germicide present. The decay rate was also determined with the germicidal spray less the active ingredient. Decay rates for various concentrations of the germicide were determined. Referring to Fig. 1, the step by step procedure for the determination of the decay rate of the bacterial aerosol was as follows. (i) With valves 1 and 2 set as in Fig. 1, the pump was operated until the vacuum gauge read 1 in. of Hg; valve 2 was turned counterclockwise 9, and the rubber tubing was disconnected from the pump intake. (ii) Valve 1 was turned 9 counterclockwise, the pump was turned on, a stop watch was started, and immediately a syringe, containing 1 ml of the test culture suspension (approximately 2 X 16 cells), was inserted into the atomizer. As soon as the suspension was aerosolized, valve 1 was turned 9 clockwise, and the pump was turned off. (iii) Valve 2 was turned 9 clockwise, allowing filtered air to flow into the chamber to atmospheric pressure. Valve 2 was then turned counterclockwise 9, and the tubing was connected to the pump intake. (iv) At 4 min, a 1-ml sample was withdrawn from the chamber with the syringe, the pump was turned on, and the sample was expelled into the intake tube of the operating sampler, followed by 1 ml of air wash. After 1 sec, the pump was shut off, and the plates were removed from the sampler. The withdrawal of samples from the chamber, expelling the sample into the sampler, and the air wash were accomplished by proper manipulation of the syringe and the three pinch clamps. (v) At 6, 14, 34, and 64 min, additional samples were likewise taken. (vi) The plates were incubated at 35 to 37 C for 2 days and counted by the positive-hole or microscope method (Andersen, 1958). The same procedure was followed when determining the effect of the germicide on the bacterial decay rate, except that prior to step iii a measured quantity of the germicide was aerosolized into the chamber after 3.5 to 4. min, and step iv was omitted. The samples thus taken were for exposures to the aerial germicide of 2, 1, 3, and 6 min. To rid the chamber of residual effect of the germicide and prevent interference with succeeding experiments, the chamber was washed twice with isopropyl alcohol, then washed with warm Tide solution, thoroughly rinsed with water, drained, and aerated for 1 hr with a vacuum cleaner. To make sure that the germicide was wielding its effect on the bacteria in the chamber rather than in the petri plates, two identical bacterial aerosol samples were generated, one after the other, and drawn through an equilibrating tube (6 ft long by 4 in. in diameter). These samples were each collected on a set of six petri plates in an Andersen sampler. One set was incubated directly to determine the number of viable staphylococcal particles in the samples. On the other set of exposed plates, an aerosol of the germicide, equivalent to from 35 to 35, times as much as in the chamber samples, was superimposed on the plates before they were removed from the sampler. Any particles of the germicide were impacted in the same identi- Downloaded from http://aem.asm.org/ on August 16, 218 by guest
VOL. 11) 1963 BACTERICIDAL SPRAY EFFECTIVE AGAINST S. AUREUS 241 cal deposit areas as the staphylococcal particles. This set of plates was then incubated, and counts were compared with the control set. RESULTS AND DIscussIoN In the first experiments, the full-strength germicidal spray was found to be so effective that even the shortest TABLE 1. Effect of germicidal spray on airborne Staphylococcus aureus and spores of Bacillus subtilis Treatment S. aureusa Control [no germicidal spray (GS)] 1% GS (2.85 mg).86 gg of active 1% GS (7.4 mg) or 2.14,ug of active 1% GS (28.5 mg) or 21.4,ug of active GS (36. mg) less active B. subtilisc Control (no GS) 1% GS (28.5 mg) or 8.6,ug of active 1% GS (28.5 mg) or 86 yg of active Full-strength GS (28.5 mg) or 86,ug of active per liter of chamber air Exposure time (min) 2 1 3 6 52,68 52,68b 52,68b 52,68b 49,24 45,43 2,88 1,54 38,58 36,53 47 22,324 2,94 2,7 15,9 14,3 (52,68b) (49,24) (47,365) (35,612) (33,748) 3,6 3, 6d 3,6d 3, 6d 29,81 28,3 29,66 28,44 19,1 12,17 8,96 6e 26,72 22,75 2,39 2 15,43 12,27 2,16 a Results expressed as number of airborne particles bearing staphylococci per liter. bbased on initial count of 52,68 per liter in control experiment. c Results expressed as number of airborne particles bearing spores of B. subtilis per liter. d Based on initial count of 3,6 per liter in control experiment. e Exposure time was 18 min. bursts from the spray can into the 35-liter chamber completely eliminated aerosols of staphylococci containing 27, particles per liter in less than 2 min. It was suspected that some of the germicide in particulate form might have been transferred with the bacterial sample to the petri plates, where it could have exerted germicidal activity. To determine whether the germicide exerted any activity in the plates, the procedure outlined above was followed. The total counts from the two sets of plates were 3,986 for the control and 4,162 for those on which the germicidal aerosol was superimposed. Despite the large amount of germicide, it appeared to exert no activity in the plates. Possible explanations for this are as follows. Either the aerosolized germicide quickly changed to gaseous form or vapor form consisting of particles too small to be impacted on the plates, or if germicidal particles were impacted on the plates they were insoluble in the aqueous agar medium and any escaping vapor, not being confined, would not build up to an effective concentration, at least against bacteria in contact with aqueous medium. Thus, the evidence from early experiments indicated that the full-strength germicidal spray had no effects on bacteria on agar plates but was so potent in the chamber that weaker preparations would be required to study minimal effective concentrations. 1 f 2 4 6 OIZERO PARTICLES AT 2 MINUTES TIME (MINUTES) FIG. 2. Effect of 2-chloro-4-phenylphenol germicidal spray on airborne Staphylococcus aureus. Downloaded from http://aem.asm.org/ on August 16, 218 by guest
242 ANDERSEN APPL. MICROBIOL. w I- bj cr w -J U 4 a. z p ril z Germicidal sprays 1 and 1 % of full strength were then prepared and supplied by the manufacturer. Data from experiments with these weaker solutions are listed in Table 1 and plotted in Fig. 2. F'or each experiment with the germicide, the initial count of the control was used as the zero time. This procedure was considered to give a more reliable initial count than would sampling from the chamber while under partial vacuum before the germicide was added. In the experiments with staphylococci, the decay rates in the two control experiments (without germicidal spray and with germicidal spray less the active constituent) were approximately the same. An amount of.1 ml of 1 % germicidal spray aerosolized into the 35-liter chamber (.86 ug of active per liter) showed a marked effect, and.25 ml of germicide in the chamber (2.14 Mug of active per liter) completely eliminated aerosols of 52, staphylococcal particles per liter in 2 min. Contrasting this minute amount of aerial germicide required (2.14,Ag per liter) and the short contact time (2 min) with the 2 to 4 mg of f-propiolactone per liter and 2- hr contact time recommended by Spiner and Hoffman (196), it is immediately obvious that Sanidril Germicidal 2 4 6 TIME (MINUTES) ZERO PARTICLES AT 6 MINUTES FIG. 3. Effect of 2-chloro-4-phenylphenol germicidal spray on airborne Bacillus subtilis spores. Spray is many times more potent than,b-propiolactone. To get a concentration of 2 to 4 mg per liter, Spiner and Hoffman (196) used 9 to 12 mg of f-propiolactone. This is even more impressive in light of the statement of Hoffman and Warshowsky (1958) that,b-propiolactone vapor is approximately 25 times more active as a vaporphase disinfectant than formaldehyde and 4, times more effective than ethylene oxide. Early in the work, it was found that there was a residual effect of the germicide in the chamber that interfered with the subsequent experiment. When the full-strength germicidal spray was used in a chamber experiment followed by 1 day or more without use and an aeration, no viable staphylococcal particles could be obtained from the chamber in the next experiment, even though no additional germicide was injected into the chamber. This necessitated adoption of the isopropyl alcohol cleaning procedure mentioned above. In Fig. 3, the data from B. subtilis var. niger strain Bg experiments are plotted. Compared with Staphylococcus the Bacillus spores are much less sensitive, in fact about 4 times less sensitive. Even so, the germicidal spray was effective against the spores and reduced 3, sporebearing particles per liter to in less than 1 hr. This small chamber and the accessory equipment described here gave a very satisfactory performance in the evaluation of the germicidal spray against airborne S. aureus and spores of B. subtilis. This apparatus, if used according to the procedure outlined, should prove equally as satisfactory for the study of other aerial disinfectants and other bacteria. The small chamber should prove particularly advantageous with highly sensitive organisms or potent germicides, or both, where it becomes necessary to determine the amount of killing during very short exposure times. The advantage of the small chamber is based on the fact that the germicide is dispersed almost instantly throughout the chamber containing a stabilized aerosol of bacteria, thereby making possible a sharp, precise starting time of germicidal activity throughout the chamber; this, in turn, makes it possible to get accurate, reliable measurements of killing even during the first 2 min of exposure. In many of the Staphylococcus experiments, the killing action was complete with 2-min exposures in the chamber and less than 1 min of additional time required to get the sample of exposed bacteria onto the nutrient medium. This would not be possible with larger chambers such as Darlow et al. (1958) used; neither would it be possible with a dynamic system such as that used by Kethley et al. (1956). The chamber and accessory equipment shown in Fig. 1, when used according to the procedure described here, are a bacteriologically closed system, safe and suitable for work with highly virulent pathogens. The apparatus can easily be set up in a bacteriological hood for additional protection. The 2-chloro-4-phenylphenol preparations evaluated in this study proved to be very effective against S. aureus, Downloaded from http://aem.asm.org/ on August 16, 218 by guest
VOL. 11, 1963 BACTERICIDAL SPRAY EFFECTIVE AGAINST S. AUREUS 243 the organism that has caused so much trouble in hospitals in the past decade or two. In areas of the hospital that could be closed for short periods, such as the operating rooms, it should be possible to eliminate or greatly reduce staphylococci with this aerial germicide. ACKNOWLEDGMENT This study was supported in part by Dumas Milner Corp., which also developed and supplied the germicidal spray designated by them as Sanidril Germicidal Spray. LITERATURE CITED ANDERSEN, A. A. 1958. A new sampler for the collection, sizing, and enumeration of viable airborne particles. J. Bacteriol. 76:471-484. COMMUNICABLE DISEASE CENTER. 1958. Selected materials on staphylococcal disease. U.S. Public Health Serv. Publ. No. 627. DARLOW, H. M., E.. POWELL, W. R. BALE, AND E. J. MORRIS. 1958. Observations on the bactericidal action of hexyl resorcinal aerosols. J. Hyg. 56:18-124. GREENE, V. W. 1961. Observations on the microbiology of the hospital environment. Chem. Specialties Mfrs. Assoc., Proc. Mid-Year Meeting 47:157-161. HOFFMAN, R. K., AND B. WARSHOWSKY. 1958. Beta-propiolactone vapor as a disinfectant. Appl. Microbiol. 6:358-362. KETHLEY, T. W., E. L. FINCHER, AND W. B. COWN. 1956. A system for the evaluation of aerial disinfectants. Appl. Microbiol. 4:237-243. SPINER, D. R., AND R. K. HOFFMAN. 196. Method for disinfecting large enclosures with 3-propiolactone vapor. Appl. Microbiol. 8:152-155. Downloaded from http://aem.asm.org/ on August 16, 218 by guest