A STUDY OF CATIOMC DESENFECTANTS WITH PARTICULAR REFERENCE TO LOW TEMPERATURE SAMTIZATION mkasis’ or the Degmecf M. S \‘V‘EIGAN STAEE CL LL 335 2331i 8.” w th‘ui 1947 THB$| L“ v-.'—.l-c-Vv This is to certify that the Q sways thesishentitigl' ' WM 9mm WWW JG- CBC-w Malta; 3 Maw presented bl] WA. W has been accepted towards fulfillment of the requirements for mrsfiw , .degree ianQL‘XQ "‘§M‘k M , 042nm, Maj r professor Date oqulk’l ,, ‘ 51-795 A Study of Cationic Disinfectants with Particular Reference to Low Temperature Sanitization by Elbert S. ggurchill A Thesis Submitted to The School of Graduate Studies of Michigan State College of Agriculture and Applied science in partial fulfillment of the requirements for the degree of Master of Science Department of Bacteriology and Public Health 1947 3HE$W§ AGKHOILEDGEIENT I wish to express my sincere appreciation to Dr.‘l; L. Isllmsnn hy those able assistance this thesis use made possible. 1 £5: 5:? ‘2) ‘ 3‘ :1 OUTLIEE Page I. Introduction A. The Use of Low Temperatures for Food Preservation ............................... l B. Significance of Low Temperatures on Bacterial Flora and Toxins ................. 2 C. The Need for Low Temperature Disinfection .... 5 D. Selection of Disinfectants for Refrigeration use 0.0... OOOOOOOOOOOOOOOOOOOO ... 000000000 O. 4 E. Cationic Disinfectants . ............... . ..... . 4 II. Experimental Procedure A. Aerosols.. ............... . ................... . 6 l. Glycol Aerosols ....... . .................. 6 2. Selection of Test Organism .............. . 7 5. The Use of Quaternary Compounds as Aerosols.. ......... . ................... 8 B. Wall Surface Disinfection .................... . 16 1. Selection of Test Organisms. ............ . 16 2. Hypochlorites Compared to Quaternary Compounds for Surface Disinfection..... l7 5. Field Tests .............................. 21 0. Effect of Low Temperature on Bactericidal Action of Quaternary Compounds.... ......... . 26 1. Selection of Test Organism. ............. . 26 2. Results of Low Temperatures in Bacteri- CidalAetionee0000000000Oeooeooeeoeoo-oo 27 D. Activity of Quaternary Ammonium Compounds on Bacterial Cells ........ . ..... .... ..... ... 28 1. Treatment of Bacterial Spores for Tests.. 28 2. Effect of Washing Treated Soores ........ . 28 III. IV. 5. Similar Tests on Vegetative Cells ..... .. E. Electrokinetic Activity of Bacterial Cells Treated with Quaternary Ammonium Compounds. 1. Preparation of Test Culture... ......... . 2. Method of Testing Electrokinetic Activity of Treated Culture.. ...... . ....... .... 5. Results. ..... .......... ................. Discussion ........ ......... ............ ......... A. Selection of Disinfectant. ......... .......... B. Residual Effect.. ....... . .......... . ........ . C. Aerosols...... ........... . ................. .. Conclusions ......... . .......................... .. Page 29 29 50 50 51 56 56 56 57 A STUDY OF CATIONIC DISINFECTANTS WITH PARTICULAR REFERENCE TO LOW TEMPERATURE SANITIZATION INTRODUCTION The processes of refrigeration have been develOped throughout the centuries from the practice of ancient peOples using the cool interiors of caves for storage of foods, to the modern cold storage plants, where thousands of tons of produce are stored. The housewife of earlier days had a Spring house in which she kept milk, butter, and eggs fresh, while the present day home has a refrigerator with compartments for fresh fruits and vegetables, milk and even a freezing compartment for frozen foods. 'The main purpose behind the use of cool temperatures, is to preserve foods from periods of plenty to a less plenti- ful season. This means the delaying of natural processes of enzymatic action, and also the slowing down of putrefaction by bacteria. This accomplished by retarding bacterial metabolism and the reduction of enzymatic activity with cold temperatures. Although low temperatures substantially decrease the rate of bacterial metabolism, there is no tem- perature which can definitely be called the end-point of all bacterial activity. While refrigeration has long been used to preserve foods, recent improvements in methods have made possible the freezing and refrigeration of many foodstuffs which formerly were either consumed fresh or preserved by other methods. The older methods of slow freezing resulted in the formation of large ice crystals within the cells of the preserved foods and the rupture of these individual cells. The newer methods of very rapid freezing produce such small crystals that the cells are morphologically unchanged and the flavor and appearance of these frozen foods are not materially altered. Since these foods will probably be treated by the housewife as fresh foods, it is essential that the microbial flora should not be materially different than that of fresh produce. With the advent of commercially frozen foods, eSpecially vegetables, the effect of low temperatures and freezing on bacteria and spores assumes a greater significance. James (1) found that repeated freezing and thawing either by the new rapid processes, or by ordinary slow freezing, had no effect on the spores or toxins of Clostridium botulinum. Wallace and Park (2) also reported that toxins of Clostrigigm thglingg types A and B were not destroyed after being frozen for a year at -l6°C. Although they saw no danger from imme - diate consumption of frozen foods, they believe that if foods containing Spores are allowed to stand at room temperatures for several days, they may become very dangerous. This is especially true of vegetables. It is readily seen that a food product must be free from bacterial contamination as possible before being stored for any length of time in a refrigerator. This is necessary to prevent deterioration of the product, and possible danger to the user at the end of the storage period. No method is better than cleanliness, in preparing and handling the material. The same thing would apply to the equipment, con- tainers, and the refrigerator itself. A good scrubbing of tables and containers should be standard procedure, at least at the end of each working day, and the refrigerator walls and floors should be thoroughly cleaned before storage of a new product. However, in addition to this it is well to use some type of disinfectant, which will finish the Job of sanitization and give complete protection to the food product from bacterial contamination. The selection of a disinfectant for use is important. Several factors must be considered before a chemical agent can be accepted for use in treatment of food storage rooms and equipment which may come in contact with foods. Toxicity is, perhaps, the most important because it is essential that the compound does not render the food unfit for consumption or irritating to the food handler. Taste and odor are also important because the food product may absorb foreign odors or tastes which would make them unacceptable to the trade. The compound must be active at low temperature and at all hu- midities. It is desirable that the compound not only kill bacteria at the time of application, but that it imparts a bacteriertatic activity to the surface treated for a long period of time. It should be easily applied and not be pro- hibitive in price. This thesis is concerned with the practical application of a new series of disinfectants and their use in the control of microbial flora in refrigeration and food preparation rooms. These new series of compounds are known specifically as quarternary ammonium salts and are classed as cationic compounds. Cationic compounds have been used for years as cleaners and detergents but it was not until 1955 that Domagk (3) showed that the detergent compound alkyl—dimethyl-benzyl- ammonium chloride was germicidally active. Due to the tre- mendous demand for improved detergents, more than a thousand detergents, interface modifiers, emulsifiers and wetting agents have been synthesized in recent years. Many of these compounds have germicidal activity and have been placed on the market as germicides. One of the aklyl-dimethyl-benzyl-ammonium chloride com— pounds, commercially named Zephiran, was the first of the cationic detergent compounds to be recommended as a bacteri- cide. Wright and Wilkinson (4) demonstrated its usefulness as a skin and injury antiseptic. Dunn (5) and White, Collins and Newman (6) also recommended the use of Zephiran as an antiseptic. In more recent times, the cationic detergents have been extensively investigated and there are now on the market con- siderable number of these compounds,wwhich are germicidal. They differ mainly in their structure, the length and compo- sition of the alkyl group, and the halide which is attached. They are alike in that they are strong electrolytes and ionize to a high degree. It is claimed that they have extremely high phenol coefficients, 500 for alkyl-dimethyl- benzyl-ammonium chloride, and no toxicity in a use dilution of 1-5000. Klarmann and Wright (7) have questioned the‘ phenol coefficients obtained for the compounds. They demon- strated that the results obtained by the FDA method of de- termining phenol coefficients does not give a true picture of the usefulness of cationic compounds. The quaternary ammonium compounds have been introduced into the field of disinfection with marked success, particular- ly the sanitization of restaurant dishes, beer glasses and dairy utensils. There is no information as yet as to how effective they would be at low temperatures and their applica— tion to the refrigeration industry. The best method of measuring the effectiveness of a com— pound to industrial use would be the actual use under field conditions and to set up laboratory demonstrations simulating field conditions as possible. The tests undertaken in this study were set up with this in mind. The thesis deals with the practical application of quaternary ammonium compounds to the field of low temperature sanitization. EXPERIMENTAL PROCEDURE Aerosols The typical refrigerator is a closed system as far as ventilation and the atmosphere within the box.are concerned. Smaller boxes depend on convection currents to circulate the air through a system of coils, while the greater majority of larger installations circulate the air by means of a blower behind the coil system, thus forcing the air to circulate throughout the box equally. The latter system gives a more constant temperature. Molds or bacteria can be eliminated by surface disinfection methods to be described later; how— ever, they could be replaced in time by any organisms in the air which is being circulated in the box, and by additional contamination from the outside whenever the box was Opened. Experiments were undertaken to determine the possible use of quaternary ammonium compounds as aerosols for the sterilization of the air in a walk-in cooler. If this could be accomplished, it would be possible to prevent recontami- nation of previously sanitized wall surfaces, and food pro- ducts, thus making possible longar storage with less spoilage. Robertson and associates (8) described the effectiveness of the glycols as aerosols, obtaining good results with all except the natural flora of the air, including spores. There are glycol aerosols on the market at the present time, and ‘Very simple and effective apparatus for diSpersing the glycol vapors into the air continuously. DeOne (9) introduced a rather complicated and costly apparatus by which air-borne bacteria could be treated with aerosols, where temperature and humidity are rigidly con- trolled. Haulton, Puck, and.Lemon (10) also have developed methods of extracting samples of air from a closed system in order to determine the bacterial content of a given volume of air. After several experiments by sampling from a small test box, it was decided that a larger test room was necessary to obtain practical conditions. A walk-in cooler of 512 cubic foot capacity was selected. A ten-inch blower circulated the air through the cooling coils, keeping the air in the box moving continuously. The temperature was maintained at 40-450F with a relative humidity of 87%. A.paint sprayer gun attached to an air supply delivering fifteen pounds per square inch pressure was used for spraying both the culture and the aerosol. The test organism used was Micrococcus caseolyticus, which is gram positive and thermoduric, selected because it is very resistant to desiccation. It will withstand pasteuri- zation temperatures, and was isolated from pasteurized milk. The small size of the organism was also considered, as a heavier organism would tend to fall more rapidly, while a Micrococcus approaches the colloidal dimensions more than any other gram positive bacteria. This organism averages one micron or less in diameter, while the dimension of a colloidal particle would begin at one half of a micron. (11) It was reasoned that in a box where the air was in motion, error due to fall of the bacterial cells of this size would be small, a fact which was proved in the experiments. In determining the number of bacteria in the air of the box, it was kept in mind that the products stored in the refrigerator would be either hanging up (such as meat) or piled on the floor. Thus it was necessary to demonstrate the difference in the number of viable organisms which would fall on the product in either location, in a treated or untreated atmOSphere. In order to carry this out, sterile Petri dishes were uncovered simultaneously at floor level and five feet above the floor, for sixty seconds at intervals of five, fifteen, thirty, forty-five and sixty minutes follow- ing the Spraying of the culture in the box. The counts at five and fifteen minutes which are recorded as innumerable, represented plates having a colony count in excess of 8,000. A total count was not recorded as any count from such a plate would be very inaccurate. Experiments were also run with the blower turned off to determine the distribution of the bacteria in the air during the period of examination. It was found that the distribu- tion at the various levels of testing was not uniform; fur— thermore, in a quiet atmosphere the bacteria would settle out more rapidly than in a moving air, introducing an addi- tional variable. The data in tables I through V contain the results of quaternary ammonium compounds used as aerosols. Table I shows the number of organisms introduced into the atmOSphere by spraying a twenty-four hour culture of Micrococcus caseolyticus into the test box, with blower running. Thirty minutes after spraying, there were over two hundred organisms at both the five foot level and on the floor; while at sixty minutes after spraying, the count was seventy and eighty-five organisms respectively. In Table II is presented the data giving the effect of introducing a water aerosol into the air followed by a Spray of organisms. This was done to show the effect of water _droplets in the possible precipitation of the bacteria from the air. Inasmuch as droplets were introduced when the disinfec— tant aerosol was added, it was necessary to know the effect of the drOplets. An examination of the data shows that some reduction was attained at all intervals of examination. After five minutes exposure, the bacterial population observed on the Petri dishes was approximately 500-600 at both times of examination, as contrasted to numbers in excess of 8,000 where no water aerosol was introduced. At the end of sixty minutes exposure, the count had been reduced from 70 and 85 (Table I) to 9 and 17 reSpectively. The total number was still high. TABLE I The distribution of bacteria in a moving, untreated air as measured by the number of bacteria falling on exposed plates in one minute. Time Following Spray Number of Bacteria Per Plate of Culture 5 Feet Above Floor Floor Level 5 minutes innumerable innumerable 15 minutes innumerable innumerable 30 minutes 211 224 45 minutes 118 100 60 minutes 70 85 512 cubic foot walk-in cooler. Temperature 55°F. Relative Humidity: 97 per cent. Blower in operation. 14 cc. of 24 hour culture g. caseolytious Sprayed into atmos- phere of box. TABLE II The distribution of bacteria in a moving, untreated air into which a water aerosol was added as measured by the number of bacteria falling on exposed plates in one minute. Time Following Spray Number of Bacteria Per Plate of Culture 5 Feet Above Floor Floor Level 5 minutes 508 762 15 minutes 952 . 252 50 minutes 91 105 45 minutes 59 26 60 minutes 9 l7 512 cubic foot walk—in cooler. Temperature 40°F. Relative humidity: 97 per cent. Blower in operation. 10 cc. of sterile water Sprayed into the box, followed by 14 cc. of 24 hour culture of M. caseolyticus fifteen minutes later. In the next series of tests, the quaternary ammonium compound was introduced in aerosol form. A 10 ml. dosage of a ten per cent solution was used. This dosage was used because in subsequent tests not reported in this thesis, lesser doses of a ten per cent solution and more dilute so- lutions were used without any appreciable reduction in bac- terial count as compared to the controls previously reported. In this series of tests, the quaternary ammonium compound was sprayed first and then, for the first trial, the organism was introduced five minutes later. The amount of culture was the same as reported in the controls. In the second trial, the organisms were introduced fifteen minutes later to see what effect the extended period of treatment would have on the bacteria introduced at a later period. The data are pre— sented in Table III. This represents a typical set of data selected from a number of experiments. It will be observed that with the introduction of the bacteria in either five or fifteen minutes after treatment, a marked reduction of bac- teria occurs. It will also be noted that a greater reduction occurred in the fifteen minute interval. This was found in all eXperiments. The most logical explanation of the more effective kill fifteen minutes after treatment, may be a better distribution of the disinfectant prior to the intro- duction of the organisms. The data show that the quaternary ammonium aerosol does effect kill of bacteria in the air. Because a better result was obtained fifteen minutes after treatment, experiments were made with still longer intervals to determine whether a still better result might be TABLE III Treatment ofggir with Ten Per Cent Quaternary Ammonium Chloride 512 cubic foot walk-in cooler. Temperature 45oF. Relative humidity: 85 per cent. Blower in Operation. 10 cc. of 10 per cent quaternary ammonium chloride Sprayed into box, followed by 14 cc. of twenty-four hour culture of E. caseolvticus. ORGANISMS INTRODUCED 5 MINUTES AFTER TREATMENT Time Following Spray Number of Bacteria Per Plate of Culture 5 Feet Above Floor Floor Level 5 minutes innumerable innumerable 15 minutes 17 4O 50 minutes 0 O 45 minutes 4 0 60 minutes 0 O L. ORGANISMS INTRODUCED 15 MINUTES AFTER TREATNENT Time Following spray Number of Bacteria Per Plate of Culture 5 Feet Above Floor Floor Level 5 minutes 0 0 15 minutes 0 O 50 minutes 1 0 60 minutes 1 O -14- TABLE Iv The Residual Capacity of a Quaternary Ammonium Chloride When Applied as an Aerosol Time Following Spray Number of Bacteria Per Plate of Culture 5 Feet Above Floor Floor Level 5 minutes 5,175 2,540 10 minutes 0 7 15 minutes 46 58 50 minutes 17 19 60 minutes 5 8 512 cubic foot walk-in cooler. Temperature 45°F. Relative humidity 87 per cent. Blower on. 10 cc. of 10 per cent quaternary ammonium chloride Sprayed into the box, followed in seventy-five minutes by 14 cc. of 24 hour culture of E. caseolyticus. obtained, as well as to determine residual capacity of the aerosol. Table IV presents the data for a typical run made seventy-five minutes after treatment. The reductions were not as great. Some reduction was still evident but the results approaches those of the controls. It is apparent from the data submitted that an aerosol treatment would have practical application in the control of air-borne bacteria. However, the quaternary ammonium com— pounds are irritating and cause coughing when inhaled in the concentrations used. In the use of such an aerosol it would be necessary to use a gas mask. The Use of anternary Ammonium Compounds for Wall Surface Disinfection It is common practice in refrigeration houses to thor- oughly clean and disinfect the storage rooms prior to use. After washing the floors and walls, a disinfectant is Sprayed on the walls to render the surfaces free of bacteria and molds. In many plants the walls are Sprayed with white wash. In some cases, a disinfectant is added to the white wash to aid in killing bacteria. The quaternary ammonium compounds have not been used for wall treatment as far as the writer knows, but from past experience with the compound it would seem that this compound would be useful. Accordingly, SXper— iments were conducted to determine their value for this purpose. To demonstrate means of application for the killing of bacteria on the walls of a refrigerator, the walk-in cooler used in the experiments on aerosols was used. Glass plates were placed on the walls for test purposes. Sampling was made from these plates, as the box had a wood surface that would be difficult to swab. The following test organisms were used: Staphylococcus aureus, Micrococcus caseolyticus, and Bacillus subtilis Spores. Staphylococcus aureus was se- lected because it is gram positive and has high resistance to most disinfectants; Micrococcus caseolyticus because it is gram positive, thermoduric, and highly resistant; and Bacillus subtilis Spores because they are extremely difficult to kill by any sanitization procedure. Staphylococcus aureus and Micrococcus caseolyticus were used in a twenty-four hour —"'"'———————_—_—"7 __ l7 _ broth culture diluted to 14 ml. Bacillus subtilis spores were grown ten days on a plain agar plate, suspended in saline, heat treated ten minutes at 80°C. to kill vegetative cells, centrifuged and resuspended in saline. Ten ml. of this treated material was used for Spraying on the surfaces. In the first experiment, the glass plates attached to the walls of the walk-in-cooler were Sprayed with the test organisms described above. Following immediately, the box was Sprayed with 10 ml. of a ten per cent solution of the quaternary compound. After exposure for one hour, the surfaces were Swabbed to determine the number of viable bacteria that survived the treatment. The data are presented in Table V. Staphylococcus aureus was completely destroyed. The Micrococcus caseolyticus and Bacillus subtilis spores were reduced to thirty and thirty- eight organisms per two inch square reSpectively. An examination of these data indicates that effective sanitization of clean surfaces can be obtained.by the applica- tion of quaternary compounds. Inasmuch as surfaces frequently become recontaminated, tests were made to determine residual effects of the quaternary compounds, as compared with the more universally used hypochlorite compounds. The hypochlorites have been used extensively for purifi— cation of water, disinfection of dishes and glasses in food establishments and the sanitization of dairy equipment, as well as many other uses. The action of these compounds de- pends upon the free chlorine available in a solution of cal- oium hypochlorite, Ca(OCl)2. One of the disadvantages of -18- these compounds is the fact that the available chlorine will be rapidly dissipated. The presence of organic matter will also tie up the chlorine, making it unavailable for action on bacteria present. For the purpose of disinfecting wall surfaces, it was anticipated that the hypochlorite would be effective only while the surface tO‘WhiCh it was applied was wet. Even then, the dissipation of the available chlorine should be \ quite rapid when Spread in a thin film on a wall surface. ' One wall surface of the walk-in-cooler was sprayed with a l-lOOO solution of a quaternary compound, while a Similar ' wall in the same cooler was Sprayed with a solution of hypo- chlorite containing ZOOppm available chlorine. Two hours after treatment the walls were Sprayed with a twenty—four hour broth culture of Micrococcus caseolyticus, diluted to 14 ml; Two inch square areas were swabbed at intervals of 5, 15,‘50, 45, 60 and 120 seconds after exposure, to deter- mine the number of surviving bacteria. As Shown in Table VI, reductions occurred on both quaternary and hypochlorite treated surfaces, although the reduction was much faster and greater in the second trial on the quaternary treated wall. This shows that there is considerable residual material left after use of the quaternary, as compared to the lack of residual effect evidenced in the case of the hypochlorite. To Show the effect of heavy impregnation of wall sur- faces over a long period of time, a smooth pine board was sprayed with a ten per cent solution of a quaternary com- Zpound. Examinations were made for continued effectiveness -19- TABLE V The effect of quaternary ammonium chloride on the bac- terial populations of refrigerator wall surfaces by spraying the compound into the air of the refrigerator. Box Sprayed with twenty-four hour culture, followed by spray of disinfectant. Glass surfaces of Walls tested. Number of Bacteria per Test Organism 2 Inch Square Glass Surface Staphylococcus aureus Control 210 Treated surface 1 hour after treatment 0 Micrococcus caseolyticus Control innumerable Treated surface 5 minutes after treatment 50 Bacillus subtilis Spores Control 4,800 Treated surface 5 minutes after treatment 58 512 cubic foot refrigerator with glass plates fastened on each wall; Sprayed with the test culture, followed by 10 ml. of ten percent solution of quaternary ammonium chloride. -29- TABLE v; Residual killing action of hypochlorite and quaternary ammonium chloride compounds on surfaces using Micrococcus caseolyticus as a test organism two hours after treatment. Sampling Time Bacteria Count of Surface Treated in Minutes After Spraying Chlorine 200 p.p.m. Quat. Amm. Chloride with Culture Trial 1 5 1,200 970 15 2,800 550 50 1,000 270 45 2,500 260 60 _ 1,700 240 120 1,200 290 Trial 2 5 . 15,000 1,500 15 15,000 660 50 5,700 560 45 2,800 0 60 14,000 0 120 ’ 15,000 0 Q of the compound at one, three and fourteen days, by Spraying a twenty—four hour culture of Micrococcus caseolyticus on a Section of the board and allowing it to remain five minutes. At the end of this time a two inch square surface was swabbed and plated to determine the number of viable organisms. The results presented in Table VII Show complete kill even four- teen days after treatment. In order to demonstrate ease of application of a quater- nary type of disinfectant, a 512 cubic foot box was sprayed with 10 ml. of ten per cent solution of quaternary ammonium compound. Four hours later the box was sprayed with a twenty- four hour culture of Micrococcus caseolyticus. These data are presented in Table VIII. There were no live organisms demonstrated on the wall surfaces five minutes after exposure. The above experiments were all laboratory tests, and because results obtained in the laboratory sometimes fail in actual usage, eXperimentS were conducted in a food prepara- tion and refrigeration plant. The worst possible conditions Were selected for test purposes. A frankfurter storage and packing room was used, one wall of which was continuously wet from condensation of moisture. The wall was heavily con- taminated with molds and bacteria.‘ Slime formation had resulted. The microbial population per four square inches ‘Was found to be twenty-eight million bacteria. Both painted and.unpainted surfaces were tested. The walls were washed imith trisodium phOSphate, rinsed with water and then swabbed \Nith l-lOOO solution of a quaternary ammonium compound. The :results obtained are recorded in Table IX. The unpainted TABLE VII Residual effect of cationic disinfectant on smooth wood surface. Half of smooth pine board treated with a ten per cent solution of disinfectant by swabbing. Other half left as control. Exposure time of organisms was five minutes. f t 1 Time of Testing Control_ Number 0 Bac er a per 2 In. Square Treated 1 day 122 0 3 days innumerable 0 14 days innumerable 0 concrete surface population dropped from 28,000,000. per two inch square to 580,000. by washing with trisodium phos- phate. Rinsing with water reduced the count to 55,000. (Swabbing with the disinfectant reduced the count to 100.) The surface had been effectively sanitized. In marked con— trast to these results were thoSe obtained on the same wall surface by swabbing with the disinfectant without previously cleaning the surface. Here the reduction was only from 28,000,000. to 11,000,000.; a reduction much less than that obtained by washing with trisodium phosphate. These data demonstrate the fact which has been repeatedly emphasized by sanitarians: that good cleaning must preceed sanitization. The quaternary ammonium compounds do not differ from other sanitizers in this reSpect. There is still no disinfectant that can effectively sanitize without the aid of cleaning first. TABLE VIII Effect of cationic disinfectant on bacteria pOpulation on wall surfaces when sprayed into air of refrigerator. Amount of disinfectant: 10 ml. ten per cent solution Box capacity: 512 cu. ft. Box Sprayed with disinfectant, followed four hours later with culture of Micrococcus caseolyticus. Number of Bacteria Time After Seeding per 2 In. Square Rough surface 5 min. after seeding 0 15 min. after seeding 0 Smooth surface 5 min. after seeding 0 15 min. after seeding 0 TABLE I_x Treatment of a badly contaminated wall in a frankfurter packing room. Number of Bacteria per 2 In. Square Before Treatment Painted surface 260,000. Unpainted concrete surface 28,000,000. Washed with trisodium phosphate solution Painted surface 250,000. Unpainted surface 580,000. LRinsed with tap water Painted surface 26,000. Unpainted surface 55,000. Swabbed with 1-1000 cationic disinfectant Painted surface 0 Unpainted surface 100 lgfeated without Cleaning 11,000,000. The Effect of Low Temperatures on Bactericidal Action of Quaternary Ammonium Compounds In the field of refrigeration it is neceSSary to have compounds which will act at low temperatures. Accordingly, studies were made with quaternary compounds to determine the effect of temperature on their germicidal or bacteriostatic action. The test organism was Spores of Bacillus subtilis prepared as follows: A seven day old growth of the organism on Petri dishes was harvested and suSpendee in saline. The suSpension was heated to 8000. for ten minutes to kill all vegetative cells, then centrifuged, and the Spores resuSpended in varying concentrations of a quaternary ammonium compound. The treated Spores were divided into three sets, each of which was held at different temperatures for fourteen days. The temperatures used were 0°, 55O,and 90°F. Selection of these temperatures would cover the ranges of frozen food, room temperature and.regular cold storage; all of which are of vital importance in storage of food products. On the first, fifth, seventh, tenth and fourteenth day, transplants were made to neutrient broth tubes which.were incubated forty-eight hours at 5700. to check for'the presence of viable spores. The results are given in Table X. An examination of these data Shows that although higher tem- peratures cause a greater activity of the compound on the organism, the difference in activity between 00 and 90°F. is not particularly significant. By special techniques to be Shown later, it was demonstrated that the action on Spores TABLE X Effect of low temperatures upon inactivation of Bacillus subtilis Spores by a quaternary ammonium chloride compound.“y Period of Exposure to Concentration Temperature Disinfectant in Days of Disinfectant of Treatment 1 5 7 10 14 1-100 00?. / f - - - 1-1000 f f f - _ 1—2ooo / f f f f 1-100 550?. - - - - - 1-1000 f - - - - 1-2000 2‘ 7‘ 7‘ 2‘ 7‘ 1-sooo / % f f % 1-1oo SOOF. - - - - - l-lOOO % - - - - 1-2000 / f f - f 1-sooo / x / x / * Inactivation or bacteriostatic action is reported because at end of five days exposure by Special laboratory technics, it was possible to demonstrate incomplete kill of l-lOO con- (Bentration at all temperatures. was not bactericidal, but bacteriostasis. However, the laboratory technique required to revive the Spores consisted of a vigorous washing of the cells, a procedure which would not be apt to occur in regular refrigeration procedure. Judging from these data, it would appear that the quaternary compounds would give satisfactory action at all temperatures. Activity of Quaternary Ammonium Compounds on Bacterial Cells It has been demonstrated that the quaternary compounds do an effective job of disinfection as far as vegetative bac- teria are concerned. However as will be noted at the bottom of Table X, the action of these compounds on bacterial Spores is bacteriostatic in nature rather than actual kill. In order to determine whether or not this would be detrimental to the usefulness of quaternaries, this question was inves- tigated further. A seven day old plate of Bacillus subtilis was washed with saline to suspend the Spores. The suSpenSion was heated ten minutes at 8000. to kill the vegetative cells, then centrifuged and the supernatant decanted. These cells were used for both tests described below. A portion of the centrifuged cells was suSpended in l—lOOO concentration of a quaternary. Ten minutes after eXposure, a loOpful was transferred to FDA broth tubes. No growth appeared in forty-eight hours incubation at 5700. The quaternary susoension of the Spores was incubated at 5000. for five days, at the end of which time it was centri- fuged and the supernatant decanted. The residue was resus- pended in sterile distilled water, centrifuged again, and the wash water decanted. The residue was then suSpended in saline, and this material used to seed FDA broth tubes, which upon incubation showed heavy growth. This would demonstrate that the action of quaternaries on spores was bacteriostatic and that by washing the cells free of the cationic, it is possible to revive them. In order to demonstrate that this bacteriostatic action is Specific for bacterial spores, but not for vegetative cells, a twenty-four hour broth culture of Escherichia coli was centrifuged, removing the cells which were then washed and resuspended in l-SOOO concentration of the same quater- nary. The same procedure was followed as above, resulting in no revival of the cells after freeing them of the quaternary. It can therefore be reasoned that in normal use of the quater— nary compounds, prOper concentrations will kill fie vegetative cells, and unless subjected to vigorous washing, the bacterial spores will be prevented from growing. Electrokinetic Activity of Bacterial Cells Treated with Quaternary Ammonium Compounds Dyar and Ordal (12) demonstrated that bacterial cells \Nhen treated with cetyl pyridinium chloride, which is a <=ationic, yield a general pattern of: (1) decrease in (Sharge on the cell, (2) reversal of charge, and (3) stabili- 23ation of charge, as the concentration of the cationic is Lincreased. Quaternary compounds are cationic in nature, carrying 51 strong positive charge on the molecule. Therefore, an experiment was undertaken to correlate the action on bacteri- al cells as described by Dyar and Ordal, with the reversible action reported previously, wherein the quaternary was re- moved by washing. Bacillus subtilis spores, heated at 80°C. for ten min- utes to kill vegetative cells were used for electrOphoretic determinations. One portion of centrifuged cells was sus- pended in sterile distilled water. Another sample was treated with the desired disinfectant for thirty minutes, centrifuged, the supernatant discarded, and the cells sus- pended in sterile distilled water. Still another portion was treatedxnith the disinfectant for thirty minutes, washed and centrifuged twiCe, then suspended in the sterile distilled water. Abramson and associates (13) claim that good results cannot be obtained in making electrophoretic determinations without using a buffer in the suSpension. However, the writer found the distilled water gave satisfactory results_ in this series of eXperiments. The method of making the electro-kinetic study was simi- lar to the method described by Falk, Tonney, White and Jensen (14). The suspended material was placed in a small églass cup with two platinum electrodes extending below the surface, and a cover glass placed over the top. The eye— g>iece of the microscOpe was fitted with a graduated disk, «and calibrated with.a 2 mm. objective to obtain the range of 'the graduations in microns. The electrical system consisted of a."B“ battery and varible resistance to give voltage of 20 f 0.04 volts and less than 0.1 milliamps, at the elec- -51- trodes. A reversible key was used in the system for revers- ing the poles instantaneously, in order to obtain the speed of migration of a single cell in both directions across the field, thus eliminating any "streaming" or "flow" error. It was also necessary to measure the depth of the glass chamber containing the suspension, and focus on a point equrdistant between the under surface of the cover slip, and the bottom of the chamber, because of the disturbance caused by the 'amraction of the cells to the glass near the surface and bottom. Measurements were made by determining in seconds the time necessary for one cell to travel from one end of the graduated field to the other and return. Five such fields were used, and an average taken. The nature of the charge On the cell was determined by the pole toward which the cells moved. Results of the electo-kinetic activity of the bacterial cells are shown in Tables Xi, XII and XIII. In all cases where quaternary ammonium chlorides were used, the charge on the cell has changed from negative to positive, but washing the cells resulted in the return to a negative charge. It is interesting to note in Table XII that Escherichia coli cells treated with quaternary ammonium chlorides did not re- turn to the negative charge when washed, although therexNas a decrease in the Speed. This was further demonstrated by showing that the Escherichia coli treated with a dilution of 1"2000 Quaternary ammonium chlorides will not grow when plaiced in nutrient media, even after washing the cells twice; Wfkereas Bacillus subtilis spores will grow upon being washed. By using 1—1000 HgClZ and 5 units per ml of penicillin, it was further demonstrated that the change of charge on the bacterial cell was a particular action of quaternary ammonium chlorides, since the charge did not change in either of these cases. TABLE XI Electrokinetic Activity of Bacillus Subtilis Spores Treated with Quaternary Ammonium Chloride Speed eXpressed in average microns per second traveled by a series of five separate organisms over a distance of 506 microns and return. milliamps of current. 20 f .4 volts; less than 0.1 Speed in Microns Treatment per Second Charge ' A. Untreated 19.58 - A. 1-500 QAC 30 minutes 52.9 f A. Washed and resuSpended 16.26 - B. Untreated 19.6 - B. l-lOOO QAC 50minutes 55.8 f B. Washed and resuSpended 15.1 - C. Untreated 19.56 - c. 1-1o,ooo QAC 50 minutes 13.3 74 C. Washed and resuSpended not determined — D. Untreated 19.58 - D. l-20u0 QAC ph adjusted to 10.5 50 minutes 25.7 f, D. Washed and resuspended 11.49 - TABLE XII Electrokinetic Activity of Escherichia Coli Treated with.anternary+§mmonium Chloride Speed eXpressed in average microns per second traveled by a series of five separate organisms over a distance of 506 microns and return. 20 f .4 volts; less than 0.1 milliamps of current. Speed in Microns C” ‘T'flo :— ireatment per Second narge A. Untreated 21.48 - A. 1-2000 QAC ph adjusted 9. 50 minutes 29.5 f A. Washed and resuspended* 12.6 f B. Untreated 21.49 — B. 1-500 QAC ph normal 50 minutes 25.5 f B. Washed and resuSpended* too slow f to count ‘ * Sample of resuspended material used to inoculate broth 'tllbes produced no growth in forty-eight hours. -55- TABLE XIII Electrokinetic Activity of Bacillus Subtilis Treated with.Mercuric Chloride and Penicillin Speed eXpressed in average microns per second traveled by a series of five separate organisms over a distance of 006 microns and return. 20 f .4 volts; less than 0.1 milliamps of current. Treatment Speed in microns per Second Charge A. Untreated A. l-lOOO HgC12 50 min.* A. Washed and resusoended* B. Untreated B. 5 u/ml. penicillin 50 minutes“ 19.58 7.97 14.84 19.58 20.44 * Samples of suspended material used to inoculate broth tLibes produced heavy growth in twenty—four hours. In selecting the proper method of sanitization for any purpose, it is essential that the conditions under which it will be used are thoroughly investigated. The two most im- portant-factors in refrigeration are the effectiveness of the compound at low temperatures, and its power to continue to give protection in high relative humidity over a period of time. Ease of application is a less important factor. It has been shown in this thesis that quaternary ammonium compounds are effective at low temperatures, although considerably retarded in their action. The slowing down is not important in refrigeration however, because: (1) the bacterial reproduction and metabolism are almost at a stand~ still (possibly explaining why the disinfection slows down), (2) the period of refrigeration usually involves holding pro- duce for a considerable length of time, long enough to allow the quaternary compound to act on the bacteria in the box. The experiments on residual effect and surface sanitiza- tion demonstrate the usefulness of these compounds for peri- odic cleaning of a refrigerator. Quaternary ammonium com- pounds could be applied easily to the clean wall surfaces ‘With.a spray gun, doing a good Job of sanitizing the wall aand remaining impregnated in the surface to continue holding clown bacterial growth. This would be most desirable in cases “filers the wall surfaces and cefling of a refrigerator or ‘3<>oler were of cement or stone, which is damp continuously. It was demonstrated that under conditions of this nature,by first doing a good Job of cleaning and rinsing, the quaternary‘will keep the contamination at a minimum. The compounds of this group react satisfactorily as aerosols showing excellent reduction of bacterial flora of the air, and remaining effective for considerable length of time after treatment. A correlation between Table IV and Tablelflll will show that by using 10 m1. of ten per cent solution of quaternary ammonium chloride, Sprayed into the atmOSphere of a 512 cubic foot refrigerator, it was possible to completely sanitize the wall surfaces, and at the same time almost entirely destroy the bacterial flora in the air. It is neceSSary to note at this time, that although the quaternary ammonium comoounds are claimed to be non-toxic and non-harmful to use in connection with food products, tests now being carried out to validate this claim are incom- plete. Until such time as the Federal Food and Drug Admin- istration approves their use in connection with foods, any wholesale use of the compounds Sprayed near or on foods might bring undesirable results to the food handler. The writer believes that the experiments on electro- liinetic activity of bacteria treated with quaternary ammon- iuntcompounds Show that bacterial Spores are inhibited to sauch a degree that they will not grow unless subjected to a Ifiather effective washing procedure, which would be difficult 't<> duplicate in any cold storage plant. It also Shows that 'tkle bacteriostatic and bactericidal action of quaternary ammonium chlorides is connected in some way to the charge 011 its molecules as well as the physical change it causes on the bacterial cell. This suggests a new field of investiga- tion which is out of the realm of this thesis. CONCLUSIONS l. Quaternary ammonium compounds are effective disin— fectants in the range of temperatures used in refrigeration. Their action is Slowed down at extremely cold temperatures, but this evidently is due to the Slowing down of cell metabolism. 2. Excellent results can be obtained by use of these compounds as disinfectants for wall, ceiling and floor surfaces. 5. The Quaternaries are good aerosols for refrigera- tors and cold storage rooms. By spraying 10 m1. of ten per cent quaternary ammonium compound into a 512 cubic foot box it was possible to sanitize not only the atmosphere, but wall surfaces as well. 4. The action of quaternary ammonium compounds on the bacterial cell is physical in nature, causing a reversal of the charge on the cell.‘ This charge is capable of being washed off bacteria in the Spore form; reviving the organism is not possible, however, in the case of vegetative cells. 5. The action of quaternary ammonium compounds on bac- ‘terial spores is bacteriostatic rather than bactericidal. LITERATURE CITED (1) James, Lawrence H.: Effects of Freezing on the Spores and Toxins of Clostridium Botulinum. Jour. Infect. Dis., 52, 257, (1955). (2) Wallace, G. 1., and Park, S. E.: Microbiology of Frozen Foods, Jour. Infect. Dis., 52, 150, (1955). (5) Domagh, G.: Eine Neue Klasse von Desinfektionsmitteln. Deut. Med. Washschr., 61, 929, (1955). (4) Wright, Louis T., and Robert S. Wilkinson.: Use of Zephirian in Injuries. Amer. Jour. Surg., XLIV, 5, (1959). (5) Dunn, C. G.: A Comparative Study of Some Antiseptics and Germicides with Special Reference to Alkyl all Methefll Benzyl Ammonium Chlorides. Amer. Jour. Surg.’ (August 1958). .JIIEE,M.3 (6) White, Charles S., J. Floyd Collins, and Howard Newman.: Clinical Use of Zephirian. Amer. Jour. Surg}]flgfl[; 607-609, (March 1958). (7) Klarmann, E. G., and Wright, E. 8.: An Inquiry into the Germicidal Performance of Quaternary Ammonium Disinfectants. Soap and Sanitary Chemicals, 22; 125, (1946). (8) Robertson, 0. H., Bigg, 0., Miller, B. F., and Baker, 2.: Sterilization of Air by Certain Glycols Employed as Aerosols. Science, 95; 215, (1941). (9) DeOne, K. B.: Effect of Temperature, Humidity, and Glycol Vapor on Viability of Air Borne Bacter- ia. Amer. Jour. Hyg.40; 259, (1944). (10) (11) (12) (15) (14) Maulton, 8., Puck, T. T., and Lemon, H. H.: An Apparatus for Determination of Bacterial Con- tent of Air. Science, 97; 51-52, (1945). Weiser, Harry Boyer: Collaidal Chemistry, 2nd Edition, John Wiley and Sons, Inc. (1941) Dyar, M. T. and Ordal, E. J.: Electrokinetic Studies on Bacterial Surfaces. Jour. Bact. 51, 149, (1946) Abramson, H. A., Moyer, L. 8., and Gorin, M. H.: Electrophoresis of Proteins. Reinhold Publish- ing Corp. New York.-(l942) Falk, I. 8., Tonney, F. 0., White, J. L. and Jensen, I. 8.: Electrical Determination of Virulence on Diphtheria Cultures. Amer. Jour. Pub. Health, XVII, 714, (1927) ROOM USE om. TEU (I WITH“)! 3 O 930 ER ll HWWITIIIWIWB l 1 3 9 64 MICIITWWIT 3 12