STUDIES ON EGG WASHING AND PRESERVATION mom for flu Degree of Ph. D. MICHIGAN STATE COLLEGE Tawfik Younis Saber I955 ffhES‘.“ This is to certify that the thesis entitled ( ~~-.". ‘ - I‘r‘" ?'-. ."'."‘ “' 1“"-‘ Lists-Ir: 03 . *- r A “ -q—. .3- . ,‘fi Into \. .ultul. presented by rfi ' 7’1. _ r ‘ . 3' .3 r- .fitJ- t (“0-1.8 m V has been accepted towards fulfillment of the requirements for 1‘?- ‘-‘ “~ n"-r\ --’ '3'} " "fir J. _] I 0 " 14 ' ,-' A‘,.-v.} _._._'“;_. degree m____ w Major professor 0-169 STUDIES ON EGG WASHING AND PRESERVATION BY TAWFIK YOUNIS SABET 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 DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1955 THESIS {T \ k.‘ ABSTRACT Present-day methods of egg washing have been more or less «I condemned because of increased susceptibility to microbiological in- vasion. This increased susceptibility of eggs to spoilage presents a major problem in cold storage, especially with regard to increasing egg production. It is the purpose, then, of this work to deve10p a method of egg washing which will properly clean the egg, prevent microbiolog- «ieal invasion, and at the same time retain the physiochemical prep- erties. During the course of this work, two artificial soils were developed as a means of determining the relative efficiency of var- ious surface ~active dete rgents. Through experimentation it—was determined-that- Pseudomonas geruginosa proved to be the most suitable organism available for this type of study. This was determined by three testing procedures de- Vised by the author. These testing methods may also have applica- tion in future work of this nature. As a consequence of experimentation concerning the original PPOblems, an oil-in-water emulsion was successfully prepared. This ii 35473 oil-in-water emulsion can be used to achieve a one-step operation for washing, sanitizing, and preserving eggs. By treating eggs with this A emulsion, their microbiological sterility and physiochemical proper- ties can be adequately preserved. It is hoped that this oil-in-water emulsion will find application and will be of value to the egg industry and its various ramifications. iii its 31cc '0 (115055 5mm Cher“: 3i v L .17. his (fl 0’ — f ACKNOWLEDGMENTS I extend my sincere gratitude to Professor W. L. Mallmann. His guidance, encouragement, and assistance during this study made its success possible. The writer wishes to express his appreciation to Professor L. E. Dawson and Professor J. A. Davidson for their patience in discussing problems encountered in this study. The assistance in supplying the eggs and taking over part of the investigation (physio- chemical studies of final emulsion) were also valuable. I am grateful to Professor H. J. Stafseth, Professor C. L. Bedford, and Professor C. A. Hoppert for their efforts in the cor- rection of this manuscript. I am indebted to Professor E. J. Miller for the preparation of the final emulsion. I also wish to extend my thanks to Mr. I. L. Dahljelm and his staff for their patience and promptness in supplying equipment and media. iv Physiochemical . Microbiological . Washing ...... Preservation . . De te rge nt Study Expe rimental Discussion . . TABLE OF CONTENTS 00000000000000000000000000000 ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo Te st Procedures ............................. Expe rimental Discussion . . Expe rimental Discussion . . OOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOO ooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooo Oil -Soluble Disinfectant ........................ Expe rimental ooooooooooooooooooooooooooooo 13 22 22 22 29 32 32 36 45 45 56 67 67 Exa rr M‘ ONC Page Percentage of Oil in Emulsion ................... 72 Experimental ............................. 72 Discussion ............................... 74 Examination of the Final Emulsion ................ 80 Microbiological Study ....................... 80 Experimental ........................... 80 Discussion ............................. 87 Physiochemical Study ....................... 94 Experimental ..... ' ...................... 94 Discussion ............................. 97 CONCLUSIONS ........................ I ........ 99 BIBLIOGRAPHY ............................... 101 vi 1947 Ah' wonh 5. There a creased due to t INT RODUC TION The egg loss due to breakage, blood spots, and Spoilage in 1947 amounted to $651 million. Inasmuch as the total egg crop was worth $2,500 million, the percentage loss would be 26 per cent (22). There are no reliable figures on the amount of spoilage due to in- creased bacterial invasion caused by washing of the eggs, but losses due to this cause are large. The washing of eggs has become an established practice so that any attempts to discourage the practice would be futile. There is a distinct need for prOper methods of washing. Although'extensive study has been made, no dependable method of washing has been developedthat-issatisfactory. The purpose of this study, therefore, is to investigate the possibility of using chem- ical sanitizers in egg washing and preservation as well as to find a simple procedure whereby the producer can wash, sanitize, and pre- serve eggs in one operation. its char Iihe 1 Virom: Rate c by ex: the :1 bTUaI an: REVIEW OF LITERATURE The egg's particular structure and the unequal distribution of its chemical constituents make it unstable. In addition, the interior of the egg is imperfectly protected and is, therefore, exposed to en- vironmental conditions. For these reasons, the egg is in a continual state of readjustment which takes place at a rate controlled largely by external factors. Physical disintegration within the egg is normal. In contrast, ‘the type of decomposition that results from microbial growth, in a broad sense, may be considered pathological. Eligiochemical ——-___————._ From the time the egg is laid, physiochemical changes start, and the rate and degree of these changes determine the keeping quality of the egg. The measurement of these changes, caused by the methods of handling the egg, can be used to assay the quality of the egg at the time of the assay and determine the ultimate storage life of the egg. The escape of moisture contained in the egg is the most im- Portant physiochemical process which gives rise to egg weight loss 2 and increased air cell height. Evaporation of water fromthe egg is a continuous process. It begins the moment the egg is laid, and it does not cease until the egg is completely dehydrated. At first, the weight loss is principally from the albumen, and later from the yolk as well. Loss of egg weight under constant environmental conditions is almost a linear relationship with respect to time (108). In terms of the egg's original weight, however, the daily loss diminishes throughout the holding period (128). The rate of weight loss is accelerated at higher temperatures, but can be lessened by maintenance of high relative humidities (108). Air movement tends to increase weight loss by the removal of higher humidities adjacent to the egg (127). Physical properties of the egg also affect the rate of egg weight loss; the less porous the shell or the larger the egg, the less the weight loss. Immediately after the egg is laid, its temperature is about 41°C (80), whereas the surrounding temperature is usually lower. When the egg cools, the contents shrink, forming the air cell. Thereafter, the air cell becomes larger as the egg‘s contents shrink due to loss of moisture. The size or height of the air cell can be ....———— -wf isfic is is prep: “e“ly la 53C) Sta revealed by candling. Since the United States standards for grading eggs take the height of the air cell into consideration, this character- istic is of significant value. The rate of air cell height increase is proportionate to the rate of egg weight loss. The albumen quality is one of the most important methods by which egg quality is determined. The process of albumen de- terioration, like all physio-chemical changes, may occur rapidly or slowly, depending upon environmental conditions. In the broken-out newly laid egg, the middle dense layer of albumen (the albuminous sac) stands up in an oval shape around the yolk and has a thick gelatinous consistency. As time passes, the albuminous sac de- creases in height at first rapidly and later slowly (66); it also loses its firmness, so that it spreads out thinly over a wide area when the egg is opened. Eventually, the dense albumen can no longer be dis- tinguished, the yolk becomes large and flattened, and if the egg has deteriorated sufficiently the vitelline membrane may rupture, and both albumen and yolk coalesce. These changes in albumen may be expressed numerically ac- cording to a set of photographic standards published by the United States Department of Agriculture (11). The thinning of albumen in- v12”. creases (i.e., the numerical number increases) as the egg becomes Older, at a rate determined by the holding temperature. n""v () IE Microbiolgical The interior of the newly laid egg is usually free from micro- organisms (123, 63, 64, 148, 156), chiefly because of the natural pro- tection provided by the egg's physical and chemical structure. Con- tamination of the contents of the egg rarely occurs before the egg is laid. It is well known that Salmonella pullorum, the causative agent of pullorum disease, may enter the yolk in the ovary. Mallmann and Moore (94) found that the incidence of infected eggs from diseased birds may run as high as 56 per cent. The bird becomes infected by way of the digestive tract (124), and the organism is tranSported to the ovary by the blood stream (100). Experiments have not shown conclusively that ovarian infection follows the feeding or inoculation of certain bacteria (100, 103), but many organisms apparently arrive at the ovary by the same avenues traveled by S. millorum. Among those found in both the ovary and the eggs are Salmonella anatum, 5’. Salmonella typhimurium, Salmonella fnteritidis (20), and Salmonella gallinarum (124). The transitory presence of harmless bacteria in the ovary is also likely (“117), for the latter tend to occur occasionally in eggs laid successively by an individual bird (99). It is also possible that eggs sometimes acquire bacteria as they pass through the oviduct. Lawson (78) stated that bacteria were often present in the oviduct. On the other hand, Rettger (123) main- tained that the oviduct is sterile. Haines and Moran (65) measured the diameter of the eggshell pores. They found that they may be as large as fifteen microns. This is large enough to permit bacterial penetration through the shell. The membranes that line the inside of the eggshell are composed of a matrix containing pores with an average diameter of one micron (157). Accordingly, bacterial penetration is mechanically possible. Wilm (152) was probably the first to demonstrate the ability of bacteria to enter the egg through the shell. By smearing the egg- shell with Vibrio comma or by immersing the egg into a liquid suspen- sion of the culture, he proved that the shell is pervious to bacteria. This has been repeatedly demonstrated with organisms such as Pseudo- monas Species (81, 126, 148, 88, 17), Escherichia coli (152, 159, 17), Proteus vulgaris (159), Micrococcuslyogenes var. aureus (117, 17), and several others (63, 103, 64, 65, 159, 17, 6). The most prevalent bacterial type found in eggs is the gram- negative short-rod group. Hadely and Caldwell (63) reported that 64 per cent of the bacterial flora of fresh eggs was very short rods. Haines (64) stated that only 32 per cent of the bacterial flora was nonspore-forming rods. By washing the eggs or holding them under cold-storage conditions, the gram-negative short rods constitute over- whelmingly the major portion of the bacterial flora (l, 87, 145, 105). It is remarkable that the interior of the egg rarely contains microorganisms, for bacteria and molds are usually present in great numbers on the surface of the eggshell. Rosser (133) reported that the surface of the shell of a fresh egg has an average of approxi- mately 1,600 mold spores and 70,000 bacteria. The maximum bac— terial number reported was 8 million (64). Molds, as well as bacteria, have been demonstrated in the interior of eggs (123, 93, 159), although they may not constitute more than 4 per cent of the total microbial flora (123). Among the various mold genera that have been found inside the egg are Penicillium, Mucor, and Aspergillus (93, 159). Experimental evidence suggests that enlargement of the pores by the mold hyphae may facilitate the entrance of bacteria (159). Eggs were smeared externally with a culture of Serratia marcescens, P. aeruginosa, P. vulgaris, or E. coli, and were kept four months at 18°C. The incidence of contamination of the contents was 70, 50, 30, and 20 per cent, respectively. When the inoculum consisted of the same organisms mixed with Aspergillus niger, the percentage of the internal contamination increased to 100, 100, 70, and 50 per cent, respectively . m2 CO I") The egg is exposed to contamination as soon as it is laid. As previously stated, the newly laid egg has a temperature of approxi- mately 41°C, whereas the outside temperature is lower. As the egg cools, bacteria and molds are possibly drawn in with the air that forms the air cell. Using the same principle of differential pres- sures, Haines and Moran (65) were able to force yeast cells through the shell pores. Regardless of all these factors, the internal contamination of fresh eggs is infrequent. The physical organization, and chemical constitution of the egg, together form a defensive system to combat invading organisms. The shell obviously provides the egg with its greatest physical protection. Normally, the eggshell pores are filled with organic sub- stances which hinder microbial penetration. It was believed that these organic substances, when dry, did not permit microorganisms to enter, and if they were dissolved or partially removed by abrasion, the pores were opened, and microbial invasion immediately became possible. By an ingenious experimentation Rievel (126) was able to dis- prove this hypothesis. He spread a dry inoculum (a loopful from a slant culture) on the shells of twenty-three eggs. Thirty-five per cent of these eggs contained the test organism after 9 to 12 days. The shell membranes, formed of interlacing fibers, may act as a filter for the removal of many organisms that might succeed in penetrating through the shell. It has also been shown that the shell membranes possess some bactericidal activity (148, 159). The most important chemical defense mechanism is offered by the albumen. Firstly, although the pH of the white of a fresh egg is about 7.6, it rapidly changes to about 9.5, and therefore be- comes unfavorable for bacterial growth. Secondly, the egg white contains lysozyme, which inhibits the growth of and may inactivate certain organisms (38). Thirdly, the natural proteins of the egg white are very resistant to bacterial attack. Bacterial decomposi- tion is only possible when the resistant albumen proteins have been already partially decomposed or when other simple nitrogen sources may be available to the organisms within the egg. Washing Pennington and Pierce (113) examined a large number of eggs in a New York. market. In one series of 258,496 dozen eggs they found 12.58 per cent were dirty, and in another series of 238,446 dozen eggs they found 13.4 per cent dirty eggs. These figures do not necessarily represent the situation on the farm at that time since r- ‘V :3. 10 some dirty eggs which may have been washed at the source were classified as clean, hence the number of dirty eggs may have actually been higher than reported. According to Brooks (15), Huttar (1928), in an investigation covering three years, found that, among the eggs produced by the Cornell poultry flock, the percentage of dirty eggs ranged from 9.8 per cent in July to 24.6 per cent in March, with an over-all average of 17.7 per cent for the year. This flock was maintained in the same manner comparable to that of the average commercial poultry man in the 1920's. Funk (41) studied the factors influencing the production of clean eggs. He found that by gathering eggs four times daily rather than at the end of each day, the total number of dirty eggs for the day's production was reduced by as much as 50 per cent; however, even under the most ideal conditions dirty eggs could not be entirely eliminated. At the present time, under field conditions, about 10 per cent of the eggs produced are dirty (43, 118, 74). The amount of soil carried on each dirty egg is quite vari- able. Soiled eggs are generally divided into three groups: "slightly dirty," "dirty," and "heavy dirty-" Dawson and Watts (29) classi- fied eggs more critically in six groups according to the amount of ll soil on the shell. For each group, they gave a numerical value; the clean eggs (no soil) are given the numerical value of l, and the extremely dirty eggs a numerical value of 6. Each year more eggs are being marketed on a graded basis. Soiled eggs, even though they may be of the highest interior quality (AA), generally are sold as C grade, and at 10 to 15 cents less per dozen than received for the highest grades (155). This fact leads to increased egg cleaning on the farm. The most generally recommended procedure for cleaning eggs has been to dry. clean the slightly dirty eggs and to wash only the badly soiled eggs. Funk (45) showed that wet cleaning is more ad- visable than dry cleaning. Winter 3531. (155) confirmed Funk's results. They used four different methods of dry cleaning. They found that all of them had the following undesirable features: 1. The abrasive removed the shell color as well as the dirt, in case of brown eggs. This left the eggs with noticeable scratches and light spots or bands on them. 2. The handling and rubbing action, necessary to remove stain and dirt by means of the abrasive, resulted in a higher percentage of cracked and broken ‘eggs than when wet methods were used. 3. Final traces of stain were not removed as effectively by dry methods of cleaning. 4. It required considerably longer time to clean by dry methods than by wet ones. 12 Some large producers now wash all the eggs gathered. Ac- cording to Black (7) and Pino (118), the eggs are washed in the wire egg baskets in which they are gathered. The baskets of eggs are immersed repeatedly in a hot (60°-70°C) detergent solution and then hosed with water of the same temperature. The procedure is said to save time and reduce breakage over that of sorting the dirty eggs and washing them separately. Several investigators (143, 84, 145, 86, 87) showed that washed eggs do not keep as well as corresponding clean or soiled untreated eggs. On the other hand, some investigators (62, 118, 106, 54, 101) found little or no harm from cleaning eggs. As a general rule most of the workers do not condemn wash- ing in itself, but claim that by washing, eggs are rendered more susceptible to microbial invasion and quality decline. In other words, eggs lose part or all of their defensive mechanism when washed. If eggs were provided with a similar or better mechanism during and after washing, washed eggs should keep at least as well if not better than the untreated eggs. 13 P re se rvation The egg has its maximum value as an article of food at the time it is laid. The start of change within the egg marks the be- ginning of its deterioration as a dietary commodity. The somewhat seasonal production, and the many phases of handling that the egg must endure before it reaches the consumer have long made its preservation desirable. Although many methods of preserving the intact egg have been attempted, none has yet been devised that can entirely prevent changes from taking place. Essentially, all that the preservation can accom- plish is to avert microbial invasion and to retard physiochemical deterioration for a reasonable length of time. These results could be achieved either by controlling the environment in which the egg is placed, or by treating the egg so that it is less easily affected by external conditions, or by both. Due to the apparent wide scope of the aforementioned points, only the treating of eggs will be dis- cussed here. It was noticed previously that, by treating the egg in such a fashion that the shell pores were completely or partially sealed, physiochemical deteriorations could be retarded. l4 Spamer (144) reported that, as early as 1807, the Dutch pre- served eggs by placing them in linseed oil for half a day, then drying them on racks. Such eggs were kept for four to five months. This process continued to be used until about 1914, when it was replaced by other processes, one of which was pickling in lime water, a proc- ess first used in Holland about 1875. Almy M. (3) recommended the use of mineral oils as a substitute for organic oils, their reason being that mineral oils were not subject to rancidity. Swenson and James (150) introduced their process in which oiling was done under vacuum. This method consists of evacuating the chamber in which eggs are immersed in oil, and then raising the eggs above the oil and releasing the vacuum in an atmosphere of carbon dioxide. McIntosh gt__a_l_. (89) determined the best time to oil-process eggs efficiently. He used physical and culinary methods as criteria. Eggs processed the day after gathering were superior to those proc- essed immediately after laying. His results were confirmed by Evans and Carver (34). Eggs oiled after periods longer than one day did not keep as well as treated day-old eggs (9). 15 Evans (33), being aware of an excessive use of oil in oiling eggs, studied the effect of diluting oils with solvents derived from the distillation of petroleum. Results were measured by changes in the albumen index and air cell diameter of eggs held for two weeks at 15.5° to 26.7°C. He concluded that oil diluted up to 50 per cent with a solvent was as effective as undiluted oil. He noted, however, that the danger of the flammable fumes from such solvents more than offset the advantages of using the solvents. A few years later, Stewart and Bose (147) studied the efficiency of different solvent-oil mixtures. They found that a 10 per cent oil and 90 per cent solvent (pentane) mixture was as effective as undiluted oil. Hearst and Hearst (68), in 1948, recommended the use of an emulsion for egg preservation. Their process, "stabilization of shell eggs," consists of using an oil—in-water emulsion of waxes having different melting points, to which is added a preservative (low molecular esters of parahydroxy benzoic acid). This emulsion is irradiated with ultraviolet light before using. The patentees Claim irradiation of the emulsion imparts unusual preservative power to the emulsion. Romanoff and Yushok (131) recommended the use of an emul- sion of stearic and lactic acids in mineral oil to treat eggs. Such 16 treatment would seal the pores and reduce the egg weight loss and pH change of albumen. The effect of this treatment on the microbial flora was not determined. Many other substances were used in sealing eggs. Funk (47) and Romanoff and Romanoff (130) presented excellent reviews on the use of these substances. Since most of the sealing substances, including oil, do not prevent microbial invasion, control of bacterial and fungal activities became of great importance. Generally eggs are treated during preservation to inactivate the contaminating microbes. After treat- ment, however, eggs would not be resistant against subsequent con— tamination. All the physical and most of the chemical methods of combating microorganisms give rise to such conditions. On the other hand, some chemical disinfectants exert their germicidal power over a long period of time. By applying such chemicals in treatment, eggs can be protected for a long period starting from the time of treatment until the germicidal activity of the disinfectant is exhausted or until the time when the eggs are consumed. Unless there is a great deal of contamination, the latter condition p re vail s . 17 The most universal physical method to combat microorgan- isms is heating. Although it is an old method, Funk (44), by intro- ducing his thermostabilization process, renewed the interest of many investigators. By this method fertile eggs were devitalized, the thick albumen was stabilized, and a pasteurization effect was obtained. These results were secured by immersing eggs in hot water or oil long enough to heat them throughout. Eggs were immersed for 10 minutes in oil held at 60°C. This process was modified later to 15 minutes at 54.4°C. Several reports have been published (9, 137, 49) confirming these results. Thermostabilization, however, has several disadvantages, Funk (47) stated: 1. The albumen of the thermostabilized eggs required more time for whipping and the volume of foam was reduced. 2. The incidence of stuck yolks was increased by the process. The adverse effect of pasteurization on egg white was con- firmed later by Goresline _e_t___a_.l. (60), Clinger _e_t___a__l.. (25), Salton _e_t___al_. (137), and Carlin and Foth (24). Scott and Vickery (138), however, were not able to confirm Funk's results concerning the effect of Pasteurization on the incidence of stuck yolk. l9 Mallmann and Davidson (91) recommended the incorporation of pentachorophenol in oil. They found that 0.25 per cent completely inhibited mold growth when incorporated in oil. They also used ortho- phenylphenol, but found it less effective. Miller e_t:___a_l_. (106) subjected Roccal and sodium pentachloro- phenol to a severe test. They broke black rot eggs in the washing water to increase the incidence of rot development. They found that sodium pentachlorophenol in concentrations up to 0.5 per cent and Roccal up to 1 per cent did not prevent rot formation. Gillespie SLEE- (54) claimed the spoilage of washed eggs is due to contamination from the egg-washing machines. By using 1 per cent calcium hypochlorite Spoilage was reduced due to inactiva- tion of rot-producing bacteria. Winter 9.331. (155) studied several egg-washing procedures. Eggs washed with warm (49°C) water containing a detergent sanitizer (Emulsept) were the only ones found to keep as well or better than the soiled untreated eggs. Forsythe e_t__a_tl. (40) recommended the use of a detergent sanitizer in washing eggs. They showed that the detergent Vel re- duced the bacterial load of the shell approximately 82.2 per cent. This reduction was due to the mechanical removal of bacteria from the surface of the egg shell. 18 Gorseline 31:31. (59) noted that egg breakage during thermo- stabilization depends on temperature; the higher the temperature, the greater the breakage, especially in day—old U. 8. Grade ”AA" eggs. Romanoff and Romanoff (129) tried a flash heat treatment using boiling water. By exposing eggs to such treatment for 5 sec- onds, a thin, protective film of coagulated albumen was found to adhere to the shell membrane. Feeny M. (36), although using temperature up to 1000°C, recommended the use of boiling water. Other physical methods of combating microbial contaminants such as X-ray radiations (9, 47) were found to be ineffective. Chemical disinfectants have been applied to eggs since egg washing became a practice. Funk (42) advocated the use of l per cent sodium hydroxide (NaOH). Due to the danger of handling and the question of effectiveness, this method was not successful. Penniston and Hedrick (114, 115, 116) recommended the use of a quaternary ammonium compound. They found fewer rots when they added the germicide to the wash water. Rosser e_t__al. (134) examined hydrogen peroxide, dimethylol urea, borax, and urea for their effect on molds. He found that dimethylol urea in a concentration of 2.8 per cent inhibited the molds completely. The rest of the compounds were less effective. 20 Conner et al. (26) washed eggs at 74°C before oiling. They incorporated 0.25 per cent pentachlorophenol in the oil as a germi- cide. By this method the number of bacteria in egg contents was kept low. n;-"‘r Winter (154) examined six different commercial preparations containing quaternary ammonium salts. The active ingredients in washing water varied from 100 to 1000 p.p.m. When dirty eggs were washed with such solutions, the percentage of rots increased to 15 to 30 per cent, compared to 4 per cent in the unwashed eggs. Funk e_t___a_l_. (49) studied the value of sanitizers in egg washing. When soiled eggs were washed in water containing 162 p.p.m. of the sanitizer, before oiling, they found 8.12 per. cent loss in treated eggs. By adding approximately 1800 p.p.m. of the sanitizer in oil, the per- centage of loss was decreased to 7.69 per cent. The untreated soiled eggs had only 2.99 per cent loss. Miller (104) studied the effect of some treatments on the inci- dence of gram-negative (spoilage) bacteria in significant numbers (more than 1,000 organisms per egg) in egg contents. When eggs were washed and rinsed in 2000 p.p.m. Roccal before being oiled, 23 per cent of the eggs contained spoilage bacteria. When 1 per cent NaOI-I was used as a disinfectant solution, 19 per cent were 21 found to contain Spoilage organisms, whereas only 8 per cent of the untreated eggs contained spoilage bacteria. In reviewing the work that has been done up to this time, the lack of adequate methods for washing and preserving eggs under field Conditions is indicated. It would seem that further investigations under carefully controlled laboratory conditions would yield a better evaluation of the various procedures available as well as new methods and reagents. Then once a method of egg washing, disinfecting, and preserving measures up to the requirements of the laboratory study study and the limitations of each process have been determined, it may then be used under field conditions with a better chance for suc- CESS. .- .4. —__~ - ...._~- _3 . . 'Lc EXPERIMENTAL PROCEDURES AND RESULTS In egg washing and oiling two procedures are generally fol- lowed. Either eggs are washed in a detergent solution, rinsed in a disinfectant, then oiled, or eggs are washed in a detergent-sanitizer and then oiled. After washing and before oiling egg shells should be dry. Any water droplets on the shell would form pockets which prevent oil from reaching the shell and sealing the pores. It was thought to combine all these steps (cleaning, disinfect- ing , and oiling) in one operation through the use of an oil-in-water emulsion containing a detergent and a disinfectant. To select each of the ingredients for such an emulsion the forthcoming experiments we re performed. Dete rge nt Study Expe rime ntal an naturally dirty eggs soil is generally a heterogeneous mix- ture of many substances with different physical and chemical charac- teriStiCS. The natural soil may be chicken manure, especially in open nest: laying houses, or it may be mud encountered frequently in 22 ’r...-~o v z-1_1 .1. U.'v‘t l’f" ' . \AOA (I) 23 rainy seasons, or it may be egg contents whenever methods of han- dling are inadequate, et cetera. It may also be a combination of two or more of these types; for example, the soil may be chicken feces mixed with feathers or egg meat covering a blood stain. The amount of soil on the naturally dirty eggs is variable. Sometimes only a small spot is found on the eggshell, and sometimes the entire sur- face is soiled. Dawson and Watts (29) classified dirty eggs into five groups according to the amount of dirt on the shell. Generally speak- ing , however, eggs are classified as ”slightly dirty," ”dirty,” and "heavy dirty." It should be understood that there is no clear-cut line se aratin these divisions, and the extent of the overla in is P g PP 8 left to human judgment. Although there has not been any work done on the incidence of dirt on each surface area of the eggshell, there is no reason to believe that one location would be more frequently soiled than another. This should not, however, subordinate the fact that individual eggs differ greatly according to location of soil on the Shell. For reasons mentioned above, it is a poor practice to use naturally dirty eggs in laboratory studies unless a large number are used to make up for these great variations. In such a case a standard soil would be desirable. tflfi‘ .. -—--;--‘*‘ '“-"* 'T‘ “H I _..--_.- W . i 24 It is obvious that the ideal standard soil should be a natural one; therefore, soils containing chicken manure alone or manure diluted with water to 25, 29, 33, 40, 50, and 67 per cent by weight we re tested. It was found that suspensions of 33 and 40 per cent gave the best results. More-concentrated soils gave a thick, lumpy, uneven film, whereas more-diluted suspensions were so light that complete coverage of the eggshell was never attained. Eggs were . w— dipped momentarily in 33 or 40 per cent soil su5pensions and dried either slowly, at room temperature for periods of 12, 24, and 36 hours, or quickly, at 65.5°C in an oven for 30, 60, and 90 minutes, or by a combination of both. It was found, however, that when these 1 eggs were passed twice through a G. L. F. washer using tap water at 55°C (in the pail), soil film was removed from all the treated eggs - This indicates that the soil was not attached strongly enough to warrant the use of a detergent. In chicken manure soil, the mucous material present acts as an adhesive agent. When it was diluted the mucous material was diluted G. L. F. egg washer, Model 210, is a rotary-type machine in WhiCh eggs are cleaned by the action of nylon brushes, wet with water Supplied from a pail placed on top of the machine. It takes 15 SecoIlds for an egg to run through the machine once. Water is used only once. 25 accordingly. The lack of sufficient concentration of the mucous ma- terial might help to explain why the films made with 33 and 40 per cent suspensions were easily removed. An attempt to develop an artificial soil using egg albumen was made. Egg-albumen powder and rhodamine B (a red fluorescent water-soluble dye) in concentrations of l and 0.025 per cent by weight, respectively, were mixed well in a Waring Blendor for two minutes, then Sprayed onto the eggs. Sprayed eggs were left at room temperature for five to six hours until dry, then heated at 65-5°C for 30 minutes. By washing soiled eggs with water as be- fore, the soil was not removed to a great extent. Anionic detergents are inactivated slowly in the presence of cations such as calcium and magnesium which constitutes the major Portion of the eggshell. For this reason it is not advisable to use anionic detergents in egg-washing solutions. Cationic detergents, on the other hand, lose some of their activity and/or interfere with the activity of several germicides (79). Since the germicide was not yet Chosen, several nonionic detergents and one cationic detergent were tested. Detergents are affected to varying degrees by water hardness; s Ome are susceptible while others are resistant. To prevent the 4.” W‘suh_u\ 26 interference of water hardness, 0.1 per cent sodium tripolyphosphate was dissolved in the water solution before the detergents were added. A series of egg-washing tests was made using eggs treated with artificial soil. Detergents which were used are presented in Table 1. Each detergent was used in a concentration of 0.1 per cent by weight in tap water at 55°C. Washing was done by the aid of a G. L. F. egg washer through which eggs were passed twice. The results of one of these tests are presented in Figures 1 to 3. As indicated by these figures, Sterox AJ and Sterox CD (nonionic deter- gents) were the most efficient in removing this artificial soil. Al- though this soil gave a measured success, it was not critical enough to show the difference in the activities between Sterox AJ and Sterox CD. A new approach was attempted to overcome the difficulty of attaching soil containing chicken manure to the eggshell. In this second artificial soil, albumen was incorporated as an adhesive agent. The soil containing 33 per cent chicken feces and one per cent albumen powder were mixed well in a Waring Blendor for three minutes. Eggs were immersed in the suspension momentarily and left to dry slowly for 15 hours at room temperature before heating at 655°C for 90 minutes. Using the same washing procedure, several tests were r“. *1: ,g 'y TABLE I DETERGENTS TESTED 27 Trade Name Chemical Name Manufacturer Alro amine 0 * Ethofat 242/25 Igepal CO - 633 Ne ut ronyx 600 Ste rox AJ Ste rox CD Synthetic s D 37 Synthetic 5 E80 Triton X100 Hete roc yclic te rtia ry amine Mono-fatty-and- resin-acid e ste rs of polyethylene glycols Alkyl phenoxy poloxy- ethylene ethanol Alkyl phenol polyglycol ether Alifalic nonionic poly- ethylene ester Polyethylene ester An ethylene oxide con- densate based on hydro- al siethyl al C ohol A rosen ethylene oxide condensate mixture Alkyl aryl polyethe r alcohol Alrose Chemical Company Armour and Com- pany Antara Chemical Division of Gen- eral Dye Stuff Co. Onyx Oil and Chem- ical Company Monsanto Chemical Company Monsanto Chemical Company He rc ule s Powde r Company He rcule s P owde r Company R ohm and Haas Company V ’4‘ Also amino 0 is the only cationic detergent; the rest are nonionic . M‘A 28 THE EFFECT OF VARIOUS DETERGENTS ON EGGS SPRAYED WITH ALBUMEN POWDER-RHODAMINE B SOIL v FAT 242’ZF ‘1 PAL (0. 633 29 performed to compare the activities of three nonionic detergent solu- tions: Sterox AJ, Sterox CD, and Triton X-100. Figures 4 and 5 show the results of a typical run where the three detergent solutions were compared with each other, and with unwashed eggs and with I‘ *1: 1 .3), eggs washed in water only. From these results it was noticed that Sterox CD was superior to the rest, followed closely by Sterox AJ. In the production of albumen powder its physiochemical prop- q! erties may have been changed. To determine if this was the case, 1 per cent albumen powder was substituted with 8.2 per cent fresh egg white (containing approximately 12.2 per cent total solids) in the second soil. By testing soil containing fresh egg white no par- ticular advantage was noticed in its use over the soil containing albumen powde r. Discus sion The selection of an artificial soil by which detergents could be evaluated according to their efficiency in cleaning eggs was found to be difficult. Natural soils such as chicken manure alone, or di- luted to various degrees with water, were tested. None of these soils was satisfactory; in the more highly concentrated soils the films obtained were uneven and lumpy, whereas in lower concentrations THE EFFECT OF VARIOUS DETERGENTS ON EGGS TREATED WITH CHICKEN MANURE SOIL Figure 4 Figure 5 30 31 they were too light to give an even coating. Although suspensions of 33 and 40 per cent gave even films, they were easily removed due to insufficient adhesive power. The addition of albumen either as a dried powder or as a fresh egg white to the 33 per cent suSpension chicken manure im- proved the adhesive power of the soil. The retention of the soil when washed was greater, thus allowing a wider range of soil re- moval for comparative purposes. A soil composed of 1 per cent albumen powder plus a dye showed some possibilities of being a good soil for the general screen- ing of detergents. This soil, beside being easy to prepare, gave more-consistent results in repeated experiments. With most of the commercial detergent mixtures presoaking is a must before an effective cleaning can be achieved (74). The mixtures which do not require presoaking need a long washing period. The longer the period of either soaking or washing the greater the chance for microorganisms to penetrate the shell (85), and thus a more prolonged activity is required of the bactericidal agent. Any contribution by which this period can be shortened has a value in reducing the extent of bacterial invasion. This increases the im- portance of the choice of detergents since they are the governing 32 factors in determining the length of time required for the effective washing period. When the albumen-dye soil was used, the two Sterox deter- gents showed the best activity. Both detergents are nonionic types. The reason for their superiority over the cationic and the other non- ionic detergents is not known. Test Procedures Expe rime ntal Although ”clean" eggs may look clean, they actually carry a large number of bacteria on the shell. According to Forsythe _e_t___al. (40), the clean eggshell has at least 10,000 microorganisms. For this reason it is very difficult to open the egg aseptically.without contamination by microorganisms. Early workers depended on disin- fectants to sterilize the eggshell before they opened it (111, 99). Due to incomplete understanding of chemical disinfection, investigators modified these procedures by flaming after disinfection. Mallmann and Moore (94), for example, submerged the eggs in 121000 HgClZ solution for 10 to 30 minutes. Then they placed them on small beakers, and while the eggs were still moist a sharp-pointed gas flame was applied to the shell covering the air sac. By this method 33 the shell was forcibly removed. Wolk still. (156) immersed eggs in HgClZ for 30 minutes, dipped them in 95 per cent ethanol, burned it off, and then carefully flamed them. They found, however, that this method was inadequate. Later investigators returned to chem- ical disinfectants without flaming. Bigland and Papas (6) washed eggs in 450 p.p.m. Roccal for one hour, followed by drying and painting with 1:1000 tincture of merthiolate. Connor _e_t__a_l_. (26) washed eggs in distilled water, followed by dipping in alcohol, drying, and then painting both ends with Churchill's iodine. In this study calcium hypochlorite solution, in a concentration of at least 1:1000 of active chlorine, was used to disinfect the egg- shell. Eggs were immersed in this solution for one to two minutes. A circle of approximately one inch in diameter was drilled with the aid of an electric hand drill on the blunt end of the egg. The shell section was removed with a sterile forceps and the egg contents were examined. When twelve 24-hour-old clean eggs were examined, as above, no bacteria were recovered from the egg contents. Four of the bacteria generally present in spoiled eggs were investigated. The speed by which each organism penetrates the egg was considered as a criterion for choosing the test organism. In testing the bacterial penetration into eggs, experiments using 24 or 36 was! -.- 34 eggs were carried out. All eggs were obtained from a White Leghorn flock at Michigan State College. Clean, white, sound-shell eggs were used when they were 24:6 hours old. Each batch was divided into four groups of six or nine eggs each. Each group was immersed into a 24—hour F.D.A. broth culture of one of the four test organ- isms (M.pyogenes var. aureus, E. cgli, P. vulgaris, and P. aeruginosa). All cultures were obtained from the stock cultures of the Department of Microbiology and Public Health, Michigan State College, with the l exception of P. aeruginosa, which was recently isolated from a spoiled egg. Cultures were transferred daily in F.D.A.. broth. Eggs were sub- merged for about two minutes in .the culture at room temperature, dried quickly in front of a fan, then held in moisture-saturated atmos- phere following the technique used by Mallmann and Davidson (91). Eggs were put in sterile 2-quart, Mason-type, wide-mouth jars. A block of wood was placed in the bottom of each jar and surrounded by water so that a saturated atmosphere would be obtained without direct contact between the water and the eggs. A square portion of an egg flat with receptacles for four eggs was placed on the wooden l Classified according to the key presented by Haynes (67), which will be used in the forthcoming seventh edition of Bergy's Manual. 35 block. Four eggs or less were put in each jar. The jars were incubated at 36°C for periods of 4, 6, 8, 10, 14, and 18 hours. Eggs were opened as above and three examinations were performed: 1. Egg-white test. By the aid of a sterile graduated 2-ml. pipette the membrane was punctured, and 0.1 ml of the egg white was transferred to a 10-ml F.D.A. broth tube. These tubes were incubated at 36°C for 48 hours. 2. Rinse test. Egg contents were removed and a Z-rnl por- tion of a saline solution was pipetted inside the shell. Saline was mixed well with the albumen adhering to the membrane by reducing and increasing the pressure through the pipette. A l-ml portion of this mixture was delivered into a 9—ml F.D.A. broth tube which was incubated as before. 3. Swab test. Eggs were emptied again and, with the narrow end up, the inner shell membrane was swabbed over an area of about one inch in diameter at the narrow end of the egg. Then the swab was broken into a lO-ml F.D.A. broth tube which was incubated as above. Aseptic techniques were followed as closely as possible through- out all these tests. In any case of doubt that the recovered organism 36 was not the one originally tested, microscopic examination and physi- ological tests were carried out. Results of these tests are compiled in Table II. No penetra- tion of the eggshell by any of the test organisms was detected after an incubation period of four hours. After six hours, P. aeruginosa was recovered from two of the twenty-four eggs tested and E. coli was recovered from only one egg. The highest number of recoveries was obtained with P. aeru- ginosa, followed by E. coli and P. vulgaris (close together). The efficiency of each of the tests was calculated as follows: the number of infected Efficiency __ eggs detected bLthe test of the test - total number of infected eggs from the three tests x 100 Table III shows the calculated efficiency of each test for each period and test organism. Discussion According to results obtained, the swab test gave the earliest and highest recovery in this particular study. This agrees with the results of Stuart and McNally (148) and Mallmann and Davidson (91), who found better recovery from the membranes than from either the whites or yolks. This can be explained by the fact that by swabbing, TABLE II SPEED OF PENETRATION OF FOUR ORGANISMS AS DETERMINED BY THREE METHODS 37 Period Number of Positive Eggs Test Organism (hours) Egg Rinse Swab White Total* Test Test Test Micrococcus pyogenes 4 0 0 0 0 var. aureus 6 0 0 0 0 8 0 0 0 0 10 0 1 3 4 14 5 6 6 8 18 13 15 16 20 Escherichia coli 4 0 0 0 0 6 0 0 l 1 8 0 3 5 5 10 l 4 5 7 14 3 13 12 15 18 16 19 20 21 Proteus vulgaris 4 0 0 0 0 6 0 0 0 0 8 0 4 6 6 10 0 7 9 10 14 4 12 14 16 18 10 16 17 20 Pseudomonas 4 0 0 0 0 aergginosa 6 0 0 2 2 8 1 4 9 10 10 3 12 13 16 14 9 15 l7 19 18 18 20 21 22 * The total of all eggs positive by one or more tests. NOTE: Twenty-four eggs were examined at the end of each period for each test organism. 38 TABLE III EFFICIENCY OF EACH OF THE THREE METHODS OF TESTING IN DETERMINING BACTERIAL PENETRATION Per Cent Efficiency of P , Total Test Organism (best-11:5) Positive Egg Rinse Swab Eggs White Test Test Test Micrococcus pytfirnes 4 0 0 0 0 var. aureus 6 0 0 0 0 8 O 0 0 0 10 4 0 25 75 14 8 63 75 75 18 20 65 75 80 Escherichia coli 4 0 0 0 0 6 1 0 0 100 8 5 0 60 100 10 7 14 57 71 14 15 20 87 80 18 21 70 90 95 Proteus vulgaris 4 0 0 0 0 6 O 0 0 0 8 6 0 67 100 10 10 0 70 90 14 16 25 75 88 18 20 50 80 85 Pseudomonas 4 0 0 0 0 aeiiginosa 6 2 0 0 100 8 10 10 40 90 10 16 19 75 81 14 19 47 79 89 18 22 82 91 95 39 the inner membrane was ruptured permitting the organisms between the membranes to be recovered giving rise to greater numbers and earlier recovery. The rinse test was less accurate than the swab test but much better than the egg-white test. This result might be expected for two reasons. The first is that the thin film of egg white adhering to the membrane was mixed with the saline. This film is the first part after the membrane that comes in contact with the invading bac- teria. The probability that this egg-white film contained more bac— teria than any other part of the white is quite understandable. The second reason, although the writer has no proof to back it up, is that rinsing or moistening the membrane changes its physiochemical characteristics in such a manner as to cause an increase in permea— bility. Stewart (146) mentioned that Nathusius in 1868 reported that the membrane fibers swell in water or glycerol and shrink in alco- hol. When Moran and Pique (108) used a saturated lime solution containing 5 per cent sodium chloride as a medium in which- to store eggs, they found that the air sac, which was originally present, was filled after storage. Since the osmotic pressure in the lime solution was higher than that inside the egg, they postulated that the lime solution attacked the egg membranes and destroyed their permeability. 40 These two reports may help to strengthen the idea that the permea- bility of the membrane may be affected when eggs are moistened. Brownwell (17) studied the bacterial penetration into eggs by testing egg whites. When he used M. pyoggnes var. aureus and stored eggs for 8, 12, 14, 16, 18, and 20 hours, he recovered the bacteria from 0, 33, 17, 83, 92, and 83 per cent of the respective periods of exposure. With E. coli after 12, 14, 16, 18, and 20 hours‘ storage his recovery was 0, 25, 58, 92, and 100 per cent. He failed, how- ever, to mention the amount of white used in sampling, which might have been larger than that used by the author and consequently could be responsible for the higher recovery. Rievel (126) opened eggs aseptically, removed the contents, and covered the interior surface with a layer of agar. The outside surface of the shell was immersed in a broth culture of a fluorescent organism and stored at 18°C. One hundred per cent pentration was noticed in two to four days. Although he examined the eggs daily he failed to mention the progressive percentage of penetration which occurred. Stuart and McNally (148) swabbed one-half of the shell of freshly laid eggs (still warm) with a 48-hour culture of P. aergginosa. Immediately after this treatment and after storage periods of 3, 6, 18, 41 24, 48, 66, 96, and 192 hours eggs were opened aseptically. Yolks, whites, and membranes were tested for the presence of the organisms. The results showed that five out of the twenty control (untreated) eggs had infected membranes. Immediately after treatment twelve eggs -.-v~ "v‘ out of twenty were positive, and an additional four were infected after three and six hours‘ storage at room temperature. Three more eggs were infected, giving the maximum of nineteen out of . 'T‘D'T‘flm—‘i twenty eggs after eighteen hours. This recovery is much higher ‘ than that obtained by the author. These data show that, of the eggs which were tested immediately (within a few minutes) after smearing with the culture, twelve had infected membranes, two had infected whites, and one had an infected yolk. It seems improbable that bacteria can penetrate through the shell, membranes, white, and vitelline membrane to the yolk in such a short time. Mallmann and Davidson (91), studying the penetration by B. aegiginosa, used the technique of Stuart and McNally (148), except for a few adaptations that were made to improve the method of cul- turing to lessen the extent of contamination. Eggs were examined after 1, 2, and 48 hours. They found the membranes, whites, and yolks to be negative. 42 In another experiment to increase the chance of bacterial con- tamination, the same authors stored eggs in a saturated atmosphere for 2, 7, and 10 days. In this case their results showed no bac- terial invasion during the 2- and 7-day storage periods. After 10 days, however, one membrane from the four eggs tested was infected. These results were much lower than those obtained by the writer. This may be due to the method of applying the bacterial culture to J *‘T’" _ . : "Mf-nfk. ri 22'» I the eggs. They swabbed the small end of the egg, whereas the author submerged the eggs in a broth culture for two minutes. By the latter method the probability of bacterial invasion is higher. The method of applying the bacteria to the eggshell is impor- tant . This has been shown by Rievel (126), since when he immersed the eggs in a broth culture, 100 per cent invasion was obtained in two to four days. When he used a dry inoculum from an agar slant culture only 35 per cent of the eggs were infected in 10 to 11 days. Mallmann and Davidson (91) also, when they swabbed the narrow end of the eggs, observed no penetration even after 36 hours, but when they immersed eggs into a bacterial suspension they found that, of the twelve eggs tested, one egg yielded organisms on the membrane and i . . n the white, and one yielded organisms on the membrane when tested irn - me diately after treatment. 43 Brownwell (17) also studied the speed of penetration of 3. Eggs were stored at 37°C and 100 per cent relative aeruginosa. humidity for periods of 4, 8, 12, 14, 16, 18, and 20 hours. Imme- diately after storage periods, eggs were opened and whites were cultured. Eggs were still free from organisms even after 8 hours‘ storage. Seven eggs out of the twelve tested showed infected whites hat 14 hours‘ storage, two more eggs were positive at 16 hours‘ storage, and at 18 hours‘ storage all eggs had bacterial invasion. His recovery was slower but the percentage of recovery was higher. This may be attributed to the stock culture strain which he used, in comparison to the freshly isolated culture used by the author. He used a small number of eggs, which was evident as in a later experi- ment he could not recover the organism except after 24 hours‘ incu- bation. In this case he obtained only one egg with infected white from the eighteen eggs tested. Lorenz et al. (88), in developing the “standard infection tech- nique_H submerged prewarmed (36°C) eggs in a suSpension of a W strain. The suspension in which eggs were kept for viable organisms. Eggs were five minutes at 15°C contained 1 x 10 allowed to dry at room temperature before being stored at 15°C. T hey detected the infected eggs by candling. using an ultraviolet 44 lamp. The first fluorescent egg was observed in less than one week. According to Buchanan and Fulmer (19), Pseudomonas excretes a leuco compound which oxides to a green pigment in the medium. To get enough pigment to be detected by the ultraviolet candler a high bacterial load is needed. The 24-hour broth culture of Pseudomonas usually contains approximately 5 x 10 viable organisms in contrast 6 . . . to 1 x 10 in the suSpenSion. These two facts may explain the very low incidence of invasion recorded by them. Recently Elliot (32) inoculated eggs in the air sac with a bacterial suspension in saline of Pseudomonas ovalis. These eggs were stored for 1, 2, 3, 6, 8, 10, and 30 days at 15°C. All the eight eggs that were tested after one day‘s storage showed infected membranes but only one had bacteria in the white. When he used a bacterial culture, however, the only egg tested had an infected membrane and white after one day's storage. Pseudomonas is known to be the single species that causes the greatest bacterial spoilage in washed or cold-stored eggs (27, 85. 145, 104). It is also known for its resistance to the bactericidal Properties of egg white (37). These characteristics, and the fact that it can penetrate into the egg very quickly, make this species the most ideal test organism for any egg-washing or egg-storage experi- mentatiOn. 45 It is important to note that, when conditions of moisture and temperature favorable for bacterial invasion prevail, eggs could be penetrated within six hours. Every precaution should be taken, therefore, in handling eggs before, during, and after storage in or- der to prevent these conditions from occurring. Wate r-Soluble Disinfectant Expe rimental As the name indicates, bisphenols are composed of two phe- nolic structures united by various linkages. Certain members of this chemical group not only possess high bacteriostatic or fungi- static powers, but also give rise to high residual values. For these reasons, they contribute in several important practical applications. The outstanding usefulness of these compounds at the present time is in antiseptic soaps (77, 70). They are also being used in the textile industry (96), in some pharmaceutical preparations (15, 5), in agriculture (58), and several other fields. Although various fields we re investigated, the application of bisphenols in egg washing has neve 1' been reported. Fahlberg, Swan, and Seaston (35) tested the retention of Z“2'‘dihydroxyu3,3',5,5',6,6‘-hexachlorphenolmethane (known as G-ll) 46 on human skin. Subjects washed their hands for six minutes with a soap containing 1 per cent G-ll for three successive days, after which the use of the antiseptic soap was stopped. For the following four days, a skin-ether extract was analyzed chemically and bacterio- logically. They recovered approximately 8.0 micrograms of G-ll from each square inch of skin immediately after the third application. On the following day they obtained 0.4 microgram; two days later, 0.08 microgram; and from the third day on it was not recoverable. The bacteriostatic activity followed the same pattern as the chemical analysis. Since bisphenols have a very low solubility in water, it was thought that the retention of G-ll by the skin is due- to the fats pres- ent on it. These workers disproved this idea by washing hands with ether acetone to remove the fats present. When antiseptic soap con- taining G-ll was used on the defatted skin it gave better residual values. This indicates that fats do not retain G—ll, but may actually interfere with its retention. To test the retention of biSphenols by the eggshell, 2,2‘- dihydroxy-3,3',5,5‘-tetrachlorphenol sulphide, having the generic Produced by Monsanto Chemical Company under the trade It ame 0f "Actamer. ‘ ‘ 47 name of bithionol, was available. The solubility of bithionol in water is very low, being in the order of 0.0004 per cent at 25°C. The addition of an alkali such as NaOH to form the monosodium salt increases the solubility markedly. During the course of this study a monosodium salt was used instead of the original compound. For brevity, monosodium bithionate will be mentioned in this paper as M.S.B. M.S.B. was prepared by dissolving the needed amount of bithionol in a concentrated solution of NaOH; the excess alkali was neutralized with dilute HCl which was added until one more drop would turn the clear solution cloudy. It was noticed that the solution starts to get cloudy when the pH drops below.10.3. Six large clean eggs were Sprayed with a 24-hour broth cul- ture of M.pypgenes var. aureus. After the eggs were dry, they were immersed into a 121000 solution of M.S.B. Eggs were disin- fected for two minutes at 50:1:1°C; then they were left to dry at room temperature for about thirty minutes. A disc, one inch in diameter, was drilled out from the blunt end of each egg. Each disc was put on a seeded plate. T. G. E. agar plates were inoculated with 1 ml of a Z4-hour broth culture of M._pyoggnes var. aureus. Plates were incubated at 36°C for 24 hours. All plates showed zones of inhibi- tion. This indicated that M.S.B. had a residual value when eggs were treated with its solution. 4.- 48 A more drastic treatment to show the extent of the retention of M.S.B. was tried. Six eggs were sprayed as before; however, after disinfection the eggs were resprayed. Using the technique de- veloped by Mallmann and Davidson (91), eggs were put in wide-mouth, Mason-type, 2-quart jars where a saturated atmosphere of moisture was attained. By this method eggs would be coated with a thin water film throughout the period during which they were in the jars. The jars were incubated at 36°C for 48 hours after which eggs were examined as before. All plates showed definite inhibition zones after this drastic treatment. From these results it is evident that M.S.B. has a high residual value when used as an egg sanitizer. When M.S.B. showed this remarkable activity against M. pytienes var. aureus, its activity was challenged against other bacteria encoun- te red frequently in spoiled eggs. Twenty-four large clean eggs were immersed in 4.5 liters of 121000 solution of M.S.B. for two minutes at 50t1°C. When the eggs were dry, discs were drilled out and placed on seeded agar plates. Plates were inoculated with 1 ml of a 24- “OUI‘ broth culture of one of the following organisms: E. coli, 1:. W, and P. aeruginosa. Each bacterial culture was used to inoCLllate eight plates. All plates were incubated at 36°C for 24 hours - No plate showed any sign of antibacterial activity. Since no 49 activity was found against the last three organisms the use of bithio- nol in egg disinfection is of questionable value. Two more bisphenols, 2,2'-dihydroxy-3,3‘,5,5‘,6,6'-hexachlor- 1 diphenolmethane, having the generic name of hexachlorophene, and 4,4'-isopropy1idene diphenol were used. The sodium salt of each compound was prepared as before. Eggs were sprayed, disinfected, and tested following the first procedure described above except that the test organism instead of being M. pyogenes var. aureus was P. aeruginosa. Neither of these two compounds showed any activity against the test organism which hinders their use as egg sanitizers. Since bisphenols did not possess the desired activities against P. aeruginosa. other chemical disinfectant groups were examined. Seven compounds representing three chemical groups were investi- gated. The quaternary ammonium surface-active compounds have attained a prominent and important place in the fields of medical and general disinfection during the past twenty years. These com- POunds are of particular interest in that, unlike most disinfectants, Produced by Sindar Corporation, under the name G-ll. 2 Produced by Dow Chemical Company, under the name of bisphe nol A. 50 they exhibit not only germicidal action but also surface-active, deter- gent, and wetting properties. In this study this group was represented by three compounds: Roccal, Arquad S, and Hyamine 2389. Halogens have been used as disinfectants since the eighteenth century. The remarkable ability of chlorine to arrest putrefaction and destroy odors attracted the attention soon after its discovery in l 774, and its use met with almost immediate approval. Chlorine is now the most widely used of all chemical disinfectants, largely due to its almost universal application in the disinfection of questionable water supplies and its ability to render sewage less objectionable. Klenzade XYIZ (sodium hypochlorite), Iodine suspensoid Merck (col- loidal iodine), and Iosan (iodophore) were investigated from the halo- gen group. Brewer (13), in developing his metal Petri dish cover with the adsorptive disc, impregnated the disc with 5 ml of 1:30 phenyl ethyl alcohol in acetone. The impregnated pad and the metal cover were placed over an agar plate that had been streaked with Proteus, P- ae ru inosa, and M. pyogenes var. aureus. After incubation, the gram-negative organisms did not overgrow the surface, whereas W3 var. aureus grew abundantly. 51 In a later publication, Brewer e_t__a_l_. (14) demonstrated the high bacteriostatic activity of phenyl ethyl alcohol against P. aerugi- 2939.. when incorporated in ophthalmic solutions. Due to these encouraging reports, phenyl ethyl alcohol was investigated for its use as an egg sanitizer. This was the only compound examined from the alcohol group. Large, clean white eggs were sprayed with a 24-hour broth culture of P. aerpginosa. After they were dry, the eggs were im- me rsed in the disinfectant solution at 40i2°C for two minutes. All disinfectants were tested at a concentration of 1000 p.p.m. active ingredients. Higher temperatures were not used as they may have a pasteurizing effect on the test organism. Each group of ten eggs was immersed in 1350 ml of disin- fectant solution at the same time (approximately the same ratio of 140 eggs in 5 gallons used in egg-washing machines). When eggs were dried, discs were cut from the blunt end of the shell as before, and Placed individually in sterile agar plates. At the end of 72 hours‘ incubaltion at 36°C, the plates were examined with the results shown in Table IV. Phenyl ethyl alcohol was the only compound that did “9t Show activity in the concentration of 1:1000. his. TABLE IV 52. GERMICIDAL ACTIVITY OF DIFFERENT COIVIPOUNDS AGAINST PSEUDOMONAS AERUGINOSA IN A CONCENTRATION OF 1000 P.P.M. ACTIVE INGREDIENTS Trade . Manu- No. of No. of Chemical Name Eggs Eggs Name facturer . , Tested P051t1ve Roccal Alkyl dimethyl ben- Winthrop- 10 O zyl ammonium Stearns chloride 10% Arquad S Alkyl (C16-C18) tri- Armour and 10 0 methyl ammonium Company chloride 50% Hyamine Alkylaled (C9-C15) Rohm and 10 O 2389 tolyl methyl tri- Haas methyl ammonium chloride 10% Kle nzade Sodium Hypochlor- Klenzade IO -0 XY 12 ite, 1% available Products chlorine IOdine Colloidal iodine, Merck and 10 0 suspensoid 20% iodine Co., Inc. Merck Iosan Nonyl phenyl ether Lazarus 10 O at polyethylene gly- Labs., Inc. coliodine complex, 1.75% available iodine --------- Phenyl ethyl alcohol ---—--—---- 10 9 _.-'., , 53 Since several disinfectants exerted high activity, a screening test was needed to evaluate these disinfectants. The same test was performed as above using a concentration of 100 p.p.m. of each com- pound. Results are presented in Table V. The three halogen com- pounds were the only active disinfectants in loo—p.p.m. concentration. For further screening, a semiexhaustion test was employed. Eggs were treated as above (ICC-p.p.m. concentration); however, after two minutes another batch of eggs (ten eggs) was treated in the same solution). The pH of each disinfectant solution was determined be‘ fore and after immersing the first batch, and after the second batch. From Table VI it can be seen that sodium hypochlorite (Klen- zade XYIZ) gave the best results since all shell discs were free from viable bacteria. In both of the iodine compounds, the second batch of eggs was not well disinfected. f.‘ )4 U —' 5' Since‘chloride was the best germicide found, the effect of Ste rox CD (the chosen detergent) on the germicidal activity of chlor- ine would be of great importance. If it antagonizes and reduces the germicidal activity of chlorine, either the amount of chlorine should be increased or another germicide should be used. If, however, the activity of chlorine was unaffected, or enhanced, the two compounds (:0 o o uld be used as a detergent-sanitizer. 54 TABLE V GERMICIDAL ACTIVITY IN lOO-P.P.M. CONCENTRATION OF ACTIVE INGREDIENTS (test organism Pseudomonas aeruginosa) Sanitizer No. of Eggs No. of Eggs Tested Positive IF Roccal ...................... 10 10 i Arquad s .................... 10 10 ! Hyamine Z389 ................. 10 8 if: Klenzade XYIZ ................ 10 O IOd1ne Suspensoid Merck ......... IO 0 Iosan ....................... IO 0 TABLE VI 55 GERMICIDAL ACTIVITY AND pH OF SOLUTIONS TESTED BY "SEMIEXHAUSTION" METHOD * No. Eggs in No. Eggs in pH First Batch Second Batch Solution After After Posi- Posi- Initial First Second t T” ed tive TeSted tive Batch Batch men- 10 O 10 O 8.3 9.0 9.2 zade XYIZ Iodine 10 0 10 8 2.2 6.8 9.1 su8pen- soid Merck Iosan 10 o 10 7 2.0 6.5 9.1 AA..." it 56 Twenty eggs were sprayed with P. aeruginosa. From these, ten were immersed in a detergent sanitizer solution containing 0.1 per cent Sterox CD and 100 p.p.m. active chlorine. After being submerged for two minutes, the eggs were dried and eggshell discs were tested as before. After two minutes from the time the first batch of eggs were removed from the solution, another batch was added. The second batch was tested in the same manner as that used for the first one. All the discs tested were free from bacteria indicating that the germicidal activity of chlorine was not destroyed in the presence of O .1 per cent Sterox CD. Discussion Bisphenols are compounds in which two phenolic bodies are lirlked together in various ways. This may involve a direct carbon- to‘Carbon bond, or there may be a connecting atom such as oxygen Or Sulphur or a connecting group such as -CHz-. The two phenolic structures may be the same (symmetrical) or different (asymmetrical). The hydroxyl groups may be in the ortho, meta, or para position to the bridge. The substitutions on the phenolic ring are several in k . . ind and position, with innumerable comb1nations. Due to the great g.» .7- 3.“ nfiiu.hg , "-‘ grim-r. :- 57 number of possibilities many compounds have been synthesized and many more could still be considered from the bisphenol group. It is fortunate that it has been possible to establish, to a great degree, a relationship between the chemical structure and the antimicrobial activity. Marsh and co-workers (97, 98) made an ex- tensive study of this group for its antifungal activity. They stated that: Activity [of bisphenols] is influenced by the following structural features: (a) The number, type, and position of halogen atoms. Two bromine or chlorine atoms per molecule increase activity above that of unhalogenated bisphenols, whereas, too high a halogen content, especially when consisting of bromine rather than chlorine and/or particularly when occupying all 4 positions ortho to the phenolic hydroxyls, leads to low activity. (b) Molecular size. Usually large molecular size leads to low activity. (c) Chemical blocking of phenolic hjdroxyls. Ether formation (but not necessarily esterification) brings about a very low potency. (d) Types of bisphenolic bridge. Bridges consisting of -CH2-, ~S-, -C-, -CH-CH3-, ~C1-1-C H5-, or -CH=CH- are com- patible with high activity; -§O-, -501. or -C-—C- are less desirable in this respect. (e) R-grouLsubstitutions on the phenolic rings. The chlor- thymol type of substitution brings about a very low activity, certain other groups are not undesirable. From what is known about the gram-negative bacteria they f . . . 0110‘” the same pattern. The gram-p051t1ve organisms, however, h ave a. slightly different relationship. The more halogen substitutions .- ’_.A~I"s" LAMI‘M “P - o 58 on the ring the more potent the compound. Moness et al. (107) found that the addition of an alkyl group on the phenolic ring increased the activity. By using M.lyog_enes var. aureus as a test organism, mono- PM. P" - ‘flnz o‘ sodium bithionate showed a remarkable residual value. This agrees with what is known about bisphenols as a group. M.S.B. in a concen- tration of 1:1000 did not exert any antibacterial activity against E. coli, r ' "‘ "“n'vtr‘ ‘3‘ “7737: P. vulgaris, and P. aeruginosa. It did show, however, a powerful activity against M. pyogenes var. aureus. The results agree partially with those obtained by Hunter et a1. (71). They found that 121000 Concentration of bithionol was not inhibitory against either E. coli or I:_.__E=leruginosa, and a concentration of 1 p.p.m. inhibited the growth of M. pyOgenes var. aureus. In the case of P. vulgaris, 100 p.p.m. Was enough to prevent’its growth. The high activity against P. vulgaris may be due to the procedure they used in their test. They dissolved bithionol with the aid of polyethylene glycol 400 as a solvent. Se- rial dilutions were prepared and inoculated with 0.05 ml of an aqueous suspension of the respective test organism. Results were read after 48 hours' incubation. The sodium salts of either hexachlorophene or bisphenol "A" It was i “ C°ncentrations of 1:1000 failed to inhibit P. aeruginosa. 59 reported (141) that 100 p.p.m. hexachlorophene is bactericidal to E. aeruggiosa. The F.D.A. procedure used in the previous report may be responsible for the difference in the results. The solution in aqueous alkali was prepared by dissolving 0.1 gram of G-ll in 1 m1 of 95 per cent alcohol and 0.75 ml of 0.5 N alcoholic potassium hy- droxide and adding water to 100 ml. Since bisphenols comprise a large group there may still be a possibility of finding a sanitizer that is active against the gram- negative organisms as well as against the gram-positive ones. Until such a compound is found, the use of bisphenols as egg sanitizers would be inadvisable. Phenyl ethyl alcohol did not show any activity against P. aeru- ginosa in 1.1000 concentration. This agrees with what was reported before. Lilly and Brewer (82) observed that 1:400 concentration, but “0t 1:800, inhibited the growth of P. aeruginosa. The three quaternary compounds tested did not show any baCtel‘iostatic or bacteriocidal activity in a concentration of 100 p.p.m., although all three inhibited the test organism in 1000 p.p.m. concen- tration_ It is very difficult to compare results obtained in this study “nth t1lose reported in the literature since the method of determining -3! 60 the germicidal activity of quaternary compounds has a marked effect on the results. In the method used in this study, the quaternary compounds' activities were not stopped when eggs were dried, but when the discs were placed on agar the activity was reduced to a minimum if it was not stopped. The effect of agar on the antibac- terial activities of quaternary compounds was reported by Tokie and Ayres (151), Hoogenheidle (69), and several others. Quinso, Gibby, and Foter (119, 120, 121) attribute this action to the physical absorp- tion of the compounds by the agar. Kivela St__al_. (75) studied the physical action of alkyl dimethyl benzyl ammonium chloride (A.D.B.) on several organisms. They noticed that at a concentration of 124000 but not 1:5000 cells of _P_. aeruginosa clump. This may explain why in a 1210,000 but not 121000 concentration the compound failed to exert effective antibacterial ac- tivity (Table V). Several investigators used A.D.B. in egg-washing experimenta- tions. As expected, they obtained contradicting results due to factors Such as concentration, kind of eggs, method of testing, et cetera. Funk and Forward (48), using hatchability as a criterion, re- pOrted that there was not too much difference between the hatchability of Lu“treated clean eggs and that of those washed in 380 p.p.m. A.D.B. 61 In a later publication, however, Funk et al. (49) used a concentration of 150 p.p.m., and immediately after washing the eggs were oiled in a preparation containing 1750 p.p.m. of A.D.B. Even after such treat- ment, they could not stop excessive bacterial invasion. Miller gt__a_l. (106) was not able to detect antimicrobial activity :81» of A.D.B. in the water in which eggs were washed until he used a concentration of 1 per cent Roccal. ,: Forsythe (39) and Forsythe iii- (40) found that 200 P.P.m. i. ,‘i A.D.B. was not harmful to the physical quality of the egg contents. At this concentration the bacterial population was reduced on the egg— shell by approximately 92.8 per cent. In their study they did not differentiate between inhibition or destruction and mechanical removal of bacteria. When they used Vel (a detergent) they obtained 82.5 per Cent reduction. From this, the high activity of A.D.B. obtained bY them may be attributed to mechanical removal of bacteria rather than inhibition and inactivation. Recently, Miller (104) reported on his experiments with dirty eggs, A solution containing 100 p.p.m. A.D.B. was not effective in washing eggs. By determining the bacterial load of egg contents, he found that washed eggs had more bacteria than untreated eggs. 62 Botwright (10) showed that Hyamine 2389 in a concentration of 130 p.p.m. was highly bactericidal against P. aeruginosa. Winter (154), however, with a concentration of 180 to 380 p.p.m., found that 15 per cent of the treated eggs had rots, whereas only 4 per cent of the untreated eggs showed rots. Why quaternary compounds failed to give as good results as halogen preparations may be explained as follows: The test used in this study was designed to be as close as possible to the practical conditions; also, the test organism was a resistant gram-negative bacterium. Generally speaking, and especially under practical con- ditions, quaternary compounds are active against gram-positive or- ganisms (31), whereas halogens are definitely more active against gram-negative bacteria (72). Mallmann _e_t_____a_._1_. (92) compared 180-p.p.m. hypochlorite with the same concentration of a quaternary compound in the disinfection of glasses in an eating establishment. Both compounds gave good comparable results. It should be realized, however, that bacteria from the gram—positive group accounted by far for the major portion of the bacterial load in their study. The three halogen preparations showed much better activity than the rest. They were active even in a concentration of 100 p.p.m. ... km.» 1.2-: u '. in I 7.1:... . 1...?“ w‘n‘.«‘ Z 63 At a relatively high temperature (40°C) and high pH, hypochlorites exerted better activity than the two iodine compounds. Mandel (95) tested three iodine preparations: Iodine suspen- soid, Lugol's solution, and Iodine Tincture. He disinfected chicken feces with concentrations up to 0.2 per cent I at a pH 5.0 for a 2 period of five minutes. The reduction of the bacterial load was identical for the three compounds. It seems, then, that iodine prep- ‘ Arm‘s” m: , "1 arations possess the same bactericidal value if they are tested at L the same pH. Wyss and Strandkov (158) reported that iodine preparations decompose very quickly at pH 8.0 or higher. Mandel (95), however, believed that loss of penetrating power is the resulting effect of high pH on iodine preparations. He showed that by increasing the pH from 5.0 to 9.0 the length of zone of inhibition decreased from 12.5 mm to zero when he used the penatube method in testing the iodine suSpensoid. Regardless of what actually happens, high pH is very detri— mental to iodine acting under practical conditions. Relatively high temperatures also adversely affect iodine com- pounds. Gershenfeld and Miller (50) reported that iodine is more effective against M. pyogenes var. aureus at 20°C than at 37°C. 64 Ostrolenk and Brewer (110) found that P.__aeruginosa was killed within five minutes by 125000 concentration of iodine at 20°C. At 37°C it required 124000 concentration to kill the same organism, whereas 15 minutes (the maximum exposure time used) were not ICE- enough at 125000 concentration. ! .1“ if i Cantor and Shelanski (23) were the first to report on the i a. i germicidal activity of the nonyl phenylether of polyethylene glycol- E iodine complex. They used their capacity test to demonstrate its ! j 3...? great germicidal power. Their test organisms were M. pyogenes var. aureus and Salmonella typhosa. They conducted all their tests at 25°C. Ostrolenk and Brewer (110) tested fourteen different or- ganisms against iodine. They found P. aeruginosa to be the most resistant; kill occurred at a concentration of 125000 at 20°C, whereas M. pypgenes var. aureus and S. tjphosa were killed at a concentra- tion of 1:8000. At 37°C P. aeruginosa, M.pyogenes var. aureus, and S. typhosa required concentrations of 1:4000, 1:8000, and 1:7000, reSpectively. Hypochlorites behave differently at relatively high tempera- tures. Rudolf and Levine (135), using Bacillus metiens, found that the killing time was reduced 60 to 65 per cent for each 10°C rise between 20°C and 50°C. At the pH of 10 and a concentration of 65 25 p.p.m. available chlorine, all spores were killed in 121 minutes at 20°C and 38.7 minutes at 35°C. Allen and Brooks (2) stated that Butterfield e.t__a_l_. (1943) showed that the bactericidal efficiency of chlorine increases with rising temperature and the effect becomes more mad1:100 <1.4 Dow Chem— 6 ophenol ical Co. Dowicide pentachlorophenol 1:400 5.7 Dow Chem- 7 ical Co. Dowicide 4 and 6-chloro-2- >1:100 <1.4 Dow Chem- 32 phenylphenol ical Co. --------- Phenoxyacetic acid >12100 <1.4 Dow Chem- ical Co. Methyl— Methyl parahydroxy- >I:100 <1.4 Heyden parasept benzoate Chemical Propyl- Propyl parahydroxy- >l:100 <1.4 Heyden parasept benzoate Chemical Ethyl— Ethyl parahydroxy- >l:100 <1.4 Heyden parasept benzoate Chemical Butyl- Butyl parahydroxy- >12100 <1.4 - Heyden parasept benzoate Chemical Benzyl- Benzyl parahydroxy- 1:100 1.4 Heyden parasept benzoate Chemical 69 For testing the antibacterial activity it was found to be much easier to examine the compounds when rendered water-soluble. This was found to be most conveniently done by adding a strong alkali (NaOH) to the phenolic compound. Kojima (76) observed that the sodium salt of a phenolic was Emm- less germicidal than the original compound. These observations if: were confirmed by Klarmann and Wright (122). In other words, if i: the sodium salt of a phenol would show activity against bacteria, the i; ,5 mg. Original compound should be expected to exert greater germicidal Potency at the same concentration and under the same conditions. The phenol coefficient was used as a criterion for determining the antibacterial activity. The United States Food and Drug Adminis- tration method (136) was applied with one modification. P. aeruginosa was used as a test organism instead of either M. pygenes var. aureus 01‘ 3. typhosa. All tests were conducted at 20°C. Experiments were repeated until three successive determinations gave the same value. The final results are tabulated in Table VII. A position of considerable importance is occupied by the este rs of parahydroxybenzoic acid, notably in the field of preserva- tion of carbohydrates, gums, proteins, and other organic materials of industrial, pharmaceutical, and cosmetic significance against spoilage 70 by air-borne microorganisms (109). A table indicating the proper concentrations of several esters required for preservation was pre- His recommendations had been confirmed by pared by Suess (149). Several investigators (52, 30). Gershenfeld and Perlstein in 1939 (52) regarded these esters as being among the most useful preservatives for pharmaceutical and related preparations available at that time. Since, in the above reports, P. aeruginosa was never used as a 'test organism, comparison of their data with those obtained in this StUdy is impossible. Recently, Sokol (142) subjected methyl, ethyl, propyl, and butyl para-hydroxybenzoate to an extensive investigation. Their anti- bacterial and antifungal properties were examined in an endeavor to find suitable test organisms and testing methods. He used twelve different test organisms, eight of which were gram-negative. It is interesting to note that in his study he found P. aeruggi_nosa ATCC 9027 to be the most resistant of all the test organisms he used. corrlplete inhibition of this organism required 0.4 per cent of either methyl or ethyl esters and 0.8 per cent of either propyl or butyl esters. The difference in results reported by the previous author and ”1038 obtained by the writer may be due to differences in the methods of testing. Sokol prepared agar plates containing the desired 71 concentration of the sodium salt of the ester. He inoculated these plates by dipping a loop in a lO-ml water suspension of a 24-hour slant of the test organism and streaking on one sector of the plate. Readings were made after 24-hour incubation at 37°C. Hearst and Hearst (68), in producing their egg-preservative emulsion, incorporated low molecular esters of parahydroxybenzoic acid. The examples they gave were the methyl, ethyl, and propyl esters. They used one or more of these esters to give a final con- centration of 4 per cent of the preservative (s). In the writer's study, Concentrations up to l per cent of the same esters did not produce any inactivation of P. aeruginosa. Phenol inactivated P. aeruginosa in a concentration of 1:70 afte r 10 minutes' eXposure but not after 5 minutes. Sodium penta- C1florophenate (sodium salt of Dowicide 7) showed the highest activity. It was effective in concentrations as low as 0.25 per cent (phenol coefficient of 5.7). As mentioned before, in selecting an oil-soluble disinfectant, its antifungal activity should be as good as its antibacterial potency. N0 antifungal examinations were performed on pentachlorophenol in this investigation since it had been thoroughly studied previously (93, 90) Mallmann and Davidson (91) recommended the use of I‘A‘“ J. 35, 'I ’7 1W; .5. 21.1.: 72 pentachlorophenol (Dowicide 7) in a concentration of 0.25 per cent to be incorporated in oil. By using such antiseptic oil, they could pre- vent mold growth inside and outside the eggs. Percentage of Oil in Emulsion Experimental In oil processing, more oil than the eggshell can retain is being used. This fact has been demonstrated by Evans (33) and Stewart and Bose (147). Both reports dealt with the use of volatile solv2nt-oil mixtures. Evans reported that 50 per cent oil was needed in a solvent-oil mixture to protect eggs as effectively as 100 per cent Oil. Stewart and Bose, however, found that 10 per cent oil in a dif- fere nt kind of solvent-oil mixture was practically as efficient as 100 per cent oil. If the right amount of oil is used in an oil-in-water emLllsion, it should give as much protection to the eggs as 100 per cent oil. In determining how much oil should be incorporated in the em"llssion, the efficiency in reducing the weight loss of eggs held at inc . . . “bation temperatures was chosen as a criterion. “as-no”: 73 A mineral oil used commercially for oiling eggs was used in all the investigations. This oil had the following physical character- istics (manufacture r's specifications): Viscosity at 37.8°C (100°F) ......... 180-190 Saybolt Specific gravity at 15.6°C (60°F) . . . . . 0.8816 maximum Open flash ..................... 190.6°C (375°F) Pour point ..................... -26.1°C (-15°F) max. Saybolt color ................... +30 The emulsions consisted of: 5.00 or 10.00 grams rm vtw—T—pm )l‘.‘ 9.. run—or,” Oil .......................... . Sterox CD ..................... 0.1 gram -' Sodium tri polyphosphate ........... 0.15 gram Active chlorine (as determined by ortho tolidine flash test) ......... 200 p.p.m. Tap water to total of ............. 100 grams In the first experiment 5 per cent emulsion was tested. Three dozen clean, white, sound-shell eggs, 241:4 hours old, were used. One dozen was left as controls, and the other two dozen were treated by imITaersion in either 5 per cent emulsion or 100 per cent oil (one d°z€n each) at 50i1°C for three minutes. After treatment the eggs were left at room temperature for 60 minutes to drain and dry. Eggs were weighed individually, put in trays, and held at 36°C for three weeks with 303:3 per cent relative humidity (as determined by dry and wet bulbs). At the end of 7, 14, and 21 days the eggs were reweighed individually. Portion cups (souffles) 5% ounces capacity we be very useful in handling eggs. ‘J— E r.- . 74 Inasmuch as the difference between the control eggs and eggs treated with 5 per cent emulsion was negligible, it was decided to try a 10 per cent emulsion. Results are summarized in Table VIII. No significant difference was found between any of the treat- ments and the control at zero days. Taking the control as standard, the re was no definite advantage in using 5 per cent emulsion, whereas the re was a decided advantage in using either the 10 per cent emul- F ' ' 2": K, . '3.i‘r‘fi.:flaa-_—- . ,. '5... \e' .._‘ — u 3' sion or the 100 per cent oil. Oiling was better than the treatment with 10 per cent emulsion as shown after the third week of holding. In other words, 10 per cent emulsion was as good as 100 per cent 011 when eggs were held for two weeks at 36°C. Discussion One of the major problems confronting the poultry industry is that of supplying consumers with eggs of high quality. The egg is a. highly perishable food product, and may lose quality rapidly afte r being laid. Just before being laid, a hen's egg is in equil- ibrium with an atmosphere having a relative humidity of 99.5 per cent (140), and containing 10 per cent CO2 at 25°C (139)- If an egg ls 1