Some Factors Influencing Bacterial Penetration of Eggs1

BACTERIAL PENETRATION OF EGGS

4. Curled tongue was not prevented by the antibiotic bacitracin or terramycin in the quantities used in the rations of this experiment. 5. Feeding poults pellets or the coarser mashes to seven weeks of age produced greater body weight and greater gain per unit of feed consumed than did the finer mashes. REFERENCES

of the tongue associated with amino acid deficiencies in the chick. Proc. Soc. Exp. Biol. Med. 59: 177-178. Hayward, J. W., 1950. Modern feeds are better. Turkey World 25(4): 11. Hudson, C. B., 1939. Curled tongue in young poults. J. Amer. Vet. Med. Assn. 47: 662. Kratzer, F. H., 1951. Personal correspondence. Robertson, E. I., 1950. The response of poults to animal protein, feed particle size, and antibiotics. Poultry Sci. 29: 777. Schweigert, B. S., 1947. Amino acid content of feeds. J. Nutrition 33: 553-559. Scott, M. L., 1951. Personal communication and correspondence. Williams, H. H., 1951. Studies of the amino acid composition of feedstuffs. Feedstuffs 23(6): 17. Ziegenhagen, E. H., L. B. Corman and J. W. Hayward. 1947. Feed particle size as a factor affecting performance of turkey poults. Poultry Sci. 26: 212-214.

Some Factors Influencing Bacterial Penetration of Eggs 1 W. A. MILLER AND L. B. CRAWFORD Department of Bacteriology, Kansas Agricultural Experiment Station, Manhattan, Kansas (Received for publication August 15, 1952)

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SIGNIFICANT percentage of commercial eggs undergo marked deterioration in quality or even complete spoilage before reaching the consumer. The prevalent opinion is that microorganisms play a dominant role in this deterioration, a concept supported by the fact that parallel with or preceding deterioration there is frequently mass growth of bacteria in the egg. Views expressed in the literature relative to the influence of various factors upon the microbial content and growth in eggs are rather divergent. The available data are fairly consistent, however, in indicating that only a small proportion of freshly-laid eggs contains living organisms, 1 Contribution No. 276, Department of Bacteriology, Kansas Agricultural Experiment Station, Manhattan, Kansas.

and that in these, the number of bacteria is relatively low (Tanner, 1944). Furthermore, it has been observed repeatedly that eggs held or stored under a variety of environmental conditions exhibit extremely wide individual variation in the rate at which deterioration in quality takes place. This is true even when the environmental conditions would seem to be almost ideal for microbial growth (Lorenz et al., 1952). These facts would indicate marked variability in one or more of the factors influencing the entrance into and subsequent development of egg-spoilage bacteria in different eggs. A number of explanations have been offered for these differences, but none has been supported by adequate experimental proof. An experimental project was initiated

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Almquist, H. J., 1951. Personal correspondence. Almquist, H. J., and C. R. Grau, 1944. The amino acid requirements of the chick. J. Nutrition 28: 325-331. Draper, C. I., 1951. Personal correspondence. Gill, V., and R. H. Phillips, 1951. Personal communication. Grau, C. R., 1951. Personal correspondence. Grau, C. R., and H. J. Almquist, 1945. Deformities

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with the objective of gaining more exact information relative to the kind, methods of gaining entrance, and factors influencing growth of spoilage microorganisms in eggs. So far, experimentation has been largely exploratory; nevertheless, the data accumulated indicate certain trends that seem worthy of recording. PROCEDURE

In a batch of 200 fresh eggs (held 1 to 7 days at 25° to 30°C), 170 failed to show any growth of bacteria while cocci developed from 27 and diphtheroids from three, when 10 ml. of the homogenized egg was cultured in broth. When 0.01 ml. was plated out, the total number of colonies appearing on plates made from positive broth culture eggs was no greater than the total number of colonies on plates from eggs yielding negative broth cultures. Therefore, the number of organisms present in the newly-laid eggs was nil or exceedingly small, and in no instance were the more common types of eggspoilage organisms cultured from fresh eggs. B. BACTERIAL CONTENT OF COMMERCIALLY DEFECTIVE EGGS

Three hundred and eleven eggs designated as "defective with shells intact" by a professional candler were received from a local packing plant over the period March to August. Defects were chiefly meat spots, blood spots, bloody whites, stuck yolks, etc. Eighty-nine percent of these eggs revealed no spoilage organisms on plates made from 0.1 and 0.01 ml. of homogenized egg. Nine percent contained large numbers of egg-spoilage bacteria—chiefly Pseudomonas, coliform, and Flavobacterium types. Approximately 2 percent contained 2,000 to 280,000 micrococci per gram, while one egg contained 14 million streptococci per gram. A considerable number of eggs having high counts were "stuck yolks." The percentage of spring eggs containing significant numbers of bacteria was lower than for summer eggs (7.2 vs. 18.4 percent). These data indicate that microorganisms are responsible for only approximately 10 percent of

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Quantitative bacterial determinations were made on individual eggs by plating appropriate dilutions of the egg on tryptone-glucose extract agar. All eggs were scrubbed with a detergent, rinsed in tap water, and dried with a clean towel. The small end of the egg was flamed until approximately 2 mm. of white was coagulated. A small amount of the shell was removed and the entire contents of the egg teased into a sterile mason jar with the aid of a sterile pipette. Whites and yolks were mixed 1 minute by means of a blender. The cutter blades of the blender employed are made to fit an ordinary mason jar. Any number of blade assemblies and jars may be used, and can be sterilized separately. To detect microorganisms which might be present in small numbers, 10 ml. of white, yolk, or mixed white and yolk were added to 300 ml. of tryptone-glucose broth. Other techniques used occasionally will be clarified when necessary. Eggs used in these studies were supplied by the Poultry Department, Kansas State College, unless otherwise specified. A distinct advantage of this arrangement was that eggs of known age could be obtained readily. Experiments have been designed to gain information relative to a number of factors, but only a summary of the data from representative experiments dealing with each of these factors will be given here.

A. BACTERIAL CONTENT OF NORMAL FRESH EGGS

BACTERIAL PENETRATION OF EGGS

the shell-intact eggs declared defective by a local packer. C. SPEED WITH WHICH SPOILAGE BACTERIA PENETRATE AND GROW IN NORMAL, FRESHLY-LAID EGGS

only in the 10 ml. broth cultures of the white. Of 95 eggs examined, during the succeeding 5 days, 53 contained from 50 to 3 million Gram negative rods (largely Pseudomonas sp.) in the mixed white and yolk, while 42 still contained no spoilage bacteria. D. BACTERIAL CONTENT OF COMMERCIAL "CHECKS" (CRACKED EGGS)

Six dozen commercial "checks" sorted from current receipts by a professional candler were obtained from a local packing plant in July, 1951. Of these, 50 visibly cracked but not leaking eggs were selected for analysis. Thin-shell eggs with no , cracks and leakers were discarded. The eggs were stored 2 to 4 days in the refrigerator at 8°C. (46.4°F.) before plating. The whole egg was removed and cultured as previously described. Seven eggs were found to contain from 3,200 to 50 million bacteria per gram (average 7 million), while no colonies developed on plates (0.01 ml. plated) from the other 43 eggs; thus, only 14 percent of these commercial checks contained significant numbers of bacteria. E. BACTERIAL GROWTH IN ARTIFICIALLY CHECKED, THICK-SHELL EGGS

The shells of six dozen clean, fresh eggs with normal shells were artificially cracked with as little effort as possible, smeared with contaminated mud (infusion of litter and chicken manure, plus known spoilage bacteria), and stored at 25°C. (77°F.) to 30°C.(86°F.). Several eggs were plated daily. Rapid penetration and growth of spoilage bacteria occurred. The lowest count observed at 24 hours was 350 per ml. and the number steadily increased during the next few days to billions per ml. No significant differences could be observed between leaking and non-leaking eggs. Comparable growth occurred in

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Results of plate and broth cultures on 200 normal commercial eggs held at 7°C. (44.6°F.) to 37°C. (98.6°F.) indicate that penetration and growth of spoilage organisms is negligible (less than 5 percent) up to 14 days, regardless of shell contamination (natural or artificial), if shells remained dry or without visible moisture adhering. Many of these eggs were subjected to relative humidities approaching saturation. Sixty percent of a similarly contaminated group of 150 eggs held 14 to 50 days contained many spoilage bacteria (gram-negative rods). Two hundred and five new-laid eggs were coated with a suspension of microorganisms isolated from high count eggs (Pseudomonas sp., Penicillium sp., Slreptomyces sp., Flavobacterium sp., and coliform types). To insure moist egg shells, these eggs were stored in wet paper egg cartons in closed containers at 35°C. (95°F.) to 37°C. (98.6°F.). Bacteriological analysis was made of whites and yolks separately on aliquots of these eggs hourly for 10 hours, then daily for 8 days. A convenient method for sampling the intact yolk remaining in the shell, after aseptically removing the white, consisted in thrusting a 1 ml. milk pipette through the vitelline membrane and withdrawing a measured quantity of yolk. One to two ml. of yolks and whites were plated separately; in addition, 10 ml. portions were cultured in broth. Of 110 eggs examined during the first 3 days, only 5 were found to contain spoilage bacteria. These were Pseudomonas sp., and at this time were detected

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another batch of eggs in which the spoilage bacteria, including coliforms, were introduced directly into the egg white.

G. BACTERIAL CONTENT OF EGGS FOLLOWING INOCULATION OF AIR-SAC One-hundred infertile, clean, fresh eggs were held 4 days at 35°C. to allow the air-sac to enlarge, after which a small area of shell was drilled away above the air-sac, exposing the outer shell membrane. A drop of a mixed culture of egg-spoilage bacteria (Pseudomonas, coliform, etc. types) was injected between the two shell membranes into the air-sac, and the opening in the shell was sealed with scotch tape. The eggs were stored at 25°C. (77°F.) to 30°C. (86°F.) and aliquots examined daily. There was no significant increase in the number of bacteria in 19 out of 27 eggs examined during the first 3 days (apparently bacteria had not spread

H. BACTERIAL CONTENT OF WASHED AND UNWASHED EGGS STORED AT A CONSTANT TEMPERATURE AND HUMIDITY During a light rainy period in September, 1949, 300 dirty eggs were collected at the Kansas State College Poultry farm, and stored in an egg cellar at 18°C. (64.6°F.) to 21°C. (69.8°F.) (summer temperature) for a period not exceeding 4 days. Approximately 50 percent of the eggs in groups 1, 2, and 3 were pullet eggs collected from the range and many were naturally smeared with ground soil. Group 4 eggs were collected in February, 1949, when chickens were running in muddy lots; they were stored (maximum 7 days) in the cellar previously menticned, at 8°C. (46.4°F.) (winter temperature). The 4 groups were stored in an experimental constant temperature humidity room at 15°C. (59°F.) to 18°C. (64.6°F) and 60 to 65 percent relative humidity. It is obvious that such a temperature is too high for practical egg storage. However, since spoilage organisms will grow more readily at this temperature than at ordinary storage temperature, use of the higher temperature made it possible to shorten the period necessary to observe the possible effect of different treatments upon the growth of spoilage bacteria in eggs. The eggs were exposed to constant external contamination, due to the presence of mice, chickens, and litter in the room. A number of eggs of each group

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F. BACTERIAL GROWTH IN ARTIFICIALLY CHECKED, THIN-SHELL EGGS Several dozen intact thin-shell eggs were secured from a local packing plant. It was reasoned that the shells might be cracked with a light blow, thereby lessening the chance of injuring the shell membranes. Proceeding on this assumption, the shells were cracked, and the surface smeared with contaminated mud. The eggs were stored at 25°C. to 30°C. and aliquots of the eggs were plated daily for 8 days. Of those examined within 3 days, 66 percent contained no bacteria, and 20 percent of those examined between 3 and 8 days were free of spoilage bacteria. An over-all 45 percent of the cracked thinshell eggs, compared with zero percent of the thicker shelled eggs, showed no significant multiplication of bacteria during the first few days. These results might be interpreted as indicating that the damage to shell membranes facilitated penetration of eggs by microorganisms.

from the air-sac to the white and yolk of these eggs), while eight eggs were found to contain 1,000 to 32 million bacteria per gram. Of 73 eggs examined on the 4th to 15th days, 64 contained spoilage bacteria ranging in number from 50,000 to 5 billion per gram. However, 9 eggs still contained fewer than 100 bacteria per gram (no colonies in 0.01 ml. plate).

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trate and grow in eggs when the conditions, presumably, were favorable for microbic growth indicated that even when the shells of eggs were coated with mixed cultures of bacteria isolated from spoiled eggs and held in a moist atmosphere at a favorable temperature, no growth was evident in any eggs prior to 72 hours and less than 5 percent of such eggs contained any spoilage bacteria even in the whites. TABLE l.-~-A pproximate percent of variously treated Approximately half the eggs examined dirty eggs containing many spoilage bacteria between the third and eighth days rePercent of eggs containing vealed no spoilage bacteria. When egg spoilage bacteria by storage intervals No. of eggs Treatment shells were heavily contaminated with 2nd 8 months 1st naturally occurring sources of infectious total 4 mo. 4 mo. material, i.e., suspension of soil and bird Washed in water Group #1 34 23 18 100 feces, and held with shells free of moisture, Group #2 Washed in water, 32 significant growth was absent from all 100 oiled eggs examined during the first two weeks. Group #3 7 27 19 1.10 Unwashed Other experiments were carried out in Group fi* 21 6 36 an effort to evaluate the significance of the Washed in water 100 egg shell and membranes as a mechanical * Winter eggs. barrier to the penetration of spoilage bacteria into eggs. When the surface of DISCUSSION The accumulated data, in part here- artifically checked normal eggs was with presented, indicate rather definitely smeared with a suspension of egg-spoilage certain facts relative to microbic penetra- bacteria, penetration and growth were tion and growth in eggs. Our data show rapid in all cases. When thin-shell eggs that the type of eggs studied and the tech- were artificially cracked and smeared nique employed in this investigation gave with spoilage bacteria, the rate of peneessentially the same results as have been tration and growth was much slower than reported by other investigators. Bushnell was the case with normal-shell eggs. A and Maurer (1914), U.S.D.A. Circular possible explanation of the difference in #583 (1941) and Tanner (1944) among the rate of penetration and growth of others have reported that a large percent- bacteria in thick- and thin-shell checks is age of freshly laid eggs contains few or no that more force is required to crack thick bacteria capable of developing in or on shells; hence, breakage may be accomordinary laboratory media. Our findings panied by greater damage to the egg in this respect substantiate those reported membranes. These data suggest (a) that by these investigators and further indicate the larger the number of spoilage bacthat" the major types of egg-spoilage bac- teria on the shell of checked eggs, the teria are absent from approximately 100 more rapid the penetration and growth of bacteria in the egg and (b) that injury to percent of freshly laid eggs. the egg membrane in the case of checks Data from experiments designed to possibly may be a factor in penetration of give information relative to the speed bacteria. Stuart and McNally (1943) with which spoilage bacteria would penewere cultured weekly for 8 months, except in Group 2. The initial data from this group of eggs suggested the desirability of more frequent analysis and hence the supply of this group was exhausted during the first 4 months. Percentage of eggs yielding significant numbers of spoilage organisms during different intervals of storage is shown in Table 1.

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When spoilage bacteria, including coliforms types, were introduced into the egg proper, large numbers of bacteria were recovered from every egg examined after 24 hours. The influence of washing dirty eggs in water and subsequently oiling them, prior to storage, upon microbic penetration and growth was studied. In order to accentuate the rate of penetration and growth of bacteria, these eggs were held at 15°C. to 18°C. rather than at the normal storage temperature of approximately 0°C. The data indicate no retardation in the penetration and growth of microorganisms as a result of removing the dirt from dirty eggs by washing. On the contrary, some of the data indicate that a combination of washing and oiling resulted in a more rapid penetration and growth of bacteria in the eggs so treated. Lorenz and associates (1952) recently reviewed the litera-

ture on the influence of washing eggs upon keeping quality, and also added considerable experimental data bearing on this point. Our data, though obtained by different methods, are in substantial agreement with theirs. It is interesting to note that 80 percent of the dirty, untreated eggs did not contain significant numbers of bacteria after six to eight months in storage at a temperature not unfavorable to the growth of the more common types of egg-spoilage bacteria. ACKNOWLEDGEMENT

The authors wish to acknowledge the interest and cooperation of Professors L. F. Payne and T. B. Avery of the Poultry Husbandry Department of Kansas State College. SUMMARY

Only 11 percent of 311 current receipt eggs declared "defective with intact shells" by a professional candler contained bacteria in significant numbers. Seven of 50 commercial "checks" (cracked eggs), selected to exclude thinshell eggs and leakers, were found to contain an average of 7,000,000 bacteria per ml., while the remaining 43 eggs yielded no bacteria on plates containing .01 ml. of mixed yolks and whites. "Checks" comparable to these are sold in retail markets. Penetration and growth of egg-spoilage bacteria were rapid in all normal eggs artificially cracked and contaminated; however, the rate of penetration and growth was much slower in similarly treated thin-shell eggs. When the air sac of eggs was heavily contaminated, no significant increase in bacteria was detected in 70 percent "of the eggs examined during the first 3 days and 13 percent resisted penetration and growth for 2 weeks or longer. The rate of penetration and growth of

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found that the shell membranes had an antibacterial action on spoilage bacteria; similarly Miller and Crawford (1948) observed that eggshell membranes in vitro when washed free of white delayed multiplication of certain egg spoilage bacteria. To gain information on the latter point, large numbers of spoilage bacteria were introduced into the air sac, in which case it was necessary for the bacteria to penetrate only the intact inner membrane. No significant increase in bacteria within the egg could be detected in 70 percent of the eggs examined during the first 3 days. Of the eggs examined during the following 12 days, 87 percent contained large numbers of bacteria; however, the remaining 13 percent resisted penetration and growth up to 2 weeks. These results were comparable to those obtained when large numbers of spoilage bacteria were smeared on intact egg shells and the eggs were stored with shells in a moist condition.

MINOR MINERAL ELEMENTS IN CANNIBALISM

spoilage bacteria in eggs was not retarded either by washing or by washing followed by oiling. REFERENCES Bushnell, L. D., and 0 . Maurer, 1914. Some factors influencing the bacterial content and keeping quality of eggs. Kansas Agric. Expt. Stat. Bull. 201. Lorenz, F. W., P. B. Starr and F. X. Ogasawara, 1952. Spoilage of washed eggs. Poultry Sci. 31 : 204-226.

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Miller, W. A., and L. B. Crawford, 1948. (Unpublished data, Department of Bacteriology, Kansas State College). Report of the Chief of the Bureau of Animal Industry Agricultural Research Administration, 1948. pp. 35. Stuart, L. S., and E. H. McNally, 1943. Bacteriological studies on the egg shell. U. S. Egg Poultry Mag. 49: 28-31. Tanner, F. W., 1944. Microbiology of Foods. Garrard Press, Champaign, Illinois. U.S.D.A. Circular No. 583, 1941. Eggs and egg products: 58-59.

CHARLES P. WILLIMON AND C. L. MORGAN Poultry Department, South Carolina Agricultural Experiment Station, Clemson, S. C. (Received for publication August 18, 1952)

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EATHER pulling, feather eating, and whole oats were fed as the sole cereal cannibalism are phases of a vice in grain as compared with barley, corn, and chickens of all ages that is a serious prob- wheat. Stuart and Charles (1931) relem in many poultry flocks. This vice de- ported that cannibalism had been contracts from the market appearance and trolled through the feeding of pine boughs tends to reduce the quality of broilers. In to chicks. Miller and Bearse (1938) relayers the loss of feather covering un- ported control of cannibalism through the doubtedly increases energy requirements use of oat hulls as a ration supplement for maintenance and egg production. Stud- but failed to reduce cannibalism through ies including both managemant and nu- the use of oat ash or oat hull ash in the trition have been made in an attempt to ration. The possibility of the production find means for the prevention and control of insoluble mineral constituents in the of this vice. While some reduction in ashing process appears not to have been feather pulling and cannibalism has been considered by these investigators. Bearse, observed following certain changes in Miller and McClary (1940) again remanagement and nutrition, a dependable ported that oat hull fiber obtained by solution for the problem has been lacking. dilute acid digestion of oats reduced canCarver (1931) reported a reduction in nibalism and improved quality of feathers feather picking among chickens in battery when used to supplement the ration. Ash brooders and fattening batteries through of the dilute acid extract and a water exthe use of ruby-colored lights. Miller tract of oat hulls was ineffective. and Bearse (1937) reported a marked Davidson, Schaible, Brant and Card reduction in feather picking and cannibal- (1941) reported a marked reduction in ism in birds to 40 weeks of age where cannibalism in White Leghorn pullets through the use of all-mash rations as Technical Contribution No. 204—South Carolina compared with feeding laying mash supAgricultural Experiment Station, Clemson, S. C. plemented with grain. Halpin (1942) rePublished with the approval of the Director.

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The Effect of Minor Nutrient Mineral Elements in the Diet of Chickens on Feather Pulling and Cannibalism