EDUCATION AND PRODUCTION

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1 EDUCATION AND PRODUCTION Hydrogen Peroxide as an Alternative Hatching Egg Disinfectant 1 B. W. SHELDON 2 and J. BRAKE 3 North Carolina State University, Raleigh, North Carolina (Received for publication June 28, 1990) ABSTRACT The present study examined the effectiveness of H2O2 at different concentrations to disinfect broiler hatching eggshell surfaces and to maintain hatching potential. Under pure culture conditions,.50% H 2 2 yielded over a 6 log kill in 30 s of three potential eggshell bacterial contaminants. Under higher H2O2 demands, such as occurs on eggshell surfaces, H2O2 concentrations of 5 % (vol/vol) were required to disinfect the shell surfaces (- 5 log reduction). Hatchability of fertile eggs from a 44-wk-old flock was significantly increased by 2% following spraying 5% H2O2 in comparison to untreated controls. Level of contaminated eggs and "early-dead" embryos were significantly reduced in the H202-treated eggs. In comparison with formaldehyde fumigation, no significant difference in hatchability due to H2O2 treatment was detected in eggs from a 30- or 56-wk-old flock. Eggshell permeability, as measured by egg moisture loss in an incubator, was not significantly affected by H 2 02 (5%) or formaldehyde fumigation when compared with untreated or watersprayed control eggs. These results demonstrated that H 2 02 compared favorably to formaldehyde as a hatching egg disinfectant without adversely affecting hatching potential. Under some conditions, H2O2 actually improved the hatching potential of fertile broiler eggs compared with hatchability of untreated eggs. (Key words: hydrogen peroxide, formaldehyde, disinfection, hatching eggs, hatchability) INTRODUCTION Over the years poultry hatchery operations have increased in size. This change has prompted increased expectations of improved hatchabilities, lower chick mortality, and better broiler performance. An effective hatchery sanitation program is critical to achieve a high level of hatchability and to ensure the production of high quality chicks. To date, formaldehyde has been the recommended fumigant used in hatcheries due to its effectiveness and ease of application (Funk and Irwin, 1955). However, the United States Environmental Protection Agency has recently moved toward regulating the use of formaldehyde under the Toxic Substances Control Act (Anonymous, 1984). In light of this potential regulatory action, alternative sanitizers, such as H2O2, 1 Paper Number FSR90-06 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, NC The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service nor criticism of similar ones not mentioned. Presented at the 79th Annual Meeting of the Poultry Science Association, Blacksburg, VA, August 6-10, Department of Food Science. 'Department of Poultry Science Poultry Science 70: must be explored to determine their suitability for application in the hatchery environment. The environment of a poultry hatchery typically is highly contaminated with a variety of microorganisms that can cause disease in chick populations. Airborne microbial populations are easily spread throughout the hatchery by employee activity and air currents, and then cycled into the setters and hatchers by the ventilation system. Newly hatched chicks may become infected with microorganisms from other hatching eggs or from surfaces within the hatcher. Sheldon and Ball (1986) determined that 75% of the microorganisms isolated during a survey of hatchery air quality were of respirable size (< 5 xm) and may be implicated in the development of avian respiratory disease epidemics. For a review of specific microorganisms associated with early chick mortality, see Magwood (1964a,b), Ernst et al. (1980), and Gardner et al. (1980). In recent studies (Sheldon and Ball, 1986), ozone was an efficacious hatchery disinfectant. Both gaseous and aqueous ozone were capable of inactivating many poultry pathogens that routinely contaminate the surfaces of eggshells, setters, and hatchers (Whistler and Sheldon, 1989a,b,c). In these studies, the microbial population of shell surfaces of fertile eggs at time of set was reduced by >2.5 log 10

2 DISINFECTION OF HATCHING EGGS 1093 following a 2-h treatment using humidified ozone gas (2.8% ozone by weight). Although these results compared favorably with the >3.0 log 10 reduction in shell surface microflora produced by formaldehyde, ozone gas yielded a 50% hatchability of fertile eggs compared with 76.5% for water-misted eggs and 87.5% for untreated eggs. In a separate trial (Whistler and Sheldon, 1989b), fertile eggs fogged with ozonated water for 30 min at time of set had significantly higher hatchabilities (94.3%) in comparison with eggs fogged with water (91.0%) or without treatment (90.9%). Although gaseous ozonation was shown to be effective in reducing microbial populations on the surfaces of hatching eggs, high embryo mortality resulted from overexposing eggs to gaseous ozonation. Hydrogen peroxide has been used successfully for many years as a disinfectant, particularly as a surface decontaminant and sterilant in industrial and commercial sanitation programs (Spaulding et al, 1977). For example, H2O2 is used for the rapid in-line sterilization of packaging materials for aseptic filling and packaging operations in the food and pharmaceutical industries. Unlike formaldehyde, H2O2 is easily evaporated or destroyed after use (i.e., H2O2 readily decomposes to water and oxygen), has no lingering unpleasant odor, and poses minimal safety problems for workers if handled properly. However, like any disinfectant, H2O2 should be handled with caution because as a strong oxidizing agent, H2O2 can irritate the skin, eyes, and mucous membranes and can discolor clothing dyes and hair. Hydrogen peroxide is significantly less expensive to use than ozone, because H2O2 does not require on-site generation, is effective at relatively low concentrations, and has similar bactericidal activities. The feasibility of an alternative disinfectant to replace formaldehyde must be evaluated not only on its disinfectant properties but also on its ability to maintain hatching potential. In order for any agent to kill microorganisms, the agent must be surface-active, but agents that kill bacteria and molds on eggshell surfaces quite probably react with and alter the external physical envelop that covers the shell (i.e., the ''Model NMC-2000, Natureform Hatchery Systems, Jacksonville, PL ^ockville, MD cuticle). The cuticle plays an active role in regulation of respiration and water loss as well as a protective role in preventing microbial invasion into the egg (Brake and Sheldon, 1990). Thus, alteration of this barrier by surface-active agents, such as disinfectants, must be examined to evaluate their effect on hatchability. The objectives of the present study were 1) to evaluate the biocidal effectiveness of H2O2 as an eggshell surface decontaminant and 2) to evaluate the potential effects of H2O2 on eggshell permeability and hatchability. General MATERIALS AND METHODS All eggs used in the present studies were from a commercial strain of broiler breeder hens (Arbor Acres slow-feathering broiler line) and were fed a standard breeder ration (16% CP, 2.89 kcal of ME/g, 3.2% calcium,.45% available phosphorus). Eggs that had been freshly laid onto nesting material (pine shavings) were collected twice daily from the North Carolina State University (NCSU) research farm and randomly assigned to individual treatments. Fecal-contaminated eggshells or eggshells with visible checks were discarded, and any remaining nesting debris was gently removed by hand from the eggshell surface. Following collection, eggs were stored for no longer than 4 days at 18.3 C and 60% relative humidity (RH) prior to initiation of an experiment. All hatchability studies were conducted using a Natureform Mini Setter/Hatcher incubator 4 set at 37.5 C and 53% RH. Test Organisms Cultures were obtained from the following sources: Salmonella typhimurium B-13, Pseudomonas fluorescens B-14, Proteus species B-13, and Aspergillus niger were from the USDA Fermentation Lab, Department of Food Science, NCSU; Staphylococcus aureus was isolated from the NCSU research unit hatchery; and Escherichia coli was from the American Type Culture Collection (ATCC 33625). 5 The microorganisms tested in the current study have either been associated with poultry diseases or could potentially limit hatchability of fertile eggs by penetration through the eggshell.

3 1094 SHELDON AND BRAKE Experimental Protocol Microbial Inactivation Studies. Eighteen- to twenty-hour trypticase soy broth 6 cultures (37 C) of each microorganism identified in the previous paragraph were harvested by centrifugation (5,000 rpm), washed twice in.1% peptone water, and resuspended in 20 ml of. 1 % peptone water to a final concentration of ca to 10 7 and 10 4 to 10 5 cfu/ml for the bacterial and fungal species, respectively. One milliliter of each suspension was transferred to either 50 ml of.5% (vol/vol) H or 50 ml of sterile water. Five-milliliter aliquots of each suspension were sampled at.5,5,10,15,20, and 25 min of exposure for bacterial cultures or extended to 35 min for fungal spores, residual H2O2 was neutralized with 56 ul of catalase (1.24 units per gram of protein), and the surviving microorganisms were enumerated on plate count agar 6 using a Spiral Plater surface plating method. 7 All plates were incubated at 37 C and colonies counted after 48 h. Aspergillus niger spore cultures were prepared using Mycophil agar (MA) plates. 8 Spore crop suspensions were prepared by flooding an MA plate having four distinct fungal colonies with 10 ml of.1% peptone water and mixing with a flamesterilized glass rod. The spore slurry was washed twice as described ana resuspended in 20 ml of.1% peptone water. Each test was replicated three times for each microorganism and a geometric mean calculated. Hatching Egg Disinfection. The effective concentration of H2O2 needed to reduce significantly the surface microflora of hatching eggs was determined in four trials. Briefly, fresh broiler hatching eggs were placed on sterilized plastic egg flats and sprayed to a drip state with either sterile water having a residual total chlorine content of less man.13 mg/l or aqueous solutions of H2O2 using a hand sprayer. 9 Following treatment, the eggs were air-dried (ca. 1 h), and the surviving surface microflora were enumerated using a whole-egg washing technique as described by Brake and Sheldon (1990). The total aerobes (37 C, 48 h), presumptive coliforms (37 C, 24 h), and yeast ^ifco Laboratories, Detroit, MI jmodel C, Spiral Systems Inc., Cincinnati, OH TBecton Dickinson & Co., Cockeysville, MD Model F80 Spray Pal, Delta Industries, Philadelphia, PA and molds (25 C, 72 h) were enumerated using plate count, violet red bile, or acidified potato dextrose agars, respectively. 6 All counts were reported on a per egg basis by multiplying the count per milliliter of wash solution times the volume of egg wash diluent used (18 ml). In Trial 1, 120 hatching eggs were divided into four groups of 30 eggs each and assigned to one of four treatments. The four treatments were an untreated control (Control 1), water spray (Control 2), and an aqueous spray of either.25% or.50% (vol/vol) H2O2. Five eggs per treatment were sampled after air-drying (Day 0) and 1 day of storage (4.4 C), and the surviving microflora (aerobes, coliforms, yeasts, and molds) were enumerated. Trial 2 was a repeat of Trial 1 with the following exceptions. Thirty eggs per treatment were hand-sprayed with either 0 (dry), 1, 5, or 10% (vol/vol) aqueous H2O2, air-dried, and the total surviving aerobes enumerated on five eggs per treatment. Trial 3 was a repeat of Trial 2 with the following exceptions. Thirty eggs per treatment were hand-sprayed with either 0 (dry), 2, 3, or 4% (vol/vol) aqueous H2O2. In Trial 4, 20 eggs per treatment were either untreated (dry control) or hand-sprayed to wetness with either sterile water (wet control) or 5% (vol/vol) H2O2 in water. After air-drying the surviving aerobic microflora and yeasts and molds were enumerated. Geometric means were calculated. Eggshell Permeability. The objective of the present study was to evaluate the effect of spraying either sterile water or H2O2 in water on eggshell permeability. Ninety eggs from each of two broiler breeder flocks, 32 and 55 wk of age, were either left untreated (dry control), handsprayed with either.25 or.33 ml per egg of sterile water (wet control) or 5% (vol/vol) H2O2 (45 eggs per application dosage), or gassed for 30 min with triple strength (3x) formaldehyde (119.8 ml formalin:59.9 g potassium permanganate per 2.83 m 3 ). All eggs were air-dried for 30 min before the start of incubation in a common setter. Shell permeability as determined by water loss was measured at 3 and 6 days of incubation and calculated as described by Brake and Sheldon (1990). Shell permeability is expressed as the milligrams of water lost per day between 72 and 144 h/torr per cm 2 of eggshell surface area. Hatchability Studies. The objective of these three trials was to evaluate the effect of hand-

4 DISINFECTION OF HATCHING EGGS 1095 spraying or fogging H2O2 in water on hatchability. Following treatment, the eggs were airdried, placed in a common setter, transferred at 18 days to a common hatcher, and the adjusted hatch of fertile eggs (minus infertile eggs) was calculated following hatching. At hatch, all of the unhatched eggs were opened and examined for evidence of embryonic development. Eggs were characterized as infertile, early-dead (1 to 7 days), late-dead (8 to 20 days), pipped (the beak had penetrated the shell), or contaminated. In Trial 1,720 eggs, representing 12 separate pens (60 eggs per pen per treatment) of a 44-wk-old, broiler-breeder flock, were either untreated (control) or hand-sprayed to wetness with 5% (vol/vol) H2O2 in water. This flock was housed in a building that showed signs of Pseudomonas contamination due to excessive nest moisture. Trial 2 was conducted in a similar fashion to Trial 1, except that 540 eggs, representing nine separate pens (60 eggs per pen) from a 56-wk-old broiler breeder flock, were treated and set. The treatments were no treatment (control), hand-spraying with 5% (vol/vol) H2O2 in water, or 3x formaldehyde gassing for 60 min. Following gassing with formaldehyde, the setter was vented for 60 min to remove any residual vapors. In Trial 3, 540 eggs, representing nine separate pens (60 eggs per pen) from a 30-wk-old, broiler-breeder flock, were either fogged for 30 min with sterile water or 5% (vol/ vol) H2O2 in water using a fogging system 10 operated at kpa or gassed with 3x formaldehyde for 30 min. All eggs were treated in Natureform setters and placed in a common setter following treatment. Statistical Analyses The data from the microbiological inactivation and eggshell disinfection studies were subjected to a one-way ANOVA and means separated by the least significant difference test (Helwig and Council, 1982). The ANOVA model used the variation within treatment as the error term. Microbial counts were transformed to log 10 prior to statistical analysis. The eggshell permeability and hatch data were subjected to a one-way ANOVA using the variation within 10 Bio-Lab, Inc., Decatur, GA treatment as the error term with the General Linear Model procedure (SAS Institute, 1982). Differences among means were partitioned using the Duncan option. Statements of statistical significance were based on P<.05 unless otherwise noted. RESULTS AND DISCUSSION Microbial Inactivation Studies With the exception of E. coli, S. typhimurium, and A. niger,.5% H2O2 in water significantly (P<.05) reduced the Pseudomonas fluorescens, Proteus species, and Staph, aureus populations to undetectable levels within 30 s of exposure (data not shown). These reductions represent over a 6 log kill. Exposure to.5% H2O2 for 5 or 10 min resulted in no culturable cells of S. typhimurium (6.9 log kill) or E. coli (6.3 log kill), respectively. Aspergillus niger spores were significantly more resistant to H2O2 than the vegetative cells of bacteria but were significantly reduced by 98.7% (1.9 logs) after 35 min of exposure to H2O2. The apparent resistance of fungal spores to H2O2 is associated with the resistant spore coat that surrounds the protoplasm (Foegeding, 1985). Although A. niger has not been associated with chick disease, several other species of this genus (i.e., Aspergillus fumigatus) are highly pathogenic to both man and poultry, resulting in pulmonary aspergillosis. Weston and Hainan (1927) determined that the growth of mold hyphae on eggshell surfaces also facilitates the enlargement of pores for the entry of potential disease causing bacteria into the egg. The results of this study demonstrated that under low H2O2 demand,.5% H2O2 in water easily destroyed bacterial populations ranging from 10 6 to 10 7 /ml. Egg Disinfection Studies Trial 1. No significant differences in the aerobic microflora, coliform, or yeast and mold eggshell counts were detected between the four treatments within 1 or 24 h of treatment (Table 1). Compared with the dry and wet control eggs, less than one log reduction (90%) for all microorganisms examined was achieved following the application of.25% and.50% H2O2 to the shell surfaces. The apparent increase of organic and inorganic loads on eggshell surfaces places a significantly higher demand for H2O2 than when tested under pure culture conditions.

5 1096 SHELDON AND BRAKE TABLE 1. Bactericidal activity (cfu per egg) of.25% and.50% H 2 Oi on the microflora of hatching eggs (geometric means logjo ± SD, n sy Treatment 3.25% H 2 C- 2.50% H % H % H % H % H h 5.27 ± ± ± ± ± ± ± ±.96 Molds 2.82 ± ± ± ±.53 Sampling timsr Aerobic 1 day plate count 5.15 ± 5.00 ± 4.77 ± 4.79 ± Coliforms 1.14 ±.33 ±.97 ±.38 ± and yeast 2.34 ± 2.02 ± 1.84 ± 1.86 ± 'Table reports results of Trial 1 of the batching egg disinfection studies. 2 One hour = 60 min after disinfection; 1 day = 24 h after disinfection. 3 = no treatment; control (wet) = handsprayed to wetness with sterile water (<13 mg chlorine/l;.25% and.50% H = hand-sprayed to wetness with either of these respective concentrations of H in water. Coliforms and yeast and mold populations constituted approximately.03 and.20%, respectively, of the total aerobic microflora. Trials 2 and 3. The effective concentration of H2O2 required to disinfect fertile eggshell surfaces was determined in these two trials. Approximately 90% of the aerobic microflora was eliminated from the shell surfaces with 1% H (Table 2). At 5% and 10%, H effectively eliminated all culturable microorgan- isms (5.3 log reduction). Compared with the untreated control eggs, significant reductions (P<.01) of the aerobic microflora of 99.8% (2.7 logs), 99.8% (2.7 logs), and 99.98% (3.9 logs) were achieved with the 2, 3, and 4% (vol/vol) H2O2 concentrations, respectively. No significant differences between the 2, 3, or 4% concentrations were detected. Five percent H2O2 appears to be the minimal concentration required to completely eliminate all surface microorganisms. The effective concentration, however, would be expected to increase as the level of organic matter increases on the shell surface. Trial 4. The washing effect of spraying eggshell surfaces with water versus 5% H2O2 (vol/vol) was tested (Table 3). Spraying with water alone significantly (P<.01) reduced the aerobic microflora by 88% (.92 logs), and spraying with 5% H2O2 reduced the aerobic microflora on an average of % (4.35 logs). Spraying with water did not significantly reduce the yeast and mold counts yet H2O2 eliminated 97.1% of these microorganisms. The reduction in shell surface microorganisms is primarily attributed to the germicidal properties of H2O2 with minor microbial reductions associated with the washing effects of spraying or residual total chlorine level (<.13 mg/l) in the sterile water. Eggshell Permeability Studies The effect of 3x formaldehyde and water or H2O2 at two dosage levels (.25 versus.33 ml per egg) on eggshell water permeability was determined for eggs from a young (32 wk) and old (55 wk) flock. No significant differences in water loss was detected between the treatments or dosages for either set of eggs (Table 4). The avian eggshell is both a conduit through which TABLE 2. Biocidal activity (cfu per egg) of H2O2 against the eggshell aerobic hatching eggs (geometric means logio ± SD) 1 surface microflora of Trial H concentration, vol/vol ±.46 A 4.34 ±.91 B 0 ± 0 C 4.81 ±.44 A 2.11 ± 2.03 B 2.12 ± 2.00 B 10 0 ± 0 C.95 ± 1.33 B A-C Means ± SD in the same row with no common superscripts are significantly different (P<01). n = 5 eggs per treatment. Surface microflora includes aerobes, coliforms, and yeast and molds. 2 Eggs were hand-sprayed to wetness with either 0, 1, 2, 3, 4, 5, or 10% H in water.

6 DISINFECTION OF HATCHING EGGS 1097 TABLE 3. Effect of hand-spraying sterile water versus 5% by volume H2O2 on the reduction of the eggshell microflora of hatching eggs (geometric means logio ± SDy Aerobic Yeast plate and Treatment 2 count molds (cfu per egg) 4.90 ±.51 A 1.62 ±.66^ 3.98 ±.51 B 1.32 ±.72 A 5% H 2 Q 2.99 ±.91 c.05 ±,21 B A-C Means in the same column with no common superscripts are significantly different (P<01). n = 20 eggs per treatment. 'Table reports results of Trial 4 of the hatching egg disinfection studies. = no treatment; control (wet) = handsprayed to wetness with sterile water (<13 mg chlorine/l); 5% H2O2 = hand-sprayed to wetness with 5% (vol/vol) H2O2 in water. diffusive water vapor, oxygen, and CO2 are permitted to pass and a barrier to the free diffusion of these same substances. This free exchange is affected by the CaC03 portion of the shell and, to a lesser extent, by the shell membranes and cuticle. The porosity of die eggshell to water vapor and vital gases during incubation influences hatchability, embryonic growth, and chick weight at hatch. Thus, any alteration or removal of the cuticle by sanitizers, bacterial growth, abrasion, storage, washing, or improper handling may have a significant impact on the hatchability and livability of the chick. Under the conditions of this study, H2O2 did not significantly affect eggshell permeability. However, due to the oxidation potential of H2O2, some modification of the proteinaceous cuticle would be expected. Hatchability Studies Trials 1 to 3. The effects of H on hatchability are shown in Table 5. Compared with untreated eggs, 5% H2O2 significantly increased the hatchability of fertile eggs from a 44-wk-old flock showing evidence of Pseudomonas contamination in the flock house. This 2.0% increase in hatchability appeared to be due to a significant reduction in the number of contaminated eggs (1.11% difference) and early embryonic mortality (2.23% difference). Vick and Brake (1986) attributed increases in water loss from eggs from young flocks to a significant reduction in early embryonic mortality. Although no significant eggshell permeability differences were detected in these studies, the increases in hatchability that may be attributed to variations in eggshell permeability cannot be ruled out. No significant differences in hatchabiuty were detected between eggs not treated (control, dry) or those fumigated with 3x formaldehyde for 1 h or hand-sprayed with 5% H2O2 (Trial 2). Fogging of eggs with either 5% H2O2 or sterile water in comparison to 3x formaldehyde fumigation yielded no significant hatchability differences although the number of late deads was significantly lower (2.45 percentage points difference) for the peroxide-treated eggs than the formaldehyde-treated eggs (Trial 3). In summary, specific concentrations of H2O2 significantly reduced the population of hatchery microbial contaminants in pure culture as well as on fertile eggshell surfaces but did not affect the functional properties of the eggshell with respect to water loss and gas exchange. Under certain conditions, such as a suspected increase in contaminated eggshell surfaces, hatchability was significantly improved by spraying H2O2 TABLE 4. Effect of spraying 5% H in water at two dosages on the water loss of hatching eggs from young and old flocks _ Shell permeability, 2 Flock T> ( I i n 3 J age Dosage of H 2 Q 2 (wk) Treatment % H Formaldehyde % H Formaldehyde 5.36 = no treatment; control (wet) = handsprayed to wetness with sterile water (<13 mg chlorine/l); 5% H2O2 = hand-sprayed to wetness with 5% (vol/vol) H2O2 in water; formaldehyde=triple strength formaldehyde fumigation for 30 min (119.8 ml formaliru59.9 g potassium permanganate/2.83 m 3 ). n = 15 eggs per treatment 2 Determined as the milligrams of H2O loss per day per torr per square centimeter as measured from 3 to 6 days of incubation. Milliliters of spray delivered per egg.

7 1098 SHELDON AND BRAKE Flock age (wk) TABLE 5. Effect of spraying 5% H2O2 on the hatchability of eggs from young and old flocks Trial 1 number Treatment 2 5% H % H Formaldehyde 5% H Formaldehyde Hatch of fertile eggs 92.7 b 94.7" a 1.94 b Earlydead Latedead * b 1.51 b 3.96* Cl!\ Pipped Contaminated a,b Means in the same column and within trials with no common superscripts are significantly different (P<05). 'Trial 1 = 720 eggs per treatment were set; Trial 2 = 540 eggs per treatment were set; Trial 3 = 540 eggs per treatment were set. ^ = no treatment; control (wet) = hand-sprayed to wetness with sterile water (<13 mg chlorine/ L); 5% hydrogen peroxide = hand-sprayed to wetness with 5% (vol/vol) hydrogen peroxide in water (Trials 1 and 2) or fogged for 30 min at kpa (Trial 3); formaldehyde = triple strength formaldehyde fumigation for 60 min (Trial 2) and 30 min (Trial 3) (119.8 ml formalin:59.9 g potassium permanganate/2.83 m 3 ). 1.25*.14" versus no treatment. As a hatching egg disinfectant, H2O2 appears to be at least equal to or perhaps slightly better than formaldehyde. ACKNOWLEDGMENTS The research reported in this publication was funded in part by the North Carolina Agricultural Research Service and Degussa Corporation. The authors appreciate the excellent technical support of Grace Brockman, Susan Creech, and Susan Hale. REFERENCES Anonymous, Formaldehyde may face regulation. Chem. Eng. News 62:8. Brake, J., and B. W. Sheldon, Effect of a quaternary ammonium sanitizer for hatching eggs on their contamination, permeability, water loss, and hatchability. Poultry Sci. 69: Ernst, R. A., A. A. Bickford, and J. Glick-Smith, Microbiological monitoring of hatcheries and hatching eggs. Poultry Sci. 59:1604.(Abstr.) Foegeding, P. M., Ozone inactivation of Bacillus and Clostridium spore populations and the importance of the spore coat to resistance. Food Microbiol. 2: Funk, E. M., and R. M. Irwin, Prevention and control of diseases in the hatchery. Pages in: Hatchery Operation and Management. John Wiley and Sons, Inc., New York, NY. Gardner, F. A., F. A. Golan, and J. H. Denton, Microbiological parameters associated with hatching turkeys. Poultry Sci. 59: Helwig, J. T., and K. A. Council, SAS User's Guide, 1982 ed. SAS Institute, Inc., Cary, NC. Magwood, S. E., 1964a. Studies in hatchery sanitation. 1. Fluctuations in microbial counts of air in poultry hatcheries. Poultry Sci. 43: Magwood, S. E., 1964b. Studies in hatchery sanitation. 2. The effect of air-borne bacterial populations on contamination of egg and embryo surfaces. Poultry Sci. 43: SAS Institute, SAS User's Guide: Statistics, 1982 ed. SAS Institute, Inc., Cary, NC. Sheldon, B. W., and H. R. Ball, Jr., Efficacy of ozone disinfection in poultry hatcheries. Industry Summary Report 119, Southeastern Poultry and Egg Association, Decatur, GA. Spaulding, E. H., K. R. Cundy, and F. J. Turner, Chemical disinfection of medical and surgical materials. Pages in: Disinfection, Sterilization and Preservation. S. S. Block, ed. Lea and Febiger, Philadelphia, PA. Vick, S. V., and J. Brake, Effect of incubation humidity on hatchability with respect to egg weight and flock age. Poultry Sci. 65(Suppl. l):130.(abstr.) Weston, W.A.R.D., and E. T. Hainan, Black spot of eggs. Poultry Sci. 6: Whistler, P. E., and B. W. Sheldon, 1989a. Biocidal activity of ozone versus formaldehyde against poultry pathogens inoculated in a prototype setter. Poultry Sci. 68: Whistler, P. E., and B. W. Sheldon, 1989b. Bactericidal activity, eggshell conductance, and hatchability effects of ozone versus formaldehyde disinfection. Poultry Sci. 68: Whistler, P. E., and B. W. Sheldon, 1989c. Comparison of ozone and formaldehyde as poultry hatchery disinfectants. Poultry Sci. 68: