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Studies on Incubator Hygiene, III. Germicidal Properties of Formaldehyde, Sulfur Dioxide, Chloropicrin and Chloracetophenone
Studies on Incubator Hygiene, I I I . Germicidal Properties of Formaldehyde, Sulfur Dioxide, Chloropicrin and Chloracetophenone ROBERT GRAHAM AND V. M.
MICHAEL
University of Illinois, Urbana (Received for Publication July 11, 1933)
" O ESULTS of studies on incubator hy-*-*• giene previously reported (1932a and b) suggest that formaldehyde fumigation conducted under proper conditions is of value in destroying S. pullorum on artificially contaminated cotton squares, chick feet, and down in two forced-draft incubatorsf. These findings further suggest that the spontaneous spread of pullorum disease in the routine of hatching chicks may be controlled by approved measures of sanitation and fumigation. In incubator hygiene studies, different disinfectants have been employed, but formaldehyde has been found the most efficient, the least expensive, and the most adaptable to forced-draft incubators. Methods of Releasing Formaldehyde Various methods of releasing formaldehyde in incubators have been employed, but only the potassium permanganate and the formolized cheesecloth methods have been found to be practical. The potassium permanganate method requires 35 cubic centimeters of formalin and 17.5 grams of potassium permanganate per 100 cubic feet of incubator space. The gas is released by pouring formalin over the permanganate crystals in a pan which may be placed under the circulating fans on the inside of the t Type A, Buckeye Mammoth, No. 44; Type B, Smith Junior. [40]
incubator or in a cabinet under the intake pipe on the outside of the incubator. The formolized cheesecloth method requires 16 to 25 cubic centimeters of formalin per 100 cubic feet of incubator space (approximately 2/3 of the amount required for the potassium permanganate method) and a strip of cheesecloth large enough to absorb all of the formalin without dripping. The cheesecloth may be formolized at the time of fumigation or it may be formolized several days or weeks beforehand and kept in an air-tight container, such as a fruit jar, until used. The formolized cloth is suspended under or near the circulation fans and the formalin allowed to evaporate. Cleanliness and Humidity With either method, efficient fumigation depends on clean incubators and a humidity of 88 to 90°F. (wet bulb). The cheesecloth method is less expensive and easier to dispatch and is more efficient per unit of formaldehyde. Increased efficiency is due in part to direct method of release in the incubator. The same cheesecloth can be used following reformolizing and no potassium permanganate is required. It has been found that chicks should remain in the incubator for three hours after the release of the formaldehyde with either method of fumiga-
JANUARY,
1934.
VOL.
X I I I , No. 1
tion. When the permanganate method is used three releases are employed during a hatching period while two releases with the cheesecloth method appear to be sufficient. On the day preceding the hatch it is advisable to release a double amount of formalin in the two forced-draft incubators referred to in this discussion in order to kill dormant infections which may have carried over from a previous hatch. Formaldehyde in amounts recommended for hatching chicks does not influence hatchability or injure the chicks at hatching time. Germicidal and Safety Range; Other Disinfectants Studies on incubator fumigation reported herein are concerned with the following questions: (a) How long is formaldehyde retained in germicidal amounts in forceddraft incubators under normal hatching conditions? (b) Can hatching chicks be safely fumigated with double the amount of formaldehyde recommended for use during the hatching period? (c) Is a massive S. pullorum droplet infection in forced-draft incubators destroyed by routine amounts of formalin recommended during the hatching period? (d) Are chemicals other than formalin, such as sulfur dioxide, chloropicrin, and chloracetophenone efficient fumigants for forced-draft incubators? In these studies eggs from non-reacting flocks were used. PART I. PERSISTENCE OF GERMICIDAL PROPERTIES OF FORMALDEHYDE IN FORCED-DRAFT INCUBATOR, TYPE B
In 13 fumigations, 33 1/3 cubic centimeters of formalin per 100 cubic feet of incubator space (the amount used on the day preceding hatching) were released in forceddraft incubator of type B by the cheesecloth method. Chicks and cotton squares contaminated with S. pullorum were placed in the incubator at the time the fumigant
41
was released. Cultures on suitable media were made from the S. pullorum contaminated squares in the doors and from contaminated chicks in the egg and hatching trays at 20 minute and 30 minute intervals for 3 hours. With 2 exceptions S. pullorum on cotton squares on the inside of the false doors was killed in less than 20 minutes. In one fumigation S. pullorum survived 90 minutes in the upper part of the front door, and in another fumigation it survived 40 minutes in one of the upper side doors. The average lethal time of S. pullorum was 21.6 minutes. When exposed on cotton squares in petri dishes placed in egg trays, S. pullorum was killed in an average time of 45+ minutes and in hatching trays in 52.5+ minutes. In the latter, S. pullorum survived the full three hours of the testing period in one fumigation, but in the majority of fumigations S. pullorum was not found on cotton squares exposed in petri dishes at the end of 30 minutes. On chick feet, S. pullorum was killed in an average time of 50+ minutes and on chick down in 32.5 minutes. In only one fumigation was S. pullorum found to be virulent on chick feet at the end of 120 minutes. Three hours after the release of the formaldehyde in type B forced-draft incubator in amounts heretofore mentioned S. pullorum contaminated cotton squares and chicks were placed in the incubator and cultures made at intervals of one hour, to determine if germicidal amounts of formaldehyde remained in the incubator. With few exceptions it was found that S. pullorum exposed on cotton squares in doors, in petri dishes in egg and hatching trays, and on chicks was still virulent at the end of three hours' exposure or a period of 6 hours after the release of the formaldehyde. Thus, germicidal amounts of formaldehyde for S. pullorum were not present in forced-draft incu-
42 bator of type B three hours after routine fumigation. PART II. EFFECT ON CHICKS OF DOUBLE AMOUNTS OF FORMALDEHYDE Chicks fumigated with 33 1/3 cubic centimeters of formalin (double amount recommended while chicks are hatching) released by cheesecloth method per 100 cubic feet of incubator space in forced-draft incubator, type B, were seriously injured. At the end of three hours' fumigation, with above amounts of formaldehyde, approximately one-half of the chicks showed heavy mouth breathing. On the day following fumigation 83 (40.8 percent) of the 203 chicks fumigated were dead. S. pullorum was not isolated from the livers of any of these chicks at autopsy. PART III. SULFUR DIOXIDE, CHLOROPICRIN, CHLORACETOPHENONE Amounts of sulfur dioxide ranging from one-fourth to five pounds were released in forced-draft incubator, type B, approximately 550 cubic feet capacity. When one pound and five pound amounts were released, S. pullorum was killed in less than 20 minutes on cotton squares on the inside of the false doors and in petri dishes in hatching trays. When one-fourth and onehalf-pound amounts were released, S. pullorum was killed in less than 60 minutes. Two chicks exposed to a five-pound release of sulfur dioxide died before all of the gas had been released. Two chicks exposed to one-half pound release died within three hours after fumigation while those exposed to one-fourth pound died within twentyfour hours after the release. None of the eggs which were fumigated with sulfur dioxide eight days before hatching time hatched, while only 14 percent of those fumigated 15 days before hatching time hatched. It appears, therefore, that the lethal properties of sulfur dioxide for chicks and embryos preclude its use as a
POULTRY
SCIENCE
fumigant notwithstanding its germicidal properties for S. pullorum. Neither chloracetophenone (10 grams in 90 cubic centimeters carbon tetrachloride) nor chloropicrin (10 cubic centimeters in 90 cubic centimeters paraffin oil) exhibited any appreciable germicidal effect when released in forced-draft incubator, type B. S. pullorum was still virulent on cotton squares exposed on the inside of the false doors and in petri dishes in egg and hatching trays at the end of 90 minutes (doors) and 180 minutes (petri dishes). Chloropicrin in amounts mentioned above caused the death of 12-week-old chicks in less than 3 hours. PART IV. EFFECT OF FUMIGATION WITH FORMALDEHYDE ON S. PULLORUM SPRAYED INTO FORCED-DRAFT INCUBATOR, TYPE B Immediately after the release of 16 2/3 cubic centimeters of formalin per 100 cubic feet in forced-draft incubator, type B, 500 cubic centimeters of broth culture 5. pullorum, approximate density of McFarland's nephelometer No. 4, were sprayed by means of an electric spray through a false door in the back of the incubator. The fumigant and culture were released when approximately one-fifth of the chicks were hatched. Following the simultaneous exposure of chicks to the fumigant and S. pullorum, chick liveability records were kept for two weeks. Three hatches (574 chicks) were exposed in this manner. One-hundred thirtyone (22.82 percent) died during the period of observation. Thirty-six or approximately one-fourth were found upon autopsy to be infected with 5. pullorum, which was not found in 36 yolks of eggs which did not hatch. Three control hatches exposed to S. pullorum spray in the manner mentioned without formalin fumigation showed a higher mortality. Of the 559 chicks exposed in this manner 195 (34.88 percent) died during the
JANUARY,
1934.
VOL.
XIII, No. 1
first two weeks after hatching. Approximately 53 percent (103) of these yielded positive cultures of S. pullorum. SUMMARY
Formaldehyde released in germicidal amounts for S. pullorum in a forced-draft incubator was not retained in the incubator in sufficient amounts three hours later to destroy the organism. Double amounts of formaldehyde employed at time of hatching proved injurious to chicks, precluding the use of larger amounts of formaldehyde than previously recommended in routine hatching.
43
A massive 5. pullorum infection introduced into the incubator at hatching time was appreciably reduced by fumigation as judged by chick liveability and bacteriological examination of contaminated swabs subjected to fumigation. Sulfur dioxide, chloropicrin, and chloracetophenone were found to be impractical for incubator disinfection. REFERENCES
Graham, Robt. and V. M. Michael, 1932a. Studies in incubator hygiene, I. Poul. Sci. 11:110-116. , 1932b. Studies in incubator hygiene, II. Poul. Sci. 11:197-207.
MICHAEL
University of Illinois, Urbana (Received for Publication July 11, 1933)
" O ESULTS of studies on incubator hy-*-*• giene previously reported (1932a and b) suggest that formaldehyde fumigation conducted under proper conditions is of value in destroying S. pullorum on artificially contaminated cotton squares, chick feet, and down in two forced-draft incubatorsf. These findings further suggest that the spontaneous spread of pullorum disease in the routine of hatching chicks may be controlled by approved measures of sanitation and fumigation. In incubator hygiene studies, different disinfectants have been employed, but formaldehyde has been found the most efficient, the least expensive, and the most adaptable to forced-draft incubators. Methods of Releasing Formaldehyde Various methods of releasing formaldehyde in incubators have been employed, but only the potassium permanganate and the formolized cheesecloth methods have been found to be practical. The potassium permanganate method requires 35 cubic centimeters of formalin and 17.5 grams of potassium permanganate per 100 cubic feet of incubator space. The gas is released by pouring formalin over the permanganate crystals in a pan which may be placed under the circulating fans on the inside of the t Type A, Buckeye Mammoth, No. 44; Type B, Smith Junior. [40]
incubator or in a cabinet under the intake pipe on the outside of the incubator. The formolized cheesecloth method requires 16 to 25 cubic centimeters of formalin per 100 cubic feet of incubator space (approximately 2/3 of the amount required for the potassium permanganate method) and a strip of cheesecloth large enough to absorb all of the formalin without dripping. The cheesecloth may be formolized at the time of fumigation or it may be formolized several days or weeks beforehand and kept in an air-tight container, such as a fruit jar, until used. The formolized cloth is suspended under or near the circulation fans and the formalin allowed to evaporate. Cleanliness and Humidity With either method, efficient fumigation depends on clean incubators and a humidity of 88 to 90°F. (wet bulb). The cheesecloth method is less expensive and easier to dispatch and is more efficient per unit of formaldehyde. Increased efficiency is due in part to direct method of release in the incubator. The same cheesecloth can be used following reformolizing and no potassium permanganate is required. It has been found that chicks should remain in the incubator for three hours after the release of the formaldehyde with either method of fumiga-
JANUARY,
1934.
VOL.
X I I I , No. 1
tion. When the permanganate method is used three releases are employed during a hatching period while two releases with the cheesecloth method appear to be sufficient. On the day preceding the hatch it is advisable to release a double amount of formalin in the two forced-draft incubators referred to in this discussion in order to kill dormant infections which may have carried over from a previous hatch. Formaldehyde in amounts recommended for hatching chicks does not influence hatchability or injure the chicks at hatching time. Germicidal and Safety Range; Other Disinfectants Studies on incubator fumigation reported herein are concerned with the following questions: (a) How long is formaldehyde retained in germicidal amounts in forceddraft incubators under normal hatching conditions? (b) Can hatching chicks be safely fumigated with double the amount of formaldehyde recommended for use during the hatching period? (c) Is a massive S. pullorum droplet infection in forced-draft incubators destroyed by routine amounts of formalin recommended during the hatching period? (d) Are chemicals other than formalin, such as sulfur dioxide, chloropicrin, and chloracetophenone efficient fumigants for forced-draft incubators? In these studies eggs from non-reacting flocks were used. PART I. PERSISTENCE OF GERMICIDAL PROPERTIES OF FORMALDEHYDE IN FORCED-DRAFT INCUBATOR, TYPE B
In 13 fumigations, 33 1/3 cubic centimeters of formalin per 100 cubic feet of incubator space (the amount used on the day preceding hatching) were released in forceddraft incubator of type B by the cheesecloth method. Chicks and cotton squares contaminated with S. pullorum were placed in the incubator at the time the fumigant
41
was released. Cultures on suitable media were made from the S. pullorum contaminated squares in the doors and from contaminated chicks in the egg and hatching trays at 20 minute and 30 minute intervals for 3 hours. With 2 exceptions S. pullorum on cotton squares on the inside of the false doors was killed in less than 20 minutes. In one fumigation S. pullorum survived 90 minutes in the upper part of the front door, and in another fumigation it survived 40 minutes in one of the upper side doors. The average lethal time of S. pullorum was 21.6 minutes. When exposed on cotton squares in petri dishes placed in egg trays, S. pullorum was killed in an average time of 45+ minutes and in hatching trays in 52.5+ minutes. In the latter, S. pullorum survived the full three hours of the testing period in one fumigation, but in the majority of fumigations S. pullorum was not found on cotton squares exposed in petri dishes at the end of 30 minutes. On chick feet, S. pullorum was killed in an average time of 50+ minutes and on chick down in 32.5 minutes. In only one fumigation was S. pullorum found to be virulent on chick feet at the end of 120 minutes. Three hours after the release of the formaldehyde in type B forced-draft incubator in amounts heretofore mentioned S. pullorum contaminated cotton squares and chicks were placed in the incubator and cultures made at intervals of one hour, to determine if germicidal amounts of formaldehyde remained in the incubator. With few exceptions it was found that S. pullorum exposed on cotton squares in doors, in petri dishes in egg and hatching trays, and on chicks was still virulent at the end of three hours' exposure or a period of 6 hours after the release of the formaldehyde. Thus, germicidal amounts of formaldehyde for S. pullorum were not present in forced-draft incu-
42 bator of type B three hours after routine fumigation. PART II. EFFECT ON CHICKS OF DOUBLE AMOUNTS OF FORMALDEHYDE Chicks fumigated with 33 1/3 cubic centimeters of formalin (double amount recommended while chicks are hatching) released by cheesecloth method per 100 cubic feet of incubator space in forced-draft incubator, type B, were seriously injured. At the end of three hours' fumigation, with above amounts of formaldehyde, approximately one-half of the chicks showed heavy mouth breathing. On the day following fumigation 83 (40.8 percent) of the 203 chicks fumigated were dead. S. pullorum was not isolated from the livers of any of these chicks at autopsy. PART III. SULFUR DIOXIDE, CHLOROPICRIN, CHLORACETOPHENONE Amounts of sulfur dioxide ranging from one-fourth to five pounds were released in forced-draft incubator, type B, approximately 550 cubic feet capacity. When one pound and five pound amounts were released, S. pullorum was killed in less than 20 minutes on cotton squares on the inside of the false doors and in petri dishes in hatching trays. When one-fourth and onehalf-pound amounts were released, S. pullorum was killed in less than 60 minutes. Two chicks exposed to a five-pound release of sulfur dioxide died before all of the gas had been released. Two chicks exposed to one-half pound release died within three hours after fumigation while those exposed to one-fourth pound died within twentyfour hours after the release. None of the eggs which were fumigated with sulfur dioxide eight days before hatching time hatched, while only 14 percent of those fumigated 15 days before hatching time hatched. It appears, therefore, that the lethal properties of sulfur dioxide for chicks and embryos preclude its use as a
POULTRY
SCIENCE
fumigant notwithstanding its germicidal properties for S. pullorum. Neither chloracetophenone (10 grams in 90 cubic centimeters carbon tetrachloride) nor chloropicrin (10 cubic centimeters in 90 cubic centimeters paraffin oil) exhibited any appreciable germicidal effect when released in forced-draft incubator, type B. S. pullorum was still virulent on cotton squares exposed on the inside of the false doors and in petri dishes in egg and hatching trays at the end of 90 minutes (doors) and 180 minutes (petri dishes). Chloropicrin in amounts mentioned above caused the death of 12-week-old chicks in less than 3 hours. PART IV. EFFECT OF FUMIGATION WITH FORMALDEHYDE ON S. PULLORUM SPRAYED INTO FORCED-DRAFT INCUBATOR, TYPE B Immediately after the release of 16 2/3 cubic centimeters of formalin per 100 cubic feet in forced-draft incubator, type B, 500 cubic centimeters of broth culture 5. pullorum, approximate density of McFarland's nephelometer No. 4, were sprayed by means of an electric spray through a false door in the back of the incubator. The fumigant and culture were released when approximately one-fifth of the chicks were hatched. Following the simultaneous exposure of chicks to the fumigant and S. pullorum, chick liveability records were kept for two weeks. Three hatches (574 chicks) were exposed in this manner. One-hundred thirtyone (22.82 percent) died during the period of observation. Thirty-six or approximately one-fourth were found upon autopsy to be infected with 5. pullorum, which was not found in 36 yolks of eggs which did not hatch. Three control hatches exposed to S. pullorum spray in the manner mentioned without formalin fumigation showed a higher mortality. Of the 559 chicks exposed in this manner 195 (34.88 percent) died during the
JANUARY,
1934.
VOL.
XIII, No. 1
first two weeks after hatching. Approximately 53 percent (103) of these yielded positive cultures of S. pullorum. SUMMARY
Formaldehyde released in germicidal amounts for S. pullorum in a forced-draft incubator was not retained in the incubator in sufficient amounts three hours later to destroy the organism. Double amounts of formaldehyde employed at time of hatching proved injurious to chicks, precluding the use of larger amounts of formaldehyde than previously recommended in routine hatching.
43
A massive 5. pullorum infection introduced into the incubator at hatching time was appreciably reduced by fumigation as judged by chick liveability and bacteriological examination of contaminated swabs subjected to fumigation. Sulfur dioxide, chloropicrin, and chloracetophenone were found to be impractical for incubator disinfection. REFERENCES
Graham, Robt. and V. M. Michael, 1932a. Studies in incubator hygiene, I. Poul. Sci. 11:110-116. , 1932b. Studies in incubator hygiene, II. Poul. Sci. 11:197-207.