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Microbiological Monitoring of Hatchery Sanitation
Microbiological Monitoring of Hatchery Sanitation KATHLEEN SOUCY, C. J. RANDALL, and R. A. HOLLEY 1 Food Production and Inspection Branch, Agriculture Canada, Ottawa, Ontario, Canada K1A 0Y9 (Received for publication July 6, 1982)
1983 Poultry Science 62:298-309 INTRODUCTION
The importance of an effective hatchery sanitation program to achieve a high level of hatchability (Hacking, 1982) and to ensure the production of high quality chicks has been firmly established (Chute and Gershman, 1961; Gentry et ai, 1962; Shane, 1981). Visual scrutiny of the environmental condition of a hatchery can be an important method of evaluating the sanitation procedures used. To assure management that their sanitation program is working effectively to prevent the build up of contamination between successive hatches, a more objective method of monitoring the level of microorganisms on surfaces and in the air is necessary. Most hatcheries have neither the facilities nor the expertise to implement routine microbiological testing; thus, there is a need for the adoption of representative and simple monitoring procedures that can be conducted and interpreted in the hatchery without recourse to an outside laboratory. An agar contact technique using Rodac plates was evaluated by Favero et al. (1968)
1 Contribution Number 505, Food Research Institute, Research Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6.
and was found to be an excellent method for use in field studies because of its portability and facility in sampling flat, smooth surfaces. This method is best adapted to measure low numbers of organisms, i.e., 5 to 50 cfu/10 cm 2 (Niskanen and Pohja, 1977). Molds, spreading colonies and debris on the agar surface, complicate the enumeration of colony forming units on Rodac plates. Niskanen and Pohja (1977) compared the recovery of bacteria from various plastic, wood, and steel surfaces using the agar contact and swab methods. Swabbing was found to be the better technique for the recovery of bacteria from uneven surfaces and heavily contaminated areas. Stinson and Tiwari (1978) compared the Millipore Swab Test Kit (MSTK), the Rodac procedure of surface sampling, and a contacttransfer method (Con-Tact-It System Bacteria Detection Unit, Birko Chemical Corporation) with the standard swab procedure as quick methods for the assessment of food plant sanitation. There was a significant correlation between results obtained from the MSTK and standard swab procedures. The MSTK was found to be more accurate than the Rodac plates. The swabbing methods recovered more contaminants than either the Con-Tact-It or Rodac procedures. 298
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ABSTRACT A comparative microbiological evaluation of sanitation practices used in six Canadian hatcheries was conducted by means of a commercially available swab kit. An assessment was made of its potential for routine use in monitoring hatchery sanitation by plant personnel. Hatcher machine inlets, exhausts, and room floors were identified as reservoirs of organisms after regular clean-up procedures had been completed. Isolated hatchery organisms were identified and the four test kit media were evaluated for their ability to exclude or support the growth of a variety of organisms. Nonselective bacteriological media in the kit were most informative for routine sanitation monitoring. A comparison between the swab kit and the direct surface agar plate techniques was undertaken in laboratory tests during which microorganisms isolated from poultry hatcheries were recovered from samples of hatchery surfaces. There was no significant difference between the two methods for recovery of Pseudomonas fragi, E, coli, Proteus, and Staphylococcus, but better recoveries were obtained for another strain of Pseudomonas and Salmonella hadar when the direct surface agar plate technique was used. Better recovery of Paecilomyces variotii was obtained when the swab kit technique was used. The lethal effect of drying on poultry microorganisms was evaluated after air drying the organisms on membrane filters. (Key words: hatchery, sanitation, surface sampling, swab kit)
299
HATCHERY SANITATION
MATERIALS AND METHODS
Hatchery
Contamination
To determine the level of microorganisms in the poultry hatcheries monitored (five commercial hatcheries and one government hatchery) the MSTK method was used in conjunction with air exposure (open) plates (Harrigan and McCance, 1976) and hatcher fluff sampling (Wright and Epps, 1958). The MSTK technique was used to collect bacteria and mold from the walls, floors, machines, and equipment within the hatchery. Air exposure plates were used to detect the level of airborne contamination in the hatchers, incubators and rooms. Hatcher fluff samples were analyzed to evaluate the sanitation procedures used in the hatcheries. Various sites in the commercial hatcheries were sampled when they were expected to be dirtiest and cleanest. (For example, hatcher machines were sampled just prior to being filled with eggs and during the hatching process.) Equipment was sampled before and after use. Swab Test Method. The MSTK system consists of four types of color coded media, which include the following: total bacteria MTSK (white), coliform MCSK (blue), yeast and mold MYSK (yellow), and for pseudomonad enumeration, MPSK (green). These were obtained from Millipore Ltd., 3610 Nashua Drive, Mississauga, Ontario L4V 1L2. The method of sampling used was the procdure outlined by the manufacturer in documents AB820, PM810, and PB427. Each site was sampled by swabbing the area within a 100 cm 2 stainless steel template by rotating the swab while swabbing at two right angle directions. The swab was then placed in a .1 mM phosphate buffer (pH 7.2) containing thiosulfate and a quaternary ammonium compound neutralizer. When dilutions were required, an aliquot of the buffer plus sample solution was added to dilution buffer without neutralizing com-
pounds. The inoculated samplers were incubated in an operating chick incubator at 37.5 C and 92% relative humidity for 48 hr to obtain bacterial counts and 5 days for mold counts. Air Sampling. Prepoured agar in petri dishes was exposed by gravity to atmospheric contamination. Dishes of solidified plate count agar (PCA) that contained 75 ppm cycloheximide (ICN Pharmaceuticals, Cleveland, OH) to inhibit mold growth, Levine eosin methylene blue agar (EMB), and potato dextrose agar (PDA) were used. These agar media were obtained from Difco Laboratories, Detroit, MI. A paper towel was placed on the area to be sampled. The petri dish lid was removed and placed beside the petri dish bottom without inversion. The plates were exposed for 5 min in the hatchers and areas where chicks and fluff were present, whereas 15 min exposures were used in all other areas. The PCA and EMB plates were incubated for 48 hr at 37 C. The PDA plates were incubated for 5 days at 3 7 C. Fluff Sampling. Wright and Epps (1958) found that the microflora of the fluff, produced by hatching chicks, was a reliable criterion of the sanitation condition of a hatchery. Sanitation ratings of A, B, C, or D were given to the hatcheries monitored based upon a standardized bacteriological technique outlined by Wright etal. (1959). Identification
of Hatchery Isolates
Organisms isolated in the hatcheries were classified according to the scheme of Vanderzant and Nickelson (1969) and Buchanan and Gibbons (1974). The general types of organisms found in hatcheries were determined and the selectivity of the MSTK's coliform, pseudomonad, and yeast and mold samplers were studied. Representative colonies isolated from the MSTK samplers and the air exposure plates were grown in nutrient broth at 37 C. After 24 hr, colonies were identified by the following methods: Gram stain, morphology, oxidase test, urea hydrolysis, the production of ammonia from arginine, the liquifaction of gelatin, the production of gas in brilliant green lactose bile broth, and motility. Laboratory Contamination of Hatchery Surfaces The MSTK and DSAP techniques were compared under laboratory conditions for their
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The purpose of this study was to evaluate the use of the MSTK system as an effective hatchery sanitation monitoring tool by comparison with the direct surface agar plate (DSAP) procedure (Angelotti and Foter, 1958; Favero et al., 1968) for their ability to recover organisms from contaminated laboratory surfaces. The MSTK was also evaluated in commercial hatcheries known to practice excellent, good, average, or poor sanitation procedures.
300
SOUCY ET AL. floor hatcher machine
floor hatcher r o o m
air-inlet hatcher machine
air-exhaust hatcher machine
outside t o p hatcher machine
107 AFTER CLEAN-UP BEFORE CLEAN-UP U
10 6
-
<
105
o o 10 4
-
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2
103
or. LU CO
D Z
102-
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 6
HATCHERY NUMBER
FIG. 1. Number of viable bacteria found on hatchery surfaces before and after clean-up using the MSTK method (total counter).
ability to recover microorganisms from samples of typical surfaces found in the hatchery. The DSAP procedure was used as the reference method because studies by Favero et al. (1968) showed the DSAP method to be an accurate laboratory technique for enumerating surface contaminants. Cultures. For this part of the study, microorganisms isolated from poultry fluff samples plus a culture of Pseudomonas fragi (ATCC 27362) were used. C. Poppe, Animal Pathology Laboratory, Agriculture Canada, Winnipeg, kindly provided isolates of Pseudomonas, Staphylococcus, Proteus, and E. coli, A mold identified as Paecilomyces variotii was obtained during this study from one of the poultry hatcheries. A culture of Salmonella hadar, isolated from a poultry hatchery, was kindly provided by N. A. Epps, Department Microbiology, University of Guelph, Guelph, Ontario. Cells were harvested in their early stationary
phase of growth. These spores were used to contaminate three smooth test surfaces: epoxy. terazzo tile (supplied by Durie Mosaic and Marble Limited, Ottawa, Ontario), glass, and a fiberglass hatcher tray (Robbins H10). Both the MSTK and DSAP methods were used to recover the organisms from the contaminated test surfaces. Growth of Test Cultures: Bacteria. Cells in their stationary phase of growth were obtained by inoculation of 10 ml of trypticase soy broth (Difco) with a loopful of growth from the surface of an agar slant stock culture of the organism ( < 3 0 days old). The subculture was incubated at 37 C for 48 hr. A .2 ml aliquot of the 48 hr culture was used to inoculate 50 ml trypticase soy broth in a 250 ml Klett sidearm flask. Another 50 ml trypticase soy broth was retained, uninoculated, in a 250 ml sidearm flask to serve as a reagent blank,. A zero reading was taken immediately after inoculation of the
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tier D en
301
HATCHERY SANITATION
107n
106
chick r o o m floor
chick vaccinating machine
chick work table
:
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hatching trays
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vacuum lift .cups
conveyor belt for chicks ^
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102
12356
123
2345
1234
3 4
5 6
HATCHERY NUMBER FIG. 2. Number of viable bacteria found on hatchery surfaces before and after clean-up using the MSTK method (total counter).
growth flask. Organisms were incubated for 16 hr at 37 C, and then hourly readings were taken during the next 8 hr period. Growth was monitored until the stationary phase, determined graphically, was reached. Growth of Test Cultures: Mold. Mold spores were obtained from a pure culture which had been incubated at 37 C for 5 days on potato dextrose agar (Difco) by washing cells from the agar surface with 5 ml peptone water. A Roux flask containing 200 ml solidified, potato dextrose agar was flooded with the cell suspension and incubated at 37 C for 7 days. Contamination of Test Surfaces: Bacteria. A 10 ml aliquot of cells in their stationary phase of growth was centrifuged for 8 min at 5000 rpm using the Spx rotor of a model GLC-1 Sorvall centrifuge. The cells were resuspended in 10 ml of sterile water, and the suspension was adjusted to 80% transmittance (Spectronic
20 colorimeter, Bausch and Lomb) by dilution. One milliliter of this cell suspension was added to a 99 ml dilution blank of sterile water and used to artificially contaminate the typical poultry hatchery surfaces described earlier. Contamination of Test Surfaces: Mold. Spores were harvested by washing the growth from the potato dextrose agar surface with 100 ml sterile water. The spore suspension was filtered through sterile glass wool and then centrifuged for 10 min at 5000 rpm using the Spx rotor of a model GLC-1 Sorvall centrifuge. The spores were resuspended in 20 ml sterile water and the suspension was diluted to a concentration of 10 4 spores per milliliter of sterile water. One milliliter of the diluted spore suspension was pipetted onto 100 cm of the test surfaces. The surfaces were then swabbed using the MSTK system at the following times: immediately after contamination, 10 min after
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E
5
SOUCYET AL.
302 1Q6 -i MOLD BACTERIA Ul o <
104-
t/i CM
Setter machine air inlet
Setter machine floor
Setter r o o m air inlet
T h e filters had been placed in e m p t y sterile petri dishes and following their inoculation were allowed to dry u n d e r the laminar flow h o o d . When dry, filters were aseptically transferred, inoculated side u p , to p r e p o u r e d plates of trypticase soy agar. T h e petri plates were incubated at 37 C for 4 8 hr before enumeration. All dilutions were tested in duplicate.
Hatchery 1 2 5 5 6
1 2 4 5 5 6 6
125
HATCHERY NUMBER
FIG. 3. Bacteria and mold contamination of hatchery surfaces using the MSTK method (total counter and yeast and mold counters).
c o n t a m i n a t i o n , and when the surfaces had completely dried ( a b o u t 4 5 min for the glass and e p o x y tile and 105 min for t h e h a t c h e r y tray). Direct Surface Agar Plate Method. Another 100 c m 2 area was inoculated as described previously and t h e surfaces were sampled by pipetting drop-wise 10 ml of 55 C trypticase soy agar (containing 30 p p m of 2, 3, 5 - trip h e n y l t e t r a z o l i u m chloride to cause colonies to become colored red) o n t o the surfaces (Chapman, 1951). After hardening, t h e agar was p r o t e c t e d from drying o u t by inverting t h e b o t t o m of a petri dish, containing a moist filter paper, over the agar. T h e test surfaces were wrapped in a plastic garbage bag and placed in a 37 C i n c u b a t o r for 4 8 hr before e n u m e r a t i o n . The h a t c h e r tray was t o o large t o place in t h e i n c u b a t o r and was therefore i n c u b a t e d at approximately 27 C (ambient) for 4 8 hr. The c o n t a m i n a t i o n , drying, and sampling of all of the test surfaces, e x c e p t t h e h a t c h e r tray, were carried o u t u n d e r a biohazard, laminar flow stainless steel h o o d (Canadian Cabinets Ltd., Ottawa). Effect
of
Drying
To evaluate the effects of drying u p o n the viability of the organisms, Millipore filters (.45 fi pore size, 4 7 m m d i a m e t e r ) were used. A .2 ml aliquot of 10 _ 1 t o 10~3 dilutions of the original stationary phase cell suspension was gently pipetted o n t o t h e surface of t h e filters.
Contamination
The number of viable microorganisms d e t e c t e d on surfaces in t h e hatcheries using the MSTK are presented in Figures 1, 2, and 3. T h e hatcheries were rated as practicing excellent, good, average, or poor sanitation procedures as follows; H a t c h e r y 2, excellent; Hatcheries 4 and 5, g o o d ; Hatcheries 1 and 6, average; Hatchery 3, p o o r . T h e hatcheries tested differed in t e r m s of age, c o n s t r u c t i o n , and materials in addition t o the rigor of sanitation practices used. In this survey t w o hatcheries, (Hatcheries 2 and 3), were n o t e w o r t h y as representing t w o extremes. Hatchery 2 was a new facility in which Chickmaster setters (Model 1025) and h a t c h e r s (Model 9 0 M - l ) were used t o set and hatch chicks from a p p r o x i m a t e l y five million eggs per year. T h e h a t c h e r y e q u i p m e n t , floors, and m a c h i n e s were in excellent condition and fluff samples o b t a i n e d from this h a t c h e r y were consistently given A ratings (Table 1). H a t c h e r y 3 was older t h a n Hatchery 2 and had cracked, rough, c e m e n t floor surfaces and old, w o o d e n , Buckeye Streamliner setters (Model 66-44) and hatchers (Model 66H-44) used to set and hatch five million chicks per year. Fluff samples o b t a i n e d from H a t c h e r y 3 were consistently given C and D ratings (Table 1). T h e surfaces sampled in Hatchery 3 revealed high n u m b e r s of microorganisms c o m p a r e d t o s o m e of the o t h e r hatcheries sampled. In several areas, such as t h e h a t c h e r machine air e x h a u s t (Fig. 1) and the chick processing r o o m floor (Fig. 2), the n u m b e r of organisms actually increased after clean-up procedures had been c o m p l e t e d . T h e widespread presence of mold was n o t e d on surfaces such as tables, trays, ceiling, air inlets, fan blades, and floors, which were swabbed t h r o u g h o u t t h e h a t c h e r y (unpublished d a t a ) . T h e surfaces sampled in Hatchery 2 had very little c o n t a m i n a t i o n in comparison with the
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RESULTS AND DISCUSSION
HATCHERY SANITATION
of contamination after cleaning exceeded those found in other hatcheries tested. The need for hatchery personnel to be conscientious in the clean-up of these areas is evident. Other areas noted to harbor bacteria included the floors of the hatch and chick processing rooms. The numbers found on clean floor surfaces varied, but it is interesting to note the similarity between the results found in Hatchery 3, where cleaning practices were considered generally poor, and those found in Hatchery 6, where cleaning was considered adequate. A routine sanitation practice carried out in Hatchery 6 involved flushing out the hatcher machine exhaust ducting with water after each hatch. The water was forced through the ducting located outside of the hatcher room and allowed to drain via a transom located at the junction between the hatcher machine and exhaust ducting. It was noted that the wash water, containing fluff and other debris, had
TABLE 1. Assessment of two hatchery operations by air exposure (open) plate data1 and by fluff sampling* Air exposure ratings Hatchery 3
Hatchery 2 Area sampled
Bacteria
Mold
Bacteria
Mold
Hatcher room
Excellent
Excellent
Unsatisfactory 3
Unsatisfactory
Excellent Excellent Excellent Excellent
Excellent Good Excellent Excellent
Excellent Poor
Unsatisfactory Unsatisfactory
Chick processing room Egg holding/receiving room Setter room
Excellent Good Excellent
Good Good Excellent
Excellent Excellent Excellent
Excellent Good Poor
Setter Setter Setter Setter
Excellent Excellent Excellent Excellent
Excellent Good Excellent Excellent
Wash room
Good
Good
Garbage room
Unsatisfactory
Good
Hatcher Hatcher Hatcher Hatcher
machine machine machine machine
machine machine machine machine
#1 #2 #3 #4
#1 #2 #3 #4
Fluff ratings Hatcher machines
Hatchery 2, Machine #3 —— Bacteria Mold Coliform Salmonella
A A A Negative
Hatchery 3, »* ,_. Bacteria Mold Coliform Salmonella
'Saddler (1975). 2
W r i g h t s al. (1959).
'The word unsatisfactory replaces the word miserable in the ratings used by Saddler (1975).
,,„
D C A Negative
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same surfaces monitored in other hatcheries with but one exception, the hatcher machine air exhaust (Fig. 1 and 2). An examination of the individual machines revealed that several had a focus of microbial contamination in the exhaust outlet prior to clean-up. The setters in Hatchery 2 contained low or no bacterial CFU on the floors and trays but molds were apparent throughout the inlet systems of all of the machines. An examination of the setter room air inlet also revealed viable molds (Fig. 3). Results from the other hatcheries tested indicated areas where bacteria accumulated and were not diminished with the cleaning systems employed. Hatchery 4 was operated under stringent sanitation practices. The areas sampled in Hatchery 4 showed a dramatic decrease in bacterial numbers after the clean-up had taken place. One exception was seen in the hatcher machine air inlet (Fig. 1) where levels
303
304
SOUCY ET AL.
By routinely monitoring the hatchery environment and comparing the results with those from the fluff test as well as production information, the hatchery manager can build an inventory of results, evaluate the sanitation procedures in use, and judge the levels of contamination that do not affect hatchery production and chick health. The benefits of such a monitoring system are well established and, most importantly, include the rapid identification and elimination of problem areas in the hatchery before production losses occur.
Using the guidelines set out by Saddler (1975) for evaluation of airborne contamination, the areas monitored in Hatcheries 2 and 3 were each given a rating (Table 1). All of the areas sampled in Hatchery 2 received excellent or good ratings based upon the numbers of bacteria and mold colonies that developed on open plates exposed to the air. In comparison, only 56% of the areas sampled in Hatchery 3 received excellent or good ratings with the remainder receiving poor or unsatisfactory ratings. The fluff test results (Table 1) indicated that Hatchery 2 operated under excellent sanitation conditions ("A" bacteria rating) whereas Hatchery 3 operated under unsatisfactory sanitation conditions ("D" bacteria rating). These results were found to substantiate the results obtained from the MSTK procedure (Fig. 1), which describe Hatchery 2 as a hatchery operating with excellent sanitation practices and Hatchery 3 as a hatchery operating with poor sanitation practices. Identification
of Hatchery Isolates
The most prevalent hatchery isolates were of the following genera: Flavobacterium, Proteus, Pseudomonas, Bacillus, Aspergillus, Paecilomyces and Penicillium, as well as coliform bacteria (Table 2). Of these microorganisms, several have been implicated as being pathogenic to the developing embryo and chick. Coliform bacteria have been associated with a disease known as omphalitis, an inflamation of the navel, which frequently results in early chick mortality (Schwartz, 1977). Wright et al, (1959) found that when 1,200 or more Aspergillus colonies per gram of fluff were detected, aspergillosis was diagnosed in chicks or poults hatched in the hatchery. Other nonspecific bacterial infections have been frequently associated with hatchery losses due to early chick mortality and poor hatchability (Watts and Rac, 1958). Thus, the types of organisms found in this study were typical of those found by others (Saddler, 1975; Wright et al., 1959) and these could certainly cause problems in the hatchery if left unchecked. Hatchery areas where a large number of microorganisms were frequently found were considered important areas to monitor routinely. These areas included: 1) room floors; 2) hatchers, floor, ceiling, air exhaust, air inlet area, and outside top of machine; and 3) setters, floor, wall, air exhaust area, and air
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been left to accumulate as a wet mass behind the hatcher machines. This practice most likely contributed to the high numbers of bacteria found on the hatcher room floor, which was monitored just prior to the transfer of eggs into the hatcher machines in Hatchery 6. The use of the MSTK in the hatcheries studied was well received by the hatchery personnel. Three of five commercial hatcheries tested routinely monitored the hatchery environment with either air exposure plates, Rodac plates, or used the services of a consulting microbiologist. It was found that the concerns of hatchery managers about using any sanitation monitoring system included the following points: 1) that the environmental sampling method used accurately reflect the microbial load in the test area; 2) that the tests be easily interpreted; 3) that guidelines be available so that realistic goals could be set; 4) that the monitoring system be economical to use. In view of these concerns the following points are noteworthy: 1) The MSTK clearly indicated areas in the hatcheries where there were reservoirs of organisms and also areas where the sanitation procedures in place were adequate. 2) The test was easy to perform and could be used on all smooth surfaces. Colonies were easily counted using the grid provided on each media filter. 3) Most hatcheries differ in the types of the machines used to set and hatch eggs. Some are old, wooden models and others are new, fiberglass types. In view of this variation, generalized guidelines for a tolerable number of organisms to be found on clean hatchery surfaces are difficult to develop. 4) The cost of material for monitoring one test site using the MSTK system was between 3 and 4 dollars. By monitoring critical areas where high microbial numbers are most likely to be found, cost can be minimized.
HATCHERY SANITATION
inlet area. Magwood and Marr (1964) found horizontal surfaces and floors in hatcheries to be very important reservoirs of microorganisms. Evaluation ofMSTK
and
DSAPMethods
well as molds. Organisms of the genus Bacillus were isolated from the four types of counters used. The most useful media strip of the MSTK system for monitoring the hatchery environment was found to be the total bacterial counter (MTSK). An examination of the specificity of the media strips prepared by Millipore revealed some additional undesirable features in the MSTK system. It was generally observed that fewer and larger colonies developed at the bottom of all media strips. The organisms growing on the MCSK varied in their appearance, with small yellow colonies observed at the top of the media strip and larger, blue-green colonies observed at the bottom of the media strip. A pure culture of E. coli was used in this part of the experiment, and microscopic examination verified that the organisms showing different pigments on the same MCSK media strip were both E. coli. The
TABLE 2. Isolation and identification of microorganisms from poultry hatcheries using the MSTK system Kit media used2 Organism'
MTSK
MCSK
Acinetobacter Alcaligenes Coliform Flavobacterium Moraxella Proteus Pseudomonas Bacillus Staphylococcus Streptococcus Aspergillus Paecilomyces Penicillium
1
MYSK
MPSK
Hatchery area sampled Chick processing room floor Hatcher machine exhaust, table used for traying eggs Chick work table, vacuum lift transfer cup, egg shell, chick processing room floor Hatcher machine floor, hatcher air inlet, chick work table, hatcher tray Egg holding room floor, hatcher machine wall Setter wall, setter floor, egg holding room floor Hatcher machine floor, hatcher tray, setter room floor, chick box, hatcher room floor Setter room floor, egg holding room floor, hatcher air inlet, setter air inlet, hatcher room floor, setter egg tray Egg shell Chick box Setter machine floor, trolley, setter machine circulating fan blade Setter room air inlet, hatcher air inlet, setter floor Chick processing room floor, fan blade in setter, hatcher air inlet and exhaust
Identification criteria, (Vanderzant and Nickelson, 1969; Buchanan and Gibbons, 1974).
2
Millipore SwabTest Kit (MSTK), Total Counter (MTSK), Coliform Counter (MCSK), Yeast and Mold Counter (MYSK), Pseudomonad Counter (MPSK). 3
+, Indicates isolation of the organism.
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Specificity. The various types of organisms isolated from the MSTK media strips used for sampling hatchery surfaces are summarized in Table 2. The total count (MTSK) counters supported the growth of a broad range of microorganisms including coliforms, staphylococci, and pseudomonads as well as molds. The coliform counters (MCSK) supported the growth of Gram negative bacteria other than coliform bacteria as well as some Gram positive bacteria. Gram negative bacteria other than pseudomonads were isolated from the pseudomonad counters (MPSK). The yeast and mold (MYSK) counters supported the growth of Gram positive and Gram negative bacteria as
305
4.23 4.70
4.75 4.40 2.87 3.32
MSTK DSAP
MSTK DSAP MSTK DSAP MSTK DSAP MSTK DSAP
Pseudomonas sp.
Staphylococcus sp.
Fiberglass hatcher tray.
NS; Not significant or P>.10.
***P<.001.
**.001
*.01
S
3.01 2.83
3.31 2.08
4.08 4.70 4.08 4.70
ND" ND
4.67 4.70 2.65 3.36
ND ND
4.28 3.43 4.38 4.70
2.86 3.11
4.34 4.70
2.03
0
Tray 3
4.26 4.70
3.00 3.18
Glass
MSTK, Millipore Swab Test Kit; DSAP, direct surface agar plate.
Surfaces had dried thoroughly.
"ND, Not done.
3
2
1
Paecilomyces variotii
Salmonella hadar
E. coli
Proteus sp.
MSTK DSAP
3.11 2.66
MSTK DSAP
Pseudomonas fragi
4.11 4.70 4.20 4.70
Epoxy
Recovery method 2
Organism
0
3.15 2.80
3.29 2.67
4.72 4.70 2.91 3.32 4.20 3.40 4.11 4.70
4.60 4.40 2.77 3.20 4.32 3.02 4.20 4.70
4.24 4.70
3.09 3.16
Glass
4.20 4.70
3.31 2.71
Epoxy
Surface ; contaminated
10
.90
3.31 2.64
4.18 3.09 4.04 4.70
2.67 3.08
ND ND
4.20 4.70
0
Tray
Minutes after surface contamination
TABLE 3. Effects of drying on the survival of microorganisms on test surfaces and their recovery usi
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307
HATCHERY SANITATION
These results showed that the ability of the MSTK to recover organisms from surfaces is realistically comparable to that of the DSAP method. Differences in recovery of organisms were exaggerated following drying of surfaces.
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location of the differently pigmented organisms suggested that the concentration of the dye in the MCSK media was not always uniform. Rehydration of the medium was, uniform. According to the MSTK operating instructions, (cat. AB820, 1981, Millipore Corp., Bedford, MA) only blue or blue-green colonies should be counted on the MCSK for coliform enumeration. In these experiments, some of the Proteus colonies did and some of the E. coli colonies did not take up the blue dye. The observations noted in these experiments further support the sole use of the MTSK media strips for monitoring the hatchery environment. The DSAP method was used in the laboratory for the accurate enumeration of surface contaminants. Favero et al. (1968) outlined the limitations of using this method in the field as 1) the inability to incubate surfaces at proper temperatures due to their fixed nature; 2) the coalescence of colonies when microbial contamination is high; 3) the inappropriateness of the method for use on surfaces containing residual amounts of chemicals that would prevent the growth of microorganisms. Recovery Efficacy. Attempts were made to choose surfaces (epoxy tile, glass, and hatcher tray) that would be representative of those found in hatcheries. Hard surfaces were chosen so that the experimental results would not be affected by differences in the porosities of the surfaces. The paired Student's t test at a .05 level of significance was used (Mendenhall, 1979) to test the hypothesis that there was not a significant difference between the number of organisms detected using the MSTK (total counter) and DSAP methods. Table 3 shows the results of the comparison between the two methods for the recovery of bacteria and one mold from deliberately contaminated surfaces. The two methods were not significantly different in their ability to recover cells of Pseudomonas fragi, Staphylococcus, Proteus, and E. coli but were significantly different in their ability to recover another strain of Pseudomonas, Salmonella hadar, and Paecilomyces variotii. The latter two bacteria were recovered in higher numbers by the DSAP method whereas MSTK gave better recovery of the mold Paecilomyces.
308
SOUCY ET AL. TABLE 5. Lethal effects of drying microorganisms on membrane filters1
Organism
1.48 3.2 6.6 2.86 1.82 1.08 1.8
X X X X X X X
102 10" 103 104 10" 103 10"
0 0 189 0 110 33 7
Percent recovery 0 0 2.86 0 .60 3.06 .04
1 Suspensions of viable microorganisms were dried on filters at 27 C in a sterile petri plate and placed on trypticase soy agar for growth at 37 C.
This was very evident in the better recovery of organisms by the DSAP method than the MSTK method after the surfaces had dried thoroughly (105 min, Time 3). The MSTK method failed to recover viable Pseudomonas and Salmonella under the same conditions. This could be a reflection of the ability of these organisms to attach themselves to surfaces and thus hinder their removal by the swab (Firstenberg-Eden, 1981). These bacteria, being attached to the surface, would develop into colonies in the agar medium provided by the DSAP method. Duncan's multiple range test at a significance level of .05 (Malik and Mullen, 1973) showed that there was no difference in the recovery of organisms when the surfaces were tested immediately after contamination (Time 1) and when the surfaces were tested 10 min after contamination (Time 2) using both the DSAP and MSTK methods of recovery. Significant differences between the recovery of organisms at Times 1 and 3 and Times 2 and 3 were apparent. Table 4 shows that at Time 3 the number of organsims recovered by both methods was very low with approximately 28% fewer organisms recovered than at Times 1 and 2. This decrease in the number of organisms enumerated was taken to mean that the organisms were unable to survive the effects of drying on hatchery surfaces. Effect of Drying, The results of drying suspensions of the test organisms on Millipore membrane filter discs are summarized in Table 5. The results seen in the comparison of the MSTK and DSAP methods, where low numbers of organisms were recovered using both the MSTK and DSAP methods of recovery on dried surfaces, are supported by the significant mortality of organisms following drying on
membrane filters. Staphylococcus, E. coli, Salmonella hadar, and Paecilomyces variotii were recovered in countable numbers after drying. Other bacteria were not detected. In similar experiments, Angelotti and Foter (1958) demonstrated the lethal effects of drying on vegetative cells. These results emphasize the importance of drying surfaces to effectively reduce the number of viable organisms. The MSTK system was evaluated and found to be a useful method of determining the microbial load on hatchery surfaces and thus is a valuable tool for routine monitoring of hatchery sanitation. The use of this method to establish the effectiveness of specific disinfectant, sanitizing, and fumigation systems used by individual hatcheries would require further study.
ACKNOWLEDGMENTS
The cooperation of the personnel of the hatcheries studied was greatly appreciated. The Ontario Ministry of Agriculture and Food, Veterinary Services Laboratory, University of Guelph, Guelph, Ontario, and Le Laboratoire Regional De L'Assomption, Division De Pathologie Animale, Ministere De L'Agriculture, Quebec, performed the laboratory analysis on the fluff samples taken from the test hatcheries. John Bissett, Biosystematics Research Institute, identified the culture of Paecilomyces variotii. REFERENCES Angelotti, R. and M. J. Foter, 1958. A direct surface agar plate laboratory method for quantitatively
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Pseudomonas fragi Pseudomonas sp. Staphylococcus sp. Proteus sp. E. coli Salmonella hadar Paecilomyces variotii
Viable organisms per filter
N u m b e r applied to each filter
HATCHERY SANITATION
Mendenhall, W., 1979. Introduction to Probability and Statistics. 4th ed. Duxbury Press, North Scituate, MA. Niskanen, A., and M. S. Pohja, 1977. Comparative studies on the sampling and investigation of microbial contamination of surfaces by the contact plate and swab methods. J. Appl. Bacterid. 4 2 : 5 3 - 6 3 . Saddler, R., 1975. Quality control of broilers from hatchery to the processing plant. Poultry Dig. 34:17-19. Schwartz, D. L., 1977. Poultry Health Handbook. 2nd ed. Coll. Agric, Pennsylvania State Univ., University Park, PA. Shane, S. M., 1981. Broiler chick mortality linked to hatchery hygiene. Poultry Dig. 40:398—400. Stinson, C. G., and N. P. Tiwari, 1978. Evaluation of quick bacterial count methods for assessment of food plant sanitation. J. Food Prot. 4 1 : 2 6 9 - 2 7 1 . Vanderzant, C , and R. Nickelson, 1969. A microbiological examination of muscle tissue of beef, pork, and lamb carcasses. J. Milk Food Technol. 32:357-361. Watts, P. S., and R. Rac, 1958. Causes of mortality in chickens up to ten days old. Br. Vet. J. 114: 396-407. Wright, M. L., G. W. Anderson, and N. A. Epps, 1959. Hatchery sanitation. Can. J. Comp. Med. 23: 288-290. Wright, M. L., and N. A. Epps, 1958. Hatchery sanitation. Can. J. Comp. Med. 22:397-399.
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detecting bacterial contamination on non-porous surfaces. Food Res. 23:170-174. Buchanan, R. E. and N. E. Gibbons, 1974. Bergey's Manual of Determinative Bacteriology. 8th ed. Williams and Wilkins Co., Baltimore, MD. Chapman, G. H., 1951. A culture medium for detecting and confirming H. coli in ten hours. Am. J.Publ. Health 41:1381. Chute, H. L., and M. Gershman, 1961. A new approach to hatchery sanitation. Poultry Sci. 40:568-571. Favero, M. S., J. J. McDade, J. A. Robertson, R. K. Hoffman, and R. W. Edwards, 1968. Microbiological sampling of surfaces. J. Appl. Bacteriol. 31:336-343. Firstenberg-Eden, R., 1981. Attachment of bacteria to meat surfaces: review. J. Food Prot. 44:602— 607. Gentry, R. F., M. Mitrovic, and G. R. Bubash, 1962. Application of Anderson sampler in hatchery sanitation. Poultry Sci. 41:794—804. Hacking, B., 1982. Sanitation for hatchery and farm. CanadaPoultryman 69:22; 3 6 - 3 7 . Harrigan, W. F., and M. E. McCance, 1976. Laboratory Methods in Food and Dairy Microbiology. 2nd ed. Academic Press, New York, NY. Magwood, S. E., and H. Marr, 1964. Studies in hatchery sanitation 2. Poultry Sci. 4 3 : 1 5 5 8 - 1572. Malik, H. J., and K. Mullen, 1973. A first course in probability and statistics. Addison-Wesley Publ. Co., Inc., Reading, MA.
309
1983 Poultry Science 62:298-309 INTRODUCTION
The importance of an effective hatchery sanitation program to achieve a high level of hatchability (Hacking, 1982) and to ensure the production of high quality chicks has been firmly established (Chute and Gershman, 1961; Gentry et ai, 1962; Shane, 1981). Visual scrutiny of the environmental condition of a hatchery can be an important method of evaluating the sanitation procedures used. To assure management that their sanitation program is working effectively to prevent the build up of contamination between successive hatches, a more objective method of monitoring the level of microorganisms on surfaces and in the air is necessary. Most hatcheries have neither the facilities nor the expertise to implement routine microbiological testing; thus, there is a need for the adoption of representative and simple monitoring procedures that can be conducted and interpreted in the hatchery without recourse to an outside laboratory. An agar contact technique using Rodac plates was evaluated by Favero et al. (1968)
1 Contribution Number 505, Food Research Institute, Research Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6.
and was found to be an excellent method for use in field studies because of its portability and facility in sampling flat, smooth surfaces. This method is best adapted to measure low numbers of organisms, i.e., 5 to 50 cfu/10 cm 2 (Niskanen and Pohja, 1977). Molds, spreading colonies and debris on the agar surface, complicate the enumeration of colony forming units on Rodac plates. Niskanen and Pohja (1977) compared the recovery of bacteria from various plastic, wood, and steel surfaces using the agar contact and swab methods. Swabbing was found to be the better technique for the recovery of bacteria from uneven surfaces and heavily contaminated areas. Stinson and Tiwari (1978) compared the Millipore Swab Test Kit (MSTK), the Rodac procedure of surface sampling, and a contacttransfer method (Con-Tact-It System Bacteria Detection Unit, Birko Chemical Corporation) with the standard swab procedure as quick methods for the assessment of food plant sanitation. There was a significant correlation between results obtained from the MSTK and standard swab procedures. The MSTK was found to be more accurate than the Rodac plates. The swabbing methods recovered more contaminants than either the Con-Tact-It or Rodac procedures. 298
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ABSTRACT A comparative microbiological evaluation of sanitation practices used in six Canadian hatcheries was conducted by means of a commercially available swab kit. An assessment was made of its potential for routine use in monitoring hatchery sanitation by plant personnel. Hatcher machine inlets, exhausts, and room floors were identified as reservoirs of organisms after regular clean-up procedures had been completed. Isolated hatchery organisms were identified and the four test kit media were evaluated for their ability to exclude or support the growth of a variety of organisms. Nonselective bacteriological media in the kit were most informative for routine sanitation monitoring. A comparison between the swab kit and the direct surface agar plate techniques was undertaken in laboratory tests during which microorganisms isolated from poultry hatcheries were recovered from samples of hatchery surfaces. There was no significant difference between the two methods for recovery of Pseudomonas fragi, E, coli, Proteus, and Staphylococcus, but better recoveries were obtained for another strain of Pseudomonas and Salmonella hadar when the direct surface agar plate technique was used. Better recovery of Paecilomyces variotii was obtained when the swab kit technique was used. The lethal effect of drying on poultry microorganisms was evaluated after air drying the organisms on membrane filters. (Key words: hatchery, sanitation, surface sampling, swab kit)
299
HATCHERY SANITATION
MATERIALS AND METHODS
Hatchery
Contamination
To determine the level of microorganisms in the poultry hatcheries monitored (five commercial hatcheries and one government hatchery) the MSTK method was used in conjunction with air exposure (open) plates (Harrigan and McCance, 1976) and hatcher fluff sampling (Wright and Epps, 1958). The MSTK technique was used to collect bacteria and mold from the walls, floors, machines, and equipment within the hatchery. Air exposure plates were used to detect the level of airborne contamination in the hatchers, incubators and rooms. Hatcher fluff samples were analyzed to evaluate the sanitation procedures used in the hatcheries. Various sites in the commercial hatcheries were sampled when they were expected to be dirtiest and cleanest. (For example, hatcher machines were sampled just prior to being filled with eggs and during the hatching process.) Equipment was sampled before and after use. Swab Test Method. The MSTK system consists of four types of color coded media, which include the following: total bacteria MTSK (white), coliform MCSK (blue), yeast and mold MYSK (yellow), and for pseudomonad enumeration, MPSK (green). These were obtained from Millipore Ltd., 3610 Nashua Drive, Mississauga, Ontario L4V 1L2. The method of sampling used was the procdure outlined by the manufacturer in documents AB820, PM810, and PB427. Each site was sampled by swabbing the area within a 100 cm 2 stainless steel template by rotating the swab while swabbing at two right angle directions. The swab was then placed in a .1 mM phosphate buffer (pH 7.2) containing thiosulfate and a quaternary ammonium compound neutralizer. When dilutions were required, an aliquot of the buffer plus sample solution was added to dilution buffer without neutralizing com-
pounds. The inoculated samplers were incubated in an operating chick incubator at 37.5 C and 92% relative humidity for 48 hr to obtain bacterial counts and 5 days for mold counts. Air Sampling. Prepoured agar in petri dishes was exposed by gravity to atmospheric contamination. Dishes of solidified plate count agar (PCA) that contained 75 ppm cycloheximide (ICN Pharmaceuticals, Cleveland, OH) to inhibit mold growth, Levine eosin methylene blue agar (EMB), and potato dextrose agar (PDA) were used. These agar media were obtained from Difco Laboratories, Detroit, MI. A paper towel was placed on the area to be sampled. The petri dish lid was removed and placed beside the petri dish bottom without inversion. The plates were exposed for 5 min in the hatchers and areas where chicks and fluff were present, whereas 15 min exposures were used in all other areas. The PCA and EMB plates were incubated for 48 hr at 37 C. The PDA plates were incubated for 5 days at 3 7 C. Fluff Sampling. Wright and Epps (1958) found that the microflora of the fluff, produced by hatching chicks, was a reliable criterion of the sanitation condition of a hatchery. Sanitation ratings of A, B, C, or D were given to the hatcheries monitored based upon a standardized bacteriological technique outlined by Wright etal. (1959). Identification
of Hatchery Isolates
Organisms isolated in the hatcheries were classified according to the scheme of Vanderzant and Nickelson (1969) and Buchanan and Gibbons (1974). The general types of organisms found in hatcheries were determined and the selectivity of the MSTK's coliform, pseudomonad, and yeast and mold samplers were studied. Representative colonies isolated from the MSTK samplers and the air exposure plates were grown in nutrient broth at 37 C. After 24 hr, colonies were identified by the following methods: Gram stain, morphology, oxidase test, urea hydrolysis, the production of ammonia from arginine, the liquifaction of gelatin, the production of gas in brilliant green lactose bile broth, and motility. Laboratory Contamination of Hatchery Surfaces The MSTK and DSAP techniques were compared under laboratory conditions for their
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The purpose of this study was to evaluate the use of the MSTK system as an effective hatchery sanitation monitoring tool by comparison with the direct surface agar plate (DSAP) procedure (Angelotti and Foter, 1958; Favero et al., 1968) for their ability to recover organisms from contaminated laboratory surfaces. The MSTK was also evaluated in commercial hatcheries known to practice excellent, good, average, or poor sanitation procedures.
300
SOUCY ET AL. floor hatcher machine
floor hatcher r o o m
air-inlet hatcher machine
air-exhaust hatcher machine
outside t o p hatcher machine
107 AFTER CLEAN-UP BEFORE CLEAN-UP U
10 6
-
<
105
o o 10 4
-
UJ rO
2
103
or. LU CO
D Z
102-
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 6
HATCHERY NUMBER
FIG. 1. Number of viable bacteria found on hatchery surfaces before and after clean-up using the MSTK method (total counter).
ability to recover microorganisms from samples of typical surfaces found in the hatchery. The DSAP procedure was used as the reference method because studies by Favero et al. (1968) showed the DSAP method to be an accurate laboratory technique for enumerating surface contaminants. Cultures. For this part of the study, microorganisms isolated from poultry fluff samples plus a culture of Pseudomonas fragi (ATCC 27362) were used. C. Poppe, Animal Pathology Laboratory, Agriculture Canada, Winnipeg, kindly provided isolates of Pseudomonas, Staphylococcus, Proteus, and E. coli, A mold identified as Paecilomyces variotii was obtained during this study from one of the poultry hatcheries. A culture of Salmonella hadar, isolated from a poultry hatchery, was kindly provided by N. A. Epps, Department Microbiology, University of Guelph, Guelph, Ontario. Cells were harvested in their early stationary
phase of growth. These spores were used to contaminate three smooth test surfaces: epoxy. terazzo tile (supplied by Durie Mosaic and Marble Limited, Ottawa, Ontario), glass, and a fiberglass hatcher tray (Robbins H10). Both the MSTK and DSAP methods were used to recover the organisms from the contaminated test surfaces. Growth of Test Cultures: Bacteria. Cells in their stationary phase of growth were obtained by inoculation of 10 ml of trypticase soy broth (Difco) with a loopful of growth from the surface of an agar slant stock culture of the organism ( < 3 0 days old). The subculture was incubated at 37 C for 48 hr. A .2 ml aliquot of the 48 hr culture was used to inoculate 50 ml trypticase soy broth in a 250 ml Klett sidearm flask. Another 50 ml trypticase soy broth was retained, uninoculated, in a 250 ml sidearm flask to serve as a reagent blank,. A zero reading was taken immediately after inoculation of the
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tier D en
301
HATCHERY SANITATION
107n
106
chick r o o m floor
chick vaccinating machine
chick work table
:
<
hatching trays
»"<
vacuum lift .cups
conveyor belt for chicks ^
—
li-
CC 3
AFTER CLEAN-UP BEFORE CLEAN-UP
C/5 CM
10
o
§ ^
10 4
rU
<
CO
LL
O
10 3 -
rr CD
D
102
12356
123
2345
1234
3 4
5 6
HATCHERY NUMBER FIG. 2. Number of viable bacteria found on hatchery surfaces before and after clean-up using the MSTK method (total counter).
growth flask. Organisms were incubated for 16 hr at 37 C, and then hourly readings were taken during the next 8 hr period. Growth was monitored until the stationary phase, determined graphically, was reached. Growth of Test Cultures: Mold. Mold spores were obtained from a pure culture which had been incubated at 37 C for 5 days on potato dextrose agar (Difco) by washing cells from the agar surface with 5 ml peptone water. A Roux flask containing 200 ml solidified, potato dextrose agar was flooded with the cell suspension and incubated at 37 C for 7 days. Contamination of Test Surfaces: Bacteria. A 10 ml aliquot of cells in their stationary phase of growth was centrifuged for 8 min at 5000 rpm using the Spx rotor of a model GLC-1 Sorvall centrifuge. The cells were resuspended in 10 ml of sterile water, and the suspension was adjusted to 80% transmittance (Spectronic
20 colorimeter, Bausch and Lomb) by dilution. One milliliter of this cell suspension was added to a 99 ml dilution blank of sterile water and used to artificially contaminate the typical poultry hatchery surfaces described earlier. Contamination of Test Surfaces: Mold. Spores were harvested by washing the growth from the potato dextrose agar surface with 100 ml sterile water. The spore suspension was filtered through sterile glass wool and then centrifuged for 10 min at 5000 rpm using the Spx rotor of a model GLC-1 Sorvall centrifuge. The spores were resuspended in 20 ml sterile water and the suspension was diluted to a concentration of 10 4 spores per milliliter of sterile water. One milliliter of the diluted spore suspension was pipetted onto 100 cm of the test surfaces. The surfaces were then swabbed using the MSTK system at the following times: immediately after contamination, 10 min after
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E
5
SOUCYET AL.
302 1Q6 -i MOLD BACTERIA Ul o <
104-
t/i CM
Setter machine air inlet
Setter machine floor
Setter r o o m air inlet
T h e filters had been placed in e m p t y sterile petri dishes and following their inoculation were allowed to dry u n d e r the laminar flow h o o d . When dry, filters were aseptically transferred, inoculated side u p , to p r e p o u r e d plates of trypticase soy agar. T h e petri plates were incubated at 37 C for 4 8 hr before enumeration. All dilutions were tested in duplicate.
Hatchery 1 2 5 5 6
1 2 4 5 5 6 6
125
HATCHERY NUMBER
FIG. 3. Bacteria and mold contamination of hatchery surfaces using the MSTK method (total counter and yeast and mold counters).
c o n t a m i n a t i o n , and when the surfaces had completely dried ( a b o u t 4 5 min for the glass and e p o x y tile and 105 min for t h e h a t c h e r y tray). Direct Surface Agar Plate Method. Another 100 c m 2 area was inoculated as described previously and t h e surfaces were sampled by pipetting drop-wise 10 ml of 55 C trypticase soy agar (containing 30 p p m of 2, 3, 5 - trip h e n y l t e t r a z o l i u m chloride to cause colonies to become colored red) o n t o the surfaces (Chapman, 1951). After hardening, t h e agar was p r o t e c t e d from drying o u t by inverting t h e b o t t o m of a petri dish, containing a moist filter paper, over the agar. T h e test surfaces were wrapped in a plastic garbage bag and placed in a 37 C i n c u b a t o r for 4 8 hr before e n u m e r a t i o n . The h a t c h e r tray was t o o large t o place in t h e i n c u b a t o r and was therefore i n c u b a t e d at approximately 27 C (ambient) for 4 8 hr. The c o n t a m i n a t i o n , drying, and sampling of all of the test surfaces, e x c e p t t h e h a t c h e r tray, were carried o u t u n d e r a biohazard, laminar flow stainless steel h o o d (Canadian Cabinets Ltd., Ottawa). Effect
of
Drying
To evaluate the effects of drying u p o n the viability of the organisms, Millipore filters (.45 fi pore size, 4 7 m m d i a m e t e r ) were used. A .2 ml aliquot of 10 _ 1 t o 10~3 dilutions of the original stationary phase cell suspension was gently pipetted o n t o t h e surface of t h e filters.
Contamination
The number of viable microorganisms d e t e c t e d on surfaces in t h e hatcheries using the MSTK are presented in Figures 1, 2, and 3. T h e hatcheries were rated as practicing excellent, good, average, or poor sanitation procedures as follows; H a t c h e r y 2, excellent; Hatcheries 4 and 5, g o o d ; Hatcheries 1 and 6, average; Hatchery 3, p o o r . T h e hatcheries tested differed in t e r m s of age, c o n s t r u c t i o n , and materials in addition t o the rigor of sanitation practices used. In this survey t w o hatcheries, (Hatcheries 2 and 3), were n o t e w o r t h y as representing t w o extremes. Hatchery 2 was a new facility in which Chickmaster setters (Model 1025) and h a t c h e r s (Model 9 0 M - l ) were used t o set and hatch chicks from a p p r o x i m a t e l y five million eggs per year. T h e h a t c h e r y e q u i p m e n t , floors, and m a c h i n e s were in excellent condition and fluff samples o b t a i n e d from this h a t c h e r y were consistently given A ratings (Table 1). H a t c h e r y 3 was older t h a n Hatchery 2 and had cracked, rough, c e m e n t floor surfaces and old, w o o d e n , Buckeye Streamliner setters (Model 66-44) and hatchers (Model 66H-44) used to set and hatch five million chicks per year. Fluff samples o b t a i n e d from H a t c h e r y 3 were consistently given C and D ratings (Table 1). T h e surfaces sampled in Hatchery 3 revealed high n u m b e r s of microorganisms c o m p a r e d t o s o m e of the o t h e r hatcheries sampled. In several areas, such as t h e h a t c h e r machine air e x h a u s t (Fig. 1) and the chick processing r o o m floor (Fig. 2), the n u m b e r of organisms actually increased after clean-up procedures had been c o m p l e t e d . T h e widespread presence of mold was n o t e d on surfaces such as tables, trays, ceiling, air inlets, fan blades, and floors, which were swabbed t h r o u g h o u t t h e h a t c h e r y (unpublished d a t a ) . T h e surfaces sampled in Hatchery 2 had very little c o n t a m i n a t i o n in comparison with the
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RESULTS AND DISCUSSION
HATCHERY SANITATION
of contamination after cleaning exceeded those found in other hatcheries tested. The need for hatchery personnel to be conscientious in the clean-up of these areas is evident. Other areas noted to harbor bacteria included the floors of the hatch and chick processing rooms. The numbers found on clean floor surfaces varied, but it is interesting to note the similarity between the results found in Hatchery 3, where cleaning practices were considered generally poor, and those found in Hatchery 6, where cleaning was considered adequate. A routine sanitation practice carried out in Hatchery 6 involved flushing out the hatcher machine exhaust ducting with water after each hatch. The water was forced through the ducting located outside of the hatcher room and allowed to drain via a transom located at the junction between the hatcher machine and exhaust ducting. It was noted that the wash water, containing fluff and other debris, had
TABLE 1. Assessment of two hatchery operations by air exposure (open) plate data1 and by fluff sampling* Air exposure ratings Hatchery 3
Hatchery 2 Area sampled
Bacteria
Mold
Bacteria
Mold
Hatcher room
Excellent
Excellent
Unsatisfactory 3
Unsatisfactory
Excellent Excellent Excellent Excellent
Excellent Good Excellent Excellent
Excellent Poor
Unsatisfactory Unsatisfactory
Chick processing room Egg holding/receiving room Setter room
Excellent Good Excellent
Good Good Excellent
Excellent Excellent Excellent
Excellent Good Poor
Setter Setter Setter Setter
Excellent Excellent Excellent Excellent
Excellent Good Excellent Excellent
Wash room
Good
Good
Garbage room
Unsatisfactory
Good
Hatcher Hatcher Hatcher Hatcher
machine machine machine machine
machine machine machine machine
#1 #2 #3 #4
#1 #2 #3 #4
Fluff ratings Hatcher machines
Hatchery 2, Machine #3 —— Bacteria Mold Coliform Salmonella
A A A Negative
Hatchery 3, »* ,_. Bacteria Mold Coliform Salmonella
'Saddler (1975). 2
W r i g h t s al. (1959).
'The word unsatisfactory replaces the word miserable in the ratings used by Saddler (1975).
,,„
D C A Negative
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same surfaces monitored in other hatcheries with but one exception, the hatcher machine air exhaust (Fig. 1 and 2). An examination of the individual machines revealed that several had a focus of microbial contamination in the exhaust outlet prior to clean-up. The setters in Hatchery 2 contained low or no bacterial CFU on the floors and trays but molds were apparent throughout the inlet systems of all of the machines. An examination of the setter room air inlet also revealed viable molds (Fig. 3). Results from the other hatcheries tested indicated areas where bacteria accumulated and were not diminished with the cleaning systems employed. Hatchery 4 was operated under stringent sanitation practices. The areas sampled in Hatchery 4 showed a dramatic decrease in bacterial numbers after the clean-up had taken place. One exception was seen in the hatcher machine air inlet (Fig. 1) where levels
303
304
SOUCY ET AL.
By routinely monitoring the hatchery environment and comparing the results with those from the fluff test as well as production information, the hatchery manager can build an inventory of results, evaluate the sanitation procedures in use, and judge the levels of contamination that do not affect hatchery production and chick health. The benefits of such a monitoring system are well established and, most importantly, include the rapid identification and elimination of problem areas in the hatchery before production losses occur.
Using the guidelines set out by Saddler (1975) for evaluation of airborne contamination, the areas monitored in Hatcheries 2 and 3 were each given a rating (Table 1). All of the areas sampled in Hatchery 2 received excellent or good ratings based upon the numbers of bacteria and mold colonies that developed on open plates exposed to the air. In comparison, only 56% of the areas sampled in Hatchery 3 received excellent or good ratings with the remainder receiving poor or unsatisfactory ratings. The fluff test results (Table 1) indicated that Hatchery 2 operated under excellent sanitation conditions ("A" bacteria rating) whereas Hatchery 3 operated under unsatisfactory sanitation conditions ("D" bacteria rating). These results were found to substantiate the results obtained from the MSTK procedure (Fig. 1), which describe Hatchery 2 as a hatchery operating with excellent sanitation practices and Hatchery 3 as a hatchery operating with poor sanitation practices. Identification
of Hatchery Isolates
The most prevalent hatchery isolates were of the following genera: Flavobacterium, Proteus, Pseudomonas, Bacillus, Aspergillus, Paecilomyces and Penicillium, as well as coliform bacteria (Table 2). Of these microorganisms, several have been implicated as being pathogenic to the developing embryo and chick. Coliform bacteria have been associated with a disease known as omphalitis, an inflamation of the navel, which frequently results in early chick mortality (Schwartz, 1977). Wright et al, (1959) found that when 1,200 or more Aspergillus colonies per gram of fluff were detected, aspergillosis was diagnosed in chicks or poults hatched in the hatchery. Other nonspecific bacterial infections have been frequently associated with hatchery losses due to early chick mortality and poor hatchability (Watts and Rac, 1958). Thus, the types of organisms found in this study were typical of those found by others (Saddler, 1975; Wright et al., 1959) and these could certainly cause problems in the hatchery if left unchecked. Hatchery areas where a large number of microorganisms were frequently found were considered important areas to monitor routinely. These areas included: 1) room floors; 2) hatchers, floor, ceiling, air exhaust, air inlet area, and outside top of machine; and 3) setters, floor, wall, air exhaust area, and air
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been left to accumulate as a wet mass behind the hatcher machines. This practice most likely contributed to the high numbers of bacteria found on the hatcher room floor, which was monitored just prior to the transfer of eggs into the hatcher machines in Hatchery 6. The use of the MSTK in the hatcheries studied was well received by the hatchery personnel. Three of five commercial hatcheries tested routinely monitored the hatchery environment with either air exposure plates, Rodac plates, or used the services of a consulting microbiologist. It was found that the concerns of hatchery managers about using any sanitation monitoring system included the following points: 1) that the environmental sampling method used accurately reflect the microbial load in the test area; 2) that the tests be easily interpreted; 3) that guidelines be available so that realistic goals could be set; 4) that the monitoring system be economical to use. In view of these concerns the following points are noteworthy: 1) The MSTK clearly indicated areas in the hatcheries where there were reservoirs of organisms and also areas where the sanitation procedures in place were adequate. 2) The test was easy to perform and could be used on all smooth surfaces. Colonies were easily counted using the grid provided on each media filter. 3) Most hatcheries differ in the types of the machines used to set and hatch eggs. Some are old, wooden models and others are new, fiberglass types. In view of this variation, generalized guidelines for a tolerable number of organisms to be found on clean hatchery surfaces are difficult to develop. 4) The cost of material for monitoring one test site using the MSTK system was between 3 and 4 dollars. By monitoring critical areas where high microbial numbers are most likely to be found, cost can be minimized.
HATCHERY SANITATION
inlet area. Magwood and Marr (1964) found horizontal surfaces and floors in hatcheries to be very important reservoirs of microorganisms. Evaluation ofMSTK
and
DSAPMethods
well as molds. Organisms of the genus Bacillus were isolated from the four types of counters used. The most useful media strip of the MSTK system for monitoring the hatchery environment was found to be the total bacterial counter (MTSK). An examination of the specificity of the media strips prepared by Millipore revealed some additional undesirable features in the MSTK system. It was generally observed that fewer and larger colonies developed at the bottom of all media strips. The organisms growing on the MCSK varied in their appearance, with small yellow colonies observed at the top of the media strip and larger, blue-green colonies observed at the bottom of the media strip. A pure culture of E. coli was used in this part of the experiment, and microscopic examination verified that the organisms showing different pigments on the same MCSK media strip were both E. coli. The
TABLE 2. Isolation and identification of microorganisms from poultry hatcheries using the MSTK system Kit media used2 Organism'
MTSK
MCSK
Acinetobacter Alcaligenes Coliform Flavobacterium Moraxella Proteus Pseudomonas Bacillus Staphylococcus Streptococcus Aspergillus Paecilomyces Penicillium
1
MYSK
MPSK
Hatchery area sampled Chick processing room floor Hatcher machine exhaust, table used for traying eggs Chick work table, vacuum lift transfer cup, egg shell, chick processing room floor Hatcher machine floor, hatcher air inlet, chick work table, hatcher tray Egg holding room floor, hatcher machine wall Setter wall, setter floor, egg holding room floor Hatcher machine floor, hatcher tray, setter room floor, chick box, hatcher room floor Setter room floor, egg holding room floor, hatcher air inlet, setter air inlet, hatcher room floor, setter egg tray Egg shell Chick box Setter machine floor, trolley, setter machine circulating fan blade Setter room air inlet, hatcher air inlet, setter floor Chick processing room floor, fan blade in setter, hatcher air inlet and exhaust
Identification criteria, (Vanderzant and Nickelson, 1969; Buchanan and Gibbons, 1974).
2
Millipore SwabTest Kit (MSTK), Total Counter (MTSK), Coliform Counter (MCSK), Yeast and Mold Counter (MYSK), Pseudomonad Counter (MPSK). 3
+, Indicates isolation of the organism.
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Specificity. The various types of organisms isolated from the MSTK media strips used for sampling hatchery surfaces are summarized in Table 2. The total count (MTSK) counters supported the growth of a broad range of microorganisms including coliforms, staphylococci, and pseudomonads as well as molds. The coliform counters (MCSK) supported the growth of Gram negative bacteria other than coliform bacteria as well as some Gram positive bacteria. Gram negative bacteria other than pseudomonads were isolated from the pseudomonad counters (MPSK). The yeast and mold (MYSK) counters supported the growth of Gram positive and Gram negative bacteria as
305
4.23 4.70
4.75 4.40 2.87 3.32
MSTK DSAP
MSTK DSAP MSTK DSAP MSTK DSAP MSTK DSAP
Pseudomonas sp.
Staphylococcus sp.
Fiberglass hatcher tray.
NS; Not significant or P>.10.
***P<.001.
**.001
*.01
S
3.01 2.83
3.31 2.08
4.08 4.70 4.08 4.70
ND" ND
4.67 4.70 2.65 3.36
ND ND
4.28 3.43 4.38 4.70
2.86 3.11
4.34 4.70
2.03
0
Tray 3
4.26 4.70
3.00 3.18
Glass
MSTK, Millipore Swab Test Kit; DSAP, direct surface agar plate.
Surfaces had dried thoroughly.
"ND, Not done.
3
2
1
Paecilomyces variotii
Salmonella hadar
E. coli
Proteus sp.
MSTK DSAP
3.11 2.66
MSTK DSAP
Pseudomonas fragi
4.11 4.70 4.20 4.70
Epoxy
Recovery method 2
Organism
0
3.15 2.80
3.29 2.67
4.72 4.70 2.91 3.32 4.20 3.40 4.11 4.70
4.60 4.40 2.77 3.20 4.32 3.02 4.20 4.70
4.24 4.70
3.09 3.16
Glass
4.20 4.70
3.31 2.71
Epoxy
Surface ; contaminated
10
.90
3.31 2.64
4.18 3.09 4.04 4.70
2.67 3.08
ND ND
4.20 4.70
0
Tray
Minutes after surface contamination
TABLE 3. Effects of drying on the survival of microorganisms on test surfaces and their recovery usi
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307
HATCHERY SANITATION
These results showed that the ability of the MSTK to recover organisms from surfaces is realistically comparable to that of the DSAP method. Differences in recovery of organisms were exaggerated following drying of surfaces.
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location of the differently pigmented organisms suggested that the concentration of the dye in the MCSK media was not always uniform. Rehydration of the medium was, uniform. According to the MSTK operating instructions, (cat. AB820, 1981, Millipore Corp., Bedford, MA) only blue or blue-green colonies should be counted on the MCSK for coliform enumeration. In these experiments, some of the Proteus colonies did and some of the E. coli colonies did not take up the blue dye. The observations noted in these experiments further support the sole use of the MTSK media strips for monitoring the hatchery environment. The DSAP method was used in the laboratory for the accurate enumeration of surface contaminants. Favero et al. (1968) outlined the limitations of using this method in the field as 1) the inability to incubate surfaces at proper temperatures due to their fixed nature; 2) the coalescence of colonies when microbial contamination is high; 3) the inappropriateness of the method for use on surfaces containing residual amounts of chemicals that would prevent the growth of microorganisms. Recovery Efficacy. Attempts were made to choose surfaces (epoxy tile, glass, and hatcher tray) that would be representative of those found in hatcheries. Hard surfaces were chosen so that the experimental results would not be affected by differences in the porosities of the surfaces. The paired Student's t test at a .05 level of significance was used (Mendenhall, 1979) to test the hypothesis that there was not a significant difference between the number of organisms detected using the MSTK (total counter) and DSAP methods. Table 3 shows the results of the comparison between the two methods for the recovery of bacteria and one mold from deliberately contaminated surfaces. The two methods were not significantly different in their ability to recover cells of Pseudomonas fragi, Staphylococcus, Proteus, and E. coli but were significantly different in their ability to recover another strain of Pseudomonas, Salmonella hadar, and Paecilomyces variotii. The latter two bacteria were recovered in higher numbers by the DSAP method whereas MSTK gave better recovery of the mold Paecilomyces.
308
SOUCY ET AL. TABLE 5. Lethal effects of drying microorganisms on membrane filters1
Organism
1.48 3.2 6.6 2.86 1.82 1.08 1.8
X X X X X X X
102 10" 103 104 10" 103 10"
0 0 189 0 110 33 7
Percent recovery 0 0 2.86 0 .60 3.06 .04
1 Suspensions of viable microorganisms were dried on filters at 27 C in a sterile petri plate and placed on trypticase soy agar for growth at 37 C.
This was very evident in the better recovery of organisms by the DSAP method than the MSTK method after the surfaces had dried thoroughly (105 min, Time 3). The MSTK method failed to recover viable Pseudomonas and Salmonella under the same conditions. This could be a reflection of the ability of these organisms to attach themselves to surfaces and thus hinder their removal by the swab (Firstenberg-Eden, 1981). These bacteria, being attached to the surface, would develop into colonies in the agar medium provided by the DSAP method. Duncan's multiple range test at a significance level of .05 (Malik and Mullen, 1973) showed that there was no difference in the recovery of organisms when the surfaces were tested immediately after contamination (Time 1) and when the surfaces were tested 10 min after contamination (Time 2) using both the DSAP and MSTK methods of recovery. Significant differences between the recovery of organisms at Times 1 and 3 and Times 2 and 3 were apparent. Table 4 shows that at Time 3 the number of organsims recovered by both methods was very low with approximately 28% fewer organisms recovered than at Times 1 and 2. This decrease in the number of organisms enumerated was taken to mean that the organisms were unable to survive the effects of drying on hatchery surfaces. Effect of Drying, The results of drying suspensions of the test organisms on Millipore membrane filter discs are summarized in Table 5. The results seen in the comparison of the MSTK and DSAP methods, where low numbers of organisms were recovered using both the MSTK and DSAP methods of recovery on dried surfaces, are supported by the significant mortality of organisms following drying on
membrane filters. Staphylococcus, E. coli, Salmonella hadar, and Paecilomyces variotii were recovered in countable numbers after drying. Other bacteria were not detected. In similar experiments, Angelotti and Foter (1958) demonstrated the lethal effects of drying on vegetative cells. These results emphasize the importance of drying surfaces to effectively reduce the number of viable organisms. The MSTK system was evaluated and found to be a useful method of determining the microbial load on hatchery surfaces and thus is a valuable tool for routine monitoring of hatchery sanitation. The use of this method to establish the effectiveness of specific disinfectant, sanitizing, and fumigation systems used by individual hatcheries would require further study.
ACKNOWLEDGMENTS
The cooperation of the personnel of the hatcheries studied was greatly appreciated. The Ontario Ministry of Agriculture and Food, Veterinary Services Laboratory, University of Guelph, Guelph, Ontario, and Le Laboratoire Regional De L'Assomption, Division De Pathologie Animale, Ministere De L'Agriculture, Quebec, performed the laboratory analysis on the fluff samples taken from the test hatcheries. John Bissett, Biosystematics Research Institute, identified the culture of Paecilomyces variotii. REFERENCES Angelotti, R. and M. J. Foter, 1958. A direct surface agar plate laboratory method for quantitatively
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Pseudomonas fragi Pseudomonas sp. Staphylococcus sp. Proteus sp. E. coli Salmonella hadar Paecilomyces variotii
Viable organisms per filter
N u m b e r applied to each filter
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detecting bacterial contamination on non-porous surfaces. Food Res. 23:170-174. Buchanan, R. E. and N. E. Gibbons, 1974. Bergey's Manual of Determinative Bacteriology. 8th ed. Williams and Wilkins Co., Baltimore, MD. Chapman, G. H., 1951. A culture medium for detecting and confirming H. coli in ten hours. Am. J.Publ. Health 41:1381. Chute, H. L., and M. Gershman, 1961. A new approach to hatchery sanitation. Poultry Sci. 40:568-571. Favero, M. S., J. J. McDade, J. A. Robertson, R. K. Hoffman, and R. W. Edwards, 1968. Microbiological sampling of surfaces. J. Appl. Bacteriol. 31:336-343. Firstenberg-Eden, R., 1981. Attachment of bacteria to meat surfaces: review. J. Food Prot. 44:602— 607. Gentry, R. F., M. Mitrovic, and G. R. Bubash, 1962. Application of Anderson sampler in hatchery sanitation. Poultry Sci. 41:794—804. Hacking, B., 1982. Sanitation for hatchery and farm. CanadaPoultryman 69:22; 3 6 - 3 7 . Harrigan, W. F., and M. E. McCance, 1976. Laboratory Methods in Food and Dairy Microbiology. 2nd ed. Academic Press, New York, NY. Magwood, S. E., and H. Marr, 1964. Studies in hatchery sanitation 2. Poultry Sci. 4 3 : 1 5 5 8 - 1572. Malik, H. J., and K. Mullen, 1973. A first course in probability and statistics. Addison-Wesley Publ. Co., Inc., Reading, MA.
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