- Email: [email protected]
A Rapid and Simple Method for the Detection and Isolation of Salmonella from Mixed Cultures and Poultry Products1
46
J. W. DEATON AND F. N. REECE
growth. The temperature used was a linear cycle on a 24-hour basis from 21° to 35° to 21°C. Birds reared from 3 to 8 weeks and receiving 12 hours of light only in the low portion of the temperature cycle weighed significantly more at 4, 6 and 8 weeks of age than birds reared from 3 to 8 weeks and receiving 12 hours of light only in the high portion of the temperature cycle. REFERENCES
A Rapid and Simple Method for the Detection and Isolation of Salmonella from Mixed Cultures and Poultry Products 1 DANIEL Y. C. FUNG 2 AND ALLEN A. KRAFT Department of Food Technology, Iowa State University, Ames, Iowa 50010 (Received for publication July 29, 1969)
R
APID and simple methods for the -detection and isolation of Salmonella from foods and feeds have been the subject of much research and investigation. A detailed discussion of the conventional biochemical methods of detecting and isolating Salmonella in foods and feeds was made by Galton et al. (1968). The use of 1 Journal Paper No. J-6168 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project 1749. Center for Agricultural and Economic Development cooperating. 2 Present address: Department of Bacteriology, Pennsylvania State University, University Park, Pa.
fluorescent antibody for rapid detection of Salmonella in animal food products was reviewed by Ayres (1967). However, all these procedures are time consuming (48-96 hr.) and laborious or require special equipment such as a microscope with fluorescence equipment. Schafer et al. (1968) reported on the use of reagent tablets for rapid biochemical confirmation of "Salmonellae after isolation on selective agar. A glass apparatus for determining the presence of Salmonella in mixed cultures was described by Banwart (1968). Also, Banwart et al. (1968) de-
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Barott, H. G., and E. M. Pringle, 1949. The effect of temperature and humidity of environment during the first 18 days after hatch. J. Nutrition, 37: 153-161. Barott, H. G., and E. M. Pringle, 1950. The effect of temperature of environment during the period from 18 to 32 days of age. J. Nutrition, 41: 25-30. Deaton, J. W., F. N. Reece and T. H. Vardaman, 1968. The effect of temperature and density on broiler performance. Poultry Sci. 47: 293-300. Duncan, D. B., 1955. Multiple range and multiple
F tests. Biometrics, 11: 1-42. Howes, J. R., W. Grub and C. A. Rollo, 1962. The effects of constant high temperature regimes upon broiler growth, feed efficiency, body composition and carcass quality. Poultry Sci. 41: 1652. Ota, H., and H. L. Garver, 1954. Studies on the growth of baby chicks to frying chickens in the poultry calorimeters. Proceedings of the Annual Meeting of the Amer. Soc. Agr. Engineers, Minneapolis, Minn. Prince, R. P., W. C. Wheeler, W. A. Junnila, L. M. Potter and E. P. Singsen, 1960. Effects of temperature on feed consumption and weight gain in broiler production. Storrs, Connecticut Agr. Exp. Sta. Progress Report 33. Reece, F. N., and J. W. Deaton, 1969. A system for control of tempertature and humidity in environmental poultry research chambers. Amer. Soc. Agr. Engineers, Paper No. 69-503. Reece, F. N., J. W. Deaton and C. W. Bouchillon, 1969. Heat and moisture production of broilers 1. Summer conditions. Poultry Sci. 48: 1297-1303. Snedecor, G. W., and W. G. Cochran, 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa.
D E T E C T I O N OF
This paper describes a simple system, combining biochemical and physical properties of Salmonella, which can detect small numbers (1 in 100 ml. of culture) of Salmonella in the presence of large numbers (1X10 7 ) of competitive organisms. Also, the system was used to detect Salmonella in turkey rolls, turkey roasts, whole egg, and commercial dried egg products in an attempt to apply the method to foods. T h e method utilizes commercially available glassware and results are obtainable within 17-36 hr. MATERIALS AND METHODS
Organisms. Salmonella lyphimurium, S. heidelberg, S. anatum 53, S. infantis, S. thompson, 20 isolates of Salmonella, Escherichia coli 2B5, Proteus sp. 2D20, Proteus sp. 2D 15, and Pseudomonas aeruginosa 2F40 (Department of Food Technology and D e p a r t m e n t of Bacteriology, Iowa State University) were used as control inocula after growth at 37°C. in nutrient broth for about 2 hr. when turbidity was evident. Three ml. aliquots of the five known Salmonella cultures in the
D
mmm
FIG. 1. Biochemical reactions of Salmonella in motility multi-layer agar flask. Flasks A and B are uninoculated. Flasks C and D show presumptive and confirmed biochemical reactions of motile HaS positive Salmonella, respectively.
logarithmic growth phase were quick frozen and stored as stock cultures. Tubes were thawed and warmed to 37°C. prior to use. Motility Flask and Media. A nepheloflask (300 ml., Bellco Glass Inc., Vineland, N. J.) was the vessel of choice for growth of Salmonella in selective liquid media for detection of motility and biochemcial reactions in solid media in the side arm (Fig. 1A). When isolation of organisms was desired, a modified nepheloflask with the tip of the side arm cut and capped with a rubber stopper was used so t h a t motile organisms could be withdrawn with a sterile syringe (Fig. I B ) . Sterile and melted triple sugar iron agar (TSI, Difco, 3 ml.), carbohydrate
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
veloped a screening method for determining Salmonella-negative samples of pasteurized dried whole egg. With most conventional methods, several subculturing steps are usually involved. Recently, a faster method (24-54 hr.) using a motility flask-fluorescent antibody (FA) staining procedure was reported by Abrahamsson el ol. (1968). Although this procedure seemed advantageous over the usual conventional methods, the motility flask illustrated seemed rather inconvenient to work with and to clean and is not commercially available, making it difficult for other workers to repeat the reported experiments. T h e FA technique is indispensible in this procedure, hence this method could not be utilized by laboratories without a fluorescence microscope.
47
SALMONELLA
48
D. Y. C. FUNG AND A. A. KRAFT
Biochemical Reactions. Turbidity of the lactose broth indicated growth of organisms in the flask. Motile Salmonella migrate through the selenite cystine agar layer and ferment the mannitol or dulcitol causing change of color from red to yellow with gas production in the carbohydrate agar layer. Other motile organisms were either inhibited or delayed in the selenite cystine layer and did not give a typical Salmonella reaction in the carbohydrate agar. This was considered as a presumptive test for Salmonella which occurred after 12 hr. of incubation. When motile Salmonella migrated to the TSI agar layer, carbohydrates were fermented and the color of the agar changed from red to yellow with the production of gas (Fig. 1C). Excessive gas production sometimes forced the selenite cystine agar layer out of the side arm into the flask. After about 4-6 hr., blackening developed at the tip of the side arm due to hydrogen sulfide production under anaerobic conditions. This stage was regarded as a confirmed test for the presence of motile-H2S producing Salmonella (Fig. ID). Further tests, including serological reactions, may be performed to complete the determination.
Detection of Salmonella in Mixed Cultures. Using the motility flask system described, an attempt was made to detect Salmonella in a mixed population containing Proteus (sp. 2D 20), Pseudomonas, and Escherichia. S. typhimurium was used as a representative organism in this study unless specified. Four control motility flasks were prepared by aseptically inoculating a 1% inoculum of each strain individually into a flask. Viable cell counts of each test organism were also made. Two other control flasks were prepared, one was inoculated with 1% Salmonella and the other with 1% inoculum of all four test organisms. At the end of the experiment, cultures were taken from the tip of the side arm of both flasks and streaked on differential media to detect the presence of Salmonella. Seven additional motility flasks were prepared; each flask was inoculated with a 1% concentration of Proteus (sp. 2D20), Pseudomonas, and Escherichia and successive decimal dilutions of Salmonella. Thus, the concentration of Salmonella in the seventh flask was a 10~6 dilution of the bacteria in the first flask. All 13 flasks were incubated at 37°C. for 36 hr. with observations made atO, 17, 20, 24, 30, and 36 hr. Effects of Sodium Lauryl Sulphate {SLS). Preliminary experiments showed the ability of Proteus (sp. 2D20) and Escherichia to travel through the above agar layers and produce atypical Salmonella reactions. In order to further eliminate these competitive organisms in this system, SLS (Jameson, 1961) was incorporated into the selenite cystine agar layer. This was done by adding appropriate percentages of sterile SLS into sterile and melted selenite cystine agar mixture. A series of motility flasks were prepared as described previously except for addition of SLS (0.1, 0.15, 0.2, 0.3, 0.5, and 1%) in the selenite cystine agar, and
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
agar (0.5% mannitol or dulcitol, 1% agar in Difco phenol red base broth, 3 ml.), and selenite cystine agar (Difco selenite cystine broth, SIM agar, prepared in accordance with the manufacturer's instructions and 1% agar, 3 ml.) were in troduced into the side arm (placed perpendicularly in a test-tube rack) of a presterilized nepheloflask. The flask was positioned so that the side arm was upright and the agars were added in succession after solidification between each addition. After solid media preparation, the nepheloflask was placed in a normal position so that the side arm was parallel to the bench top before 250 ml. of sterile lactose broth (Difco lactose broth) and 1% inoculum were added.
DETECTION or SALMONELLA
49
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
substitution of mannitol for dulcitol in the motility agar flasks containing turkey carbohydrate layer for all subsequent roast homogenate. The flasks were inexperiments. The flasks were inoculated cubated at 37°C. and readings were at a level of 1% with cultures of Proteus taken at 0, 24, and 36 hr. of incubation. (sp. 2D 15), Escherichia, and Salmonella Detection of Salmonellae in chicken individually to determine the effects of was also investigated. Chicken wings different concentrations of SLS on the were purchased from a retail store on the migration of these organisms in this day the poultry was delivered to the multi-layer agar system. The 18 flasks store. The packaged chicken was stored were incubated at 37°C. for 36 hr. with at about 4°C. for 2 or 12 days and then readings taken at 0, 17, 24, 30, and 37. examined for Salmonellae by the new Tests of Different Strains of Salmonella. method and the conventional method Five known strains and 20 unknown described by Galton et al. (1968). For Salmonella isolates were tested in this sampling, each chicken part was rinsed system by introducing one percent in- individually in 200 ml. of sterile lactose oculum into 250 ml. lactose broth con- broth; a total of 29 chicken wings were tained in multi-layer motility agar flasks. tested for Salmonellae as they might occur The selenite cystine agar was fortified in a natural mixed population of organwith 0.15% SLS. The flasks were incu- isms. bated at 37°C. for 42 hr. with readings Detection of Salmonella in Egg Products. taken at 0, 17, 24, 36, and 42 hr. of in- For the detection of Salmonella in whole cubation. The Salmonella isolates were egg, one whole egg was aseptically intropreviously obtained from chicken meat cuced into a motility flask containing by use of the conventional procedures and sterile lactose broth to give a total volume tested with polyvalent Group O serum. of 250 ml. The egg shell was disinfected byPoultry Product Preparations. Fifty dipping the egg in alcohol, draining and grams of commercially prepared turkey flaming the air cell end of the egg. A small roll were homogenized aseptically with portion of the egg shell was then cracked 100 ml. each of sterile distilled water and and the contents were carefully emptied sterile lactose broth in an Osterizer (John into the flask. One set of flasks was preOster Mfg. Co., Racine, Wise.) then in- pared with one egg and lactose broth troduced into a multi-layer motility agar added to each flask. The mixture was then flask. For this test a total of ten flasks inoculated with a constant number of containing turkey roll homogenate were competitive organisms and serial dilutions prepared. One control flask was inoculated of Salmonella. Another set of motility with Salmonella, and another with Pro- flasks was prepared with the first flask teus. Eight test flasks were inoculated only containing one egg and lactose broth with constant numbers of competitive with Salmonella. Decimal dilutions of organisms (Escherichia, Proteus, and Pseu- Salmonella from this flask were made to domonas) and decimally diluted Sal- the next three flasks each of which conmonella. A similar experiment, with no tained 250 ml. of sterile lactose broth. A competitive organisms, was performed by constant number of competitive organinoculating, in duplicate, three dilutions isms was added to all four flasks. Detec(at low concentrations) of five known tion of Salmonella in whole egg was also strains of Salmonella. Inoculations were performed by preparing twelve motility made in 30 motility agar flasks containing flasks with one egg added aseptically to turkey roll homogenate and another 30 each flask but without added inocula.
50
D. Y. C. FUNG AND A. A. KRAFT
RESULTS AND DISCUSSION
The results for detection of Salmonella in mixed cultures are presented in Table 1. Typical complete Salmonella reactions
were obtained in the Salmonella control flask as early as 17 hrs. of incubation. Other control flasks showed no typical Salmonella reactions. The Escherichia control demonstrated acid production but no gas formation. The Proteus (sp. 2D20) control showed acid but no gas formation after 24 hr. incubation, while the flask containing only Pseudomonas gave no biochemical changes. In the modified nepheloflasks, typical Salmonella reactions were obtained in about 20 hr. of incubation. Salmonella only was recovered when organisms were drawn from the tip of the side arm by a sterile syringe and streaked on differential media (brilliant green agar, EMB agar, and Pseudomonas P agar). In the test flasks, Salmonella was present in small numbers (2.1 X 10_1 to 2.1 X105 per flask) compared with the competitive organisms (3.9X10 7 per flask). Typical Salmonella reactions progressed in direct relation to the initial numbers of these organisms. The greatest inoculum of Salmonella initially showed typical complete reactions first. Of significance was the observation that flask no. 10 showed complete Salmonella reactions after 36 hr. with initially only 2.1 Salmonella present in 250 ml. of medium in the presence of 3.9 X10 7 competitive organisms. No typi-
TABLE 1. Detection of Salmonella in mixed cultures Incubation time in hr.
No. of organisms in flask
Flask 0 1 2 3 4 5 6 7 8 9 10 11
R R R R R R R R R R R
17 B R R Y G G G G G R R
20 B R R Y B B G G G Y Y
24 B R Y Y B B B G G G Y
30 B R Y Y B B B B B G Y
36 B R Y Y B B B B B B Y
R—red mannitol layer and red TSI layer. Y—yellow in both layers. G—gas formation in agar layer(s)—presumptive test. B—blackening in TSI layer—confirmed test.
Others
Salmonella 2.1X10
— — —
5
2.1X10 5 2.1X10 4 2.1X10 3 2.1X10 2 2.1X10 1 2.1X10° 21.X10" 1
— 3. 8X10' 1. IX10 5 1.1X10 4 3.9X10 7 3.9X10 7 3.9X10 7 3.9X10' 3.9X10 7 3.9X10 7 3.9X10 7
{Pseudomonas) {Proteus sp. 2D20) {Escherichia) {Combined)
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
These control flasks contained eggs having only the native or "wild" bacterial populations normally present. The flasks were incubated at 37°C. with readings taken at suitable intervals up to 40 hr. Similar experiments were performed using commercial egg yolk solids, egg white solids and whole egg solids in order to test the applicability of this system for detection of Salmonella in processed egg products. Fifty grams of egg solids were added aseptically to a series of motility flasks and dispersed in sterile lactose broth to give a total liquid volume of 250 ml. Again the Salmonella culture was decimally diluted in the flask with constant numbers of competitive organisms. Viable cell counts of all inocula were made for each experiment. The flasks, prepared and inoculated, were incubated at 37°C. for a maximum of 48 hr. with readings usually taken at 0, 17, 20, 24, and 36 hr. Control flasks contained uninoculated egg solids, and were used for testing the organisms native to the products.
D E T E C T I O N OF
51
SALMONELLA
TABLE 3.—Test of different strains of Salmonella
TABLE 2.—Effects of SLS concentrations ][ncubation time in hr.
Percentage of SLS
0.10 0.15 0.20 0.30 0.50 1.00
Proteus sp . 2D15
0
17
24
30
36
0
17
24
30
36
R K R R R R
G
B B
B K B B
B K B B B R
R R H R K R
R R R R R R
Y R R R R R
Y R R R R R
B R R R R R
Y Y Y R R
G G R R
<; R
Incubation time in hrs.
Organisms
Salmonella typh tmunum
R—Red dulcitol layer and red TSI layer. Y—Yellow agar layer(s). G—Gas formation in agar layers—presumptive test. B—Blackening in TSI layer—confirmed test.
Tests of 25 strains of Salmonella in this system are recorded in Table 3. T h e 5 known Salmonella strains gave positive results as early as 17 hr. of incubation with the production of acid, gas, and H 2 S. Eighteen of the 20 isolates gave positive results after 24 hr. of incubation with production of gas and acid b u t no H 2 S. After 36 hr. of incubation, one additional isolate (no. 2) provided a positive result. The other isolate (no. 1) did not show
17
24
36
42
R R R R R R R R
B Y Y B B R R R
B B B B B R R G
B B B B B R G G
B B B B B R G G
typical reactions after prolonged incubation. These isolates were found to be H 2 Snegative by the conventional procedure. Thus, of 25 strains of Salmonella tested, 24 gave positive results in the specified time limit of 36 hr. I t is possible t h a t the organism giving negative results may have been non-motile. D a t a for the detection of Salmonella in the turkey roll homogenate are presented in Table 4. The Salmonella control showed a confirmed Salmonella reaction in 24 hr. while the Proteus control remained unchanged at 36 hr. Control flasks for Escherichia and Pseudomonas were not prepared because these organisms were previously shown not to penetrate the selenite cystine agar layer. Confirmed Salmonella reactions were obtained in all flasks containing Salmonella (at concentrations of 7.5 to 7.5 X10 6 cells per flask) grown in the presence of large numbers of competitive organisms (6.5 X 1 0 8 cells per flask). Of significance was the observation T A B L E 4.—Detection of Salmonella
in turkey roll Incubation time in hrs. 1 2 3 4 5 6
/
S 0
10
homogenate
No. of organisms in flask
0
24
36
Salmonella
R K R R R R K K R R
B R G G Y Y R R R R
B R B B B B B B B R
7.5X10«
—
7.5X10« 7.5X105 7.5X10" 7.5X10* 7.5X102 7.5X101 7.5X10" 7.5X10-1
Others 1 . 5 X 1 0 ' (Proteus) 6 . 5 X 1 0 ' (combined) 6.5X10* 6.5X108 7.5X108 6.5X108 6.5X108 6.5X10' 6.5X10S
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
cal Salmonella reactions were demonstrated in the next dilution flask (0.21 Salmonella calculated per 250 ml.) after 26 hr. Prolonged incubation to 96 hr. also yielded negative results. Effects of SLS on motility of organisms are presented in Table 2. D a t a for Escherichia were not included because all 6 concentrations of SLS showed inhibitory effects. Proteus (sp. 2D15) was delayed a t the 0.10% SLS level and gave atypical Salmonella reactions (no acid and gas in dulritol layer) in the side a r m after 36 hr. of incubation. At an SLS level of 0.15% or more, Proteus (sp. 2D 15) was completely inhibited even after 36 hr. of incubation. Salmonella, on the other hand, were not inhibited by SLS concentrations up to 0.30%. However, 0.50% and 1.00% SLS produced delayed or inhibitory effects, respectively. T h u s , 0.15% SLS was chosen to be used in future experiments to produce a higher degree of selectivity for Salmonella in this system.
S. typhimurium S. heidelberg S. analum 53 5. infantis S. thompson Isolate 1 Isolate 2 Isolates 3 to 20
0
52
D. Y. C. FUNG AND A. A. KRAFT TABLE 5.—A. Detection of different strains of Salmonella in turkey products
Organisms
No of organisms in flask"
Turkey roast homogenate 0
24
Turkey roll homogenate
36
24
36
S typhimurium
30 3 0.3
R R R
B B R
B G R
B B R
S. Heidelberg
33 3.3 0.33
R R R
B B R
G G R
9.5 0.95 0.095
B B R
G G R
5. infotitis
8.5 0.85 0.085
B B R
G G R
'S. thompson
5 0.5 0.05
B B R
G G R
R R R
B. Comparison of two methods for detection of Salmonellae on chicken Sample No.
No.. of days stored a t 4°C.
Flash method
Conventional method
Table 6 shows results for detection of Salmonella in whole egg and egg yolk solids. Flasks 1 to 8 demonstrated the ability of this system to detect as few as 1 Duplicate analyses for each dilution. 3 Salmonella per egg. Complete reactions of the first series (flasks 1 to 4) with one that flask no. 9 showed a confirmed egg in each flask, were delayed for several Salmonella reaction after 36 hr. with only hours compared to the second series 7.5 Salmonella present initially in the tur- (flasks 5 to 8) which had one egg in the key roll homogenate, although the level of first flask only and serial dilutions of egg competitive bacteria was high (6.5 X10 8 in the next three flasks. These results may cells per 250 ml.). No reactions were ob- have been caused by more complete disserved in the next dilution flask (0.75 persion of Salmonella in the lower conSalmonella calculated per 250 ml. turkey centrations of egg material and possible roll homogenate) after 36 hr. dilution of antibacterial agents in the The data for the detection of the five second series. The Salmonella control strains of Salmonella in turkey roast and (flask 9) demonstrated a complete reacturkey roll homogenates are presented in tion after 36 hr. of incubation. Similar Table 5A. Again, low levels of all five data were obtained for egg yolk solids Salmonella strains were detected in both (flask 10 to 13) in which relatively small 4 turkey products within 36 hr. The fact numbers of Salmonella (12 to 1.2 X10 that positive results were obtained from cells per flask) were detected in the pressome flasks containing theoretically less ence of large numbers of competitive 8 than one Salmonella may be the result of organisms (1.7X10 cells per flask). No dilution variations within the limit of reaction was observed after 36 hr. of incubation of the twelve flasks containing experimental error. Fifteen of 17 chicken wings stored at one whole egg each without inocula, inl t o 14 15 16 & 17 18 to 24 25 26 to 29
2 2 2 12 12 12
+ + + +
+ — + —
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
S. anatum 53
4°C. for 2 days showed positive results for Salmonella by the multi-layer motility agar method, while 14 were Salmonellapositive after testing by the conventional method. Similar results were obtained when tests were performed after 12 days of storage; 8 of 12 samples demonstrated Salmonella by the new method, and 7 of 12 by the usual testing procedure (Table 5B). The possibility that samples 15 and 25 gave false positive results by the motility agar procedure was investigated by picking organisms from the tip of the modified nepheloflask and performing serological tests. In both instances, agglutination with Polyvalent O serum (Difco) was observed, and it is suggested that serological testing be performed with the flask method just as it is with conventional screening techniques.
53
DETECTION OF SALMONELLA TABLE 6.—Detection of Salmonella in whole egg and egg yolk solids
1 2 3 4 5 6 7 8 9
No. of organisms in flask
Incubation time in hrs.
Flask 0
17
24
30
36
40
Salmonella
Others
R R R R R R R R R
R R R R R Y Y Y R
R R R R G G Y G Y
G Y Y R G G Y G G
B Y B Y B B B B B
B B B B B B B B B
3,000 300 30 3 3,000 300 30 3 3,000
1.2X10 9 1.2X10 9 1,2X10 9 1.2X10 9 1.2X10, 1.2X10 9 1.2X10 9 1.2X10 9
Amount of whole egg
0.1 0.01 0.001 1 Amount of egg yolk solids
10 11 12 13 12 controls (no inoculum)
R R R R R
R R R R R
Y Y R R R
Y G G G R
Y G G G R
G G G G R
dicating the absence of Salmonella in the eggs tested. Detection of Salmonella in whole egg solids and egg white solids are presented in Table 7. Again, small numbers of Salmonella (3.7 to 37 cells per flask) in both products were detected in the presence of large numbers of competitive organisms (2.1 X107 cells per flask). Control samples containing only natural contaminants demonstrated no Salmonella reactions. These data demonstrated the ability of the multi-layer-motility agar system to detect small numbers of Salmonella in mixed cultures and poultry products in the presence of large numbers of competitive organisms.
12,000 1,200 120 12
1.7X10 8 1.7X10 8 1.7X10 8 1.7X10*
—
—
50 50 50 50 50
gm. gm. gm. gm. gm.
The major advantages of this system compared to the conventional procedure are: 1) rapid (17-36 hr.) detection of Salmonella in food and culture media; 2) sensitivity of the system by its ability to detect one Salmonella in 100 ml. of medium; 3) savings of time, labor, and material in operation; 4) test of relatively large quantities (100-300 ml.) of food in commercially available motility flasks. Further confirmation of Salmonella by agglutination reactions or other biochemical tests may be performed rather easily. This system has the potential as a simple and rapid method for large scale screening test for Salmonella in a variety of commercial food products.
TABLE 7.—Detection of Salmonella in whole egg solids and egg white solids Incubation time in hrs. No. of added to flask Salmonella Others
0
2.1X10 7 37 3.7 2.1X10 7 0.37 2.1X10 7 None (natural contaminants)
R R R R
Whole egg solids 24 36
0
B B R R
R R R R
G Y R R
Egg white solids 24 36 G Y R R
B B R R
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
—
54
D. Y. C. FUNG AND A. A. KRAFT ACKNOWLEDGMENTS
We thank Paul Hartman for suggestitins and for bacterial cultures and Carmen Rey for the chicken isolates. This investigation was supported in part by Public Health Service research grant UI 00263-02 from the National Center for Urban and Industrial Health. REFERENCES
Lysine, Methionine and Glycine Requirements of Japanese Quail to Five Weeks of Age ANNA SVACHA, C. W. WEBER AND B. L. REID Department of Poultry Science, University of A rizona, Tucson, A rizona 85721 (Received for publication July 31, 1969)
INTRODUCTION
F
EW studies of the amino acid requirements of Japanese quail have been reported. Donaldson (1967) found the requirements for the total sulfur amino acid to be 0.78% in a practical diet containing 25% protein, the glycine requirement to be 1.28% in a purified diet containing 29% protein, and the lysine requirement was met at 1.35% of a practical diet containing 25% protein. Amino acid requirements of Bob white quail and Ringneck pheasants, members of the same family as Coturnix, have been reported; Baldini et al. (1953) suggested a Arizona Agricultural Experiment Station Journal Article No. 1528.
lysine requirement for young Bobwhite quail of 1.3% of the diet when the protein level was 20-24%. Scott et al. (1963) showed the total sulfur amino acid requirements of Bobwhite quail and Ringneck pheasants to be 3.66% of the protein in a practical corn-soybean meal diet. Scott and Reynolds (1949) found in studies with pheasant chicks that the glycine requirement was met in a basal diet containing 1.7% glycine and 29.1% protein. The following experiments were designed to estimate minimum dietary lysine, methionine, and glycine requirements of Japanese quail for optimum growth, feed conversion, nitrogen retention, and protein efficiency ratio during
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Abrahamsson, K., G. Patterson and H. Riemann, 1968. Detection of Salmonella by a single-culture technique. Appl. Microbiol. 16: 1695-1698. Ayres, J. C , 1967. Use of fluorescent antibody for the rapid detection of enteric organisms in egg, poultry and meat products. Food Technol. 21: 145-154. Banwart, G. J., 1968. Glassware apparatus for de-
termining motile bacteria. 1. Salmonella. Poultry Sci. 47: 1209-1212. Banwart, G. J., A. J. Mercuri and T. R. Ryan, 1968. Screening method for determining SalraoneKa-negative samples of pasteurized dried whole egg. Poultry Sci. 47: 598-603. Galton, M. M., G. K. Morris and W. T. Martin, 1968. Salmonellae in foods and feeds: review of isolation methods and recommended procedures. PHS, Communicable Disease Center, Atlanta, Georgia. Jameson, J. E., 1961. A study of tetrathionate enrichment techniques, with particular reference to two new tetrathionate modifications used in isolating salmonellae from sewer swabs. J. Hyg. 59: 1-13. Schafer, W. J., R. E. Anderson, R. A. Morck and W. E Cassidy, 1968. Use of reagent tablets for rapid biochemical identification of Salmonellae and other enteric bacteria. Appl. Microbiol. 16: 1629-1630.
J. W. DEATON AND F. N. REECE
growth. The temperature used was a linear cycle on a 24-hour basis from 21° to 35° to 21°C. Birds reared from 3 to 8 weeks and receiving 12 hours of light only in the low portion of the temperature cycle weighed significantly more at 4, 6 and 8 weeks of age than birds reared from 3 to 8 weeks and receiving 12 hours of light only in the high portion of the temperature cycle. REFERENCES
A Rapid and Simple Method for the Detection and Isolation of Salmonella from Mixed Cultures and Poultry Products 1 DANIEL Y. C. FUNG 2 AND ALLEN A. KRAFT Department of Food Technology, Iowa State University, Ames, Iowa 50010 (Received for publication July 29, 1969)
R
APID and simple methods for the -detection and isolation of Salmonella from foods and feeds have been the subject of much research and investigation. A detailed discussion of the conventional biochemical methods of detecting and isolating Salmonella in foods and feeds was made by Galton et al. (1968). The use of 1 Journal Paper No. J-6168 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project 1749. Center for Agricultural and Economic Development cooperating. 2 Present address: Department of Bacteriology, Pennsylvania State University, University Park, Pa.
fluorescent antibody for rapid detection of Salmonella in animal food products was reviewed by Ayres (1967). However, all these procedures are time consuming (48-96 hr.) and laborious or require special equipment such as a microscope with fluorescence equipment. Schafer et al. (1968) reported on the use of reagent tablets for rapid biochemical confirmation of "Salmonellae after isolation on selective agar. A glass apparatus for determining the presence of Salmonella in mixed cultures was described by Banwart (1968). Also, Banwart et al. (1968) de-
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Barott, H. G., and E. M. Pringle, 1949. The effect of temperature and humidity of environment during the first 18 days after hatch. J. Nutrition, 37: 153-161. Barott, H. G., and E. M. Pringle, 1950. The effect of temperature of environment during the period from 18 to 32 days of age. J. Nutrition, 41: 25-30. Deaton, J. W., F. N. Reece and T. H. Vardaman, 1968. The effect of temperature and density on broiler performance. Poultry Sci. 47: 293-300. Duncan, D. B., 1955. Multiple range and multiple
F tests. Biometrics, 11: 1-42. Howes, J. R., W. Grub and C. A. Rollo, 1962. The effects of constant high temperature regimes upon broiler growth, feed efficiency, body composition and carcass quality. Poultry Sci. 41: 1652. Ota, H., and H. L. Garver, 1954. Studies on the growth of baby chicks to frying chickens in the poultry calorimeters. Proceedings of the Annual Meeting of the Amer. Soc. Agr. Engineers, Minneapolis, Minn. Prince, R. P., W. C. Wheeler, W. A. Junnila, L. M. Potter and E. P. Singsen, 1960. Effects of temperature on feed consumption and weight gain in broiler production. Storrs, Connecticut Agr. Exp. Sta. Progress Report 33. Reece, F. N., and J. W. Deaton, 1969. A system for control of tempertature and humidity in environmental poultry research chambers. Amer. Soc. Agr. Engineers, Paper No. 69-503. Reece, F. N., J. W. Deaton and C. W. Bouchillon, 1969. Heat and moisture production of broilers 1. Summer conditions. Poultry Sci. 48: 1297-1303. Snedecor, G. W., and W. G. Cochran, 1956. Statistical Methods. The Iowa State College Press, Ames, Iowa.
D E T E C T I O N OF
This paper describes a simple system, combining biochemical and physical properties of Salmonella, which can detect small numbers (1 in 100 ml. of culture) of Salmonella in the presence of large numbers (1X10 7 ) of competitive organisms. Also, the system was used to detect Salmonella in turkey rolls, turkey roasts, whole egg, and commercial dried egg products in an attempt to apply the method to foods. T h e method utilizes commercially available glassware and results are obtainable within 17-36 hr. MATERIALS AND METHODS
Organisms. Salmonella lyphimurium, S. heidelberg, S. anatum 53, S. infantis, S. thompson, 20 isolates of Salmonella, Escherichia coli 2B5, Proteus sp. 2D20, Proteus sp. 2D 15, and Pseudomonas aeruginosa 2F40 (Department of Food Technology and D e p a r t m e n t of Bacteriology, Iowa State University) were used as control inocula after growth at 37°C. in nutrient broth for about 2 hr. when turbidity was evident. Three ml. aliquots of the five known Salmonella cultures in the
D
mmm
FIG. 1. Biochemical reactions of Salmonella in motility multi-layer agar flask. Flasks A and B are uninoculated. Flasks C and D show presumptive and confirmed biochemical reactions of motile HaS positive Salmonella, respectively.
logarithmic growth phase were quick frozen and stored as stock cultures. Tubes were thawed and warmed to 37°C. prior to use. Motility Flask and Media. A nepheloflask (300 ml., Bellco Glass Inc., Vineland, N. J.) was the vessel of choice for growth of Salmonella in selective liquid media for detection of motility and biochemcial reactions in solid media in the side arm (Fig. 1A). When isolation of organisms was desired, a modified nepheloflask with the tip of the side arm cut and capped with a rubber stopper was used so t h a t motile organisms could be withdrawn with a sterile syringe (Fig. I B ) . Sterile and melted triple sugar iron agar (TSI, Difco, 3 ml.), carbohydrate
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
veloped a screening method for determining Salmonella-negative samples of pasteurized dried whole egg. With most conventional methods, several subculturing steps are usually involved. Recently, a faster method (24-54 hr.) using a motility flask-fluorescent antibody (FA) staining procedure was reported by Abrahamsson el ol. (1968). Although this procedure seemed advantageous over the usual conventional methods, the motility flask illustrated seemed rather inconvenient to work with and to clean and is not commercially available, making it difficult for other workers to repeat the reported experiments. T h e FA technique is indispensible in this procedure, hence this method could not be utilized by laboratories without a fluorescence microscope.
47
SALMONELLA
48
D. Y. C. FUNG AND A. A. KRAFT
Biochemical Reactions. Turbidity of the lactose broth indicated growth of organisms in the flask. Motile Salmonella migrate through the selenite cystine agar layer and ferment the mannitol or dulcitol causing change of color from red to yellow with gas production in the carbohydrate agar layer. Other motile organisms were either inhibited or delayed in the selenite cystine layer and did not give a typical Salmonella reaction in the carbohydrate agar. This was considered as a presumptive test for Salmonella which occurred after 12 hr. of incubation. When motile Salmonella migrated to the TSI agar layer, carbohydrates were fermented and the color of the agar changed from red to yellow with the production of gas (Fig. 1C). Excessive gas production sometimes forced the selenite cystine agar layer out of the side arm into the flask. After about 4-6 hr., blackening developed at the tip of the side arm due to hydrogen sulfide production under anaerobic conditions. This stage was regarded as a confirmed test for the presence of motile-H2S producing Salmonella (Fig. ID). Further tests, including serological reactions, may be performed to complete the determination.
Detection of Salmonella in Mixed Cultures. Using the motility flask system described, an attempt was made to detect Salmonella in a mixed population containing Proteus (sp. 2D 20), Pseudomonas, and Escherichia. S. typhimurium was used as a representative organism in this study unless specified. Four control motility flasks were prepared by aseptically inoculating a 1% inoculum of each strain individually into a flask. Viable cell counts of each test organism were also made. Two other control flasks were prepared, one was inoculated with 1% Salmonella and the other with 1% inoculum of all four test organisms. At the end of the experiment, cultures were taken from the tip of the side arm of both flasks and streaked on differential media to detect the presence of Salmonella. Seven additional motility flasks were prepared; each flask was inoculated with a 1% concentration of Proteus (sp. 2D20), Pseudomonas, and Escherichia and successive decimal dilutions of Salmonella. Thus, the concentration of Salmonella in the seventh flask was a 10~6 dilution of the bacteria in the first flask. All 13 flasks were incubated at 37°C. for 36 hr. with observations made atO, 17, 20, 24, 30, and 36 hr. Effects of Sodium Lauryl Sulphate {SLS). Preliminary experiments showed the ability of Proteus (sp. 2D20) and Escherichia to travel through the above agar layers and produce atypical Salmonella reactions. In order to further eliminate these competitive organisms in this system, SLS (Jameson, 1961) was incorporated into the selenite cystine agar layer. This was done by adding appropriate percentages of sterile SLS into sterile and melted selenite cystine agar mixture. A series of motility flasks were prepared as described previously except for addition of SLS (0.1, 0.15, 0.2, 0.3, 0.5, and 1%) in the selenite cystine agar, and
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
agar (0.5% mannitol or dulcitol, 1% agar in Difco phenol red base broth, 3 ml.), and selenite cystine agar (Difco selenite cystine broth, SIM agar, prepared in accordance with the manufacturer's instructions and 1% agar, 3 ml.) were in troduced into the side arm (placed perpendicularly in a test-tube rack) of a presterilized nepheloflask. The flask was positioned so that the side arm was upright and the agars were added in succession after solidification between each addition. After solid media preparation, the nepheloflask was placed in a normal position so that the side arm was parallel to the bench top before 250 ml. of sterile lactose broth (Difco lactose broth) and 1% inoculum were added.
DETECTION or SALMONELLA
49
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
substitution of mannitol for dulcitol in the motility agar flasks containing turkey carbohydrate layer for all subsequent roast homogenate. The flasks were inexperiments. The flasks were inoculated cubated at 37°C. and readings were at a level of 1% with cultures of Proteus taken at 0, 24, and 36 hr. of incubation. (sp. 2D 15), Escherichia, and Salmonella Detection of Salmonellae in chicken individually to determine the effects of was also investigated. Chicken wings different concentrations of SLS on the were purchased from a retail store on the migration of these organisms in this day the poultry was delivered to the multi-layer agar system. The 18 flasks store. The packaged chicken was stored were incubated at 37°C. for 36 hr. with at about 4°C. for 2 or 12 days and then readings taken at 0, 17, 24, 30, and 37. examined for Salmonellae by the new Tests of Different Strains of Salmonella. method and the conventional method Five known strains and 20 unknown described by Galton et al. (1968). For Salmonella isolates were tested in this sampling, each chicken part was rinsed system by introducing one percent in- individually in 200 ml. of sterile lactose oculum into 250 ml. lactose broth con- broth; a total of 29 chicken wings were tained in multi-layer motility agar flasks. tested for Salmonellae as they might occur The selenite cystine agar was fortified in a natural mixed population of organwith 0.15% SLS. The flasks were incu- isms. bated at 37°C. for 42 hr. with readings Detection of Salmonella in Egg Products. taken at 0, 17, 24, 36, and 42 hr. of in- For the detection of Salmonella in whole cubation. The Salmonella isolates were egg, one whole egg was aseptically intropreviously obtained from chicken meat cuced into a motility flask containing by use of the conventional procedures and sterile lactose broth to give a total volume tested with polyvalent Group O serum. of 250 ml. The egg shell was disinfected byPoultry Product Preparations. Fifty dipping the egg in alcohol, draining and grams of commercially prepared turkey flaming the air cell end of the egg. A small roll were homogenized aseptically with portion of the egg shell was then cracked 100 ml. each of sterile distilled water and and the contents were carefully emptied sterile lactose broth in an Osterizer (John into the flask. One set of flasks was preOster Mfg. Co., Racine, Wise.) then in- pared with one egg and lactose broth troduced into a multi-layer motility agar added to each flask. The mixture was then flask. For this test a total of ten flasks inoculated with a constant number of containing turkey roll homogenate were competitive organisms and serial dilutions prepared. One control flask was inoculated of Salmonella. Another set of motility with Salmonella, and another with Pro- flasks was prepared with the first flask teus. Eight test flasks were inoculated only containing one egg and lactose broth with constant numbers of competitive with Salmonella. Decimal dilutions of organisms (Escherichia, Proteus, and Pseu- Salmonella from this flask were made to domonas) and decimally diluted Sal- the next three flasks each of which conmonella. A similar experiment, with no tained 250 ml. of sterile lactose broth. A competitive organisms, was performed by constant number of competitive organinoculating, in duplicate, three dilutions isms was added to all four flasks. Detec(at low concentrations) of five known tion of Salmonella in whole egg was also strains of Salmonella. Inoculations were performed by preparing twelve motility made in 30 motility agar flasks containing flasks with one egg added aseptically to turkey roll homogenate and another 30 each flask but without added inocula.
50
D. Y. C. FUNG AND A. A. KRAFT
RESULTS AND DISCUSSION
The results for detection of Salmonella in mixed cultures are presented in Table 1. Typical complete Salmonella reactions
were obtained in the Salmonella control flask as early as 17 hrs. of incubation. Other control flasks showed no typical Salmonella reactions. The Escherichia control demonstrated acid production but no gas formation. The Proteus (sp. 2D20) control showed acid but no gas formation after 24 hr. incubation, while the flask containing only Pseudomonas gave no biochemical changes. In the modified nepheloflasks, typical Salmonella reactions were obtained in about 20 hr. of incubation. Salmonella only was recovered when organisms were drawn from the tip of the side arm by a sterile syringe and streaked on differential media (brilliant green agar, EMB agar, and Pseudomonas P agar). In the test flasks, Salmonella was present in small numbers (2.1 X 10_1 to 2.1 X105 per flask) compared with the competitive organisms (3.9X10 7 per flask). Typical Salmonella reactions progressed in direct relation to the initial numbers of these organisms. The greatest inoculum of Salmonella initially showed typical complete reactions first. Of significance was the observation that flask no. 10 showed complete Salmonella reactions after 36 hr. with initially only 2.1 Salmonella present in 250 ml. of medium in the presence of 3.9 X10 7 competitive organisms. No typi-
TABLE 1. Detection of Salmonella in mixed cultures Incubation time in hr.
No. of organisms in flask
Flask 0 1 2 3 4 5 6 7 8 9 10 11
R R R R R R R R R R R
17 B R R Y G G G G G R R
20 B R R Y B B G G G Y Y
24 B R Y Y B B B G G G Y
30 B R Y Y B B B B B G Y
36 B R Y Y B B B B B B Y
R—red mannitol layer and red TSI layer. Y—yellow in both layers. G—gas formation in agar layer(s)—presumptive test. B—blackening in TSI layer—confirmed test.
Others
Salmonella 2.1X10
— — —
5
2.1X10 5 2.1X10 4 2.1X10 3 2.1X10 2 2.1X10 1 2.1X10° 21.X10" 1
— 3. 8X10' 1. IX10 5 1.1X10 4 3.9X10 7 3.9X10 7 3.9X10 7 3.9X10' 3.9X10 7 3.9X10 7 3.9X10 7
{Pseudomonas) {Proteus sp. 2D20) {Escherichia) {Combined)
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
These control flasks contained eggs having only the native or "wild" bacterial populations normally present. The flasks were incubated at 37°C. with readings taken at suitable intervals up to 40 hr. Similar experiments were performed using commercial egg yolk solids, egg white solids and whole egg solids in order to test the applicability of this system for detection of Salmonella in processed egg products. Fifty grams of egg solids were added aseptically to a series of motility flasks and dispersed in sterile lactose broth to give a total liquid volume of 250 ml. Again the Salmonella culture was decimally diluted in the flask with constant numbers of competitive organisms. Viable cell counts of all inocula were made for each experiment. The flasks, prepared and inoculated, were incubated at 37°C. for a maximum of 48 hr. with readings usually taken at 0, 17, 20, 24, and 36 hr. Control flasks contained uninoculated egg solids, and were used for testing the organisms native to the products.
D E T E C T I O N OF
51
SALMONELLA
TABLE 3.—Test of different strains of Salmonella
TABLE 2.—Effects of SLS concentrations ][ncubation time in hr.
Percentage of SLS
0.10 0.15 0.20 0.30 0.50 1.00
Proteus sp . 2D15
0
17
24
30
36
0
17
24
30
36
R K R R R R
G
B B
B K B B
B K B B B R
R R H R K R
R R R R R R
Y R R R R R
Y R R R R R
B R R R R R
Y Y Y R R
G G R R
<; R
Incubation time in hrs.
Organisms
Salmonella typh tmunum
R—Red dulcitol layer and red TSI layer. Y—Yellow agar layer(s). G—Gas formation in agar layers—presumptive test. B—Blackening in TSI layer—confirmed test.
Tests of 25 strains of Salmonella in this system are recorded in Table 3. T h e 5 known Salmonella strains gave positive results as early as 17 hr. of incubation with the production of acid, gas, and H 2 S. Eighteen of the 20 isolates gave positive results after 24 hr. of incubation with production of gas and acid b u t no H 2 S. After 36 hr. of incubation, one additional isolate (no. 2) provided a positive result. The other isolate (no. 1) did not show
17
24
36
42
R R R R R R R R
B Y Y B B R R R
B B B B B R R G
B B B B B R G G
B B B B B R G G
typical reactions after prolonged incubation. These isolates were found to be H 2 Snegative by the conventional procedure. Thus, of 25 strains of Salmonella tested, 24 gave positive results in the specified time limit of 36 hr. I t is possible t h a t the organism giving negative results may have been non-motile. D a t a for the detection of Salmonella in the turkey roll homogenate are presented in Table 4. The Salmonella control showed a confirmed Salmonella reaction in 24 hr. while the Proteus control remained unchanged at 36 hr. Control flasks for Escherichia and Pseudomonas were not prepared because these organisms were previously shown not to penetrate the selenite cystine agar layer. Confirmed Salmonella reactions were obtained in all flasks containing Salmonella (at concentrations of 7.5 to 7.5 X10 6 cells per flask) grown in the presence of large numbers of competitive organisms (6.5 X 1 0 8 cells per flask). Of significance was the observation T A B L E 4.—Detection of Salmonella
in turkey roll Incubation time in hrs. 1 2 3 4 5 6
/
S 0
10
homogenate
No. of organisms in flask
0
24
36
Salmonella
R K R R R R K K R R
B R G G Y Y R R R R
B R B B B B B B B R
7.5X10«
—
7.5X10« 7.5X105 7.5X10" 7.5X10* 7.5X102 7.5X101 7.5X10" 7.5X10-1
Others 1 . 5 X 1 0 ' (Proteus) 6 . 5 X 1 0 ' (combined) 6.5X10* 6.5X108 7.5X108 6.5X108 6.5X108 6.5X10' 6.5X10S
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
cal Salmonella reactions were demonstrated in the next dilution flask (0.21 Salmonella calculated per 250 ml.) after 26 hr. Prolonged incubation to 96 hr. also yielded negative results. Effects of SLS on motility of organisms are presented in Table 2. D a t a for Escherichia were not included because all 6 concentrations of SLS showed inhibitory effects. Proteus (sp. 2D15) was delayed a t the 0.10% SLS level and gave atypical Salmonella reactions (no acid and gas in dulritol layer) in the side a r m after 36 hr. of incubation. At an SLS level of 0.15% or more, Proteus (sp. 2D 15) was completely inhibited even after 36 hr. of incubation. Salmonella, on the other hand, were not inhibited by SLS concentrations up to 0.30%. However, 0.50% and 1.00% SLS produced delayed or inhibitory effects, respectively. T h u s , 0.15% SLS was chosen to be used in future experiments to produce a higher degree of selectivity for Salmonella in this system.
S. typhimurium S. heidelberg S. analum 53 5. infantis S. thompson Isolate 1 Isolate 2 Isolates 3 to 20
0
52
D. Y. C. FUNG AND A. A. KRAFT TABLE 5.—A. Detection of different strains of Salmonella in turkey products
Organisms
No of organisms in flask"
Turkey roast homogenate 0
24
Turkey roll homogenate
36
24
36
S typhimurium
30 3 0.3
R R R
B B R
B G R
B B R
S. Heidelberg
33 3.3 0.33
R R R
B B R
G G R
9.5 0.95 0.095
B B R
G G R
5. infotitis
8.5 0.85 0.085
B B R
G G R
'S. thompson
5 0.5 0.05
B B R
G G R
R R R
B. Comparison of two methods for detection of Salmonellae on chicken Sample No.
No.. of days stored a t 4°C.
Flash method
Conventional method
Table 6 shows results for detection of Salmonella in whole egg and egg yolk solids. Flasks 1 to 8 demonstrated the ability of this system to detect as few as 1 Duplicate analyses for each dilution. 3 Salmonella per egg. Complete reactions of the first series (flasks 1 to 4) with one that flask no. 9 showed a confirmed egg in each flask, were delayed for several Salmonella reaction after 36 hr. with only hours compared to the second series 7.5 Salmonella present initially in the tur- (flasks 5 to 8) which had one egg in the key roll homogenate, although the level of first flask only and serial dilutions of egg competitive bacteria was high (6.5 X10 8 in the next three flasks. These results may cells per 250 ml.). No reactions were ob- have been caused by more complete disserved in the next dilution flask (0.75 persion of Salmonella in the lower conSalmonella calculated per 250 ml. turkey centrations of egg material and possible roll homogenate) after 36 hr. dilution of antibacterial agents in the The data for the detection of the five second series. The Salmonella control strains of Salmonella in turkey roast and (flask 9) demonstrated a complete reacturkey roll homogenates are presented in tion after 36 hr. of incubation. Similar Table 5A. Again, low levels of all five data were obtained for egg yolk solids Salmonella strains were detected in both (flask 10 to 13) in which relatively small 4 turkey products within 36 hr. The fact numbers of Salmonella (12 to 1.2 X10 that positive results were obtained from cells per flask) were detected in the pressome flasks containing theoretically less ence of large numbers of competitive 8 than one Salmonella may be the result of organisms (1.7X10 cells per flask). No dilution variations within the limit of reaction was observed after 36 hr. of incubation of the twelve flasks containing experimental error. Fifteen of 17 chicken wings stored at one whole egg each without inocula, inl t o 14 15 16 & 17 18 to 24 25 26 to 29
2 2 2 12 12 12
+ + + +
+ — + —
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
S. anatum 53
4°C. for 2 days showed positive results for Salmonella by the multi-layer motility agar method, while 14 were Salmonellapositive after testing by the conventional method. Similar results were obtained when tests were performed after 12 days of storage; 8 of 12 samples demonstrated Salmonella by the new method, and 7 of 12 by the usual testing procedure (Table 5B). The possibility that samples 15 and 25 gave false positive results by the motility agar procedure was investigated by picking organisms from the tip of the modified nepheloflask and performing serological tests. In both instances, agglutination with Polyvalent O serum (Difco) was observed, and it is suggested that serological testing be performed with the flask method just as it is with conventional screening techniques.
53
DETECTION OF SALMONELLA TABLE 6.—Detection of Salmonella in whole egg and egg yolk solids
1 2 3 4 5 6 7 8 9
No. of organisms in flask
Incubation time in hrs.
Flask 0
17
24
30
36
40
Salmonella
Others
R R R R R R R R R
R R R R R Y Y Y R
R R R R G G Y G Y
G Y Y R G G Y G G
B Y B Y B B B B B
B B B B B B B B B
3,000 300 30 3 3,000 300 30 3 3,000
1.2X10 9 1.2X10 9 1,2X10 9 1.2X10 9 1.2X10, 1.2X10 9 1.2X10 9 1.2X10 9
Amount of whole egg
0.1 0.01 0.001 1 Amount of egg yolk solids
10 11 12 13 12 controls (no inoculum)
R R R R R
R R R R R
Y Y R R R
Y G G G R
Y G G G R
G G G G R
dicating the absence of Salmonella in the eggs tested. Detection of Salmonella in whole egg solids and egg white solids are presented in Table 7. Again, small numbers of Salmonella (3.7 to 37 cells per flask) in both products were detected in the presence of large numbers of competitive organisms (2.1 X107 cells per flask). Control samples containing only natural contaminants demonstrated no Salmonella reactions. These data demonstrated the ability of the multi-layer-motility agar system to detect small numbers of Salmonella in mixed cultures and poultry products in the presence of large numbers of competitive organisms.
12,000 1,200 120 12
1.7X10 8 1.7X10 8 1.7X10 8 1.7X10*
—
—
50 50 50 50 50
gm. gm. gm. gm. gm.
The major advantages of this system compared to the conventional procedure are: 1) rapid (17-36 hr.) detection of Salmonella in food and culture media; 2) sensitivity of the system by its ability to detect one Salmonella in 100 ml. of medium; 3) savings of time, labor, and material in operation; 4) test of relatively large quantities (100-300 ml.) of food in commercially available motility flasks. Further confirmation of Salmonella by agglutination reactions or other biochemical tests may be performed rather easily. This system has the potential as a simple and rapid method for large scale screening test for Salmonella in a variety of commercial food products.
TABLE 7.—Detection of Salmonella in whole egg solids and egg white solids Incubation time in hrs. No. of added to flask Salmonella Others
0
2.1X10 7 37 3.7 2.1X10 7 0.37 2.1X10 7 None (natural contaminants)
R R R R
Whole egg solids 24 36
0
B B R R
R R R R
G Y R R
Egg white solids 24 36 G Y R R
B B R R
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
—
54
D. Y. C. FUNG AND A. A. KRAFT ACKNOWLEDGMENTS
We thank Paul Hartman for suggestitins and for bacterial cultures and Carmen Rey for the chicken isolates. This investigation was supported in part by Public Health Service research grant UI 00263-02 from the National Center for Urban and Industrial Health. REFERENCES
Lysine, Methionine and Glycine Requirements of Japanese Quail to Five Weeks of Age ANNA SVACHA, C. W. WEBER AND B. L. REID Department of Poultry Science, University of A rizona, Tucson, A rizona 85721 (Received for publication July 31, 1969)
INTRODUCTION
F
EW studies of the amino acid requirements of Japanese quail have been reported. Donaldson (1967) found the requirements for the total sulfur amino acid to be 0.78% in a practical diet containing 25% protein, the glycine requirement to be 1.28% in a purified diet containing 29% protein, and the lysine requirement was met at 1.35% of a practical diet containing 25% protein. Amino acid requirements of Bob white quail and Ringneck pheasants, members of the same family as Coturnix, have been reported; Baldini et al. (1953) suggested a Arizona Agricultural Experiment Station Journal Article No. 1528.
lysine requirement for young Bobwhite quail of 1.3% of the diet when the protein level was 20-24%. Scott et al. (1963) showed the total sulfur amino acid requirements of Bobwhite quail and Ringneck pheasants to be 3.66% of the protein in a practical corn-soybean meal diet. Scott and Reynolds (1949) found in studies with pheasant chicks that the glycine requirement was met in a basal diet containing 1.7% glycine and 29.1% protein. The following experiments were designed to estimate minimum dietary lysine, methionine, and glycine requirements of Japanese quail for optimum growth, feed conversion, nitrogen retention, and protein efficiency ratio during
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Abrahamsson, K., G. Patterson and H. Riemann, 1968. Detection of Salmonella by a single-culture technique. Appl. Microbiol. 16: 1695-1698. Ayres, J. C , 1967. Use of fluorescent antibody for the rapid detection of enteric organisms in egg, poultry and meat products. Food Technol. 21: 145-154. Banwart, G. J., 1968. Glassware apparatus for de-
termining motile bacteria. 1. Salmonella. Poultry Sci. 47: 1209-1212. Banwart, G. J., A. J. Mercuri and T. R. Ryan, 1968. Screening method for determining SalraoneKa-negative samples of pasteurized dried whole egg. Poultry Sci. 47: 598-603. Galton, M. M., G. K. Morris and W. T. Martin, 1968. Salmonellae in foods and feeds: review of isolation methods and recommended procedures. PHS, Communicable Disease Center, Atlanta, Georgia. Jameson, J. E., 1961. A study of tetrathionate enrichment techniques, with particular reference to two new tetrathionate modifications used in isolating salmonellae from sewer swabs. J. Hyg. 59: 1-13. Schafer, W. J., R. E. Anderson, R. A. Morck and W. E Cassidy, 1968. Use of reagent tablets for rapid biochemical identification of Salmonellae and other enteric bacteria. Appl. Microbiol. 16: 1629-1630.