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Membrane Filter Method for the Detection and Enumeration of Salmonella in Egg Albumen1
Membrane Filter Method for the Detection and Enumeration of Salmonella in Egg Albumen 1 WlLLET K . KlRKHAM 2 AND PAUL A. HAETMAN Department of Bacteriology, Iowa State University, Ames, Iowa (Received for publication November 10, 1961)
RITICAL studies outlining techniques for isolating Salmonella from dried egg products were reported in 1953 (Ayres, 1953; Banwart and Ayres, 1953; Hurley and Ayres, 1953; North and Bartram, 1953). Although these methods have been reasonably effective and are in general use today, they are not without certain shortcomings. The analyses are time consuming and recovery of Salmonella is less than 100%. Efforts to improve the efficacy of the culture medium have been more or less successful (Byrne, Rayman and Schneider, 1955; Montford and Thatcher, 1961; North, 1961; Osborne and Stokes, 1955; Stokes and Osborne, 1955; Taylor, 1961; Taylor and Silliker, 1961; Taylor, Silliker and Andrews, 1958; and Wells, Bergquist and Forsythe, 1958). The major difficulty lies in the fact that addition of large quantities of some food materials, such as egg albumen, to selective enrichment broths greatly diminishes the performance of these media (Banwart and Ayres, 1953; Hurley and Ayres, 1953; North, 1961; Silliker and Taylor, 1958). 1
Journal paper no. J-3952 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project no. 1379. This investigation was supported (in part) by research grant No. E-1141 from the Institute of Allergy and Infectious Diseases of the National Institute of Health, Public Health Serivce. 2 Hall Township High School, Spring Valley, Illinois. A portion of this research was conducted by the senior author while a National Science Foundation Summer Research Participant for Teacher Training.
Lactose pre-enrichment (North, 1960, 1961; Taylor, 1961; Taylor and Silliker, 1961) is helpful in increasing the counts of Salmonella obtained. A centrifugation-enrichment method (Silliker and Taylor, 1958) and an antiserum-enrichment procedure with or without pre-centrifugation (Sugiyama, Dack and Lippitz, 1960) apparently also result in higher yields of Salmonella. Success of the latter techniques is due to concentration of the Salmonella from a large quantity of material, with subsequent culture in an environment which is much less inhibitory than a primary enrichment culture using a selective medium. All of these methods, however, are not without certain disadvantages. Pre-enrichment introduces one more step to an already laborious procedure. In like manner, centrifugation or use of antiserum results in substantial increases of effort and materials needed to perform the analyses. This report describes a simplified method for the detection and enumeration of Salmonella in egg albumen. Bacteria are removed from treated egg white by nitration of the white through a membrane filter, upon which the organisms may then be cultured on a selective medium. MATERIALS AND METHODS
Egg albumen resists ultrafiltration even though the size of the molecular units in this colloid are sufficiently small to pass a filter of 0.45 JA. pore size. Preliminary investigations were conducted to study the mechanism of ultrafiltration resistance, and to discover treatment(s) which might hold promise in effecting ultrafiltration of the
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DETECTION OF SALMONELLA
albumen. Reconstituted albumen (in 4% aqueous solution) was subjected to various treatments, as listed in Table 1, then was tested for filterability. Additional levels of reagents and combinations of treatments were also examined; however, these are not included in the table for the sake of brev-
1. Dialysis: Overnight against distilled water at 21° C , then 1 day against dilute acetic acid solution (pH 6.0) at 4° C. 2. Ion exchange resin: Strong cationic-anionic resin removed from a Bantam Demineralizer Cartridge (Barnstead Still and Sterilizer Co., Inc., Boston 31, Mass.). 3. Cations and anions: Sodium chloride, ammonium chloride, calcium chloride, ammonium phosphate, sodium ammonium tartrate, sodium citrate. 4. Chelating agents: Di-sodium Perma Kleer-50, Tri-sodium Perma Kleer 50, and Perma Kleer-80 beads (Refined Products Corp., Lyndhurst, N. J.); sodium citrate. 5. Surface active agents: Triton X-100 and Triton WR-1339 (Rohm and Haas Co., Philadelphia 5, Pa.); Tween 80 (Chemicals Dept., Atlas Powder Co., Wilmington, Del.); Antifoam A Emulsion (Dow Corning Corp., Midland, Mich.); Ucon Oil LB-525-43 (Carbide and Carbon Chemicals Co., New York 17, N. Y.); Antifoam 60 (Silicone Products Dept., General Electric Co., Waterford, N. Y.); and Antifoam "GY" (Hodag Chemical Corp., Chicago, 111.). 6. Reducing agents: Sodium thioglycollate, thioglycollic acid, sodium sulfite, and sodium thiosulfite. 7. Hydrogen bond breakers: Urea and guanidinium hydrochloride. 8. Silicone-coated filters: Desicote (Aloe Scientific Div., A. S. Aloe Co., St. Louis 12, Mo.) and Siliclad (Clay-Adams, Inc., New York 20, N. Y.). 9. Enzymes: Pepsin 1:3,000 and 1:10,000 (Cudahy Laboratories Div., Cudahy Packing Co., Omaha 7, Nebr.); Pancreatin, N. F. (Cudahy); Ficin (Merck and Co., Rahway, N. J.); Protease P-1S concentrate (now Protease W-15: Miles Chemical Co., Miles Laboratories, Clifton, N. J.); Rhozyme B-6 (Rohm and Haas Co., Philadelphia 5, Pa.); and Trypsin (Difco Laboratories, Detroit 1, Mich.). 10. Absorbants and filter aids: Diatomaceous earth (Celite Analytical Filter Aid: Johns-Manville Products Corp., Chicago 90, 111.) and alumina. 11. Adjustment of pH with dilute sodium hydroxide or hydrochloric acid. 12. Filtration at elevated temperatures. 13. Clarification through fluted Whatman no. 1, 4, or 12 paper prior to filtration.
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ity. Treatments which substantially increased the filterability of liquid albumen were: mixture with an ion-exchange resin; urea (3 M); use of surface active agents; enzymic hydrolysis; clarification prior to filtration; and filtration at elevated temperatures. A procedure was developed whereby the albumen was hydrolyzed with 1% pepsin, 1:3000, (pH 3.0), 1% ficin (pH 7.4), and protease P-lS in succession for 1 hr. each at 45°C, then clarified before filtration. This procedure, as with several other treatments developed previously which had successfully enabled filtration of the albumen, resulted in considerable loss of bacterial viability. Studies by Weiss and Ayres (unpublished) indicated that the reaction temperature of 45°C. was too high, however reduction of either hydrolysis time or temperature resulted in greatly decreased filtration rates. It was later found that filtration of some samples of commercial albumen could be effected simply by mixing the dried egg powder with water and Celite, and filtering after the Celite had settled for a sufficient period of time so that the resultant filter cake would be negligible in size. Other commercial samples could not be filtered when treated in the same manner; pepsin treatment prior to addition of the Celite, however, did render these latter samples filterable. The following procedure was finally adopted: 20 g. of dried egg albumen were blended for 10 to 20 sec. in a one liter Waring Blendor container with 460 ml. of sterile water and 20 ml. of 10% pepsin (1:3,000) solution (pH 3.5). The mixture was placed in a 40°C. water bath and held for 30 min., then 3 g. of Celite were added to the mixture, and the mixture was blended for an additional 10 to 20 sec. The container was again placed in a 40°C. water bath and allowed to settle for IS
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TABLE 1.—Treatments examined for their eject on the filtration rate of solutions of four percent egg albumen
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W . K . KlRKHAM AND P . A. HARTMAN
filtered pepsin solutions were plated on trypticase soy agar.4 Pepsin solutions could be stored in the refrigerator (4°C.) for periods of at least a week. During final trials, commercial egg albumen5 known to contain low numbers of Salmonella were used. In some instances, typical colonies were tested with Lederle polyvalent Salmonella antiserum. Difficulty was experienced in obtaining positive agglutination reactions using material taken directly from the membrane filters; however, the suspect colonies could be picked and streaked over an area of 10 to 15 mm2. on a sterile portion of the same plate. An incubation period of from 3 to 6 hrs. was all that was necessary to provide better isolation and an adequate supply of cells for agglutination tests (Taylor, 1958). As controls, samples were analyzed by the most probable number (MPN) method (Ayres, 1953), using the cysteine-selenite F enrichment medium of North and Bartram (1953). A brilliant green agar streak plate was made using a loopful of material from each MPN sample tube and bottle after an incubation period of 20 hr. and again following incubation for 44 hr. at 37°C. An MPN container was considered to be positive for Salmonella if either of the two plates yielded typical colonies; however, it should be noted that there were frequent occasions when an MPN container was apparently negative for Salmonella as indicated by one plate, yet was positive as indicated by the other. This composite-positive system did serve to eliminate many of the "skips" referred to by North (1960, 1961), and to result in higher MPN counts than would be ordinarily encountered using streak plates 5
3
Millipore Filter Corp., Bedford, Mass.; Gelman Instrument Co., Chelsea, Mich. "Baltimore Biological Laboratories, Baltimore 18, Md.
Many of the samples were kindly supplied by John C. Ayres, Dept. of Diary and Food Industries, Iowa State University, and Margaret Lally, Laboratory Director, Institute of American Poultry Industries, Chicago, Illinois.
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min., then 250 ml. of the clear portion of the hydrolyzate, followed by 20 ml. of wash water, were filtered through a 0.45 \i. membrane filter.3 Filtration time, for a 250 ml. sample (equivalent to 10 gm. of dried albumen), using a water aspirator or vacuum pump (0.02 mm. Hg, 33.4 liters per min.), was about 15 min. The filter was transferred to the center of a brilliant green agar4 plate. After incubation in an inverted position for 24 hrs. at 37°C, the filter was observed for the appearance of typical pink colonies. Filter assemblies were immersed in boiling water for 15 minutes between use, cooled, and a sterile filter placed in position. Care must be taken to assure that the pepsin used for hydrolysis does not contain salmonellae. Sterilization using ethylene oxide (Griffith and Hall, 1940) was unsuccessful. The pepsin used in these studies was freed of Salmonella by centrifuging a 10% solution (pH 3.5) at 5,000 rpm. in a Sorvall centrifuge for 30 min. followed by filtration through a 0.45 u,. pore diameter membrane filter. The pH of the pepsin solution, which became higher during filtration, was adjusted to 3.5 after filtration because pepsin is relatively unstable in other than acid menstruum, and because no other adjustment of pH was necessary during the hydrolysis step when acidified pepsin solutions were used. Such solutions were apparently Salmonella-free when utilized in control runs where egg albumen was omitted from the assay procedure, or when several different quantities of pepsin solution were filtered and the filters were incubated on brilliant green agar plates. In addition, no colonies were formed when
DETECTION OF SALMONELLA
made after only one incubation time (see also Taylor and Silliker, 1961). RESULTS AND DISCUSSION
TABLE 2.—Salmonella per 100 g. of albumen, as determined by two methods of enumeration
Sample No.
M P N per 100 g.
Membrane Filter Count per 100 g.
1
0
0
2 3 4 5 6 7
0 30 300 250 300 450
30 50 70 20 120 100
8
0
0
9 10
0 0
0 over 100
TABLE 3.—Reproducibility of the membrane filter method for detection and enumeration of Salmonella in egg albumen Count per 100 g.
Sample No.
rep 1
rep 2
rep 3
2 3 4 6
30 50 70 120
70 100 40 0
0 100 80 20
one filter, counts obtained on the highercount products (Table 2) were lower than would be anticipated. In addition, the low counts might result from antagonisms due to the close proximity of Salmonella and lactose or sucrose fermenting organisms, the latter organisms which usually crowded the filter. The MPN method, therefore, seems to possess a wider range than the membrane filter method for enumeration of Salmonellae, unless several filters are made from each lot of egg, using a different sample size for each filter. On the other hand, the membrane filter technique may be more applicable as a convenient test for detection, and possibly for enumeration, of Salmonellae in very low-count product. The shorter incubation period required to complete the membrane filter test is a distinct advantage of this method over most MPN methods, particularly if detection of Salmonella is the primary objective. Salmonella colonies were more clearly differentiated when the filters were incubated on agar plates than on absorbent pads saturated with double strength broth. In addition, use of agar plates was more convenient, since the rest of the plate was readily available for confirmation of suspect colonies. Bismuth sulfite apparently can be used satisfactorily with the membrane filter for selective growth of Salmonella (Clark et al., 1951). This medium was not tested extensively in the present studies, but results of preliminary work indicated that brilliant green agar was more
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As is shown in Table 2, use of the membrane filter method resulted in recovery of Salmonella from a larger proportion of the samples examined than did the most probable numbers (MPN) method. In addition, the direct enumeration of Salmonella by the membrane filter method usually allowed completion of presumptive tests within 24 hr., although one sample was encountered where no pink colonies were noted at 24 hrs., but 2 colonies could be observed after incubation for an additional 24 hours. If serological tests were not conclusive, Salmonella suspect colonies from the filter could be streaked on a sterile portion of the brilliant green agar plate to obtain better isolation and larger inocula for serological and physiological tests. Reproducibility of the membrane filter method (Table 3) was comparable to conventional techniques. The presence of Salmonella was frequently indicated by red or pink colonial areas (often more pronounced on the reverse than on the surface of the filter), rather than by distinct, pink colonies surrounded by brilliant red zones. Because enumeration of Salmonella on the filters was difficult when about 5 or more red areas were present on
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W . K . KlRKHAM AND P . A. HARTMAN
Whether or not treatment with pepsin is necessary must be determined by conditions of use of the millipore method. Commercial albumen varies considerably in ease of handling. Several samples required only pre-filtration treatment with Celite; other samples could be filtered only when pepsin was used prior to Celite treatment, and one sample required additional pepsin treatment. In the latter case, the hydrolyzate was quite different in appearance from that observed with the other samples: there was considerably less flocculation after incubation at 40°C. for 30 min. With experience the user can easily alter the procedure at this point by lengthening the incubation period with or without the addition of more pepsin solution. No explanation can be given for the great increase in filterability of albumen when treated with Celite. Whether the mechanism is similar to one factor which contributes to egg white thinning in shell eggs is not known, but it might be pointed out that diatomaceous earth possesses negative charges at some points and can absorb positively charged particles (Alexander and Johnson, 1949), including animal lysozyme (Petersdorf, Ribble and Shulman, 1961). Quite a few reagents caused apparent thinning of the albumen without affecting the filtration rate. The addition of various cations and anions did not appreciably alter the action of Celite, nor did the addition of bovine submaxillary gland mucin.6 In addition, dialysis to precipitate ovomucin (Table 1) did not affect filterability of an albumen solution. Since Celite was effective on several samples of albumen only subsequent to treatment with pepsin, some form of protein degradation may be a necessary predecessor of Celite action. In fact, filterability "Wilson Laboratories, Wilson and Co., Inc., Chicago 9, 111.
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satisfactory (see also, Banwart and Ayres, 1953). A pre-enrichment period prior to use of the selective medium (Clark et al., 1951) was not examined as a means of increasing the recovery of Salmonella. In the light of recent studies by North (1960, 1961) Taylor (1961) and Taylor and Silliker (1961), however, it might be of benefit to use lactose broth as a wash solution, thus giving the Salmonella on the filter a brief enrichment period. Such treatment would not entail any increased effort on behalf of the analyst. The temperature of treatment and filtration (40°C.) was selected so that enzyme action and filtration would be rapid (Fifield, Hoff and Proctor, 1957), yet most strains of Salmonella should neither proliferate nor die rapidly at this temperature in the presence of egg albumen. Lysozyme and other enzymes act synergistically on Salmonella which have been heated (Becker and Hartsell, 1955). Organisms in commercially dried albumen have undoubtedly received a certain degree of heat treatment during if not before drying, yet no detrimental effect of the pepsin used in the millipore procedure on bacteria was noted using commercially dried egg albumen or liquid egg albumen which had been prepared in the laboratory and inoculated with Salmonella gallinarum. Celite treatment, however, resulted in considerable loss of viable bacteria. Several experiments, using Serratia marcescens, indicated that only about 10% of the cells introduced into albumen prior to Celite and pepsin treatment subsequently formed colonies when the filter was incubated on trypticase soy agar at 25°C. The organisms were not necessarily carried down with the Celite in the settling process, since no difference was found in plate counts of the uppermost and lower portions of Celite-treated suspensions of S. marcescens in albumen.
DETECTION OF SALMONELLA
SUMMARY
A membrane filter method for the detection and enumeration of Salmonella in egg albumen is described. Reconstituted albumen is hydrolyzed with pepsin and treated with diatomaceous earth in order to affect filtration, then the membrane filter is incubated on a plate of brilliant green agar. The membrane filter technique required less effort and incubation time than commonly used most probable numbers methods to obtain presumptive evidence for the presence of Salmonella in egg albumen. ACKNOWLEDGMENTS
We are indebted to John C. Ayres and Karl F. Weiss for their interest, encouragement, and assistance, and to Allen A. Kraft, Richard H. Forsythe and Owen J. Cotterill for their suggestions. REFERENCES Alexander, A. E., and P. Johnson, 1949. Colloid Science. Clarendon Press, Oxford, pp. 706-715. Ayres, J. C , 1953. Methodology for isolating Salmonella from dried egg products. Iowa State J. Sci. 27: 479-489. Banwart, G. J., and J. C. Ayres, 1953.. Effect of various enrichment broths and selective agars upon the growth of several species of Salmonella. Appl. Microbiol. 1: 296-301. Becker, M. E., and S. E. Hartsell, 1955. The synergistic action of lysozyme and trypsin in bacterioloysis. Arch. Biochem. Biophys. 55: 257269. Byrne, A. F., M. M. Rayman and M. D. Schneider, 1955. Methods for the detection and estimation of numbers of Salmonella in dried
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egg and other food products. Appl. Microbiol. 3 : 368-372. Clark, H. F., E. E. Geldreich, H. L. Jeter and P. W. Kabler, 1951. The membrane filter in sanitary bacteriology. Publ. Health Repts. 66: 951-977. Fifield, C. W., J. E. Hoff and B. E. Proctor, 1957. The millipore filter for enumerating coliform organisms in milk. J. Dairy Sci. 40: 588589. Griffith, C. L., and L. A. Hall, 1940. Sterilization of pancreatin. TJ. S. Patent 2,189,948, Feb. 13, 1940. Hurley, N. A., and J. C. Ayres, 1953. A comparison of six enrichment media for isolating Salmonella pullorum from egg-products. Appl. Microbiol. 1: 302-306. Montford, J., and F. S. Thatcher, 1961. Comparison of four methods of isolating salmonellae from foods, and elaboration of a preferred procedure. J. Food Sci. 26: 510-517. North, W. R., Jr., 1960. Use of crystal violet or brilliant green dyes for the determination of Salmonellae in dried food products. J. Bacteriol. 80: 861. North, W. R., Jr., 1961. Lactose pre-enrichment method for isolation of Salmonella from dried egg albumin. Appl. Microbiol. 9: 188-195. North, W. R., and M. T. Bartram, 1953. The efficiency of selenite broth of different compositions in the isolation of Salmonella. Appl. Microbiol. 1: 130-134. Osborne, W. W., and J. L. Stokes, 1955. A modified selenite brilliant-green medium for the isolation of Salmonella from egg products. Appl. Microbiol. 3 : 295-299. Petersdorf, R. G., J. L. Ribble and J. A. Shulman, 1961. Absorption of endogenous pyrogens by bentonite. Proc. Soc. Exptl. Biol. Med. 106: 234-237. Silliker, J. H., and W. I. Taylor, 1958. Isolation of Salmonellce from food samples. II. The effect of added food samples upon the performance of enrichment broths. Appl. Microbiol. 6 : 228-232. Stokes, J. L., and W. W. Osborne, 1955. A selenite brilliant green medium for the isolation of Salmonella. Appl. Microbiol. 3 : 217-220. Sugiyama, H., G. M. Dack and G. Lippitz, 1960. Agglutinating antiserum for the isolation of Salmonella with special reference to isolation from egg alubumin. Appl. Microbiol. 8: 205209. Taylor, W. I., 1958. A simple, rapid technique for increasing the recognition of Salmonella-
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after Celite treatment alone may be a method of estimating the quantity of protein denaturation in a product. One sample, however, could be filtered after treatment with Celite alone, but not following peptic hydrolysis and Celite treatment unless the pepsin treatment was extensive. These aspects of the study have not been examined further.
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W . K. KlRKHAM AND P . A. HARTMAN Taylor, W. I., J. H. Silliker and H. P. Andrews, 1958. Isolation of Salmonellae from food samples. I. Factors affecting the choice of media for the detection and enumeration of Salmonella. Appl. Microbiol. 6: 189-193. Wells, F., D. Bergquist and R. H. Forsythe, 1958. A comparison of selective media for the isolation of Salmonella from commercial egg white solids. Appl. Microbiol. 6: 198-201.
Citrus Bioflavonoids in Broiler Diets C. W. DEYOE, 1 L. E. DEACON2 AND J. R. COUCH Department of Poultry Science, Texas A & M College, College Station, Texas (Received for publication November 10, 1961)
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ITRUS bioflavonoids are naturally occurring plant pigments characterized by two aromatic rings linked together by a three carbon aliphatic chain (Horowitz, 1957). The various flavonoids may be sugar free aglycones or glycosides characterized by a substituent group of either a mono or disaccharide made up of rhamnose or glucose or both. The classification of these compounds are further governed by the oxidized or reduced state of the third carbon in the aliphatic chain. A number of bioflavonoid materials have been evaluated for their effect on capillary fragility. Warter et al. (1948) reported that the administration of hesperidin (a bioflavonoid) and ascorbic acid was effective in reducing capillary fragility in patients with rheumatoid arthritis. Capillary fragility could be expected to lead to an increase in bruising. Ringrose (1953) and Kaiser and Smith (1958) reported that bruising is one of the major causes of downgrading of broilers. In view of the condemnations due to bruising in the 1 Present address: Dept. of Flour & Feed Milling, Kansas State University, Manhattan, Kansas. 2 Present address: B & D Mills, Grapevine, Texas.
broiler industry it is desirable to study the effect of citrus bioflavonoids on growth and feed utilization. These compounds, if well tolerated, might be considered for their effectiveness in reducing bruising and condemnations in market broilers. EXPERIMENTAL PROCEDURE Two experiments have been conducted to study the effect of feeding citrus bioflavonoids to broiler chicks. In Experiment 1 replicate groups of 250 Arbor Acres X Vantress broiler chicks received the following treatments: basal, basal + 1 % citrus bioflavonoids, basal + 2^% citrus bioflavonoids, and basal + 5 % citrus bioflavonoids. In Experiment 2, 6 pens containing 1,000 Arbor Acres X Vantress broiler chicks received the basal diet shown in Table 1 for the first 6 weeks of the test. From 6-8 weeks duplicate groups of 1,000 birds were placed on the following 3 treatments: basal, basal + i % citrus bioflavonoids and basal + 1 % citrus bioflavonoids. The basal starter and finisher diets used in both experiments are given in Table 1. The starter diet was supplied for the first 6 weeks of the experiment and the finisher diet was used from the 6th to the 8th week of the experiment. The citrus bioflavonoids
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suspect colonies. Am. J. Clin. Pathol. 30: 361— 363. Taylor, W. I., 1961. Isolation of Salmonellae from food samples. V. Determination of the method of choice for enumeration of Salmonella, Appl. Microbiol. 9: 487-490. Taylor, W. I. and J. H. Silliker, 1961. Isolation of Salmonellae from food samples. IV. Comparison of methods of enrichment. Appl. Microbiol. 9: 484-486.
RITICAL studies outlining techniques for isolating Salmonella from dried egg products were reported in 1953 (Ayres, 1953; Banwart and Ayres, 1953; Hurley and Ayres, 1953; North and Bartram, 1953). Although these methods have been reasonably effective and are in general use today, they are not without certain shortcomings. The analyses are time consuming and recovery of Salmonella is less than 100%. Efforts to improve the efficacy of the culture medium have been more or less successful (Byrne, Rayman and Schneider, 1955; Montford and Thatcher, 1961; North, 1961; Osborne and Stokes, 1955; Stokes and Osborne, 1955; Taylor, 1961; Taylor and Silliker, 1961; Taylor, Silliker and Andrews, 1958; and Wells, Bergquist and Forsythe, 1958). The major difficulty lies in the fact that addition of large quantities of some food materials, such as egg albumen, to selective enrichment broths greatly diminishes the performance of these media (Banwart and Ayres, 1953; Hurley and Ayres, 1953; North, 1961; Silliker and Taylor, 1958). 1
Journal paper no. J-3952 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project no. 1379. This investigation was supported (in part) by research grant No. E-1141 from the Institute of Allergy and Infectious Diseases of the National Institute of Health, Public Health Serivce. 2 Hall Township High School, Spring Valley, Illinois. A portion of this research was conducted by the senior author while a National Science Foundation Summer Research Participant for Teacher Training.
Lactose pre-enrichment (North, 1960, 1961; Taylor, 1961; Taylor and Silliker, 1961) is helpful in increasing the counts of Salmonella obtained. A centrifugation-enrichment method (Silliker and Taylor, 1958) and an antiserum-enrichment procedure with or without pre-centrifugation (Sugiyama, Dack and Lippitz, 1960) apparently also result in higher yields of Salmonella. Success of the latter techniques is due to concentration of the Salmonella from a large quantity of material, with subsequent culture in an environment which is much less inhibitory than a primary enrichment culture using a selective medium. All of these methods, however, are not without certain disadvantages. Pre-enrichment introduces one more step to an already laborious procedure. In like manner, centrifugation or use of antiserum results in substantial increases of effort and materials needed to perform the analyses. This report describes a simplified method for the detection and enumeration of Salmonella in egg albumen. Bacteria are removed from treated egg white by nitration of the white through a membrane filter, upon which the organisms may then be cultured on a selective medium. MATERIALS AND METHODS
Egg albumen resists ultrafiltration even though the size of the molecular units in this colloid are sufficiently small to pass a filter of 0.45 JA. pore size. Preliminary investigations were conducted to study the mechanism of ultrafiltration resistance, and to discover treatment(s) which might hold promise in effecting ultrafiltration of the
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albumen. Reconstituted albumen (in 4% aqueous solution) was subjected to various treatments, as listed in Table 1, then was tested for filterability. Additional levels of reagents and combinations of treatments were also examined; however, these are not included in the table for the sake of brev-
1. Dialysis: Overnight against distilled water at 21° C , then 1 day against dilute acetic acid solution (pH 6.0) at 4° C. 2. Ion exchange resin: Strong cationic-anionic resin removed from a Bantam Demineralizer Cartridge (Barnstead Still and Sterilizer Co., Inc., Boston 31, Mass.). 3. Cations and anions: Sodium chloride, ammonium chloride, calcium chloride, ammonium phosphate, sodium ammonium tartrate, sodium citrate. 4. Chelating agents: Di-sodium Perma Kleer-50, Tri-sodium Perma Kleer 50, and Perma Kleer-80 beads (Refined Products Corp., Lyndhurst, N. J.); sodium citrate. 5. Surface active agents: Triton X-100 and Triton WR-1339 (Rohm and Haas Co., Philadelphia 5, Pa.); Tween 80 (Chemicals Dept., Atlas Powder Co., Wilmington, Del.); Antifoam A Emulsion (Dow Corning Corp., Midland, Mich.); Ucon Oil LB-525-43 (Carbide and Carbon Chemicals Co., New York 17, N. Y.); Antifoam 60 (Silicone Products Dept., General Electric Co., Waterford, N. Y.); and Antifoam "GY" (Hodag Chemical Corp., Chicago, 111.). 6. Reducing agents: Sodium thioglycollate, thioglycollic acid, sodium sulfite, and sodium thiosulfite. 7. Hydrogen bond breakers: Urea and guanidinium hydrochloride. 8. Silicone-coated filters: Desicote (Aloe Scientific Div., A. S. Aloe Co., St. Louis 12, Mo.) and Siliclad (Clay-Adams, Inc., New York 20, N. Y.). 9. Enzymes: Pepsin 1:3,000 and 1:10,000 (Cudahy Laboratories Div., Cudahy Packing Co., Omaha 7, Nebr.); Pancreatin, N. F. (Cudahy); Ficin (Merck and Co., Rahway, N. J.); Protease P-1S concentrate (now Protease W-15: Miles Chemical Co., Miles Laboratories, Clifton, N. J.); Rhozyme B-6 (Rohm and Haas Co., Philadelphia 5, Pa.); and Trypsin (Difco Laboratories, Detroit 1, Mich.). 10. Absorbants and filter aids: Diatomaceous earth (Celite Analytical Filter Aid: Johns-Manville Products Corp., Chicago 90, 111.) and alumina. 11. Adjustment of pH with dilute sodium hydroxide or hydrochloric acid. 12. Filtration at elevated temperatures. 13. Clarification through fluted Whatman no. 1, 4, or 12 paper prior to filtration.
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ity. Treatments which substantially increased the filterability of liquid albumen were: mixture with an ion-exchange resin; urea (3 M); use of surface active agents; enzymic hydrolysis; clarification prior to filtration; and filtration at elevated temperatures. A procedure was developed whereby the albumen was hydrolyzed with 1% pepsin, 1:3000, (pH 3.0), 1% ficin (pH 7.4), and protease P-lS in succession for 1 hr. each at 45°C, then clarified before filtration. This procedure, as with several other treatments developed previously which had successfully enabled filtration of the albumen, resulted in considerable loss of bacterial viability. Studies by Weiss and Ayres (unpublished) indicated that the reaction temperature of 45°C. was too high, however reduction of either hydrolysis time or temperature resulted in greatly decreased filtration rates. It was later found that filtration of some samples of commercial albumen could be effected simply by mixing the dried egg powder with water and Celite, and filtering after the Celite had settled for a sufficient period of time so that the resultant filter cake would be negligible in size. Other commercial samples could not be filtered when treated in the same manner; pepsin treatment prior to addition of the Celite, however, did render these latter samples filterable. The following procedure was finally adopted: 20 g. of dried egg albumen were blended for 10 to 20 sec. in a one liter Waring Blendor container with 460 ml. of sterile water and 20 ml. of 10% pepsin (1:3,000) solution (pH 3.5). The mixture was placed in a 40°C. water bath and held for 30 min., then 3 g. of Celite were added to the mixture, and the mixture was blended for an additional 10 to 20 sec. The container was again placed in a 40°C. water bath and allowed to settle for IS
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TABLE 1.—Treatments examined for their eject on the filtration rate of solutions of four percent egg albumen
IN ALBUMIN
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W . K . KlRKHAM AND P . A. HARTMAN
filtered pepsin solutions were plated on trypticase soy agar.4 Pepsin solutions could be stored in the refrigerator (4°C.) for periods of at least a week. During final trials, commercial egg albumen5 known to contain low numbers of Salmonella were used. In some instances, typical colonies were tested with Lederle polyvalent Salmonella antiserum. Difficulty was experienced in obtaining positive agglutination reactions using material taken directly from the membrane filters; however, the suspect colonies could be picked and streaked over an area of 10 to 15 mm2. on a sterile portion of the same plate. An incubation period of from 3 to 6 hrs. was all that was necessary to provide better isolation and an adequate supply of cells for agglutination tests (Taylor, 1958). As controls, samples were analyzed by the most probable number (MPN) method (Ayres, 1953), using the cysteine-selenite F enrichment medium of North and Bartram (1953). A brilliant green agar streak plate was made using a loopful of material from each MPN sample tube and bottle after an incubation period of 20 hr. and again following incubation for 44 hr. at 37°C. An MPN container was considered to be positive for Salmonella if either of the two plates yielded typical colonies; however, it should be noted that there were frequent occasions when an MPN container was apparently negative for Salmonella as indicated by one plate, yet was positive as indicated by the other. This composite-positive system did serve to eliminate many of the "skips" referred to by North (1960, 1961), and to result in higher MPN counts than would be ordinarily encountered using streak plates 5
3
Millipore Filter Corp., Bedford, Mass.; Gelman Instrument Co., Chelsea, Mich. "Baltimore Biological Laboratories, Baltimore 18, Md.
Many of the samples were kindly supplied by John C. Ayres, Dept. of Diary and Food Industries, Iowa State University, and Margaret Lally, Laboratory Director, Institute of American Poultry Industries, Chicago, Illinois.
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min., then 250 ml. of the clear portion of the hydrolyzate, followed by 20 ml. of wash water, were filtered through a 0.45 \i. membrane filter.3 Filtration time, for a 250 ml. sample (equivalent to 10 gm. of dried albumen), using a water aspirator or vacuum pump (0.02 mm. Hg, 33.4 liters per min.), was about 15 min. The filter was transferred to the center of a brilliant green agar4 plate. After incubation in an inverted position for 24 hrs. at 37°C, the filter was observed for the appearance of typical pink colonies. Filter assemblies were immersed in boiling water for 15 minutes between use, cooled, and a sterile filter placed in position. Care must be taken to assure that the pepsin used for hydrolysis does not contain salmonellae. Sterilization using ethylene oxide (Griffith and Hall, 1940) was unsuccessful. The pepsin used in these studies was freed of Salmonella by centrifuging a 10% solution (pH 3.5) at 5,000 rpm. in a Sorvall centrifuge for 30 min. followed by filtration through a 0.45 u,. pore diameter membrane filter. The pH of the pepsin solution, which became higher during filtration, was adjusted to 3.5 after filtration because pepsin is relatively unstable in other than acid menstruum, and because no other adjustment of pH was necessary during the hydrolysis step when acidified pepsin solutions were used. Such solutions were apparently Salmonella-free when utilized in control runs where egg albumen was omitted from the assay procedure, or when several different quantities of pepsin solution were filtered and the filters were incubated on brilliant green agar plates. In addition, no colonies were formed when
DETECTION OF SALMONELLA
made after only one incubation time (see also Taylor and Silliker, 1961). RESULTS AND DISCUSSION
TABLE 2.—Salmonella per 100 g. of albumen, as determined by two methods of enumeration
Sample No.
M P N per 100 g.
Membrane Filter Count per 100 g.
1
0
0
2 3 4 5 6 7
0 30 300 250 300 450
30 50 70 20 120 100
8
0
0
9 10
0 0
0 over 100
TABLE 3.—Reproducibility of the membrane filter method for detection and enumeration of Salmonella in egg albumen Count per 100 g.
Sample No.
rep 1
rep 2
rep 3
2 3 4 6
30 50 70 120
70 100 40 0
0 100 80 20
one filter, counts obtained on the highercount products (Table 2) were lower than would be anticipated. In addition, the low counts might result from antagonisms due to the close proximity of Salmonella and lactose or sucrose fermenting organisms, the latter organisms which usually crowded the filter. The MPN method, therefore, seems to possess a wider range than the membrane filter method for enumeration of Salmonellae, unless several filters are made from each lot of egg, using a different sample size for each filter. On the other hand, the membrane filter technique may be more applicable as a convenient test for detection, and possibly for enumeration, of Salmonellae in very low-count product. The shorter incubation period required to complete the membrane filter test is a distinct advantage of this method over most MPN methods, particularly if detection of Salmonella is the primary objective. Salmonella colonies were more clearly differentiated when the filters were incubated on agar plates than on absorbent pads saturated with double strength broth. In addition, use of agar plates was more convenient, since the rest of the plate was readily available for confirmation of suspect colonies. Bismuth sulfite apparently can be used satisfactorily with the membrane filter for selective growth of Salmonella (Clark et al., 1951). This medium was not tested extensively in the present studies, but results of preliminary work indicated that brilliant green agar was more
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As is shown in Table 2, use of the membrane filter method resulted in recovery of Salmonella from a larger proportion of the samples examined than did the most probable numbers (MPN) method. In addition, the direct enumeration of Salmonella by the membrane filter method usually allowed completion of presumptive tests within 24 hr., although one sample was encountered where no pink colonies were noted at 24 hrs., but 2 colonies could be observed after incubation for an additional 24 hours. If serological tests were not conclusive, Salmonella suspect colonies from the filter could be streaked on a sterile portion of the brilliant green agar plate to obtain better isolation and larger inocula for serological and physiological tests. Reproducibility of the membrane filter method (Table 3) was comparable to conventional techniques. The presence of Salmonella was frequently indicated by red or pink colonial areas (often more pronounced on the reverse than on the surface of the filter), rather than by distinct, pink colonies surrounded by brilliant red zones. Because enumeration of Salmonella on the filters was difficult when about 5 or more red areas were present on
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IN ALBUMIN
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W . K . KlRKHAM AND P . A. HARTMAN
Whether or not treatment with pepsin is necessary must be determined by conditions of use of the millipore method. Commercial albumen varies considerably in ease of handling. Several samples required only pre-filtration treatment with Celite; other samples could be filtered only when pepsin was used prior to Celite treatment, and one sample required additional pepsin treatment. In the latter case, the hydrolyzate was quite different in appearance from that observed with the other samples: there was considerably less flocculation after incubation at 40°C. for 30 min. With experience the user can easily alter the procedure at this point by lengthening the incubation period with or without the addition of more pepsin solution. No explanation can be given for the great increase in filterability of albumen when treated with Celite. Whether the mechanism is similar to one factor which contributes to egg white thinning in shell eggs is not known, but it might be pointed out that diatomaceous earth possesses negative charges at some points and can absorb positively charged particles (Alexander and Johnson, 1949), including animal lysozyme (Petersdorf, Ribble and Shulman, 1961). Quite a few reagents caused apparent thinning of the albumen without affecting the filtration rate. The addition of various cations and anions did not appreciably alter the action of Celite, nor did the addition of bovine submaxillary gland mucin.6 In addition, dialysis to precipitate ovomucin (Table 1) did not affect filterability of an albumen solution. Since Celite was effective on several samples of albumen only subsequent to treatment with pepsin, some form of protein degradation may be a necessary predecessor of Celite action. In fact, filterability "Wilson Laboratories, Wilson and Co., Inc., Chicago 9, 111.
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satisfactory (see also, Banwart and Ayres, 1953). A pre-enrichment period prior to use of the selective medium (Clark et al., 1951) was not examined as a means of increasing the recovery of Salmonella. In the light of recent studies by North (1960, 1961) Taylor (1961) and Taylor and Silliker (1961), however, it might be of benefit to use lactose broth as a wash solution, thus giving the Salmonella on the filter a brief enrichment period. Such treatment would not entail any increased effort on behalf of the analyst. The temperature of treatment and filtration (40°C.) was selected so that enzyme action and filtration would be rapid (Fifield, Hoff and Proctor, 1957), yet most strains of Salmonella should neither proliferate nor die rapidly at this temperature in the presence of egg albumen. Lysozyme and other enzymes act synergistically on Salmonella which have been heated (Becker and Hartsell, 1955). Organisms in commercially dried albumen have undoubtedly received a certain degree of heat treatment during if not before drying, yet no detrimental effect of the pepsin used in the millipore procedure on bacteria was noted using commercially dried egg albumen or liquid egg albumen which had been prepared in the laboratory and inoculated with Salmonella gallinarum. Celite treatment, however, resulted in considerable loss of viable bacteria. Several experiments, using Serratia marcescens, indicated that only about 10% of the cells introduced into albumen prior to Celite and pepsin treatment subsequently formed colonies when the filter was incubated on trypticase soy agar at 25°C. The organisms were not necessarily carried down with the Celite in the settling process, since no difference was found in plate counts of the uppermost and lower portions of Celite-treated suspensions of S. marcescens in albumen.
DETECTION OF SALMONELLA
SUMMARY
A membrane filter method for the detection and enumeration of Salmonella in egg albumen is described. Reconstituted albumen is hydrolyzed with pepsin and treated with diatomaceous earth in order to affect filtration, then the membrane filter is incubated on a plate of brilliant green agar. The membrane filter technique required less effort and incubation time than commonly used most probable numbers methods to obtain presumptive evidence for the presence of Salmonella in egg albumen. ACKNOWLEDGMENTS
We are indebted to John C. Ayres and Karl F. Weiss for their interest, encouragement, and assistance, and to Allen A. Kraft, Richard H. Forsythe and Owen J. Cotterill for their suggestions. REFERENCES Alexander, A. E., and P. Johnson, 1949. Colloid Science. Clarendon Press, Oxford, pp. 706-715. Ayres, J. C , 1953. Methodology for isolating Salmonella from dried egg products. Iowa State J. Sci. 27: 479-489. Banwart, G. J., and J. C. Ayres, 1953.. Effect of various enrichment broths and selective agars upon the growth of several species of Salmonella. Appl. Microbiol. 1: 296-301. Becker, M. E., and S. E. Hartsell, 1955. The synergistic action of lysozyme and trypsin in bacterioloysis. Arch. Biochem. Biophys. 55: 257269. Byrne, A. F., M. M. Rayman and M. D. Schneider, 1955. Methods for the detection and estimation of numbers of Salmonella in dried
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egg and other food products. Appl. Microbiol. 3 : 368-372. Clark, H. F., E. E. Geldreich, H. L. Jeter and P. W. Kabler, 1951. The membrane filter in sanitary bacteriology. Publ. Health Repts. 66: 951-977. Fifield, C. W., J. E. Hoff and B. E. Proctor, 1957. The millipore filter for enumerating coliform organisms in milk. J. Dairy Sci. 40: 588589. Griffith, C. L., and L. A. Hall, 1940. Sterilization of pancreatin. TJ. S. Patent 2,189,948, Feb. 13, 1940. Hurley, N. A., and J. C. Ayres, 1953. A comparison of six enrichment media for isolating Salmonella pullorum from egg-products. Appl. Microbiol. 1: 302-306. Montford, J., and F. S. Thatcher, 1961. Comparison of four methods of isolating salmonellae from foods, and elaboration of a preferred procedure. J. Food Sci. 26: 510-517. North, W. R., Jr., 1960. Use of crystal violet or brilliant green dyes for the determination of Salmonellae in dried food products. J. Bacteriol. 80: 861. North, W. R., Jr., 1961. Lactose pre-enrichment method for isolation of Salmonella from dried egg albumin. Appl. Microbiol. 9: 188-195. North, W. R., and M. T. Bartram, 1953. The efficiency of selenite broth of different compositions in the isolation of Salmonella. Appl. Microbiol. 1: 130-134. Osborne, W. W., and J. L. Stokes, 1955. A modified selenite brilliant-green medium for the isolation of Salmonella from egg products. Appl. Microbiol. 3 : 295-299. Petersdorf, R. G., J. L. Ribble and J. A. Shulman, 1961. Absorption of endogenous pyrogens by bentonite. Proc. Soc. Exptl. Biol. Med. 106: 234-237. Silliker, J. H., and W. I. Taylor, 1958. Isolation of Salmonellce from food samples. II. The effect of added food samples upon the performance of enrichment broths. Appl. Microbiol. 6 : 228-232. Stokes, J. L., and W. W. Osborne, 1955. A selenite brilliant green medium for the isolation of Salmonella. Appl. Microbiol. 3 : 217-220. Sugiyama, H., G. M. Dack and G. Lippitz, 1960. Agglutinating antiserum for the isolation of Salmonella with special reference to isolation from egg alubumin. Appl. Microbiol. 8: 205209. Taylor, W. I., 1958. A simple, rapid technique for increasing the recognition of Salmonella-
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after Celite treatment alone may be a method of estimating the quantity of protein denaturation in a product. One sample, however, could be filtered after treatment with Celite alone, but not following peptic hydrolysis and Celite treatment unless the pepsin treatment was extensive. These aspects of the study have not been examined further.
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W . K. KlRKHAM AND P . A. HARTMAN Taylor, W. I., J. H. Silliker and H. P. Andrews, 1958. Isolation of Salmonellae from food samples. I. Factors affecting the choice of media for the detection and enumeration of Salmonella. Appl. Microbiol. 6: 189-193. Wells, F., D. Bergquist and R. H. Forsythe, 1958. A comparison of selective media for the isolation of Salmonella from commercial egg white solids. Appl. Microbiol. 6: 198-201.
Citrus Bioflavonoids in Broiler Diets C. W. DEYOE, 1 L. E. DEACON2 AND J. R. COUCH Department of Poultry Science, Texas A & M College, College Station, Texas (Received for publication November 10, 1961)
C
ITRUS bioflavonoids are naturally occurring plant pigments characterized by two aromatic rings linked together by a three carbon aliphatic chain (Horowitz, 1957). The various flavonoids may be sugar free aglycones or glycosides characterized by a substituent group of either a mono or disaccharide made up of rhamnose or glucose or both. The classification of these compounds are further governed by the oxidized or reduced state of the third carbon in the aliphatic chain. A number of bioflavonoid materials have been evaluated for their effect on capillary fragility. Warter et al. (1948) reported that the administration of hesperidin (a bioflavonoid) and ascorbic acid was effective in reducing capillary fragility in patients with rheumatoid arthritis. Capillary fragility could be expected to lead to an increase in bruising. Ringrose (1953) and Kaiser and Smith (1958) reported that bruising is one of the major causes of downgrading of broilers. In view of the condemnations due to bruising in the 1 Present address: Dept. of Flour & Feed Milling, Kansas State University, Manhattan, Kansas. 2 Present address: B & D Mills, Grapevine, Texas.
broiler industry it is desirable to study the effect of citrus bioflavonoids on growth and feed utilization. These compounds, if well tolerated, might be considered for their effectiveness in reducing bruising and condemnations in market broilers. EXPERIMENTAL PROCEDURE Two experiments have been conducted to study the effect of feeding citrus bioflavonoids to broiler chicks. In Experiment 1 replicate groups of 250 Arbor Acres X Vantress broiler chicks received the following treatments: basal, basal + 1 % citrus bioflavonoids, basal + 2^% citrus bioflavonoids, and basal + 5 % citrus bioflavonoids. In Experiment 2, 6 pens containing 1,000 Arbor Acres X Vantress broiler chicks received the basal diet shown in Table 1 for the first 6 weeks of the test. From 6-8 weeks duplicate groups of 1,000 birds were placed on the following 3 treatments: basal, basal + i % citrus bioflavonoids and basal + 1 % citrus bioflavonoids. The basal starter and finisher diets used in both experiments are given in Table 1. The starter diet was supplied for the first 6 weeks of the experiment and the finisher diet was used from the 6th to the 8th week of the experiment. The citrus bioflavonoids
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suspect colonies. Am. J. Clin. Pathol. 30: 361— 363. Taylor, W. I., 1961. Isolation of Salmonellae from food samples. V. Determination of the method of choice for enumeration of Salmonella, Appl. Microbiol. 9: 487-490. Taylor, W. I. and J. H. Silliker, 1961. Isolation of Salmonellae from food samples. IV. Comparison of methods of enrichment. Appl. Microbiol. 9: 484-486.