A Comparison of Various Methods for Recovery of Salmonella Derby from Egg Shell Surfaces1,2

A Comparison of Various Methods for Recovery of Salmonella Derby from Egg Shell Surfaces1*2 D. B. MELLOE 3 AND G. J. BAN WART4 Animal Sciences Department, Purdue University, Lafayette,

Indiana

(Received for publication December 4, 1964)

T

1 Journal Paper No. 2459, Purdue Agricultural Experiment Station, Lafayette, Indiana. 2 This investigation was supported in part by Public Health Research Grant EF-00416 from the Division of Environmental Engineering and Food Protection. 'Present address: USAID/ARD, APO, New York 90676. ••Present address: TJSDA/ARS, Rm. 203, West Bldg., Plant Industry Station, Bellsville, Maryland 20705.

shell surfaces, however, interferring organic matter is not of concern. The growth of interfering organisms and the possibility of low numbers of Salmonella are important, however. During a study of hens inoculated with S. derby (Mellor and Banwart, 1964), it was apparent that all methods currently in use for examination for Salmonella did not give the same opportunities for recovery from egg shell surfaces. The present study was undertaken to evaluate three media commonly used in the examination of egg shell surfaces for the presence of salmonellae. METHODS AND MATERIALS

Fresh, nest clean, unwashed eggs were obtained from the Purdue University Poultry Farm. The eggs were inoculated with one of the following cultures: (1) Salmonella derby, (2) Pseudomonas aeruginosa, (3) Proteus vulgaris, and (4) a mixed inoculum of these 3 cultures. The 5. derby was isolated at Purdue University and typed at the Indiana Department of Health Laboratories. The other 2 cultures were obtained from the culture collection, Department of Biological Sciences, Purdue University. The media employed in this study were: (a) lactose broth (LB, Difco) for the method of North (1961); (b) selenite Fcystine (SFC, BBL); (c) tetrathionate broth (Difco) with 1 ml. of a 1/100 brilliant green solution added per liter (TBG), and brilliant green (BG) and brilliant green sulfa (BGS) agars (Difco). Three trials were made of the basic design shown in Table 1. For each trial 30

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HE examination of food and food products for the presence of salmonellae has been receiving considerable attention. Methods for examination of foods for salmonellae were reviewed by Galton and Boring (1964). When food samples are analyzed, changes in media have been made to compensate for the addition of the large amount of organic matter, and, generally, a lower number of salmonellae (Montford and Thatcher, 1961; Silliker and Taylor, 1958; Taylor et al., 1958). In the lactose preenrichment method of North (1961) for the enumeration of salmonellae in dried egg albumen, for example, the suspect food is incubated in the non-inhibitory lactose broth at 37°C, a temperature favorable to many bacteria. A minute amount of the lactose broth, with organisms that have grown in it, is then transferred to the selective medium. This assures the smallest interference of the food materials with the balance of the medium and also that any Salmonella has a chance to develop before being subjected to a medium which is even slightly inhibitory to Salmonella (Banwart and Ayres, 1953). When isolating Salmonella from egg

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TABLE 1.—Design of experiment for inoculation of eggs with three species of bacteria and recovery in three enrichment broths and two agars. Number of eggs (samples) for each trial is shown under each treatment Enrichment Agar

LB BG

Inoculation Organism S. derby 10 P. vulgaris 10 P. aeruginosa 10 Mixture 10

TBG

SFC

BGS

BG

BGS

BG

BGS

10 10 10 10

10 10 10 10

oooo

10 10 10 10

10 10 10 10

LB =Lactose broth preenrichment-f-selenite F-cystine broth enrichment. SFC =Selenite F-cystine broth T B G = T e t r a t h i o n a t e brilliant green broth BG=Brilliant green agar BGS =Brilliant green sulfa agar.

broth containing gas fermentation tubes. Since neither P. vulgaris nor P. aeruginosa ferment mannitol this eliminated the need for further confirmation on a large number of samples. When required, further testing consisted of inoculation of Triple Sugar Iron Agar tubes and confirmation by agglutination using Group B Salmonella O antiserum (Difco). To further confirm the results of the studies with eggs, pure culture tests were performed with the 3 cultures used previously, 3 unknown "aerobacter group" cultures isolated in a previous study (Mellor and Banwart, 1964) and three additional cultures obtained from the Purdue culture collection: Proteus rettgeri, Aerobacter aerogenes and Escherichia coli. Pure lactose broth cultures (or dilutions therefrom) were inoculated into 5 tubes each containing 10 ml. of the three enrichment broths LB, SFC, TBG. These were treated as the egg samples except that streaking was done only at 24 hrs. RESULTS AND DISCUSSION

S. derby was recovered more often when LB was used than when SFC was used as the initial enrichment medium. Both of these media gave better recoveries than TBG as is shown in Table 2. The time of streaking or types of solid media, BG or BGS agar, did not affect the recovery of 5. derby. When we speak of LB and SFC liq-

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eggs were dipped in an inoculum consisting of 2000 ml. of sterile distilled water containing 15 gm. of NaCl and a dilution of a lactose broth culture of each organism. Each culture was incubated at 37°C. for 24 hours, counted by standard plate count techniques and then refrigerated at 4°C. until used. The inoculum level was determined for (1), (2), and (3) by standard plate count techniques. For each trial the mixed cultures (4) contained the same quantities of each organism as were present in the individual inoculum. Inocula were normally at room temperature (72°F.) and eggs were approximately SS°F. No attempt was made to achieve or prevent shell penetration. The eggs were left in the inoculum for IS minutes, removed and dried for 2 hours on autoclaved filler flats. Eggs that had been spray oiled at the nest were used in trial 2 only. For each trial, 10 eggs from each inoculation group were placed individually in jars containing 100 ml. of 1 of the 3 enrichment media using sterile techniques throughout. The soak technique of Solowey et al. (1946) was adapted for this study to facilitate handling and also since a small preliminary trial had shown no difference between positives and negatives when the soak method (IS min.) was compared with a Waring Blendor wash (2 min.) (Rosser, 1942). After 1 hr. the eggs were removed and the jars containing the broths were incubated at 37°C. A loopful of broth (using a 7 mm. loop) was removed from each LB jar at the end of 16 hr. and placed in a tube containing 2 ml. of SFC. The tubes were incubated at 37°C. and this was still considered the LB sample. At 24 hr. and 40 hr. periods a loopful of each individual sample was streak-plated on BG and BGS agars, and incubated at 37°C. for 24 hrs. Agar surfaces which contained colonies typical of Salmonella were recorded as positive. Screening of the suspect cultures was accomplished by inoculation of mannitol

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D. B. MELLOR AND G. J. BANWART

onto egg shells it passed through the enrichment broths and showed as apparent Salmonella on all media tested (Table 3). More contamination seemed to be present when streaking was done after 40 hr. rather than after 24 hr. incubation of the broth. All plates containing red colonies were not recorded in Table 3 since some colonies were definitely "spreader" colonies. These were not recorded as "typical" Salmonella. The results of the pure culture tests (Table 7) indicate that BGS agar was beneficial in reducing the number of false positives which were produced by P. vulgaris. The shorter, 8 hr. SFC enrichment following the 16 hr. LB preenrichment was more effective than the longer 24 hr. SFC enrichment in reucing false positives due to P. vulgaris. Galton et al. (19S4) added sodium sulfadiazine to BG agar to suppress the growth of large numbers of Pseudomonas in meat samples. They reported no inhibition of Salmonella. Osborne and Stokes (1955) added 0.1% sodium sulfapyridine to BG agar to enhance Salmonella selectivity. The data of Table 4 shows that P. aeruginosa is suppressed by BGS agar.

TABLE 2.—Number of plates showing typical Salmonella appearing colonies from eggs Salmonella derby following enrichment in three broths and streak-plated on two agars Enriichment broth 1 Agar1 Trial 1 2 3

Inoculation level 110,000/ml. 700/ml. 1,700/ml.

Total 1 2 3 Total 1

110,000/ml. 700/ml. 1,700/ml.

LB BG

TBG

SFC BGS

8 9 10

8 9 10

27

27

8 9 10

8 9 10

27

27

For identification of media see Table 1,

BG

BGS

24 hours of incubation 4 4 8 8 8 8 20

20

40 hours of incubation 4 4 9 9 8 8 21

with

21

BG

BGS

1 3 0

1 2 0

4

3

1 6 2

1 6 2

9

9

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uid media we are not referring to different inhibitory media, but to the effects of preenrichment in LB and incubation time in SFC. Since TBG is the medium recommended for analyzing food samples for Salmonella (Galton and Boring, 1964; I.A.P.I., 1962), much better growth of the organisms was expected than was found (Table 2). Leistner et al. (1963) isolated more serotypes with tetrathionate (without BG) than with selenite-F (without cystine). They were able to isolate S. derby more frequently with selenite F, however. A possible explanation for this may be the fact that brilliant green is extremely inhibitory (Miller and Banwart, 1965). No inhibition was evident with pure cultures, however, when S. derby was inoculated in TBG broth (Table 7). S. derby growth on BGS agar was not as profuse as on BG agar. For this reason isolated colonies could be picked much easier for transfer to other media. The BGS agar did not prove to be too restrictive as it was in the study of Smyser et al. (1963) in the recovery of S. typhimurium from feedstuffs. When the P. vulgaris was inoculated

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TABLE 3.—Number of plates showing typical Salmonella appearing colonies from eggs inoculated with Proteus vulgaris following enrichment in three broths and streak-plated on two agars

Trial 1 2 3

Inoculation level 130,000/ml. SOO/ml. 1,200/ml.

Total

.

130,000/ml. SOO/ml. 1,200/ml.

Total

TBG

SFC

BG

BGS

0 0 2

0 0 1

2

1

0 2 3

1 1 3

5

5

BGS

BG

24 hr. of incubation 1 1 0 0 2 3 4

3

40 hr. of incubation 0 1 0 2 7 7 9

8

BG

BGS

0 0 4

0 0 3

4

3

0 0 5 5

0

For identification of media see Table 1.

This suppression is far more definite with P. aeruginosa than with P. vulgaris both on the egg shells (Tables 3 and 4 )and in pure culture (Table 7). Since Pseudomonas species are common on eggs, their suppression is important to the recovery of salmonellae. When Pseudomonas grow on BG agar the colonies resemble Salmonella so closely that additional testing is necessary to distinguish between them. When isolating from BGS agar it was found useful to allow the plates to remain at room temperature for 24 hr. after original incu-

bation. This made isolation easier and revealed several positive plates which were missed after the incubation for 24 hr. at 37°C. Only plates showing S. derby were recorded in Table 5. The LB method has a slight advantage over the use of only SFC. Either of these enrichment methods are superior to TBG. As was the case with the eggs inoculated with only S. derby, the BG agar did not give any more or less recoveries than did BGS agar. Colony growth was less profuse on BGS agar and individual

TABLE 4.—Number of plates showing typical Salmonella appearing colonies from eggs inoculated with Pseudomonas aeruginosa following enrichment in three broths and streak-plated on two agars

Trial 1 2 3

Inoculation level 90,000/ml. 500/ml. 850/ml.

Total 1 2 3 Total 1

90,000/ml. SOO/ml. 850/ml.

LB

SFC

BG

BGS

1 9 7

0 0 3

17

3

2 9 9

2 0 1

20

3

For identification of media see Table 1.

BG

TBG BGS

24 hr. of incubation 0 3 0 10 9 7 21

7

40 hr. of incubation 3 3 3 1 9 0 15

4

BG

BGS

0 0 3

OOO

Enriichment broth 1 Agar1

3

0

0 5 8

0 0 1

13

1

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1 2 3

LB

OOO

Enr:ichment broth 1 Agar1

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D. B. MELLOR AND G. J. BANWART

TABLE S.—Number of plates containing Salmonella derby colonies from eggs inoculated with Salmonella derby, Proteus vulgaris, and Pseudomonas aeruginosa following enrichment in three broths and streak-plating on two agars Enrichment broth 1 Agar1 Trial 1 2 3

Inoculation level 110,000+130,000+90,000/ml. 7OO+5OO+5O0/ml. 1,700+1,200+850/ml.

Total 110,000+130,000+90,000/ml. 700+5O0+5O0/ml. l,7O0+l,20O+85O/ml.

Total 1

BG

SFC BGS

BG

TBG BGS

BG

BGS

24 hr. of incubation 10 7

10 7

4 9 9

4 9 9

0 5 6

25

25

22

22

11

10 7

10 7

25

25

40 hr. of incubation 4 4 9 9 9 9 22

22

0 6 6

0 6 6

12

12

For identification of media see Table 1.

colonies were smaller. The unusually low number of recoveries with SFC in trial 1, compared to trial 2 and trial 3, cannot be explained, especially since S. derby, P. aeruginosa and P. vulgaris all grew in the pure culture test in the SFC medium (Table 7). False positives were recorded several times with the mixed culture inoculation. In the first trial one of the S. derby SFCBGS agar plates appeared to be positive but further biochemical tests proved the culture to be other than Salmonella. The

false positives for the mixed culture inoculations are shown in Table 6. It is recognized that the recording of false positives can be subjective. At times the lack of inhibition would allow organisms to spread and make recognition as other than Salmonella evident. This accounted for the recording of two false positives of BGS agar at 24 hr. for TB enrichment in trial 3. More frequently though the greater restriction of the BGS agar did not allow "typical" colonies to develop for organisms which gave "typical" colonies on BG agar.

TABLE 6.—False positive plates recorded after mixed culture inoculation of eggs with S. derby, Proteus vulgaris, Pseudomonas aeruginosa following enrichment in three broths and streak-plated on two agars

Trial 1 2 3

Inoculation level 110,000+130,000+90,000/ml. 700+500+500/ml. 1,700+1,200+850/ml.

Total 1 2 3

110,000+130,000+90,000/ml. 700+500+500/ml. 1,700+1,200+850/ml.

Total 1

For identification of media see Table 1.

SFC

LB BG

BGS

0 0 1

0 0 0

1

0

0 0 2

0 0 1

2

1

BG

TBG BGS

24 hr. incubation 0 1 1 0 1 0 2

1

40 hr. of incubation 2 2 1 0 1 1 4

3

BG

BGS

OOO

Enrichment broth 1 Agar1

0 0 2

0

2

0 3 2

0 1 1

5

2

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1 2 3

LB

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TABLE 7.—Number of plates showing growth of various pure bacterial cultures following enrichment in

three broths and streak-plated ion two agars Enrichment 1jroth 1 Agar1 Culture M-13622 M-157 2

TBG

SFC

BG

BGS

BG

BGS

BG

BGS

+5 05 +5 +5 + 1 +5 — 05

+5 05 +5 +5 — —

+5 05 +5 +5 +2 +5 — 05

+5 05 +5 +5 +2 — — 05

+5 05 + 1 +5 +4 +2 — 05

+5 05 — +5 — — — 05

+

+

•

—

•

05 —

•

+ Number of plates showing typical Salmonella colonies. 0 Number of plates showing growth other than typical Salmonella colonies. — No growth. 1 For identification of media see Table 1. 2 Unknown "aerobacter group" species isolated from poultry feces, egg shell and commercial feed.

Montford and Thatcher (1961), after comparing 4 methods for detection of Salmonella in food products, recommended SFC enrichment and streaking on BGS agar. From the results of the trials presented here with recovery of 5. derby from egg shells, it appears that a lactose broth preenrichment for 16 hr. followed by an 8 hr. enrichment in selenite F-cystine with streaking on brilliant green sulfa agar gives best results. The work presented here does not show BGS agar to be too restrictive as stated by Smyser et al, (1963). Elimination of more of the interferring organisms by a restrictive medium permits a better opportunity for recovery of Salmonella, especially when they are present in low numbers. The results of the inoculation of various pure cultures into the test media are given in Table 7. E. coli did not grow on either of the solid media from any of the enrichment broths. A. aerogenes grew profusely in all media but with the typical yellow-green growth of a lactose fermenter when streaked on BG or BGS agars. P. rettgeri did not grow well in any media. P. vulgaris, however, grew in all

media and grew very well in TBG when streaked on BG agar. A difference in response to the agar was noted depending on whether the organism spent 24 or 8 hours in SFC following LB. When grown in LB for 16 hours and then placed in SFC for only 8 hours no growth was evident in BGS agar and 1 positive BG agar plate. When enriched in SFC for 24 hr. there were 2 positive plates on both BG and BGS agars. The three "aerobacter group" cultures (M-191, M-157, and M-93) were originally isolated as possible Salmonella. Culture M-136 appeared to be a typical Salmonella on all culture plates. Culture M-157 had a slight yellow tinge on the edge of each colony on both agars. The M-93 culture did not grow well in TBG. SUMMARY AND CONCLUSIONS Recovery of S. derby, P. vulgaris, and P. aeruginosa from inoculated egg shell surfaces was compared using 2 variations of each of 3 enrichment procedures: 1. Lactose broth 16 hr. plus selenite Fcystine for 8 and 24 hr. 2. Selenite F-cystine for 24 and 40 hr.

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M-93 5. derby P. vulgaris P. aeruginosa E. coli Aerobacler aerogenes Proteus rettgeri

Inoculation (organisms) 50 13 100 15 27 40 24 13 45

LB

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REFERENCES 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. Galton, M. M., and J. R. Boring, III, 1964. Methods for Isolation of Salmonellae and Shigellae from Food Products. Public Health Service, Washington, D.C. 20201. pp. 26-32. Galton, M. M., W. D. Lowery, and A. V. Hardy, 19 54. Salmonella in fresh and smoked pork sausage. J. Infectious Diseases, 95: 232-235. Institute of American Poultry Industries, 1962. Qualitative Procedure for Salmonella. Chemical and Bacteriological Methods for the Examination of Eggs and Egg Products, p. 12. Leistner, L., R. H. Deibel, J. Johantges and C. F.

Niven, Jr., 1963. Contribution to the methodology of Salmonella detection. Am. Meat Inst. Found. Bull. No. 56. Mellor, D. B., and G. J. Banwart, 1964. Salmonella derby contamination of eggs from inoculated hens. J. Food Sci. (in review). Miller, V. R., and G. J. Banwart, 1965. The effect of various concentrations of brilliant green and bile salts on salmonellae and other microorganisms. Appl. Microbiol, (in press). 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., 1961. Lactose pre-enrichment method for isolation of Salmonella from dried egg albumen. Appl. Microbiol. 9: 188-195. Osborne, W. W., and J. L. Stokes, 1955. A modified selenite brilliant-green medium for the isolation of Salmonella from egg prolucts. Appl. Microbiol. 3 : 295-299. Rosser, F. T., 1942. Preservation of eggs. II. Surface contamination on egg-shell in relation to spoilage. Can. J. Research, D. 20: 291-296. Silliker, J. H., and W. I. Taylor, 1958. Isolation of salmonellae from food samples. II. The effect of added food samples upon performance of enrichment broths. Appl. Microbiol. 6: 228-232. Smyser, C. F., J. Bacharz and H. Van Roekel, 1963. Detection of Salmonella typhimurium from artificially contaminated poultry feed and animal by-products. Avian Diseases, 7: 423-434. Solowey, M., E. H. Spaulding and H. E. Goresline, 1946. An investigation of a source and a mode of entry of Salmonella organisms in spray dried whole-egg powder. Food Research, 11: 380-390. Taylor, W. I., J. H. Silliker and H. P. Andrews, 1958. Isolation of salmonellae from food samples. 1. Factors affecting the choice of media for the detection and enumeration of Salmonella. Appl. Microbiol. 6: 189-193.

NEWS AND NOTES (Continued from page 979) icity program involves the development of rapid, toxic chemicals in mammals, their action on emsensitive and reliable tests for detecting and mea- bryos as well as on young and adult birds, and the comparative effects of herbicides on proliferating suring very small amounts of toxicants in lower aquatic animals and their correlation with tests plant and animal tissues. The Environmental Fate Program will be deusing mammals. The Morphological, Physiological and Biochemi- signed to survey the decomposition of pesticides by cal Aspects of Chronic Toxicity program will in- temperature, light and air; to chemically identify the breakdown products and determine the toxicity clude research into the storage and elimination of (Continued on page 1019)

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3. Tetrathionate broth + 1/100,000 Brilliant green for 24 and 40 hr. and two plating media: a. Brilliant green agar b. Brilliant green sulfa agar S. derby recovery was shown to be best with the use of the lactose broth 16 hr., selenite F-cystine 8 hr. enrichment and subsequent streak-plating on brilliant green sulfa agar. Selenite F-cystine enrichment for 24 hr., streak-plated on brilliant green sulfa agar was slightly inferior to the lactose preenrichment procedure but vastly superior to tetrathionate brilliant green enrichment. The selenite F-cystine enrichment was considered better when streakplated on brilliant green sulfa agar than when brilliant green agar was used, mainly because of the occurrence of interfering organisms.