Journal ofApplied Bacteriology 1919,46,567-569
495/091/78
A Note on the Comparison of two Modifications of Rappaport's Medium with Selenite Broth in the Isolation of Salmonellas P. VASSILIADIS, D. TRICHOPOULOS, G. PAPOUTSAKIS AND E. PALLANDIOU The Hellenic Pasteur Institute, Athens 618, Greece and the Department of Hygiene and Epidemiology of the University ofAthens, Athens 609, Greece Received 26 September 1978 and accepted 2 1 November 1978 Two modified Rappaport's enrichment broths were compared with selenite broth in the isolation of salmonellas from pork sausages. It was found that: (1) both modifications of Rappaport's broth were significantly superior to selenite broth, and (2) one of the modifications (R10/43"C), had a remarkable inhibitory effect on the competing bacteria, including those which produce salmonella-like colonies.
WE HAVE COMPARED the standardized procedure for the isolation of salmonellas (Edel & Kampelmacher 1969, 1973; Anon. 1975) with two new procedures, involving enrichment from buffered peptone water (BPW) in two modifications of Rappaport's broth (R25 and R10) incubated at 37 "C (R25/37 "C) and 43 "C (R10/43 "C). As a rule more positive samples containing salmonellas were found with R25/37 "C and R10/43 "C methods than with the standardized procedure involving enrichment in Muller-Kauffmann tetrathionate broth at 43 "C (MK/43 "C). In addition, the R10/43"C method, when compared with all other procedures, had a stronger inhibitory effect on the competing bacteria, including those that are lactose- and sucrose-negative and produce salmonella-like colonies on brilliant green deoxycholate agar plates (Vassiliadis etal. 1977, 1978~). The extension of our comparison of R25/37 OC and R10/43 "C with enrichment in selenite broth is discussed in this communication. The selenite broth is widely used, and was found (Roberts et al. 1975) to be superior to Muller-Kauffmann broth in the isolation of salmonellas from pork sausages, but inferior to Rolfe's (1946) tetrathionate broth A incubated at 37" and 43 "C.
Materials and Methods Three hundred and seventeen samples of fresh pork sausages were examined. The pre-enrichment medium was the buffered peptone water of Edel & Kampelmacher (1973). The Rappaport et al. (1956) media (R media) were prepared from solutions A, B and C. In modification R25, only 25 ml of solution C (0.4% (w/v)malachite green oxalate in distilled water) were present in 1125 ml of the final medium; in modification R10, only 10 ml in 1110 ml (Vassiliadis et al. 1976, 1977, 1978b), as against 30 ml in 1130 ml in the original formulation (Rappaport et al. 1956). The concentration of malachite green in the original medium was thus 106 mg/l; in R25, 89 mg/l; and in R10, 36 mg/l. The three enrichment broths S , R25 and R10 were distributed (10 ml) in test tubes. 0021-8847/79/030567 +03%01.00/0
[5671
@ 1979 T h e Society for Applied Bacteriology
P. VASSILIADIS ET AL.
568
Mannitol selenite broth (S broth) was prepared with a commercial dehydrated broth base (Oxoid CM399) and sodium biselenite (Oxoid L 121). The preparation and sterilization of this medium were done according to the manufacturer's instructions and it was used within 24 h of preparation. Brilliant green deoxycholate agar (BGD agar) was prepared as described by Vassiliadis et al. (1977, 19786). Jars containing finely minced pork sausage (1 5 g) and BPW (100 ml) were incubated at 37 "C for 18-22 h. The enrichment media R25 and R10 were inoculated with 0.1 ml of the BPW pre-enrichment culture. The R25 medium was incubated at 37 "C, and the R10 medium at 42.9 "C (k0.25 "C), for 48 h. The S medium was inoculated with 1 ml of the BPW pre-enrichment and incubated at 42.9 "C (k0.25 "C) for 48 h (S/43 "C). After 24 and 48 h incubation of the three enrichment media, subcultures were made on BGD agar in Petri dishes which were incubated at 37°C for 24 h. If available, four suspicious colonies from each positive BGD agar plate were examined. The growth of competing organisms on BGD agar was also recorded subjectively: confluent or almost confluent growth on approximately one quarter of the agar was classified as light growth, on a half as moderate, on three-quarters as heavy, and over the whole surface of the agar as very heavy.
Results and Conclusions Of the 3 17 samples examined, 122 (38.5%) were found to contain salmonellas when the R25/37OC or the R10/43OC procedures were used; only 57 samples (18.0%) were positive with the S/43 "C method. With the R25/37 OC procedure 129 strains belonging to 27 serotypes were isolated, with the R10/43 OC procedure 151 strains of 29 serotypes, and with the S/43 "C method only 60 strains belonging to 16 serotypes. With these three enrichment procedures 29 different serotypes were isolated, three, Salmonella caen, Salm. kedougou and Salm. worthington, for the first time in Greece.
TABLE1 Growth of competing organisms on brilliant green deoxycholate agar after subculture from the three enrichment broths, inoculated from pre-enrichments, in buflered peptone water
-
Enrichment procedures. Growth of competing organisms Absent-light Moderate fieavy-very heavy
R25/3 7 O C (h) ---4---
24
116(37)t 58(18)
143(45)
R10/43 "C (h)
48
84(26) 42( 13) 191(60)
24
48
302(95) 6(2) 9(3)
285(90) 15(5) 17(5)
s/43 "C (h)
* 24
48
89(28) 63(20) 165(52)
123(39) 60(19) 134(42)
*R25/37 "C, enrichment at 37 "C in Rappaport's broth containing 25 ml of the 0.4% (w/v) solution of malachite green oxalate (Merck)/ll25 ml of medium; R10/43 "C, enrichment at 43 OC in Rappaport's broth containing only 10 ml of malachite green solution/l 1 10 ml of medium; and S/43 OC, enrichment at 43 OC in mannitol-selenite broth. +Percentages in parentheses.
569
ENRICHMENT BROTHS F O R SALMONELLAS
Procedure R10/43 OC was more inhibitory to competing organisms (either negative or positive to lactose and sucrose), than the methods S/43 OC and R25/37 OC (Table 1). The number of lactose- and sucrose-negative (‘false positive’) colonies on BGD agar which were not salmonellas is given in Table 2. It can be seen that procedure R10/43 OC was superior to R25/31 OC and even more so to the S/43 “C method, as it allowed the growth of fewer ‘false positive’ colonies. A similar observation was made before when the comparison was done with procedure MK/43OC (Vassiliadis et al. 19783). This feature was important as time, labour and media can be saved when procedure R10/43 OC is used. TABLE2 Isolation of Salmonella from brilliant green deoxycholate agar streaked from three enrichment media Enrichment procedures* r
No. of samples with suspicious colonies No. of colonies examined No. of Salmonella colonies No. of ‘false positive’ colonies
A
R25l37 O
C
152 1059 893 (84.3)t 166 (15.7)
\
R10l43 OC
S143 OC
130 980 946 (96.5) 34 (3.5)
112 692 334 (48.3) 358 (51.7)
*tSee footnotes Table 1.
References ANON. 1975 Meat and meat products-Detection oj- salmonellae (Reference method). International Standard IS0 3565. Geneva, Switzerland: International Organization for
Standardization. EDEL,W. & KAMPELMACHER, E. H. 1969 Salmonella isolation in nine European laboratories using a standardized technique. Bulletin of the World Health Organization 41,297-306. EDEL,W. & KAMPELMACHER, E. H. 1973 Comparative studies on the isolation of sublethally injured salmonellae in nine European laboratories. Bulletin of the World Health Organization 48,167-174. RAPPAPORT, F., KONFORTI,N. & NAVON,B. 1956 A new enrichment medium for certain salmonellae.Journal of Clinical Pathology 9,26 1-266. ROBERTS, D., BOAG,K., HALL,M. L. M. & SHIPP,C. R. 1975 The isolation of salmonellas from British pork sausages and sausage meat. Journal of Hygiene, Cambridge 75,173-184. ROLFE,V. 1946 A note on the preparation of tetrathionate broth. Monthly Bulletin of Ministry of Health and the Emergency Public Health Laboratory Service 5 , 158-159. VASSILIADIS, P., PATERAKI, E., PAPA~CONOMOU, N., PAPADAKIS, J. A. & TRICHOPOULOS, D. 1976 Nouveau prockdi d’enrichissement de Salmonella. Annales de Microbiologie, Institut Pasteur 127B, 195-200. VASSILIADIS, P., KALANDIDI, A., XIROUCHAKI, E., PAPADAKIS, J. & TRICHOPOULOS, D. 1977 Isolement de salmonelles a partir de saucisses de porc en utilisant un nouveau procedi d’enrichissement (R 10/43O). Recueil de Mkdecine Vderinaire, Alfort 153,489-494. VASSILIADIS,P., TRICHOPOULOS, D., PATERAKI, E. & PAPAYCONOMOU, N. 1978a Isolation of Salmonella from minced meat by the use of a new procedure of enrichment. Zentralblatt fur Bakteriologie Originale B. Hygiene, Praventive Medizin 166,8 1-86. VASSILIADIS, P., TRICHOPOULOS, D., KALANDIDI, A. & XIROUCHAKI, E. 19786 Isolation of salmonellae from sewage with a new procedure of enrichment. Journal of Applied Bacteriology 44,233-239.
495/091/78
A Note on the Comparison of two Modifications of Rappaport's Medium with Selenite Broth in the Isolation of Salmonellas P. VASSILIADIS, D. TRICHOPOULOS, G. PAPOUTSAKIS AND E. PALLANDIOU The Hellenic Pasteur Institute, Athens 618, Greece and the Department of Hygiene and Epidemiology of the University ofAthens, Athens 609, Greece Received 26 September 1978 and accepted 2 1 November 1978 Two modified Rappaport's enrichment broths were compared with selenite broth in the isolation of salmonellas from pork sausages. It was found that: (1) both modifications of Rappaport's broth were significantly superior to selenite broth, and (2) one of the modifications (R10/43"C), had a remarkable inhibitory effect on the competing bacteria, including those which produce salmonella-like colonies.
WE HAVE COMPARED the standardized procedure for the isolation of salmonellas (Edel & Kampelmacher 1969, 1973; Anon. 1975) with two new procedures, involving enrichment from buffered peptone water (BPW) in two modifications of Rappaport's broth (R25 and R10) incubated at 37 "C (R25/37 "C) and 43 "C (R10/43 "C). As a rule more positive samples containing salmonellas were found with R25/37 "C and R10/43 "C methods than with the standardized procedure involving enrichment in Muller-Kauffmann tetrathionate broth at 43 "C (MK/43 "C). In addition, the R10/43"C method, when compared with all other procedures, had a stronger inhibitory effect on the competing bacteria, including those that are lactose- and sucrose-negative and produce salmonella-like colonies on brilliant green deoxycholate agar plates (Vassiliadis etal. 1977, 1978~). The extension of our comparison of R25/37 OC and R10/43 "C with enrichment in selenite broth is discussed in this communication. The selenite broth is widely used, and was found (Roberts et al. 1975) to be superior to Muller-Kauffmann broth in the isolation of salmonellas from pork sausages, but inferior to Rolfe's (1946) tetrathionate broth A incubated at 37" and 43 "C.
Materials and Methods Three hundred and seventeen samples of fresh pork sausages were examined. The pre-enrichment medium was the buffered peptone water of Edel & Kampelmacher (1973). The Rappaport et al. (1956) media (R media) were prepared from solutions A, B and C. In modification R25, only 25 ml of solution C (0.4% (w/v)malachite green oxalate in distilled water) were present in 1125 ml of the final medium; in modification R10, only 10 ml in 1110 ml (Vassiliadis et al. 1976, 1977, 1978b), as against 30 ml in 1130 ml in the original formulation (Rappaport et al. 1956). The concentration of malachite green in the original medium was thus 106 mg/l; in R25, 89 mg/l; and in R10, 36 mg/l. The three enrichment broths S , R25 and R10 were distributed (10 ml) in test tubes. 0021-8847/79/030567 +03%01.00/0
[5671
@ 1979 T h e Society for Applied Bacteriology
P. VASSILIADIS ET AL.
568
Mannitol selenite broth (S broth) was prepared with a commercial dehydrated broth base (Oxoid CM399) and sodium biselenite (Oxoid L 121). The preparation and sterilization of this medium were done according to the manufacturer's instructions and it was used within 24 h of preparation. Brilliant green deoxycholate agar (BGD agar) was prepared as described by Vassiliadis et al. (1977, 19786). Jars containing finely minced pork sausage (1 5 g) and BPW (100 ml) were incubated at 37 "C for 18-22 h. The enrichment media R25 and R10 were inoculated with 0.1 ml of the BPW pre-enrichment culture. The R25 medium was incubated at 37 "C, and the R10 medium at 42.9 "C (k0.25 "C), for 48 h. The S medium was inoculated with 1 ml of the BPW pre-enrichment and incubated at 42.9 "C (k0.25 "C) for 48 h (S/43 "C). After 24 and 48 h incubation of the three enrichment media, subcultures were made on BGD agar in Petri dishes which were incubated at 37°C for 24 h. If available, four suspicious colonies from each positive BGD agar plate were examined. The growth of competing organisms on BGD agar was also recorded subjectively: confluent or almost confluent growth on approximately one quarter of the agar was classified as light growth, on a half as moderate, on three-quarters as heavy, and over the whole surface of the agar as very heavy.
Results and Conclusions Of the 3 17 samples examined, 122 (38.5%) were found to contain salmonellas when the R25/37OC or the R10/43OC procedures were used; only 57 samples (18.0%) were positive with the S/43 "C method. With the R25/37 OC procedure 129 strains belonging to 27 serotypes were isolated, with the R10/43 OC procedure 151 strains of 29 serotypes, and with the S/43 "C method only 60 strains belonging to 16 serotypes. With these three enrichment procedures 29 different serotypes were isolated, three, Salmonella caen, Salm. kedougou and Salm. worthington, for the first time in Greece.
TABLE1 Growth of competing organisms on brilliant green deoxycholate agar after subculture from the three enrichment broths, inoculated from pre-enrichments, in buflered peptone water
-
Enrichment procedures. Growth of competing organisms Absent-light Moderate fieavy-very heavy
R25/3 7 O C (h) ---4---
24
116(37)t 58(18)
143(45)
R10/43 "C (h)
48
84(26) 42( 13) 191(60)
24
48
302(95) 6(2) 9(3)
285(90) 15(5) 17(5)
s/43 "C (h)
* 24
48
89(28) 63(20) 165(52)
123(39) 60(19) 134(42)
*R25/37 "C, enrichment at 37 "C in Rappaport's broth containing 25 ml of the 0.4% (w/v) solution of malachite green oxalate (Merck)/ll25 ml of medium; R10/43 "C, enrichment at 43 OC in Rappaport's broth containing only 10 ml of malachite green solution/l 1 10 ml of medium; and S/43 OC, enrichment at 43 OC in mannitol-selenite broth. +Percentages in parentheses.
569
ENRICHMENT BROTHS F O R SALMONELLAS
Procedure R10/43 OC was more inhibitory to competing organisms (either negative or positive to lactose and sucrose), than the methods S/43 OC and R25/37 OC (Table 1). The number of lactose- and sucrose-negative (‘false positive’) colonies on BGD agar which were not salmonellas is given in Table 2. It can be seen that procedure R10/43 OC was superior to R25/31 OC and even more so to the S/43 “C method, as it allowed the growth of fewer ‘false positive’ colonies. A similar observation was made before when the comparison was done with procedure MK/43OC (Vassiliadis et al. 19783). This feature was important as time, labour and media can be saved when procedure R10/43 OC is used. TABLE2 Isolation of Salmonella from brilliant green deoxycholate agar streaked from three enrichment media Enrichment procedures* r
No. of samples with suspicious colonies No. of colonies examined No. of Salmonella colonies No. of ‘false positive’ colonies
A
R25l37 O
C
152 1059 893 (84.3)t 166 (15.7)
\
R10l43 OC
S143 OC
130 980 946 (96.5) 34 (3.5)
112 692 334 (48.3) 358 (51.7)
*tSee footnotes Table 1.
References ANON. 1975 Meat and meat products-Detection oj- salmonellae (Reference method). International Standard IS0 3565. Geneva, Switzerland: International Organization for
Standardization. EDEL,W. & KAMPELMACHER, E. H. 1969 Salmonella isolation in nine European laboratories using a standardized technique. Bulletin of the World Health Organization 41,297-306. EDEL,W. & KAMPELMACHER, E. H. 1973 Comparative studies on the isolation of sublethally injured salmonellae in nine European laboratories. Bulletin of the World Health Organization 48,167-174. RAPPAPORT, F., KONFORTI,N. & NAVON,B. 1956 A new enrichment medium for certain salmonellae.Journal of Clinical Pathology 9,26 1-266. ROBERTS, D., BOAG,K., HALL,M. L. M. & SHIPP,C. R. 1975 The isolation of salmonellas from British pork sausages and sausage meat. Journal of Hygiene, Cambridge 75,173-184. ROLFE,V. 1946 A note on the preparation of tetrathionate broth. Monthly Bulletin of Ministry of Health and the Emergency Public Health Laboratory Service 5 , 158-159. VASSILIADIS, P., PATERAKI, E., PAPA~CONOMOU, N., PAPADAKIS, J. A. & TRICHOPOULOS, D. 1976 Nouveau prockdi d’enrichissement de Salmonella. Annales de Microbiologie, Institut Pasteur 127B, 195-200. VASSILIADIS, P., KALANDIDI, A., XIROUCHAKI, E., PAPADAKIS, J. & TRICHOPOULOS, D. 1977 Isolement de salmonelles a partir de saucisses de porc en utilisant un nouveau procedi d’enrichissement (R 10/43O). Recueil de Mkdecine Vderinaire, Alfort 153,489-494. VASSILIADIS,P., TRICHOPOULOS, D., PATERAKI, E. & PAPAYCONOMOU, N. 1978a Isolation of Salmonella from minced meat by the use of a new procedure of enrichment. Zentralblatt fur Bakteriologie Originale B. Hygiene, Praventive Medizin 166,8 1-86. VASSILIADIS, P., TRICHOPOULOS, D., KALANDIDI, A. & XIROUCHAKI, E. 19786 Isolation of salmonellae from sewage with a new procedure of enrichment. Journal of Applied Bacteriology 44,233-239.
