A comparisoin of the R25 modification of Rappaport s enrichment medium with strontium chloride B for salmonella isolation from sewage polluted natural water

The relation of salmonella isolation efficiency and the size of inoculum introduced from a buffered peptone water culture of sewage polluted water into strontium chloride B medium was investigated. Two separate studies were made, one using enrichment at 37 degrees C, the other at 43 degrees C. From these trials, two inocula suitable for efficient salmonella isolation were determined. Using this information, strontium chloride B medium was compared with modified Rappaport's broth (R25). The inoculum used with R25 was 0.005 ml, determined in an earlier study. Two incubation temperatures were employed with strontium chloride enrichment (37 and 43 degrees C). Rappaport's medium was incubated at 37 degrees C only. Elevated temperature enrichment at 43 degrees C improved the performance of strontium chloride B, but Rappaport's broth still gave significantly better results. This supports earlier studies on simplification of salmonella isolation and standardization of routine technique on a single enrichment medium: Rappaport broth (R25) incubated at 37 degrees C.     

Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for salmonella isolation from chicken giblets after pre-enrichment in buffered peptone water.

Six hundred and eighty three samples of chicken giblets were examined for salmonellas. Three hundred and forty nine of these were neck and crop specimens and 224 were combined liver and heart samples. Two hundred and ten, in all, contained salmonellas. The technique of examination included pre-enrichment in buffered peptone water at 37 degrees C for 18 h and subculture to three enrichment media: Muller-Kauffmann tetrathionate, selenite F and Rappaport''s magnesium chloride malachite green broth. Inocula from buffered peptone water to 10 ml of tetrathionate and selenite were 1 ml in each case. The inoculum from the pre-enrichment medium to 10 ml of Rappaport was 0.005 ml. Tetrathionate and selenite were incubated at 43 degrees C for 48 h. Rappaport''s medium was incubated at 37 degrees C for 48 h. Subcultures from all three enrichment broths were made at 24 h and 48 h to brilliant green MacConkey agar. Selective agars were incubated at 37 degrees C for 24 h. The most successful technique for salmonella isolation used Rappaport''s medium, which was significantly more efficient than either tetrathionate or selenite. This finding reinforces results obtained using sewage polluted natural water as test material and it is suggested that routine examination of environment samples for salmonellas could be based on Rappaport''s medium alone. If S. typhi, S. dublin or subgenus III salmonellas were likely to be present in the sample, the technique described here would require modification.     

A comparison of two modifications of Rappaport's enrichment medium (R25 and RV) for the isolation of salmonellas from sewage polluted natural water.

The development of Rappaport''s enrichment medium and elevated temperature incubation as methods of salmonella isolation is traced. The recent recording of a merger of the two techniques by means of Rappaport--Vassiliadis medium is noted (RV medium). In Cardiff, we have found an earlier modification of Rappaport''s enrichment (R25) by Vassiliadis to be efficient in salmonella recovery from environment samples. The current study compares the two media using sewage polluted natural water as test material. Under the conditions of experiment, R25 was more successful in salmonella isolation than RV, although the later medium inhibited competitive organisms better. R25 is a convenient enrichment broth for routine use. In combination with pre-enrichment it allows the use of a loop for subculture rather than a pipette. This increases safe manipulation. It also produces a high proportion of positive isolations at the 24 h subculture time in contrast to other enrichment broths. For these reasons it forms an integral part of salmonella isolation methodology in our laboratory.     

A comparison of enrichment media for the isolation of salmonellae from seagull cloacal swabs.

The relative efficiency of three selective enrichment broths (Muller-Kauffmann tetrathionate. Rappaport''s and selenite F) was investigated for the isolation of salmonellae from seagull cloacal swabs. Pre-enrichment in buffered peptone water was employed throughout the study, which involved the examination of 560 gulls, sixty (10.7%) of which were found to be carrying salmonellae. Rappaport''s broth as modified by Vassiliadis for incubation at 43 degrees C (Vassiliadis et al. 1976) yielded the highest number of positive swabs (57) and the widest range of serotypes. It was significantly more efficient that either selenite F or tetrathionate broth, although the results obtained with Rappaport''s broth incubated at 37 and 43 degrees C did not differ significantly (P greater than 0.5). Eleven serotypes were isolated during the study, the most prevalent being Salmonella virchow.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios.

The ability of malachite green/magnesium chloride broth (Rappaport''s medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport''s broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

A comparison of the original Rappaport medium (R medium) and the Rappaport-Vassiliadis medium (RV medium) in the isolation of salmonellae from meat products.

The Rappaport-Vassiliadis enrichment medium (RV medium) in 10 ml quantities (RV/43 degrees C, 10 ml) inoculated with 0.1 ml of pre-enrichment medium (P medium) was found more efficient in the isolation of salmonellae from 409 pre-enriched samples (mainly meat products), than the original Rappaport medium incubated at 43 degrees C (R/43 degrees C) and the RV medium in 5 ml quantities (RV/43 degrees C, 5 ml) inoculated with 0.01 ml of P medium (P less than 0.001, in both instances). Therefore, the inoculum from pre-enriched foods should not be less than 0.1 ml in 10 ml of RV medium. The RV/43 degrees, 10 ml was also better (P less than 0.01) in detecting samples containing salmonellas than the original Rappaport medium incubated at 37 degrees C (R/37 degrees C, 10 ml) and the modification R25 of R medium incubated at 37 degrees C. The R25 modification was used in 10 ml quantities (R25/37 degrees C, 10 ml) inoculated with 0.1 ml of P medium and in 5 ml quantities (R25/37 degrees, 5 ml) inoculated with 0.01 ml of P medium. The last two R25 procedures were of the same efficiency in isolating salmonellas from meat products.     

Isolation of Salmonella with the use of 100 ml of the R10 modification of Rappaport's enrichment medium.

One hundred and eighty samples of pork sausages were examined after pre-enrichment in buffered peptone water (P medium), for the presence of salmonellas. From each pre-enrichment four enrichments were made: (1) o.1 ml of P medium was inoculated into 10 ml of Rappaport''s medium formula R 10 (R 10/43 degrees C), /2) 1 ml of the P medium was added to 100 ml of R10 broth (R10/100 ml/43 degrees C), (3) 1 ml of P medium was inoculated into 10 ml of Muller-Kauffmann tetrathionate broth (MK medium) prepared in accordance with the International Standards Organization document ISO 3565 (MK/43 degrees C) and (4) 10 ml of P medium were added to 100 ml of MK broth (MK/100 ml/43 degrees C). All the enrichments were incubated at 43 degrees C for 48 h. Forty-six and 47 samples were found positive with the first two enrichment methods (R10/43 degrees C and R10/100 ml/43 degrees C), while only 16 samples were found positive with the method MK/43 degrees C, and 27 with the methods MK/100ml/43 degrees C. The superiority of either one of the two R10 procedures over either one of the two MK methods is statistically highly significant (paired Chi2; P less than 0.001 in all four comparisons). The superiority of procedures MK/100 ml/43 degrees C over the method MK/43 degrees C is also statistically significant (P less than 0.005). Both R10/43 degrees C and R10/100 ml/43 degrees C procedures had a much stronger inhibitory effect on the competing organisms (lactose- and sucrose negative) than the two MK methods.     

Influence of multiple plating from fluid media on salmonella isolation from animal feeding stuffs.

The influence of multiple plating of fluid cultures on salmonella isolation from animal feeding stuffs was examined. Four plating were made from broth culture after 24 h at 37 degrees C and four platings from selenite enrichment from 24 h at 43 degrees C. Selenite enrichment followed broth culture which was used as a pre-enrichment stage. Brilliant green MacConkey agar plates were employed for broth subculture and brilliant green MacConkey and desoxycholate citrate agars for selenite subculture. The eight brilliant green plates subcultured from broth and selenite were examined for salmonellas after incubation for 24 h at 37 degrees C. The four desoxycholate citrate agars after 24 h at 37 degrees C were used for motility enrichment. The food sample size was a single 100 g instead of 4 x 25 g cultured in an earlier study. This pooling of samples aimed at technical economy. Quadruple plating played an important part in salmonella isolation from 100 g specimens. The combination of multiple plating with motility enrichment was the most successful technique used.     

The isolation of salmonellas from British pork sausages and sausage meat.

Between 1969 and 1974, 1467 packets (3309 samples) of pork sausages and sausage meat produced by two large and two medium sized manufacturers and several local butchers were examined for the presence of salmonellas. Of these, 435 packets (786 samples) were found to contain salmonellas, but there was a wide variation in the isolation rates according to the producer. The salmonella incidence in samples from several small and two medium sized producers was low (0-11%) while the results from the two large producers investigated showed a striking difference, the rate of salmonella contamination in the product of one was low (about 2%) and in that of the other consistently high (40-60%). A comparison of liquid enrichment media, incubation temperatures and selective agar media was also carried out to determine the most efficient combination for the isolation of salmonellas from minced meat products. The results showed that (a) incubation of enrichment cultures at 43 degrees C. yielded a consistently greater number of salmonella isolations that at 37 degrees C., regardless of plating medium, (b) tetrathionate broth A (Rolfe) was superior to selenite broth as en enrichment medium at both 37 and 43 degrees C. and (c) brilliant green agar gave better results than deoxycholate citrate sucrose agar and bismuth sulphite agar as a selective medium.     

A comparison of two modifications of Rappaport's enrichment medium (R25 and RV) for the isolation of salmonellas from sewage polluted natural water

The development of Rappaport's enrichment medium and elevated temperature incubation as methods of salmonella isolation is traced. The recent recording of a merger of the two techniques by means of Rappaport--Vassiliadis medium is noted (RV medium). In Cardiff, we have found an earlier modification of Rappaport's enrichment (R25) by Vassiliadis to be efficient in salmonella recovery from environment samples. The current study compares the two media using sewage polluted natural water as test material. Under the conditions of experiment, R25 was more successful in salmonella isolation than RV, although the later medium inhibited competitive organisms better. R25 is a convenient enrichment broth for routine use. In combination with pre-enrichment it allows the use of a loop for subculture rather than a pipette. This increases safe manipulation. It also produces a high proportion of positive isolations at the 24 h subculture time in contrast to other enrichment broths. For these reasons it forms an integral part of salmonella isolation methodology in our laboratory.     

Studies on the isolation of Salmonella dublin.

Abattoir drain swabs, bovine faeces and a few other veterinary samples were examined for the presence of Salmonella dublin. Three selective agar media and four enrichment broths were investigated. The two most efficient plating media were deoxycholate citrate agar and brilliant green MacConkey agar. Wilson and Blair''s bismuth sulphite agar (de Loureiro''s modifications) was least successful. Selenite F broth, whether incubated at 37 or 43 degrees C., was better than the other enrichment broths used which contained a triphenyl methane dye as one of the selective ingredients.     

The effect of the use of different selective media on the ability to recover salmonellae from seagull faeces.

Solid media were compared for their ability to recover salmonellae from seagull faecal material after pre-enrichment in buffered peptone water and enrichment in Rappaport''s broth. Of the 847 specimens examined 96 were found to be positive for salmonellae. Use of Brilliant Green agar containing sulphamandelate supplement resulted in the detection of salmonellae from each of the 96 samples found to be positive and was the most efficient medium tested. Brilliant Green agar lacking the supplement was the least effective medium, salmonellae being isolated from only 80 samples using this medium. All of the media tested were shown to support the growth of a wide range of salmonella serotypes, although Salmonella typhi and S. dublin did not form colonies on those media which contained Brilliant Green. Hynes'' modification of deoxycholate citrate agar was shown to be considerably less inhibitory to salmonellae after ageing for four days. Ageing of other media had no significant affect on their ability to support the growth of salmonellae.     

A comparison of enrichment media for the isolation of salmonellae from seagull cloacal swabs

The relative efficiency of three selective enrichment broths (Muller-Kauffmann tetrathionate. Rappaport's and selenite F) was investigated for the isolation of salmonellae from seagull cloacal swabs. Pre-enrichment in buffered peptone water was employed throughout the study, which involved the examination of 560 gulls, sixty (10.7%) of which were found to be carrying salmonellae. Rappaport's broth as modified by Vassiliadis for incubation at 43 degrees C (Vassiliadis et al. 1976) yielded the highest number of positive swabs (57) and the widest range of serotypes. It was significantly more efficient that either selenite F or tetrathionate broth, although the results obtained with Rappaport's broth incubated at 37 and 43 degrees C did not differ significantly (P greater than 0.5). Eleven serotypes were isolated during the study, the most prevalent being Salmonella virchow.     

Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for the isolation of salmonellas from sewage-polluted natural water using a pre-enrichment technique.

Three enrichment broths, selenite F, Muller-Kauffmann tetrathionate and Rappaport, were examined for their efficiency in salmonella isolation. The three media, prepared from single ingredients in the laboratory, were compared with their commercial equivalents. Laboratory-prepared media were more efficient for isolating salmonellas from sewage-polluted natural water samples. A pre-enrichment stage using buffered peptone water was employed throughout the investigation. The size of inoculum from the pre-enrichment medium was relevant to successful salmonella isolation. Inocula studied were 1 ml and one loopful (3 mm diameter loop). The smaller inoculum gave better results with Rappaport, the larger with selenite and tetrathionate. Using the optimal inocula, Rappaport was the most efficient enrichment broth of the three fluid media in this study.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios

The ability of malachite green/magnesium chloride broth (Rappaport's medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport's broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

The use of Preston enrichment broth for the isolation of 'thermophilic' campylobacters from water.

The application of Preston enrichment broth to isolation of ''thermophilic'' campylobacters from water has been investigated. Enrichment substantially increased the yield of such organisms. The optimum timing for subculturing the broth was after 48 h incubation. Despite the addition of aerotolerant supplement to the broth the number and variety of isolates was greater when the broth was incubated microaerically than when it was incubated in air.     

Salmonella isolation from reptilian faeces: a discussion of appropriate cultural techniques

A short study of salmonella isolation from reptile faeces is described. The samples came from the reptile house at the Bristol Zoological Gardens. The wide range of salmonella serotypes present in the material, including representatives of all four subgenera, is noted. The important factors in the technique of isolation are discussed. These are choice of inoculum, enrichment medium, and plating medium, use of multiple subculture and serological isolation. If the number of samples available is scanty, an extended technique will probably yield more information than a simple method.     

Salmonella isolation from reptilian faeces: a discussion of appropriate cultural techniques.

A short study of salmonella isolation from reptile faeces is described. The samples came from the reptile house at the Bristol Zoological Gardens. The wide range of salmonella serotypes present in the material, including representatives of all four subgenera, is noted. The important factors in the technique of isolation are discussed. These are choice of inoculum, enrichment medium, and plating medium, use of multiple subculture and serological isolation. If the number of samples available is scanty, an extended technique will probably yield more information than a simple method.     

Recovery of Campylobacter from human faeces stored at 4 degrees C

Six hundred and thirteen fresh diarrhoeal faeces were inoculated on Skirrow blood agar (SK), on Preston blood free agar (PBF), and in Campy-thioglycolate broth (CT). After 24 h of storage at 4 degrees C, specimens were again inoculated on SK and PBF, and in Campylobacter enrichment broth (CEB). CT tubes were placed overnight at 4 degrees C. Plates and CEB tubes were incubated at 43 degrees C in microaerophilic conditions. A total of 68 specimens was positive for campylobacter on direct plating. Sixty-four of them were also recovered after subculturing from CT, and only 51 from CEB. Delayed inoculation of plates after storage of samples at 4 degrees C yielded 57 isolates. The storage of faeces at 4 degrees C for 24 h significantly reduces the number of campylobacter isolates. When samples are not plated immediately we recommend inoculating a CT tube maintained at 4 degrees C overnight as a holding medium.     

Salmonellas in Danish pigs: a comparison of three isolation methods.

Caecal samples from 350 Danish bacon pigs were investigated for salmonella using three methods of isolation. (1) Direct inoculation of 1 g of faeces into 10 ml of Muller-Kaufmann medium (MK medium) with addition of 0.3% Teepol 610 and subculture on Brilliant Green lactose sucrose phenol-red agar (BLSF agar) with 0.3% Teepol 610. (2) Pre-enrichment of 5 g of faeces into buffered peptone water with addition of 1% Teepol 610 followed by enrichment of 1 ml in 10 ml MK medium with 1% Teepol 610 and subculture on BLSF agar with 0.3% Teepol. (3) Incubation of 0.1 ml of the pre-enrichment (2) into 10 ml Rappaport-Vassiliadis medium (RV 10 medium) incubated at 43 degrees C, subculture on BLSF agar. The MK media with and without pre-enrichment yielded higher findings than the RV 10 media. In total, 28 (8%) of the pigs were found positive, representing 11 (7.4%) of a total of 142 herds investigated. Lymph glands were collected at a later date from six of the positive herds. Five of the herds were found positive. The number of salmonellas in the glands was low, probably less than ten per gram.