Salmonella isolation with Rappaport-Vassiliadis enrichment medium seeded with different sized inocula of pre-enrichment cultures of meat products and sewage polluted water.

A total of 574 samples, of seven different types, were examined for the presence of salmonellas. All the specimens were pre-enriched in buffered peptone water and enriched in Rappaport-Vassiliadis medium (RV medium). In one trial 0.1 ml of pre-enrichment culture of 497 samples (79 chicken carcasses, 228 specimens of minced meat, 100 pork sausages, 19 samples of dried powdered chicken meat, 11 specimens of faeces of healthy pigs and 60 samples of sewage polluted natural sea water) was seeded to 10 ml as well as to 100 ml of RV medium. With the first inoculum (ratio 0.1:10), 111 samples were found to contain salmonellas, while with the second inoculum (ratio 0.1:100), only 102 positive samples were detected. This difference is marginally significant (P less than 0.05). In another trial, 0.1 ml, 0.2 ml and 0.5 ml of pre-enrichment culture of 162 specimens (71 chicken carcasses, 40 samples of sewage polluted sea water and 51 samples of sewage polluted river water) were in turn introduced to 10 ml of RV medium. With the 0.1 ml inoculum 93 positive samples were detected, while with the 0.2 and 0.5 ml inocula 93 and 88 positive samples were found. The differences are not statistically significant. In these trials the growth of competing organisms was minimal with ratios of inocula 0.1:10 or 0.1:100.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparison of procedures based upon Rappaport-Vassiliadis medium with those using Muller-Kauffmann medium containing Teepol for the isolation of Salmonella sp

A total of 308 samples of different types were examined for the presence of salmonellas by means of three different procedures. The first consisted of pre-enrichment in buffered peptone water followed by enrichment in Rappaport-Vassiliadis medium (P/RV). The second differed only in that 1% Teepol was added to the pre-enrichment medium (PT/RV). In the third, buffered peptone water with 1% Teepol was followed by enrichment in Muller-Kauffmann tetrathionate broth also containing 1% Teepol (PT/MKT). The first of these combinations (P/RV) proved superior to the others both in terms of isolation rates and in the appearance of suspicious colonies.     

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.     

Quality control tests of two salmonella enrichment media using different inocula.

Inocula of polluted water naturally infected with salmonellas effectively distinguished six brands of selenite and six brands of tetrathionate enrichment media into satisfactory and unsatisfactory categories. Minimal inocula of pure cultures differentiated the tetrathionates, but not the selenites. Inocula of naturally infected chicken giblets suggested that there was a difference between two comparable brands of tetrathionate, but this was not statistically significant. The difference was, however, clearly demonstrated by minimal inocula of pure cultures. Intensive investigation of two inferior tetrathionates revealed inhomogeneity in the distribution of brilliant green in one bottle of one brand. The importance of the salmonella serotype and even the colonial variant used for the pure culture inoculum was also demonstrated.     

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 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.     

Effect of preincubation time and concentration of selective media on the effectiveness of salmonella organism isolation from frozen meat homogenates

Double concentrated RV and SC media were used for selective multiplication of frozen meat homogenates preincubated in buffered peptone water during 6 and 20 hours. It was found that cultures of 2 ml of homogenate preincubated during 6 hours in 10 ml of twofold concentrated RV medium, and selective multiplication during 20 hours made possible obtaining of a greater number of Salmonella isolates as compared to samples preincubated during 20 hours and transferred in volumes of 0.1 ml into 10 ml of normal concentration medium. On the other hand, the growth of 1 ml of a culture preincubated during 6 hours transferred into 10 ml of twofold concentrated SC medium yielded a similar number of salmonella isolates as in the case of samples preincubated during 24 h and transferred in 1 ml volumes into 10 ml of the SC medium without concentration change.     

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.     

The occurrence and significance to animal health of salmonellas in sewage and sewage sludges.

A total of 882 samples of settled sewage, sewage sludges and final effluents from eight sewage treatment plants were examined for the presence of salmonellas. Of these samples 68% were positive, isolations being made most frequently from settled sewage (85%), raw sludge (87%) and anaerobically digested sludge (96%). Fewer isolations were made from final effluent (24%) and processed sludges (58%). Samples usually contained less than 200 salmonellas/100 ml and arguments are presented that such concentrations should not lead to disease in animals if suitable grazing restrictions are followed.     

Detection of salmonella in food products]

The Rappaport-Vassiliadis (RV) medium prepared under laboratory conditions from its different components was found to be equally suitable as the commercial RV Oxoid medium for routine detection of Salmonella in food products. The best detection (30 of 113 examined samples) was obtained using 0.1 ml of culture and 10 ml of medium. In case of 1 ml of culture and 10 ml of medium Salmonella was isolated only from 3 samples. Only 7 positive samples were obtained using MK medium. Necessity of preliminary toxicity verification for malachite green oxalate colour used in RV medium, to standard Salmonella strains, prior to routine food products tests, was found.     

Factors affecting the toxicity of rotting carcasses containing Clostridium botulinum type C

Mice killed shortly after receiving 1300-3000 spores of Clostridium botulinum type C per os were incubated at one of four chosen temperatures together with bottles of cooked meat medium seeded with a similar inoculum. After incubation the rotting carcasses were homogenized. Sterile membrane filtrates of the homogenates (10-20.8%, w/v) and pure cultures were then titrated for toxicity. A temperature of 37 degrees C produced less toxicity in most carcasses than in cultures. At 30 degrees C, however, toxicity (often 2 X 10(5) to 2 X 10(6) mouse intraperitoneal LD/g or ml) was roughly similar in both systems, and some carcasses and cultures were still toxic (2 X 10(4) to 2 X 10(5) LD/g or ml) after 349 days. Surprisingly, at 23 degrees C, through greatly reduced in the cultures, toxicity was high in many carcasses for at least 28 days. Little or no toxin was produced in either system at 16 degrees C. Unfiltered homogenates (17.8-22.5%, w/v; dose 0.25 ml per os) of toxic carcasses incubated at 30 degrees C for 7 days invariably produced death from botulism, often within as little as 4 h, but a 1 in 10 dilution produced less than 100% mortality.     

A standard technique for the isolation of Salmonella from animal feeds.

Two trials were carried out by 9 and 47 laboratories to evaluate the reproducibility and sensitivity of a standard technique for the isolation of salmonellas from animal feeding stuffs. The trials involved the examinations of test samples containing known numbers of salmonellas by a buffered peptone water pre-enrichment technique. Statistical analysis of the results of the trials showed that the method was capable of detecting salmonellas in most probable numbers as low as 2.3 per 100 g and that no significant differences in performance were observed between laboratories. The standard technique described is recommended for the examination of animal feed and conforms to international recommendations.