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Faeces samples enriched for salmonellas in selenite F broth incubated at 43 degrees C were subcultured to brilliant green MacConkey agar after 6 h. Four platings were made. As a control technique, each sample was enriched in selenite F incubated at 37 degrees C for 24 h and subcultured once to brilliant green MacConkey agar. Of the specimens plated at 6 h, out of a total of 25 positive samples, 6 required more than a single subculture to reveal the presence of salmonellas. Twenty-four positive isolations were made from the selenite broths incubated at 37 degrees C for 24 h and subcultured once only. All faecal samples positive by enrichment were also positive by direct plating. Nine samples seeded by 6 h produced only 1-2 salmonella colonies per plate. Convalescent specimens are, therefore, unlikely to be efficiently diagnosed by early subculture even if it is combined with multiple plating.River Taff samples, after pre-enrichment in buffered peptone water, were enriched in Rappaport's medium at 37 degrees C for 6 h. Subcultures were made to four plates of brilliant green MacConkey agar. A control subculture was made at 24 h to a single plate of brilliant green MacConkey. Although multiple subculture at 6 h improved salmonella isolation, the method was not as efficient as a single plating from the same enrichment culture at 24 h. There was no evidence that subculture at 6 h combined with multiple plating had a value either for faecal examination or for the isolation of salmonellas from sewage-polluted natural water.Samples of other natural waters submitted routinely by Environmental Health Officers were pre-enriched in buffered peptone water before subculture to Rappaport's medium. The enrichment cultures were incubated at 37 degrees C for 24 h and duplicate platings were made to brilliant green MacConkey agar. By double plating at 24 h, a gain of 16% in salmonella recovery was made. This compares favourably with the gain obtained by subculture from Rappaport after 24 and 48 h incubation. It also saves 24 h in examination time.

Selenite F broth and a modified Muller-Kauffmann tetrathionate broth were investigated using sewage-polluted natural water as inocula. The modification of the tetrathionate medium was necessary as commercial alternatives would not allow multiplication of small numbers of salmonellas. The wide range of molar concentrations in different tetrathionate broths was emphasized. Selenite F broth was more efficient than Muller-Kauffmann tetrathionate broth, in our hands, with the material tested. Direct inoculation of the enrichment media was used. Each of the two media examined had a bias towards selection of certain serotypes. If possible, both enrichment broths should be used to obtain maximum information.

Stools, known to have contained salmonellas were cultured in three enrichment media on arrival at the laboratory. The three media were laboratory prepared selenite F, laboratory prepared Muller-Kauffmann tetrathionate and commercially obtained Oxoid Muller-Kauffmann tetrathionate (CM343). Where faecal samples were sufficiently large, they were stored at room temperature and re-examined using the same three enrichment broths. Whether fresh or stored stools were investigated, the laboratory prepared media were significantly more efficient than the commercial medium. In a straight comparison of the two laboratory prepared media, no significant difference in efficiency was evident with fresh stool samples. With stored specimens, however, Muller-Kauffmann tetrathionate was significantly better than selenite F. This finding might be relevant in examining samples delayed in the post.

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.

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 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.

Four microbiologists from four European laboratories came together to a single laboratory for two weeks and during that period examined 100 samples of minced meat artificially contaminated with salmonellae and 100 naturally contaminated samples using a standardized technique. No significant differences were found between the results obtained by the different analysts with 92 to 97 of the artificially contaminated and 30 to 42 of the naturally contaminated samples being found positive. Over a further period of eight weeks, these analysts examined the same number of samples in their own laboratories again using an identical procedure. There were no significant containing approximately 100 salmonellae per gram but significant differences (43 to 93) were found in isolation rates from the artificially contaminated samples copntaining approximately 1 salmonella per gram. The differences in findings between the first and the second part of the experiments may have been caused fairly by variations in media preparation, temperature of incubators, storage conditions of media and thawing conditions of samples etc., and secondly by differences in working conditions such as performing only one task during two weeks in contrast to examining the samples as part of the daily routine. At high levels of contamination, these factors do not appear to have an important influence on the performance of a laboratory.

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.

An area of the laboratory bench on which slide agglutinations were performed in the diagnosis of salmonella and shigella infection was examined for these organisms. Impression plates and broth-moistened swabs were used for sampling. Both techniques gave satisfactory results, but the contact plates provided positive results a day earlier than the swabs. Suitable precautions to minimize contamination of the bench surface are discussed.

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 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 suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4 degrees C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4 degrees C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure.

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.

Evidence of the presence of salmonellas in a paediatric ward, a special care baby unit, a maternity unit and a hospital kitchen was obtained by culture of sewer swabs, faeces and food samples. The survey was designed to cause as little administrative interference as possible. The technical aspects of the survey did not strain laboratory facilities. Minimal secondary spread of salmonella infection was experienced.

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.

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.

H phase II --> phase I conversion of Salmonella thompson was studied at different temperatures. All strains were isolated from one outbreak, and a convenient method of phase conversion was sought. In our hands this was best effected at 20 degrees C.