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 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 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.     

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.     

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 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.     

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 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.     

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.     

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.     

Observations on the occurrence of Salmonella cholerae-suis and other salmonellas in two herds of feeder pigs.

Modified brilliant green agar (BGA), Muller-Kauffmann tetrathionate, Rappaport''s and selenite F broths were compared for their efficiency in isolating salmonellas from pigs and their excreta. It was concluded that BGA and Rappaport''s broth were the media of choice. Where searches were made for Salmonella cholerae-suis alone, the use of a trehalose McConkey agar provided a rapid method of differentiating S. cholerae-suis, which does not ferment trehalose, from the majority of other salmonellas, which do ferment trehalose. Casualties were collected from two farms where infection with S. cholerae-suis was endemic. The isolation rates of S. cholerae-suis from different carcase sites were compared in order to determine the relative importance of the salivary, upper respiratory and faecal routes of excretion. S. cholerae-suis was isolated from numerous carcase sites in carriers including the salivary glands, tonsils, trachea and lungs. However, isolations from the nasal passages, mouth, pharynx and gastro-intestinal tract of carriers were either infrequent or absent. When, in a further study, S. cholerae-suis was isolated from only 3/414 faeces, 1/170 nasal swabs and not at all from 170 oral swabs taken from live pigs, it was concluded that there must be more significant modes of transmission than from the salivary glands, upper respiratory or gastro-intestinal tracts. Cannibalism was considered to be a possibility. In contrast to S. cholerae-suis, other salmonellas were frequently isolated from randomly collected faeces and from the gastro-intestinal tract as well as other sites in casualties.     

A medium for the rapid enumeration of Escherichia coli in the presence of other faecal coliforms in tropical waters.

A selective membrane filtration medium is described for use in the rapid assessment of water quality in tropical countries where the incidence of faecal coliforms other than E. coli presents problems in the interpretation of results. The medium gives comparable results to MPN values obtained in the multiple tube dilution test using modified Gray''s glutamate medium, and to membrane filtration counts obtained using M-FC broth and membrane-enriched Teepol broth, whilst differentiation of E. coli is enhanced.     

A most probable number method for estimating small numbers of campylobacters in water.

A most probable number (MPN) method capable of estimating as few as ten campylobacters per 100 ml of water is described. The method gave results close to those obtained by the viable count method of Miles, Misra & Irwin (1938) with graded suspensions of Campylobacter jejuni. The method was used to test raw water samples: counts were obtained ranging from 10 to 230 campylobacters per 100 ml for 11 49 coastal and estuary water samples, and from 10 to 36 campylobacters per 100 ml for 7 of 44 river samples. Campylobacters were isolated from an additional 24 of the ''negative'' samples by testing 200 ml volumes by glass microfibre filtration and enrichment culture methods. The MPN method should prove to be a useful epidemiological tool particularly suited to the enumeration of campylobacters in particulate fluids.     

Spot Sample Urine Specific Gravity Does Not Accurately Represent Small Decreases in Plasma Volume in Resting Healthy Males

Background: Urine specific gravity (U_SG) is often used to assess hydration status, particularly around athletic competition, but it is unknown whether high U_SG is indicative of plasma volume (PV) reduction (i.e., hypohydration). We tested the hypothesis that if high U_SG is reflective of reduced PV, subsequent fluid ingestion would increase PV.

Purpose: The purpose of this study was to examine 24-hour changes in U_SG and PV in individuals presenting with high and low spot U_SG.

Methods: Nineteen healthy males were provided food and water over 24 hours with a total water volume of 35 ml·kg^?1 body mass. Absolute PV and blood volume (BV), measured using the CO-rebreathe technique, along with U_SG were measured before and after a 24-hour intervention period. Based on a preintervention morning spot U_SG, subjects were post hoc assigned to groups according to U_SG (≤1.020 or >1.020; low and high U_SG, respectively).

Results: Despite presenting with an elevated spot U_SG (1.026 ± 0.004), subsequent fluid ingestion over 24 hours did not lead to changes (?) in PV (?75 ± 234 ml) or BV (?156 ± 370 ml) in the high U_SG group (p > 0.05). However, a spot U_SG after the 24-hour intervention in this group decreased (p = 0.018) to a level indicating improved hydration status (1.017 ± 0.007). In the low U_SG group, there were no changes in PV (?39 ± 274 ml), BV (?82 ± 396 ml), or U_SG (0.003 ± 0.007) over the 24-hour fluid intervention (all p > 0.05).

Conclusions: Despite a high preintervention U_SG and subsequent decrease after 24-hour fluid intake, measures of PV and BV were not indicative of this seemingly improved hydration status. This suggests that a highly concentrated spot sample U_SG and subsequent changes are not accurately representative of PV or BV.     

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.     

Stimulating effect of pyruvate on the growth of Mycobacterium leprae in cellfree, semisynthetic, soft agar medium

A bacterial suspension prepared from a nodule from a patient with lepromatous leprosy was inoculated into the cell-free, semisynthetic, semiliquid agar medium M—Y 11d—modified from the authors'' medium M-Y 1c by omitting glycerol and adding pyruvate—and incubated at 37°C. The growth of the organism was stimulated so markedly that after about 50 weeks'' incubation of the primary culture microcolonies could be seen floating in the agar layer as tiny white particles, while the medium fluid assumed a yellow tinge. These macroscopic colonies were sucked up by pipette and subjected to microscopic examination. A bacterial suspension prepared from the first subculture of this strain by centrifugation elicited the same skin reactions in leprosy patients as did the standard Mitsuda''s antigen. The organism in the culture was therefore identified as M. leprae.     

Continuous nasogastric tube feeding: monitoring by combined use of refractometry and traditional gastric residual volumes

BACKGROUND & AIMS: Traditional use of gastric residual volumes (GRVs) is insensitive and cannot distinguish retained enteral formula from the large volume of endogenous secretions. We designed this prospective study to determine whether refractometry and Brix value (BV) measurements could be used to monitor gastric emptying and tolerance in patients receiving continuous enteral feeding. METHODS: Thirty-six patients on continuous nasogastric tube feeding were divided into two groups; patients with lower GRVs (<75 ml) in Group 1, patients with higher GRVs (>75 ml) in Group 2. Upon entry, all gastric contents were aspirated, the volume was recorded (Asp GRV), BV measurements were made by refractometry, and then the contents were reinstilled but diluted with 30 ml additional water. Finally, a small amount was reaspirated and repeat BV measurements were made. Three hours later, the entire procedure was repeated a second time. The BV ratio, calculated (Cal) GRV, and volume of formula remaining were calculated by derived equations. RESULTS: Mean BV ratios were significantly higher for those patients in Group 2 compared to those in Group 1. All but one of the 22 patients (95%) in Group 1 had a volume of formula remaining in the stomach estimated on both measurements to be less than the hourly infusion rate (all these patients had BV ratios <70%). In contrast, six of the 14 patients in Group 2 (43%) on both measurements were estimated to have volumes of formula remaining that were greater than the hourly infusion rate (all these patients had BV ratios >70%). Three of the Group 2 patients (21%) whose initial measurement showed evidence for retention of formula, improved on repeat follow-up measurement assuring adequate gastric emptying. The remaining five patients from Group 2 (35%) had a volume of formula remaining that was less than the hourly infusion rate on both measurements. The pattern of Asp GRVs and serial pre- and post-dilution BVs failed to differentiate these patients in Group 2 with potential emptying problems from those with sufficient gastric emptying. CONCLUSIONS: Refractometry and measurement of the BV may improve the clinical utilization of GRVs, by its ability to identify the component of formula within gastric contents and track changes in that component related to gastric emptying.     

我国西部地区个人现金卫生支出致贫影响研究——基于少数民族边远地区的调查

目的:分析2011年和2014年我国西部少数民族边远地区农村居民现金卫生支出(out-of-pocket,ap)对贫困的影响。方法:利用2011年和2014年家庭入户调查数据,比较国家贫困标准下当地居民OOP发生前后的贫困发生率和贫困差距等指标。结果:2011年,OOP前后贫困发生率为31.07%和51.70%,致贫影响为20.63%,平均贫困差距增加284.24元,相对贫困差距增加183.73元;2014年,OOP前后贫困发生率为29.40%和44.66%,致贫影响为15.26%,平均贫困差距增加242.68元,相对贫困差距增加240.41元。结论:当地居民贫困发生率和平均贫困差距有所下降,但相对贫困差距上升;需要进一步调整新型农村合作医疗补偿方案,提高居民受益水平;重点关注低收入人群,减少因病致贫、因病返贫的现象。     

Single tube confirmatory tests for Escherichia coli.

In a multi-laboratory trial, lauryl tryptose mannitol broth (LTMB) and minerals-modified glutamate medium with added tryptophan (MMGM + T) were compared as single tube tests for the confirmation of Escherichia coli in water; the confirmed results were also compared with the production of gas from minerals-modified glutamate medium without added tryptophan (MMGM). LTMB and MMGM + T gave similar gas and indole results with about 90% of the water samples in most of the laboratories. When compared with the ''correct'' results as judged by acid and gas production from lactose peptone water and indole from tryptone water, the difference in the rate of false positive reactions between LTMB and MMGM + T was insignificant; but LTMB gave a significantly lower rate of false negative reactions than MMGM + T. Gas production from MMGM without added tryptophan gave significantly higher rates of both false positive and false negative reactions. Lauryl sulphate is therefore a suitable inhibitory surfactant for use in single tube media for the confirmation of E. coli, which can be recommended.