Evaluation of Culture Media for the Isolation of Salmonellas from Sewage Sludge

Ten samples of sewage sludge were examined by various methods for the isolation of salmonellas using three types of enrichment broth: Muller-Kauffmann Tetrathionate Broth (MKTB), Selenite F Broth (SFB), and Brilliant Green Broth (BGB), two temperatures (37°C and 43°C) and three selective media: Deoxycholate Citrate Agar incubated aerobically (DCA), and anaerobically (DCA(N)), Brilliant Green Agar (BGA), and Bismuth Sulphite Agar (BSA). The results suggest that a combination of pre-enriched MKTB incubated at 37°C and plated on to BGA at 24 and 48 h was the best method, but when examining contaminated material such as sewage sludge, it appears unwise to rely on one single method.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

The efficiency of Rappaport's broth ( RB10 ) and Rappaport's broth containing novobiocin ( NRB10 ) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used: no significant difference in their ability to recover salmonellae was demonstrated.     

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.
A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow , and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81–92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 micrograms of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 μg of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

A note on the use of Rappaport-Vassiliadis medium (RIO/RV) for the isolation of salmonellas from sewage and sewage sludges

The use of a modified Rappaport broth for the selective enrichment of salmonellas in sewage sludge is described. Comparative trials were carried out using Muller Kauffman—Tetrathionate (MKT) broth and Rappaport-Vassiliadis (R10/RV) medium for selective enrichment. Results have indicated that R10/RV broth is more selective and is to be preferred for routine monitoring.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Comparative study on Salmonella isolation from sewage—contaminated natural waters

A lcaide , E., M artinez , J.P. & G aray , E. 1984. Comparative study on Salmonella isolation from sewage contaminated natural waters. Journal of Applied Bacteriology 56 , 365–371.
A comparative study of five factors influencing the isolation of salmonellas from sewage-contaminated natural waters was carried out. The effect of pre-enrichment in buffered peptone water was compared with single-step enrichment in NR10 broth incubated at 43C. A modification of NR10 has been compared with the original composition. Bismuth sulphite agar (BSA), Hektoen enteric agar (HE) and brilliant green agar (BGA) have been used as plating media. Other factors considered have been temperature of the water and sampling site. A total of 759 salmonella strains belonging to 36 different serotypes has been recovered in a two-year study. All five factors considered in the study have shown a significant effect on the recovery of salmonellas. The combination of direct enrichment in NR10, followed by BSA or HE as plating media was most effective for the isolation of Salmonella . The influence of water temperature and characteristics of the sampling sites have also been discussed.     

Growth of salmonellas in different enrichment media

The usefulness-of selenite-F (S-F), tetrathionate (MKT) and Rappaport-10 (R-10) broths as enrichment media to support growth of salmonellas either alone or in the presence of other competing organisms was studied. Their ability to support the growth of stressed salmonellas from water was also investigated. It was observed that R-10 was more inhibitory to competing organisms than MKT and S-F. It strongly inhibited the growth of Pseudomonas aeruginosa, Citrobacter freundii and Proteus vulgaris though not of Escherichia coli and Enterobacter aerogenes. It was more toxic, however, to small numbers of salmonellas than MKT and S-F. Tetrathionate was strongly inhibitory for E. coli and Ent. aerogenes but much less so for Proteus and Pseudomonas species. Selenite-F was much less inhibitory than MKT to Ps. aeruginosa and it did not inhibit growth of E. coli and Ent. aerogenes as much as MKT. Salmonellas were inhibited by all three enrichment media and none of them is ideally suited for direct use. Of the three media, R-10 was much more inhibitory to stressed organisms than S-F or MKT.     

Growth of salmonellas in different enrichment media

The usefulness of selenite-F (S-F), tetrathionate (MKT) and Rappaport-10 (R-10) broths as enrichment media to support growth of salmonellas either alone or in the presence of other competing organisms was studied. Their ability to support the growth of stressed salmonellas from water was also investigated. It was observed that R-10 was more inhibitory to competing organisms than MKT and S-F. It strongly inhibited the growth of Pseudomonas aeruginosa, Citrobacter freundii and Proteus vulgaris though not of Escherichia coli and Enterobacter aerogenes. It was more toxic, however, to small numbers of salmonellas than MKT and S-F. Tetrathionate was strongly inhibitory for E. coli and Ent. aerogenes but much less so for Proteus and Pseudomonas species. Selenite-F was much less inhibitory than MKT to Ps. aeruginosa and it did not inhibit growth of E. coli and Ent. aerogenes as much as MKT. Salmonellas were inhibited by all three enrichment media and none of them is ideally suited for direct use. Of the three media, R-10 was much more inhibitory to stressed organisms than S-F or MKT.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42 degrees C) were higher even when MKT (43 degrees C) performance was improved by the use of brilliant green sul-phamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42°C) were higher even when MKT (43°C) performance was improved by the use of brilliant green sulphamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Some modification to the media for rapid automated detection of salmonellas by conductance measurement

A selenite medium for the automated detection of salmonellas by conductance measurements has been modified to eliminate the negative results given by some dulcitol-negative strains. The dulcitol is replaced with mannitol and pre-enrichment is best done in buffered peptone water containing mannitol and dimethylsulphoxide. It is suggested that both versions of the selenite medium be used initially.     

Studies on the Multiplication of Salmonellae in Various Enrichment Media at Different Incubation Temperatures

Several selected Salmonella strains did not multiply in tetrathionate brilliant green bile medium when the inoculum was small and the medium was incubated at 43° C. Gradual heating from 20° to 43° C and dilution of the medium with buffered peptone water (1:10) containing egg yolk did not decrease its inhibitory properties. It became less inhibitory, however, after the growth of other Enterobacteriaceae. These findings stress the advantage of using non-selective pre-enrichment since in that case the Muller-Kauffmann tetrathionate broth will be inoculated with a large number of salmonellae and other Enterobacteriaceae thus facilitating the isolation of the former.     

Dynamics of salmonella isolation with modified Rappaport's medium (R10)

Enhanced growth of salmonellas in Rappaport's medium as modified by Vassiliadis et al. (1976) after pre-enrichment in buffered peptone water during the first 6 h was obtained by replacement of tryptone by soya peptone. The competing bacteria, i.e. those which grow on brilliant green agar and which may interfere with the isolation of salmonellas when Rappaport's medium (R10) incubated at 43°C is used for enrichment were inhibited or reduced in numbers when the normal amount of 5 g soya peptone/litre was used. When the amount was increased to 10 g/l, growth occurred, mainly of Enterobacter and Klebsiella species. The isolation of salmonellas was found to be largely dependent on the number and the ratio of their competitors. Every measure taken to reduce the number of competitive bacteria increases the possibility of isolating salmonellas. This explains the effect of improved selectivity of Rappaport's medium when small inocula are used. Rapid onset of growth of salmonellas by employing soya peptone introduces the possibility of using shorter incubation times than 48 h.     

Importance of pre-enrichment media for isolation of Salmonella spp. from swine and poultry

The performance of two new (1-day) culture methods, Salmonella Enrichment Broth (SEB) and Revive, and an alternative pre-enrichment broth, designated Universal pre-enrichment broth (UB), was compared to the internationally accepted buffered peptone water (BPW). The study was directed towards detection of Salmonella in 100 faecal samples from porcine and 100 neck-skin samples from poultry. The sensitivity (number of positive cases per method among all the positive cases) of the conventional pre-enrichment in BPW was found to be 0.77 for swine and 0.66 for poultry samples, while a combination of the BPW method with parallel pre-enrichment of the same sample in UB resulted in high sensitivity for swine (0.92) and poultry (0.95) samples. A 2-h pre-enrichment in the non-selective Revive, followed by overnight enrichment in selective broth, resulted in a low sensitivity, particularly for the neck-skin samples (0.16, P=0.001). The SEB method in the porcine samples resulted in a sensitivity (0.71) comparable to the standard method (P=0.31). In conclusion, additional pre-enrichment of samples in UB may substantially increase the culture sensitivity. During routine screening of large numbers of samples, it may be advantageous to use SEB rather than standard culturing.     

A Modification of Brilliant Green Agar for Improved Isolation of Salmonella

Five organisms commonly found to be capable of growth on commercial Brilliant Green Agar (BGA) after enrichment in Muller-Kauffman Tetrathionate broth (MKT) were tested for sensitivity to 18 antimicrobial agents. The sensitivities of two Salmonella serotypes to these agents were also tested. A combination of sulphacetamide (at 1.0 mg/ml) and mandelic acid (at 0.25 mg/ml) incorporated into BGA was found to give maximum recovery of salmonellas from MKT broth enrichment whilst giving maximum suppression of contaminating organisms. More importantly, this Antibiotic-enriched Brilliant Green Agar (ABG) gave a lower incidence of false positive results when compared with commercial BGA. Increasing the incubation temperature from 35 to 43°C was found to accentuate the selectivity of ABG without inhibiting the growth of salmonellas. A total of 31 Salmonella serotypes were tested for their ability to grow on ABG at 43°C; all produced typical colonies.     

Improvement in Salmonella detection in milk and dairy products: comparison between the ISO method and the Oxoid SPRINT Salmonella test

The Oxoid SPRINT Salmonella test was compared with the ISO method (ISO 6579: 1993) for the detection of Salmonella in milk and dairy products. Samples were artificially contaminated, in some cases with sublethally injured salmonellas. Experiments with raw milk, soft cheese made from heat-treated milk (mould-ripened and with smear) and soft cheese with smear made from raw milk showed no significant differences between the SPRINT and ISO methods. With dried milk products and mould-ripened soft cheese made from raw milk the reference method gave significantly more positive results. The addition of ferrioxamine E to pre-enrichment (ISO) or pre-enrichment/enrichment broth (SPRINT test) did not improve Salmonella detection.