Inhibitory Action of Various Plating Media on the Growth of Certain Salmonella Serotypes

The growth of 68 strains of Salmonella typhi , 697 other Salmonella strains and 213 strains of other Gram negative intestinal bacteria on 8 plating media was assessed semi-quantitatively. These media were found to be differentially inhibitory to different Salmonella serotypes. The combined use of two plating media, brilliant green MacConkey agar and deoxycholate citrate agar, allowed potentially the recovery of the maximum number of Salmonella strains. If only one medium was used, brilliant green MacConkey agar would appear to be the best plating medium for the isolation of non-typhoid salmonellas in general and S. choleraesuis in particular. Xylose lysine deoxycholate agar, on which a certain proportion of salmonellas failed to yield typical, recognizable colonies, was found not to be a good plating medium for their isolation.     

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.     

A study of isolation procedures for multiple infections of Salmonella and Arizona in a wild marsupial, the quokka (Setonix brachyurus)

Rectal swabs and faeces were used in the regular sampling for salmonellas and Arizonas from a heavily-infected population of a marsupial, the quokka ( Setonix brachyurus ). The media used were strontium selenite A and strontium chloride B enrichment broths, with subculture onto modified bismuth sulphite agar and deoxycholate citrate agar. A study of sampling, enrichment, sub-culture and colony selection procedures produced an optimal scheme giving high yields but consistent with reasonable economy of time and materials. A three-swab sample was taken and inoculated into the two enrichment media, and with each enrichment subjected to three subcultures. The absolute efficiency of this procedure was greater than 80% (and confirmed by a serological method), compared with only 67% for a single swab in a single enrichment. Recovery of some serotypes depended on the media used; e.g. Arizonas could not be recovered satisfactorily from strontium chloride B enrichment. Faeces samples were found to be greatly superior to rectal swabs for detecting salmonellas and arizonas but they were less convenient in field studies. In a comparison of rectal swabs and faeces samples where the actual concentration of salmonellas was known, it was found that the efficiency of rectal swabs approached 100% if there were more than 10³ salmonellas/g faeces, but this declined to approximately 50% if there were 10²-10³ salmonellas/g faeces and only 25% if there were less than 10² salmonellas/g faeces. A new statistical procedure was introduced for comparing the number of isolations from two methods, and this should be of use in similar methodological studies.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10⁸/ml or more. During incubation in TBB at 43C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10³–10⁷/ml in BPw and after transfer to TBB slowly reached 10⁴/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10(8)/ml or more. During incubation in TBB at 43 degrees C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10(3)-10(7)/ml in BPw and after transfer to TBB slowly reached 10(4)/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Evaluation of different plating media used in the isolation of salmonellas from environmental samples

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24h incubation in any of the media, but after 48h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes.
Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 μg/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.     

Evaluation of different plating media used in the isolation of salmonellas from environmental samples

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24 h incubation in any of the media, but after 48 h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes. Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 micrograms/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.     

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.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat.

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Observations on Pre-enrichment for Isolating Salmonellas from Sewage Polluted Natural Water Using Muller-Kauffmann Tetrathionate Broth Prepared with Fresh and Desiccated Ox Bile

Desiccated bile (Oxoid L50) was substituted for fresh bile obtained from the abattoir in Muller-Kauffmann Tetrathionate Broth. Two versions of the enrichment medium were prepared, one with desiccated bile the other with fresh bile. The efficiency of these two media was compared using 25 ml quantities of sewage polluted natural water as inocula. Direct enrichment and preenrichment techniques were used. With direct enrichment the Muller-Kauffmann Tetrathionate Broth prepared from fresh bile was more efficient for salmonella isolation. With the pre-enrichment technique using preliminary culture of the water sample in buffered peptone water prior to enrichment, there was no significant difference between the efficiency of the two versions of tetrathionate. Comparison of direct enrichment with the pre-enrichment method for isolating salmonellas from sewage polluted water clearly demonstrated the advantages of pre-enrichment. This would certainly have a bearing on the quantitative estimation of salmonellas in polluted water and might increase the number of serotypes isolated from a sample divided into subsamples.     

Methods for isolating Campylobacter jejuni from low-turbidity water.

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

Methods for isolating Campylobacter jejuni from low-turbidity water

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

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.     

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.     

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 degrees 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 48 h.