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.     

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.     

Thermal Injury and Recovery of Salmonella typhimurium and Its Effect on Enumeration Procedures

Exposure of Salmonella typhimurium 7136 to sublethal heating produced a temporary change in the tolerance of the organism to a particular stress medium. After sublethal heat treatment at 48 C for 30 min, greater than 90% of the viable population was unable to reproduce on Levine Eosin Methylene Blue Agar containing 2% NaCl. This sensitivity was dependent on the pH of the heating menstruum. In addition, the heated cells displayed a sensitivity to Brilliant Green Agar, Levine Eosin Methylene Blue Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. Unheated cells displayed a sensitivity to Brilliant Green Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. When the injured cells were placed in a suitable medium (Trypticase Soy Broth), they recovered and grew at a rate equal to that of normal cells. Recovery was also possible in Nutrient Broth, Lactose Broth, and Lauryl Tryptose Broth. Although recovery of the injured cell occurred in Tetrathionate Broth and Selenite F Broth, they were less than ideal growth media for the organism.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.     

Loss of Viability and Metabolic Injury of Staphylococcus aureus Resulting from Storage at 1°, 3°, 5° and 7°C

Staphylococcus aureus incubated in Tryptic Soy Broth at 1°, 3°, 5° and 7°C became increasingly sensitive to Mannitol Salt Agar. Injury, as measured by salt sensitivity, decreased with increasing temperature from 1° to 7°C.     

Isolation and Enumeration of Listeria monocytogenes from Sewage, Sewage Sludge and River Water

Listeria monocytogenes in sewage, sewage sludge and river water was isolated by enrichment at 4°C with subculture and enrichment in thiocyanate, naladixic acid broth and plating on to Tryptose Agar. The results indicated that L. monocytogenes is present in sewage and sewage sludge in considerable numbers and that this organism survives longer than Salmonella spp. on land sprayed with sewage sludge.     

The microflora of soak water during natural fermentation of coarsely ground chickpea (Cicer arietinum) seeds

K. KATSABOXAKIS AND K. MALLIDIS. 1996. The microflora of soak water was studied during natural fermentation of coarsely ground chickpea seeds at 32°, 37° and 42°C. The soak water was slightly salted with NaCl 0.5% (w/v) and its proportion to sample was 10 : 1 (v/w). A gas-producing Clostridium species isolated from Reinforced Clostridium Agar (RCA) plates incubated anaerobically, was found to dominate this fermentation system particularly at the higher temperatures. Gram-negative bacteria and yeasts were not found. Bacillus species were isolated from Plate Count Agar (PCA) plates and Lactobacillus, Corynebacterium, Micrococcus and Pediococcus spp. from RCA plates were incubated aerobically. Butyric was the main acid produced at 37° and 42°C and CO₂ with hydrogen were identified as main fermentation gases.     

Isolation of Salmonellae from Sewage with a New Procedure of Enrichment

Forty samples of sewage on Moore's swabs were examined for the presence of salmonellae. They were first pre-enriched in buffered peptone water. From each pre-enrichment, three enrichments were made: (1) in a new, considerably modified, formula of Rappaport medium (R 10) incubated at 43 °C (R 10/43 °C), (2) in the usual formula (R25) of the same medium at 37 °C (R25/37 °C) and (3) in Muller-Kauffmann's tetrathionate broth at 43 °C (MK/43 °C). Practically the same numbers of swabs were found positive by the first two enrichment procedures, 38 and 39 respectively, while only 17 were found positive by the MK procedure. The R10/43 °C method was superior to the two other procedures; it yielded 103 strains of salmonellae as against 82 with the second Rappaport procedure, and only 25 with the MK/43 °C technique. A similar observation was made concerning the frequency of isolation of different serotypes by the three procedures; the number of the isolated serotypes was 24, 19 and 11, respectively. The new R 10/43 °C method of enrichment had also a much stronger inhibitory effect on the competing bacteria than the two other procedures of enrichment used.     

Isolation of Salmonella from environmental samples collected in the reptile department of Antwerp Zoo using different selective methods

AIMS: To evaluate the environmental spread of Salmonella strains in the reptile department of Antwerp Zoo and to compare different isolation methods for Salmonella. METHODS AND RESULTS: One hundred environmental samples were collected in the service sections and public spaces of the reptile department. After pre-enrichment in buffered peptone water (BPW), selective enrichment was performed in Rappaport Vassiliadis Single Component Enrichment Broth (RVS), Selenite Cystine Broth (SEL) and Mueller Kauffman Tetrathionate Broth (MKTTn). Subculturing on Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, and the combined use of immunomagnetic separation (IMS) and RVS was evaluated. The isolation media used were Hektoen Enteric Agar (HE), Phenol Red Brilliant Green Agar (BG) and Xylose Lysine Decarboxylase Agar (XLD). Salmonella strains were found in 47 samples (47.0%). Most isolations were made on HE after combined IMS/RVS enrichment. Sixty-six Salmonella strains were serotyped, 29 belonged to Salmonella enterica ssp. enterica (I), 3 to ssp. salamae (II), 29 to ssp. arizonae or diarizonae (IIIa/b), 4 to ssp. houtenae (IV) and 1 strain showed autoagglutination. In addition, a 10-year survey (1995-2004) of Salmonella serovars isolated from reptiles at Antwerp Zoo is presented. CONCLUSIONS: A high prevalence of Salmonella strains was noted in the service sections of the reptile department. Only a few isolations were made in the public spaces. Selective enrichment in RVS was the most efficient. In combination with IMS, this method gave an even higher isolation rate than the International Standard method (ISO 6579:2002). SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the importance of reptiles as spreaders of Salmonella in their surroundings. The possible infectious risks for zoo personnel and visitors are evaluated. Improved laboratory protocols for the isolation of Salmonella from the environment are suggested.     

Rapid Quantitative Method for Salmonella Detection in Polluted Waters

A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 +/- 1 hr), plating on Brilliant Green Sulfa Agar (20 +/- 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 +/- 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges.     

Elevated-Temperature Technique for the Isolation of Salmonella from Streams

With a modified Moore swab for sampling bacteria, Salmonella organisms were recovered consistently from surface waters when the enrichment broths of SBG-sulfa and tetrathionate and Brilliant Green Agar plates were incubated at 41.5 C. When an equal portion of the same swab sample was incubated at 37.0 C, no salmonellae were detected. By use of the elevated-temperature technique, salmonellae were recovered from stream sites having relatively low total coliform densities of 2,200 per 100 ml and fecal coliform densities of 220 per 100 ml. These pathogens were isolated in water sampled as far as 73 river miles and 4 days time of travel downstream from the source of pollution under an ice cover that averaged 2.5 ft (76.2 cm) in thickness.     

Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics

Aims:  To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells.
Methods:  Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates.
Results:  Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37°C; on SDA plates, one fungal colony was found at 30°C and one at 37°C. The SDA agar plate incubated at 30°C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h.
Conclusions:  As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points.
Significance and Impact of the Study:  This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.     

Sensitivity and Specificity of Different Methods for the Isolation of Salmonella from Pigs

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.     

Effect of Storage at 1° and 4°C on Viability and Injury of Staphylococcus aureus, Escherichia coli and Streptococcus faecalis

Staphylococcus aureus and Escherichia coli incubated at 1° or 4°C became increasingly sensitive to Mannitol Salt Agar and Violet Red Bile Agar respectively. The increased sensitivity was most marked with exponential phase cultures. Streptococcus faecalis was refractory to similar treatments. The viability of all 3 organisms on selective and non-selective media is reported.     

New Method of Isolating Salmonellae from Milk

The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations.     

Influence of Incubation Temperature and Sodium Heptadecyl Sulfate (Tergitol No. 7) on the Isolation of Salmonellae from Pork Sausage

Cultures of 68 samples of fresh pork sausage purchased locally were incubated at 37 and 43 C, with and without Tergitol No. 7 (sodium heptadecyl sulfate) added to the tetrathionate-Brilliant Green enrichment broth. The results indicated an advantage in incubating the tetrathionate broth at 43 C rather than 37 C in attempting to isolate salmonellae from pork sausage. Without Tergitol, more samples were positive at 43 C than at 37 C, but with Tergitol there was no difference. The higher temperature suppressed the competing gram-negative bacteria and permitted Salmonella to grow in relatively pure culture, thus providing an advantage for isolating and identifying the organisms. Tergitol dispersed and emulsified the fat which improved the isolation of Salmonella when the cultures were incubated at 37 C but not at 43 C. Brilliant Green-sulfadiazine agar was superior to bismuth sulfite agar for isolating salmonellae from tetrathionate broth cultures of fresh pork sausage.     

The effect of sewage sludge treatment processes on oocysts of Cryptosporidium parvum

T.N. WHITMORE AND L.J. ROBERTSON. 1995. The effect of common sewage sludge treatment processes on oocysts of the coccidian protozoan Cryptosporidium was evaluated in laboratory simulations. The ability of primary sewage sedimentation to remove Cryptosporidium oocysts was found to be poor. Thermophilic (55°C) aerobic digestion and sludge pasteurization at the same temperature were found to be effective treatments to inactivate Cryptosporidium oocysts. Approximately 10% of the oocyst population were found to be viable after 18 d exposure to mesophilic (35°C) anaerobically digesting sludge. The viability of Cryptosporidium oocysts decreased within the range 20–40% in sludge-treated soil mesocosms over 30 d. The survival results obtained, however, indicated that oocysts would survive well beyond this period.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.     

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective-diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.