Effect of sample preparation and preenrichment media on the recovery of Salmonella from cantaloupes, mangoes, and tomatoes

Studies were conducted to determine the relative effectiveness of buffered peptone water (BPW), lactose (LAC) broth, and Universal Preenrichment (UP) broth for the recovery of Salmonella organisms from fruit rinses, whole fruit, and comminuted fruit. In the first phase, the relative effectiveness of the rinse and soak methods for the recovery of Salmonella from surface-contaminated mangoes and tomatoes was examined. Fruits were spot inoculated with single Salmonella serovars and held for 4 days at 2-6 degrees C before analysis was initiated. The contaminated fruit was rinsed in portions of BPW, LAC broth, or UP broth. Portions from each rinse were added to its respective broth (e.g., BPW to BPW). Individual whole fruit, in their remaining broth rinses (soak method), and the fruit rinse/broths (rinse method) were incubated for 24 h at 35 degrees C. The Bacteriological Analytical Manual (BAM) Salmonella culture method was followed thereafter. The soak method produced significantly greater numbers (P < 0.05) of positive test portions than did the rinse method for the analysis of mangoes (93 versus 12) and tomatoes (85 versus 34). The 3 broths were comparable for the recovery of Salmonella for both the soak and the rinse methods for mangoes. For tomatoes, there were no significant differences among the broths for the soak method, but BPW and UP broth were significantly more productive (P < 0.05) than LAC broth by the rinse method. In the second phase, the relative effectiveness of LAC broth, BPW, and UP broth for the recovery of Salmonella from comminuted fruit was examined. Fruits were contaminated with single Salmonella serovars and aged for 4 days at 2-6 degrees C. Twenty 25 g test portions were preenriched in each of the following broths: BPW, LAC broth, and UP broth. The BAM Salmonella culture method was followed thereafter. For cantaloupes, significantly more (P < 0.05) Salmonella-positive test portions were recovered with UP broth (96 Salmonella-positive test portions) and BPW (87 Salmonella-positive test portions) than with LAC broth (57 Salmonella-positive test portions). For mangoes, BPW recovered an arithmetically larger number of Salmonella-positive test portions (27 Salmonella-positive test portions) than did either LAC broth (14 Salmonella-positive test portions) or UP broth (18 Salmonella-positive test portions). For tomatoes, there were no significant differences among the broths: BPW recovered 65 Salmonella-positive test portions, UP broth recovered 62 Salmonella-positive test portions, and LAC broth recovered 60 Salmonella-positive test portions. For the analysis of whole fruit, it is recommended that the soak method be used. For whole fruit analyzed with the soak method, UP broth should be used for tomatoes and BPW should be used for mangoes. It is further recommended that UP broth be used for the analysis of comminuted cantaloupes and that BPW be used for the analysis of comminuted mangoes and tomatoes.     

The effect of preenrichment and selective enrichment media on recovery of Salmonella Typhi from the tropical fruit mamey

The Bacteriological Analytical Manual (BAM) Salmonella culture method did not detect Salmonella Typhi in mamey, the tropical fruit that was implicated in a 1999 typhoid outbreak. The relative effectiveness of BAM's nonselective preenrichment and selective media for the recovery of S. Typhi from mamey was examined to determine if the BAM's preenrichment/selective enrichment strategy was the cause of the method's failure with this food. The preenrichment media were lactose broth, buffered peptone water (BPW), and universal preenrichment (UP) broth. The selective enrichment media were selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium. UP broth was significantly more effective (P < 0.05) than either lactose broth or BPW for the recovery of 2 different S. Typhi strains from mamey. Of 120 test portions tested, 105 S. Typhi-positive test portions were recovered using UP broth, whereas only 1 S. Typhi-positive test portion was recovered using BPW, and no S. Typhi-positive test portions were recovered using lactose broth. SC and TT broths were both significantly more effective (P < 0.05) than RV medium. Of 105 S. Typhi-positive test portions, SC broth recovered 80, TT broth recovered 67, and RV medium recovered 9. After the above comparison, an incomplete UP (UPI) broth formulation was found to be significantly more effective (P < 0.05) than the complete formulation (UPC). Of 80 total positive test portions, UPI recovered 71, whereas UPC recovered only 48. The following UP broth formulations were compared to determine if any of the components of the UP broth formulation were inhibitory to S. Typhi: UPC, UP broth without sodium pyruvate (UPS), UP broth without ferric ammonium citrate (UPF), UP broth without MgSO4 (UPM), and UPI. It was found that none of the ingredients were inhibitory to S. Typhi and that, out of 140 total test portions, UPI and UPF, with 108 and 103 positive test portions, respectively, were significantly more effective (P < 0.05) than the other UP broth formulations (82 for UPC, 74 for UPS, and 60 for UPM). Rather than being inhibitory to the growth of S. Typhi, it appears that ferric ammonium citrate enhanced the growth of competitors which suppressed the growth of S. Typhi. These results demonstrate that UPI and UPF are effective for the recovery of S. Typhi from mamey.     

Enhanced recovery of Salmonella from apple cider and apple juice with universal preenrichment broth

A comparison was made of the relative efficiencies of Universal Preenrichment (UP) broth and lactose broth for the recovery of a variety of Salmonella serovars from pasteurized and unpasteurized apple cider and pasteurized apple juice. Bulk portions of juice were contaminated with single Salmonella serovars at high and low levels of 0.4 and 0.04 CFU/mL, respectively. The juice was aged for a minimum of 5 days at 2-5 degrees C. On the day analysis was initiated, each of 20 test portions (25 mL) of the contaminated juice was preenriched in UP broth and in lactose broth. The Bacteriological Analytical Manual Salmonella culture method was followed thereafter. For pasteurized apple cider, UP broth recovered significantly (p < 0.05) more Salmonella-positive test portions than did lactose broth (112 and 75, respectively). For unpasteurized apple cider, UP broth recovered significantly more Salmonella-positive test portions than did lactose broth (326 and 221, respectively). For pasteurized apple juice, UP broth recovered more Salmonella-positive test portions than did lactose broth (93 and 81, respectively). However, this difference was not statistically significant. These results indicate that UP broth should replace lactose broth for the analysis of pasteurized and unpasteurized apple cider and pasteurized apple juice.     

Evaluation of TA10 broth for recovery of heat- and freeze-injured Salmonella from beef

The Bacteriological Analytical Manual (BAM) Salmonella pre-enrichment broth [lactose (LAC) broth], buffered peptone water, and universal pre-enrichment (UP) broth were compared with TA10 broth, developed in our laboratory, for recovery of heat- and freeze-injured Salmonella (55 degrees C for 2-20 min and -20 degrees C for 2 months, respectively) from beef. Beef samples were contaminated with single Salmonella serovars, and contamination levels of 0.44 to <0.001 most probable number (MPN)/g and 0.74 to 0.14 MPN/g were used for heat- and freezing-induced injury studies, respectively. Twenty test portions (25 g) of the contaminated beef were pre-enriched in each broth, and the BAM Salmonella culture method was used thereafter. There was a significant difference (chi2 = 7.73) in recovery of heat-injured Salmonella between TA10 broth and LAC broth, 189 (67.5%) versus 156 (55.7%) positive samples, respectively, determined by plating onto selective agars and identification by biochemical tests. For the recovery of freeze-injured Salmonella, there was a significant difference (chi2 = 24.7) between TA10 and LAC broth, 189 (72.7%) versus 133 (51.2%) positive samples, respectively. TA10 broth was more effective than LAC broth and UP broth for recovery of freeze-injured Salmonella. The results indicate that TA10 broth should be used instead of LAC broth for testing of beef that may be contaminated with heat- and freeze-injured Salmonella spp.     

Rappaport-Vassiliadis medium for recovery of Salmonella spp. from low microbial load foods: collaborative study

Twenty-three laboratories participated in a collaborative study to compare the relative effectiveness of Rappaport-Vassiliadis (RV) medium incubated at 42 degrees C, selenite cystine (SC) broth (35 degrees C), and tetrathionate (TT) broth (35 and 43 degrees C) for recovery of Salmonella from the following foods with a low microbial load: dried egg yolk, dry active yeast, ground black pepper, guar gum, and instant nonfat dry milk. For dry active yeast, lauryl tryptose (LT) broth, incubated at 35 degrees C, was used instead of SC broth. All of the foods were artificially inoculated with single Salmonella serovars, that had been lyophilized before inoculation, at high and low target levels of 0.4 and 0.04 colony forming units/g food, respectively. For analysis of 870 test portions, representing all of the foods except yeast, 249 Salmonella-positive test portions were detected by RV medium, 265 by TT broth (43 degrees C), 268 by TT broth (35 degrees C), and 269 by SC broth (35 degrees C). For analysis of 225 test portions of yeast, 79 Salmonella-positive test portions were detected by RV medium, 79 by TT broth (43 degrees C), 84 by TT broth (35 degrees C), and 68 by LT broth (35 degrees C). RV medium was comparable to, or even more effective than, the other selective enrichments for recovery of Salmonella from all of the foods except guar gum. It is recommended that RV (42 degrees C) and TT (35 degrees C) be used with foods that have a low microbial load, except for guar gum for which SC (35 degrees C) and TT (35 degrees C) are recommended.     

Evaluation of culture media for selective enrichment and isolation of Salmonella in seafood

Seafood, including fish, shrimp, clam, crab, mussel, oyster, lobster, squid, octopus, and cuttlefish samples, was used to compare the recovery of Salmonella serovars by different selective enrichment and isolation media. The samples were selectively enriched in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT), followed by selective isolation on Hektoen enteric (HE) agar, xylose lysine desoxycholate (XLD) agar, bismuth sulfite (BS) agar, and Brilliant Green (BG) agar media. Of 443 seafood samples analyzed, 108 were found to be contaminated with Salmonella. The role of selective enrichment in Salmonella spp. recovery with RV medium was distinctly high (70%) compared to TT broth (30%). The selective enrichment in RV broth followed by selective isolation on XLD, HE, BS, and BG agar recovered Salmonella at levels of 56, 41, 28, and 16%, respectively. Similarly, after enrichment in TT broth, XLD and HE agars recovered 27 and 23% respectively. The recovery of Salmonella with enrichment in TT followed by isolation on BS and BG was abysmally low at 4.6 and 5%, respectively. There was no significant difference (P > 0.05) in the recovery of Salmonella using the combinations of XLD and HE media with selective enrichment in RV broth. However, performance difference (P < 0.05) was observed in the recovery when BS and BG with RV, and XLD, HE, BS, and BG agars with TT broth were used. The present study showed that the combination of RV with XLD was the most efficient media for isolation of Salmonella from seafood when compared to other isolation media combinations.     

Salmonella in foods: new enrichment procedure for TECRA Salmonella Visual Immunoassay using a single rv(R10) only, TT only, or dual rv(R10) and TT selective enrichment broths (AOAC official method 998.09): collaborative study

A collaborative study was conducted to compare a new enrichment procedure for the TECRA Salmonella Visual Immunoassay (TSVIA) with the reference method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (7th Ed.). Three food types (milk powder, pepper, and soy flour) were analyzed in Australia and 3 food types (milk chocolate, dried egg, and raw turkey) were analyzed in the United States. Thirty-eight collaborators participated in the study. The TECRA method was evaluated using both Rappaport-Vassiliadis R10 (RV(R10)) and tetrathionate (TT) broths for selective enrichment. M broth cultures arising from each of the 2 selective enrichment broths were tested in the TSVIA using 2 individual wells, one for each selective broth, and a single well to test the pooled selective enrichment broths. The results for the pooled enrichment broths were reported elsewhere. This study presents the results for the use of single enrichment broths, i.e., RV(R10) only or TT only, with the TSVIA. No significant differences (p > 0.05) were observed for the pairwise comparison of the proportion of positive samples for either RV(R10) or TT used as a single enrichment broth for the TSVIA with that for the reference method.     

Evaluation of sample preparation methods for the isolation of Salmonella from alfalfa and mung bean seeds with the Bacteriological Analytical Manual's Salmonella culture method

Five pre-enrichment methods were evaluated for effectiveness with the U.S. Food and Drug Administration's Bacteriological Analytical Manual Salmonella culture method in recovering S. Stanley, S. Poona, and S. Muenchen from artificially contaminated alfalfa seeds, and S. Saintpaul, S. Anatum, and S. Infantis from artificially contaminated mung bean seeds. The methods included: (1) Soak.--Test portions were inoculated into pre-enrichment media; (2) Rinse.--Test portions were rinsed with pre-enrichment media, and the media was decanted from the test portions; (3) Rinsed seed.--Pre-enrichment media was added to the test portions that were rinsed in the rinse method; (4) Wet blend.--Test portions were blended with the pre-enrichment media; and (5) Dry blend.--Test portions were blended prior to pre-enrichment. The methods of pre-enrichment were also evaluated for effectiveness in recovering Pantoea agglomerans from alfalfa and mung bean seeds with a modified culture method for the recovery of Enterobacteriaceae from foods. The purpose of these studies was to provide a model for the recovery of Salmonella that may occur in seeds as a natural contaminant. The relative effectiveness of the soak method was consistently superior to the rinse method in isolating the selected Salmonella serovars from both seed types. Statistically, the rinsed seed method was as effective as the soak method in all trials, except 1 of 3, with S. Muenchen and alfalfa seeds (P > 0.05). The relative effectiveness of the methods in isolating P. agglomerans from alfalfa and mung bean seeds was similar to that observed with the artificially contaminated test portions. The soak method was consistently the most effective method and the rinse method was consistently the most ineffective method. The rinsed seed, wet blend, and dry blend methods were also as effective as the soak method in all 3 trials with each seed type (P > 0.05).     

Validation study to demonstrate the equivalence of a minor modification (TECRA ULTIMA protocol) to AOAC Method 998.09 (TECRA Salmonella Visual Immunoassay) with the cultural reference method

The TECRA Salmonella Visual Immunoassay (VIA) using Rappaport-Vassiliadis RV[R10] as a single selective enrichment broth has Final Action approval (AOAC Method 998.09). TECRA has recently developed a protocol (TECRA ULTIMA), which involves the addition of a new additive to a 1 mL aliquot of the RV[R10] broth, prior to the heat-killing step, thereby allowing the RV[R10] broth to be tested directly in the kit and thus eliminating the need for the 2 h post-enrichment in M broth. An in-house validation study was conducted to compare the modified AOAC Method 998.09 to the reference culture method. Three foods were used in the study: Naturally contaminated raw ground poultry at high (10-50 cells/25 g), and low (1-5 cells/25 g) levels; and milk powder and peanut butter, artificially inoculated at low and high levels with Salmonella bovismorbificans and S. enterica Mbandaka, respectively. Twenty test portions were analyzed for each level with 10 uninoculated control samples per food. Overall, no significant differences (p <0.05) were observed when the proportion of positive test portions for the modified VIA were compared with that for the reference method. This minor modification, which employs the additive (provided in the TECRA ULTIMA SALMONELLA Test Kit) to permit the direct analysis of RV[R10] broth has demonstrated the utility of the TECRA ULTIMA SALMONELLA protocol. It is recommended that the minor modification to Method 998.09 be approved First Action as an additional option within the method.     

Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the latter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth).     

Comparison of assurance gold salmonella EIA, BAX for screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays for detection of Salmonella in alfalfa sprouts and sprout irrigation water

The Assurance Gold Salmonella EIA, BAX for Screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays were compared with official cultural methods described in the Bacteriological Analytical Manual (BAM) for analysis of alfalfa sprouts and sprout irrigation water for the presence of Salmonella. The lower limits of detection of 4 serovars of Salmonella cells (S. tennessee, S. muenchen, S. mbandanka, and S. cubana) in pure culture were determined as approximately log10 2, 5, and 6 for the BAX, GENE-TRAK, and Gold EIA, respectively. Despite its low detection limit, the BAX did not perform as well as the other assays in analyzing contaminated sprouts and sprout irrigation water. For 4 different lots of sprouts and sprout irrigation water samples inoculated with the 4 serovars at low [1-2 colony forming units (CFU/g)] and high (68-180 CFU/g) levels, the BAX detected Salmonella in 58/64 (90.6%) of the samples, compared with 64/64 (100%) by the GENE-TRAK, Gold EIA, and BAM methods. Assay performance was also compared for analysis of naturally contaminated sprouts and sprout irrigation water with 3 lots of alfalfa sprouted seeds associated with different salmonellosis outbreaks. Positive assay results for the naturally contaminated samples were Gold EIA 41, GENE-TRAK 36, BAM 33, and BAX 13.     

Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.     

Method extension study to validate applicability of AOAC Official Method 996.14 Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Assurance Listeria Polyclonal Enzyme Immunoassay (EIA) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 23 collaborators, representing government agencies, as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 550 test portions and controls was analyzed and confirmed, of which 207 were positive and 336 were negative by both methods. Six test portions were positive by culture, but negative by the EIA. Three test portions were negative by culture, but positive by the EIA. Two test portions were negative by EIA and by culture, but confirmed positive when EIA enrichment broths were subcultured to selective agars. The data reported here indicate that the Assurance Listeria EIA method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

Method extension study to validate applicability of AOAC Official Method 997.03 visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Visual Immunoprecipitate assay (VIP) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 27 laboratories, representing government agencies as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 615 test portions and controls was analyzed and confirmed, of which 227 were positive and 378 were negative by both methods. Nine test portions were positive by culture, but negative by the VIP. Five test portions were negative by culture, but positive by the VIP. Four test portions were negative by VIP and by culture, but confirmed positive when VIP enrichment broths were subcultured to selective agars. The data reported here indicate that the VIP method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of VIDAS Salmonella (SLM) immunoassay method with Rappaport-Vassiliadis (RV) medium for detection of Salmonella in foods: collaborative study

A collaborative study was conducted to compare the VIDAS Salmonella (SLM) with Rappaport-Vassiliadis (RV) method for detection of Salmonella in foods to the current standard method presented in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the culture method presented in AOAC's Official Methods of Analysis. The VIDAS SLM with RV method uses tetrathionate broth in combination with RV medium in place of selenite cystine broth for selective enrichment, thereby eliminating the hazardous waste issue for laboratories. Twenty five laboratories participated in the evaluation, each testing one or more of 8 test products: nonfat dry milk, dried egg, soy flour, lactic casein, milk chocolate, raw ground pork, raw ground turkey, and raw peeled shrimp. Results of the study showed no significant differences in the numbers of confirmed positive samples with the VIDAS SLM with RV procedure and the BAM/AOAC culture procedure. The VIDAS SLM with RV method was effective for rapid detection of Salmonella in foods. It is recommended that AOAC INTERNATIONAL modify the VIDAS Salmonella SLM procedure to include the RV method.     

Evaluation of the use of liquid dishwashing compounds to control bacteria in kitchen sponges

A test procedure for evaluating the effect of adding commercial liquid hand dishwashing detergents to kitchen sponges to control microbial growth is described. Claims for this type of application are being made on dishwashing detergents throughout the world. In this evaluation, commercially available kitchen sponges were stripped of antimicrobial compounds. Sponges were then inoculated with a pool of 7 microorganisms which consisted of gram positives, gram negatives, and yeast. Inoculated sponges were treated with the detergent as recommended by the manufacturer and allowed to incubate for 16 h at ambient temperature. Surviving microorganisms were then quantitated using either the spiral or pour plate method. Tests were run using both clean sponges and sponges soiled with 0.5% nonfat dry milk (NFDM). Untreated sponges showed stasis or slightly increased bacterial populations after the incubation period in the absence of NFDM. Significant increases of up to 3 log cfu/mL were observed for untreated sponges when soiled with NFDM. Statistically significant reductions were observed for clean sponges (99.8-99.9998%) and sponges soiled with NFDM (87.6-99.9%) when detergents making "antibacterial sponge" claims were added to the inoculated sponges. Statistically significant differences between detergents making "antibacterial sponge" claims were also observed.     

New role of antibody in bacterial isolation

To eliminate the interference caused by Pseudomonas aeruginosa in the isolation of Salmonella, a rabbit polyclonal antibody against P. aeruginosa was prepared by inoculating four New Zealand rabbits with the pathogen. The antiserum was purified using saturated ammonium sulfate and added into Rappaport-Vassiliadis medium with soya (RVS) broth and Muller-Kauffmann tetrathionate novobiocin broth (MKTTn broth) to evaluate whether it could inhibit the growth of P. aeruginosa. Observations by scanning electron microscopy demonstrated that P. aeruginosa was attacked and destroyed by the antibody when incubated for 10 min at 37 degrees C. The activity of the antibody was also effective against 11 other strains of P. aeruginosa. Twenty-six strains of Salmonella were mixed with P. aeruginosa in RVS and MKTTn broth at 37 degrees C for 12 h, respectively, and the cultures were plated on Salmonella chromogenic medium (SCM; Oxoid, Basingstoke, UK). Only Salmonella grew on SCM; five colonies were randomly selected for identification by VITEK 2 (bioMérieux, Lyon, France). Additionally, when mixed with two strains of Enterobacter cloacae (ATCC 700323 and YG001), the prepared antibody did not affect the growth of E. cloacae. The results demonstrated that the microbicidal activity of the antibody did not affect the tested Salmonella sp. or E. cloacae strains. Therefore, the antibody generated could be used to increase the accuracy of Salmonella isolation.     

Singlepath Salmonella. Performance-Tested Method 060401

Singlepath Salmonella is an immunochromatographic (lateral flow) assay for the presumptive qualitative detection of Salmonella spp. in food. The AOAC Performance-Tested Method study evaluated Singlepath Salmonella as an effective method for the detection of Salmonella spp. in the following selected foods: dried skimmed milk, black pepper, dried pet food, desiccated coconut, cooked peeled frozen prawns, raw ground beef, and raw ground turkey. When the foods were inoculated with Salmonella spp. at levels ranging from low [0.23-1.08 colony forming units (CFU)/25 g] to high (2.3-6.0 CFU/25 g), a Chi-square value of 0.9 indicated that there was no significant difference between Singlepath Salmonella and the ISO 6579:2002 reference method. Singlepath Salmonella gave a false-positive rate of 7.3% and a false-negative rate of 2.5%. For the inclusivity study, all 105 Salmonella serovars reacted with Singlepath Salmonella. For the exclusivity study, 58 non-Salmonella spp. were tested. There were no cross-reactions with Singlepath Salmonella from these strains.     

Determination of beta-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection

This paper describes a novel and sensitive pre-column derivatisation method for the detection and quantitation of beta-lactams and their biosynthetic precursors at trace levels in fermentation media. Filtered broths from fermentations of strains of Penicillium chrysogenum and Cephalosporium acremonium, after deproteination and centrifugation, were incubated with 9-fluorenylmethylchloroformate for 5 min at 20 degrees C in 0.2 M borate buffer at pH 7.7. Following two-fold pentane extraction of the reagent hydrolysis product, the aqueous layer was injected directly onto a C18 reversed-phase column, and products were detected spectrofluorimetrically with excitation and emission wavelengths of 260 and 313 nm, respectively. Detection limits of 0.01 and 0.05 micrograms ml-1 were achieved for both 6-aminopenicillanic acid (6-APA) and isopenicillin N in borate buffer and filtered fermentation broths, respectively, using a 10-microliter injection volume. A linear calibration for 6-APA in fermentation broth was obtained for a very wide concentration range (0.05-100 micrograms ml-1). Detection limits for solutions of cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C in broth were all 0.25 micrograms ml-1. The detection limit for the beta-lactam precursor delta-(L-aminoadipyl)-L-alpha-cysteinyl-D-valine (ACV) dimer in borate buffer was 0.5 microgram ml-1. The cephalosporins and ACV dimer gave linear plots in the ranges 3-25 and 1-100 micrograms ml-1, respectively. Repeated analysis of 6-APA at a concentration of 10 micrograms ml-1 in filtered broth gave a mean peak area of 2.5.10(6) with a standard deviation of 2.6.10(5) using a 10-microliter injection volume. Ampicillin spiked into deproteinated blood serum gave a linear calibration in the concentration range 2-100 micrograms ml-1.