bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOSENSORS: BLESSING OR BANE?
Authors: GOLDSCHMIDT MILLICENT C.
Published: May 2007

Biosensor technology is changing the methodology used to detect or characterize many microorganisms and/or their metabolites of importance to food microbiologists and the food industry. Biosensors have been developed to monitor the freshness of meat and fish. ATP and glucose concentrations have been monitored as well as continuous control operations in food processing. Enzyme‐substrate transformations, DNA or RNA hybridizations and antibody‐antigen interactions are examples of the types of molecules used in biosensor systems. Instrumentation coupled to the biological molecules and measuring the changes that occur include reactions on simple ion‐sensing electrodes, as well as complex chips, optical fibers or piezoelectric crystals. In most cases, data can be obtained within a few minutes on very small amounts of compounds. However, the long term stability of the biological molecules involved in these procedures presents a major stumbling block. Partially or completely disposable devices are under consideration.
SIMPLE AND RAPID METHODS FOR IDENTIFICATION OF VIRULENT PLASMID‐BEARING CLONES OF YERSINIA ENTEROCOLITICA1
Authors: BHADURI SAUMYA
Published: May 2007

The detection of cold‐tolerant plasmid‐bearing human pathogenic strains of Yersinia enterocolitica depends on the expression of various plasmid‐associated characteristics. However, techniques based on these characteristics have limited reliability and are impractical for diagnostic use. Two rapid, reliable, and simple methods based on the binding of crystal violet and Congo red for identification and differentiation of Y. enterocolitica containing virulent plasmid‐bearing strains are discussed. These dye binding assays can be used to assess the pathogenicity of Y. enterocolitica isolates from a variety of sources for identification of individual plasmid‐bearing clones.
FLUORESCENCE CONCENTRATION IMMUNOASSAY FOR RAPID DETECTION OF CAMPYLOBACTER SPP. IN CHICKEN RINSE WATER
Authors: TSAI HSIU‐CHUAN SONIA , SLAVIK MICHAEL F.
Published: May 2007

A total of 215 freshly processed post‐chill whole chicken carcasses were assessed for Campylobacter spp. contamination by a fluorescence concentration immunoassay (FCIA) procedure. Whole chicken carcasses were sampled with low volume water rinses from which 5 ml portions were enriched with brucella enrichment broth with or without oxyrase supplement in a test tube enrichment system. After a 24h stationary incubation at 42C, each sample was assayed using a FCIA procedure for the presence or absence of campylobacters. The FCIA procedure indicated Campylobacter spp. contamination in 84% of carcasses using oxyrase supplemented enrichment, while only 47% of the chicken carcasses were positive from nonsupplemented enrichment. The corresponding incidence rates detected by culture method were 92% and 87% for oxyrase supplemented and unsupplemented samples, respectively. The FCIA procedure can be completed in less than 1 h with 48 samples including a positive and a negative control assayed on one plate. In summary, the test tube oxyrase‐supplemented stationary enrichment system followed by the use of the FCIA procedure was found to be an effective, rapid method for the detection of Campylobacter spp. in chicken rinse water.
COMPARISON OF RAPID TECHNIQUES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7
Authors: NIROOMAND FAHIEMH , LORD CATHY
Published: May 2007

Four different commercial kits (EHEC‐TEK of Organon Teknika, Durham, NC; HEC O157 of 3M, St. Paul, MN; and Coline dipstick and Coline one‐step of AMP‐COR Inc., Camden, NJ) were evaluated for the detection and recovery of E. coli O157:H7from 75 fresh meat samples and 23 artificially inoculated beef and pork samples. Of the total 75 samples tested, 21 (28%) were presumptive positive by HEC O157 and Coline dipstick, 18 (24%) by Coline one‐step, and 12 (16%) by EHEC‐TEK. None of the presumptive positive samples by any of the methods was confirmed as E. coli O157:H7 (false positive). Of the 23 positive spiked samples tested, 23 were recovered by Coline dipstick and one‐step (100%), 22 (95.6%) by HEC O157, and 20 (86.9) were recovered by EHEC‐TEK. In the confirmation step 17 of the 23 spiked samples produced characteristic colonies on MacConkey sorbitol agar (Difco) with 5‐bromo‐4‐chloro‐3‐indoxy‐β‐D‐glucuronide (Biosynth International) (MSA‐BCIG) and were confirmed as E. coli O157:H7. No characteristic colonies on MSA‐BCIG were detected for 6 of the spiked samples with initial inoculum levels of between 2 to 70 cells/g and, therefore, were not confirmed as E. coli O157:H7. A better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli O157:H7 and provide lower detection levels.
A POLYMYXIN‐CLOTH ENZYME IMMUNOASSAY SYSTEM FOR SALMONELLA O SEROGROUP IDENTIFICATION 4
Authors: WANG HAIYAN , BLAIS BURTON W. , YAMAZAKI HIROSHI
Published: May 2007

A simple method is described for the serological identification of Salmonella O antigens by capture of the lipopolysaccharide antigens on polymyxin‐coated polyester cloth followed by immunoenzymatic detection of the captured O antigens using rabbit Salmonella O antisera and an anti‐rabbit IgG‐peroxidase conjugate. By using a strip of polymyxin‐coated cloth, it was possible to characterize a given isolate with multiple antisera dot blotted on the strip. Conversely, the same format enabled the simultaneous characterization of multiple isolates with a given antiserum on a single strip. This method was much less cumbersome and time‐consuming than the traditional agglutination technique, and utilized very little of the antisera.
USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE
Authors: NIELSEN K. , TSANG R. , D'AOUST J.‐Y. , GARCIA M. , SURUJBALLI O. , HENNING D. , BROOKS B. , KELLY W.
Published: May 2007

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.

The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.

The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.
EFFECT OF ENRICHMENT PROCEDURES ON THE STABILITY OF THE VIRULENCE PLASMID AND VIRULENCE‐ASSOCIATED CHARACTERISTICS IN YERSINIA ENTEROCOLITICA1
Authors: BHADURI SAUMYA
Published: May 2007

This report presents information on the effects of five current enrichment procedures including trypticase soy broth, phosphate buffered saline, peptone enriched phosphate buffered saline, phosphate buffered saline with sorbitol and bile salts and MacConkey broth on the stability of virulence plasmid in Yersinia enterocolitica. Virulence assays using crytal violet binding, low calcium response, Congo red uptake, hydrophobicity by latex particle agglutination, and autoagglutination as well as plasmid DNA analysis showed that Y. enterocolitica did not lose the virulence plasmid during isolation at 28C and the cells remained virulent. Moreover, postenrichment treatment with alkali to reduce competing microflora, when used in conjunction with these five enrichment techniques, did not cause the loss of the virulence plasmid, thus providing a way to selectively isolate this pathogen.
A METHOD FOR ISOLATION OF CHROMOSOMAL AND PLASMID DNA FROM YERSINIA ENTEROCOLITICA FOR SIMULTANEOUS AMPLIFICATION BY POLYMERASE CHAIN REACTION: A POSSIBLE MODEL FOR OTHER BACTERIA
Authors: BHADURI SAUMYA , PICKARD ALLAN R.
Published: May 2007

A commercial DNA isolation kit was evaluated for simultaneous isolation of chromosomal and plasmid DNA from Yersina enterocolitica for polymerase chain reaction amplification. The genomic and plasmid DNA samples obtained by use of the kit were suitable for use in polymerase chain reactions both individually and in multiplex reactions. The results obtained by the use of the kit suggest that this kit may be applied to isolate both genomic and plasmid DNA from other microorganisms for polymerase chain reaction amplification.
A SIMPLE AND RAPID METHOD FOR THE DETECTION AND ENUMERATION OF PHAGES OF BACTEROIDES FRAGILIS HSP40 IN FOODS AND SEWAGE1
Authors: KONE KARIM , FUNG D.Y.C.
Published: May 2007

The phage‐host model of Bacteroides fragilis HSP40 has been documented as a potential index of human sewage and enteroviruses in sewage samples. However, this organism is an obligate anaerobe, which significantly limits the convenience of this model. The objectives of this study were to develop a simple method for the detection and enumeration of phages of B. fragilis HSP40 without using cumbersome anaerobic methods, to compare the novel method to the standard plaque assay (PA), and to enumerate phages naturally occurring in foods and sewage using both methods. Oxyrase enzymeTM (OE) was used at 0.5 U/ml in the growth medium of B. fragilis HSP40 before aerobic incubation. A double tube for phage (DTP) was developed and compared to the standard PA throughout the study. An enrichment procedure for samples was tested using 0.5 U/ml OE. Results indicated that the DTP could detect plaque forming units (PFU) of phages of B. fragilis HSP40 within 12–18h, with a sensitivity of 1 PFU/ml. The need for anaerobic incubation was totally eliminated by using OE and applying the DTP. Phage counts using DTP and PA were highly correlated (R2= 0.99–1.00) in spiked peptone water and ground beef. Enrichment of samples with OE enhanced phage recovery from spiked ground beef. With the DTP and PA, phages of B. fragilis HSP40 were detected from sewage, oysters, mushrooms and chicken. No such phage was detected from fresh ground beef before and after enrichment with either method. The DTP method was simpler than the PA, and it can be recommended for rapid screening of foods for fecal contamination and potential presence of viruses.
MEMBRANE LIFTING TECHNIQUE FOR RAPID DETECTION OF ESCHERICHIA COLI 0157:H7 IN INOCULATED GROUND MEATS
Authors: TSAI HSIU‐CHUAN SONIA , SLAVIK MICHAEL F.
Published: May 2007

A membrane lifting technique was developed for direct rapid detection of Escherichia coli 0157:H7 in inoculated ground meats. Duplicate groups of 2 meatballs were inoculated with volumes of 0.1‐ml of a serial dilution (1:10) of E. coli 0157:H7 or a mixed culture containing one strain of E. coli 0157:H7 and a non‐0157:H7E. coli serotype (E. coli ATCC 25922). Each meatball was sampled by sandwiching with 2 pieces of nitrocellulose membranes and pressing against each other to the center of the meatball. The membranes were in contact with the meats for 10 min to lift the bacteria. The membranes were removed and incubated on MacConkey‐sorbitol agar plates with the meat contact side up. After 18 h incubation at 37C, an immunostain was performed directly on the membranes for detection of the presence or absence of E. coli 0157:H7. This method was found to be sensitive enough to detect as few as 1 to 2 cells of E. coli 0157:H7 inoculated on surfaces of 18‐g meatballs. This method might be used as a rapid presumptive test for E. coli 0157:H7.
GLUCOSE FERMENTATION AND GROWTH OF SELENOMONAS SPUTIGENA ON A MINIMAL MEDIUM
Authors: RICKE S.C. , SCHAEFER D.M.
Published: May 2007

Very little is known about the growth physiology and metabolic niche of the human oral isolate Selenomonas sputigena. The objective of this study was to devise a minimal medium for comparing growth rates and fermentation of rumen Selenomonas ruminantium strains with S. sputigena. When anaerobically grown on a minimal glucose medium containing yeast extract as the only chemically undefined component, S. sputigena produced acetate, propionate, and succinate while S. ruminantium strains produced primarily lactate. When strains were compared (P < 0.05) for each carbon source that yielded growth, rumen strain HD4 grew faster than all other strains on glucose, cellobiose and glycerol while strain GA192 grew faster on trehalose. Rumen strains GA192, PC18, and HD4 grew faster on mannitol than rumen strains D and GA31. S. sputigena grew faster on lactate (0.38 ± 0.04) than any of the S. ruminantium strains. The minimal medium developed in this study should be useful for jurmer physiological studies on fermentation and metabolism in S. sputigena.
RAPID PROCEDURE FOR DETECTING CIGUATOXINS IN FISH
Authors: DIXON LURLENE G. , HAMDY MOSTAFA K. , HAYDEN MARSHA A. , LANCETTE GAYLE A.
Published: May 2007

Flesh and viscera/gill tissues of six amberjacks (Seriola dumerilii), suspected positive for ciguatoxins, were each extracted and the toxins partially purified. Both flesh and viscera/gill of only five fish were toxic to mice exhibiting ciguatoxins (CTX) symptoms. The methanol extracts of the five fish were pooled and concentrated, the volume of flesh extract was 50.0 mL (129.4 mg toxins/mL) and viscera/gill had 25.0 mL (25.5 mg toxins/mL). Pooled extracts exhibited CTX symptoms in mice but only flesh killed mice in 6 h and the LD50 was 1.72 mg toxins. The lethal potencies of the pooled flesh killed mice in 6 h and the LD50 was 1.72 mg toxins. The lethal potencies of the pooled flesh was 198.17 g fish, equivalent to 58.3 mouse unit. An efficient fractionation and purification procedure was developed for the extracts using an HPTLC and silica gel 60 plate with a chromatographic solvent mixture of chloroform:methanol:water (60:35:8, v/v). The system yielded 10 fractions for flesh and 9 for viscera/gill. Scanned plates were subdivided into three equal zones, each scraped, methanol extracted and tested in mice. The 2nd zone (Rf fractions between 0.40 and 0.66) was very toxic to mice compared to 1st or 3rd zones and the mice had CTX symptoms. The scanner for this 2nd zone had a cluster of minor peaks on both sides of the major one with a sum total area of 62.47% indicating multiplicity of CTX in amber‐jack fish. The major peak, at retention time of 1.48 s and a single area of 43.28%, is believed to be the main ciguatoxins present. The HPTLC is a rapid and sensitive procedure for ciguatoxins in fish flesh extracts with a detection limit of 40.0 ± 1.9 picogram toxins.
EVALUATION OF CANDIDA ISOLATION MEDIUM IN FOOD HYGIENE IN HUNGARY
Authors: FUNG DANIEL Y.C. , DEÁK TIBOR , VASAVADA P.C. , PÉTER GÁBOR
Published: May 2007

Aniline Blue containing Yeast and Mold agar, or Candida Isolation Agar (CIA, DIFCO) was evaluated and found successful in presumptive identification of 8 strains of laboratory cultures of Candida albicans based on the ability of C. albicans to grow and give characteristic fluorescence at 365 nm UV light. C. tropicalis (3 strains) was able to grow but did not fluoresce. The CIA medium was also useful for the recovery of C. albicans from inoculated milk and orange juice. The recovery was complete when peptone water was used as the diluent, however, when milk or orange juice were used as the diluent of the respective food, recovery was reduced to about 40%. No C. albicans was recovered in this study from hands of factory workers (dairy, brewery and canning) or from 12 types of foods tested as well as from food processing environment. More samplings are needed to further ascertain the role of C. albicans in Food Microbiology.
DEVELOPMENT AND EVALUATION OF A RAPID ENZYME‐LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME‐LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B
Authors: VALDIVIESO‐GARCIA ALFONSO , SURUJBALLI OM , HABIB KAMAL D. , ABUBAKAR OMAR , BROOKS BRIAN W.
Published: May 2007

An enzyme‐linked immunofiltration assay (ELIFA) and a microtitre plate enzyme‐linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre‐coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.
A COMPARISON STUDY BETWEEN OXYRASE ANAEROBIC AGAR PLATES AND CONVENTIONAL ANAEROBIC CHAMBER FOR THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM CLINICAL INFECTIONS
Authors: THURSTON MURIEL , GANNON COLLEEN K.
Published: May 2007

Misplaced confidence in the broad‐spectrum antibiotics, increased resistance among previously predictable anaerobic antibiograms, and the push to maximize productivity of available space and downsizing trends has created a need for a simplified cost‐effective, and superior method for the isolation and identification of anaerobic bacteria. In this study, the Oxyrase anaerobic plate system which requires no extraneous apparatus to create an anaerobic environment was compared to an anaerobic chamber in the isolation of anaerobic bacteria from 212 consecutive wound specimens. Brucella blood agar and KVL agar plates were used in this comparison study. RapID ANA II, AP120A, special potency disks, and GLC were used for identification. Of the 212 specimens cultured, 87 yielded anaerobic bacteria comprising 182 strains. Thirty‐nine strains failed to grow in the anaerobic chamber but grew on the OxyPlates(TM). These strains were predominantly Peptostreptococcus species (28%), Eubacterium species (20%), and Propionibacterium species (20%). Fourteen strains failed to grow on the OxyPlates, but grew in the anaerobic chamber. No trend was noted and all organisms in this category grew on the OxyPlates from other specimens. In conclusion, the Oxyrases anaerobic plate system appears to be an excellent alternative to the conventional anaerobic chamber in the isolation and identification of clinically significant anaerobes found in human samples, obviating the need for separate anaerobic‐aerobic workstations, expensive anaerobic apparatus, and additional incubator space.
EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES
Authors: BAUTISTA D.A. , KOZUB G. , JERICHO K.W.F. , GRIFFITHS M.W.
Published: May 2007

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm2) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 104, 1 × 105 and 1 × 106 CFU/cm2, there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k‐statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.
DNA AMPLIFICATION USING TABLETED PCR REAGENTS FOR IDENTIFICATION OF ESCHERICHIA COLI O157:H7 ISOLATED FROM FOODS1
Authors: DENG MING Y. , FRATAMICO PINA M. , JENSEN MARK A. , TICE GEORGE
Published: May 2007

A DNA amplification method using tableted multiplex polymerase chain reaction (PCR) reagents was developed for identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples. A suspect colony from MacConkey sorbitol agar containing 5‐bromo‐4‐chloro‐3‐indoxyl‐6‐D‐glucuronide (MSA‐BCIG) was used to perform the PCR. Three different DNA sequences of E. coli O157:H7 were amplified simultaneously in the PCR: a specific fragment of an attaching and effacing gene (eae gene), conserved sequences of Shiga‐like toxins (SLT) I and II, and a fragment of the 60‐MDa plasmid. This method detected all reference strains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, and O157:H, and negative results were obtained with all strains of nontoxigenic E. coli serogroup O157, other serotypes of E. coli, and other bacterial species. The detection limit of the method was 950 colony forming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serological tests were positive in the PCR. After a 6‐h enrichment, EHEC O157:H7 was identified from all experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (mL). This method offers significant advantages in terms of technical simplicity and reproducibility of test results over conventional PCR and other assays for detecting E. coli serotype O157 and should be suitable for use in routine examination of food and other samples for the presence of the pathogen.
ETHYL ALCOHOL REDUCTION OF SALMONELLA TYPHIMURIUM IN POULTRY FEED
Authors: HA S.D. , MACIOROWSKI K.G. , RICKE S.C.
Published: May 2007

This study was designed to evaluate the effect of ethyl alcohol as a potential treatment for reduction of Salmonella populations in poultry feed. Growth rate of S. typhimurium in tryptic soy broth was significantly reduced by addition of greater than 0.3% volume/volume of ethyl alcohol and growth was completely inhibited by addition of 5% ethyl alcohol. Ethyl alcohol concentrations of 20% volume/weight and greater significantly reduced initial S.typhimurium populations in poultry feed (for 20% treated, 2.31 ± 0.31 vs 3.39 ± 0.29 for untreated; P < 0.05). When feed treatment was administered either before or after inoculation of S. typhimurium with 60% ethyl alcohol or 0.04% buffered propionic acid, populations in feeds treated after inoculation were decreased to a nondetection level (< 1.0 log10 CFU/g) by ethyl alcohol treatment but not by other treatments. Ethyl alcohol treatment may have the potential for reducing Salmonella spp. contamination in poultry feed.
AN INDIRECT ENZYME‐LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA
Authors: SURUJBALLI OM P.
Published: May 2007

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra‐centrifugation. The antigen showed little cross‐reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross‐reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.
A COLLABORATIVE STUDY FOR DIRECT ENUMERATION OF LOW LEVELS OF LISTERIA MONOCYTOGENES FROM VARIOUS FOODS
Authors: DALEY E.F. , FARBER J.M. , WARBURTON D.W.
Published: May 2007

A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 102 and 103/g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa‐lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide.
EFFECT OF AN OXYGEN‐REDUCING MEMBRANE FRACTION ON HELICOBACTER PYLORI
Authors: COHEN ALICE , HIRSHFIELD IRVIN
Published: May 2007

The emerging gastric pathogen Helicobacter pylori is an oxygen‐sensitive fastidious microaerophile. Culturability of this organism is rapidly lost in oxygen levels present in the atmosphere due to its morphological transformation into a viable but not culturable state. The effect of the OxyraseTM system of oxygen‐reducing membrane fragments on H. pylori was evaluated at levels ranging from 0.1 to 0.6 Units/mL in Brucella broth supplemented with 5% horse serum. Duplicate sets of OxyraseTM dilutions inoculated with H. pylori were incubated at 35C aerobically and microaerobically. At these OxyraseTM levels, a logarithmic loss of H. pylori viability was evident in the aerobic cultures. The inoculum remained recoverable for 24 h in the presence of OxyraseTM, whereas recovery of inoculum in untreated broth was greatly reduced after 8 h of aerobic incubation, and the organism was unrecoverable after 24 h. OxyraseTM‐containing broth cultures of H. pylori incubated microaerobically showed a similar drop in viable counts for the first 48 h of incubation; however, at the lower levels of OxyraseTM, some cells survived, and resumed logarithmic growth at 96 h. To explore the effects of short term aerobic incubation in the presence of 0, 0.005, 0.05, and 0.5 Units OxyraseTM, cultures were examined microscopically after 4, 8, and 24 h. In the OxyraseTM‐containing broths, <90% of the cells exhibited rod shape morphology after 8 h, whereas in the untreated broth, most cells appeared coccoid. After 24 h, all cells exhibited coccoid morphology.
MONOCLONAL ANTIBODY‐BASED ELISAS FOR THE DETECTION OF BACTERIAL SPORES 1
Authors: QUINLAN JENNIFER J. , FOEGEDING PEGGY M.
Published: May 2007

Abstract Monoclonal antibodies against bacterial spores were used to develop ELISAs to detect a range of spores. ELISAs utilizing anti‐Bacillus spore antibodies detected spores of Bacillus megaterium ATCC 12872, Bacillus stearothermophilus ATCC 7953, Bacillus subtilis var. globigii and Bacillus cereus T. They did not detect Bacillus subtilis A, Bacillus coagulans ATCC 56177 or Bacillus licheniformis ATCC 9789. One ELISA utilizing anti‐Clostridium spore antibodies detected spores of Desulfotomaculum nigrificans ATCC 7946, Clostridium perfringens ATCC 3624 and B. stearothermophilus ATCC 7953, but not spores of Clostridum sporogenes PA3679 or Clostridium botulinum 62A. Whole milk, skim milk, gelatin and starch all interfered to some degree with the abilities of the assays to detect spores. Neither centrifugation nor immunomagnetic bead separation provided sufficient concentration of spores from the food matrix to be applicable to the ELISAs developed.

These results demonstrate the ability to detect a range of spores using monoclonal antibody‐based ELISAs. Such assays may, however, be limited by sensitivity and interference from food components.
THE PULSIFIER®: A NEW INSTRUMENT FOR PREPARING FOOD SUSPENSIONS FOR MICROBIOLOGICAL ANALYSIS 1
Authors: FUNG D.Y.C. , SHARPE A.N. , HART B.C. , LIU Y.
Published: May 2007

Abstract We evaluated a new sample processor, the PULSIFIER®, for preparing food suspensions for microbiological analysis. In a comparative study on 96 samples of representative foods, Total Aerobic Counts (TACs) from “pulsified'’samples equaled or exceeded those from a Stomacher® 400 at levels up to 107 CFU/g, ranging up to 2x higher at low count levels. Except for already comminuted products, pulsified suspensions always contained much less food debris. For example, for 8 replicate samples of celery and carrot, average TACs of pulsified samples were higher (1.3x and 2.5x, respectively) although pulsified suspensions were clear while stomachered suspensions contained enough debris to interfere with pipetting. The superior quality of microbial suspensions from the Pulsifier has implications for general analysis as well as techniques such as PCR and ATP bioluminescence, which are easily perturbed by tissue extracts.
DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)
Authors: MALLE P. , VALLÉ M. , DEMARQUE P. , EB P. , TAILLIEZ R.
Published: May 2007

H2S+ bacteria responsible for the degradation of sulfur‐containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H2S+ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.
APPLICATION OF POLYMERASE CHAIN REACTION‐OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS
Authors: STONE G.G. , CHENGAPPA M.M. , HAYS M.P. , OBERST R.D.
Published: May 2007

We have tested a rapid and sensitive DNA‐based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella‐specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA‐based microtiter plate format. The combined cultivation and PCR‐OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty‐one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR‐OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR‐OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR‐OLA (96.1% specificity). All positive PCR‐OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR‐OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR‐OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.
OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH
Authors: MALLE P. , VALLE M. , EB P. , TAILLIEZ R.
Published: May 2007

H2S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA‐H2S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H2S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H2S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H2S bacteria at the fifth day of storage and 100% at the fifteenth.
COMPARISON OF THE MILLIPORE DIGITAL TOTAL COUNT SYSTEM AND STANDARD MEMBRANE FILTRATION PROCEDURE TO ENUMERATE MICROORGANISMS IN WATER SAMPLES FROM COSMETIC/PHARMACEUTICAL ENVIRONMENTS
Authors: SCALICI C. , SMALLS S. , BLUMBERG S. , ENGLISH D. , JIMENEZ L.
Published: May 2007

A study was performed to compare the Millipore Digital Total Count System with a standard membrane filtration procedure in enumerating the number of microorganisms present in several types of water samples (e.g., Hot/Cold Deionized, Tap, and RO/Ultra Filtration). Water samples were collected over a 4 month period. Statistical data analysis demonstrated an overall correlation of greater than 82% between the two test methodologies. The linearity of the microbial counts between both test methods was compared by artificially contaminating sterile water samples with Pseudomonas aeruginosa. The linearity of the microbial counts between both methods was found to be greater than 96%. The Millipore Digital Total Count System was found to be comparable to the standard membrane filtration method in determining the number of microorganisms in a water sample. In conclusion, the Millipore Digital Total Count System was able to provide a 24 h enumeration of microorganisms present in a water sample. This rapid enumeration allows for a faster quality evaluation of water samples from an industrial water system that is used in the manufacture of cosmetic/pharmaceutical products.
APPLICABILITY OF RAPID METHODS FOR DETECTION OF SALMONELLA SPP. IN POULTRY FEEDS: A REVIEW
Authors: RICKE S.C. , PILLAI S.D. , NORTON R.A. , MACIOROWSKI K.G. , JONES F.T.
Published: May 2007

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.
DETECTION OF TYPE A BOTULINAL TOXIN‐PRODUCING ORGANISMS SUBCULTURED FROM CHEESE USING AN AMPLIFIED ELISA SYSTEM
Authors: FERREIRA JOSEPH L. , CRAWFORD RONALD G.
Published: May 2007

Mascarpone cheese implicated in a botulism outbreak was examined for preformed and cultural botulinal toxins using the mouse bioassay. The cheese was also assayed for cultural toxins and for the most probable number (MPN) of toxin‐producing organisms/g using an amplified ELISA. No preformed botulinal toxins were discovered in the cheese samples (pH range 5.84‐5.86) using the mouse bioassay. However, after cheese subculture in tryptone‐peptone‐glucose‐yeast extract broth, type A botulinal toxin‐producing organisms that formed more than 10,000 MLD (mouse lethal dose)/mL in culture were detected. The ELISA results also revealed that type A toxin was present in the culture with a sensitivity of ∼ 10 MLD/mL. The MPN of type A toxin‐producing organisms/g in 12 cheese samples examined ranged from < 0.3‐9.33. No ELISA cross‐reactivity was noted between the type A toxic cultures and other types (B, E, or F). The ELISA sensitivity was ∼5 MLD/mL casein buffer using purified type A neurotoxin. The advantages of the ELISA test are that the toxin type and approximate lethal dose can be determined within one day compared to the mouse bioassay which takes 3–5 days.
IMMUNOMAGNETIC‐ELECTROCHEMILUMINESCENT DETECTION OF E. COLI O157:H7 IN GROUND BEEF1
Authors: CRAWFORD C.G. , WIJEY C. , FRATAMICO P. , TU S.I. , BREWSTER J.
Published: May 2007

An immunomagnetic electrochemiluminescence approach using a commercially available instrument was developed to detect the presence of E. coli O157:H7 in samples of ground beef. The approach can detect E. coli O157:H7 that have been incurred with 0.05 CFU of the bacterium per gram of beef after an enrichment for 18 h at 37C. Results of comparison tests indicate that the approach is at least 100 times more sensitive than the Reveal dipstick method with a probable false positive rate less than 4–5%. To enhance the adaptability of the approach, a heat treatment was introduced to eliminate the possibility of generating aerosols of live pathogens during the sample loading process. To minimize the carryover of contaminants associated with the capture of E. coli O157:H7 by immuno magnetic beads (IMB), an immuno magnetic separation procedure was developed. The procedure involved the use of a magnetic field to first retain the IMB together with captured bacteria on a paramagnetic column. After washing away the contaminants, the target bacteria were then released from the column by removing the magnetic field. With the proven sensitivity and improved sample treatment procedures, the ECL approach could be considered as an attractive alternative for screening the presence of E. coli O157:H7 in foods.
AGITATION DURING INCUBATION REDUCES THE TIME REQUIRED FOR A LYSINE MICROBIOLOGICAL GROWTH ASSAY USING AN ESCHERICHIA COLI AUXOTROPHIC MUTANT
Authors: LI X. , ERICKSON A.M. , RICKE S.C.
Published: May 2007

Microbiological assays involving Escherichia coli lysine auxotrophs must be optimized to facilitate routine use. Our objectives in this study were to characterize growth of an auxotrophic E. coli lysine mutant (American Type Culture Collection strain #23812) and examine the effect of agitation on E. coli mutant growth. A defined minimal salts basal medium was used and supplemented with various lysine concentrations. The E. coli lysine auxotroph responded to increasing lysine concentration with increasing optical density. When maximum optical density (MOD) was determined for the auxotroph, a linear increase was obtained as lysine concentrations were increased (R2± 0.96) for both agitation and static cultures. Growth rates were not significantly (p > 0.05) affected by lysine concentrations, cultural conditions or their combined effect. However, growth with agitation significantly (p < 0.05) reduced the assay time by shortening the lag phase and causing stationary phase to occur earlier. The values of R2 (± 0.96) relatively remained constant over the range while the bacterial population were in the stationary phase. In conclusion, the lysine growth assay using the E. coli lysine auxotroph can be made more rapid by agitating the culture during incubation.
EVALUATION OF THIN AGAR LAYER METHOD FOR RECOVERY OF COLD‐INJURED FOODBORNE PATHOGENS1
Authors: WU V.C.H. , FUNG D.Y.C. , KANG D.H.
Published: May 2007

The thin agar layer (TAL) method was used to enumerate cold‐injured (‐20C for 2 h and then 25C for 2 h) foodborne pathogens. This method involves overlaying 14 mL of nonselective medium (Tryptic Soy Agar, TSA) onto a prepoured, pathogen‐specific, selective medium. The microbial recovery with the TAL method was compared with the recovery for TSA and pathogen‐specific, selective media. No significant difference occurred between TSA and TAL (P< 0.05) for enumeration of cold‐injured Listeria monocytogenes, Salmonella typhimurium, and Yersinia enterocolitica, and they recovered significantly higher numbers than selective media (P<0.05). No statistical differences occurred among TSA, TAL, and selective media (P>0.05) for recovery of cold‐injured Escherichia coli O157:H7 and Staphyococcus aureus, but the numbers of cells recovered were lower in selective media than TSA or TAL. The TAL method has better recovery capacity than selective media and provides better selectivity than TSA to differentiate target microorganisms.
COMPARISON OF PULSIFIERTM AND STOMACHERTM TO DETACH MICROORGANISMS FROM LEAN MEAT TISSUES1
Authors: KANG DONG‐HYUN , DOUGHERTY RICHARD H. , FUNG DANIEL Y.C.
Published: May 2007

A new sample processor, the PulsifierTM was evaluated for detaching bacteria from lean meat tissue (LMT) and compared with stomacher. In a comparative study on 50 samples of LMT, Total Aerobic Counts (TAC) and coliforms from pulsified samples were not statistically different from stomached samples (R2= 0.971; y = 1.0274x −0.1694 and R2= 0.959; y = 1.0073 ×+ 0.188, respectively). Even though the detaching capacity of microorganisms from LMT was similar between Pulsifier and Stomacher, pulsified suspensions always contained much less meat particles than stomached samples. In the case of stomached samples, many meat particles were present to interfere with even regular pour plating. The superior quality of microbial suspensions from the Pulsifier has implications for general analysis as well as techniques such as ATP bioluminescence, DNA, or enzymatic assay, which may be interfered by tissue extracts.
A SIMPLE MAXIMUM PROBABILITY RESOLUTION ALGORITHM FOR MOST PROBABLE NUMBER ANALYSIS USING MICROSOFT EXCEL1
Authors: IRWIN P. , FORTIS L. , TU S.
Published: May 2007

Traditional most probable number (MPN) methods are executed using one of two schemes. The direct maximum probability resolution (DMPR) technique involves calculating the binomial probability distribution array, P (= IIi Pi; i dilutions), as a function of cell density (A) and finding the value of δ which corresponds to the maximum in P (the MPN). Alternatively, indirect MPR methods seek the solution to a nonlinear equation, related to Pi, by altering δ. We describe herein a simple maximum probability resolution (SMPR) method of the second type which involves the repeated calculation (j cycles) of δj+1 by the addition of a term, related to the partial first derivative of Pi with respect to δ, to δj until the MPN (δfinal) is reached. Using this SMPR algorithm and comparing our results with a DMPR procedure (n = 5, 10, or 96; 10,000 points per Pi), another indirect computer‐based MPR method (n = 10 or 96), or published MPN tables (n = 5) we found that there was agreement to 3–5 significant figures. The SMPR approach also outperformed all other computer‐based MPR techniques tested.
ADAPTATION OF NUCLEIC ACID EXTRACTION METHODS FOR ANIMAL FEEDS
Authors: MACIOROWSKI K.G. , PILLAI S.D. , RICKE S.C.
Published: May 2007

Animal feeds contain a wide variety of microorganisms that may cause animal and human health problems. It would be desirable to rapidly detect as many of these organisms as possible. The objective of this study was the evaluation of nucleic acid extraction protocols to determine an optimal sample processing strategy for DNA isolation from different animal feeds. DNA extraction methods were applied to selected animal feeds to determine which could consistently provide DNA visible by gel electrophoresis. Comparable recovery of DNA extraction protocols was achieved after 20 g of each feed samples were mixed with 0.2% peptone containing 0.01% Triton X‐100. For 9 different feeds visible DNA could be consistently detected for each feed indicating that a detergent‐based dilution step prior to DNA extraction recovered sufficient DNA regardless of the feed matrix.
RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION
Authors: YANG LIJU , RUAN CHUANMIN , LI YANBIN
Published: May 2007

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 103 105 cfu/mL, with R2= 0.99. The detection limit of this method was 1.09 × 103 cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.
USE OF SILICONE GREASE TO AVOID “SMILING EFFECT” IN DENATURING GRADIENT GEL ELECTROPHORESIS
Authors: BRINKHOFF THORSTEN , VAN HANNEN ERIK J.
Published: May 2007

A common problem in DGGE analysis is the distortion of the denaturant gradient in the outer lanes of the gel. The resulting smiling effect, which hampers the analysis of these outer lanes, can simply be avoided by applying silicone grease between the spacers and the glass plates.