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.     

Should the AOAC use-dilution method be continued for regulatory purposes?

Despite its very poor reproducibility, AOAC INTERNATIONAL's use-dilution method (UDM) for bactericidal activity (AOAC Methods 964.02, 955.14, and 955.15) has been required by the U.S. Environmental Protection Agency (EPA) since 1953 for regulatory purposes, while methods with better reproducibility have been adopted in Canada and Australia. This study reviews UDM from a statistical perspective. Additionally, the test's expected results were compared to those obtained from actual evaluation of several formulations. Significant gaps have been identified in the reproducibility of the test data as predicted by statistical analysis and those presented to the EPA for product registration. UDM's poor reproducibility, along with its qualitative nature, requires the concentration of the active ingredient to be high enough to ensure all or most carriers to be free of any viable organisms. This is not in accord with the current trends towards sustainability, human safety, and environmental protection. It is recommended that the use of the method for regulatory purposes be phased out as soon as possible, and methods with better design and reproducibility be adopted instead.     

Matching reference materials with AOAC International methods of analysis

Proper implementation and use of validated analytical methodology with use of appropriate reference materials (RM) is a preferred means of helping to ensure equivalent analytical method performance in diverse laboratories. Choice of an appropriate RM that not only matches the analyte and matrix of the required determination, but also has been demonstrated to be within the applicability of a specific analytical method, are key factors. In response to numerous requests since its founding in 1993, the Technical Division on Reference Materials (TDRM), AOAC International is implementing a program for recognizing the matching of specific reference materials to specific AOAC methods of analysis. This recognition is accomplished by means of a thorough peer-reviewed selection system, under the auspices of the AOAC official methods board and the executive committee of the TDRM. Potential RM/method matching (RM/MM) proposals will be submitted to an RM/MM committee. After technical review of the suitability of the proposed RM by the RM/MM committee, acceptable matches are recommended for review by the current AOAC process responsible for review and recognition of new methods and modifications to existing AOAC methods of analysis. Several trial matches have been used to develop and test this system. The end product of this effort will ultimately be made available as either a stand-alone document, a section of the AOAC Official Methods of Analysis, or a site within the AOAC web site listing recognized matches.     

Simultaneous determination of p-phenylenediamines and aminophenols in hair dye products: interlaboratory study

An isocratic HPLC method routinely used in the National Laboratory for Food and Drug Analysis of Taiwan was validated for the simultaneous determination of six aminophenols and phenylenediamines in commercial hair dyes. After extraction of the commercial hair dye product, the dye intermediates were determined by HPLC. Recoveries from the extraction were between 91.6 and 96.5%. The method was then evaluated in an interlaboratory collaborative study according to AOAC guidelines. Five laboratories in Taiwan participated in the study that analyzed the test product, which was preanalyzed by two laboratories to ensure acceptable homogeneity. The RSD(r) and RSD(R) values of the measurements obtained for the dye intermediates in the product were < or = 3.75 and < or = 5.95%, respectively. The method demonstrated acceptable reproducibility, as evidenced by HorRat values of 0.82- 0.97. The applicability of the method to the determination of oxidative hair dye components was further demonstrated in analyses of two different products. The method is thus proposed to be used by manufacturers and laboratories to evaluate the quality of commercial hair dyes containing the six aminophenols and phenylenediamines.     

Studies of polyester fiber as carrier for microbes in a quantitative test method for disinfectants

Tests were conducted by a Task Force on Disinfectant Test Methods that was appointed to investigate controversies regarding the accuracy of AOAC test methods for disinfectants as presented in AOAC's Official Methods of Analysis, Chapter 6. The general principles for new and improved AOAC tests are discussed, and a disinfectant test using microbes labeled onto a polyester fiber surface is described. The quantitative test measures the survival of test microbes as a function of exposure time as well as the exposure conditions required to kill 6 log10 of the test microbes. The time required was similar to that for the kinetics of the kill of Bacillus subtilis-labeled cylinders as tested by methods of the AOAC Sporicidal Test 966.04.     

In-house validation study for salmonella unique testing of juice (modification of AOAC official method 2000.07)

Following an industry request, a study was undertaken to validate a minor change to the Unique method for testing fruit juice. Twenty foods were tested in the original precollaborative study for TECRA Unique Salmonella test (2000.07). To validate the modification for juice, both the modified method (42 degrees C module incubation with a 5 h replication step) and the current AOAC Method 2000.07 (37 degrees C incubation with a 4 h replication step) were compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM; 8th Ed., 1998) reference method, which uses lactose broth as pre-enrichment medium. Twenty uninoculated replicates, 20 replicates with low-level inoculum (target 1-5 cells/25 g), and 20 replicates with high-level inoculum (target 10-50 cells/25 g) were tested for a single batch of fresh orange juice in accordance with AOAC requirements. There was exact agreement between the 2 Unique methods for all samples and exact agreement between the 2 Unique methods and the BAM method for the uninoculated and high-level inoculum samples. For low-level inoculum, 17 samples were confirmed positive with the new Unique method, 17 with AOAC Method 2000.07, and 14 with the BAM method.     

Reveal for Salmonella test system.

The Reveal for Salmonella (RSS) test system is a presumptive qualitative test that detects the presence of Salmonella organisms in foods within 21 h total testing time, allowing the user to release negative products 24 h earlier than when using other rapid test kits. Foods are enriched with a proprietary resuscitation medium called Revive and then selectively enriched with either Selenite Cystine or Rappaport-Vassiliadis selective media. The enriched culture is used to inoculate the RSS detection device, which initiates a lateral flow through a reagent zone containing anti-Salmonella antibodies conjugated to colloidal gold particles that capture antigens present in the culture. The antigen-antibody complex migrates farther and is captured by an additional anti-Salmonella antibody, causing the colloidal gold to precipitate and form a visual line, indicating a positive result. A procedural control line also will form regardless of the presence of Salmonella organisms to indicate the test is working properly. Existing AOAC Official Methods for Salmonella organisms require a 48 h enrichment before testing. Hence, a food product has to be held before release, adding extra cost to the company and the consumer. The RSS test system was evaluated by quantitative spiking studies. Although AOAC encourages inclusion of naturally contaminated foods, almost all microbiological AOAC validation studies have been performed with artificially contaminated foods for absolute control over the study. The RSS test system is designed to test many food types for Salmonella organisms and has a limit of detection of 5-10 colony-forming units (cfu)/25 g with a false-negative rate of < 1% and a false-positive rate of < 5.0%. It showed an 81% overall agreement with the traditional procedure of the U.S. Department of Agriculture's Food Safety Inspection Service.     

Efficacy testing of disinfectants

The U.S. Environmental Protection Agency's (EPA) Office of Pesticide Programs (OPP) has the responsibility for regulating antimicrobial products, including sporicides, used to treat and decontaminate inanimate surfaces. In response to the anthrax (Bacillus anthracis) attacks of 2001 and the associated need for verifying the performance of chemicals for building decontamination, the EPA initiated research in late 2003 to evaluate and improve efficacy test methods for sporicides. The OPP Microbiology Laboratory located at the Environmental Science Center, Ft. Meade, MD is the lead laboratory. Through funding provided by EPA's Office of Research and Development (Safe Buildings Program), a collaborative research plan has been established to address several key issues. Research is currently being conducted on 2 fronts: (1) the evaluation of quantitative methodology for assessing the efficacy of sporicides, and (2) the development and comparative testing of selected modifications to improve the AOAC Sporicidal Activity Test (AOAC Method 966.04). Future studies will include the evaluation of candidate surrogates of B. anthracis using a quantitative method, and a multilaboratory validation study of a quantitative method-surrogate combination. The General Referee is serving as the Principal Investigator for all research described in this report, and has the overall responsibility for the technical conduct of the projects. In cases where the General Referee has oversight of projects that involve official collaborative studies and validation support from AOAC INTERNATIONAL, AOAC officials and the Committee Chair will determine the appropriate mechanism for formal study review. The 2003 General Referee report provides the background on the development and direction of the research projects. The preliminary data, general conclusions, next steps, and recommendations are provided in this report.     

Quantitative assessment of hard surface disinfectant activity against the foodborne pathogen Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen that must be controlled to ensure food safety. For the years 2003 and 2004, L. monocytogenes caused 20 deaths per 100 listeriosis cases and was responsible for most food recalls for pathogen contamination. The objective of this work was to develop a quantitative method to assess disinfectant activity against L. monocytogenes. Standard procedures for testing disinfectants against 3 bacteria are described in the AOAC Official Methods of Analysis as use-dilution methods. No standard methods are provided for L. monocytogenes. In this study, preliminary efficacy of a quaternary ammonium compound with hydroperoxide ion was determined for 25 bacterial strains. The zones of inhibition ranged from 7.0 to 12.5 mm, and the minimum inhibitory concentration ranged from 5 to 250 ppm. For final efficacy, stainless steel carriers were contaminated with L. monocytogenes and tested separately for 5, 10, and 15 min in disinfectant or phenol. After exposure, the carriers were placed into 2 series of D/E neutralization broth. For 3 replications with duplicate samples, the phenol coefficient was 3.3. This research presents a technique-sensitive method that provides quantitative data for comparison and analysis of disinfectant activity against L. monocytogenes.     

Enumeration procedure for monitoring test microbe populations on inoculated carriers in AOAC use-dilution methods

The AOAC Use-Dilution methods do not provide procedures to enumerate the test microbe on stainless steel carriers (penicylinders) or guidance on the expected target populations of the test microbe (i.e., a performance standard). This report describes the procedures used by the U.S. Environmental Protection Agency to enumerate the test microbe (carrier counts) associated with conducting the Use-Dilution method with Staphylococcus aureus (Method 955.15) and Pseudomonas aeruginosa (Method 964.02) and the examination of historical data. The carrier count procedure involves the random selection of carriers, shearing bacterial cells from the carrier surface through sonication, and plating of serially diluted inoculum on trypticase soy agar. For each Use-Dilution test conducted, the official AOAC method was strictly followed for carrier preparation, culture initiation, test culture preparation, and carrier inoculation steps. Carrier count data from 78 Use-Dilution tests conducted over a 6-year period were compiled and analyzed. A mean carrier count of 6.6 logs (approximately 4.0 x 10(6) colony-forming units/carrier) was calculated for both S. aureus and P. aeruginosa. Of the mean values, 95% fell within +/- 2 repeatability standard deviations. The enumeration procedure and target carrier counts are desirable for standardizing the Use-Dilution methods, increasing their reproducibility, and ensuring the quality of the data.     

Performance of three pneumatic probe samplers and four analytical methods used to estimate aflatoxins in bulk cottonseed

The requirement by the U.S. Food and Drug Administration that agricultural products susceptible to aflatoxin contamination contain aflatoxin at levels < or =20 parts per billion for consumer-ready products has led to the establishment of inspection programs by various industries. In Arizona, cottonseed samples from 100 ton piles are collected by an accumulation of 3 or more probings with a pneumatic probe. When sampling compacted cottonseed piles, the large official pneumatic probe (7.6 x 127 cm) decreases in efficiency. Two smaller probes (1.9 x 127 cm and 1.9 x 254 cm ) were therefore developed and tested for their suitability for sampling cottonseed piles. Three rapid analytical methods (one thin-layer chromatographic and 2 immunochemical) were tested for suitability as on-site assay systems. An analysis of variance of the analytical test results showed no differences between the various probes tested. Of the rapid methods, however, only the AflaTest-P immunoaffinity column gave results similar to those of the official AOAC thin-layer chromatography method. In terms of safety, however, all methods prevent material contaminated above regulatory limits from reaching the consumer.     

Determination of insoluble, soluble, and total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study

A method for the determination of insoluble (IDF), soluble (SDF), and total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official Methods 985.29, 991.43, 2001.03, and 2002.02, the method quantitates water-insoluble and water-soluble dietary fiber. This method extends the capabilities of the previously adopted AOAC Official Method 2009.01, Total Dietary Fiber in Foods, Enzymatic-Gravimetric-Liquid Chromatographic Method, applicable to plant material, foods, and food ingredients consistent with CODEX Definition 2009, including naturally occurring, isolated, modified, and synthetic polymers meeting that definition. The method was evaluated through an AOAC/AACC collaborative study. Twenty-two laboratories participated, with 19 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 10.45 to 29.90%. Digestion of samples under the conditions of AOAC 2002.02 followed by the isolation, fractionation, and gravimetric procedures of AOAC 985.29 (and its extensions 991.42 and 993.19) and 991.43 results in quantitation of IDF and soluble dietary fiber that precipitates (SDFP). The filtrate from the quantitation of water-alcohol-insoluble dietary fiber is concentrated, deionized, concentrated again, and analyzed by LC to determine the SDF that remains soluble (SDFS), i.e., all dietary fiber polymers of degree of polymerization = 3 and higher, consisting primarily, but not exclusively, of oligosaccharides. SDF is calculated as the sum of SDFP and SDFS. TDF is calculated as the sum of IDF and SDF. The within-laboratory variability, repeatability SD (Sr), for IDF ranged from 0.13 to 0.71, and the between-laboratory variability, reproducibility SD (SR), for IDF ranged from 0.42 to 2.24. The within-laboratory variability Sr for SDF ranged from 0.28 to 1.03, and the between-laboratory variability SR for SDF ranged from 0.85 to 1.66. The within-laboratory variability Sr for TDF ranged from 0.47 to 1.41, and the between-laboratory variability SR for TDF ranged from 0.95 to 3.14. This is comparable to other official and approved dietary fiber methods, and the method is recommended for adoption as Official First Action.     

Microbiological assay-trienzyme procedure for total folates in cereals and cereal foods: collaborative study

In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. A microbiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSD(R)) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11-20%. RSD(R) values were higher (22.7-52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSD(R) ranged from 1.8 to 11.2% for 8 fortified products. RSD(R) values were higher (27.9-28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.     

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

Modification to the AOAC Sporicidal Activity of Disinfectants Test (Method 966.04): collaborative study

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 microg/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 microg/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCI resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCI resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.     

Precollaborative study of the GeneQuence Salmonella assay using 24-hour enrichment protocols for detection of Salmonella spp. in select foods

New enrichment protocols are described for use with a DNA hybridization (DNAH) method for detection of Salmonella spp. in select foods. GeneQuence Salmonella, in its original version, utilized a 3-stage enrichment of minimum 42 h duration. New 2-stage procedures of 24-28 h duration are described for raw poultry, raw beef, pasteurized egg products, milk chocolate, and dry pet food. In the validation study described here, a total of 345 samples were tested by the abbreviated DNAH method in parallel with either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedures. Results showed an overall sensitivity for the DNAH method of 97.1% (false-negative rate 2.9%). There were no false-positive results by the DNAH method; therefore the specificity was 100%. Overall agreement between the DNAH and reference culture methods was 98.5%. There were no significant differences in performance between the DNAH and reference methods for any of the foods tested as determined by Chi-square analysis. It is recommended that the DNAH method be subjected to AOAC collaborative study.     

Determination of vitamin A (retinol) in infant and medical nutritional formulas with AOAC method 992.06 using a modified extraction procedure: single-laboratory validation

The applicability of AOAC Official Method 992.06, vitamin A (retinol) in milk-based infant formula can be extended to specialty infant formulas, and medical and adult nutritional products with a few minor modifications to the sample preparation procedure. Currently, AOAC Official Method 992.06 is only applicable to milk-based infant formulas containing >500 IU vitamin A per reconstituted quart. When this method is used as written to test specialty infant formulas, vitamin A recoveries are low compared to results generated with alternate validated vitamin A methods. AOAC Method 992.06 vitamin A recoveries can be improved significantly in specialty infant formulas if the amount of potassium hydroxide used during the saponification step is doubled. With this one minor modification to the sample preparation procedure, AOAC Method 992.06 demonstrates acceptable precision and accuracy for the quantitation of vitamin A (retinol) in specialty infant formulas, milk- and soy-based infant formulas, and adult and medical nutritionals. Because increasing the amount of potassium hydroxide can cause emulsions to form, 2-4 mL aliquots of reagent alcohol may need to be added to some samples to separate the organic and aqueous layers during the extraction step. A single-laboratory validation of these modifications was completed. During validation, 15 different product matrixes were analyzed. The intermediate precision averaged 2.70% RSD, and spike recovery data averaged 96.3%.     

Antimicrobial Thiol–ene–acrylate Photosensitive Resins for DLP 3D Printing

Designing digital light processing (DLP) 3D printable photosensitive resins with antibacterial properties is especially vital because of their potential applications in various biomedical fields. In this contribution, a thiol–ene–acrylate ternary system with reduced volume shrinkage and fast photopolymerization rate was chosen as the antibacterial 3D printing matrix resin. Two quaternary ammonium salt‐type antibacterial agents (QAC and SH‐QAC) with different molecular weight were designed and prepared, which can participate in the curing of matrix resin to achieve contact antibacterial effect. The effects of antibacterial agent content on the photopolymerization kinetics and on thermal and mechanical properties were discussed in detail. When the amount of added QAC is 4wt%, the antibacterial rate is almost 100% for Escherichia coli and Staphylococcus aureus, and when the amount of SH‐QAC is 10wt%, the antibacterial rate against S. aureus is also essentially 100%. Both antibacterial photosensitive resins have been successfully applied in DLP technology to fabricate tooth model with high precision.     

AOAC method 966.04: preliminary evaluation of cooked meat medium with manganese sulfate for the cultivation of Clostridium sporogenes: precollaborative study

AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. For regulatory purposes, Method 966.04 is accepted by the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) for the generation of product performance data for sporicides and sterilants. In this study, we report on findings associated with proposed improvements (modifications) to the Clostridium component of the method. Egg meat medium (EMM), the culture medium for C. sporogenes currently specified in the method, is no longer commercially available and finding a suitable replacement is critical. In addition, the use of a nonstandardized extract of raw soil as an amendment to EMM, as stipulated in the current method, may result in a highly variable spore suspension. The primary focus of this study was to find replacements for EMM and soil extract. A carrier count procedure, the establishment of target carrier counts (spores/carrier), and a neutralization confirmation procedure were also evaluated. The study was limited to liquid products tested against Clostridium on a hard surface carrier (porcelain penicylinder). Spore suspensions of C. sporogenes were generated using: (1) EMM with soil extract (EMM/SE), (2) cooked meat medium with soil extract (CMM/SE), and (3) cooked meat medium with 5 microg/mL manganese sulfate (CMM/MnSO4). The titer of the spore suspension, carrier counts, resistance to hydrochloric acid (HCI), and efficacy against 3 liquid sporicidal agents were used to evaluate the potential of CMM and MnSO4 as replacements. The study was performed by the EPA Office of Pesticide Programs Microbiology Laboratory, Fort Meade, MD. Use of CMM/SE and CMM/MnSO4 resulted in comparable results for titer of spore suspensions (approximately 10(8) spores/mL) and carrier counts (approximately 3 x 10(6) spores/carrier). The carrier counts for the EMM/SE were approximately 1 log lower than CMM-based treatments; however, no attempt was made to dilute the CMM spore suspensions prior to carrier inoculation to reduce the carrier counts for CMM. Resistance of spores to 2.5 M HCI was acceptable across the 3 media types. Treatments for comparative efficacy testing were designed to provide a range of sporicidal activity, i.e., high and low efficacy treatments. Sodium hypochlorite (bleach), hydrogen peroxide/peracetic acid, and glutaraldehyde were used as test chemicals. The number of carriers resulting in growth (positive) for the low treatments for all 3 chemicals ranged from 9 to 59 out of 60 across the 3 media types--EMM exhibited fewer positives overall. The high efficacy treatments for sodium hypochlorite and hydrogen peroxide/peracetic acid yielded a range of 0 to 2 positives out of 60 across the 3 media. However, the high glutaraldehyde treatment generated 3, 20, and 20 positives out of 60 for the EMM/SE, CMM/SE, and CMM/MnSO4, respectively. The lower number of positive carriers for EMM/SE may be due to the reduced carrier counts. CMM, either with SE or MnSO4, appears to be a suitable replacement for EMM/SE. On the basis of the results of this study, the Study Director recommends that CMM/MnSO4 and the spore enumeration target carrier count and neutralization procedures be considered for collaborative study to officially modify the Clostridium x porcelain component of Method 966.04.     

Validation study of a rapid ELISA for detection of histamine in tuna

Neogen Corp. has developed an improved Veratox histamine test kit for the detection of histamine in tuna tissue. The purpose of this study was to validate the method under the requirements of AOAC Research Institute (RI) Performance Tested Methods. Three AOAC Official Methods for histamine (954.04, 957.07, and 977.13) and one ELISA method have been performance tested by the AOAC RI. The most popular is AOAC Official Method 977.13, the fluorometric method, which is considered the reference method, but is complicated and time-consuming. The need for a rapid ELISA test kit to be validated by the AOAC RI exists.