Evaluation of enrichment and plating media for isolating Listeria monocytogenes

We compared selective enrichment broths used by the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service, for their efficiency in the quantitative recovery of Listeria monocytogenes from a naturally contaminated Brie cheese that was obtained as part of an epidemic investigation. Quantitative recovery of Listeria in FDA broth (greater than 2.4 x 10(5) colony forming units/mL) was significantly better than recovery in USDA broth (9.3 x 10(3) colony forming units/mL). When USDA broth was supplemented with D-glucose and Phytone (papaic digest of soy protein), its recovery efficiency improved but did not equal that of FDA broth for isolating L. monocytogenes from Brie cheese. A comparison of 4 selective plating media [modified McBride's agar, gum base nalidixic acid agar, lithium chloride-phenylethanol-moxalactam agar (LPM), and acriflavine-ceftazidime agar (AC)] showed that 3 L. monocytogenes strains belonging to serotype 1/2a were partially or completely inhibited on LPM and AC agars. One strain of serotype 1/2a formed microcolonies on modified McBride's agar after 48 h of incubation.     

Identification and enumeration of beta-hemolytic Listeria monocytogenes in naturally contaminated dairy products

A DNA probe was used to identify hemolytic Listeria monocytogenes in naturally contaminated dairy products: unpasteurized milk, ricotta cheese, and imported semisoft cheeses. Of 34 milk samples, 12 were suspected to contain hemolytic L. monocytogenes; 1 contained greater than 6000 viable organisms/g. The ricotta cheese, although temperature-abused, had a titer of 3.6 x 10(6) beta-hemolytic L. monocytogenes cells/g, whereas the semisoft cheeses reached a maximum of 5.6 x 10(6) cells/g. Pure cultures of L. monocytogenes isolated from both types of cheese were found positive by the CAMP test and the DNA probe.     

Direct plating method for enumeration of Staphylococcus aureus: collaborative study.

Considerable evidence has been published regarding the adverse effect of sodium chloride on physiologically impaired cells of Staphylococcus aureus, such as are to be expected in processed foods. A direct plating method for enumeration of S. aureus eliminating the use of sodium chloride was devised and subjected to collaborative study by 16 analysts. Results obtained by the direct plating method were compared to those obtained by the AOAC official first action method (46.036--46.040). Participating analysts examined duplicate samples at population levels of 91, 34, and 20 S. aureus/g. Coefficients of variation among analysts were considerably lower for the direct plating method (31, 81, and 48%, respectively) than for method 46.040 (59, 156, and 150%, respectively) at all 3 population levels. High coefficients of variation for the direct plating method at 2 of the 3 levels were due principally to low populations of S. aureus. The direct plating method has been adopted as official first action for general purpose use and use of method 46.036--46.040 has been restricted to raw food ingredients and nonprocessed foods.     

Comparative studies of nucleic acid hybridization assay for Listeria in foods

A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a 32P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in a filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.     

Isolation and identification of Listeria monocytogenes in dairy products

After an outbreak of listeriosis in Massachusetts in 1983, the ability of Listeria monocytogenes to survive in raw and pasteurized milk was investigated. An enrichment broth (EB) containing acriflavine, nalidixic acid, and cycloheximide was used to eliminate overgrowth of the culture by competing organisms, and a modification of McBride's agar (MMA) was used as the isolation medium. The culture was incubated 24 h at 30 degrees C. To isolate Listeria from soft cheese, the incubation period was lengthened to 1 week, and the EB culture was streaked to MMA at 1 and 7 days. Physical and biochemical patterns, the CAMP test, serological tests, and mouse pathogenicity studies were helpful in determining the identity of L. monocytogenes.     

Dry rehydratable film for enumeration of total aerobic bacteria in foods: collaborative study

A rehydratable dry-film plating procedure for aerobic plate counts has been compared to the standard agar plate method (966.23B and C, 15th ed.; 46.014-46.015, 14th ed.) in a collaborative study by 12 laboratories. Each laboratory analyzed the normal microflora of 3 samples in duplicate for 6 products. The aerobic plate counts ranged from 1.0 x 10(3) to 1.0 x 10(8) cfu/g. The products were flour, nuts, frozen raw shrimp, spice, frozen raw ground turkey, and frozen and refrigerated vegetables. Repeatability standard deviations of the 2 methods did not differ significantly for 13 of 18 test samples. For 1 shrimp and 2 turkey samples, the dry-film method had lower repeatability variances (P less than 0.05) and for 1 spice sample the agar method had lower repeatability variances (P less than 0.05). Relative standard deviations of repeatability were between 1.7 and 15.5% for the dry-film method and 1.2 and 16.0% for the agar method. Relative standard deviations of reproducibility ranged from 2.4 to 23.4% for the dry-film method and 2.3 to 18.8% for the agar method. The dry rehydratable film method has been adopted official first action for determination of the aerobic plate count.     

Dry rehydratable film for enumeration of total coliforms and Escherichia coli in foods: collaborative study

Rehydratable dry-film plating methods for total coliforms and Escherichia coli in foods have been compared to the AOAC most probable number methods. Fourteen laboratories participated in the collaborative study. Three coliform and E. coli levels in 6 samples of 4 product types (flour, nuts, cheese, and beef with gravy) and in 3 samples of 2 product types (mushrooms and raw turkey) were tested in duplicate by the participants. The mean log counts for the 3 methods were comparable. In general, the repeatability and reproducibility variances of the plating methods were as good as or better than that of the MPN method. The method has been adopted official first action by AOAC.     

Most probable number method for isolation and enumeration of Staphylococcus aureus in foods: collaborative study

Enumeration of Staphylococcus aureus in foods was collaboratively studied by comparing the present AOAC final action method, 46.062, which uses trypticase soy broth with 10% NaCl to a proposed replacement method which uses the same broth with 1% sodium pyruvate added. Fifteen collaborators analyzed uninoculated samples of milk, tuna salad, and ground turkey, as well as samples inoculated with low (10(2) cells/g), middle (10(4) cells/g), and high (10(6) cells/g) levels of S. aureus. The samples were frozen immediately to maintain the inoculated level of S. aureus in the food. A different strain of S. aureus was used for each food; heat-stressed S. aureus cells were used to inoculate the milk samples. The pyruvate-amended broth significantly (alpha = 0.05) increased enumeration of low, middle, and high levels of S. aureus from milk and ground turkey, and from tuna salad at middle and high levels. The pyruvate-amended media method has been adopted official first action to replace method 46.062.     

叶类蔬菜中单核细胞增生李斯特氏菌PCR快速检测方法的建立

为建立一种无需样品前增菌即可快速检测叶类蔬菜中单核细胞增生李斯特氏菌的聚合酶链式反应(PCR)方法,本试验将β-环糊精和牛乳蛋白包被活性炭结合用于去除叶类蔬菜基质中的PCR反应抑制因子,从而促进该菌的回收,随后以iap为靶基因,进行PCR检测,确定该方法的特异性、灵敏度和实用性,并评估该方法所用主要试剂在冷冻条件下的稳定性。结果表明,与prfA序列相比,该方法检测78株目标菌和63株非目标菌后,无假阳性或假阴性结果,特异性为100%;该方法无需前增菌,可在4 h内完成检测,灵敏度为10¹ CFU·25g^-1;该方法与常规培养法在实际叶类蔬菜样品中目标菌的检出率、活体菌检测率均一致(符合率100%);所用的主要试剂在-20℃冰箱中保存至少12个月后仍可正常使用,稳定性较好。综上,该方法可快速、灵敏、特异地检测叶类蔬菜中的单核细胞增生李斯特氏菌,且实用性较好。本研究结果为降低单核细胞增生李斯特氏菌对消费者造成的安全风险提供了技术支持。     

Collaborative study of an improved method for the enumeration and confirmation of Clostridium perfringens in foods.

A collaborative study was conducted in 10 laboratories to evaluate the performance of a new method for the enumeration of vegetative cells of Clostridium perfringens in foods. Results obtained by the new method were compared with results from the official first action method, 46.049-46.053. Per cent recoveries of 4 C. perfringens strains from inoculated roast beef samples were higher and more consistent in tryptose-sulfite-cycloserine (TSC) agar with or without added egg yolk than in sulfite-polymyxin-sulfadiazine (SPS) agar, specified in the official first action method. The confirmatory technique utilized in the new method was also found to be more reliable than the technique described in the official first action method. Based on the collaborative results, the new method with TSC agar for enumeration and a modified motility-nitrate medium together with a lactose-gelatin medium for confirmation of C. perfringens has been adopted as official first action to replace 46.049-46.053.     

Direct plating versus dilution plating in qualitatively determining the mold flora of dried beans and soybeans

Two methods presently used for examining whole foods and feeds for viable molds were evaluated for their relative effectiveness in the qualitative determination of the total number of mold species present in soybeans and dried beans: the direct plating method and the serial dilution method. Sixty-nine soybean samples and 40 dried bean samples were examined. Although the quantitative results of this study were inconclusive, the qualitative results show that the direct plating method was substantially more effective in detecting individual mold species. An average of 12.9 and 10.9 species was detected by the direct plating method in whole soybean and dried bean samples, respectively. An average of 4.4 and 2.8 species was detected by the dilution method in ground soybean and dried bean samples, respectively. A total of at least 37 mold species were found in the study, including 10 toxicogenic species. With few exceptions, detection rates of the 37 individual species were substantially greater among the samples examined by direct plating than those examined by serial dilution.     

Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry

A method was developed specifically to detect naturally occurring Listeria monocytogenes in meat because the traditional cold enrichment procedure was extremely slow and other procedures were ineffective. This method could identify beta-hemolytic Listeria colonies in 3-4 days. The use of a 2-stage enrichment, highly selective LPM agar, and a thin-layer horse blood agar plate for the detection of beta-hemolytic Listeria isolates are the important steps of this method. L. monocytogenes was recovered from 20 of 41 samples of frozen ground beef, 12 of 23 samples of pork sausage, and 7 of 22 samples of poultry. These results indicate that L. monocytogenes is common in raw meat and that this method is effective for its recovery.     

Rapid fluorogenic enumeration of Escherichia coli in selected, naturally contaminated high moisture foods

An assay for the enzyme glucuronidase was used to determine the presence of Escherichia coli in selected, naturally contaminated high moisture foods. Raw pork sausage, ground turkey, and ground beef were inoculated into tubes containing the substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) in lauryl tryptose (LT) medium. After incubation at 35 degrees C for 24 h, the inoculated LT-MUG tubes were examined under longwave ultraviolet light for the presence of a fluorogenic glucuronidase end product. A fluorescing tube indicated the presumptive presence of E. coli. The 10 day most probable number method of the AOAC and the LT-MUG procedure gave comparable recoveries of E. coli.     

Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study

Twenty-four laboratories participated in a collaborative study to validate a hydrophobic grid membrane filter (HGMF) method incorporating the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) for enumeration of total coliform and Escherichia coli bacteria in foods by comparing its performance against the AOAC 3-tube MPN method (46.013-46.016). Raw milk, raw ground poultry, whole egg powder, cheese powder, and ground black pepper were included in the study. The total coliform methods did not differ significantly, except that the 3-tube method detected a significantly higher level of total coliforms than did the HGMF method in the ground black pepper. Conversely, the HGMF/MUG E. coli method detected significantly higher numbers of E. coli present in the egg powder, cheese powder, and ground black pepper samples, while not differing significantly from the 3-tube method for the raw milk and raw ground poultry samples. The overall confirmation rate of MUG-positive colonies isolated using the HGMF method was 99.5%. The hydrophobic grid membrane filter/MUG method has been adopted official first action as an additional method to AOAC official final action method 46.030-46.034.     

Efficiency of two commercial ELISA kits compared with the BAM culture method for detecting Listeria in naturally contaminated foods.

The efficiency of 2 commercial enzyme-linked immunosorbent assay (ELISA) kits (Listeria-Tek and Tecra) for detecting Listeria in naturally contaminated foods was evaluated and compared with that of the culture method described in the Bacteriological Analytical Manual (BAM). Both ELISAs use modified University of Vermont (UVM-1) medium as a primary enrichment; the BAM method uses Listeria enrichment broth. Secondary enrichments for Listeria-Tek and Tecra, respectively, were Fraser broth and UVM-2, which contains additional acriflavin-HCl. When ELISA test results differed, secondary enrichments were tested against the other ELISA; Fraser broth was used to determine recovery rates because of its superiority over UVM-2. Of the 178 food samples examined, the presence of Listeria was detected and culturally confirmed in 38, 37, and 40 samples by the BAM, Listeria-Tek, and Tecra methods, respectively. Differences in results of the ELISAs compared with those of the BAM method were not statistically significant; however, differences between results of the 2 ELISA methods were significant. It was concluded that as rapid screening methods, the Listeria-Tek and the Tecra kits qualify as alternative methods to the BAM cultural method.     

Discrimination of intact and injured Listeria monocytogenes by Fourier transform infrared spectroscopy and principal component analysis

Fourier transform infrared spectroscopy (FT-IR, 4000-600 cm(-)(1)) was used to discriminate between intact and sonication-injured Listeria monocytogenes ATCC 19114 and to distinguish this strain from other selected Listeria strains (L. innocua ATCC 51742, L. innocua ATCC 33090, and L. monocytogenes ATCC 7644). FT-IR vibrational overtone and combination bands from mid-IR active components of intact and injured bacterial cells produced distinctive "fingerprints" at wavenumbers between 1500 and 800 cm(-)(1). Spectral data were analyzed by principal component analysis. Clear segregations of different intact and injured strains of Listeria were observed, suggesting that FT-IR can detect biochemical differences between intact and injured bacterial cells. This technique may provide a tool for the rapid assessment of cell viability and thereby the control of foodborne pathogens.     

State programs for pesticide residues in foods

Two U.S. data collection and dissemination programs, FEEDCON and FOODCONTAM, are described. FEEDCON provides information on contamination levels in animal feeds of toxic chemical residues (pesticides, industrial chemicals, heavy metals, mycotoxins, natural plant toxins, salmonella, and therapeutic drug cross-contaminations). FEEDCON data are collected from approximately 40 state feed regulatory agencies, feed manufacturers, and related groups who subscribe ($100-$200 per year) to the program, which is sponsored by the Association of American Feed Control Officials. FOODCONTAM provides similar information, but is limited to pesticides, heavy metals and industrial chemicals (polychlorinated and polybrominated biphenyls, etc.) in human foods. Both programs have been developed and initiated under U.S. Food and Drug Administration contracts with the Mississippi State Chemical Laboratory. Program structures of both are outlined conceptually, and FOODCONTAM is described in detail. FOODCONTAM data-sharing program development is essentially complete, but expansion by incorporating FDA data with State Laboratory data is nearing reality.