Evaluation of Plastic Multi-Well Plates for Serological Screening of Salmonella Cultures with Spicer-Edwards Pooled Antisera

We compared the relative advantages of using glass test tubes and plastic multi-well plates in the serological identification of Salmonella cultures by the Spicer-Edwards method, and we conclude that the advantages of multi-well plates outweigh those of test tubes.     

Three-Tube Method for Screening Stool Cultures for Salmonella and Shigella

A three-tube method developed is described as a screening test for non-lactose-fermenting organisms isolated from stool cultures. To evaluate the method, 976 strains of gram-negative bacilli were tested. All strains of Salmonella and Shigella were correctly identified generically.     

Accelerated Procedure for Salmonella Detection in Dried Foods and Feeds Involving Only Broth Cultures and Serological Reactions

A procedure has been developed in which Salmonella can be detected in dried foods and feeds within 50 hr. This includes preenrichment (18 hr), selective enrichment (24 hr), elective enrichment (6 to 8 hr), and serological testing (2 hr). The procedure is as sensitive as the more time-consuming, traditional (plating, biochemical, and serological) procedure which may require at least 4 to 5 days. The new procedure is rapid and accurate in comparison to traditional procedures. Moreover, it is simple and inexpensive to perform. As described, the procedure does not necessitate the isolation of pure cultures, but this step is performed easily if desired.     

Detection of Nuclease Activity in Semisolid and Broth Cultures

A method for the detection of deoxyribonuclease activity in semisolid agar cultures was investigated. When cultures were overlaid with an acridine orangedeoxyribonucleate-agar (ADA) mixture, incubated for 1 to 3 hr, and observed under ultraviolet light, clear halos developed around colonies that produced deoxyribonuclease. A variation of the method for use with broth cultures involved impregnation of filter-paper discs with a small portion of the culture and overlaying the discs with the ADA mixture. This alteration has the advantage that the test tube cultures are easily heat-treated prior to assay to determine the heat resistance of the enzyme.     

Pooled Screening for Synergistic Interactions Subject to Blocking and Noise

The complex molecular networks in the cell can give rise to surprising interactions: gene deletions that are synthetically lethal, gene overexpressions that promote stemness or differentiation, synergistic drug interactions that heighten potency. Yet, the number of actual interactions is dwarfed by the number of potential interactions, and discovering them remains a major problem. Pooled screening, in which multiple factors are simultaneously tested for possible interactions, has the potential to increase the efficiency of searching for interactions among a large set of factors. However, pooling also carries with it the risk of masking genuine interactions due to antagonistic influence from other factors in the pool. Here, we explore several theoretical models of pooled screening, allowing for synergy and antagonism between factors, noisy measurements, and other forms of uncertainty. We investigate randomized sequential designs, deriving formulae for the expected number of tests that need to be performed to discover a synergistic interaction, and the optimal size of pools to test. We find that even in the presence of significant antagonistic interactions and testing noise, randomized pooled designs can significantly outperform exhaustive testing of all possible combinations. We also find that testing noise does not affect optimal pool size, and that mitigating noise by a selective approach to retesting outperforms naive replication of all tests. Finally, we show that a Bayesian approach can be used to handle uncertainty in problem parameters, such as the extent of synergistic and antagonistic interactions, resulting in schedules for adapting pool size during the course of testing.     

Estimation of the Relative Sensitivity of qPCR Analysis Using Pooled Samples

The high sensitivity of qPCR makes it a desirable diagnostic method in epidemiological surveillance programs. However, due to high costs, the use of pooling has been suggested. In this paper, an algorithm based on the Montecarlo method has been designed and implemented. The algorithm had been tested in many different situations, and finally it was validated with a real dataset. Moreover, based on the results obtained and depending on pooling conditions, a drastic decrease of sensitivity is observed.     

Isolation of Salmonellae From Food Samples: VI. Comparison of Methods for the Isolation of Salmonella From Egg Products

The recovery of salmonellae from egg products was studied, by use of three different enrichment procedures: (i) selenite broth, (ii) selenite broth containing 10% sterile feces, and (iii) the lactose pre-enrichment procedure. Brilliant Green Agar was used throughout as the recovery medium. Although the lactose pre-enrichment methodology promoted Salmonella recovery from samples containing small numbers of dormant organisms, the efficiency of this enrichment method is adversely affected by unfavorable coliform-Salmonella ratios. Under such conditions, early subculture of lactose broth into selenite broth is indicated. Selenite broth containing 10% sterile feces was more efficient than the lactose pre-enrichment methodology in promoting the growth of “dormant” salmonellae. Albumen adversely affected recovery of salmonellae from selenite broth, whereas whole egg and egg yolk enhanced Salmonella recovery from this medium. The selenite-feces medium presents a solution to the major problems encountered in the detection of salmonellae in egg products and offers an approach to a single medium in which food-borne salmonellae will manifest themselves with a minimum of laboratory manipulation.     

Development of Radiation Resistance in Salmonella Cultures

Cultures of Salmonella subjected to repeated cycles of gamma-irradiation and subculture developed radiation resistance sooner at a low cycling dose ( approximately 1% survival) compared to a high cycling dose ( approximately 0.001% survival). Radioresistant cells in a population of radiosensitive cells of Salmonella newport or S. typhimurium could be selected by the double-irradiation plate method. The frequency of radioresistant cells in a population of S. newport was found to be about 1 per 8.9 million. Radio-resistant cells obtained by cyclic irradiation and subculture were larger (plumper) than the parent strain and showed a marked pleomorphism.     

Device for Turbidity Standardizing of Cultures for Antibiotic Sensitivity Testing

A specially designed metal rack which is illuminated by a modified Rh-typing view box is described. This device facilitates the handling and reduces the time involved in standardizing cultures.     

Detection and Identification of Salmonella enterica,Escherichia coli,and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay''s ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.     

Nalidixic Acid for Enrichment of Auxotrophs in Cultures of Salmonella typhimurium

Nalidixic acid kills only growing cells of Salmonella typhimurium and can be used to enrich for auxotrophs in populations where prototrophs predominate.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

A Pooled Response Strategy for Combining Multiple Lines of Evidence to Quantitatively Estimate Impact

The impacts of sediment contaminants can be evaluated by different lines of evidence, including toxicity tests and ecological community studies. Responses from 10 different toxicity assays/tests were combined to arrive at a “site score.” We employed a relatively simple summary measure, pooled P-values where we quantify a potential decrement in response in a contaminated site relative to nominally clean reference sites. The response-specific P-values were defined relative to a “null” distribution of responses in reference sites, and were then pooled using standard meta-analytic methods. Ecological community data were also evaluated using an analogous strategy. A distribution of distances of the reference sites from thecentroid of the reference sites was obtained. The distance from each of the test sites from the centroid of the reference sites was then calculated, and the proportion of reference distances that exceed the test site difference was used to define an empirical P-value for that test site. A plot of the toxicity P-value versus the community P-value was used to identify sites based on both alteration in community structure and toxicity, that is, by weight-of-evidence. This approach provides a useful strategy for examining multiple lines of evidence that should be accessible to the broader scientific community. The use of a large collection of reference sites to empirically define P-values is appealing in that parametric distribution assumptions are avoided, although this does come at the cost of assuming the reference sites provide an appropriate comparison group for test sites.     

Diagnostic Real-Time PCR for Detection of Salmonella in Food

A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10³ CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10⁴ CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

Simple Method for Collection of Samples from a Frozen Food

The conical end of a plastic funnel can be sterilized and used for aseptic collection, transfer, and distribution of shaving samples drilled from a frozen-food product.