Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

A replica plating technique for the study of plaque-forming centers

Mouse spleen cells were cultured on Millipore filters supported on the surface of Eagle's agarose medium. The secretory products of individual plaque-forming centers (PFC) revealed after diffusion into the agarose, were studied over an extended period of time by transferring the Millipore filters to fresh agarose surfaces repeatedly in a manner analogous to the replica plating technique employed with bacteria.     

A new technique for selective identification and mapping of enhancers within long genomic sequences

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

A double filter paper technique for plating cultured plant cells

Summary An inexpensive method for achieving quantitative clonal growth of single cells and small clumps of cells ofHaplopappus gracilis (Nutt.) Gray on filter paper discs is described. Plating efficiencies of 55–80% are routinely obtained. This method depends upon the presence of a feeder layer of cells only in the initial phases of the plating, allows easy counting of colonies and permits rapid, convenient transfer of groups of colonies to other media without loss of individual colony identity or spatial distribution. This work was supported by Grant PCM75-21779 from the National Science Foundation and by funds from the Agricultural Experiment Station, University of California, Riverside.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods.

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

Detection of listeria monocytogenes in foodstuffs using chromogenic isolation media

A study was carried out to compare two selective plating media commonly used to detect Listeria monocytogenes in food samples (Palcam and Oxford agar) with two more recently formulated chromogenic media (ALOA and Rapid'L.mono). A comparison was also made between the identification systems API-Listeria and Monocytogenes ID Discs. The results obtained from the analysis of 132 food samples showed that the chromogenic media allowed Listeria monocytogenes to be detected more rapidly and with higher levels of specificity and sensitivity. The efficiency of the Monocytogenes ID Discs test was confirmed, giving results comparable to those obtained with API-Listeria, but in a simpler and quicker manner.     

A profiling platform for the identification of selective metalloprotease inhibitors

Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.     

Development of a selective plating technique for the recovery of Escherichia coli O157: H7 after heat stress

The use of Sorbitol MacConkey Agar supplemented with 4-methylumbelliferyl β-D-glucuronide (MSMA), which is commonly used in the isolation of Escherichia coli O157: H7, has been shown to perform poorly when stressed cells of the pathogen are present. The incorporation of a resuscitation period (2 h at 25°C) on Trypticase Soy Agar (TSA) before overlay with MSMA was found to significantly ( P 0·01) improve recovery of heat-stressed (52°C/60 min) cells. Maximal recovery was, however, obtained by adding catalase (1000 U) to the TSA before overlaying with MSMA. This recovery protocol was shown not to result in the loss of the major known virulence factors of E. coli O157: H7 (genes encoding eae , VT1 and VT2).     

Photographic amplification of the replica plating technique

Antonie van Leeuwenhoek -     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

A microsatellite-based multilocus screen for the identification of local selective sweeps

With the availability of completely sequenced genomes, multilocus scans of natural variability have become a feasible approach for the identification of genomic regions subjected to natural and artificial selection. Here, I introduce a new multilocus test statistic, ln RV, which is based on the ratio of observed variances in repeat number at a set of microsatellite loci in two groups of populations. The distribution of ln RV values captures demographic history of the populations as well as variation in microsatellite mutation among loci. Given that microsatellite loci associated with a recent selective sweep differ from the remainder of the genome, they are expected to fall outside of the distribution of neutral ln RV values. The ln RV test statistic is applied to a data set of 94 loci typed in eight non-African and two African human populations.     

A direct observation technique for studies on rhizoplane and rhizosphere colonization

Summary A slide incubation chamber was described which allowed small plants to be grown from seed and the root systems to be observed microscopically. A fluorescence stain, the ammonium salt of 8-anilino-1-naphthalene sulfonic acid, was applied to the soil in which the roots were growing and the stained microorganisms on the roots and in the rhizosphere were counted. A statistical pattern analysis technique, the two-within-four randomization test, was used to analyze the data obtained from quadrats on the roots. Distinct colonization patterns and colony growth, especially of bacteria, were easily distinguished with the technique.     

A selective isolation technique for determining Chaetomium in soil

Summary It was shown that Chaetomium was isolated from four types of soil; Cinebar, Ephrata, Milville, and quartz sand, more frequently when using alcohol agar as the growth medium than it (Chaetomium) was isolated from these soils when using either Czepak's-Dox agar or peptone-dextrose agar plus rose bengal and streptomycin. This isolation method proved to be one which permits identification of the Chaetomium species without further culturing.This paper is based on work performed under United States Atomic Energy Commission Contract AT (45-1)-1830.