Standard Plate Method Research Articles

Survival of Gastric and Enterohepatic Helicobacter spp. in Water: Implications for Transmission

Part of the reason for rejecting aquatic environments as possible vectors for the transmission of Helicobacter pylori has been the preference of this microorganism to inhabit the human stomach and hence use a direct oral-oral route for transmission. On the other hand, most enteric bacterial pathogens are well known for being able to use water as an environmental reservoir. In this work, we have exposed 13 strains of seven different Helicobacter spp. (both gastric and enterohepatic) to water and tracked their survival by standard plating methods and membrane integrity assessment. The influence of different plating media and temperatures and the presence of light on recovery was also assessed. There was good correlation between cultivability and membrane integrity results (Pearson's correlation coefficient = 0.916), confirming that the culture method could reliably estimate differences in survival among different Helicobacter spp. The species that survived the longest in water was H. pylori (>96 h in the dark at 25 degrees C), whereas H. felis appeared to be the most sensitive to water (<6 h). A hierarchical cluster analysis demonstrated that there was no relationship between the enterohepatic nature of Helicobacter spp. and an increased time of survival in water. This work assesses for the first time the survival of multiple Helicobacter spp., such has H. mustelae, H. muridarum, H. felis, H. canadensis, H. pullorum, and H. canis, in water under several conditions and concludes that the roles of water in transmission between hosts are likely to be similar for all these species, whether enterohepatic or not.

Susceptibility of microcosm subgingival dental plaques to lethal photosensitization

Photodynamic therapy (PDT) offers potential as a non-invasive treatment of periodontal disease. In this study, microcosm biofilmswere grown in vitro under conditions designed to mimic subgingival plaques typically found in patients with periodontitis. To investigate potential PDT modalities, biofilms were exposed to light from a helium/neon laser in conjunction with a photosensitizer, toluidine blue O (TBO), at varying output and concentration, respectively. To determine cytotoxic effects, viability profiling was undertaken on whole biofilms using standard plating methods, and on horizontal cross-sections of biofilms using confocal laser-scanning microscopy (CLSM) in conjunction with a differential viability stain. A light energy dose of 94.5 J in combination with 81.7 µM TBO was found to be optimal, achieving significant kills of over 97%. CLSM enabled visualization of the effects of PDT in three dimensions. Viability profiling of the CLSM images revealed that lethal photosensitization was most effective in the upper layers of biofilm. PDT was found to reduce the viability of subgingivally modelled plaques in vitro by a magnitude similar to that of chlorhexidine digluconate, which is commonly used to treat periodontal disease. The findings of this study indicate that PDT may be an effective alternative to conventional modalities in the treatment of periodontal disease.

Cleaning and Handling Implements as Potential Reservoirs for Bacterial Contamination of Some Ready-to-Eat Foods in Retail Delicatessen Environments

This study assessed the association of bacteria with cleaning tools, such as floor mops (n = 25) and cleaning cloths (n = 39), and handling devices, such as disposable plastic gloves (n = 20), used during filled baguette and assorted salad preparation in four selected retail delicatessens in Johannesburg, South Africa. Samples of each cleaning or handling tool were prepared for aerobic (APC), coliform (CC), Escherichia coli (EC), Bacillus cereus (BCC), and Staphylococcus aureus (SAC) counts, as well as tested for the incidence of Listeria monocytogenes (LM) and Salmonella (SALM) by standard plating methods. Bacterial populations attached to the cleaning and handling tools were observed by scanning electron microscopy (SEM). Ten selected gram-positive isolates were further analyzed by 16S rRNA sequence analysis and compared with isolates from filled baguettes and assorted salads. The floor mops consistently yielded the highest APCs, CCs, and ECs (5.7, 4.1, and 3.0 log CFU/g, respectively), while gloves had the lowest corresponding counts (3.6, 2.0, and 1.0 log CFU/g, respectively). Low BCCs and SACs were recorded in this study (ca. 1.2 log CFU/g), while SALM and LM were each detected in five cleaning tool samples. SEM showed rods and cocci attached to handling and cleaning tools. Furthermore, results of 16S rRNA sequence analysis indicated that several gram-positive isolates were identified as S. aureus, Staphylococcus pasteuri, Staphylococcus sciuri, and Enterococcus faecalis. Genetically similar strains (100% similarity) were isolated from cleaning and handling tools and associated ready-to-eat (RTE) foods. Cleaning and handling tools may act as reservoirs of contamination for RTE foods during preparation in retail delicatessens in South Africa. The transfer of potential pathogens, such as S. aureus, to foods from cleaning and handling tools may hold food safety implications.

Feasibility of Utilizing Bioindicators for Testing Microbial Inactivation in Sweetpotato Purees Processed with a Continuous‐Flow Microwave System

Continuous-flow microwave heating has potential in aseptic processing of various food products, including purees from sweetpotatoes and other vegetables. Establishing the feasibility of a new processing technology for achieving commercial sterility requires evaluating microbial inactivation. This study aimed to assess the feasibility of using commercially available plastic pouches of bioindicators containing spores of Geobacillius stearothermophilus ATCC 7953 and Bacillus subtilis ATCC 35021 for evaluating the degree of microbial inactivation achieved in vegetable purees processed in a continuous-flow microwave heating unit. Sweetpotato puree seeded with the bioindicators was subjected to 3 levels of processing based on the fastest particles: undertarget process (F(0) approximately 0.65), target process (F(0) approximately 2.8), and overtarget process (F(0) approximately 10.10). After initial experiments, we found it was necessary to engineer a setup with 2 removable tubes connected to the continuous-flow microwave system to facilitate the injection of indicators into the unit without interrupting the puree flow. Using this approach, 60% of the indicators injected into the system could be recovered postprocess. Spore survival after processing, as evaluated by use of growth indicator dyes and standard plating methods, verified inactivation of the spores in sweetpotato puree. The log reduction results for B. subtilis were equivalent to the predesigned degrees of sterilization (F(0)). This study presents the first report suggesting that bioindicators such as the flexible, food-grade plastic pouches can be used for microbial validation of commercial sterilization in aseptic processing of foods using a continuous-flow microwave system.

Irradiation D Values ofSalmonellaspp. in Diced Tomatoes Dipped in 1% Calcium Chloride

Outbreaks of salmonellosis have been associated with eating raw domestic tomatoes. In this study, we examined the efficiency of combined irradiation and a 1% calcium chloride dip to reduce the population of Salmonella enterica strains on diced tomatoes. Tomatoes were contaminated with nalidixic acid-resistant strains of S. Hartford, S. Montevideo, or a mixture of 5 strains (S. Hartford, S. Montevideo, S. Poona, S. Michigan, S. Gaminara). We irradiated tomatoes at various doses up to 0.9 kGy from an electron beam source to conduct a D-value study (decimal reduction time required to eliminate 90% of the organism). Surviving Salmonella populations were detected by standard and recovery plating methods. D-value results ranged from 0.26 to 0.39 kGy, indicating that a 5 log(10) CFU/g reduction in Salmonella spp. in diced tomatoes would require a dose of 1.3-1.95 kGy.

Field Tests of the Soil Heat Flux Plate Method and Some Alternatives

Heat flux plates are commonly used to measure soil heat flux, a component of the surface energy balance. The plate method is simple and precise, but several previous studies have demonstrated the potential for relatively large errors. Here we present the results of in situ tests of the plate method, and we describe some promising alternative methods. Summertime soil heat flux was measured with heat flux plates and with two alternative methods at each of three sites. In total, three alternative methods were used: a single probe gradient method, a three needle gradient method, and a self‐calibrating plate method. The standard plate method underestimated the magnitude of the heat flux by 18 to 66% depending on the site and type of plate. Agreement between the alternative methods was good with discrepancies ranging from 2 to 6%. The plates underestimate flux apparently due to a combination of low plate thermal conductivity, thermal contact resistance, and latent heat transfer effects. The three needle gradient method for measuring heat flux performed well at all three sites, providing a good alternative to the standard plate method. The self‐calibrating plate method performed well at the one site where it was tested and may also be a good alternative. Increased adoption of these methods should lead to more accurate soil heat flux and surface energy balance data.

Activation of Fibrinolysis (Nattokinase) Induced by Dipicolinic Acid and Related Compounds

Culture medium containing dipicolinic acid at final concentrations of 0, 1, 2 and 4mM were inoculated with the Miyagino strain of Bacillus subtilis natto, the bacteria used for the production of natto, and subjected to stationary culture for 24 hours, after which the activity of nattokinase was investigated using two methods : a standard fibrin plate method, and the synthetic substrate Suc-Ala-Ala-Pro-Phe-pNA. Using the standard fibrin plate method, it was confirmed that nattokinase activity rose to 12.5FU/ml in the samples containing 2mM dipicolinic acid, a value 5.6 times greater than that of the sample containing no dipicolinic acid (2.25FU/ml). Colorimetric measurements using the synthetic substrate showed that the activity level was highest (168.5nmol/min/ml) with addition of 2mM dipicolinic acid, about 4.3 times the activity of the sample with no dipicolinic acid (39.6nmol/min/ml). The effects of dipicolinic acid were also clear for the Naruse strain of Bacillus subtilis natto. Based on the results of comparison tests using DPA and nine structurally related compounds, including nicotinic acid, it appears that dipicolinic acid is capable of enhancing specific nattokinase production to a considerable extent.

Fungal contamination of some imported spices

Seventeen imported raw spice samples obtained from retail outlets were examined for spoilage mould profile. A total of 665 fungal isolates, representing 14 species, were recovered and identified from dried and ground spice samples on several media using standard dilution plate method. Moisture content varied greatly among various samples and were generally high. The most heavily contaminated spice samples examined were observed in red chili and black pepper in order of magnitude of 1580 and 1120 cfu/g, respectively. The most predominant fungal genera encountered were Aspergillus, Penicillium, Rhizopus, Cladosporium and Trichoderma. Yeasts were also frequently recovered, but not identified. Relative occurrence values of taxa disclosed ranged between 36.4% for A. flavus and 0.6% for A. parasiticus and Absidia corymbifera. Samples obtained from gunny bags encounter higher colony counts and contamination frequency than other packing methods. The present magnitude of contamination and spectra of mycobiota approximate those reported for similar spice samples. Although several potentially mycotoxigenic fungi were isolated during the present study, neither the foodstuff nor the fungi were assayed for the presence of these toxins.

Physical Properties of Three Sand Size Classes Amended with Inorganic Materials or Sphagnum Peat Moss for Putting Green Rootzones

Modern putting green rootzones are typically constructed using sands to avoid compaction and facilitate rapid drainage. Sands are often amended with organic matter (OM) such as sphagnum peat moss (SP) to increase moisture holding capacity. However, OM decomposition into finely divided material may negatively affect long‐term soil physical properties. Inorganic amendments (IAs) having high water retention may be more suitable because of their resistance to biodegradation. A laboratory study determined the physical properties [bulk density, saturated hydraulic conductivity (Ksat), water retention, and pore size distribution] of three USDA sand size classes (fine, medium, and coarse) with and without amendment. Amendments used were calcined clay, vitrified clay, extruded diatomaceous earth, a processed zeolite, and SP. Amendments were tested at two incorporation rates (10 and 20% v/v), and in situ in 30‐cm‐deep rootzones at two incorporation depths (15 and 30 cm). Bulk density decreased, total porosity increased, and Ksat declined with amendment rate, but varied considerably depending on amendment, sand size, and incorporation depth. The Ksat was high for all mixtures, averaging 250 cm h−1, probably because of the very uniform sands. On the basis of standard pressure plate methods, IAs increased total water holding capacity (WHC) of all three sands but did not increase available water. However, a unique bioassay for available water indicated that porous IAs may contain appreciably more available water than measured by the pressure plate technique. Although the IAs significantly altered the physical properties of the three sands, they were not as effective as SP at improving water retention in coarse‐textured, drought‐prone sands.

Isolation and Anti-fungal Activities of 2-Hydroxymethyl-chroman-4-one Produced by Burkholderia sp. MSSP

In the course of screening for biological active compounds from microbial origins, various strains were isolated from roots of Mimosa pudica using standard dilution agar plate method. One isolate was selected for further studies of bacterial anti-fungal properties. It was designated MSSP and identified as Burkholderia sp. by many biological characteristics such as carbohydrate utilization, biochemical and physiological test and 16S rDNA sequence homology search. To investigate antagonistic principle, anti-fungal compounds were extracted and fractionated by different solvent systems. An anti-fungal compound was isolated from its culture filtrate using organic solvent extraction, column chromatography and thin layer chromatography and pure solid form compound (MSSP2) was obtained. The chemical structure of anti-fungal compound was identified as 2-Hydroxymethyl-chroman-4-one based on the data obtained from spectroscopic analysis such as mass spectral analyses and NMR spectral data. The compound 2-Hydroxymethyl-chroman-4-one exhibited good activities against phytopathogen such as Pythium ultimum, Phytophthora capsici and Sclerotinia sclerotiorum.

Physical Properties of Three Sand Size Classes Amended with Inorganic Materials or Sphagnum Peat Moss for Putting Green Rootzones

Modern putting green rootzones are typically constructed using sands to avoid compaction and facilitate rapid drainage. Sands are often amended with organic matter (OM) such as sphagnum peat moss (SP) to increase moisture holding capacity. However, OM decomposition into finely divided material may negatively affect long-term soil physical properties. Inorganic amendments (IAs) having high water retention may be more suitable because of their resistance to biodegradation. A laboratory study determined the physical properties [bulk density, saturated hydraulic conductivity (Ksat), water retention, and pore size distribution] of three USDA sand size classes (fine, medium, and coarse) with and without amendment. Amendments used were calcined clay, vitrified clay, extruded diatomaceous earth, a processed zeolite, and SP. Amendments were tested at two incorporation rates (10 and 20% v/v), and in situ in 30-cm-deep rootzones at two incorporation depths (15 and 30 cm). Bulk density decreased, total porosity increased, and Ksat declined with amendment rate, but varied considerably depending on amendment, sand size, and incorporation depth. The Ksat was high for all mixtures, averaging 250 cm h−1, probably because of the very uniform sands. On the basis of standard pressure plate methods, IAs increased total water holding capacity (WHC) of all three sands but did not increase available water. However, a unique bioassay for available water indicated that porous IAs may contain appreciably more available water than measured by the pressure plate technique. Although the IAs significantly altered the physical properties of the three sands, they were not as effective as SP at improving water retention in coarse-textured, drought-prone sands.

Bioremediation of oily sea water by bacteria immobilized in biofilms coating macroalgae

Using the standard plate method and a solid mineral medium containing crude oil as a sole source of carbon and energy, 10 different macroalgae from the Arabian Gulf were found associated with large numbers of oil-utilizing bacteria. Each gram fresh alga was associated with about two to about 30 million cells of bacteria predominantly belonging to the nocardioforms and the genus Acinetobacter. Shaking macroalgal samples in sea water batches containing known amounts of individual hydrocarbons led to considerable attenuation of these compounds as measured by GLC. Thus, bacteria associated with macroalgae consumed about 64–98% of n-octadecane and about 38–56% phenanthrene from medium aliquots containing 0.03% of the test hydrocarbon after 2 weeks. Meanwhile, the oil-utilizing bacteria, especially the nocardioforms, associated with the macroalgae increased in number by about 32–490 fold, depending on the macroalgae and hydrocarbons studied. On the other hand, relatively negligible numbers of bacteria were released into the sea water compared with the numbers immobilized on the macroalgal surfaces. Individual bacterial isolates could grow on a wide range of pure alkanes and aromatic hydrocarbons as sole sources of carbon and energy. It was concluded that macroalgae submerged in the sea waters are coated with biofilms rich in oil-utilizing bacteria, that contribute to hydrocarbon attenuation in water. These natural biological consortia represent valuable tools that could be of high potential for phytoremediation of oily sea water.

Comparison of the Compact Dry TC and 3M Petrifilm ACP Dry Sheet Media Methods with the Spiral Plate Method for the Examination of Randomly Selected Foods for Obtaining Aerobic Colony Counts

Two hundred thirty-six randomly selected food and milk samples were examined to obtain aerobic colony counts by two dry sheet media methods and a standard Public Health Laboratory Service spiral plate method. Results for 40 samples were outside the limits of detection for one or more of the tested methods and were not considered. (The limits of detection for the spiral plate method were 200 to 1 × 108 CFU/ml for the spiral plate method and 100 to 3 × 106 CFU/ml for the dry sheet media methods.) The remaining 196 sets of results were analyzed further. When the results from the three methods were compared, correlation coefficients were all >0.80 and slopes and intercepts were close to 1.0 and 0.0, respectively. Mean log values and standard deviations were very similar for all three methods. The results were evaluated according to published UK guidelines for ready-to-eat foods sampled at the point of sale, which include a quality acceptability assessment that is based on aerobic colony counts. Eighty-six percent of the comparable results gave the same verdict with regard to acceptability according to the aerobic colony count guidelines. Both dry sheet media methods were comparable to the spiral plate method and can be recommended for the examination of food.

Assessment of the efficiency of SimPlate™ total plate count color indicator (TPC CI) to quantify mesophilic aerobic microorganisms in pasteurized milk

The SimPlate™ TPC CI system is a rapid method to count mesophilic aerobic microorganisms (MAM) in foods, based on the use of resazurine to indicate bacterial growth. Its efficiency in pasteurized milk was evaluated using 142 pasteurized milk samples (38 type A, 43 type B and 61 type C) collected in Londrina, PR. The standard plating method, using Plate Count Agar (PCA) was used for comparison. The plates of both systems were incubated at 35oC and read after 24h and 48h. The occurrence of false-positive and false-negative wells and the predominant microorganisms in them were also evaluated. The results were compared by simple correlation and mean variance analyses. The correlation (r) and mean variance values were 0.6811 and 0.7583 for the results obtained after 24h, respectively, and 0.9126 and 0.0842 for the results obtained after 48h, respectively. These results indicate that the performance of the system increases when the plates are incubated for 48h. When the three types of milk were evaluated separately, these values were 0.9285 and 0.0817 for type A milk, 0.9231 and 0.0466 for type B milk and 0.7209 and 0.1082 for type C milk. These results indicate that the better the quality of the milk the better the performance of SimPlate™ TPC CI. False-negative wells, found more frequently in samples with high MAM counts, were caused by Gram positive microorganisms, poorly detected by the SimPlate™ TPC CI system because they grew slowly and had low reduction capacity. The results indicated a higher efficiency of the SimPlate™ TPC CI system in the reading at 48h.

How to optimize the drop plate method for enumerating bacteria

The drop plate (DP) method can be used to determine the number of viable suspended bacteria in a known beaker volume. The drop plate method has some advantages over the spread plate (SP) method. Less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar. By distributing the sample in drops, colony counting can be done faster and perhaps more accurately. Even though it has been present in the laboratory for many years, the drop plate method has not been standardized. Some technicians use 10-fold dilutions, others use twofold. Some technicians plate a total volume of 0.1 ml, others plate 0.2 ml. The optimal combination of such factors would be useful to know when performing the drop plate method. This investigation was conducted to determine (i) the standard deviation of the bacterial density estimate, (ii) the cost of performing the drop plate procedure, (iii) the optimal drop plate design, and (iv) the advantages of the drop plate method in comparison to the standard spread plate method. The optimal design is the combination of factor settings that achieves the smallest standard deviation for a fixed cost. Computer simulation techniques and regression analysis were used to express the standard deviation as a function of the beaker volume, dilution factor, and volume plated. The standard deviation expression is also applicable to the spread plate method.

Multiregional Evaluation of the SimPlate Heterotrophic Plate Count Method Compared to the Standard Plate Count Agar Pour Plate Method in Water

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0. 95; y = 0.99X + 0.06).

LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.

EFFECT OF ELECTRICAL TREATMENT ON SALMONELLA TYPHIMURIUM IN POULTRY SCALDER WATER USING A FLOW-THROUGH TREATMENT SYSTEM

A flow-through electrical treatment system was evaluated as a method to eliminate S typhimurium in poultryscalder water. The flow-through treatment system consisted of an inlet tank, a treatment cylinder, and an outlet tank. A dcpower supply provided electrical signals to platinum wire electrodes in the treatment cylinder. With 0.15 M NaCl added asan electrolyte, a 2-A electrical current was applied to poultry scalder water flowing through the treatment cylinder at a rateof 5 L/min. Residence time in the cylinder was 1.82 min. Samples were tested for salmonellae using standard platingmethods as well as pre-enrichment/enrichment techniques. All salmonellae were eliminated from the treated poultry scalderwater while the untreated scalder water contained large numbers of salmonellae.

A simple microtiter plate screening assay for bacterial invasion or adherence

Many bacteriologic studies, including cellular invasion and adherence assays, require enumeration of viable organisms or colony forming units. When attempting to screen large numbers of clinical or environmental isolates or laboratory-derived mutants for differences in invasion or adherence phenotype, standard plating methods can be cumbersome and severely limit the number of organisms or conditions which can be tested. As a potential alternative, we describe a simple, rapid and inexpensive soft agar-based technique for semi-quantitative determination of bacterial colony counts directly within the wells of a 96-well microtiter plate.

Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods

A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.