Standard Plate Method Research Articles

Isolation and characterization of novel marine luminescent bacteria from Diu beach, India

AimTo carry out the isolation and characterization of novel marine luminescent bacteria from Diu beach, India. Materials & methodsIn the present study attempt has been made to isolate luminescent bacterial species from marine water sampled from Diu beach, India, using standard plating method on nutrient medium. Obtained isolate was analyzed susceptibility against antibiotics and heavy metals in order to use them in bio-sensing system. Isolated bacterium was amplified for 16S rRNA gene by PCR and further identified by aligning through BLAST and MEGA software. ResultThe Vibrio rotiferianus when growing prominently at 22 °C shown tolerance to low concentration of heavy metals & resistance to few antibiotics which makes isolate, a potential bio-sensing agent. Further, molecular characterization including 16S rRNA gene and its phylogenetic analysis confirmed that the isolated strain as V. rotiferianus. ConclusionThe use of pharmaceuticals products is on the rise and in turn its pollution is rising in an ecosystem. The isolated strain may act as a potential biosensing agent as it responded to change in luminescence for even nanomolar quantities of several heavy metals/antibiotics. In conclusion, study highlighted V. rotiferianus isolated from Diu beach could be utilized in detection and quantitative estimation of pollutant by change in luminescence level of V. rotiferianus.

ANTIMICROBIAL EFFECTS OF ASCORBIC ACID AND CALCIUM LACTATE IN FRESHCUT JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAM.)

ISHS Southeast Asia Symposium on Quality Management in Postharvest Systems and Asia Pacific Symposium on Postharvest Quality Management of Root and Tuber Crops ANTIMICROBIAL EFFECTS OF ASCORBIC ACID AND CALCIUM LACTATE IN FRESHCUT JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAM.)

Resazurin-based assay for screening bacteria for radiation sensitivity

We report a simple and efficient colorimetric method to screen large numbers of bacterial strains for UV- and X-radiation sensitivity. We used reference radiation-sensitive and control strains of Escherichia coli K-12 to compare our colorimetric method to a standard clonogenic plating method. Our colorimetric method was as accurate as the standard method and was superior in terms of savings in supplies and man-hours.

Rapid enumeration of viable bacterial cells in processed soy products using an automated cell counting system

A fluorescent filter method is an alternative culture-independent microbial test. The applicability of an automated cell counting system based on a fluorescent filter method was evaluated for processed soy products. To overcome the disadvantage of the filter method for colloidal and protein suspension samples such as soy milk and soy protein isolates, soy protein matrices were eliminated by isoelectric precipitation and bacterial cells in sample aliquots were enumerated using an automated cell counting system (Bioplorer). Bacterial counts with the Bioplorer method were well correlated with those obtained by the standard plate method. Coliform detection in soy products was possible within 7 h by this method, including 6 h required for culture enrichment, compared with more than 24 h required for confirmation by conventional plate methods. These results suggested that the Bioplorer method is a rapid and efficient method for the enumeration of viable bacterial cells in processed soy products.

A Biosurfactant-Sophorolipid Acts in Synergy with Antibiotics to Enhance Their Efficiency

Sophorolipids (SLs), biosurfactants with antimicrobial properties, have been tried to address the problem of antibiotic resistance. The synergistic action of SL and antibiotics was checked using standard microdilution and spread plate methods. With Staphylococcus aureus, SL-tetracycline combination achieved total inhibition before 4 h of exposure while tetracycline alone couldnot achieve total inhibition till the end of 6 h. The inhibition caused by exposure of bacterium to SL-tetracycline mixture was ~25% more as compared to SL alone. In spite of known robustness of gram-negative bacteria, SL-cefaclor mixture proved to be efficient against Escherichia coli which showed ~48% more inhibition within 2 h of exposure as compared to cefaclor alone. Scanning electron microscopy of the cells treated with mixture revealed bacterial cell membrane damage and pore formation. Moreover, SLs being a type of asymmetric bola, they are expected to form self-assemblies with unique functionality. This led to the speculation that SLs being amphiphilic in nature can span through the structurally alike cell membrane and facilitate the entry of drug molecules.

Application of Proanthocyanidins from Peanut Skins as a Natural Yeast Inhibitory Agent

Proanthocyanidins were extracted from peanut skins and investigated for their antimicrobial activity against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus in traditional growth media (Sabouraud Dextrose and Maltose broth) and a simulated apple juice beverage. Peanut skins extracts (PSE) were prepared through a multisolvent extraction procedure. The PSE extended the lag phase growth of the 3 yeasts studied at a concentration of 1 mg/mL and at 10 mg/mL yeast growth was totally inhibited for 120 h. PSE was fractionated by normal phase high performance liquid chromatography and the active components/fractions were determined. Compounds present in the fractions were identified by liquid chromatography-mass spectrometry to determine the compounds responsible for inhibition. Fractions consisting mostly of A-type proanthocyanidin dimers, trimers, and tetramers showed the highest percent inhibition toward the yeasts tested in this study. Both optical density (OD) and standard enumeration plating methods were performed in this study. The OD method led to an overestimation of the inhibitory effects of PSE, the 2 methods agreed in respect to treatment effects but not the severity of the inhibition. There is a growing consumer demand for "fresh like" products containing reduced amounts of chemical preservatives without compromising food safety and quality. Therefore, the goal of this study was to determine if an extract of peanut skins containing flavonoid rich compounds could function as a natural antimicrobial in a model beverage system. Proteins were removed through the process of producing the peanut skin extract, thus it is unlikely to contain peanut allergens. The antimicrobial compounds mentioned in this study were successfully integrated into a model beverage system, and were found to have antimicrobial effect. However, the incorporation of these compounds would likely lead to negative sensory attributes at the concentration needed to achieve an appreciable antimicrobial effect alone.

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication. Recently, thermal inkjet printing has also been used for gene transfection. The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm. The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells. Cell viability after printing has been shown to be similar to standard cell plating methods. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. A standard HP DeskJet 500 printer was modified to allow for cell printing. The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%–100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%–100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.

Enumeration of micro-organisms in processed soy products with an automated most probable number method compared with standard plate method

The automated TEMPO system (bioMerieux) is based on the most probable number (MPN) method for the enumeration of micro-organisms in foods. In this study, we evaluated the performance of the TEMPO system as a diagnostic tool in comparison with the standard method in processed soy products. A verification study was conducted using artificially contaminated soy product samples such as soy protein isolate, water-soluble soy polysaccharides, soy milk and processed soy food. Five types of micro-organisms were analysed using the automated MPN method (total aerobic bacteria, total coliforms, Enterobacteriaceae, yeast and mould and Staphylococcus aureus) vs the standard plate method. The results from each of the methods were highly correlated (r > 0·95). Naturally contaminated processed soy products on the market were also studied. There were no discrepancies observed between the respective methods. TEMPO methods were equivalent to the corresponding standard plate methods with very good rates of agreement. The automated MPN method is more practical and reliable for in-house microbiological testing in processed soy products.

Frequency distribution of Candida albicans blastospores adhered to mucosal epithelial cells in-vitro.

Although several methods are available for examination of microbial adherence to epithelial cells, these do not distinguish between adherence of viable and non-viable micro-organisms. This study reports the use of acridine orange-stained blastospores of Candida albicans in conjunction with direct epifluorescence microscopy to determine viable (orange-fluorescing) and non-viable (green-fluorescing) blastospore adherence to buccal epithelial cells. The method was also employed to examine the effects of chlorhexidine treatment at subminimum inhibitory concentrations on the adherence of viable and non-viable blastospores. There was good correlation in the assessment of blastospore viability between the direct epifluorescence microscopy technique and the standard serial dilution and plating method for viable counting, confirming the reliability of direct epifluorescence microscopy. Chlorhexidine treatment before acridine orange staining did not alter this assessment of viability. Blastospore adherence to buccal epithelial cells resulted in a similarly skewed distribution whether examined using a crystal violet stain in conjunction with light microscopy or using direct epifluorescence microscopy, therefore validating the direct epifluorescence microscopy technique for the enumeration of blastospore adherence. Chlorhexidine treatment (0.0005% v/v, 30 min) of either blastospores or buccal epithelial cells altered the distribution of adherent blastospores per epithelial cell by increasing the number of epithelial cells having no adherent blastospores. No differences in adherence were, however, observed between blastospore or epithelial cells after treatment with this agent. Examination of the adherence of viable and non-viable blastospores to buccal epithelial cells using direct epifluorescence microscopy revealed a greater adherence capacity of non-viable than viable blastospores for buccal epithelial cells. Treatment of blastospores with chlorhexidine altered the frequency distributions of viable and non-viable blastospores with lower numbers of blastospores adherent per epithelial cell. The larger reduction in adherent viable blastospores in comparison with their non-viable counterparts is, however, an important observation which might have clinical relevance. Microbial cells adhere to epithelial cells resulting in a skewed distribution; study of this distribution gives useful information about the adherence process. Viable and non-viable components of a microbial population have different adherence capabilities and treatment of such populations with an antimicrobial agent exerting anti-adherent activity at sub-minimum inhibitory concentrations reduces the amount of adherence of these viable/non-viable components to different extents.

Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.

EVALUATION OF TWO COMMERCIAL REAL‐TIME PCR ASSAYS FOR DETECTING CAMPYLOBACTER IN BROILER CARCASS RINSES*

ABSTRACT Traditional plating methods are reliable means for Campylobacter identification from poultry samples but automated gene‐based detection systems now available can reduce assay time, data collection and analysis. Bio‐Rad and DuPont Qualicon recently introduced Campylobacter assays for their real‐time polymerase chain reaction (PCR) instruments. We evaluated the utility of these assays compared with standard plating and enumeration methods routinely used in our laboratory. Two replicates of 40 broiler carcass rinses collected before and after defeathering at a commercial processing plant were tested. All samples were positive for Campylobacter by direct plating of rinses: log10 cfu values ranged from 0.24 to 4.61. In contrast, the Bio‐Rad iQ‐Check assay returned 60–72.5% positives on direct rinses; the Qualicon BAX Q7 test produced 60–85% positives with direct rinse samples. Using aliquots of 24 h enrichment broth from rinses in the real‐time assays significantly improved detection: the Bio‐Rad test had 95–100% positive while the Qualicon assay found 90–95% positive.PRACTICAL APPLICATIONSTraditional detection methods for Campylobacter are time‐consuming, requiring as much as 5 days to complete. The recent introduction of commercial real‐time PCR instruments for the identification of Campylobacter offer more rapid, simplified alternatives to standard culture‐based techniques.

A multiparametric PCR-based tool for fast detection and identification of spore-forming bacteria in food

The presence of psychrotrophic or highly thermoresistant spore-forming bacteria in food and feedstuff responsible for food poisoning and spoilage raises major safety and economical issues. The aim of this study was to evaluate the performances of a ready-to-use PCR assay (alternative method) in comparison with the standard microbiological plating method regarding spore-forming bacteria detection in food samples. An overnight sample enrichment was selected to increase sporeformer diversity recovery, spore germination, bacterial growth and favour DNA extraction. A total of 180 sporeformer isolates representing 38 different species and 8 genera were tested in the PCR assays. Inclusivity and exclusivity results ensured specific detection and identification of the majority of targeted genera and species. Validation studies carried on artificially contaminated food samples showed detection of the inoculated contaminants in most cases, with increased detection limit for the alternative method which enabled detection with up 1 spore of B. cereus in 25 g food sample. Using naturally contaminated food samples, standard method comforted the alternative method. In a number of cases, the alternative method was able to identify species not detected with the standard method. In addition, identification and discrimination between the B. cereus group members was possible. Thus, associated to a key element, i.e., the enrichment step, the developed multiparametric PCR-based assays reported in this study provide a fast, sensitive and reliable detection and identification tool for mostly encountered spore-forming food contaminants.

Synthesis and SAR of Some New 4 Substituted 3H-1,2,3,5-oxathiadiazole 2-Oxides as Antifungal Agents

A new series of 4-substituted 3H-1,2,3,5-oxathiadiazole 2-oxides bearing 1,2,3 triazole and piperidine ring has been synthesized in one step from amidoxime using thionyl chloride (SOCl2) and Triethylamine (TEA). All the synthesized compounds (10a-10l) are new and evaluated for their in vitro antifungal activities using standard agar plate method. SAR for the series has been developed by comparing their MIC values with miconazole and fluconazole. Compound 10j from the series was equipotent with miconazole against C. albicans and F. oxysporum whereas activity of compound 10i was comparable to miconazole against F. oxysporum and C.neoformans (MIC-30 μg/mL). Keywords: 1,2,3,5-Oxathiadiazole 2-oxides, Amidoxime, 1,2,3 triazole, Piperidine, Antifungal activity, MIC values, SAR

Contamination of common spices in Saudi Arabia markets with potential mycotoxin-producing fungi

Fifteen spices obtained from common markets were examined for their mould profile. A total of 520 fungal isolates, representing 57 species, were recovered and identified from dried and ground spice samples on three different media using standard dilution plate method. The most heavily contaminated spice samples examined were observed in ginger in order of magnitude of 5325–6800 cfu/g. The most predominant fungal genera encountered were Aspergillus, Penicillium, and Rhizopus. Relative occurrence values of taxa disclosed ranged between 80% for Aspergillus flavus, Aspergillus niger and Penicillium arenicola, and 10% for some species. Samples obtained from sumac encountered very rare colony counts indicating its antifungal prosperities. The present magnitude of contamination and spectra of mycobiota approximate those reported for similar spice samples. Several potentially mycotoxigenic fungi were isolated from the majority of samples. The present study attracts the attention to potential risk for mycotoxins contamination may be caused as a result of using these spices, especially in great quantities. The study strongly recommends reduction in application of heavily contaminated spices like ginger in food processing and using some others like clove and sumac due to their antimicrobial properties.

Rapid qualitative and quantitative detection of beef fillets spoilage based on Fourier transform infrared spectroscopy data and artificial neural networks

Abstract A machine learning strategy in the form of a multilayer perceptron (MLP) neural network was employed to correlate Fourier transform infrared (FTIR) spectral data with beef spoilage during aerobic storage at chill and abuse temperatures. Fresh beef fillets were packaged under aerobic conditions and left to spoil at 0, 5, 10, 15, and 20 °C for up to 350 h. FTIR spectra were collected directly from the surface of meat samples, whereas total viable counts of bacteria were obtained with standard plating methods. Sensory evaluation was performed during storage and samples were attributed into three quality classes namely fresh, semi-fresh, and spoiled. A neural network was designed to classify beef samples to one of the three quality classes based on the biochemical profile provided by the FTIR spectra, and in parallel to predict the microbial load (as total viable counts) on meat surface. The results obtained demonstrated that the developed neural network was able to classify with high accuracy the beef samples in the corresponding quality class using their FTIR spectra. The network was able to classify correctly 22 out of 24 fresh samples (91.7%), 32 out of 34 spoiled samples (94.1%), and 13 out of 16 semi-fresh samples (81.2%). No fresh sample was misclassified as spoiled and vice versa. The performance of the network in the prediction of microbial counts was based on graphical plots and statistical indices (bias and accuracy factors, standard error of prediction, mean relative and mean absolute percentage residuals). Results demonstrated good correlation of microbial load on beef surface with spectral data. The results of this work indicated that the biochemical fingerprints during beef spoilage obtained by FTIR spectroscopy in combination with the appropriate machine learning strategy have significant potential for rapid assessment of meat spoilage.

The study of pathogenic microbial communities in graywater using membrane bioreactor

Wastewater originating from any source in the residence except for the toilet is defined as graywater. If graywater is treated appropriately, it can be used as reused water. However, wastewater reclamation carries certain health risks, and hence this study is an analysis of pathogenic microorganism and microbial communities in treated graywater for the reuse of water treated by MBR. To reuse graywater, MBR system of a lab scale was constructed with sediment, anaerobic, anoxic and oxic reactors. In the oxic reactor, a submerged MF (pore size is 0.45 μm) membrane was installed to maintain activated sludge biomass. For the quantification of pathogenic organisms, a standard spread plate method was modified using the selective medium plates. Analysis of 16S rDNA was conducted to detect microbial community. Pathogenic microorganisms such as Escherichia coli, Coliform, Staphylococcus aureus and Salmonella were detected in effluent. According to analysis of phylum and class levels, species of microorganism become simplified through membrane. This suggests that the MF membrane in the MBR system could not perfectly remove microorganisms and further research in diverse pathogens is needed for wastewater reclamation.

Synthesis of novel 3-(1-(1-substituted piperidin-4-yl)-1 H-1,2,3-triazol-4-yl)-1,2,4-oxadiazol-5(4 H)-one as antifungal agents

A novel series of 1,2,3 triazole compounds possessing 1,2,4 oxadiazole ring were efficiently synthesized. Synthesized compounds were evaluated for their in vitro antifungal activities using standard cup plate method. SAR for the series has been developed by comparing their MIC values with miconazole and fluconazole. Compound 11a from the series was more potent than miconazole against Candida albicans (MIC-20) and Aspergillus flavus (MIC-10) whereas equipotent with miconazole against Fusarium oxysporum (MIC-25) and Aspergillus niger (MIC-12.5). Also compound 11h was more potent than miconazole against Candida albicans (MIC-20) and Aspergillus niger (MIC-10) and equipotent with miconazole against Fusarium oxysporum. Compound 11h was equipotent with fluconazole against Aspergillus niger (MIC-10).

Innate immunity is initially effective in killing Mycobacterium tuberculosis (129.20)

Abstract Mycobacterium tuberculosis (MTB) is a slow growing pathogen often resulting in lifelong infection. To investigate whether slow replication is an immune evasion strategy for MTB, we developed a replication tracking tool consisting of an unstable plasmid that is lost from replicating MTB at a constant rate. Mice were infected with MTB harboring this plasmid and at various times total and plasmid bearing bacteria in lungs were enumerated. MTB replication and death rates were derived using a mathematical model. Lung bacterial numbers increase for 21 days until antigen specific T cells arrive, after which time they remain constant. Using the unstable plasmid, we observed that replication and death rates were highest early and then slowed during chronic infection. Closer analysis of the acute phase revealed the dynamics of MTB replication and death. High replication and death rates were observed in the first 5 days and then both abruptly slowed. Killing was also observed in Rag1-/- mice indicating that innate immunity is effective in killing MTB. Our studies demonstrate that standard plating methods provide an incomplete picture. Using a replication tracking tool, we found that innate immunity is initially effective in killing MTB but that this is short lived. Which innate immune response mechanisms are at work and why both MTB replication and death so rapidly decrease are topics of ongoing studies. This work was funded by the Paul G Allen Family Foundation.

Comparison of TEMPO®EC and TBX medium for the enumeration ofEscherichia coliin cheese

TEMPO EC (Escherichia coli) is based on glucuronidase activity and is a test for use with the TEMPO system for the automated 24 h enumeration of E. coli in food products. In this study, TEMPO EC was compared with TBX medium, the current standard plate method for the enumeration of E. coli in cheese. For comparative purposes, both naturally (92) and artificially contaminated (31) cheese samples were studied. Pearson correlation coefficients were determined as 0.954 and 0.978 between the two methods for naturally and artificially contaminated samples, respectively. Regression analysis yielded the following equations: log(10) TEMPO EC = 0.340 + 0.889 log(10) TBX medium and log(10) TEMPO EC = 0.174 + 0.899 log(10) TBX medium for naturally and artificially contaminated samples, respectively. In general, absolute differences did not exceed one log between results obtained by the two methods. Statistical analysis of the results showed good agreement between the two enumeration methods. TEMPO EC is a practical and reliable alternative to the current standard plate method for the enumeration of E. coli in foods.