Showing papers on "Agar plate published in 2012"

Automated Counting of Bacterial Colony Forming Units on Agar Plates

Abstract: Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates. A colony counter hardware was designed and a novel segmentation algorithm was written in MATLAB. In brief, pre-processing with Top-Hat-filtering to obtain a uniform background was followed by the segmentation step, during which the colony images were extracted from the blood agar and individual colonies were separated. A Bayes classifier was then applied to count the final number of bacterial colonies as some of the colonies could still be concatenated to form larger groups. To assess accuracy and performance of the colony counter, we tested automated colony counting of different agar plates with known CFU numbers of S. pneumoniae, P. aeruginosa and M. catarrhalis and showed excellent performance.

Effects of Solid-Medium Type on Routine Identification of Bacterial Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract: Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

Abstract: The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.

Inaccuracy of the Disk Diffusion Method Compared with the Agar Dilution Method for Susceptibility Testing of Campylobacter spp.

Abstract: The agar dilution method has been standardized by the CLSI for the susceptibility testing of Campylobacter species, and according to these standards, the disk diffusion method should be used only in screening for macrolide and ciprofloxacin resistance. Nevertheless, the disk diffusion test is currently widely used, since it is easy to perform in clinical microbiology laboratories. In this study, the disk diffusion method was compared to the agar dilution method by analyzing the in vitro activities of seven antimicrobial agents against 174 Campylobacter strains collected in Finland between 2003 and 2008. Recommendations of the CLSI were followed using Mueller-Hinton agar plates with 5% of sheep blood. For each strain, the disk diffusion tests were performed two to four times. Of the 33 erythromycin-resistant strains (MIC, ≥16 μg/ml), 24 (73%) constantly showed a 6-mm erythromycin inhibition zone (i.e., no inhibition), while for seven strains the inhibition zone varied from 6 to 44 mm in repeated measurements. Among the 141 erythromycin-susceptible strains (MIC, <16 μg/ml), erythromycin inhibition zones varied between 6 and 61 mm. Of the 87 ciprofloxacin-resistant strains, 47 (54%) showed 6-mm inhibition zones, while 40 strains showed inhibition zones between 6 and 60 mm. Significant differences between the repetitions were observed in the disk diffusion for all antimicrobial agents and all strains except for the macrolide-resistant strains regarding the macrolides. For 17 (10%) strains, the variation in repeated measurements was substantial. These results show that the disk diffusion method may not be a reliable tool for the susceptibility testing of Campylobacter spp. Further studies are needed to assess whether the disk diffusion test could be improved or whether all susceptibilities of campylobacters should be tested using an MIC-based method.

Antimicrobial effect of conventional root canal medicaments vs propolis against Enterococcus faecalis, Staphylococcus aureus and Candida albicans.

Abstract: Aim To evaluate and compare antimicrobial effect of various root canal medicaments against Enterococcus faecalis, Staphylococcus aureus and Candida albicans. Materials and methods Six root canal medicaments: 2% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) Calcium hydroxide (Ca(OH)2), EDTA, MTAD and propolis and three microorganisms: Staphylococcus aureus, Enterococcus faecalis and Candida albicans were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 hours. For the agar diffusion test (ADT), petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were spread on the medium, obtaining growth injunction. Paper disks were immersed in the experimental solutions for 1 minute. Subsequently, four papers disks containing one of the substances were placed on the BHI agar surface in each agar plate. The plates were incubated at 37°C for 48 hours. The diameter of microbial inhibition was measured around the papers disks containing the substances. One way ANOVA followed by Tukey's post-hoc test were used. p-value >0.05 was considered statistically significant. Results Propolis and other irrigants were found to be effective on C. albicans, S. aureus and E. faecalis. CHX and MTAD were found to be most effective amongst all the materials tested followed by propolis. Conclusion Propolis showed antimicrobial activity against E. faecalis, S. aureus, C. albicans. It appears that propolis is an effective intracanal irrigant in eradicating E. faecalis and C. albicans. Clinical significance Propolis is an effective intracanal irrigant in eradicating E. faecalis and C. albicans. It could be used as an alternative intracanal medicament.

Bacterial mutagenicity assays: test methods.

Abstract: The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems.

Screening for phosphate solubilizing bacteria inhabiting the rhizoplane of rice grown in acidic soil in bangladesh

Abstract: The objectives of the research were to isolate phosphate solubilizing bacteria (PSB) from the rhizoplane of rice (Oryza sativa L.) cv. BRRIdhan 29 cultivated in acidic soils of Tangail in Bangladesh and evaluate their performances in phosphate solubilization in both in vitro and in vivo conditions. A total of 10 bacterial strains were isolated and purified by repeated streak culture on nutrient agar medium. Upon screening, five isolates (OS01, OS03, OS07, OS08 and OS10) showed varying levels of phosphate solubilizing activity in agar plate and broth assays. Among them, the strain OS07 (B1) and two previously isolated PSB strains B2 and B3 were selected for evaluation for their performances in rice alone or in combination of TSP (triple super phosphate: P1) and rock phosphate (P2). Plant height and the number of tillers per plant were significantly increased by all PSB isolates when used in combination with TSP but PSB alone did not influence much on plant height and the number of tillers except B1. The le...

Inaccuracy of the Disk Diffusion Method Compared with the Agar Dilution Method for Susceptibility Testing of Campylobacter spp.

Abstract: The agar dilution method has been standardized by the CLSI for the susceptibility testing of Campylobacter species, and according to these standards, the disk diffusion method should be used only in screening for macrolide and ciprofloxacin resistance. Nevertheless, the disk diffusion test is currently widely used, since it is easy to perform in clinical microbiology laboratories. In this study, the disk diffusion method was compared to the agar dilution method by analyzing the in vitro activities of seven antimicrobial agents against 174 Campylobacter strains collected in Finland between 2003 and 2008. Recommendations of the CLSI were followed using Mueller-Hinton agar plates with 5% of sheep blood. For each strain, the disk diffusion tests were performed two to four times. Of the 33 erythromycin-resistant strains (MIC, ≥16 μg/ml), 24 (73%) constantly showed a 6-mm erythromycin inhibition zone (i.e., no inhibition), while for seven strains the inhibition zone varied from 6 to 44 mm in repeated measurements. Among the 141 erythromycin-susceptible strains (MIC, <16 μg/ml), erythromycin inhibition zones varied between 6 and 61 mm. Of the 87 ciprofloxacin-resistant strains, 47 (54%) showed 6-mm inhibition zones, while 40 strains showed inhibition zones between 6 and 60 mm. Significant differences between the repetitions were observed in the disk diffusion for all antimicrobial agents and all strains except for the macrolide-resistant strains regarding the macrolides. For 17 (10%) strains, the variation in repeated measurements was substantial. These results show that the disk diffusion method may not be a reliable tool for the susceptibility testing of Campylobacter spp. Further studies are needed to assess whether the disk diffusion test could be improved or whether all susceptibilities of campylobacters should be tested using an MIC-based method.

Effects of cholesterol on Helicobacter pylori growth and virulence properties in vitro.

Abstract: Background: Colonization of the gastric mucosa by Helicobacter pylori is often associated with chronic gastric pathologies in humans. Development of disease correlates with the presence of distinct bacterial pathogenicity factors, such as the cag type IV secretion system (cag-T4SS), the vacuolating cytotoxin (VacA), or the ability of the bacteria to acquire and incorporate cholesterol from human tissue. Materials and Methods: The in vitro growth of H. pylori requires media (Brucella broth) complemented with vitamins and horse serum or cyclodextrins, prepared as blood agar plates or liquid cultures. Liquid cultures usually show a slow growth. Here, we describe the successful growth of H. pylori strains 26695, P217, P12, and 60190 on serum-free media replacing serum components or cyclodextrins with a commercially available cholesterol solution. Results: The effects of cholesterol as a substitute for serum or cyclodextrin were rigorously tested for growth of H. pylori on agar plates in vitro, for its general effects on bacterial protein synthesis (the proteome level), for H. pylori’s natural competence and plasmid DNA transfer, for the production of VacA, and the general function of the cag-pathogenicity island and its encoded cag-T4SS. Generally, growth of H. pylori with cholesterol instead of serum supplementation did not reveal any restrictions in the physiology and functionality of the bacteria except for strain 26695 showing a reduced growth on cholesterol media, whereas strain 60190 grew more efficient in cholesterol- versus serum-supplemented liquid medium. Conclusions: The use of cholesterol represents a considerable option to serum complementation of growth media for in vitro growth of H. pylori.

Rapid identification of yeast isolates from clinical specimens in critically ill trauma ICU patients.

Abstract: Purpose: The purpose was to evaluate the performance of a commercially available chromogenic Candida speciation media and the Vitek 2 ID system for the identification of medically important yeasts and yeast-like organisms in a routine clinical microbiology laboratory. Materials and Methods: A total of 429 non duplicate, consecutive yeast strains were included during the 3.5-year study period. The performance of the Vitek 2 ID system and a chromogenic agar medium was evaluated against the gold standard conventional phenotypic and biochemical identification method for speciation of yeast isolates from trauma patients. Results: Candida tropicalis (64%) was the most common Candida species, followed by Candida albicans (14%), Candida rugosa (7%), and Candida parapsilosis (6.5%). Of the 429 isolates, 183 could be identified to species level by all the three methods. Agreement between the chromogenic agar method and conventional methods was 80% for Candida tropicalis , 100% for Candida rugosa , 89% for Candida albicans, and 77% for Candida parapsilosis . Vitek 2 had lower sensitivity, with agreement of 49% for Candida tropicalis , 100% for Candida rugosa , 39% for Candida albicans , and 31% for Candida parapsilosis . Conclusion: Thus, in long-term ICU patients, an increasing trend of isolating nonalbicans Candida spp. continues. The chromogenic agar medium is a convenient and economic method to identify commonly isolated species in busy clinical microbiology laboratories.

Construction of the mutant strain in Aspergillus oryzae 3.042 for abundant proteinase production by the N+ ion implantation mutagenesis

Abstract: Summary Because of secreting large amounts of enzymes, Aspergillus oryzae was widely used in the fermentation of soy sauce in many Asian countries. Here, N+ ion implantation mutagenesis was applied to improve the ability of A. oryzae to secrete acid protease. Several mutants were screened using three kinds of plates (Casein medium, Czapek’s medium and carboxymethyl cellulose medium agar plates). Compared with the other mutants, the mutant A100-8 could secrete the most protease. Acid protease activity of A100-8 was enhanced about 44.1% at 36 h by koji fermentation test. In addition, A100-8 was stable by periodically subculturing and maintaining on the agar slant. The mRNA expression levels of two kinds of acid protease (serine carboxypeptidase and aspartyl protease) were measured by RT-PCR at different periods. Serine carboxypeptidase and some kinds of aspartyl protease of A100-8 were significantly (P < 0.01) expressed higher than the control at all times.

Suitability of MRS-bile agar for the selective enumeration of mixed probiotic bacteria in presence of mesophilic lactic acid cultures and yoghurt bacteria

Abstract: Measuring the viability of probiotic microorganisms in food products using plate count methodology is a common practice due to the simplicity (ease of performance), inexpensive and routine testing characters of this method. In present study, the suitability of de man rogosa and sharpe agar (MRS) bile agar medium for the selective enumeration of mixed probiotic bacteria (Lactobacillus acidophilus LA-5, L. casei 431 and Bifidobacterium lactis BB-12) in presence of mesophilic lactic cultures (Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris) and yoghurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) was investigated. Yoghurt bacteria did not grow neither in presence of 0.15% nor 0.30% of bile salts, as was expected. Mesophilic lactic starters could grow at both concentrations of bile salts at all incubation temperatures except 37°C. According to these results, MRS-bile agar (0.15 bile salts) could be successfully used for selective enumeration of mixed probiotic cultures in presence of mesophilic culture and/or yoghurt bacteria when plates were incubated at 37°C for 72 h.

Screening of chitinolytic actinomycetes for biological control of Sclerotium rolfsii stem rot disease of chilli

Abstract: Two hundred and eighty three strains were isolated from rhizoshere-associated soils, from Ubon Ratchathani and Srisaket province, using Enrichment Media for isolation of Chitinase-producing Actinomycetes agar (EMCA agar). All strains were screened for chitinolytic activity and sixty eight strains gave significant clear zone on EMCA agar plates. The selected chitinolytic strains were assayed for in vitro antagonism against Sclerotium rolfsii using cornmeal agar (CMA agar) assay procedure and the result showed that thirteen isolates have remarkable inhibiting the growth of the fungus and the top five antagonistic actinomycetes were PACCH 277, PACCH129, PACCH225, PACCH24 and PACCH246, respectively. The result indicated that these actinomycetes produce chitinase which catalyze the degradation of chitin, resulting in inhibition of S. rolfsii growth. Their abilities to control the disease development were tested for in vivo biocontrol assay on chilli seedlings. Two out of thirteen candidate, PACCH24 and PACCH225, antagonists reduced the disease development at 90%. It was suggested that the ability to inhibit the growth of pathogen in vitro was not related to the disease reduction in vivo. The strain PACCH24 was further identified as Streptomyces hygroscopicus according to morphological characteristic, cell wall and cellular sugar analysis and 16S rDNA sequencing. The study implies a novel chitinolytic actinomycete which could be developed to be a biological agent which would be included as a complement with organic fertilizers in order to control stem rot disease and promote growth of chilli.

Proteolytic activity of alkaliphilic, salt-tolerant actinomycetes from various regions in Saudi Arabia

Abstract: Actinomycetes were isolated from various desert soil samples of Saudi Arabia using alkaline and normal pH media. A total of 42 akaliphilic actinomycetes isolated from yeast extract-soluble starch (YS) agar media (pH 11.0 ± 1) and 102 actinomycetes were isolated from tap water agar media (pH 7.0 ± 1) for comparison. Aerial mycelium colours of actinomycetes were observed for the detection of strain varieties in the different soil samples from different areas. Colour types were more abundant in isolates grown tap water agar (TWA) than in isolates grown on YS agar medium (pH 11.0). On the TWA medium, there were 18 colour types of actinomycetes and on YS agar medium (pH 11.0) there were 10 colour types. The number of neutrophilic actinomycetes isolated in this study is higher than the alkaliphilic actinomycetes. Among the 42 alkaliphilic actinomycetes isolates, 30 isolates were salt tolerant alkaliphilic. Later about 16 alkaliphilic actinomycetes were screened on skim milk agar for preliminary proteolytic activity. A total of 10 isolates showed proteolytic activity. Of the 10 isolates, three isolates were alkaliphilic and seven isolates were salt-tolerant alkaliphilic. All the isolates need to be further studied for the ability of their potential protease enzyme production.

Biodegradation of poly(β-hydroxybutyrate) by a novel isolate of Streptomyces bangladeshensis 77T-4.

Abstract: A thermophilic Streptomyces bangladeshensis isolate designated 77T-4 that can degrade poly(β-hydroxybutyrate) (PHB) was isolated from a soil sample in Taiwan. Isolate 77T-4 grew well in urea fructose oatmeal medium and produced a clear zone around the colony when grown on an agar plate containing emulsified PHB, indicating the presence of an extracellular PHB depolymerases. A PHB-degrading enzyme was purified to homogeneity from the culture supernatant. The molecular weight of the PHB-degrading enzyme was estimated to be approximately 40 kDa. A small portion of the N-terminal sequence (Ala-Val-Pro- Leu-Thr-Arg) of the purified enzyme was determined and found to share a significant homology with that of the PHB depolymerase of Streptomyces sp. MG.

Seed borne mycoflora of legume seeds

Abstract: S The seed mycoflora of different varieties of Legumes was screened by standard blotter paper, agar plate and seed washates methods. Among the three methods, the agar paper method was found to be suitable as in less incubation; there was higher percent incidence of seed mycoflora. The different cultivars of Legumes screened for seed mycoflora were TLG - 45, LGN -1-1, BDN - 9 - 4, N - 59, BSMR - 853, BSMR - 736, BPMR-145, S-8, S-1-1 and T - 9. The Cv. TLG- 45 shows higher percent seed mycoflora with sixteen fungi as compared to other cultivars.

Evaluation of Plant Extracts as Antifungal Agents Against Fusarium solani (Mart.) Sacc.

Abstract: Experiments were carried out to test the aqueous extracts of twenty plants for their antifungal activity against Fusarium solani the causal of dry rot disease of potato. The obtained results showed a differential activity of the plant extracts against the mycelium growth. The combined leaf extracts of Lawsonia alba and stem extracts of Acacia catechu in general showed a strong enhancement in activities over the individual extract of each against the mycelium growth. The seed extracts of Dedonia viscosa also showed strong inhibitory effect against the test fungi. The petal extracts of Mimosa hamata, leaf extracts of Acacia arabicae, Jacranda mimosaefolia and Ocimum sanctum showed appreciable good inhibitory effect against the test fungi. Potato (Solanum tuberosum L.) is the world's fourth kept in dark between the filter papers at room temperature important food crop after wheat, rice and maize because of till completely dry. Each plant sample was individually its great yield potential and high nutritive value for grounded into powder for preparation of extract. The human. FAO data show that in 2005, for the first time, the fungus Fusarium solani (IARI 5155 F) used for the study developing world's potato production exceeded that of was obtained from the Division of Plant Pathology, IARI, the developed world and India ranks third contributing New Delhi. The fungal cultures were maintained at 4°C on around 7.5% to the world's production. Ever increasing Yeast Glucose Agar medium with periodic sub-culturing. world population requires the production of huge quantity of potato but our efforts are hampered due to Antifungal Activity Test: The plant part extract (15% w/v) various biotic as well as abiotic factors. Among the biotic was prepared by brewing in hot water. Fifteen g dry factors, Fusarium solani (Mart.) Sacc. was first described powder of each plant sample was weighed and put in a by C.F.P. Von Martius in 1842 from rotted tubers of potato cheesecloth bag and suspended in 100ml of boiling (1). Dry rot of potato caused by Fusarium solani is an distilled water for 20 minutes. The extract was allowed to internationally important disease of potato resulting in stand for some time and decanted off into the flask and about 25 to 60% loss in yield in different countries and final volume was raised to 100ml by adding boiled distilled attempts have been made to manage the disease by water. The supernatant was used for assay. The treating with chemical compounds, biological agents as antifungal activity of each plant part extract was reported by Wharton and Kirk (2). In the present study, determined by measuring the mycelia growth inhibition of efficacy of twenty plant extracts including combined tested fungi as described by Bragulat et al. (3). A known plants extracts for antifungal activity against dry rot volume of 15% plant sample extract was supplemented pathogen was tested. with yeast extract, glucose and agar. The medium was

Antibacterial and antifungal activities of Actinobacteria isolated from Rathnagiri hills

Abstract: Article history: Received on: 18/08/2012 Revised on: 29/08/2012 Accepted on: 05/09/2012 Available online: 29/10/2012 This study was performed to isolate actinomycete colonies having antibacterial and antifungal activity from soil samples. A total of 27 actinomycete colonies were isolated in pure culture from five soil samples using Starch casein agar medium. Entire isolates were screened for their antimicrobial activity by agar plug method against five each of human pathogenic bacteria and fungi. Of this, 7 strains inhibits B. substilis, 3 strains inhibits Klebseilla sp, 6 strains inhibits B. cerus, 5 strains inhibits S. aureus and only 2 strains inhibits E. coli. Incase of fungi all the actinobateria has moderate activity with less fungal strains, only 1 strain (RA 5) inhibits entire fungus except Penicillium sp. The metabolites from potent strain was produced by fermentation, separated by centrifugation, it was tested for their antimicrobial activity against the test bacterial and fungal strains by well diffusion and disc diffusion method. In this study, the metabolites from RA5 (identified as Streptomyces sp.) have showed good antibacterial and antifungal activity. Since many isolates showed inhibitory activity against indicator bacteria, it is suggestive that Rathnagiri hill’s soil could be an interesting source to explore for antibacterial secondary metabolites.

Antimicrobial Activity of Chestnut Extracts for Potential Use in Managing Soilborne Plant Pathogens.

Abstract: Chestnut extracts were studied for antimicrobial activity against selected microorganisms, including plant pathogens. Chestnut extract on paper discs was applied to an agar medium to evaluate the inhibition to multiple microorganisms or the extract was added at various concentrations to a culture medium to evaluate the growth of target microorganisms. Chestnut type, tissue of plants (shell, pellicle, and leaf), extraction methods, and physical characteristics were studied to determine antimicrobial activity. Most test microorganisms were inhibited by the extracts at different effective concentrations for 50% growth inhibition (EC50). Pseudomonas fluorescens was the most sensitive (EC50 = 4.4 μg/μl), Phytophthora cambivora was one of the least inhibited (EC50 = 185 μg/μl), and Cryphonectria parasitica was not inhibited. Extracts of the Japanese × European chestnut (Castanea crenata × C. sativa) 'Colossal' showed a greater inhibition than those of wild trees of the Chinese species (C. mollissima). High temperature did not affect the inhibitory effect. Extracts from chestnut pellicle had the highest concentration of antimicrobial compound, compared with leaf and shell. The active fraction contained several substances with molecular masses consistent with one flavonol glycoside and several terpenoid substances. Pellicle and shell tissue reduced radish scab disease caused by Streptomyces scabies in the greenhouse.

Investigation of Probiotic Chocolate Effect on Streptococcus mutans Growth Inhibition

Abstract: Background: One of the most important factors in inducing the logarithmic growth of Streptococcus mutans, is a diet containing fermentable carbohydrates such as sucrose. Objectives: The aim of the current research was to compare the ability of ordinary and probiotic chocolate to induce or inhibit the growth of S. mutans. Materials and Methods: Lactobacillus rhamnosus, L. plantarum and L. acidophilus as probiotic strains, were cultivated on MRS agar for 24 hours at 35 C in 5% CO2. S. mutans which is a dominant factor in causing dental plaque, was isolated from 20 samples of dental plaque and caries lesions in adults on Streptococcus selective agar medium, and diagnosed by routine biochemical tests. The antimicrobial effect of three probiotic strains on S. mutans was evaluated by the deferred cross-streak method and susceptibility through the disk diffusion test. The antimicrobial effect of the probiotic supernatant powder was determined by a dilution method. Probiotic strains were added to dark chocolate with a concentration of 108 CFU/mL and their antimicrobial effect on S. mutans was evaluated by the disk diffusion susceptibility method. Survival of the probiotic strains in chocolate and pH shifts were studied in different environmental storage conditions. Results: The results showed that S. mutans was the dominant strain in all of the 20 dental plaque samples. L. plantarum showed the most antimicrobial effect on S. mutans with the maximum diameter of growth inhibitory zone, 35 mm and 78 78 mm in the disk diffusion method and deferred cross-streak method, respectively. Probiotic supernatant powder inhibited S. mutans strains in concentrations of 500-700 and 100-300 mg/ml at t = 0 and t = 24, respectively. Comparing the results in terms of maintenance and storage of probiotic chocolate, it showed that the best condition to keep this chocolate is at 4? C (refrigerated) with a probiotic survival of 25 to 30 days. The pH level during this period decreased from a pH of 5 to 4. Probiotic chocolate containing L. rhamnosus was shown to have the greatest antimicrobial effect on S. mutans with a maximum diameter of growth inhibitory zone of 75 mm during 59 days storage at an ambient temperature and 4 C. Conclusions: These results suggest that probiotic chocolate is able to inhibit the growth of S. mutans rather than ordinary chocolate.

Detection of Legionella spp. in Natural and Man-made Water Systems Using Standard Guidelines

Abstract: Infections caused by Legionella spp. are considered at the present time, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of water from 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to char- acterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the tech- nique of cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, for detection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtrate was resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria was performed. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days) at 37 ℃. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agar for verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYE agar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as be- longing to the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, corresponding therefore to "false-positive" results. The methods used in this study allowed the isolation of a number of isolates (40), which form an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic dis- tance might be a genus not yet described and therefore a new species. The results obtained, highlighted the importance of using culture and genetic methods in parallel for the proper identification of microorganisms. Keywords Water, Legionella, ISO 11731:1998, False positives, 16S rRNA gene sequencing

Trace Amounts of Furan-2-Carboxylic Acids Determine the Quality of Solid Agar Plates for Bacterial Culture

Abstract: Background Many investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed. Methodology/principal findings According to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L(-1) (13 and 21 nmol L(-1)), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium. Conclusions/significance Agar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.

Screening of alkaline protease production by fungal isolates from different habitats of Sagar and Jabalpur district (M.P)

Abstract: Fungal isolates were screened for alkaline protease production on casein containing agar plates and identified by clear zones of casein hydrolysis around colonies. Totally 141 fungal isolates were used in primary screening of alkaline protease and out of these, only 108 test fungi were found to produce alkaline protease activity as indicated by production of zone of casein hydrolysis around the fungal colonies grown in pH 9 after 5 d of incubation at 28C. Thirty three test fungi were found to grow on casein containing medium (pH 9) but were unable to produce zone of hydrolysis. For the production of extracellular alkaline proteases in liquid cultures, a total of 10 proteolytic fungi were selected which showed relative enzyme activity (REA) in the range of ≥ 2.29–3 based on the screening results of their REA on solid media. Maximum enzyme activity of 119.48 U/mL was found in Aspergillus versicolor PF/F/107 at pH 9 and temperature of 28oC after an incubation of 96 h in orbital shaker (150 rpm). Enzymes produced from microorganisms that can survive under extreme pH could be particularly useful for commercial applications under high alkaline conditions.