Showing papers on "Agar plate published in 2006"
TL;DR: The characteristic morphology is not evident, and hemolysis is poor on HuBA, so HuBA is not recommended for use for the isolation or the susceptibility testing of any of these organisms.
Abstract: Human blood agar (HuBA) is widely used in developing countries for the isolation of bacteria from clinical specimens. This study compared citrated sheep blood agar (CSBA) and HuBA with defibrinated horse blood agar and defibrinated sheep blood agar (DSBA) for the isolation and antibiotic susceptibility testing of reference and clinical strains of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Reference and clinical strains of all organisms were diluted in brain heart infusion and a clinical specimen of cerebrospinal fluid and cultured on all agars. Viable counts, colony morphology, and colony size were recorded. Susceptibility testing for S. pneumoniae and S. pyogenes was performed on defibrinated sheep blood Mueller-Hinton agar, citrated sheep blood Mueller-Hinton agar (CSB MHA), and human blood Mueller-Hinton agar plates. For all organisms, the colony numbers were similar on all agars. Substantially smaller colony sizes and absent or minimal hemolysis were noted on HuBA for all organisms. Antibiotic susceptibility results for S. pneumoniae were similar for the two sheep blood agars; however, larger zone sizes were displayed on HuBA, and quality control for the reference strain failed on HuBA. For S. pyogenes, larger zone sizes were demonstrated on HuBA and CSBA than on DSBA. Poor hemolysis made interpretation of the zone sizes difficult on HuBA. CSBA is an acceptable alternative for the isolation of these organisms. The characteristic morphology is not evident, and hemolysis is poor on HuBA; and so HuBA is not recommended for use for the isolation or the susceptibility testing of any of these organisms. CSB MHA may be suitable for use for the susceptibility testing of S. pneumoniae.
79 citations
TL;DR: The most productive strain, identified as Colletotrichum gloesporioides, produced 27,700 U/l of lipase under optimized conditions and the crude lipase preparation was capable of hydrolysing a broad range of substrates including lard, natural oils and tributyrin.
Abstract: Fifty-nine lipase-producing fungal strains were isolated from Brazilian savanna soil by employing enrichment culture tecniques. An agar plate medium containing bile salts and olive oil emulsion was employed for isolating and growing fungi in primary screening assay. Twenty-one strains were selected by the ratio of the lipolytic halo radius and the colonies radius. Eleven strains were considered good producers under conditions of submerged liquid fermentation (shaken cultures) and solid-state fermentation. The most productive strain, identified as Colletotrichum gloesporioides, produced 27,700 U/l of lipase under optimized conditions and the crude lipase preparation was capable of hydrolysing a broad range of substrates including lard, natural oils and tributyrin.
76 citations
TL;DR: To compare agar plate and real‐time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces.
Abstract: Aims: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces.
Methods and Results: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g−1 faeces) for 48-h incubation at 37°C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp® DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTectTM SYBR® green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0·78, P < 0·01) and total anaerobic bacteria (R2 = 0·21, P < 0·05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0·83 × log(CFU) + 1·43 and log(DNA copy) = 1·62 × log(CFU) − 6·32 respectively.
Conclusions: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5–6 days.
Significance and Impact of the Study: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.
69 citations
TL;DR: A simple method for the analysis of aerobic bacteria in complex samples such as broth and food homogenates using commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers.
Abstract: A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given.
60 citations
TL;DR: It was found that fungal growth rate on straw-based agar plates per se was indicative of the ability of white-rot fungi to establish in the presence of a competing fungus, but was a poor indicator of the fungus’ ability to degrade PAHs.
60 citations
TL;DR: The objective of this study was to compare, by two experimental methods, the antimicrobial efficacy of different root canal filling pastes used in pediatric dentistry.
Abstract: The objective of this study was to compare, by two experimental methods, the antimicrobial efficacy of different root canal filling pastes used in pediatric dentistry. The tested materials were: Guedes-Pinto paste (GPP), zinc oxide-eugenol paste (OZEP), calcium hydroxide paste (CHP), chloramphenicol + tetracycline + zinc oxide and eugenol paste (CTZP) and Vitapex. Fiven microbial strains (S. aureus, E. faecalis, P. aeruginosa, B. subtilis and C. albicans) obtained from the American Type Culture Collection were inoculated in Brain Heart Infusion (BHI) and incubated at 37 degrees C for 24 h. For the direct exposure test (DET), 72 paper points were contaminated with the standard microbial suspensions and exposed to the root canal filling pastes for 1, 24, 48 and 72 h. The points were immersed in Letheen Broth (LB), followed by incubation at 37 degrees C for 48 h. An inoculum of 0.1 mL obtained from LB was then transferred to 7 mL of BHI, under identical incubations conditions and the microbial growth was evaluated. The pastes showed activity between 1 and 24 h, depending on the material. For the agar diffusion test (ADT), 30 Petri plates with 20 mL of BHI agar were inoculated with 0.1 mL of the microbial suspension, using sterile swabs that were spread on the medium. Three cavities were made in each agar plate (total = 90) and completely filled with one of the filling root canal pastes. The plates were pre-incubated for 1 h at room temperature and then incubated at 37 degrees C for 24 to 48 h. The inhibition zone around each well was recorded in mm. The complete antimicrobial effect in the direct exposure test was observed after 24 h on all microbial indicators. All root canal filling materials induced the formation of inhibition zones, except for Vitapex (range, 6.0-39.0 mm).
59 citations
TL;DR: Evaluated parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.
Abstract: Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin. The performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay. The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.
54 citations
TL;DR: The ethyl-cyanoacrylate has bacteriostatic and bactericidal action in vitro, mainly against gram-positive microorganism tested and showed bactericidal effect over 70% for all of them.
Abstract: Purpose To analyze the antimicrobial properties of ethyl-cyanoacrylate (Superbonder, Loctite, Brazil) in vitro in different microorganisms related to corneal infections. Methods We analyzed the following microorganisms: (1) Staphylococcus aureus (multiresistant); (2) S. aureus (ATCC 25923); (3) coagulase-negative Staphylococcus; (4) Streptococcus pyogenes; (5) Streptococcus pneumoniae; (6) Pseudomonas aeruginosa (multiresistant); (7) P. aeruginosa (ATCC 27853); (8) Escherichia coli (ATCC25922); and (9) Enterococcus faecalis (ATCC 29212). One drop of the glue was dropped directly into the nutrient broth. The plates were incubated at 35 +/- 2 degrees C and its growth examined after 24 hours. Bactericidal activity of the glue was verified by sampling inhibition zones when present. The samples were plated in blood agar an analyzed after 24 and 48 hours. Results The ethyl-cyanoacrylate inhibited the growth of every gram-positive microorganism tested and showed bactericidal effect over 70% for all of them. Among the gram-negative microorganisms, only the E. coli and the E. faecalis had its growth inhibited, and the bactericidal effect was 60% and 40%, respectively. Conclusion The ethyl-cyanoacrylate has bacteriostatic and bactericidal action in vitro, mainly against gram-positive microorganisms.
50 citations
Journal Article•
TL;DR: To evaluate the occurrence of mycotic and protohecal mastitis in herds in south-eastern part of Poland, milk samples from udder quarters with clinical and subclinical mastitis from 29 dairy herds was investigated.
Abstract: The aim of the study was to evaluate the occurrence of mycotic and protohecal mastitis in herds in south-eastern part of Poland. A total of 3091 milk samples from udder quarters with clinical and subclinical mastitis from 29 dairy herds was investigated in this survey. Milk samples were plated as soon as possible on blood agar (BA), Mac Conkey agar, aesculin-tallium acetate-crystal violet agar, and Sabouraud agar. A hundred and thirty one yeast (4.23%) and eleven Protoheca zopfii (0.35%) strains were isolated from cows with clinical and subclinical mastitis. All the isolated fungi were the yeast classified into 4 genera (Candida, Trichosporon, Saccharomyces and Rhodotorula). The most frequently isolated yeasts were Candida sp., C. kefyr, C. humicola, C. rugosa and C. inconspicua. Both fungi and algae were isolated first of all during a confinement-housing season.
48 citations
TL;DR: Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.
47 citations
TL;DR: Xanthan gum, microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used alone and in combination with agar for microbial culture media and growth of most of the fungi was better on xanthan and xantha + agar media than agar medium.
Abstract: Xanthan gum, microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used alone and in combination with agar for microbial culture media. As illustrative examples, eight bacteria and eight fungi were grown on media solidified with either agar (A, 1.5%), xanthan gum (X, 1%), or combinations of both (0.9% X + 0.1% A, 0.8% X + 0.2% A, 0.7% X + 0.3% A, 0.6% X + 0.4% A). All fungi and bacteria exhibited normal growth and differentiation in all these treatments. Rather, growth of most of the fungi was better on xanthan (alone) and xanthan + agar media than agar medium. As the media gelled with xanthan gum alone flow, it was not possible to incubate Petri plates in inverted position. Moreover, because of the softness, streaking of bacteria was difficult on such media. However, these problems could be overcome by partially replacing xanthan gum with 0.3% agar. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method on agar (1.5%), 0.7%/0.6% X + 0.3%/0.4% A yielded similar counts. Selective media, succinate medium for Pseudomonas sp., and MacConkey broth medium for Escherichia coli gelled with 0.7%/0.6% X + 0.3%/0.4% A did not support growth of other bacteria when inoculated along with the above-mentioned bacteria. Likewise, differential medium, CRMA (Congo red mannitol agar) gelled with xanthan-agar combination could differentiate between Agrobacterium tumefaciens and Rhizobium sp.
Journal Article•
TL;DR: The effect of crude extracts of neem (Azadirachta indica) leaf, neem seed and garlic (Allium sativum) at concentrations ranging from 5% to 30% of the material in 100 ml of Potato Dextrose Agar on mycelial growth of Fusarium oxysporum f.
Abstract: The effect of crude extracts of neem (Azadirachta indica) leaf, neem seed and garlic (Allium sativum) at concentrations ranging from 5% to 30% of the material in 100 ml of Potato Dextrose Agar on mycelial growth of Fusarium oxysporum f. sp. lycopersici was assessed. All the extracts inhibited mycel- lial growth at various levels. Dry neem seed extract gavel 100% inhibition of mycelial growth. Fresh neem leaf extract reduced mycelial growth with increasing concentration while in garlic there were no differences in growth inhibition among the various concentrations used. However garlic extracts de- creased sporulation with increasing concentration and cultures grown on extract amended agar plates remained viable.
TL;DR: A hyaluronic acid (HA)-producing strain, Streptococcus zooepidemicus NJUST01, can grow in a serum-free agar medium, with starch as exclusive carbon source, but not glucose, sucrose, dextrine, xylose, or lactose, but colonies of the strain after 60th generation could not produce a clear hemolytic zone on sheep blood agar plates.
Abstract: A hyaluronic acid (HA)-producing strain, Streptococcus zooepidemicus NJUST01, can grow in a serum-free agar medium, with starch as exclusive carbon source, but not glucose, sucrose, dextrine, xylose, or lactose. In this starch medium, the strain NJUST01 reproduced successively at 37°C for 60 generations, with no obvious variation on morphology and physiology, but colonies of the strain after 60th generation could not produce a clear hemolytic zone on sheep blood agar plates. Hyaluronic acid production by the strain NJUST01 was analyzed relative to the starch medium. Employing a multifactor cross experiment, an optimum medium revealed for hyaluronic acid production was composed of 5% starch, 0.3% glucose, 0.5% peptone, 0.15% MgSO4, and 2.0% K2HPO4. The amount of HA 6.7 g/l was obtained in batch fermentation on a rotary shaker at 37°C, 220 rpm for 36 h.
TL;DR: This study shows that development of larvae of C. macellaria does not depend on bacteria and that some bacterial isolates negatively impact larval development.
Abstract: Bacteria were isolated and identified from the digestive tract of the secondary screwworm, Cochliomyia macellaria (F.) (Diptera: Calliphoridae), and their role in the larval development of this insect was assessed in laboratory bioassays. The analysis of 16S rDNA sequences revealed that the bacterial isolates represented four species: Providencia sp., Escherichia coli O157:H7 (Escherich), Enterococcus faecalis (Orla-Jensen), and Ochrobactrum sp. (Holmes). Developmental assays demonstrated that C. macellaria larvae are able to develop on a sterile blood agar, and no bacteria are required to complete larval development. Indeed, developmental times were shorter and survival rates of C. macellaria on a sterile blood agar and the modified Harris rearing diet were greater compared with that on the blood agar inoculated with individual and mixed bacterial isolates. The cultures of Ochrobactrum sp. and E. faecalis supported larval development to a significantly greater extent than those of Providencia sp. and E. coli O157:H7. The presence of bacteria in newly emerged C. macellaria adults also was assessed and revealed that the bacteria in the gut of larvae can survive pupation and colonize the gut of adult flies. This study shows that development of larvae of C. macellaria does not depend on bacteria and that some bacterial isolates negatively impact larval development.
01 Jan 2006-Food Additives and Contaminants Part A-chemistry Analysis Control Exposure & Risk Assessment
TL;DR: The proposed method detects β-lactams, sulfonamides, tetracyclines, aminoglycosides, macrolides and quinolones at MRL concentrations and reliably indicates the inhibitor family.
Abstract: This paper proposes an improved high throughput microbial method for the simultaneous performance of first and second level screening for antibacterial residues in meat. It is based on growth inhibition of B. subtilis on agar medium pH 6, 7.2 and 8, of B. cereus on agar medium pH 5.9, of M. luteus on agar medium pH 8 and of E. coli on agar medium pH 7.2 (research or first level screening) and on the use of confirmatory solutions (Pase, Paba, MgSO4) for the identification or second level screening. In kidney control samples, dialysis membranes were interposed between samples and the agar surface to both prevent the action of lysozyme and reduce false positive results. The proposed method detects beta-lactams, sulfonamides, tetracyclines, aminoglycosides, macrolides and quinolones at MRL concentrations and reliably indicates the inhibitor family. Results are obtained in 18-24 h.
TL;DR: This study characterises the bacteria associated with a marine hatchery in Tunisian coastal marine waters and found V. alginolyticus was the dominant bacteria found in rotifers (Brachionus plicatilis) andArtemia cultures (Artemia salina), and an increase ofVibrionaceae was noted after larvae were fed withArtemia.
Abstract: This study characterises the bacteria associated with a marine hatchery in Tunisian coastal marine waters. Presumptive vibrios (TCBS agar) and heterotrophic aerobic microflora (CFU) were studied at different stages within the hatchery: seawater, batches of algal cultures, rotifers andArtemia culture tanks. The bacterial strains were isolated on TCBS Agar plates and described using different bacteriological tests (standardised micromethods “API 20 E Strips”, exoenzymes production, growth at different temperatures, pH and salinity, vibriostatic agent O/129 and antibiotics susceptibility). Two dominant genera of bacteria were found (Vibrio andAeromonas) associated with some strains of thePseudomonadaceae family.Vibrio alginolyticus was the dominant bacteria (75% of total isolates) found in rotifers (Brachionus plicatilis) andArtemia cultures (Artemia salina). In larvae rearing tanks, an increase ofVibrionaceae was noted after larvae were fed withArtemia. Most of the studied bacteria used the skin mucus ofSparus aurata larvae as their sole source of carbon. All theV. alginolyticus strains were β-haemolytic, hydrolyse the DNA and were susceptible to several tested antibiotics.
TL;DR: Test medium-dependent variation of MICs and the applicability of LSM+cys for antimicrobial susceptibility testing of bifidobacteria is described.
TL;DR: High-throughput genotyping of Aspergillus oryzae was achieved using an FTA card for the extraction of a genomic DNA template for polymerase chain reaction from a fungal colony growing on an agar plate.
TL;DR: Issues with the identification of Burkholderia pseudomallei with the new VITEK 2 GN card are highlighted and Laboratory personnel need to be aware of the current limitations with this GN card and the software and rely on clinical history, a high index of suspicion, and basic microbiology tests to confirm the identification.
Abstract: The use of automated systems in the modern microbiology laboratory is becoming commonplace as the pressure of cost containment impacts on staff resources. With increased international travel and threats of bioterrorism, recognition and accurate identification of organisms such as Burkholderia pseudomallei is important. In areas where this organism is endemic, identification is not usually problematic. This study evaluates the performance of the new VITEK 2 colorimetric GN card for the identification of this organism. A total of 103 previously identified clinical isolates were tested with the new card with isolates taken from MacConkey agar, Columbia horse blood agar, Columbia sheep blood agar, and Trypticase soy agar in order to determine identification performance and to see if there was any variability in results due to the agar. Columbia horse blood agar produced the highest rates of identification (81%), followed by Trypticase soy agar (78%), Columbia sheep blood agar (75%), and MacConkey agar (63%). There was considerable variability in some of the reactions obtained. Seven isolates failed to identify from any of the agars used. This study highlights issues with the identification of this organism with the new VITEK 2 GN card. Enhancements of the algorithm parameters for the GN card are warranted and are in progress. Laboratory personnel need to be aware of the current limitations with this GN card and the software (version 4.02 or older for the VITEK 2 60/XL and version 1.02 or older for VITEK 2 Compact) and rely on clinical history, a high index of suspicion, and basic microbiology tests to confirm the identification of this organism.
TL;DR: Antimicrobial susceptibilities of Staphylococcus aureus strains can be determined accurately by using isolates from mannitol salt agar, and yellow isolates onMannitol Salt agar at quantities of >1+ can be reported as S. a Aureus.
Abstract: Antimicrobial susceptibilities of Staphylococcus aureus strains can be determined accurately by using isolates from mannitol salt agar, and yellow isolates on mannitol salt agar at quantities of >1+ can be reported as S. aureus. These methods decrease the time to identification/antimicrobial susceptibility testing of S. aureus and decrease costs through eliminating additional testing.
TL;DR: Monitoring of growth and production of metabolite in submerged fermentation showed that metabolite production peaked on the fourth day in Russula sp.
Abstract: Russula sp. and Pycnoporus cinnabarinus (wild mushrooms) were subjected to laboratory cultivation by spore germination and tissue culturing on Sabouraud dextrose agar plates. Subsequently, the growth and production of metabolite(s) were monitored in submerged fermentation for 7days using agar diffusion method. The result obtained showed that metabolite production peaked on the fourth day in Russula sp. and on the fifth day in Pycnoporus cinnabarinus with subsequent decrease in activity of the fermentation extract. Dry weight increases with fermentation time in both mushrooms.
TL;DR: An attempt is made to evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women.
Abstract: Aims: To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women. Methods and Results: The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar. Conclusions: The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples. Significance and Impact of the Study: This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.
TL;DR: Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3‐phosphate dehydrogenase based on their N‐terminal amino acid sequences derived by Edman degradation.
Abstract: Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.
TL;DR: There was a significant reduction in colony-forming units when the cultures were subjected to photodynamic therapy, and the antibacterial effect of diode laser light of wavelength 650 nm, in association with the photosensitive substance toluidine blue, on the bacteria in infected skin ulcers.
Abstract: PURPOSE: To evaluate in vitro the antibacterial effect of diode laser light of wavelength 650 nm, in association with the photosensitive substance toluidine blue, on the bacteria in infected skin ulcers. METHODS: Samples were collected by means of swabs containing a medium for transporting infected material from skin ulcers. The material was inoculated into culturing medium containing azide blood agar for the growth of Gram-positive bacteria, and MacConkey agar for Gram-negative bacteria, and incubated for 48 hours. The results obtained from counting the colony-forming units were correlated and subjected to statistical analysis, adopting the significance level of p³ 0.05. RESULTS: From analysis of variance (ANOVA), the result for the general mean was p = 0.0215. Using the t test with post-hoc test, the result for TBO vs. Control was p = 0.0186, and for TBO + Laser vs. Control it was p = 0.0039. CONCLUSION: There was a significant reduction in colony-forming units when the cultures were subjected to photodynamic therapy.
TL;DR: For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 was constructed and green fluorescent protein (GFP) was displayed on T4e - small outer capside (SOC) protein to detect E. bacteria in the sewage influent.
TL;DR: This study describes a rapid method for the isolation and detection of Escherichia coli O157:H7 present at low levels in fresh salad produce, linked to real-time polymerase chain reaction and/or selective agar plating and has the potential to provide a result within 7 h.
Abstract: This study describes a rapid method for the isolation and detection of Escherichia coli O157:H7 present at low levels (0.04–0.4 cfu/g) in fresh salad produce. Following a short enrichment (5 h at 42C), samples were processed using Pathatrix, a novel recirculating immunomagnetic capture system, linked to real-time polymerase chain reaction and/or selective agar plating. Processing of post-enrichment sample aliquots (10 mL diluted 1:25) and the use of preblocked paramagnetic anti-E. coli O157:H7 beads enhanced target pathogen recovery on selective agar plates in cases where a high microbial load of nontarget microflora was present. This is of particular relevance to the testing of bean sprouts. This method has the potential to provide a result within 7 h. The speed of result offers significant benefits for outbreak investigations and allows the rapid screening of salad produce for E. coli O157:H7 contamination prior to product entering the distribution chain.
TL;DR: The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively, and less than 10% of all species were misidentified using both nutrient media.
TL;DR: A simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment and eliminates the need for streaking cells onto agar, which affects the invasion of cells into the agar.
Abstract: Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8 The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar. Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.
TL;DR: A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp.
Abstract: Hydrocarbon-degrading microorganisms from natural environments have been isolated and identified using culture-dependent or molecular techniques. However, there has been little research into the occurrence of microorganisms incorporated into crude oil in the initial steps of extraction and handling, which can reduce the quality of stored petroleum. In the present study, a packed-column reactor filled with autoclaved perlite soaked with crude oil was subjected to a continuous flow of sterile medium in order to determine the presence of potential hydrocarbon degraders. Microorganisms developed on the surface of the perlite within a period of 73 days. DNA was extracted from the biofilm and then PCR-amplified using 16S rRNA bacterial and archaeal primers and 18S rRNA eukaryotic primers. No amplification was obtained using archaeal primers. However, denaturing gradient gel electrophoresis (DGGE) revealed the presence of unique bands indicating bacterial and eukaryotic amplification. Excision of these bands, sequencing, and subsequent BLAST search showed that they corresponded to Bacillus sp. and Aspergillus versicolor. The fungus was later isolated from intact perlite in agar plates. A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp. Analysis of the petroleum components by gas chromatography showed that there n-alkanes, aromatic hydrocarbons, and carbazoles were degraded.
TL;DR: Repeated testing of different batches of mustard seed agar confirmed the ability of the new medium to support growth and pigment production of C. neoformans, and the first report on the prospective usage of a mustard seed-based agar in the identification of the fungus.
Abstract: Cryptococcosis due to Cryptococcus neoformans has emerged as the fourth most common lethal infection among AIDS patients (2). The formation of brown pigment due to phenol oxidase activity on medium containing seed extracts of Guizotia abyssinica (Staib’s niger seed agar) or Helianthus annus (Pal’s sunflower seed agar) or on medium containing chemicals such as caffeic acid or L-3,4-dihydroxyphenylalanine (L-DOPA) has been widely used as a criterion for the identification of this fungus (1, 3). In a search for a simple alternative seed-based medium for identification of C. neoformans, mustard seed extracts were found to induce the brown color effect (BCE) in C. neoformans. Brassica juncea or brown mustard is a cash crop widely cultivated, especially in India, for oil extraction and culinary purposes. Preparation of mustard seed agar involved procurement of dried mustard seeds from the local market followed by grinding in a domestic blender (Preethi Chefpro, Chennai, India). First, an aqueous extract of ground seeds (including kernels and shells) was prepared by adding the ground seeds to distilled water at a ratio of 1:20 (wt/vol), followed by boiling for 30 min. Next, the seed extract was cooled and filtered through gauze. The volume was then readjusted to 1 liter, and 20 g of agar (HiMedia, India) was added before the mixture was autoclaved at 121°C for 15 min. The medium was allowed to cool to between 45 and 55°C and dispensed into sterile 90-mmdiameter petri dishes (25 ml agar per plate). Five isolates of C. neoformans (consisting of three isolates of C. neoformans var. gatti and two isolates of C. neoformans var. neoformans) and five isolates of Candida albicans were inoculated on the mustard seed agar and incubated at 30°C on the laboratory bench. In addition, two strains comprising C. neoformans var. grubii (H-99) and C. neoformans var. neoformans (B-3501), kindly provided by J. Kwon-Chung (National Institutes of Health, Bethesda, Md.), were used as reference strains. At 48 h postinoculation, all seven isolates of C. neoformans could be easily identified by the BCE on mustard agar and differentiated from the white colonies of C. albicans. Mustard agar plates were held for up to 7 days to check for any variations in the colony color. The BCE was observed to intensify progressively during the entire period of incubation. Strains were also seeded on Pal’s agar and were observed to produce BCE. Repeated testing of different batches of mustard seed agar confirmed the ability of the new medium to support growth and pigment production of C. neoformans. Studies using mustard seed agar prepared according to Staib’s recommended formula with 0.1% glucose–0.1% creatinine– 0.1% KH2PO4 were also attempted. Growth of C. neoformans was observed within 48 h, but pigment production appeared to be delayed (72 h). Hence, mustard seed agar without supplements appeared to be more suitable for differentiation of C. neoformans. Thus, to the best of our knowledge, this is the first report on the prospective usage of a mustard seed-based agar in the identification of C. neoformans. The BCE of C. neoformans on media such as Staib’s or Pal’s agar is attributed to the presence of diphenolic substrates such as L-DOPA. However, mustard does not contain such compounds. Hence, the BCE on mustard agar may not be the same as that one observes on standard L-DOPA-containing media. This phenomenon has to be analyzed further for the elucidation of the exact mechanism of BCE on mustard agar. Furthermore, evaluation of this medium with more strains, including melanin-deficient mutants, would confirm its utility in diagnostic laboratories.