Showing papers on "Agar plate published in 2002"

Evaluation of a New Etest for Detecting Metallo-β-Lactamases in Routine Clinical Testing

Abstract: Several Etest (AB BIODISK, Solna, Sweden) gradient formats were developed for detection of metallo-β-lactamases based on the reduction of imipenem (IP) or ceftazidime (TZ) MICs in the presence of EDTA or 2-mercaptopropionic acid (MPA). The Etest metallo-β-lactamase (Etest MBL) strips consisted of a double-sided seven-dilution range of IP or TZ (4 to 256 μg/ml) and IP or TZ (1 to 64 μg/ml) overlaid with a constant concentration of EDTA or MPA. The prototype strips were evaluated on several agar media (brain heart infusion agar, Isosensitest agar, nutrient agar, and Mueller-Hinton agar for aerobes and brucella blood agar for anaerobes) with 138 challenge strains: Acinetobacter spp. (n = 9), Aeromonas spp. (n = 8), Chryseobacterium spp. (n = 28), Escherichia coli (n = 1), Klebsiella pneumoniae (n = 4), Pseudomonas aeruginosa (n = 14), Proteus mirabilis (n = 3), Serratia spp. (n = 10), Stenotrophomonas maltophilia (n = 43), Sphingobacterium spp. (n = 3), and Bacteroides fragilis group (n = 15). PCR analysis using specific primers for IMP-1, L1, CcrA, and blaB/C confirmed the presence of the metallo-β-lactamase genes. Enzyme assays were also performed with IP as an indicator substrate followed by EDTA inhibition profiles. EDTA was found to be a better inhibitor of metallo-β-lactamases, especially for anaerobes. IP was a better than TZ. Mueller-Hinton agar was the preferred medium, particularly when compared to Isosensitest agar, which frequently produced falsely low MICs for IP. Etest IP plus IP-EDTA with Mueller-Hinton agar had a sensitivity of 94% (79 of 84) and specificity of 95% (124 of 130). The Etest MBL strip appears to be an acceptable diagnostic reagent to detect metallo-β-lactamase phenotypes in the clinical microbiology laboratory.

In vitro efficacy of plant volatiles for inhibiting the growth of fruit and vegetable decay microorganisms.

Abstract: The effects of acetaldehyde, benzaldehyde, cinnamaldehyde, ethanol, benzyl alcohol, nerolidol, 2-nonanone, beta-ionone, and ethyl formate vapors on the growth of Rhizopus stolonifer, Penicillium digitatum, Colletotrichum musae, Erwinia carotovora, and Pseudomonas aeruginosa on agar medium were evaluated. The aldehydes were found to be the strongest growth inhibitors and the most lethal to the fungal spores and mycelia and bacterial cells. The average minimum inhibitory concentrations (MICs) of aldehydes that were germicidal to decay microorganisms were 0.28, 0.49, and 0.88 mmol per Petri dish, for cinnamaldehyde, benzaldehyde, and acetaldehyde, respectively. Ethanol also inhibited growth completely, but the MIC, which was 14.6 mmol per Petri dish, was significantly higher than those of the aldehydes. Ethanol can be considered germistatic because the alcohol does not inhibit germination of spores completely; it completely controlled only mycelial growth. The ketones tended to be effective only on P. digitatum and C. musae, whereas ethyl formate was not effective except on P. digitatum. The concentration of a volatile compound in the headspace of the Petri dish and its diffusion into the medium largely determined its efficacy against decay microorganisms.

The effectiveness of processed grapefruit-seed extract as an antibacterial agent: I. An in vitro agar assay.

Abstract: Objectives: Grapefruit-seed extract (GSE®) Citricidal® has, in recent reports, been reported to be successful in combating a variety of common infectious agents. In our study, drops of concentrated grapefruit-seed extract were tested for antibacterial properties against a number of gram-positive and gram-negative organisms. Design: Sixty-seven (67) distinct biotypes were tested for their susceptibilities to the GSE as well as to 5 other topical antibacterials (Silvadene,® Sulfamylon,® Bactroban,® Nitrofurazone,® and Silvadene,® Nystatin). Wells were punched into Mueller-Hinton agar plates, which were then inoculated with the organism to be tested; each well was then inoculated with one of the antibacterial agents. After an overnight incubation period, the plates were checked for zones of bacterial susceptibility around the individual wells, with a measured susceptibility zone diameter of 10 mm or more considered a positive result. Results: The GSE was consistently antibacterial against all of the biotypes...

Antagonism of Bacillus species (strain BC121) towards Curvularia lunata.

Abstract: A soil bacterium, Bacillus sp. strain BC121, isolated from the rhizosphere of sorghum, showed high antagonistic activity against Curvularia lunata. A clear inhibition zone of 0.5-1 cm was observed in dual plate assay. After 10 days of incubation, the bacterial strain grew over the fungal mycelial surface and multiplied extensively on it. Scanning electron microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual cultures, the Bacillus strain BC121 inhibited the C. lunata up to 60% in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5% colloidal chitin, indicating that it excretes chitinase. The role of the Bacillus strain BC121 in suppressing the fungal growth in vitro was studied in comparison with a mutant of that strain, which lacks both antagonistic activity and chitinolytic activity. The extra-cellular protein precipitate from Bacillus strain BC121 culture filtrate had significant growth-retarding effect and mycolytic activity on C. lunata. The protein extract from the wild strain, when tested on SDS-PAGE gel showed a unique band corresponding to the molecular mass of 25 kDa, which could be the probable chitinase protein.

Poly(L‐lactide)‐Degrading Activity in Various Actinomycetes

Abstract: The poly(L-lactide) (PLA)-degrading ability of actinomycetes obtained from culture collections was examined by the formation of clear zones on PLA-emulsified agar plates. Using 41 genera. (105 strains) of actinomycetes with phylogenetic affiliations based on 16S rRNA sequences, PLA degraders were found to be limited to members of the family Pseudonocardiaceae and related genera. They included Amycolatopsis, saccharothrix, Lentzea, Kibdelosporangium, and Streptoalloteichus. A large number of PLA degraders were widely distributed within the genus Saccharothrix. Most strains forming clear zones on PLA-emulsified agar plates also formed clear zones on silk fibroin agar plates. Saccharothrix species showed an ability to degrade PLA films and assimilate degradation products in liquid cultures. No significant change of the molecular weight and polydispersity (M w /M n ) of the remaining film fragments was confirmed. After cultivation for two weeks, many irregular holes/pits on the surface of the film due to the colonization of microorganisms were observed by scanning electron microscopy.

Production of trichothecenes and other secondary metabolites by Fusarium culmorum and Fusarium equiseti on common laboratory media and a soil organic matter agar: an ecological interpretation.

Abstract: Fusarium culmorum and F. equiseti were characterized with regard to production of trichothecenes and other secondary metabolites. Results following growth on laboratory media are interpreted with the aim of increasing the understanding of fungal metabolism in the field environment. While trichothecene production was detected for 94 of 102 F. culmorum isolates, only 8 of 57 F. equiseti isolates were positive. Profiles of secondary metabolites were compared by following growth on yeast extract sucrose agar (YES), potato sucrose agar (PSA), and an agar medium, prepared from soil organic matter (SOM), which was included to simulate growth conditions in soil. SOM supported the production of chrysogine by F. culmorum. The two species utilized the media differently. F. culmorumproduced zearalenone (ZEA) on YES, whereas some F. equiseti isolates produced ZEA on PSA. Other F. equiseti isolates produced equisetin. These differences may reflect that F. culmorum depends on a pathogenic life style while F. equiseti has a more saprotrophic mode of existence.

Symbiotic Relations of Sediment‐Agglutinating Nematodes and Bacteria in Detrital Habitats: The Enzyme‐Sharing Concept

Abstract: A new concept ('enzyme sharing') concerning the interaction of marine nematodes and microbes in the degradation of sedimentary detritus is presented. Elements of this concept are (1) the notorious tendency of many aquatic nematodes to agglutinate detrital particles by mucus secretions, (2) new observations on the stimulation of microbial growth by nematodes in agar plates, and (3) literature data on limited endogenous proteolytic capacities of aquatic nematodes.Observations on nematode-microbe associations in agar plates prompted the conceptual synthesis. In agar medium without the addition of any nutrients a spectacular growth of bacteria was visible on the sinusoidal crawling trails of nematodes only 2-3 days after introduction of the worms (species of Adoncholaimus, Anoplostoma and Sabatieria). Juveniles of Anoplostoma that hatched in the agar cultures left their minute trails in the medium and these were rapidly occupied by bacteria. The nematodes repeatedly visited their bacterial trails, which persisted as a peculiar biotic structure for more than one year and survived the nematodes.In sterile agar preparations containing the fluorogenic methylumbelliferyl-beta-glucoside in the presence of the nematode Adoncholaimus an enhanced fluorescence of the medium was visible indicating beta-glucosidase activity. We therefore assume that oncholaimid nematodes discharge enzymes that alone, or in concert with microbial activities, contribute to the hydrolytic cleavage of refractory polysaccharides containing beta-glucosidic bonds such as agar components and cellulose. The sugars thus produced may then be taken up by the nematodes and concomitantly support the conspicuous growth of microbes. Since we did not observe any feeding of the nematodes on the associated microbes in agar plates, we question the nutritive potential of intact microbial cells for a number of nematodes abounding in detrital habitats, and call attention to the significance of ambient dissolved or adsorbed organic monomeric nutrients.Consequently, we perceive the puzzling perpetual accretion of detrital organic particles to sediment agglutinations by nematodes as an adaptation for operating an 'enzymatic reactor' for the production of dissolved nutrients. We hypothesize a relationship of mutual commensalism of nematodes and heterotrophic microbes in detrital habitats and propose the term 'enzyme sharing' for this relationship. Both parties invest in a common enzyme pool that decomposes organic detritus for their nutrition. We present here evidence that nematodes contribute beta-glucosidase that is involved in the cellulase system. Data from the literature suggest that microbial enzymatic processing of detrital proteins results in amino acids available to nematodes which apparently have no efficient proteolytic enzyme system in their intestines.

Helicobacter pylori susceptibility testing by disc diffusion

Abstract: The bacterium Helicobacter pylori is found in c. 40% of the population and is responsible for the development of duodenal disease. Triple treatment with a proton-pump inhibitor or bismuth salt plus two antibiotics is now commonplace in all patients diagnosed. As antibiotic resistance reduces treatment efficacy, it is time to consider routine susceptibility testing to guide individual patient treatment and surveillance of antibiotic resistance. There are no published nationally agreed standards for disc diffusion testing of H. pylori. After reviewing the literature, we recommend the following method for disc diffusion tests. A suspension of cultures 21 mm susceptible. We suggest that isolates in the intermediate zone should be re-tested by Etest. Zone sizes with the Columbia agar base for metronidazole testing are or = 10 mm susceptible. Co-infection with two strains, which may be a mixture of isolates susceptible and resistant to metronidazole leading to conflicting susceptibility results, occurs in 5-10% of patients. Zone sizes with Mueller-Hinton agar and Columbia blood agar for clarithromycin testing are resistant no zone and susceptible any zone.

New Chromogenic Agar Medium for the Identification of Candida spp.

Abstract: A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.

Performance of Four Chromogenic Urine Culture Media after One or Two Days of Incubation Compared with Reference Media

Abstract: Four chromogenic urine culture media were compared to culture on blood agar, MacConkey agar, and CLED (cysteine-, lactose-, and electrolyte-deficient) agar for detection of uropathogens in 1,200 urine specimens. After 2 nights of incubation, 96% of all isolates were recovered on blood agar, 96% were recovered on CLED agar, 92% were recovered on CPS ID2, 96% were recovered on CHROMagar Orientation from BBL, 95% were recovered on CHROMagar Orientation from The CHROMagar Company, and 95% were recovered on Chromogenic UTI Medium.

Recognition of anaerobic bacterial isolates in vitro using electronic nose technology.

Abstract: Aims: Use of an electronic nose (e.nose) system to differentiation between anaerobic bacteria grown in vitro on agar media. Methods and Results: Cultures of Clostridium spp. (14 strains) and Bacteroides fragilis (12 strains) were grown on blood agar plates and incubated in sampling bags for 30 min before head space analysis of the volatiles. Qualitative analyses of the volatile production patterns was carried out using an e.nose system with 14 conducting polymer sensors. Using data analysis techniques such as principal components analysis (PCA), genetic algorithms and neural networks it was possible to differentiate between agar blanks and individual species which accounted for all the data. A total of eight unknowns were correctly discriminated into the bacterial groups. Conclusions: This is the first report of in vitro complex volatile pattern recognition and differentiation of anaerobic pathogens. Significance and Impact of the Study: These results suggest the potential for application of e.nose technology in early diagnosis of microbial pathogens of medical importance.

Stimulation of nodulation and plant growth of chickpea (Cicer arietinum L.) by Pseudomonas spp. antagonistic to fungal pathogens

Abstract: Two strains of Pseudomonas MRS23 and CRP55b showed antagonistic activity towards the pathogenic fungi Aspergillus sp., Fusarium oxysporum f. sp. ciceri, Pythium aphanidermatum and Rhizoctonia solani under culture conditions. Larger growth inhibition zones were obtained on nutrient agar (NA) and King's B media in comparison to potato dextrose agar and pigment production media. Both the strains produced siderophore in agar plates as well as in liquid cultures. Fungal inhibition zones were reduced in size and abolished in iron-supplemented NA medium by Pseudomonas strains MRS23 and CRP55b, respectively, indicating that some other metabolites along with siderophores are involved in growth inhibition of fungi by strain MRS23, whereas CRP55b produced only siderophores. Only Pseudomonas strain MRS23 was found to produce hydrocyanic acid (HCN). Seed bacterization with Pseudomonas strains of two chickpea (Cicer arietinum L.) cultivars, H8618 and C235, showed root-stunting effects at 5 days, whereas this inhibitory effect was overcome at 10 days of seedling growth in cv. H8618. Coinoculation of chickpea with Pseudomonas strains MRS23 and CRP55b, and Mesorhizobium sp. Cicer strain Ca181 resulted in the formation of 68.2–115.4% more nodules at 80 and 100 days after planting as compared to single inoculation with the Mesorhizobium strain under sterile conditions. The shoot dry weight ratios of coinoculated treatments at different stages of plant growth varied from 1.18 to 1.35 times that of Mesorhizobium-inoculated and 3.25 to 4.06 times those in uninoculated controls. The plant N contents were also increased significantly on coinoculation. Coinoculation effects of HCN-producing strain MRS23 were significantly lower than those of non-HCN-producing strain CRP55b in terms of shoot dry weight and shoot N. The results demonstrated the potential benefits of using rhizosphere bacteria as coinocula in nodule promotion and plant growth in chickpea.

Comparison of antibacterial activity of simplified adhesive systems.

Abstract: PURPOSE To determine and compare the intrinsic antibacterial activity of commercially available simplified adhesive systems. MATERIALS AND METHODS The antibacterial activity of five self-etching/priming one-step adhesives and three priming/bonding adhesives against Streptococcus mutans, Lactobacillus casei, and Actinomyces viscosus was assessed by the agar disc-diffusion test and determination of the minimum inhibitory/bactericidal concentration (MIC/MBC). Twenty microl of each adhesive was impregnated into a paper disc and placed on an agar plate inoculated with a bacterial suspension, with or without light-irradiation. The size of inhibition zones produced around the specimens was measured after 48 hours of incubation. The MIC values were measured by serial microdilution assays, visually examining the growth of bacteria after incubation with each adhesive for 24-48 hours. The subcultures were made on BHI agar plates from the wells showing no visible growth of bacteria, and the MBC values were determined based on production of colonies. Two primers in two-step self-etching systems and an experimental primer containing chlorhexidine were tested for reference. The results of disc-diffusion tests were analyzed by ANOVA and Fisher's PLSD test. RESULTS The size of inhibition zones produced by adhesives varied among the brands. None of the available commercial adhesives showed significant inhibition against all three of the bacterial species tested. Light-irradiation reduced the size of inhibition zones for a few materials, but a similar trend to the effectiveness of non-irradiated specimens was observed. The antibacterial activity of commercial products determined in terms of MIC/MBC values were different from the results by disc-diffusion tests. The self-etching adhesives with low pH were not necessarily more effective than priming/bonding solutions, and no significant relationships between the acidity and antibacterial effects were found. Compared with commercial products, an experimental primer containing chlorhexidine produced significantly larger inhibition zones against all species (P < 0.05) and exhibited greater bacteriostatic/bactericidal activity, demonstrating lower MIC/MBC values.

Pathogenesis of Streptoverticillium albireticuli on Caenorhabditis elegans and its antagonism to soil-borne fungal pathogens.

Abstract: Aims: To examine the biological activity of Streptoverticillium albireticuli. Methods: Isolation of S. albireticuli was carried out using the dry-heat technique. Nematicidal and pathogenic activity on Caenorhabditis elegans was measured by mortality in metabolites and colonization rate on fishmeal extract agar. Antifungal and enzymatic activities of S. albireticuli were measured by the agar plate method and the semidefined solid media method, respectively. Results:S. albireticuli showed strong nematicidal activity against C. elegans . Pathogenic activity was also evident with the colonized nematode by the isolate on fishmeal extract agar. It also showed antifungal activity against certain fungal pathogens such as Rhizoctonia solani , Phytophthora cinnamomi and Fusarium oxysporum . Significance and Impact of the Study: The discovery of an actinomycete showing pathogenic activity against the nematode may indicate the potential for it to be used as a biocontrol agent of parasitic nematodes, in addition to its ability to suppress fungal pathogens.

Evaluation of surface disinfectants utilized in dentistry

Abstract: Surface disinfection is a procedure carried out on the external parts of the dental equipment as well as on other items of the dental office The aim of this study was to analyze the efficacy of 4 surface disinfectants utilized in dentistry: 77°GL alcohol, phenolic compound (Duplofen), iodophor (PVP-I) and 77°GL alcohol with 5% of chlorhexidine Four surfaces of the equipment were analyzed in the study (the carter, the washbasin for hand-washing, the headrest of the chair and the external surface of the reflector), and the spray-wipe-spray procedure was carried out From each surface, samples were collected by means of surface plates containing Mitis Salivarius bacitracin sucrose agar, Sabouraud Dextrose agar with chloramphenicol, MacConkey agar and blood agar, for counting mutans streptococci, Candida yeasts, gram-negative bacteria and total microorganisms, respectively (ufc/plate) The results were statistically analyzed by means of the Student's t test in order to compare the mean ufc/plate values The most effective disinfectant was 77°GL alcohol with 5% of chlorhexidine, mainly against gram-positive bacteria Iodophor and phenolic compound were also effective in microbial reduction 77°GL alcohol was the least effective product - however, although it is not considered as a surface disinfectant, it produced, in this study, statistically significant microbial reduction after the disinfecting procedure

Comparative Evaluation of Two Commercial Chromogenic Media for Detection and Presumptive Identification of Urinary Tract Pathogens

Abstract: The performance of two commercial chromogenic media for the isolation and presumptive identification of urinary tract pathogens, the CPS ID2 (bioMerieux, France) and the CHROMagar Orientation (BBL Becton Dickinson, USA), was evaluated and compared with that of cystine-lactose-electrolyte-deficient agar and tryptic soy agar with 5% sheep blood. The detection, determination of bacterial counts, and presumptive identification of bacteria causing urinary tract infections were evaluated in 3,000 urine specimens. The two chromogenic media showed excellent correlation with the standard media for the detection and the bacterial count of urinary pathogens. The Escherichia coli strains produced the expected colour on the CHROMagar Orientation and the CPS ID2 media in 99% and 90% of the cases, respectively. The Klebsiella-Enterobacter-Citrobacter and the Proteus-Morganella-Providencia groups were easily identified on both chromogenic media, but further biochemical tests were needed to differentiate them to a species level. Both media enabled the differentiation, with varying degrees of difficulty, of Pseudomonas spp. strains from members of the family Enterobacteriaceae. All isolates of Enterococcus spp. were correctly identified and were easily distinguished from the Streptococcus agalactiae isolates. Staphylococcus saprophyticus isolates were easy to identify only on the CHROMagar Orientation medium. No substantial difference was observed when comparing the results of the susceptibility tests, which were performed according to the standardized disk diffusion method as described by the National Committee for Clinical Laboratory Standards, for colonies recovered from the blood agar versus those recovered from the chromogenic media. In conclusion, the CPS ID2 and CHROMagar Orientation media enabled excellent detection, count determination, and presumptive identification of urinary pathogens, both in pure and mixed cultures, and reliable and accurate antimicrobial susceptibility testing directly from primary isolates. Moreover, these media allowed a remarkable reduction in the workload and a significant savings of time. On the basis of their performance, these media can replace the standard primary plating media used in the routine diagnosis of urinary tract infections.

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

Abstract: The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.

Biodegradation of triazine herbicides on polyvinylalcohol gel plates by the soil yeast Lipomyces starkeyi.

Abstract: The soil yeast Lipomyces starkeyi was tested for its ability to degrade triazine herbicides. Polyvinylalcohol (PVA) was employed as a solid medium in culture plates instead of agar. The cell sizes of the control (without nitrogen source) on the PVA gel plate were much smaller than those on the agar gel plate. The difference between the diameters of the sample and control colonies on the PVA gel plate were almost twice those of the colonies on the agar gel plate (1.9 and 1.0 mm, respectively). Thus, the PVA gel plate is much better than the agar plate for evaluating the degree of utilization of a sole nitrogen source. The yeast grew well (more than 4 mm in diameter) with 1,3,5-triazine or cyanuric acid as nitrogen source. In addition, melamine and thiocyanuric acid inhibited growth of the yeast, and the sizes of colonies were smaller than those of the control. All triazine herbicides tested (simazine, atrazine, cyanazine, ametryn, and prometryn) could be degraded and assimilated by L. starkeyi.

Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields.

Abstract: A new selective myclobutanil agar medium for the detection of Fusarium species is proposed. Ten media formulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, ma...

Rapid detection of myrosinase-producing fungi: a plate method based on opaque barium sulphate formation

Abstract: A simple and rapid technique to assess the capability of fungi to produce myrosinase is reported. This was carried out by growing the tested fungi in sinigrin–barium agar plates. Strains capable of producing myrosinase were indicated by an opaque barium sulphate zone forming underneath and/or surrounding their colonies. This simple test has been confirmed by determination of myrosinase activity in liquid culture. In positive isolates, enzyme acitivity was detected in cell-free extracts, not in culture filtrates. In the case of non-myrosinase-producing strains, no opaque zone was observed and the enzyme was not detected either in cell-free extracts or in culture filtrates.

Individual based simulations of bacterial growth on agar plates

Abstract: The individual based simulator, INDividual DIScrete SIMulations (INDISIM) has been used to study the behaviour of the growth of bacterial colonies on a finite dish. The simulations reproduce the qualitative trends of pattern formation that appear during the growth of Bacillus subtilis on an agar plate under different initial conditions of nutrient peptone concentration, the amount of agar on the plate, and the temperature. The simulations are carried out by imposing closed boundary conditions on a square lattice divided into square spatial cells. The simulator studies the temporal evolution of the bacterial population possible by setting rules of behaviour for each bacterium, such as its uptake, metabolism and reproduction, as well as rules for the medium in which the bacterial cells grow, such as concentration of nutrient particles and their diffusion. The determining factors that characterize the structure of the bacterial colony patterns in the presents simulations, are the initial concentrations of nutrient particles, that mimic the amount of peptone in the experiments, and the set of values for the microscopic diffusion parameter related, in the experiments, to the amount of the agar medium.

Relevance of incubation temperature for Vibrio salmonicida vaccine production.

Abstract: Aims: To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). Methods and Results: The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10°C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15°C on solid media and 10°C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10°C was not found to stimulate a specific humoral response. Conclusions: Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15°C. High yield broth cultures for vaccine production should be incubated at 10°C or lower. Significance and Impact of the Study: This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs.

Rapid detection of pathogenic bacteria by volatile organic compound (VOC) analysis

Abstract: Developments in rapid detection technologies have made countless improvements over the years. However, because of the limited sample that these technologies can process in a single run, the chance of capturing and identifying a small amount of pathogens is difficult. The problem is further magnified by the natural random distribution of pathogens in foods. Methods to simplify pathogenic detection through the identification of bacteria specific VOC were studied. E. coli O157:H7 and Salmonella typhimurium were grown on selected agar medium to model protein, and carbohydrate based foods. Pathogenic and common spoilage bacteria (Pseudomonas and Morexella) were screened for unique VOC production. Bacteria were grown on agar slants in closed vials. Headspace sampling was performed at intervals up to 24 hours using Solid Phase Micro-Extraction (SPME) techniques followed by GC/MS analysis. Development of unique volatiles was followed to establish sensitivity of detection. E. coli produced VOC not found in either Trypticase Soy Yeast (TSY) agar blanks or spoilage organism samples were - indole, 1-decanol, and 2-nonanone. Salmonella specific VOC grown on TSY were 3-methyl-1-butanol, dimethyl sulfide, 2-undecanol, 2-pentadecanol and 1-octanol. Trials on potato dextrose agar (PDA) slants indicated VOC specific for E. coli and Salmonella when compared to PDA blanks and Pseudomonas samples. However, these VOC peaks were similar for both pathogens. Morexella did not grow on PDA slants. Work will continue with model growth mediums at various temperatures, and mixed flora inoculums. As well as, VOC production based on the dynamics of bacterial growth.

Isolation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) from Reticulitermes flavipes (Isoptera: Rhinotermitidae) with convenient methods for its culture and collection of conidia

Abstract: A virulent strain (pathotype) of the entomogenous fungus causing green muscardine disease in insects, Metarhizium anisopliae, was isolated from the eastern subterranean termite, Reticulitermes flavipes, in Toronto, Ontario. The method of culturing the fungus involved the following steps: 1) dusting termites with conidia by swirling them in a glass petri plate for several minutes with conidia 2) allowing 2-4 days for the conidia dusted termites to die in a clean dry petri dish and for conidia to germinate and invade the termite body, 3) transferring the petri dish to a humidity chamber to promote growth of aerial hyphae, 4) transferring hyphae covered corpses to 2% potato dextrose agar or Sabouroud's dextrose agar with 0.05% ampicillin 5) followed by sequential daily transfer to new agar plates if necessary until all contaminant bacteria and fungi are removed. With spacing of corpses 1 cm apart the mycelium will grow over the whole plate producing a solid 0.5 mm spore mass within about 4 weeks. Conidia so grown are dark green rather than grey when grown on media alone. Conidia are harvested by gently loosening them with a sterile probe and then inverting the culture dish over a sterilized glass lid and applying an electric vibrator to the plate to dislodge them onto the lid. The pure conidia can then be weighed and applied to termites by swirling then in a dish with the termites. The dry conidia are adsorbed onto the epicuticle of the termites.