Showing papers on "Agar plate published in 2009"

Poly(ethylene terephthalate) polymer surfaces as a substrate for bacterial attachment and biofilm formation.

Abstract: Plastic debris causes extensive damage to the marine environment, largely due to its ability to resist degradation. Attachment on plastic surfaces is a key initiation process for their degradation. The tendency of environmental marine bacteria to adhere to poly(ethylene terephthalate) (PET) plastic surfaces as a model material was investigated. It was found that the overall number of heterotrophic bacteria in a sample of sea water taken from St. Kilda Beach, Melbourne, Australia, was significantly reduced after six months from 4.2-4.7×10(3) cfu mL(-1) to below detectable levels on both full-strength and oligotrophic marine agar plates. The extinction of oligotrophs after six months was detected in all samples. In contrast, the overall bacterial number recovered on full strength marine agar from the sample flasks with PET did not dramatically reduce. Heterotrophic bacteria recovered on full-strength marine agar plates six months after the commencement of the experiment were found to have suitable metabolic activity to survive in sea water while attaching to the PET plastic surface followed by the commencement of biofilm formation.

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Abstract: Summary Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow-growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet-to-be cultured microorganisms.

Comparative evaluation of antimicrobial activity of three cements: new endodontic cement (NEC), mineral trioxide aggregate (MTA) and Portland

Abstract: Using the agar diffusion method, we conducted an in vitro study to evaluate the antimicrobial activity of mineral trioxide aggregate (MTA), new endodontic cement (NEC) and Portland cement at different concentrations against five different microorganisms. A base layer was made using Muller-Hinton agar for Escherichia coli (ATCC 10538) and Candida (ATCC 10231). For Actinomyces viscosus (ATCC 15987), Enterococcus faecalis (ATCC 10541) and Streptococcus mutans (ATCC 25175) blood agar medium was used. Wells were formed by removing the agar, and the materials were placed in the well immediately after manipulation. The plates were kept at room temperature for 2 h for prediffusion, and then incubated at 37 degrees C for 72 h. The inhibition zones were then measured. The data were analyzed using ANOVA and the Tukey test to compare the differences among the three cements at different concentrations. The positive controls showed bacterial growth, while the negative controls showed no bacterial growth. All materials showed antimicrobial activity against the tested strains except for Enterococcus faecalis. NEC created larger inhibition zones than MTA and Portland cement. This difference was significant for Portland cement (P 0.05). Among the examined microorganisms, the largest inhibition zone was observed for Actinomyces group (P < 0.05). The antimicrobial activity of the materials increased with time and concentration (P < 0.05). It was concluded that NEC is a potent inhibitor of microorganism growth.

Fimbriation and colony type of moraxella bovis in relation to conjunctival colonization and development of keratoconjunctivitis in cattle

Abstract: Morphological variants of four Moraxella bovis strains were studied, using different cultural conditions. On bovine blood agar medium two colony types could be distinguished: 1) large, flat, rough and agar-corroding, and 2) most often smaller, convex, smooth and non-corroding forms. When transferred to human blood agar, the flat type formed convex, spreading-corroding colonies (SC type) and the other type remained convex, non-spreading and non-corroding (N type). Some cell lines lost the original flat colony appearance on bovine blood agar after passages for a longer period on human blood agar; on bovine blood agar these SC cells now formed convex, but still corroding colonies. By electron microscopy it was observed that cells from corroding colonies on either medium were strongly fimbriated, whereas preparations from non-corroding populations had non-fimbriated cells or contained only an occasional fimbria. Conjunctival infection experiments in calves revealed that only fimbriated variants of each strain were able to colonize the conjunctival mucosa. Ultraviolet irradiation of the eye prior to inoculation facilitated the bacterial colonization, with respect to initiation and duration. Non-fimbriated M. bovis could not be reisolated even after irradiation. Keratitis and conjunctivitis only occurred in irradiated eyes inoculated with cell lines yielding typically flat, corroding colonies on bovine blood agar. The possible function of fimbriae for attachment of M. bovis to the mucous membranes of the eye is discussed.

A novel poly (L-lactide) degrading actinomycetes isolated from Thai forest soil, phylogenic relationship and the enzyme characterization.

Abstract: Thirteen poly (L-lactide)-degrading microorganisms were isolated and selected based on their ability of clear zone formation on an emulsified PLA agar plate and the enzyme activity in culture broth. According to phenotypic properties and 16S rRNA gene sequence, these strains were classified to various families such as Thermomonosporaceae, Micromonosporaceae, Streptosporangiaceae, Bacillaceae and Thermoactinomycetaceae. Strain T16-1, identified as Actinomadura sp., demonstrated the highest PLA-degrading activity in the liquid culture using PLA film as a carbon source. A PLA-degrading enzyme produced by the strain was purified to homogeneity shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with specific activity of 38.3 unit/mg protein. The optimum pH and temperature were 10.0 and 70 masculineC, respectively, which are higher than previously reported among PLA-degrading enzyme. The enzyme was stable at pH 11-12. However, the enzyme activity remained at 70% when kept at 70 masculineC for 1 h. The molecular weight of purified PLA-degrading enzyme from the strain T16-1 was 30 kDa. The purified enzyme was inhibited by 5 mM EDTA and 5 mM phenylmethylsulfonyl fluoride and diisopropyl fluorophosphates, strongly hydrolyzed Suc-(Ala)(3)-pNA, gelatin and PLA, but showed low activity on casein. The results indicated the PLA-degrading enzyme produced by the strain Actinomadura sp. T16-1 should be classified as serine protease.

The hemolytic and cytolytic activities of Serratia marcescens phospholipase A (PhlA) depend on lysophospholipid production by PhlA

Abstract: Serratia marcescens is a gram-negative bacterium and often causes nosocomial infections. There have been few studies of the virulence factors of this bacterium. The only S. marcescens hemolytic and cytotoxic factor reported, thus far, is the hemolysin ShlA. An S. marcescens shlAB deletion mutant was constructed and shown to have no contact hemolytic activity. However, the deletion mutant retained hemolytic activity on human blood agar plates, indicating the presence of another S. marcescens hemolytic factor. Functional cloning of S. marcescens identified a phospholipase A (PhlA) with hemolytic activity on human blood agar plates. A phlAB deletion mutant lost hemolytic activity on human blood agar plates. Purified recombinant PhlA hydrolyzed several types of phospholipids and exhibited phospholipase A1 (PLA1), but not phospholipase A2 (PLA2), activity. The cytotoxic and hemolytic activities of PhlA both required phospholipids as substrates. We have shown that the S. marcescens phlA gene produces hemolysis on human blood agar plates. PhlA induces destabilization of target cell membranes in the presence of phospholipids. Our results indicated that the lysophospholipids produced by PhlA affected cell membranes resulting in hemolysis and cell death.

Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests.

Abstract: Background Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis [1], [2]. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies. Methods and Findings Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test. Conclusions The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

Measuring minimum inhibitory concentrations in mycobacteria.

Abstract: An agar dilution method for measuring minimum inhibitory concentrations (MICs) of Mycobacterium tuberculosis, based on the method of proportion, is described. Mycobacterium strains are grown on Middlebrook 7H10 (or 7H11) agar medium with twofold serially diluted drug concentrations in order to determine specific inhibitory values. The proportion of bacilli resistant to a given drug is determined by comparing the number of colony-forming units (CFU) on a drug-free control with those growing in the presence of drug within a specific concentration range. The MIC is defined as the lowest drug concentration that inhibits growth of more than 99% of a bacterial population of M. tuberculosis on solid Middlebrook medium within 21 days of incubation at 37 degrees C. The proportion method, the absolute concentration method, and the resistant ratio method have traditionally been used as standard procedures for antimycobacterial drug-susceptibility testing (DST), and reference data are mainly based on these methods. DST concepts and alternative procedures that have been adopted for DST are also briefly discussed.

High Rate of N2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

Abstract: For evaluating N(2) fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N(2)-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N(2) fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N(2)-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N(2) fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.

The haemolytic activity of Haemophilus species.

Abstract: The importance of the species of blood employed for detection of haemolysis in seventy-seven Haemophilus strains of human and porcine origin was studied. Significant differences in the visibility of haemolytic zones obtained on the different blood agar media were observed. In decreasing order, the suitability of the species of blood was: calf, sheep, human, rabbit, poultry and horse blood. On plates containing washed sheep or calf red cells the haemolysin of all 36 strains of Haemophilus pleuropneumoniae acted synergistically with the beta-toxin of the Staphylococcus aureus strain used as "feeder strain", giving rise to a lytic phenomenon resembling the CAMP reaction.

Rapid identification of Enterobacteriaceae. II. Use of a beta-glucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples.

Abstract: beta-glucuronidase activity is an exclusive characteristic of E. Coli and some shigellae among Enterobacteriaceae and Vibrionaceae. An agar medium (PGUA agar) which permits the detection of bacteria with beta-glucuronidase activity in mixed cultures was evaluated as a primary culture medium for clinical samples of urine. The medium was selective for enterobacteria and yielded significantly higher recoveries than MacConkey agar. Based on the examination of 3,460 urine samples, it was found that the use of the PGUA agar has several advantages over conventional methods: 1) 94% of all E. coli cultures could be identified on the basis of their appearance on the primary plates; 2) The use of the PGUA method did not result in any misidentidications as compared to 1% of cultured misidentified by the conventional procedure; 3) Approximately one-half of the urine samples which contained E. coli as the sole organism could be reported following the reading of primary culture plates; 4) The application of the PGUA medium resulted in a 46% reduction in the cost of media employed and a 67% reduction in the time required for the processing of urine samples.

Application of PCR-RFLP to rapid identification of the main pathogenic dermatophytes from clinical specimens.

Abstract: Background: In the present study, a PCR-RFLP based molecular technique was designed to rapid identification of der­matophytes in clinical specimens. Skin scrapings obtained from human cases suspected to dermatophytosis were studied in or­der to identify involved etiological fungi. Methods: In this experimental study, the specimens (skin scrapings) of patients referred to Mycology Department of Pas­teur Institute of Iran were inoculated on Petri dishes contained selective agar for pathogenic fungi (SAPF) and incubated at 25o C until visible growth of fungal colonies. The colonies were examined for standard morphological characteristics after visi­ble growth on the agar medium. A small portion of each fungal colony was further studied by restriction fragment length poly­morphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). PCR amplicons were electrophoresed on 2% agarose gel after digesting by different restriction enzymes including Mva I, Hinf I and Hae III. Results: Among 160 clinical samples examined, 6 dermatophyte species including Trichophyton mentagrophytes, T. ru­brum, T. verrucosum, T. tonsurans, Microsporum canis and Epidermophyton floccosum were finally identified based on the col­ony morphology and microscopic criteria. Specific PCR products and RFLP patterns for Mva I, Hinf I and Hae III en­zymes allowed the rapid identification and reliable differentiation of isolated dermatophytes at the genus or species level for 5-10 day-old colonies. Conclusions: The results showed that PCR-RFLP analysis of the ITS region of rDNA is a rapid and reliable tool which al­lows identification of major pathogenic dermatophytes isolated in this study at species level in young 5-10 day-old colonies.

A comparison of different culture methods for the recovery of Campylobacter species from pets.

Abstract: Five culture methods for the recovery of Campylobacter species (spp.) were evaluated on 361 rectal swabs collected from cats and dogs in Ireland. Speciation using PCR methods was performed on all isolates to assess the sensitivity of each culture method for isolation of Campylobacter spp., and to establish the prevalence of Campylobacter jejuni, C. coli, C. upsaliensis, C. lari and C. helveticus. Overall 163 of 361 (45.2%) samples were confirmed Campylobacter spp. positive. Direct plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA) with cefoperazone, amphotericin and teicoplanin (CAT) selective supplement yielded a significantly higher prevalence of Campylobacter spp. (33.0%) than each of the other four methods (P < or = 0.05). This method was also the most sensitive method for isolation of C. upsaliensis compared with any of the other four methods used in the current study (P < or = 0.05). A direct plating method onto mCCDA agar with CCDA selective supplement and a filtration method onto blood agar after pre-enrichment in CAT supplemented broth yielded lower Campylobacter spp. prevalences of 19.7% and 17.5% respectively. A filtration method onto CAT agar and pre-enrichment in Preston broth before plating onto mCCDA agar were less sensitive for the isolation of Campylobacter spp. Speciation results of Campylobacter isolates revealed the majority of Campylobacter isolates were C. upsaliensis (50.0%) and C. jejuni (41.9%). A small number of isolates were C. coli (2.6%), C. lari (1.5%) and C. helveticus (1.1%). The overall detection of Campylobacter spp. in the 361 pets sampled was significantly increased by using a combination of isolation methods (P < or = 0.05), producing a more accurate determination of the prevalence of Campylobacter spp. in pets in Ireland and of the actual Campylobacter species. As the majority of Campylobacter spp. were recovered by direct plating onto mCCDA agar with CAT supplement, this method is the method of choice if only a single method is selected for isolation of the most common Campylobacter spp. detected in pets and humans.

Bioaccumulation of Arsenic by Fungi

Abstract: Problem statement: Arsenic is a known toxic element and its presence and toxicity in nature is a worldwide environmental problem. The use of microorganisms in bioremediation is a potential method to reduce as concentration in contaminated areas. Approach: In order to explore the possible bioremediation of this element, three filamentous fungi-Aspergillus niger, Serpula himantioides and Trametes versicolor were investigated for their potential abilities to accumulate (and possibly solubilize) arsenic from an agar environment consisting of non buffered mineral salts media amended with 0.2, 0.4, 0.6 and 0.8% (w/v) arsenopyrite (FeAsS). Growth rates, dry weights, arsenic accumulation and oxalate production by the fungi as well as the pH of the growth media were all assessed during this study. Results: There was no visible solubilization of FeAsS particles underneath any of the growing fungal colonies or elsewhere in the respective agar plates. No specific patterns of growth changes were observed from the growth ratios of the fungi on agar amended with different amounts of FeAsS although growth of all fungi was stimulated by the incorporation of varying amounts of FeAsS into the agar with the exception of A. niger on 0.4% (w/v) amended agar and T. versicolor on 0.8% (w/v) amended agar. The amounts of dry weights obtained for all three fungi also did not follow any specific patterns with different amounts of FeAsS and the quantities obtained were in the order A. niger > S. himantioides > T. versicolor. All fungi accumulated as in their biomasses with all amounts of FeAsS although to varying levels and T. versicolor was the most effective with all amounts of FeAsS while A. niger was the least effective. Conclusion: The accumulation of arsenic in the biomasses of the test fungi as shown in this study may suggested a role for fungi through their bioaccumulating capabilities as agents in the possible bioremediation of arsenic contaminated environments.

Interpretation of MRSASelect Screening Agar at 24 Hours of Incubation

Abstract: An incubation time of 24 h at 35 to 38°C is recommended for the optimal performance of MRSASelect (Bio-Rad) chromogenic screening agar. An additional 24 h is required to capture slow-growing methicillin-resistant Staphylococcus aureus (MRSA). However, the normal hours of operation for most laboratories cannot reliably accommodate 24-h interpretation intervals. Daily agar plate interpretations are more likely to occur around 18 h and 42 h of incubation, which may compromise the performance of the chromogenic agar and negatively impact patient infection control efforts. In order to validate the importance of stringent incubation times to plate performance, we evaluated MRSASelect medium at controlled intervals of 24 and 48 h of incubation, using clinical MRSA-screening swabs. A total of 1,071 MRSA-positive and 2,733 MRSA-negative cultures were selected for analysis. Compared to 48-h-incubation results, the sensitivity and specificity of MRSASelect at 24 h were 98.3% and 98.2%, respectively. Only 19 of 1,071 (1.8%) MRSA-positive isolates required 48 h for detection. Holding 24-h negative plates an additional 24 h resulted in the workup of 253 (6.6%) pink, yet non-MRSA, colonies. The 24-h positive and negative predictive values of MRSASelect, assuming MRSA prevalences of 1, 5, and 10%, were 35.5 and 99.98%, 74.2 and 99.9%, and 85.9 and 99.8%, respectively. MRSASelect medium held for 24-h incubation is a highly sensitive and specific MRSA-screening tool. Further incubation prolongs the turnaround time for results and creates a significant amount of unnecessary work in the laboratory.

Stable isotope probing reveals Trichosporon yeast to be active in situ in soil phenol metabolism

Abstract: The aim of this study was to extend the results of our previous stable isotope probing (SIP) investigation: we identified a soil fungus involved in phenol biodegradation at an agricultural field site. DNA extracts from our previous study were examined using fungi-specific PCR amplification of the 18S-28S internal transcribed spacer (ITS) region. We prepared an 80-member clone library using PCR-amplified, (13)C-labeled DNA derived from field soil that received 12 daily doses of (13)C-phenol. Restriction-fragment-length-polymorphism screening and DNA sequencing revealed a dominant clone (41% of the clone library), the ITS sequence of which corresponded to that of the fungal genus Trichosporon. We successfully grew and isolated a white, filamentous fungus from site soil samples after plating soil dilutions on mineral salts agar containing 250 p.p.m. phenol. Restreaking on both yeast extract-peptone-galactose and Sabouraud dextrose agar plates led to further purification of the fungus, the morphological characteristics of which matched those of the genus Trichosporon. The ITS sequence of our isolated fungus was identical to that of a clone from our SIP-based library, confirming it to be Trichosporon multisporum. High-performance liquid chromatography and turbidometeric analyses showed that the culture was able to metabolize and grow on 200 p.p.m. phenol in an aqueous mineral salts medium within 24 h at room temperature. Gas chromatography-mass spectrometry analysis of (13)CO(2) respiration from laboratory soil incubations demonstrated accelerated phenol mineralization in treatments inoculated with T. multisporum. These findings show that T. multisporum actively degraded phenol in our field-based, soil experiments.

Antibacterial effect of silver-zeolite containing root-canal filling material

Abstract: The aim of this study was to determine the in vitro antibacterial effect of two experimental glass ionomer cements (GICs) on Streptococcus milleri, Staphylococcus aureus, and Enterococcus faecalis after 24 and 48 h incubation by using the agar diffusion inhibitory test. Silver zeolite (SZ) was added at 0.2 and 2% mass fraction concentration to GIC (Endion). The control group was Endion with no SZ. Each of them were prepared to uniform size using a custom-made Teflon mold, and the GIC materials were prepared to form disks (n = 5 per group). The effect of these materials on the growth of three bacteria associated with endodontic infections was determined using the agar diffusion inhibitory test. The amounts of silver ion release from these materials were measured with atomic absorption spectrophotometry at 10 min, 24- and 48-h periods. The pH of samples was measured with a pH-meter at 10 min, 24- and 48-h periods. After the incubation period, the agar plates were evaluated and the degrees of bacterial inhibition were measured in millimeters. A comparison of the mean of the test materials was statistically different in each group of specimens (p < 0.05). Between the two tested materials 2% SZ containing GIC showed the largest zone of inhibition on the agar plates of all the tested strains (p < 0.05). The most inhibition in bacterial growth occurred in E. faecalis. Adding 2% SZ to GIC resulted in a significant increase in the silver release into deionized water. This study demonstrated that GIC had an inhibitory affect on Streptococcus milleri, Staphylococcus aureus, and Enterococcus faecalis and that adding SZ increases that affect proportional to its concentration.

Production of Alkaline Xylanase by an Alkalo-thermophilic Bacteria, Bacillus halodurans, MTCC 9512 isolated from Dung

Abstract: An alkalo-thermophilic bacteria from dung has been isolated using Emerson medium in the agar plates. The bacteria has growth at the pH 10 and temperature 55°C. The bacteria was screened for the xylanase activity using Congo red dye followed by wash out by 1 mM sodium chloride. A clear zone around the colony in the replica plate was considered to have xylanase activity. The suspected colony in another replica plate was grown in Emerson broth and extracellular xylanase enzyme activity was analyzed by the colorimetric method using dinitro salicylic acid for estimation of reducing power. The morphological study of the bacteria was done after Gram stain and using 40x amplification in the phase contrast microscope. The isolated bacteria retained violet color after washing with acetone. Therefore, it is gram positive. Further characterization using various morphological, physiological and biochemical tests confirmed the bacteria as Bacillus halodurans and was given Accession number MTCC 9512 by IMTECH, Chandigarh. Growth conditions for the bacteria were optimized for maximum production of xylanase. The maximum amount of xylanase activity was found at the pH 9.5 and temperature 55°C. The growth of the bacteria and enzyme production were monitored up to 52 hours and it was found that the bacteria grew logarithmically up to 30 hours. Different carbon sources viz. xylan, sucrose, glucose, starch individually at 0.5% concentration were used in the Emerson growth medium. Maximum biomass growth was found with xylan whereas xylanase was maximally produced with glucose as carbon source. Therefore, glucose was considered to be the best inducer followed by xylan among the various carbon sources used. The enzyme was enriched by using 0–80% ammonium sulfate precipitation followed by desalting through Sephadex-G-25 gel filtration. The results indicated inhibitory nature of ammonium sulfate.

Evaluation and Implementation of a Chromogenic Agar Medium for Salmonella Detection in Stool in Routine Laboratory Diagnostics

Abstract: We evaluated which chromogenic agar medium for Salmonella detection in stool would be most sensitive and specific in our culture protocol. The use of BBL CHROMagar Salmonella chromogenic medium combined with xylose-lysine-deoxycholate agar yielded a sensitivity of 100% and also reduced workload and costs.

Colony variations in Candida species.

Abstract: Summary: One hundred isolates of seven Candida species and three isolates of Saccharomyces cerevisiae were plated on phloxine B agar and examined for variations in the morphology or colour of colonies that developed. Colony variations were found in 9 of 12 C. parapsilosis isolates, 8 of 13 C. tropicalis isolates, 4 of 9 C. krusei isolates and 12 of 30 C. albicam isolates. None of 23 C. glabrata isolates grew on the test medium. Variant colonies often generated further different colony forms on secondary subculture. The rate of production of fimbriate and rhizoid colonies by two C. albicam isolates varied with agar thickness and the nutrient content of the medium. These results suggest that colony variation is a common property among isolates of many Candida species and that strict control of agar medium thickness and composition is essential for reproducible screening of isolates for colony variations. Zusammenfassung: Einhundert Isolate von 7 Candida-Arten und 3 Isolate von Saccharomyces cerevisiae wurden auf Phloxin-B-Agar ausgeimpft und auf morphologische und farbliche Variationen der Kolonien untersucht. Kolonie-Variationen wurden gefunden in 9 von 12 C. parapsilosis-Isolaten, 8 von 13 C. tropicalis-Isolaten, 4 von 9 C. krusei-Isolaten und 12 von 30 C. albicans-Isolaten. Keines der 23 C. glabrate-Isolate wuchs auf dern Testmedium. Kolonie-Varianten wuchsen oft in sekundaren Subkulturen zu weiteren unterschiedlichen Kolonieformen aus. Die Bildungsrate fimbriater und rhizoider Kolonien von 2 C. albicans-Isolaten varriierte mit der Agarschichtdicke und dem Nahrstoffgehalt des Mediums. Diese Ergebnisse lassen vermuten, das die Kolonie-Variationen eine verbreitete Eigenschaft von Isolaten vieler Candida-Arten ist und das die strikte Einhaltung von Agarschichtdicke und Nahrmedum-Zusammensetzung fur reproduzierbare Untersuchungsresultate uber Kolonie-Variationen an Hefeisolaten essentiell ist.

Disk diffusion test and E-test with enriched Mueller-Hinton agar for determining susceptibility of Candida species to voriconazole and fluconazole.

Abstract: BACKGROUND AND PURPOSE A simplified antifungal disk diffusion test using Mueller-Hinton agar containing 2% glucose and methylene blue 5 microg/mL (GM-MH, Clinical and Laboratory Standards Institute [CLSI] M44-A) has proved to correlate well with the standard reference test. A new azole, voriconazole, has recently been approved for clinical therapy in Taiwan. This study investigated the reliability of the disk diffusion test with GM-MH agar and compared the results with those of the E-test using GM-MH agar to determine the voriconazole and fluconazole susceptibility of Candida isolates. METHODS The antimicrobial susceptibility of Candida isolates were evaluated by E-test and disk diffusion test in accordance with the guidelines of the CLSI, and compared with the reference antifungal macrodilution susceptibility test (CLSI M27-A). RESULTS The antifungal disk diffusion test and the E-test using GM-MH agar plate provided a sufficiently accurate, time-efficient, and cost-effective way to determine the susceptibility of 182 Candida spp. to voriconazole and fluconazole. There was a high correlation between the test results of the E-test using the GM-MH agar plate and those obtained by the reference antifungal macrodilution susceptibility test (CLSI M27-A). The results of the E-test and those of the 1-microg voriconazole disk diffusion test on the GM-MH agar plate at 24 h had a high correlation. All the minimal inhibitory concentrations of voriconazole for all Candida spp. were <8 microg/mL. The positive predictive value of the susceptible disk test of voriconazole on the GM-MH agar plate was 100% at 24 h for C. albicans and other Candida spp. CONCLUSION The disk diffusion test and the E-test using the GM-MH agar plate can be performed quickly, simply, and cost-effectively, and are practicable methods for the initial testing of the susceptibility of Candida spp. to voriconazole and fluconazole.