Showing papers on "Agar plate published in 2010"

Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms

Abstract: Objectives: This study was carried out to elucidate the role of reduced antibiotic penetration in the resistance of Staphylococcus aureus and Staphylococcus epidermidis biofilms to different antibiotics. Methods: The biofilms of S. aureus ATCC 29213 and S. epidermidis ATCC 35984 were grown on black, polycarbonate membranes (diameter, 13 mm; pore size, 0.4 mm) placed on tryptic soy agar plates at 378C for 48 h. The penetration of oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin through the biofilms was determined by measuring the diameter of zones of growth inhibition (of S. aureus ATCC 25923, a quality control strain) on Mueller‐Hinton agar plates following diffusion of each antibiotic from an overlying antibiotic disc through the biofilm to the agar medium versus the respective control assemblies. Results: The penetration of oxacillin and cefotaxime (b-lactams) and vancomycin (a glycopeptide) was significantly reduced through S. aureus and S. epidermidis biofilms whereas that of amikacin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone) was unaffected. Conclusions: The results of this study indicate that the role of reduced antibiotic penetration in the drug resistance of S. aureus and S. epidermidis biofilms may vary with the antibiotic being used.

Killing of adherent oral microbes by a non-thermal atmospheric plasma jet.

Abstract: Atmospheric plasma jets are being intensively studied with respect to potential applications in medicine. The aim of this in vitro study was to test a microwave-powered non-thermal atmospheric plasma jet for its antimicrobial efficacy against adherent oral micro-organisms. Agar plates and dentin slices were inoculated with 6 log(10) c.f.u. cm(-2) of Lactobacillus casei, Streptococcus mutans and Candida albicans, with Escherichia coli as a control. Areas of 1 cm(2) on the agar plates or the complete dentin slices were irradiated with a helium plasma jet for 0.3, 0.6 or 0.9 s mm(-2), respectively. The agar plates were incubated at 37 degrees C, and dentin slices were vortexed in liquid media and suspensions were placed on agar plates. The killing efficacy of the plasma jet was assessed by counting the number of c.f.u. on the irradiated areas of the agar plates, as well as by determination of the number of c.f.u. recovered from dentin slices. A microbe-killing effect was found on the irradiated parts of the agar plates for L. casei, S. mutans, C. albicans and E. coli. The plasma-jet treatment reduced the c.f.u. by 3-4 log(10) intervals on the dentin slices in comparison to recovery rates from untreated controls. The microbe-killing effect was correlated with increasing irradiation times. Thus, non-thermal atmospheric plasma jets could be used for the disinfection of dental surfaces.

Annual Variations in the Diversity, Viability, and Origin of Airborne Bacteria

Abstract: The presence of bacteria in aerosols has been known for centuries, but information on their identity and role in dispersing microbial traits is still limited. This study monitored the airborne bacterial community during an annual survey using samples collected from a 25-m tower near the Baltic Sea coast. The number of CFU was estimated using agar plates while the most probable number (MPN) of viable bacteria was estimated using dilution-to-extinction culturing assays (DCAs). The MPN and CFU values produced quantitatively similar results, displaying a pronounced seasonal pattern, with the highest numbers in winter. The most dominant bacteria growing in the DCAs all formed colonies on agar plates, were mostly pigmented (80%), and closely resembled (>97%) previously cultured bacteria based on their 16S rRNA gene sequences. 16S rRNA gene clone libraries were constructed on eight occasions during the survey; these revealed a highly diverse community with a few abundant operational taxonomic units (OTUs) and a long tail of rare OTUs. A majority of the cloned sequences (60%) were also most closely related to previously “cultured” bacteria. Thus, both culture-dependent and culture-independent techniques indicated that bacteria able to form colonies on agar plates predominate in the atmosphere. Both the DCAs and clone libraries indicated the dominance of bacteria belonging to the genera Sphingomonas sp. and Pseudomonas sp. on several sampling occasions. Potentially pathogenic strains as well as sequences closely resembling bacteria known to act as ice nuclei were found using both approaches. The origin of the sampled air mass was estimated using backward trajectories, indicating a predominant marine source.

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women.

Abstract: Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS. A total of 300 swabs was taken from 100 pregnant women at 35-37 weeks of gestation. For each subject, one rectovaginal, one vaginal and one rectal ESwab were collected. Plating onto Columbia CNA agar (CNA), group B streptococcus differential agar (GBSDA) (Granada Medium) and chromID Strepto B agar (CA), with and without Lim broth enrichment, were compared. The isolates were confirmed as S. agalactiae using the CAMP test on blood agar and by molecular identification with tDNA-PCR or by 16S rRNA gene sequence determination. The overall GBS colonization rate was 22%. GBS positivity for rectovaginal sampling (100%) was significantly higher than detection on the basis of vaginal sampling (50%), but not significantly higher than for rectal sampling (82%). Direct plating of the rectovaginal swab on CNA, GBSDA and CA resulted in detection of 59, 91 and 95% of the carriers, respectively, whereas subculturing of Lim broth yielded 77, 95 and 100% positivity, respectively. Lim broth enrichment enabled the detection of only one additional GBS positive subject. There was no significant difference between GBSDA and CA, whereas both were more sensitive than CNA. Direct culture onto GBSDA or CA (91 and 95%) detected more carriers than Lim broth enrichment and subculture onto CNA (77%). One false negative isolate was observed on GBSDA, and three false positives on CA. In conclusion, rectovaginal sampling increased the number GBS positive women detected, compared to vaginal and/or rectal sampling. Direct plating on CA and/or GBSDA provided rapid detection of GBS that was at least as sensitive and specific as the CDC recommended method of Lim broth subcultured onto non chromogenic agar.

Isolation and Partial Characterization of Bacterial Strains on Low Organic Carbon Medium from Soils Fertilized with Different Organic Amendments

Abstract: A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml−1) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml−1). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a “broad range” of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.

Plant Growth Promoting Characteristics in Some Flavobacterium spp. Isolated from Soils of Iran

Abstract: Plant growth promoting rhizobacteria (PGPR) is referred to a heterogeneous group of beneficial rhizospherebacteria that could enhance plant yield through one or more mechanisms. Flavobacterium has been noted asPGPR in almost all review articles. However, there are a few studies regarding plant growth promotion imposedby them. Some of Plant growth promoting characteristics such as Phosphate solubilizing capacity, ability to useof 1-Amino Cyclopropan-1-Carboxylate (ACC) as sole nitrogen source and production of auxin, siderophore,salicylic acid, chitinase and hydrogen cyanide were evaluated in forty-four flavobacteria isolated fromrhizosphere of wheat. Results showed that none of the isolates were able to produce siderophore, salicylic acidand chitinase and they were not able to use ACC as well. Determining the siderophore showed that none isolatesdid not grow on Chrome Azurol S (CAS) Agar medium. The results of this part were further analyzed usingCAS Agar Diffusion (CASAD) method, but the results were also negative. HCN production was observed in allisolates, but in lowest limit. Thirty-four isolates were capable to solubilize insoluble inorganic Phosphate (P)sources. The average rate of P-solubilization was 3.54 ?g Pml-1, ranging from zero to 37.48 ?gPml-1. There wasa significant negative correlation (r = -0.81**) between solubilized P and the final pH of the growth medium. Inthis study, all the isolates were able to produce auxin, ranging from 0.27 to 12.03 ?gml-1 averaged by 2.03.Considering the ability of the isolates to produce auxin and for P-solubilization, it is necessary to evaluate theireffect on growth and yield of different crop plants.

McKay agar enables routine quantification of the 'Streptococcus milleri' group in cystic fibrosis patients.

Abstract: The 'Streptococcus milleri' group (SMG) has recently been recognized as a contributor to bronchopulmonary disease in cystic fibrosis (CF). Routine detection and quantification is limited by current CF microbiology protocols. McKay agar was developed previously for the semi-selective isolation of this group. Here, McKay agar was validated against a panel of clinical SMG isolates, which revealed improved SMG recovery compared with Columbia blood agar. The effectiveness of this medium was evaluated by appending it to the standard CF sputum microbiology protocols in a clinical laboratory for a 6-month period. All unique colony types were isolated and identified by 16S rRNA gene sequencing. Whilst a wide variety of organisms were isolated, members of the SMG were the most prevalent bacteria cultured, and McKay agar allowed routine quantification of the SMG from 10(3) to >10(8) c.f.u. ml(-1) directly from sputum. All members of the SMG were detected [Streptococcus anginosus (40.7 %), Streptococcus intermedius (34.3 %) and Streptococcus constellatus (25 %)] with an overall prevalence rate of 40.6 % in our adult CF population. Without exception, samples where SMG isolates were cultured at 10(7) c.f.u. ml(-1) or greater were associated with pulmonary exacerbations. This study demonstrates that McKay agar can be used routinely to quantify the SMG from complex clinical samples.

Biochemical and Molecular Characterization of a New Local Keratinase Producing Pseudomomanas sp., MS21

Abstract: This study aimed to isolate and identify a new local bacterial strain, able to completely degrade keratin-rich wastes into soluble and useful materials which can be used in many proposes. Bacterial keratinases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. These enzymes have been studied for de-hairing processes in the leather industry and hydrolysis of feather and keratin. Samples from poultry industry wastes, soil, water, fodder and feather were collected from different places in Jeddah, Saudi Arabia. Each sample was plated on feather meal agar plates containing 5 g L feather as the sole carbon and nitrogen source and 1 the obtained colonies were selected, purified and their growth were detected on skimmed milk agar and feather meal broth media. The well grown isolates on feather meal agar which producing the largest clearing zone on skimmed milk plate were selected for keratinase assays. Out of 23 bacterial isolates, 7 isolates were selected. The best keratinase producing bacterium kera MS21 was selected and identified based on morphological, physiological and some biochemical characteristics. It was recorded as a species belonging to the genus Pseudomonas and identified as Pseudomonas sp. The results of identificatio n were confirmed by 16S rDNA studies. Precipitation and purification of the keratinase enzyme in addition to factors affecting enzyme activity were studied. The enzyme molecular weight was determined to be of 30 KDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The optimum temperature and pH were determined to be 37°C and pH 8.0, respectively. The effect of some proteases inhibitors and activators were also studied.

Heavy metal resistance by two bacteria strains isolated from a copper mine tailing in China

Abstract: Two highly heavy metal resistant indigenous bacterial strains, DX-T3-01 and DX-T3-03, were isolated from the biggest tailing in Asia-Dexing copper mine 4# tailing. The strain DX-T3-01 exhibited high tolerance to cadmium: 10 mM Cd2+ with yeast tryptophan peptone glucose (YTPG) agar plates and 18 mM in liquid medium. The strain DX-T3-03 was highly resistant to zinc and could endure 35 mM Zn2+ with YTPG agar plates and 40 mM in liquid medium. They also showed tolerance to other heavy metals, such as copper, lead and nickel. The morphology, physiological and biochemical characteristics of the two strains were examined by scanning electron microscope (SEM) and BIOLOG. The strains showed different metabolic patterns of carbon sources. The strain DX-T3-03 had a larger range of antibiotic resistance than DX-T3-01. On the basis of 16S rDNA sequencing, the two strains were identified as Ralstonia pickettii strain DX-T3-01 and Sphingomonas sp. strain DX-T3-03, respectively. This study supplied potential bacterial materials for tailing bioremediation in the future. Key words: Heavy metals, tolerant bacteria, isolation, mine tailing.

Cultivation of hitherto-uncultured bacteria belonging to the Verrucomicrobia subdivision 1 from the potato (Solanum tuberosum L.) rhizosphere

Abstract: Purpose The role of dominant bacterial groups in the plant rhizosphere, e.g., those belonging to the phyla Acidobacteria and Verrucomicrobia, has, so far, not been elucidated, and this is mainly due to the lack of culturable representatives. This study aimed to isolate hithertouncultured bacteria from the potato rhizosphere by a combination of cultivation approaches. Materials and methods An agar medium low in carbon availability (oligotrophic agar medium) and either amended with potato root exudates or catalase or left unamended was used with the aim to improve the culturability of bacteria from the potato rhizosphere. The colony forming unit numbers based on colonies and microcolonies were compared with microscopically determined fluorescence-stained cell numbers. Taxonomical diversity of the colonies was compared with that of library clones made from rhizosphere DNA, on the basis of 16S rRNA gene comparisons. Results and discussion The oligotrophic media amended or not with catalase or rhizosphere extract recovered up to 33.6% of the total bacterial numbers, at least seven times more than the recovery observed on R2A. Four hithertouncultured Verrucomicrobia subdivision 1 representatives were recovered on agar, but representatives of this group were not found in the clone library. Conclusions The use of oligotrophic medium and its modifications enabled the growth of colony numbers, exceeding those on classical agar media. Also, it led to the isolation of hitherto-uncultured bacteria from the potato rhizosphere. Further improvement in cultivation will certainly result in the recovery of other as-yet-unexplored bacteria from the rhizosphere, making these groups accessible for further investigation, e.g., with respect to their possible interactions with plants.

Effect of reactor surface on production of bacterial cellulose and water soluble oligosaccharides by Gluconacetobacter hansenii PJK

Abstract: The effect of agar plates on the bacterial cellulose (BC) production in a static culture was investigated in order to find the role of agar component as a surface modifying agent. Two types of surface modified reactors (SMRs: SMRD and SMRB) were prepared by coating the bottom of the reactors with agar dissolved in distilled water and basal medium, respectively. The SMRs were used for BC and water soluble oligosaccharides (WSOS) production. Control was done by the same procedure using reactors without agar plate. In both types of SMRs, the maximum production rate was observed after the second day of cultivation compared to third day of cultivation in the case of the control. The maximum productions of BC 5.308 and 5.472 g/L were observed at the first batch using SMRs prepared with agar dissolved in distilled water (SMRDs) and SMRs prepared with agar dissolved in a basal medium (SMRBs), respectively. Similarly, in the daily-culture and successive batch strategy experiments the maximum amount of WSOS produced in the SMRs was almost double that of the control. The highest water holding capacity value 92.21 g/g was observed for BC formed in the SMRs prepared with 3.0% of agar. FTIR and XRD analyses were carried out to study the structural features of the prepared BC.

Detection of Campylobacter colonies using hyperspectral imaging

Abstract: The presence of Campylobacter in foods of animal origin is the leading cause of bacterially induced human gastroenteritis. Isolation and detection of Campylobacter in foods via direct plating involves lengthy laboratory procedures including enrichments and microaerobic incubations, which take several days to a week. The incubation time for growing Campylobacter colonies in agar media usually takes 24–48 h. Oftentimes the problem is the difficulty of visually differentiating Campylobacter colonies from non-Campylobacter contaminants that frequently grow together with Campylobacter on many existing agars. In this study, a new screening technique using non-destructive and non-contact hyperspectral imaging was developed to detect Campylobacter colonies in Petri dishes. A reflectance spectral library of Campylobacter and non-Campylobacter contaminants was constructed for characterization of absorption features in wavelengths from 400 to 900 nm and for developing classification methods. Blood agar and Campy-Cefex agar were used as culture media. The study found that blood agar was the better culture medium than Campy-Cefex agar in terms of Campylobacter detection accuracy. Classification algorithms including single-band thresholding, band-ratio thresholding and spectral feature fitting were developed for detection of Campylobacter colonies as early as 24 h of incubation time. A band ratio algorithm using two bands at 426 and 458 nm chosen from continuum-removed spectra of the blood agar bacterial cultures achieved 97–99% of detection accuracy. This research has profound implications for early detection of Campylobacter colonies with high accuracy. Also, the developed hyperspectral reflectance imaging protocol is applicable to other pathogen detection studies.

Evaluation of Blood Agar Microtiter Plates for Culturing Leishmania Parasites To Titrate Parasite Burden in Spleen and Peripheral Blood of Patients with Visceral Leishmaniasis

Abstract: Serial dilution of blood and spleen biopsy specimens, plated on Novy-MacNeal-Nicolle (NNN) blood agar using microtiter culture plates, is a sensitive and reproducible method for detection and growth of Leishmania parasites. Plates could be easily monitored, and growth could be rapidly detected. Moreover, parasite number may be estimated using this technique.

Inhibitory effect of quercetin on periodontal pathogens in vitro

Abstract: Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) are bacteria strongly associated with early onset, progressive and refractory periodontal disease and associated alveolar bone loss. Quercetin is a flavonoid found in many foods including apples, onions and tea. The aim of this study was to evaluate the effect of quercetin on in vitro growth of periodontal pathogens Aa and Pg. For comparison, quercetin's effect on several oral microbes was also evaluated. Different concentrations of quercetin solution were added to calibrated suspensions of Aa and Pg. All suspensions were incubated for 1, 3, 6, and 24 h in an anaerobic chamber at 37 degrees C. At each time point, selected dilutions from each culture broth were plated on blood agar plates. Colonies appearing on blood agar plates were visually counted on 3 days for Aa and 5 days for Pg. Minimum inhibitory concentrations of both periodontal pathogens were also determined. Both periodontal bacteria showed a significant decrease (p < 0.05) in viable counts after 1 h. No colony forming units of Pg could be observed after 24 h. The results suggest that quercetin possesses significant antimicrobial properties on periodontal pathogens in vitro.

Underestimation of Vancomycin and Teicoplanin MICs by Broth Microdilution Leads to Underdetection of Glycopeptide-Intermediate Isolates of Staphylococcus aureus

Abstract: Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus. Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 10(6) CFU, were significantly higher (2 microg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 microg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 microg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 microg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.

Susceptibility of Metarhizium spp. and other entomopathogenic fungi to dodine-based selective media

Abstract: The fungicide dodine has been widely used in selective media to isolate entomopathogenic fungi (EF) from contaminating microorganisms, primarily bacteria and non-entomopathogenic fungi. In order to isolate the fungus Metarhizium acridum from soil for grasshopper and Mormon cricket control in the western USA, the susceptibility of M. acridum was compared with two Metarhizium spp. and other EF species. The isolates were inoculated onto mycological media with concentrations of dodine ranging from 0.0001 to 0.03% active ingredient (A.I.). In addition, susceptibilities of five Metarhizium spp. isolates to two sources of dodine, Syllit® commercial fungicide (65% A.I.) and Sigma® reagent grade (99% A.I.), were compared using Czapek agar medium. Responses to the two dodine sources were virtually identical. Accordingly, subsequent experiments used the less expensive Syllit dodine. Three media [Czapek, potato dextrose agar plus yeast extract (PDAY) and oatmeal agar] were evaluated for appropriateness as th...

Culture and Identification of Candida Albicans from Vaginal Ulcer and Separation of Enolase on SDS-PAGE

Abstract: Candida albicans were isolated from patients with clinical symptoms of vaginal ulcer. Culture test for vaginal swab andscrapings were conducted on Sabouraud’s dextrose broth and Sabouraud’s dextrose agar plate respectively. Hichromecandida agar culture was used for differential identification of Candida. Smears from vaginal scrapings were preparedfor gram staining. The suspected strain of Candida was inoculated on corn meal agar medium for chlamydosporeformation. The suspected strain of Candida was inoculated in human serum for germ tube formation. Carbohydrateassimilation and fermentation tests were also conducted. The selected Candida colony was inoculated in YEPD mediumfor subculture and the cultured organism was harvested. The organisms were homogenized, centrifuged and thesupernatant was filtered. The filtrate was extracted in chloroform. The extract was centrifuged and the aqueous phasewas dialyzed. The dialyzed crude enolase was subjected to SDS-PAGE. The Sabouraud’s dextrose broth inoculatedwith vaginal swab showed turbid growth. The scraping from vagina showed typical smooth creamy white colonies witha characteristic yeast odour on Sabouraud’s dextrose agar plate. On Hichrome candida agar the Candida growthappeared as glistening green colored. Gram stained smears from vaginal scraps showed appearance of fungus as yeast budding. On corn meal agar the suspected Candida growth showed the formation of large, highly refractive, thickwalled terminal chlamydospores. Germ tubes were seen as long tube like projections extending from the yeast cells onhuman serum inoculated with suspected strain of Candida. The carbohydrate assimilation tests were positive fordextrose, maltose, sucrose, galactose, xylose and trehalose, and negative for lactose, melibiose, ellobiose, inositol,reffinose and dulcitol. The carbohydrate fermentation tests showed positive for dextrose, maltose, galactose andtrehalose, and negative for sucrose and lactose. SDS-PAGE for enolase from C. albicans showed a single polypeptideband of around 46 – 48 kDa.

Mango Malformation Disease in South China Caused by Fusarium proliferatum

Abstract: A total of 13 Fusarium isolates were obtained from samples of malformed mango seedlings from Yunnan and Sichuan provinces in China, and five morphologically similar isolates were confirmed causing the disease by satisfying Koch’s postulates. One typical isolate (MG4) was selected for detailed morphological and molecular studies. Based on the following morphological characteristics, isolate MG4 was identified as Fusarium proliferatum: white aerial mycelium on PSA (potato sucrose agar: potato 200 g; sucrose 15 g; agar 18 g; distilled water 1000 ml) medium; hyaline reverse of colonies on PSA; production of pink pigment on rice medium and the production of conidia on branched conidiophore with monophialides bearing false heads of conidia. On carnation leaf agar medium, the microconidia were ovate to elongated ovoid, 0–1 septate, 3.1–10.2 × 1.5–2.2 μm; the macroconidia were fusiform, 3–5 septate, 18–38 × 1.8–2.4 μm, whereas chlamydospores and sexual structures were absent on all media used. The identity of the pathogen was confirmed by its high similarity (99.8–100%) in the sequence alignment of rDNA-ITS 1 and 4 with both isolates of F. proliferatum in the GenBank database.

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

Abstract: A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression.

Abstract: Aims: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. Methods and Results: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. Conclusion: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. Significance and Impact of the Study: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Production of Polyhydroxybutyrate (Bioplastic) and its Biodegradation by Pseudomonas Lemoignei and Aspergillus Niger

Abstract: The biodegradation of polyhydroxybutyrate (PHB) and its copolymer polyhydroxy butyrate-co-hydroxyvalerate (PHB-co-HV) was studied. Bacterial as well as fungal isolates were isolated from the local industrial ecosystem. All these isolates were tested for the degradation of the above polymers in assay agar medium as well in liquid cultures. The culture biomass and the clear zone around the colonies were measured to evaluate the activity of these isolates. In all, the fungal isolates were found to degrade these polymers more rapidly when compared to bacteria, due to their versatile depolymerase activities.

Antimicrobial activity of some selected vegetables

Abstract: The aqueous and methanolic extracts of spinach (Spinacea oleracia), pumpkin (cucurbita pepo), suran (Amorphophalus campanulatus) and ghuiya (Colocasia esculenta) were evaluated for antimicrobial activity against bacterial strains (Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Enterobacter agglomerans, Salmonella enteritidis, Salmonella cholerasius, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Penicillium chrysogenum, Enterobacter faecalis, Klebsiella pneumonia, B.sphericus, B.thruengenesis and Cryptococcus meningitis). The in vitro antimicrobial activity was performed by Agar well diffusion method on Nutrient Agar medium and Muller Hinton Agar medium. The methanolic extracts of all the vegetables showed moderate to high activity against all the investigated microbial strains. Methanolic extract of spinach was most effective among all extracts (32mm inhibition zone against E. coli). Methanolic extracts were more effective than

Evaluation of microbial contamination associated with different preparation methods for neonatal intravenous fat emulsion infusion.

Abstract: Purpose Microbial contamination associated with different methods of neonatal intravenous fat emulsion (IVFE) preparation and delivery was evaluated. Methods Sterility testing was performed on IVFE dispensed via three different methods: (1) in the original container ( n = 60), (2) repackaged into a syringe ( n = 90), and (3) drawdown of the original container ( n = 60). At the end of each infusion (24 hours for methods 1 and 3, 12 hours for method 2), a sample of the IVFE was withdrawn from the container using a sterile syringe in an International Organization for Standardization class 5 hood and sent to the hospital microbiology laboratory, where the samples were introduced into blood culture bottles and incubated for five days. Each sample was then subcultured on a blood agar plate with olive oil and left for an additional two days in a carbon dioxide incubator to assess for Malassezia furfur . Results None of the samples from the original containers showed bacterial or fungal growth. Three of the samples from syringes had bacterial growth (two samples contained coagulase-negative staphylococcus and one contained both Klebsiella oxytoca and Citrobacter freundii ), yielding a contamination rate of 3.3%. The number of contaminated samples did not significantly differ among the three preparation methods ( p = 0.13). Conclusion Repackaging IVFE into sterile syringes resulted in bacterial contamination and should be avoided in clinical practice. IVFE samples obtained using the drawdown procedure under sterile conditions for infusion over 24 hours revealed no microbial contamination.

Isolation of two Kocuria species capable of growing on various polycyclic aromatic hydrocarbons

Abstract: Different samples collected from crude oil contaminated beach were enriched for isolation of bacterial strains capable of growing on naphthalene, phenanthrene and fluoranthene. Respiratory reduction of WST-1{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} to a colored formazan showed that one isolated strain CMG2028, identified as Kocuria flava by 16s rRNA, grew on naphthalene and phenanthrene while CMG2042, identified as Kocuria rosea grew on all three polycyclic aromatic hydrocarbons (PAHs). In naphthalene degradation test, 64 and 47% residual naphthalene was extracted after ten days of incubation from culture medium of K. rosea CMG2042 and K. flava CMG2028, respectively, when provided with 0.5 mg ml-1 concentration as sole carbon source. Due to addition of 0.5 mg ml-1 yeast extract as additional carbon source, residual naphthalene extracted was 41 and 55% from K. rosea CMG2042 and K. flava CMG2028, respectively. Both strains exhibited growth on 0.01 mg ml-1 phenanthrene and fluoranthene in yeast extract added or omitted medium but only K. rosea CMG2042 degraded 9% phenanthrene as a sole carbon source. Both strains had growth on minimal agar plates coated with Iranian light crude oil as sole carbon source and on agar medium added with yeast extract.

Evaluation of Spectra VRE, a New Chromogenic Agar Medium Designed To Screen for Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium

Abstract: Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h.

Is inclusion of Sabouraud dextrose agar essential for the laboratory diagnosis of fungal keratitis

Abstract: Purpose: To determine whether the inclusion of Sabouraud dextrose agar (SDA) is essential in the diagnosis of fungal keratitis. Materials and Methods: Corneal scrapings of 141 patients with microbial keratitis were smeared and cultured. Sheep blood agar (BA), chocolate agar (CA), SDA, non-nutrient agar (NNA) with Escherichia coli overlay, and brain heart infusion broth (BHI) were evaluated for time taken for growth and cost. The media were also evaluated experimentally for rate of growth and time taken for identification. Results: Twenty-six of 39 patients positive for fungus in corneal scrapings by microscopy were culture-positive. Fungus grew on BA in 22/39, on CA in 18/39, on SDA in 17/39, on NNA in 17/39, and on BHI in 13/39 cases. Growth on SDA was higher in ulcers with larger infiltrate (6/18 versus 9/13, P = 0.04). Estimated saving with inclusion of only BA/CA was Rs. 600 per patient. Performance of all media was similar in in vitro experiment although the characteristic spores and color were seen earlier on SDA. Conclusion: Fungal keratitis can be reliably confirmed on BA or CA, which support growth of both bacteria and fungus.