Showing papers on "Agar plate published in 2019"

Antibacterial efficacy of cold atmospheric plasma against Enterococcus faecalis planktonic cultures and biofilms in vitro.

Abstract: Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC.

Metabolic characterization of supernatants produced by Lactobacillus spp with in vitro anti-Legionella activity

Abstract: Legionella pneumophila is an organism of public health interest for its presence in water supply systems and other humid thermal habitats. In this study, ten cell-free supernatants produced by Lactobacillus strains were evaluated for their ability to inhibit L. pneumophila strains isolated from hot tap water. Production of antimicrobial substances by Lactobacillus strains were assessed by agar well diffusion test on BCYE agar plates pre-inoculated with L. pneumophila. Cell-free supernatants (CFS) showed antimicrobial activity against all Legionella strains tested: L. rhamnosus and L. salivarius showed the highest activity. By means of a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy, we detected and quantified the Lactobacillus metabolites of these CFSs, so to gain information about which metabolic pathway was likely to be connected to the observed inhibition activity. A panel of metabolites with variations in concentration were revealed, but considerable differences among inter-species were not showed as reported in a similar work by Foschi et al. (2018). More than fifty molecules belonging mainly to the groups of amino acids, organic acids, monosaccharides, ketones, and alcohols were identified in the metabolome. Significant differences were recorded comparing the metabolites found in the supernatants of strains grown in MRS with glycerol and the same strains grown in MRS without supplements. Indeed, pathway analysis revealed that glycine, serine and threonine, pyruvate, and sulfur metabolic pathways had a higher impact when strains were grown in MRS medium with a supplement such as glycerol. Among the metabolites identified, many were amino acids, suggesting the possible presence of bacteriocins which could be linked to the anti-Legionella activity shown by cell-free supernatants.

Automated Detection of Streptococcus pyogenes Pharyngitis by Use of Colorex Strep A CHROMagar and WASPLab Artificial Intelligence Chromogenic Detection Module Software.

Abstract: Colorex Strep A agar (CHROMagar, Paris, France) was evaluated with PhenoMATRIX chromogenic detection module (CDM) software (Copan Diagnostics Inc., Murrieta, CA) to detect group A Streptococcus (GAS) from throat specimens. The software results were compared to those of manual plate image reading. In addition, GAS PCR testing was performed on all specimens. True-positive specimens were defined as culture-positive (by either PhenoMATRIX CDM or manual reading) specimens confirmed as GAS by matrix-assisted laser desorption ionization-time of flight mass spectrometry plus any culture-negative specimens that were positive by both initial and repeat PCR testing. Of 480 specimens, 96 were considered true-positive specimens. Software reading of the chromogenic agar for suspected colonies detected 110 orange colonies, whereas technologist reading interpreted only 93/110 specimens (84.5%) as positive. None of the 361 cultures interpreted as negative by the PhenoMATRIX CDM software was positive by manual reading. In comparison with true-positive results, the sensitivity and specificity were 96.9% and 100% for PCR testing, 87.5% and 97.7% for technologist reading of chromogenic agar, 90.6% and 94.0% for software reading of chromogenic agar, 83.3% and 97.7% for technologist reading for β-hemolysis on blood agar, and 39.5% and 83.1% for technologist reading for β-hemolysis on blood agar accompanied by any zone of inhibition around a bacitracin-impregnated disk, respectively. The software had the most accurate results of the non-molecular testing methods, detecting all suspected colonies on the chromogenic agar and identifying 3 additional true-positive specimens that were missed by manual reading. The PhenoMATRIX CDM software and the Colorex Strep A agar can improve detection of GAS from throat specimens, and they compared favorably to molecular testing.

Effect of cosmetic chemical preservatives on resident flora isolated from healthy facial skin.

Abstract: Background/aims Healthy skin harbors numerous microbes known to maintain its health and prevent attacks from external pathogens. The influence of chemical preservatives commonly used in cosmetic products on facial resident flora remains poorly characterized. In this study, we aimed to investigate the antibacterial activity of five such preservatives on in vitro cultivated skin-resident bacteria. Methods Both Gram-positive and Gram-negative bacteria were isolated on blood agar, tryptic soy agar, and nutrient agar; Gram-negative bacteria were then selected on Hank's balanced salt solution containing antibiotics and Reasoner's 2A. The minimum inhibitory concentrations (MICs) of methylisothiazolinone (MTI), iodopropynyl butylcarbamate (IPBC), ethylhexylglycerin (EHG), methylparaben (MP), and phenoxyethanol (PE) were estimated for nine facial resident bacteria, Escherichia coli, and Staphylococcus aureus using serial broth dilution in vitro. Results The maximum test concentrations coincided with the upper limits set by the "Cosmetic Safety and Technical Specification" (2015 edition, China). Nine facial resident bacteria were isolated from 14 healthy adults: Staphylococcus epidermidis, Staphylococcus capitis, Kocuria, Micrococcus luteus, Bacillus, Acinetobacter, Pseudomonas parafulva, Pseudomonas oleovorans, and Roseomonas cervicalis. MTI and IPBC displayed the strongest effect on all tested strains (MICs ≤0.01%), followed by EHG and MP (MICs ≤0.3%), and finally PE with the weakest effect (MIC ≤1%). Conclusion The five chemical preservatives assayed inhibited survival of the nine facial resident bacteria isolates, when tested at the maximum allowed limit. The corresponding MICs will provide a reference for the effective utilization of these compounds in product formulations.

Antibiotic susceptibility of bacterial pathogens recovered from the hand and mobile phones of university students.

Abstract: Introduction This study aimed to isolate bacterial pathogens from the dominant hand and mobile phones and to determine their antibiotic susceptibility profiles. The dominant hand and mobile surfaces were swabbed to detect the transmission of bacterial pathogens among university students. Methods Two hundred and twenty hand and mobile phone swabs were collected from the students of four different colleges in a Jordanian university between October and December 2017. The swabs were collected and transported to the Microbiology laboratory within one hour. At the lab, swabs were inoculated on nutrient agar, MacConkey agar, blood agar and mannitol salt agar. The subsequent bacterial isolates were identified by their cultural, morphological and biochemical characteristics. Results Eight bacterial species were isolated and identified in the current study, namely Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, Micrococcus spp. and Escherichia coli. The percentage of isolated bacteria was 54.5%, 25.5%, 14.5% and 5.5% from veterinary, biology, biomedical engineering and chemistry students, respectively. Many isolates were highly resistant to most tested antibiotics. Conclusions Pathogenic bacteria were detected with multiple antibiotic resistance indexes. Hands and mobile phones can act as carriers for infectious agents, suggesting the need for proper hand hygiene and disinfecting mobile phones surfaces.

Deciphering rhizosphere microbiome for the development of novel bacterial consortium and its evaluation for salt stress management in solanaceous crops in India

Abstract: Eighty bacteria were isolated from the salt affected solanaceous crops (potato, tomato, chilli and brinjal) rhizosphere and fallow land in Mau, Faizabad, Ballia, Gazipur and Varanasi districts of Uttar Pradesh, India. All the bacterial isolates showed morphological variation on Nutrient agar, Jenson’s medium, Modified Czapek-Dox medium, Mannitol egg yolk polymyxin agar, Pseudomonas isolation agar medium, Luria Bertni agar, Tryptic soya agar, King B agar and Nutrient broth yeast extract. Majority of the isolates showed creamy colony (32.5%), spherical (66.25%), shiny (88.7%), raised (62.25%), translucent (71.25%) and entire type of surface margin (78.75%). The gram staining, catalase test, glucose fermentation, IAA test, H2S production, starch hydrolysis, tween 80, gelatin hydrolysis, casein hydrolysis, P solubilization, Zn solubilization and ammonia production tests revealed that 26, 28, 13, 3, 11, 10, 18, 10, 17, 24, 23 and 16 isolates were positive for these tests respectively. The molecular characterization of bacterial isolates Brevibacillus fluminis, Brevibacillus agri, Bacillus paralicheniformis, and Microbacterium arborescens revealed that these are highly salt tolerant (18%) produced 37 alleles using box primer. The number of bands (400–3190 bp) varied from 1 to 10. A cluster analysis with unweighted pair-group method with arithmetic means (UPGMA) based on Jaccard’s similarity coefficient divided the bacterial isolates into two major and three minor clusters with coefficient of similarities ranging from 1 to 23%. Phylogenetic analysis of salt tolerant bacterial isolates showed that out of 22 isolates, 17 (77.27%) were found to be phylogenetically closely related to Bacillus, with 99–100% similarity in their 16S rDNA sequences, making Bacillus the most dominant genus among salt tolerant bacteria in the study.

Surface mould growth on wood: a comparison of laboratory screening tests and outdoor performance

Abstract: Laboratory screening tests are commonly used to indicate wood materials’ resistance or susceptibility to surface mould growth, but the results can deviate from what happens during outdoor exposure. In this study, the aim was to investigate how well agar plate screening tests and water uptake tests can predict mould growth on exterior wooden claddings. The tested wood materials included Norway spruce heartwood (Picea abies), sapwood and heartwood of Scots pine (Pinus sylvestris), aspen (Populus tremula), acetylated Radiata pine (Pinus radiata) and DMDHEU-modified Scots pine sapwood. The agar plate test included four inoculation methods (two monoculture spore suspensions of Aureobasidium species, one mixed-culture spore suspension, and inoculation from outdoor air) and three incubation temperatures (5, 16 and 27 °C). Inoculation method and incubation temperature had significant effects on the mould rating in the agar plate screening test, but none of the agar plate test combinations gave good indications of outdoor performance. Results from the agar plate test gave significantly negative correlations or no significant correlation with results from the outdoor test. However, the water uptake test gave significantly positive correlations with outdoor mould rating, and could be a useful indicator of susceptibility of uncoated wooden claddings to surface mould growth.

Isolation and Characterization of Cellulase-producing Bacteria from Sugar Industry Waste

Abstract: Cellulases are inducible enzymes that are synthesized by a large number of microorganisms during their growth on cellulosic materials. This study focuses on the isolation and screening of cellulase-producing bacteria from sugar industry waste (molasses) and characterization by morphological and biochemical analysis. Further, purification of cellulase was carried by ammonium sulfate precipitation and followed by column chromatography and molecular weight determined by SDS-PAGE. The isolated bacterial strains were grown on carboxymethyl cellulose (CMC) agar plate at various optimum conditions like pH, temperature, incubation period, carbon and nitrogen sources and substrate concentration. Three isolated strains showed clear hydrolyzing zone on agar plates containing CMC agar after Congo-red staining were identified as cellulase-producing bacteria. Based on cultural, morphological, and biochemical characteristics, the isolated strains were identified as Paenibacillus sp., Aeromonas sp., and Bacillus sp. Among the isolated strains, Paenibacillus sp. showed the capability for highest cellulase production (0.89 µmol ml-1 min-1) at optimal pH 7.0 and 40°C temperature on 24 hour of the incubation period at 1% CMC substrate concentration and was selected for further cellulase purification. In the final step of cellulase purification, the specific activity, purification fold and recovery were 1720 U/mg, 9.74 and 35.6%, respectively. The molecular weight of the purified enzyme was determined 66.9 kDa and the enzyme showed a high specificity to CMC substrate. The bacterial strains present in molasses have the potential for cellulase production. Substrate specificity of the purified cellulase indicates it to be an endo-β-1, 4-glucanase. The cellulase produced from the selected strain may benefit for industrial application.

Isolation and identification of marine strains of Stenotrophomona maltophilia with high chitinolytic activity.

Abstract: Chitin is the second most abundant organic compound in nature and represents a rich carbon and nitrogen source that is primarily transformed by bacterial communities. Bacteria capable of gradually hydrolyzing chitin into N-acetylglucosamine monomers can have applications in the transformation of residues from shrimp and other crustaceans. The objective of the present study was to isolate, characterize and identify microorganisms with high chitinolytic activity. These microorganisms were isolated and characterized based on macro- and microscopic morphological traits. Strains were selected on colloidal chitin agar medium primarily based on a hydrolysis halo larger than 2 mm and a growing phase no longer than 6 days. Secondary selection consisted of semi-quantitative evaluation of chitinolytic activity with a drop dilution assay. From the above, ten strains were selected. Then, strain-specific activity was evaluated. The B4 strain showed the highest specific activity, which was 6,677.07 U/mg protein. Molecular identification indicated that the isolated strains belong to the species Stenotrophomonas maltophilia.

Growth of E. coli on Solid Media

Abstract: We provide protocols for titering and isolating bacterial colonies from single cells by serial dilutions, for streaking agar plates, and for spreading suspensions of cells on plates. Support protocols describe replica plating and methods for storing strains as agar stabs and frozen stocks. © 2018 by John Wiley & Sons, Inc.

Bacterial isolation from internal organs of rats (Rattus rattus) captured in Baghdad city of Iraq.

Abstract: Aim Rats are accused in disseminating many zoonotic diseases. This study aimed to isolate and identify bacteria from internal organs of rats captured in Baghdad City, Iraq. Materials and methods A total of 120 black rats (R. rattus) were trapped from different areas in Baghdad city. Rats were kept in individual plastic cages for 3 h before euthanizing. Deep pharyngeal swab, intestinal content, urine, and pieces of the liver and spleen, lung, kidney, and brain were obtained aseptically. The specimens were inoculated into peptone water and incubated at 37°C for 24 h for enrichment. A loopful of each specimen was then subcultured onto MacConkey Agar, Blood Agar, and Mannitol Salt Agar. CHROMagar O157 H7 and CHROMagar Listeria were used to detect Escherichia coli 157:7 and Listeria spp., respectively. Biochemical tests on analytical profile index, microscopic examination, and commercial kit for latex agglutination test for serotyping E. coli O157:H7 were used. Results Mixed bacterial isolates were recorded as 116, 52, 36, 28, 18, 6, and 4 from intestinal contents, deep pharyngeal, liver and spleen, urine, lung, brain, and kidney, respectively. Microorganisms included E. coli, Staphylococcus aureus, Streptococcus spp., Bacillus spp., Pseudomonas aeruginosa, Citrobacter freundii, Proteus vulgaris, E. coli O157:H7, Enterobacter cloacae, Listeria spp., Klebsiella spp., Ochrobactrum anthropi, Aeromonas spp., Brucella spp., Pseudomonas fluorescens, Escherichia fergusonii, Micrococcus spp., Morganella spp., Proteus mirabilis, Pseudomonas luteola, and Streptobacillus spp. The highest bacterial prevalence (88; 73.33%) was recorded for E. coli, where 68 isolates were identified from the intestinal contents. Of these, four isolates were E. coli O157:H7. Conclusion Rats are important carriers and transmitters of a number of pathogens and can disseminate these microorganisms to humans and animals.

Neisseria chenwenguii sp. nov. isolated from the rectal contents of a plateau pika (Ochotona curzoniae).

Abstract: Two Gram-stain negative, catalase positive, coccus shaped bacteria, designated 10023T and 10010, were isolated from the rectal contents of a plateau pika (Ochotona curzoniae) in Qinghai–Tibet Plateau, China. Based on 16S rRNA gene sequence analysis, phylogenetic trees showed that these two isolates (10023T, 10010) group with members of the genus Neisseria. Additionally, these two isolates exhibited high 16S rRNA gene sequence similarity with Neisseria zalophi CSL 7565T (96.98%), Neisseria wadsworthii WC 05-9715T (96.92%) and Neisseria canis ATCC 14687T (96.79%). Further phylogenetic analysis based on the rplF gene showed that these two novel strains can be easily discriminated from phylogenetically closely related species. Optimal growth was found to occur on BHI agar with 5% defibrinated sheep blood at 37 °C and growth was also observed on nutrient agar, Columbia blood agar and chocolate agar plates; however, growth was not observed on MacConkey agar after 7 days. The major cellular fatty acids of these strains were identified as C16:0 and C16:1ω7c/C16:1ω6c. The complete genome size of the type strain 10023T is 2,496,444 bp, with DNA G+C content of 54.0 mol %. The average nucleotide identity values were 73.5–79.3% between isolate 10023T and reference Neisseria spp. Based on polyphasic analysis, these isolates (10023T and 10010) are considered to represent a novel species in the genus Neisseria, for which the name Neisseria chenwenguii sp. nov. is proposed. The type strain is 10023T (= DSM 103440T = CGMCC 1.15736T).

The Growth of Staphylococcus aureus in the blood agar plate media of sheep blood and human blood groups A, B, AB, and O

Abstract: BAP media is a medium used to distinguish pathogenic bacteria based on their hemolytic power on red blood cells. Staphyllococcus aureus is a bacterium that is able to emolate red blood cells with 3 types of hemolysis, namely α, β, γ, and δ. Usually BAP media is made by adding 5-10% sheep blood. Making BAP media using sheep blood has become a problem for several laboratories today, including health education laboratories. This is because the health education laboratory does not yet have a sheep farm, so it has not been able to procure sheep blood. The use of human blood as a substitute for sheep blood in making BAP media may be a solution, but it is not yet known whether there are differences in the growth and hemolysis of S. aureus bacteria on BAP media in sheep's blood and human blood. This research is an experimental study with a completely randomized design (CRD) of 3 replications which aims to determine whether there are differences in growth and hemolysis of bacteria S. aureus in BAP media of sheep blood and human blood groups A, B, AB, and O. The results showed that S. aureus bacteria could grow and show hemolysis in BAP media in sheep blood and human blood in groups A, B, AB, and O. The results of subsequent studies analyzed ANOVA using the software spss for windows with a significant level of 0.05. From the results of research and data analysis it can be concluded that S. aureus bacteria can grow and show hemolysis in BAP media of sheep blood and human blood groups A, B, AB and O, but there are significant differences in the number of S. aureus bacteria colonies grown in BAP media of sheep's blood and human blood groups A, B, AB and O.

Lysogeny in the Lactic Acid Bacterium Oenococcus oeni Is Responsible for Modified Colony Morphology on Red Grape Juice Agar.

Abstract: Oenococcus oeni is the lactic acid bacterium (LAB) that most commonly drives malolactic fermentation in wine. Although oenococcal prophages are highly prevalent, their implications on bacterial fitness have remained unexplored and more research is required in this field. An important step toward achieving this goal is the ability to produce isogenic pairs of strains that differ only by the lysogenic presence of a given prophage, allowing further comparisons of different phenotypic traits. A novel protocol for the rapid isolation of lysogens is presented. Bacteria were first picked from the center of turbid plaques produced by temperate oenophages on a sensitive nonlysogenic host. When streaked onto an agar medium containing red grape juice (RGJ), cells segregated into white and red colonies. PCR amplifications with phage-specific primers demonstrated that only lysogens underwent white-red morphotypic switching. The method proved successful for various oenophages irrespective of their genomic content and attachment site used for site-specific recombination in the bacterial chromosome. The color switch was also observed when a sensitive nonlysogenic strain was infected with an exogenously provided lytic phage, suggesting that intracolonial lysis triggers the change. Last, lysogens also produced red colonies on white grape juice agar supplemented with polyphenolic compounds. We posit that spontaneous prophage excision produces cell lysis events in lysogenic colonies growing on RGJ agar, which, in turn, foster interactions between lysed materials and polyphenolic compounds to yield colonies easily distinguishable by their red color. Furthermore, the technique was used successfully with other species of LAB.IMPORTANCE The presence of white and red colonies on red grape juice (RGJ) agar during enumeration of Oenococcus oeni in wine samples is frequently observed by stakeholders in the wine industry. Our study brings an explanation for this intriguing phenomenon and establishes a link between the white-red color switch and the lysogenic state of O. oeni It also provides a simple and inexpensive method to distinguish between lysogenic and nonlysogenic derivatives in O. oeni with a minimum of expended time and effort. Noteworthy, the protocol could be adapted to two other species of LAB, namely, Leuconostoc citreum and Lactobacillus plantarum It could be an effective tool to provide genetic, ecological, and functional insights into lysogeny and aid in improving biotechnological processes involving members of the lactic acid bacterium (LAB) family.

Screening of lindane degrading bacteria isolated from soil for their plant growth promoting attributes

Abstract: Pesticide degrading bacteria that are isolated from agricultural soils are known to harbor multiple auxiliary characteristics such as production of phytohormone, solubilization of inorganic phosphate, nitrogen fixation, biosurfactant production etc., which are essential for plant growth promotion. In this study, twenty bacterial cultures were isolated from different agricultural fields of Odisha, West Bengal and Manipur (three States of eastern India) by enrichment culture method. These bacteria were isolated based on their ability to grow in media containing mineral salt with lindane at the concentration of 100 mg L−1 as the only carbon utilization source. Along with lindane degradation capacity, the ability of the isolates to promote plant growth was characterized by evaluating their ability for production of ammonia, phytohormones and biosurfactants, solubilization of inorganic phosphates and nitrogen fixation. Preliminary identification of all the isolates was done by morphological as well as biochemical tests. Among the 20 isolates, 8 were found to be Gram−positive and 12 were Gram-negative. The percentage of isolates positive for solubilization of inorganic phosphate, nitrogen fixation, production of indole-3-acetic acid, biosurfactant (in CTAB-methylene blue agar and by hemolysis test in blood agar) and ammonia were 40, 70, 90, 70, 85 and 100% respectively. Finally, three isolates were selected which showed positive results for majority of the tested attributes and identified on the basis of their 16S rRNA gene sequences. It was observed that all the three isolates belonged to the genus Ochrobactrum. Hence it was concluded that along with pesticide utilization these isolates have properties for plant growth promotion and can be potent agents for raising the yield of crops in pesticide-contaminated soils by bioaugmentation.

Fungal strain selection for protease production by solid-state fermentation using agro-industrial waste as substrates

Abstract: Screening of the fungal strains, Aspergillus niger, Trichoderma harzianum, and Rhizopus microsporus var. chinensis for protease production was realised by monitoring the presence of clear zones on skimmed milk agar plates. Additionally, screening of the radial growth on vanilla waste, cane bagasse, and pineapple crown plates was performed. Radial growth data of the fungal strains on skimmed milk agar and agro-industrial waste plates were adjusted to the Gompertz model. The results indicate that R. microsporus var. chinensis and pineapple crown were adequate for the production of milk-clotting enzymes by solid-state fermentation. Crude enzyme extract was produced via solid-state fermentation using two particle sizes [2–2.6 mm (SSF1) and a mixture of less than 1 mm with 2–2.6 mm, at a ratio of 1:1(SSF2)]. The highest milk-clotting and proteolytic activities were shown within 12 h and 48 h of fermentation for SSF1 and SSF2, respectively. The optimum milk-clotting activity presented by the crude enzyme extract was at pH 6.5, 40 °C, and 0.04 M CaCl2. The ratio of milk-clotting activity to proteolytic activity (9.7) presented by the crude enzyme extract indicates potential for use as a calf rennet substitute.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples.

Abstract: Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 °C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a "sandwiched", anaerobic environment. Agar plates are incubated overnight at 37 °C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 °C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15-20% of samples test positive for C. perfringens.

Evaluation of various methods of selection of B. subtilis strains capable of secreting surface-active compounds.

Abstract: The aim of the study was the evaluation of a three-step method for the selection of bacterial strains capable of producing surfactin. The procedure consisted of the following steps: 1.blood agar test, 2. measurement of the surface tension (ST) of the medium using the du Nouy method before and after submerged culture, 3. qualitative and quantitative assessment of surfactin by HPLC. Forty five Bacillus subtilis natto strains producing haemolysis zones (≥3mm) were selected. Nineten of them reduced ST of the medium to ≤ 40 mN/m; in six cases, the reduction was as much as 50%. All indicated strains produced surfactin. Positive correlations (p <0.5) between the percentage reduction of ST of the medium and surfactin concentration (r = 0.44), indicate that this parameter is determinant of the ability to synthesize this compound. The blood agar test has been shown to be useful only as a pre-selection criterion for surfactin producers (18 strains selected by this method reduced ST by only ≤30%). The proposed selection strategy proved effective and made it possible to select the BS15 strain that reduced the ST of the medium to 30.56 ± 0.15 mN/m and simultaneously provided a high concentration of surfactin compared to other strains.

Legionella qingyii sp. nov., isolated from water samples in China.

Abstract: Three Legionella -like strains, designed km488T, km489 and km521, were isolated from freshwater samples in China. Cells were Gram-stain-negative, rod-shaped and non-spore-forming. Growth was observed on BCYEα agar, but not on BCYEα agar without l-cysteine, chocolate agar with PolyViteX or Columbia blood agar. The major fatty acids (>5 %) of strains km488T, km489 and km521 were C16 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The mip gene sequences (574 nt) showed the isolates were almost identical with more than 99.7 % sequence similarities, and closely matched to L. gormanii ATCC 33297T with 95.4–95.6 % sequence similarities. Phylogenetic analyses based on concatenated gene (16S rRNA, mip, rpoB and rnpB) sequences indicated that the isolates formed a distinct cluster along with L. gormanii within the genus Legionella . Matrix-assisted laser desorption ionization time-of-flight analyses also demonstrated a clear separation between the isolates and other closely and distantly related Legionella species. DNA–DNA hybridization studies demonstrated that the isolates were closely related (92.0 –95.0 % DNA-DNA relatedness) but differentiated from their phylogenetic neighbours (<70 % DNA–DNA relatedness). The whole genome of km488T was sequenced, and showed a G+C content of 37.8 mol%. Based on the findings from this polyphasic taxonomic study, the isolates are considered to represent a single novel species, for which the name Legionella qingyii sp. nov. is proposed. The type strain is km488T (KCTC 15636T=CCTCC AB 2018025T=NRBC 113223T).

Microbial contamination of multiple‐use bottles of fluorescein ophthalmic solution

Abstract: Background The contamination of ophthalmic solutions in ophthalmic practices remains an important cause of a myriad of secondary eye infections and a source of aggravation of ocular disorders such as corneal ulcers and keratitis. The aim of this study was to investigate the possible microbial contamination of fluorescein sodium dye solutions used in eye clinics in Ghana. Methods Fluorescein sodium solutions were collected from various eye clinics in Ghana. Twenty-one samples of multiple-use fluorescein ophthalmic solutions were collected from various regions in Ghana. Eighteen unopened bottles yet to be used were also collected to serve as controls from the same facilities. The solutions were inoculated in different culture plates (blood agar, MacConkey agar, Sabouraud dextrose agar and plate count agar). The resulting microbial growth was identified using standard microbial identification techniques. Susceptibility tests were performed to ascertain the clinical importance of the organisms identified. Results Positive cultures were recorded for all 21 multiple-use bottles (in-use) collected, but there were no positive cultures for the unopened bottles (yet to be used). Six different genera of bacteria were identified from fluorescein solutions, including resistant strains of Staphylococci spp., Bacillus spp., Klebsiella spp., Pseudomonas spp., Haemophilus spp. and Bordetella spp. Pseudomonas spp. were the most common bacterial contaminants. For fungi contaminations, Aspergillus spp., Penicillium spp. and Cladosporium spp. were isolated. The most common fungal contaminants were Aspergillus spp. Conclusions Multiple-use bottles of fluorescein solution used in eye clinics in Ghana were contaminated with clinically important strains of bacteria and fungi.

Screening of growth phases of Antarctic algae and cyanobacteria cultivated on agar plates by chlorophyll fluorescence imaging

Abstract: Recently, chlorophyll fluorescence imaging is frequently used non-invasive method to monitor the metabolic state and photosynthetic activities of vascular plants and other autotrophic organisms. In our study, we used the measurements of chlorophyll fluorescence kinetics to follow the development of culture of Antarctic algae ( Macrochloris rubrioleum , Zygnema sp.) and cyanobacteria ( Hassalia antarctica , Nostoc commune ). On the cultures grown on agar plates, Bold´s Basal Medium (BBM), slow Kautsky kinetics supplemented with saturation pulses were measured repeatedly in a week interval. On the kinetics, typical points (OPSMT) were distinguished and species-specific and time of cultivation-dependent differences in shape of the OPSMT kinetics evaluated. We tested sensitivity of various chlorophyll fluorescence parameters to cultivation time on agar plates. In the algae, the most pronounced changes were the decrease in maximum quantum yield of photosystem II (F V /F M ) and quenching of basal chlorophyll fluorescence qF 0 ( M. rubrioleum , Zygnema sp.). In cyanobacteria, chlorophyll fluorescence parameters did not show clear trends with the time of cultivation. F 0 quenching (qF 0 ) reached positive values in H. antarctica , while it was negative in N. commune . In both cases, however, qF 0 showed an increase with cultivation time. The differences are discussed as well as the potential of the emerging area of the application of chlorophyll fluorescence imaging for evaluation of photosynthetic performance of algal/cyanobacterial cultures on agar plates.

Escherichia coli Culture Filtrate Enhances the Growth of Gemmata spp.

Abstract: Background: Planctomycete bacteria are known to be difficult to isolate, we hypothesized this may be due to missing iron compounds known to be important for other bacteria. We tested the growth-enhancement effect of complementing two standard media with Escherichia coli culture filtrate on two cultured strains of Gemmata spp. Also, the acquisition of iron by Gemmata spp. was evaluated by measuring various molecules involved in iron metabolism. Materials and Methods: Gemmata obscuriglobus and Gemmata massiliana were cultured in Caulobacter and Staley's medium supplemented or not with E. coli culture filtrate, likely containing siderophores and extracellular ferrireductases. We performed iron metabolism studies with FeSO4, FeCl3 and deferoxamine in the cultures with the E. coli filtrate and the controls. Results and Discussion: The numbers of G. obscuriglobus and G. massiliana colonies on Caulobacter medium or Staley’s medium supplemented with E. coli culture filtrate were significantly higher than those on the standard medium (p<0.0001). Agar plate assays revealed that the Gemmata colonies near E. coli colonies were larger than the more distant colonies, suggesting the diffusion of unknown growth promoting molecules. The inclusion of 10-4 to 10-3 M FeSO4 resulted in rapid Gemmata spp. growth (4-5 days compared with 8-9 days for the controls), suggesting that both species can utilize FeSO4 to boost their growth. In contrast, deferoxamine slowed down and prevented Gemmata spp. growth. Further studies revealed that the complementation of Caulobacter medium with E. coli culture filtrate and 10-4 M FeSO4 exerted a significant growth-enhancement effect compared with that obtained with Caulobacter medium supplemented with E. coli culture filtrate alone (p<0.0122). Moreover, the intracellular iron concentrations in G. obscuriglobus and G. massiliana cultures in iron-depleted broth supplemented with the E. coli filtrate were 0.63 ± 0.16 µmol/L and 0.78 ± 0.12 µmol/L, respectively, whereas concentrations of 1.72 ± 0.13 µmol/L and 1.56 µmol/L ± 0.11 µmol/L were found in the G. obscuriglobus and G. massiliana cultures grown in broth supplemented with the E. coli filtrate and FeSO4. The data reported here indicated that both E. coli culture filtrate and FeSO4 act as growth factors for Gemmata spp. via a potentiation mechanism.

Quantity changes in Pseudomonas species in dairy manure during anaerobic digestion at mesophilic and thermophilic temperatures

Abstract: Anaerobic digestion is known to eliminate many kinds of microorganisms in livestock wastes. In this study, the quantitative changes of Pseudomonas species during mesophilic and thermophilic anaerobic digestions were investigated with focus on P. aeruginosa and P. fluorescens, which are known as a pathogen and a plant growth-promoting rhizobacterium, respectively. Furthermore, quantitative changes in antimicrobial-resistant Pseudomonas species against cefazolin were also investigated as a representative of antimicrobial resistance in dairy farms. The quantitative measurement of Pseudomonas species was performed by a plating method with modified Pseudomonas-selective agar medium. The colonies on the agar plates were classified into three groups by their colour development and constitutional fluorescence, and the group to which P. aeruginosa and P. fluorescens belong was confirmed by colony PCR using species-specific primers. The results revealed that while the number of total Pseudomonas decreased, the number of fluorescent Pseudomonas including P. fluorescens increased after anaerobic digestion. The abundance of cefazolin-resistant Pseudomonas also decreased, and no P. aeruginosa could be detected in any samples. These results suggested that the digestate has benefits such as the absence of pathogenic risks led by the Pseudomonas species and is expected to have a plant growth-promoting effect facilitated by fluorescent Pseudomonas.

The effect of shock freezing on physiological properties and consequent growth of Antarctic filamentous (Stigeoclonium sp.) and coccal alga (Diplosphaera chodatii) on agar plates

Abstract: In this study, we investigated the effects of shock freezing on physiological properties and consequent growth of in the Antarctic alga Stigeoclonium sp. and comparative coccal alga Diplosphaera chodatii on agar plates. Culture of algae grown in liquid medium were used to study subzero temperatures on the species resistance to shock freezing. Then, microalgae were frozen in liquid nitrogen and inoculated on BBM agar after thawing. Physiological status of algae was evaluated by chlorophyll fluorescence parameters during 28 days. The results showed that interspecific differences existed in their tolerance to shock freezing, as well as their consequent growth rate on agars. Direct effects of freezing in liquid nitrogen was demonstrated in chlorophyll fluorescence parameters recorded immediately after thawing the samples (in liquid medium). In spite of the fact that majority of cells was destroyed by shock freezing, the potential of photochemical processes in PS II (FV/FM) remained constant in D. chodatii. It may indicate high resistance of the species to freezing/thawing cycles and a capability of the surviving cells, core chlorophylls in PS II respectively, to perform photosynthetic processes related to PS II. Contrastingly, Stigeoclonium sp. showed a shock freezing-dependent decrease in FV/FM. When shock-frozen, thawed and inoculated on agar plates, the culture of D. chodatii, and Stigeoclonium sp. showed cultivation time-dependent increase in chlorophyll fluorescence parameters (FV/FM, FS).

Calcite formation induced by Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens and Rheinheimera texasensis

Abstract: A wide range of bacterial species are able to induce calcium carbonate precipitation. Using our own laboratory-preserved strains, we have newly discovered that Ensifer sp. MY11e, Microbacterium sp. TMd9a1, Paeniglutamicibacter sp. MSa1a, Pseudomonas sp. GTc3, and Rheinheimera sp. ATWe6 can induce the formation of calcite crystals on an agar medium. Type strains of their closely related species (Ensifer adhaerens, Microbacterium testaceum, Paeniglutamicibacter kerguelensis, Pseudomonas protegens, and Rheinheimera texasensis) could also induce calcite formation. Although the initial pH value of the agar medium was 6.1, the pH of the agar media containing calcite, induced by cultivation of the 10 bacterial strains, increased to 8.0–8.4. The ammonification (oxidative deamination) of amino acids may been responsible for this increase in pH. The crystals formed both on and around the bacterial colonies. Furthermore, when these strains (excepting two Microbacterium strains) were cultivated on a cellulose acetate membrane filter (0.20 μm pore size) resting on the surface of the agar medium (i.e., in the membrane filter culture method), the crystals formed on the agar medium separate from the bacterial cells. These results indicate that the bacterial cells did not necessarily become nucleation sites for these crystals. We also investigated whether the studied strains could be applied to the biocementation of sand, and found that only two Ensifer strains were able to form large sand lumps.