An improved growth medium for Flavobacterium psychrophilum

Supplementing cytophaga agar and broth with 0.5 g l-1 each of D(+) galactose, D(+) glucose, L-rhamnose and skimmed milk gave a dramatic improvement in the isolation of the fish pathogen Flavobacterium psychrophilum. By means of spread-plating, approximately double the number of colonies of larger size were obtained on the improved medium compared to cytophaga agar alone. In supplemented cytophaga broth, growth of Fl. psychrophilum was more rapid and generated greater biomass.     

Application of activated charcoal and nanocarbon to callus induction and plant regeneration in aromatic rice (Oryza sativa L.)

The investigations of nanotechnology with the application on agricultural products also have been few reported, especially the plant regeneration. The effects of activated charcoal and nanocarbon on the callus induction and plant regeneration of aromatic rice were studied. Activated charcoal was added into the callus induction and regeneration medium. The presence of activated charcoal in the callus induction medium (100–500 mg L^?1), activated charcoal significantly reduced the percentage of the callus induction and biomass accumulation (fresh weight, dry weight and size). Whereas, the regeneration medium supplemented with 100 mg L^?1 of activated charcoal showed the highest percentage of plant regeneration (61.90%) and the ratio of the number of seedlings to the number of regenerated calli (RSR; 3.06) that derived from the callus induction medium (without activated charcoal). Moreover, the induced calli derived from the callus induction medium supplemented with nanocarbon at 5 mg L^?1 showed the highest percentage of callus induction (94.70%), the percentage of green spots (95.83%), the percentage of plant regeneration (60.42%) and the RSR (3.12) when transferred the calli into the regeneration medium (without nanocarbon). After that, nanocarbon was also added into the regeneration medium. The percentage of green spots (96.08%), the percentage of plant regeneration (62.75%) and the RSR (3.16) obtained from the regeneration medium supplemented with 20 mg L^?1 of nanocarbon showed the highest values. This experiment showed that the optimum concentration of activated charcoal and nanocarbon had potential to enhance the callus induction and plant regeneration frequencies in tissue culture medium of aromatic rice.     

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

Influence of the toxic compounds present in brewer's spent grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion by Candida guilliermondii

Fermentation media containing different concentrations of toxic compounds were prepared from brewer's spent grain (BSG) hemicellulosic hydrolysate, and used for xylose-to-xylitol bioconversion by Candida guilliermondii. Such fermentation media were composed of the hydrolysate in the following ways: raw (RH); concentrated four-fold (CH); concentrated and treated with activated charcoal (TCH); raw supplemented with sugars until a concentration four-fold higher (SRH); concentrated and subsequently diluted but supplemented with sugars until a concentration four-fold higher (SDCH). All media presented an initial xylose concentration of 85 g/l, except RH, which contained 23 g/l xylose. Fermentation results revealed that the sugars supplementation to raw hydrolysate favored the xylitol production. Nevertheless, xylitol production from CH was negatively affected due to the high concentration of toxic compounds present in the medium. The hydrolysate treatment with activated charcoal partially removed the toxic compounds, and the xylitol production was higher than in CH, but not so efficient as in SRH. It was thus concluded that to obtain an efficient xylose-to-xylitol bioconversion from BSG hydrolysate, the sugars concentration must be increased, but the toxic compounds concentration must be reduced to the same level present in the raw hydrolysate.     

A proposed serotyping system for Flavobacterium psychrophilum

AIMS: To develop a common serological system for rapid and routine identification of the fish pathogen Flavobacterium psychrophilum. METHODS: Thirty-four isolates of Fl. psychrophilum from different fish species and different geographical areas were typed using a slide agglutination test and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven host-dependent serovars (1: salmon; 2: trout; 3: trout; 4: eel; 5: carp; 6: tench; 7: ayu) were found. Serovar 2 was divided into two antigenic subgroups (2a and 2b). The results achieved by both slide agglutination and ELISA methods were totally consistent with each other. Although both techniques proved to be simple to carry out and useful, only the ELISA allowed identification of Fl. psychrophilum serovars using unabsorbed antiserum and whole-cells as antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proposes a harmonized scheme for serological identification of Fl. psychrophilum to be used for diagnostic and seroepidemiological studies of the diseases it causes.     

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools.

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow trout

AIMS: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. METHODS AND RESULTS: The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the 'OmpA/MotB' domain/signature and five putative 'Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. CONCLUSIONS: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.     

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.     

An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish

AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2-13 bacteria x g(-1)), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.     

Growth Factors of Bacterial Origin for the Culture of the Rumen Oligotrich Protozoon, Entodinium caudatum

Attempts were made to develop an artificial medium suitable for axenic culture of Entodinium caudatum. Agnotobiotic cultures of the protozoon were established as stock cultures for testing the suitability of various growth media. A cell-free extract of mixed bacteria isolated from the rumen was shown to contain one or more growth factors for the protozoon when supplied with activated charcoal as a carrier. The medium (CYSE medium), which supported the growth of the protozoon in the presence of 50 μg/ml each of penicillin and chloramphenicol, consisted of activated charcoal (20 mg), heat-treated yeast (Y) (80 mg), 13%β-sitosterol-coated rice starch (S) (120 mg), and cell-free extract of rumen bacteria (1 ml) in 40 ml buffer solution. When culturing the protozoon, the CYSE medium was supplemented daily with 20 mg each of Y and S and half of the medium was replaced with fresh medium once every 5 d. The possible use of this method to establish an axenic culture of E. caudatum is discussed.     

Adhesion of the fish pathogen Flavobacterium psychrophilum to unfertilized eggs of rainbow trout (Oncorhynchus mykiss) and n-hexadecane

AIMS: The ability of Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS) in fish, to attach to unfertilized rainbow trout (Oncorhynchus mykiss) eggs and to hydrocarbon n-hexadecane was examined in the present study. METHODS AND RESULTS: Five different isolates of Fl. psychrophilum obtained from a variety of origins were compared. The effect of the age of the bacterium and conditions of starvation on the ability of the bacterium to adhere, were also evaluated. CONCLUSION: The different isolates were found to exhibit a similar ability to attach to both substrates. Increased surface hydrophobicity and a greater ability to attach to the surface of the eggs were observed with bacteria aged for one month, compared to bacteria cultured in Cytophaga agar for only three days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide useful information regarding the pathogenicity of RTFS, especially during the initial steps of infection.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

Chloramphenicol and florfenicol susceptibility of fish-pathogenic bacteria isolated in France: comparison of minimum inhibitory concentration, using recommended provisory standards for fish bacteria

AIM: To investigate the distribution of antimicrobial resistance to phenicols in the fish pathogenic bacteria Aeromonas salmonicida, motile Aeromonas, Yersinia ruckeri, lactic bacteria and the nutritionally fastidious Flavobacterium psychrophilum. The last species was screened on two media (diluted Mueller-Hinton and peptone-enriched Anacker and Ordal), both supplemented with horse serum. METHODS AND RESULTS: Minimal inhibitory concentration (MIC) assessment, using the agar dilution method according to proposed standards, confirmed that chloramphenicol resistance was more frequent and expressed at higher levels than florfenicol resistance. A significant resistant population, highlighted by the bimodal distribution of MICs, was detected only for chloramphenicol in A. salmonicida. No link could be found with the geographical origin of the isolates or fish species. Other cases of resistance appeared randomly distributed or related to the natural properties of the bacterial species. Although the two media used for testing F. psychrophilum resulted in comparable performances in dilution methods, Anacker and Ordal was more adapted to disc diffusion tests. CONCLUSION: Despite wide use, resistance to florfenicol does not seem to occur frequently in French fish farms. SIGNIFICANCE AND IMPACT OF THE STUDY: It is important to maintain a surveillance, as development of florfenicol resistance has occasionally been documented. For this purpose, and for the species studied in this work, the recently proposed standards appear generally well-adapted.     

Effects of activated charcoal effects on induction and development of microtubers in potato (Solanurn tuberosum L.)

The aim of this research was to compare hormone-free medium with media with regulator substances (activated charcoal, cytokinins, polyamine biosynthesis inhibitor and chlorocholine chloride) used for microtuber induction and development. Explants of cvs Monalisa, Primura and Spunta were multiplied subculturing nodal segments on plant growth regulator-free Murashige & Skoog (1962) (MS) medium. When the plantlets had 6–8 nodes, single-node stem segments were excised and transferred to eight tuberisation media, each consisting of MS basal components supplemented with sucrose (8% w/v) and various regulator substances. The control was a regulator-free medium including only sucrose. Results were expressed as the number and weight of microtubers per nodal explant.
The cultivars showed wide variations in the mean weight of microtubers, ranging from 44.6 mg (Primura) to 77.5 mg (Spunta), and nearly all plants produced tubers. Medium containing activated charcoal gave the highest rate of tuberisation and the largest microtubers. It thus played a role in optimising conditions for rapid, mass tuberisation of these cultivars, and produced large microtubers for field planting.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery.

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.