Rhythmic Conidiation in Constant Light in Vivid Mutants of Neurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).     

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Assay and Characteristics of Circadian Rhythmicity in Liquid Cultures of Neurospora crassa

Previous work on circadian rhythms of Neurospora crassa has been done almost exclusively with cultures expressing rhythmic conidiation and growing on solid agar medium. Such conditions severely restrict the kinds of biochemical experiments that can be carried out. We have now developed systems which allow indirect assay of circadian rhythmicity in liquid culture. Neurospora was grown in glucose and acetate liquid media under conditions which result in a range of growth rates and morphologies. Liquid media were inoculated with conidia and the cultures were grown in constant light for 33 or 48 hours, by which time floating mycelial pads had formed. Experimental pieces of mycelium then were cut and placed in fresh new liquid medium. As controls, other pieces of mycelium were cut and put directly on solid agar medium in race tubes. All cultures were transferred to constant darkness at this time. This light-to-dark transition set the phase of the circadian clock of both the liquid and solid cultures. At various times after the light-to-dark transition, the mycelial pieces in the liquid were transferred in the dark to solid medium in race tubes, where they grew normally and conidiated rhythmically. Comparison of the phase of the rhythm in these race tubes to the controls demonstrated that, under appropriate conditions, the circadian clock of the liquid cultures functions normally for at least two cycles in constant conditions. Using these culture systems, a significantly greater variety of biochemical studies of circadian rhythmicity in Neurospora is now possible.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Dietary Effects on the Composition of Fecal Flora of Rats

A long-term animal feeding experiment was conducted to compare the effect of meat and Wayne laboratory chow diets on the composition of rat fecal flora. Fecal bacteria were enumerated on selective media under both aerobic and anaerobic conditions. Intrarectal administration of N-methyl-N′ -nitro-N-nitro-soguanidine (MNNG) affected the count only on the phenylethylalcohol and veillonella-neomycin agars, whereas a slightly higher number of anaerobes appeared in the feces of rats that were treated with MNNG as compared with those obtained in the feces of untreated rats on the meat diet. In the absence of MNNG, feces of meat-fed rats yielded higher bacterial counts on aerobically incubated MacConkey agar, deoxycholate agar, and Pfizer selective enterococcus agar as well as higher numbers of clostridia on anaerobic egg yolk agar than did feces of rats on the Wayne diet. Feces of the group fed the Wayne diet produced more colonies on aerobic mitis-salivarius agar and lactobacillus agar as well as on anaerobically incubated phenylethylalcohol agar, veillonellaneomycin agar, bifidobacteria agar, fusobacterium (Nissui) agar, and kanamycin-vancomycin blood agar. These differences were consistent throughout the 1-year feeding period.     

A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: I. Phasing in Relation to the Light-Off Signal

Evidence is presented of an endogenous rhythm in flowering response to far-red (FR) irradiation, with a period of about 12 h (hence semidian rhythm), which persists through at least three cycles in constant conditions of continuous light at 27°C and has a marked influence on the flowering response in Pharbitis nil to a subsequent inductive dark period. The phase of the rhythm is not influenced by real time nor by the time from imbibition or from the beginning of the light period. Rather, it is fed forward from the beginning of the FR interruption to the beginning of the inductive dark period. The period of the rhythm is not affected by irradiance but is longer at cooler temperature. When there are two FR interruptions during the preceding light period, it is primarily the later one which determines the phase of the rhythm, although some interactions are evident. There appears to be an abrupt rephasing of the rhythm at the beginning of the inductive dark period. No overt rhythms which could be used as “clock hands” for the semidian rhythm were detected in photosynthesis, stomatal opening, or translocation.     

Influence of Structural Properties and Kinetic Constraints on Bacillus cereus Growth

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter⁻¹. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.     

Isubgol as an Alternative Gelling Agent for Microbial Culture Media

Isubgol, the mucilaginous husk derived from the seeds of Plantago ovata, has been successfully used as a gelling agent for microbial culture media. As illustrative examples, fast growing symbiotic bacterium, Rhizobium meliloti and saprophytic fungi, Aspergillus flavus and Penicillium chrysogenum were cultured on media gelled with either Isubgol or agar. All the three microbes employed in the study exhibited normal growth when cultured on their respective media gelled with Isubgol. Rather, Isubgol gelled medium appears to promote the growth of bacterial cultures as the colonies on this medium were denser than the corresponding ones on the medium gelled with agar. Likewise, on Isubgol gelled medium, sporulation in both the fungi took place earlier than on the medium gelled with agar, thus indicating the promotive influence of the former gelling agent.     

Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates

On agar media the maximum conidia production of Exserohilum monoceras occurred on V-8 juice agar (VA) or centrifuged V-8 juice agar, whereas the optimal radial mycelial growth occurred on Czapek-Dox agar. The optimal temperatures for radial mycelial growth and conidia production were 28 and 27°C respectively. Light prohibited E. monoceras conidia production. The best sporulation occurred under continuous dark conditions. Echinochloa leaf decoction significantly increased conidia production on potato dextrose agar (PDA) and VA, and significantly increased germ tube length on PDA, lima bean agar and VA, but did not affect conidia germination. No conidia were produced in liquid media. Of 22 agricultural-based products evaluated as solid substrates, the most abundant sporulation (1.8 × 10⁶ conidia g^-1 of dry weight) occurred on corn leaves. The conidia production of E. monoceras on corn leaves was affected by incubation period, moisture content and substrate quantity. There were no differences in germination rate, germ tube length and virulence of conidia produced on agar media or corn leaves.     

Influence of the medium-solidifying agent, the nutrient, and the genotype on the production of gametangia by Phytophthora ramorum in vitro

The effect of different parameters, including the type of nutrients, the quality of the gelling agent, and the genotype of the strain, were evaluated in the production of gametangia by Phytophthora ramorum in vitro. By comparing different agar sources on a carrot-based medium, a delay or a failure in the production of oospores was observed in pairings carried out on media supplemented with technical agar. In contrast, oospores were produced on other agar types, the production on media supplemented with agarose being slightly higher. The formation of gametangia was also influenced by the genotype of the strains involved in the pairing. A European A1 strain producing very few chlamydospores was found to be a better mating partner than other A1 strains. Using a carrot-agarose medium and selected genotypes, all European isolates were characterized in terms of mating type. A macroscopic experiment highlighted a particular spatial distribution of P. ramorum oospores in vitro. A method using polycarbonate membrane was evaluated to assess the selfing ability of P. ramorum.     

Enumeration of Bifidobacteria in Gastrointestinal Samples from Piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.     

Galactose, aspartate crossway and rhythm inhibition in Aspergillus niger

When the glucose/K⁺ balance in the medium is favourable to rhythm expression in Aspergillus niger Van Tieghem, galactose has an inhibitory effect on amplitude but not on period length of the rhythm. Galactose is active when its level reaches 1/10 of that of glucose. On liquid media a small quantity of glucose is necessary to start growth on galactose. Under these conditions U¹⁴C-galactose is rather slowly metabolized. After 6 h of feeding on the labelled galactose medium, this sugar is converted into glucose, which is used both for the synthesis of compounds derived from C₃ and C₄ units and for synthesis of polysaccharides and perhaps small peptides. The labelling of the macromolecules always remains low. The insoluble carbohydrates of the mycelium are little affected by the type of sugar supplied to the fungus. The metabolism on galactose differs from the metabolism on glucose mainly in a decrease of the free asparagine pool and a simultaneous equivalent increase of the free aspartate pool; such an effect could not be correlated with an increase of the aspartate aminotransferase activity. Supply of aspartate but not of gluta-mate into the agar medium inhibits the rhythm amplitude. So, the damping effect of galactose on the rhythm might be at least partly due to its effect on the regulation of the aspartate cross-way.     

The wetting agent required for swarming in Salmonella enterica serovar typhimurium is not a surfactant

We compared the abilities of media from agar plates surrounding swarming and nonswarming cells of Salmonella enterica serovar Typhimurium to wet a nonpolar surface by measuring the contact angles of small drops. The swarming cells were wild type for chemotaxis, and the nonswarming cells were nonchemotactic mutants with motor biases that were counterclockwise (cheY) or clockwise (cheZ). The latter strains have been shown to be defective for swarming because the agar remains dry (Q. Wang, A. Suzuki, S. Mariconda, S. Porwollik, and R. M. Harshey, EMBO J. 24:2034-2042, 2005). We found no differences in the abilities of the media surrounding these cells, either wild type or mutant, to wet a low-energy surface (freshly prepared polydimethylsiloxane); although, their contact angles were smaller than that of the medium harvested from the underlying agar. So the agent that promotes wetness produced by wild-type cells is not a surfactant; it is an osmotic agent.     

Efficacy of seed-based media for the mould-yeast conversion of Blastomyces dermatitidis

The efficacy of 20 seed-based media is reported for the in vitro mould-yeast conversion of Blastomyces dermatitidis, employing pharmamedia agar, peptone glucose agar, glucose agar and water agar as controls. The mould-yeast conversion varied significantly according to the culture medium, fungal strains and incubation period (p<0.01). Garden-pea, chick-pea, cow-pea, soyabean, peanut, green gram, French bean, lentil, okra and cottonseed converted all of the 7 B. dermatitidis test strains after 5 days of incubation at 37 ° C. Although the efficacy of many of these seed media was found to be at par with pharmamedia agar — a commercial cottonseed embryo-derived protein, garden-pea seed agar is adopted because of the wider availability and low fat content of this seed. The recommended composition of the medium comprises 2% aqueous seed extract, 2% glucose and pH 6–7. Only nigerseed and sunflower seeds failed to support the conversion of B. dermatitidis. Of the control media, peptone glucose agar, glucose agar and water agar did not support the conversion of 2 of the B. dermatitidis test strains. The mechanism underlying variable mould-yeast conversion of B. dermatitidis on seed-based media is not clearly understood. However, most of the seeds supporting excellent mould-yeast conversion are known for their high protein content. The conversion was apparently not affected by the fat content of the seeds or by incorporation of glucose in the medium.     

Pollinia-borne chemicals that induce early postpollination effects in Dendrobium flowers move rapidly into agar blocks and include ACC and compounds with auxin activity

The early visible effects of pollination in orchids are likely due to pollinia-borne chemicals. In Dendrobium we tested whether such compounds were water soluble and would diffuse in solid-aqueous phase, and determined both 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations and auxin activity. Following pollination, the flower peduncle showed epinastic movement, followed by yellowing of the flower lip, flower senescence and ovary growth. Placing pollinia on agar blocks for 3, 6, 9 or 12 h, prior to transferring them to the stigma, increased the time to these early postpollination effects or prevented them. Placing agar blocks that had been used for contact with the pollinia on the stigma also induced the early postpollination effects. The concentrations of ACC, the direct precursor of ethylene, in pollinia was lower the longer the pollinia had been in contact with the agar blocks, whilst the ACC content in the agar blocks increased with the period of contact. The auxin activity of the agar blocks also increased with the time of contact with pollinia. It is concluded that chemicals in the pollinia are responsible for the early visible postpollination effects, and that these (a) rapidly diffuse in aqueous media, and (b) comprise at least ACC and compounds with auxin activity. The idea is discussed that ACC plus auxin is adequate for the production of the early postpollination effects.     

Independence of genetic variation between circadian rhythm and development time in the seed beetle, Callosobruchus chinensis

A positive genetic correlation between periods of circadian rhythm and developmental time supports the hypothesis that circadian clocks are implicated in the timing of development. Empirical evidence for this genetic correlation in insects has been documented in two fly species. In contrast, here we show that there is no evidence of genetic correlation between circadian rhythm and development time in the adzuki bean beetle, Callosobruchus chinensis. This species has variation that is explained by a major gene in the expression and period length of circadian rhythm between strains. In this study, we found genetic variation in development time between the strains. The development time was not covaried with either the incidence or the period length of circadian rhythm among the strains. Crosses between strains suggest that development time is controlled by a polygene. In the F₂ individuals from the crosses, the circadian rhythm is attributable to allelic variation in the major gene. Across the F₂ individuals, development time was not correlated with either the expression or the period length of circadian rhythm. Thus, we found no effects of major genes responsible for variation in the circadian rhythm on development time in C. chinensis. Our findings collectively give no support to the hypothesis that the circadian clock is involved in the regulation of development time in this species.     

A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter⁻¹. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C₈ fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.