Low Agar Concentration Research Articles

Analysis of biofilm expansion rate of Bacillus subtilis (MTC871) on agar substrates with different stiffness.

The surface morphology of mature biofilms is heterogeneous and can be divided into concentric rings wrinkles (I), labyrinthine networks wrinkles (II), radial ridges wrinkles (III), and branches wrinkles (IV), according to surface wrinkle structure and distribution characteristics. Due to the wrinkle structures, channels are formed between the biofilm and substrate and transport nutrients, water, metabolic products, etc. We find that expansion rate variations of biofilms growing on substrates with high and low agar concentrations (1.5, 2.0, 2.5 wt.%) are not in the same phase. In the first 3 days' growth, the interaction stress between biofilm and each agar substrate increases, which makes the biofilm expansion rate decreases before wrinkle pattern IV (branches) comes up. After 3 days, in the later growth stage after wrinkle pattern IV appears, the biofilm has larger expansion rate growing on 2.0 wt.% agar concentration, which has the larger wrinkle distance in wrinkle pattern IV reducing energy consumption. Our study shows that the stiff substrate does not always inhibit the biofilm expansion, although it does in the earlier stage; after that, mature biofilms acquire larger expansion rate by adjusting the growth mode through the wrinkle evolution even in nutrient extremely depletion.

Rapid Bacterial Motility Monitoring Using Inexpensive 3D-Printed OpenFlexure Microscopy Allows Microfluidic Antibiotic Susceptibility Testing.

Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance.

Effects of culture medium composition and PEG on hyperhydricity in Dendrobium officinale

Hyperhydricity is a physiological disorder during plant tissue culture that seriously affects regeneration and micropropagation. In this study, Dendrobium officinale plantlets were cultured on solid Murashige and Skoog (MS) medium supplemented with plant growth regulators and various concentrations of sucrose, agar, and polyethylene glycol (PEG)-6000 to explore the effect of osmotic stress on hyperhydricity. The results show that low concentrations of sucrose or agar, as well as PEG-6000 at various concentrations, significantly increase the hyperhydric rate of D. officinale, whereas high concentrations of sucrose or agar did not. Furthermore, high concentrations of PEG-6000 significantly increase total water content, free-water content, relative electrical conductivity, and peroxidase (POD) activity of D. officinale plantlets, whereas they significantly decrease bound-water content, proline content, soluble protein content, soluble sugar content, and superoxide dismutase (SOD) activity. These results indicate that PEG-6000 disrupts the antioxidant system and water metabolism in D. officinale plantlets, as well as increases cell membrane permeability, which might be the key factors for the occurrence of hyperhydricity in this species.

Improved in vitro propagation of Rose cultivars against varying concentrations of carbon source, agar and plant growth regulators

The present study was conducted to improve upon the micropropagation protocol of Rose cultivars by modification of the MS medium through variations in sucrose and agar concentrations thereby enhancing the shoot multiplication and rooting efficiency of the nodal explants. High sucrose concentrations and low agar concentrations favoured shoot mutiplication during the in vitro stages. Enhanced multiplcation and growth was observed on sub culturing the mother explants with regenerated shoots on fresh MS medium containing sucrose (3.5%, w/v) and agar (0.6%, w/v), supplemented with lower concentrations cytokinin combination of BAP and KN (2.5 mgl-1+1.5 mgl-1) respectively. Half strength MS medium containing sucrose (2.0%, w/v) and agar (0.3%, w/v) with NAA and BAP (2.0 mgl-1+0.5 mgl-1) in combination was most effective for rooting.

The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

ABSTRACTInvasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis.

Swarming motility by Photorhabdus temperata is influenced by environmental conditions and uses the same flagella as that used in swimming motilityThis is scientific contribution No. 2431 from the New Hampshire Agricultural Experiment Station.

Photorhabdus temperata, an insect pathogen and nematode symbiont, is motile in liquid medium by swimming. We found that P.temperata was capable of surface movement, termed swarming behavior. Several lines of evidence indicate that P. temperata use the same flagella for both swimming and swarming motility. Both motility types required additional NaCl or KCl in the medium and had peritrichous flagella, which were composed of the same flagellin as detected by immunoblotting experiments. Mutants defective in flagellar structural proteins were nonmotile for both motility types. Unlike swimming, we observed swarming behavior to be a social form of movement in which the cells coordinately formed intricate channels covering a surface. The constituents of the swarm media affected motility. Swarming was optimal on low agar concentrations; as agar concentrations increased, swarm ring diameters decreased.

Spreading versus biomass production by colonies of the fish pathogen Flavobacterium psychrophilum: role of the nutrient concentration.

Colonies of the fish pathogen Flavobacterium psychrophilum have gliding motility in media with low agar concentrations. Although gliding motility, particularly in Flavobacterium johnsoniae, has been well-studied, little is known about its regulation by environmental factors. The work described here shows that the ability of F. psychrophilum to spread over surfaces depends on nutrient availability. In fact, as the nutrient contents of the medium decreased, spreading was favored and the diameter of the colonies increased. Macroscopy examination revealed modifications in colony morphology as nutrient depletion increased: from a dense and defined colony to the formation of microcolonies inside a general colony structure. Additionally, colony expansion dynamics and population density across the colony radius varied inversely with bacterial biomass production. Motility was an immediate response when bacteria were transferred from a rich to a more diluted medium. Our results suggest that, when nutrients are limiting, F. psychrophilum activates a specific growth mode that enables it to colonize surfaces by means of gliding motility. The use of diluted media allowed the differentiation, among previously isolated F. psychrophilum non-gliding mutants, of those completely unable to glide and those with only partially impaired gliding ability.

Strain competition and agar affect the interaction of rhizobia with rice

Competition assays with Sinorhizobium meliloti 1021 and its GFP-labelled pSymA cured and deleted derivatives, SmA818 and SmA146, demonstrated that Sm1021 could still inhibit rice seedling growth even when outnumbered by a large excess of the noninhibitory cured or deleted strain. The wild-type strain Sm1021 also inhibited the growth of its noninhibitory pSymA-cured strain SmA818(gfp) and its pSymA-deleted strain SmA146(gfp) in a manner suggesting that Sm1021 produced a bacteriocin-like substance. The production of, and resistance to, this substance seemed to be pSymA-associated, but it was not the cause of killing in competition experiments on rice, suggesting that the killing of SmA818(gfp) and SmA146(gfp) was medium dependent. The addition of agar in liquid F10 medium at concentrations < or = 0.4% (m/v) abolished the rice growth inhibition of strain Sm1021 and Sm1021(gfp). The increased medium viscosity at higher agar concentrations decreased the diffusion of gases and small molecules through the media. Thus, the low agar concentrations may mimic waterlogged soil conditions leading to the production of inhibitory compounds by the bacterial strains under microaerobic conditions.

Sub-cellular location of H 2O 2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

In carnation shoots ( Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.

Keeping your lawn wet

Even before the existence of experimental proof, it was thought that the length of the bacterial flagellum was controlled by capillary action. Flagellin subunits travel through the hollow structure of the flagellum and assemble at the growing tip outside the cell body. The growth rate of a flagellum has been shown to decrease as its length increases, with growth stopping at a flagellum length of approximately 15 μm (Iino, 1969). The same growth rate and terminal length was observed for a flagellum that was sheared off by mechanical forces. This would suggest that length is indeed limited by capillary action. Recent work by Wang and colleagues reports a relatively simple assay system showing that the length of Salmonella flagella is dependent directly on the degree of hydration in their external environment (Wang et al , 2005). The authors take advantage of earlier discoveries by Harshey and colleagues that Salmonella typhimurium , Escherichia coli and Serratia marcescens can switch from a swimming phenotype, in which they use flagellar rotation to propel themselves (swim) through liquid environments, to a swarming phenotype, in which the bacteria use flagellar rotation to crawl (swarm) across surfaces (Alberti & Harshey, 1990; Harshey & Matsuyama, 1994). Furthermore, the bacteria undergo a morphogenetic change when they are taken from a swimming environment and placed on a hydrated surface. A low agar concentration in Petri dishes (0.3%) provides enough hydration to allow the bacteria to swim through the agar. Bacteria isolated from these …

Characterization of swimming motility and identification of flagellar proteins in Salmonella pullorum isolates

Abstract Objective To identify swimming motility in Salmonella pullorum isolates and to characterize the flagellar proteins produced by motile isolates. Sample population 30 S pullorum isolates and isolates of 7 other Salmonella sp. Procedure Salmonella pullorum isolates were inoculated into high motility medium to evaluate swimming motility. Putative flagellar proteins were purified from the organisms and analyzed by means of gel electrophoresis and western blotting procedures, using various antisera specific for flagellar proteins. Antisera shown to be reactive with putative flagellar proteins were incorporated into the growth medium to examine their effects on motility of the isolates. Results All S pullorum isolates had evidence of swimming motility. Two putative flagellar proteins were purified from 2 of the S pullorum isolates: a 60 to 62 kd protein shown to react with antiserum specific for type y flagellar protein, and a 58 to 59 kd protein shown to react with antiserum specific for type d flagellar protein and with antibody reactive to a highly conserved flagellar epitope found on various Enterobacteriaceae. Antiserum specific for type d flagellar protein inhibited swimming motility of S pullorum isolates, but antiserum specific for type y flagellar protein did not. Conclusions Results suggest that S pullorum isolates can be induced to manifest swimming motility when grown on medium with a low agar concentration and possess a 58 to 59 kd protein of d serotype and a second protein of 60 to 62 kd that also may be a flagellar protein. (Am J Vet Res 1999;60:1322–1327)

The distribution of gentamicin in the rabbit cornea following iontophoresis to the central cornea.

The purpose of this study was to evaluate the penetration of gentamicin into the central, midperipheral and peripheral cornea of rabbits following iontophoresis to the central 3 mm of the cornea. Four groups (groups 1-4) of five rabbits (one eye per rabbit) underwent corneal iontophoresis using gentamicin dissolved in agar. Low (1 mg/ml) and high (10 mg/ml) concentrations of gentamicin in agar were used for one or ten minutes. Two control groups (groups 5 and 6) of five eyes each underwent mock iontophoresis with low and high concentrations of agar-gentamicin mixture. Following sacrifice of the rabbits, the central, midperipheral and peripheral parts of each cornea were excised. Gentamicin concentration was determined in each part of every cornea. High concentrations of gentamicin (951.6 +/- 369.4 microg/ml to 26.6 +/- 41.34 microg/ml) were obtained in the central parts of all the iontophoresis-treated corneas. In each group, except group 6, central corneas had higher concentrations of gentamicin compared to midperipheral corneas (p = 0.038 to p = 0.021), and midperipheral corneas had higher levels than peripheral corneas (p = 0.038 to p = 0.021). Following iontophoresis, gentamicin is found in all portions of the corneas; however, the highest concentration of the drug remains in the central cornea.

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.

An in vitro model for the study of hydroponic forcing in chicory (Cichorium intybus)

An in vitro model for studying the influence of different factors on chicon formation during hydroponic forcing has been developed. The shoot apex was isolated from the chicory roots and cultured on a gelled nutrient medium. This medium was considered as a replacement of the root. Small chicons (5 g) were produced. Water and, more importantly, sucrose availability had important influences on the outgrowth of the chicons. When sucrose was added to the medium the chicon-weight increased two-fold. On a medium with low agar concentration (0.3% (w/v)), heavier chicons were produced compared with a medium with agar at 1.2% (w/v). Browning of the pith tissue (= flowering stem) decreased with increased agar concentration. The results presented indicate that the in vitro system can be used as a research model to study chicon development in relation to root functioning and composition.

The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.

The growth and morphology of submerged bacterial colonies was investigated. Five separate colonial forms were recognized depending both on species and on agar concentration. These were (i) branched, dendritic structures seen only with Bacillus cereus; (ii) lenticular colonies for all other species at high agar concentrations; (iii) small lobed to spherical colonies for non-motile organisms at low agar concentrations; (iv) and (v) large diffuse spherical colonies which can be further subdivided into 'snowball' or 'wispy' types for motile bacteria growing at agar concentrations below about 0.65% w/v. Viable count determinations suggested that agar concentration had little effect in the early stages of growth but that motile cells at low agar concentrations achieved higher cell numbers than did those in concentrations greater than 0.65% w/v. Transmission electron microscopy indicated that bacteria in lenticular colonies were tightly packed within lens-shaped splits in the agar whilst at low agar concentrations motile cells were well separated and appeared to move through the agar matrix.

Initial Process of Colony Forming in Bacillus subtilis: Relationships between Colony Morphology and Agar Concentration.

Bacillus subtilisのある種の株は,分裂の初期の段階において一本のひものように伸び複雑な曲折を示す。この曲折は,固形培地の堅さや摩擦係数などの培地表面の性質によって影響を受けることが予想された。そこで培地の寒天濃度を変え,細胞がどのような増殖を示すかを観察した。その結果,寒天濃度が低いときはツイストが生じることが少なく低い密度の増殖をしたが,寒天濃度が高くなると細胞のひも同士が平行に寄り添いコンパクトなコロニーとなった。またどの濃度でも初期コロニーにおいては一本のひものまま増殖し,切断が生じたことは認められなかった。さらに細胞のひものツイストが各濃度で認められたがその様式には濃度による差があるように思われた。

Holotransformations of bacterial colonies and genome cybernetics

We present a study of colony transformations during growth of Bacillus subtilis under adverse environmental conditions. It is a continuation of our pilot study of “Adaptive self-organization during growth of bacterial colonies” (Physica A 187 (1992) 378). First we identify and describe the transformations pathway, i.e. the excitation of the branching modes from Bacillus subtilis 168 (grown under diffusion limited conditions) and the phase transformations between the tip-splitting phase (phase T) and the chiral phase (phase C) which belong to the same mode. This pathway shows the evolution of complexity as the bacteria are exposed to adverse growth conditions. We present the morphology diagram of phases T and C as a function of agar concentration and pepton level. As expected, the growth of phase T is ramified (fractal-like or DLA-like) at low pepton level (about 1 g/1) and turns compact at high pepton level (about 10 g/1). The growth of phase C is also ramified at low pepton level and turns denser and finally compact as the pepton level increases. Generally speaking, the colonies develop more complex patterns and higher micro-level organization for more adverse environments. We use the growth velocity as a response function to describe the growth. At low agar concentration (and low pepton level) phase C grows faster than phase T, and for a high agar concentration (about 2%) phase T grows faster. We observe colony transformations between the two phases (phase transformations). They are found to be consistent with the “fastest growing morphology” selection principle adopted from azoic systems. The transformations are always from the slower phase to the faster one. Hence, we observe T→C transformations at low agar concentrations and C→T transformations at high agar concentrations. We have observed both localized and extended transformations. Usually, the transformations are localized for more adverse growth conditions, and extended for growth conditions close to the boundaries between morphologies. We have observed also transformations between different branching modes, as well as transformations via virtual states. Motivated by the contemporary knowledge about phages and plasmids, we postulate a theoretical framework to comply with our experimental findings. We explain our observations using these assumptions as well as our proposal of co-mutations and auto-catalytic mutations as presented in the above mentioned pilot paper. This theoretical framework is a part of the new evolving picture of genome cybernetics. We also discuss the concept of adaptive genome changes which are based on pre-existing knowledge as well as the concept of genetic learning. i.e. changes (in response to a new problem) which develop the potential for adaptive genome changes. These concepts follow naturally if the picture of genome cybernetics is accepted. We conclude with a discussion of the implications and with further predictions (to be tested experimentally) derived from our assumptions.

FORMATION OF RAMIFIED COLONY OF FUNGUS ASPERGILLUS ORYZAE ON AGAR MEDIA

Ramified colonies of fungus Aspergillus oryzae have been found to grow at a low growth rate on “liquid-like” agar media with low concentrations of agar and glucose. Box-counting fractal dimensions of the individual colony branches have been found to decrease with the time of incubation. Addition of glucose solution in the interior of branched colonies has brought about the production of the hyphal filaments almost only at the apical region of the colony branches. Active growth of the ramified colonies is localized in the peripheral zone, and this growth manner implies that the fungus is exhibiting a positive exploitation.

In vitro rearing of Bracon mellitor and Catolaccus grandis with artificial diets devoid of insect components

AbstractBracon mellitor Say (Hymenoptera: Braconidae), and Catolaccus grandis Burks (Hymenoptera: Pteromalidae), ectoparasitoids of the boll weevil, Anthonomus grandis Boheman, were reared in vitro for the first time on artificial diets devoid of insect components. The duration of the life cycles from egg to adult when these parasitoids were reared in vivo on boll weevil larvae, on semi‐artificial diets retained with cotton fabric pads, or on diets with low agar concentrations (0.7%) were ca. 15.5, 16.5, and 15.0 days, for B. mellitor and 16.0, 17.5, and 15.5 days, respectively, for C. grandis. Percent adult emergence obtained with the same treatments was 58, 25, and 65 percent, for B. mellitor and 50, 28, and 60 percent, respectively, for C. grandis. Adult morphological characteristics and mating appeared normal. Male to female sex ratios were ca. 45:55 for both species.

Apparent Rejuvenation of Mature Wild Cherry ( Prunus avium L.) during Micropropagation

Summary Shoot tips from mature trees of the cherry rootstocks F12/1( Prunus avium L.), Colt ( P. avium × P. pseudocerasus ) and an accession of P. avium from British woodland were placed onto a medium previously published for use with cv. F 12/1. Reduction of pH to 5.0, a lower benzyladenine concentration and omission of GA 3 , low agar concentration and addition of phloroglucinol each increased shoot production on this culture medium. With cv. F12/1, number of shoots per culture and ability of shoots to produce adventitious roots increased with age of the culture line. Phloroglucinol added to the rooting medium significantly improved the proportion of shoots that rooted, but only during the first 700d of the culture lines. Phloroglucinol did not increase the number of roots per rooted shoot. The results are discussed as evidence for rejuvenation in vitro of mature P. avium shoots. Suggestions are made to improve the reporting of micropropagation data from shoot cultures derived from mature tissues and to explain variation in rooting results with phloroglucinol.