Showing papers on "Agar plate published in 1985"

Enhanced Chlorine Resistance ofTapWater-Adapted Legionella pneumophila asCompared withAgarMedium-Passaged Strains

Abstract: Previous studies haveshownthatbacteria maintained ina low-nutrient "natural" environment suchas swimming poolwaterare muchmore resistant todisinfection byvarious chemical agentsthanstrains maintained on richmedia. Inthepresent study a comparison was madeofthechlorine (Cl2) susceptibility of hot-water tankisolates ofLegionella pneumophila maintained intapwaterandstrains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Ourearlier workhasshownthat environmental andclinical isolates ofL.pneumophila maintained on agarmediumaremuchmore resistant toCl2thancoliforms are.Underthepresent experimental conditions (21°C, pH 7.6to8.0,and0.25mg offreeresidual Cl2per liter, we foundthetapwater-maintained L. pneumophila strains tobeevenmore resistant thantheagar-passaged isolates. Underthese conditions, 99%kill oftapwater-maintained strains ofL.pneumophila was usually achieved within 60to90mincompared with10 minforagar-passaged strains. Samples fromplumbing fixtures ina hospital yielded legionellae which were "super"-chorine resistant whenassayed undernatural conditions. After one agar passagetheir resistance dropped tolevels ofcomparable strains whichhadnotbeenpreviously exposed toadditional chlorination. Thesestudies more closely approximate natural conditions thanour previous workandshowthattap water-maintained L.pneumophila isevenmore resistant toCl2thanits already resistant agarmedium-passaged counterpart.

Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains.

Abstract: Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.

Determination of ansamycin MICs for Mycobacterium avium complex in liquid medium by radiometric and conventional methods.

Abstract: A radiometric method to determine the MIC of ansamycin (LM427) for Mycobacterium avium complex clinical isolates has been developed. It is based on a comparison of the conventional growth curve determination and the radiometric detection of growth (growth index) in the same liquid medium (7H12 broth). This new method requires less time and labor than does a conventional determination of MIC in liquid medium (CFU). Other advantages of this method include relatively short periods of exposure of the drug to 37 degrees C and the composition of 7H12 broth, which has practically no substrates which could absorb or bind the drug. Thus, a more accurate estimation of the MIC in this medium can be expected than by the conventional agar dilution (proportion) method. The MICs of ansamycin appeared to be higher in agar plates than in 7H12 broth. More than 70% of the isolates had a broth-determined MIC one to three times lower than the average peak concentration of ansamycin achieved in sera of patients. The wide range of MICs suggests the importance of testing susceptibility in broth with many concentrations in addition to, or rather than in, agar plates with concentrations of 2.0 or 1.0 micrograms/ml only. Taking into account relatively low levels of ansamycin in sera of patients, it would be appropriate to compare the MICs with the levels in serum to make the outcome of chemotherapy more predictable.

Comparative in vitro studies with 4-quinolone antimicrobials.

Abstract: The minimal inhibitory concentrations (MICs) of nalidixic acid, pipemidic acid, cinoxacin, oxolinic acid, flumequine, pefloxacin, acrosoxacin, amifloxacin, norfloxacin, enoxacin, ofloxacin and ciprofloxacin were determined for a range of clinical isolates. MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood. The inoculum used was approximately 10(4) colony forming units, contained in 10 microliters Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator. Following inoculation, plates were incubated in conditions appropriate for the organisms under investigation. The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organisms examined were also determined. All of the more recently synthesised 4-quinolones showed considerably greater activity than the parent compounds, nalidixic acid, pipemidic acid and cinoxacin, against the range of organisms used in this study. Ciprofloxacin and ofloxacin were the two most active of the 4-quinolones examined.

Isolation of Bacteroides fragilis from the feces of diarrheic calves and lambs

Abstract: A selective medium was developed for the isolation of Bacteroides fragilis directly from ovine and bovine fecal samples. The medium (tryptose blood agar plus polymyxin B, triclosan (Irgasan), novobiocin, and nalidixic acid) permitted growth of B. fragilis and several other species of Bacteroides but inhibited growth of most other intestinal bacteria.

Competition between different strains of Streptococcus cremoris

Abstract: Streptococcus cremoris strain HP was found to grow poorly on agar plates under aerobic conditions in comparison to several other strains of S. cremoris (Wg2, ML1, AM1, E8). This made it possible to determine the numbers of strain HP in mixed cultures with other strains under different culture conditions. None of the mixtures was stable in batch cultures as a result of differences in the maximum specific growth rate. In continuous culture under lactose limitation strain HP outcompeted strains E8 and ML1 at low dilution rates, but at high dilution rates and in batch culture the reverse was observed. This represents another example of crossing μ-s curves in anaerobic bacteria.

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus.

Abstract: Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp.

Abstract: Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO2/C2H2 ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by 15N2 incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

Abstract: The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and D...

Growth of Aeromonas spp. on cefsulodin-Irgasan-novobiocin agar selective for Yersinia enterocolitica.

Abstract: Twenty-eight strains of Aeromonas spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan (triclosan; Ciba-Geigy AG, Basel, Switzerland)-novobiocin (CIN) agar containing 15 or 4 mg of cefsulodin per ml and on inositol-bile salts-brilliant green (IBB) agar. Relative to blood agar, 68% of the strains were inhibited by more than 2 logs (i.e., less than 1% growth) at 37 degrees C (39% at 25 degrees C) on CIN I (high cefsulodin concentration), 7% were inhibited at either temperature on CIN II (low cefsulodin concentration), 4% were inhibited on IBB agar at 37 degrees C, and none were inhibited on IBB agar at 25 degrees C. These results reflect the MICs of cefsulodin on CIN Base: the MIC for 50% of the strains was 8 mg/liter at 37 and 25 degrees C, and the MICs for 90% of the strains were 16 mg/liter at 37 degrees C and 64 mg/liter at 25 degrees C. The MICs of Irgasan and novobiocin were far beyond the concentrations used in CIN media. We argue that CIN agar containing 4 mg of cefsulodin per ml (CIN II) can be used for the simultaneous detection of Aeromonas spp. and Yersinia spp.

Screening Procedures to Identify Soil-Borne Actinomycetes That Can Produce Herbicidal Compounds'

Abstract: An isolation and screening procedure was de- veloped to identify soil actinomycetes that can produce germination and growth inhibitors on agar plates. Eight of 120 isolates tested demonstrated severe toxicity on cress (Lepidium sativum L. 'Curly') and barnyardgrass (Echino- cbloa crus-galli (L.) Beauv. #3 ECHCG). Several isolates were observed to stimulate growth. In replicated tests using the agar screening procedure, compounds produced by micro - organisms significantly inhibited germination and growth of indicator plants. Organisms shown to produce toxins in agar media did not always produce toxins when grown in submerged culture. Additional index words. Microbial herbicides, natural product

Heterokaryon compatibility and genetic recombination within a host plant between hop wilt isolates of Verticillium albo–atrum

Abstract: Heterokaryon compatibility was tested by pairing complementary auxotrophic mutants of three fluctuating (M18, M33 and M50) and three progressive (PV1, PV2 and PV3) hop wilt isolates of Verticillitim albo–atrum. The criteria of compatibility adopted were prototrophic growth on a glucose minimal medium at 26°C and the presence of diploid conidia. Most pairings produced at least some heterozygous diploids, showing there was no complete incompatibility barrier to hyphal and nuclear fusion on an agar medium. The incidence of prototrophic diploidy was greater within paired mutants of progressive isolates, and between PV1 and the three fluctuating isolates. A recombinant haploid prototroph was re–isolated following inoculation of Antirrhinum plants with a pair of complementary auxotrophs (M18 nic-4 cob-26 and PV3 arg-8 pyr-2). In hop, this recombinant was of intermediate pathogenicity compared with the two parental wild–type isolates. Four large–spored, diploid isolates were obtained from Antirrhinum following inoculation with heterozygous diploid conidia (M18 nic-4 cob-26/PV3 arg-8 pyr-2) from diploids synthesized on agar. All four diploids remained stable on agar and one showed moderately high pathogenicity in hop, from which it was re–isolated as a stable diploid 10 weeks later.

Isolation of Capnocytophaga species with a new selective medium.

Abstract: A selective medium (CAP) composed of a GC agar base supplemented with 1% hemoglobin, 1% Polyvitex, and an antibiotic mixture of polymyxin B (15 U/ml), vancomycin (5 micrograms/ml), trimethoprim (25 micrograms/ml), and amphotericin B (25 micrograms/ml) was compared with another selective medium (TBBP) and two nonselective media--a blood agar and a chocolate agar--to isolate Capnocytophaga species from 725 clinical specimens These included sputa (467 specimens), throat swabs (116 specimens), oral ulcerations (35 specimens), and periodontal pockets (107 specimens) The recovery rate of Capnocytophaga species was significantly higher with the CAP medium (96%) than with the selective TBBP medium (522%), the nonselective blood agar (62%), and the chocolate agar (46%) Growth of the normal flora was best inhibited on CAP medium Colony size and yellow-brown pigment formation were maximally expressed on chocolate agar and CAP medium, but gliding motility was mostly absent We conclude that the CAP medium is an excellent medium for the recovery of Capnocytophaga species from contaminated clinical specimens

Identification and characterization of antimicrobial activity in two yeast genera.

Abstract: A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.

Isolation ofCapnocytophaga Species witha New Selective Medium

Abstract: A selective medium(CAP)composed ofa GC agarbasesupplemented with1% hemoglobin, 1% Polyvitex, andan antibiotic mixture ofpolymyxin B (15U/ml), vancomycin (5,ug/ml), trimethoprim (2.5,ug/ml), and amphotericin B (2.5,ig/ml) was compared withanother selective medium(TBBP)andtwononselective media-ablood agaranda chocolate agar-toisolate Capnocytophaga species from725clinical specimens. Theseincluded sputa(467specimens), throat swabs(116specimens), oralulcerations (35specimens), and periodontal pockets (107specimens). TherecoveryrateofCapnocytophaga species wassignificantly higher with theCAPmedium(96%)thanwiththeselective TBBPmedium(52.2%), thenonselective blood agar(6.2%), andthechocolate agar(4.6%). Growthofthenormalflora was bestinhibited onCAPmedium.Colony size and yellow-brown pigment formation were maximally expressed on chocolate agarandCAPmedium,butgliding motility was mostly absent. We conclude thattheCAP mediumisan excellent mediumfortherecoveryof Capnocytophaga species fromcontaminated clinical specimens. Thegenus Capnocytophaga hasbeenidentified aspartof thenormalgingival flora inhumansandappearstobe important intheetiology oflocalized juvenile periodontitis (17). Several reports indicate thatCapnocytophaga species can causesepsis inimmunocompromised

Comparison of basal media for culturing Campylobacter jejuni and Campylobacter coli.

Abstract: Four strains of Campylobacter jejuni and four strains of Campylobacter coli were used to compare the quantitative growth of Campylobacter cells on blood agar base no. 2 (Oxoid), brucella agar (BBL Microbiology Systems and Difco Laboratories), campylobacter agar base (Difco), Columbia blood agar base (Difco and Oxoid), and Mueller-Hinton agar (Difco and Oxoid). Columbia blood agar base and blood agar base no. 2 were inhibitory to most of the strains tested, as evidenced by reduced (10- to 1,000-fold) colony counts compared with other basal media. One of the brucella agars was inhibitory to two of the C. coli strains. The inhibitory effect of these media could be eliminated by addition of FBP (0.05% each ferrous sulfate hydrate, sodium metabisulfite, and sodium pyruvate) or 7% defibrinated sheep blood. However, addition of FBP or blood to brucella agar, campylobacter agar base, or Mueller-Hinton agar did not significantly affect the count, indicating that supplements are not required in these media for growth of Campylobacter in pure culture.

The comparative activity of twelve 4-quinolone antimicrobials against Haemophilus influenzae and Streptococcus pneumoniae.

Abstract: The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for 100 isolates of Haemophilus influenzae (including 30 beta-lactamase producing strains) and 100 isolates of Streptococcus pneumoniae. MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood. The inoculum used was approximately 10(4) colony-forming units, contained in 10 microliters of Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator. Following inoculation, plates were incubated at 37 degrees C for 18 h in an atmosphere enriched to 10% carbon dioxide. The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organisms examined were also determined. The more recently synthesised 4-quinolones showed considerably greater activity than nalidixic acid and pipemidic acid against clinical isolates of Haemophilus influenzae and Streptococcus pneumoniae. There was no apparent difference between the MICs observed for beta-lactamase producing and non-beta-lactamase producing strains of Haemophilus influenzae. Ciprofloxacin was the most active 4-quinolone examined (MIC90 for Haemophilus influenzae 0.008 microgram/ml; Streptococcus pneumoniae 2 micrograms/ml). Clinical studies on a possible role for some of the more recently synthesised 4-quinolones in the management of patients with respiratory infection are indicated.

A note on a selective agar medium for the enumeration of Flavobacterium species in water

Abstract: A selective nutrient agar medium containing kanamycin at 50 micrograms/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium. Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.

Analysis by gas liquid chromatography of production of volatile fatty acids by anaerobic bacteria grown on solid medium.

Abstract: Volatile fatty acids produced in Robertson's cooked meat medium by a range of clinically relevant anaerobes were compared by gas liquid chromatography with those produced in blood agar. The same volatile fatty acid profiles were obtained in both media, although the concentration of acids was lower in blood agar. We conclude that detection of volatile fatty acids from a pure culture of an organism on solid medium is practicable and offers advantages over the conventional technique.

Production of ethanol from biomasses. Part II. Preparation of mutants resistant to catabolite repression of Trichoderma reesei.

Abstract: The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth's cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (K.DD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

Abstract: We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.

Isolation and detection of multiple yeasts from a single clinical sample by use of Pagano-Levin agar medium.

Abstract: A total of 15,234 clinical samples were tested on modified Pagano-Levin agar medium to detect multiple yeast species within a single sample. Samples containing more than one yeast species were estimated to be 8.0% of the total. The most frequent combination of different yeasts was Candida albicans and Torulopsis glabrata.

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Abstract: The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible).

Fish flesh agar medium — a suitable experimental medium for the detection of spoilage bacteria

Abstract: The spoilage characteristics of bacterial strains were studied by growing them at 28 +/- 2 degrees C in agar and broth media prepared with sterile fish and prawn flesh homogenates. The percentage of spoilers found among the bacterial isolates tested, as shown by odour production and halo zone formation, was independent of the source of flesh used. Indole and fluorescent pigment production were also observed in the broth. Pseudomonas, Vibrio and Acinetobacter exhibited faster growth in flesh media than in the usual artificial media. Decrease of protein and lipid concentration in the clear zone of agar media suggests the utilization of the available substrate by spoilage bacteria.

Visual detection of β-fructofuranosidase (inulase) —Regulatory mutants of Kluyveromyces fragilis

Abstract: Inulase constitutive mutant cells of the yeastKluyveromyces fragilis were enumerated in continuous culture cell populations. After cloning and growth on glycerol agar plates, mutant colonies stained red when exposed to a mixture of sucrose and a chromogenic reagent for glucose. Mutants with improved inulase production on glucose were isolated from opaque agar plates containing undissolved inulin. Mutant colonies were surrounded with clearing zones. Attempts to isolate similar mutants by selection for 2-deoxyglucose resistance proved unsuccessful withK. fragilis.