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

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.     

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

A selective nutrient agar medium containing kanamycin at 50 μg/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.     

Aberrant Growth and Conidiation in Wild-Type Cultures of Fusarium Species from Wheat

Fusarium avenaceum, F. graminearum, F. poae and F. tricinctum showed abnormal growth, morphology and conidiation, and a tendency to produce crystals, inclusion bodies and sclerotia when freshly isolated from wheat stem bases or kernels onto low‐carbon potato dextrose agar (PDA). Observations of alterations in conidiation and conidium morphology are particularly significant, as these are the principal morphological diagnostic characteristics for Fusarium species. The fungi had normal growth when sub‐cultured onto standard PDA, suggesting that a balance of nutrients was responsible for the effects. Specific causes are discussed in detail in relation to published information. The importance of standard media in the identification of Fusarium species is emphasized, whilst non‐standard media may be useful for specific purposes, including routine isolation of fungi from mixed communities of species with different nutrient requirements.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals.

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.     

Comparison of Media for Detection of Fungi on Spacecraft

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.     

苹果盘二孢的分离培养研究

苹果盘二孢Marssonina coronaria引起的褐斑病是造成我国苹果树早期落叶的主要原因,由于该病菌分离培养困难,阻碍了对其生物学特性的研究,进而影响了对其防治机理和流行规律的研究。本研究应用4种培养基质,探索了3种方法对苹果盘二孢的分离效果。结果表明,3种方法均可分离到病原菌,但组织块分离法和分生孢子团分离法成功率仅有10%左右,而单孢分离法污染少,成功率高达到90%以上,明显优于其他两种方法。不同培养基上菌落形态、大小和产孢情况差异也很大,培养1个月(25℃)后PDA上菌落黑褐色隆起,表面蚯蚓粪状,无气生菌丝,无子实体和基内菌丝;10%V8培养基上菌落中央隆起,黑褐色,表面生少量气生菌丝,边缘放射状,基内菌丝深褐色,有子实体;苹果叶片葡萄糖琼脂培养基(LDA)上菌落平坦,黄褐色,表面生茂密的金黄色气生菌丝,基内菌丝深褐色,有子实体;苹果叶片煎汁葡萄糖琼脂培养基(LEDA)上菌落有明显的不规则隆起,黄褐色至黑褐色,表面生少许气生菌丝,菌落生长缓慢,无基内菌丝,分生孢子盘菌落表面生,菌落直径仅2mm左右,而在其他培养基上的菌落直径可达6-8mm,说明培养基质、分离方法均对苹果盘二孢的分离培养和生长发育有明显的影响。     

不同培养基对兰科药用植物手参原球茎共生真菌的分离效果

兰科植物手参Gymnadenia conopsea作为国家二级重点保护野生植物具有重要的药用价值。目前手参还未实现人工栽培,但其种子的真菌共生萌发已获成功。为明确除促萌发真菌外,还有哪些土著真菌参与了手参种子的萌发过程,本研究在自然条件下采用促萌发真菌伴播手参种子,获得了种子萌发形成的原球茎,进而对比了6种常见的培养基PDA (马铃薯葡萄糖琼脂培养基)、MMN (改良Melin-Norkrans培养基)、FIM (真菌分离培养基)、MEA (麦芽浸膏琼脂培养基)、CAM (胡萝卜葡萄糖琼脂培养基)和CMA (玉米粉琼脂培养基)对手参原球茎共生真菌分离效果的影响。共从6种培养基上分离获得了75个菌株,其中MMN、CAM、PDA、FIM、MEA、CMA培养基依次分离得到20株、16株、15株、11株、8株、5株菌。此外,真菌的多样性分析结果表明,MMN培养基的Chao 1、Shannon-Wiener和Simpson多样性指数最高,CAM和PDA培养基次之,CMA培养基最低。综上所述,真菌分离效果最好的是MMN培养基,其次是CAM和PDA培养基,而FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。本研究结果可为其他兰科植物原球茎共生真菌的分离提供借鉴,所获得的菌株也有望进一步应用于功能菌剂的研发。     

Performance of Selective and Differential Media in the Primary Isolation of Yeasts from Different Biological Samples

In view of the increase in yeast infections, especially polymicrobial ones, differential culture media have acquired increasing importance. The present study evaluated the Sabouraud chloramphenicol, Biggy agar, Pagano Levin agar and CHROMagar Candida media in terms of isolation, number of yeast colony forming units per plate, and inhibition of bacteria and filamentous fungi. To this end, we used 223 biological samples, including feces, and oral, vaginal and anal mucosae from 86 patients presenting or not symptoms of fungal infections. The four media did not differ significantly in terms of detection of yeast-positive cultures. The number of colony forming units per plate ranged from zero to 2.380, with a predominance of counts of 1 to 9 colonies per plate. No significant differences were observed among the four culture media in terms of number of colonies counted, for each kind of biological material. Fifteen species belonging to the genera Candida, Saccharomyces, Cryptococcus, Trichosporon and Rhodotorula were isolated, with C. albicans being the predominant species, followed by C. parapsilosis and R. rubra. CHROMagar Candida and Biggy agar were complementary in the isolation of the different species and favored a greater recovery of polymicrobial cultures. Pagano Levin agar isolated the smallest variety of species. Sabouraud chloramphenicol agar was the least effective in terms of bacterial inhibition and favored a greater development of filamentous fungi. The results suggest that more than one culture medium should be used for an adequate primary isolation.     

A glass fiber filter technique for studying nutrient uptake by fungi: The technique used on colonies grown on nutrient gradients of carbon and phosphorus

In most natural environments supporting fungal growth, nutrients are heterogeneously distributed in space. Growth of a fungus will thus take place in an environment characterized by gradients. A system has been developed for growth of fungi on opposing carbon and mineral nutrient gradients, present in liquid medium in glass fiber filters. By labeling the carbon or phosphorus in the medium, the amount of carbon or phosphorus accumulated inside hyphae ofRhizopus nigricans and the amount still present outside the hyphae were determined. The distribution of labeled C and P in the medium and in the colonies of the fungus grown in the presence and absence of initial gradients in the medium was compared. In both cases, little carbon or phosphorus was found remaining in the medium after fungal growth. With colonies grown on media without an initial gradient, two peaks of carbon and phosphorus accumulation were found, but when there was a gradient there was only one such peak. These peaks coincided with the regions of greatest sporulation. It was concluded, by comparing the distribution of the total amount of carbon inside and outside mycelium grown on gradients with that in control media which was uninoculated, that the translocation of carbon inside the mycelium could have been brought about by simple diffusion.     

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen? AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen? AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen? AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen? AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.     

Efficiency of various selective media in determining Pythium population in soil

Of 15 selective media recommended for isolation and enumeration ofPythium spp. directly from soil, corn meal agar (CMA) supplemented with agar, sucrose, minor elements, thiamine, rose bengal, pimaricin, pentachloronitrobenzene and vancomycin (MPVM) was the most efficient. Streptomycin (30–50 ppm) and rose bengal (33–60 ppm) as used in certain tested media effectively suppressed development of bacteria and actinomycetes. However, these chemicals adversely affected germination of spores and mycelial growth and thereby the recovery ofPythium spp. from soil. Media containing pimaricin (5 to 100 ppm) were more effective than those with nystatin (40 ppm) in suppressing development of nonphycomycetous fungi on isolation plate. MPVM with pimaricin at 5 ppm was more efficient than that with 10 ppm of the antibiotic in recoveryingPythium from soil. However, there was no difference in recovery ofPythium by this medium containing rose bengal at 5 ppm or at 10 ppm, butPythium colonies were more dense and better delineated when the medium contained 10 ppm of rose bengal. CMA containing pimaricin (5–100 ppm) and vancomycin (200 ppm) permitted occasionally development of a large number ofMortierella and bacterial colonies from certain soils, that interfered with accurate determination of colonies of certainPythium spp. on the plates. Vancomycin at 300 ppm, as used in MPVM, substantially reduced development of bacterial colonies compared to 200 ppm of the antibiotic. Surface-soil dilution-plate was more effective than the soil-dilution-plate method in reducing bacteria andMortierella colonies on isolation plates without affecting recovery ofPythium. The importance of basal medium, complement of antimicrobial agents, and isolation methods for efficiency of selective medium in recovery ofPythium spp. directly from soil is discussed.     

A Rapid Method for Isolating Mutants of Bacillus subtilis Producing Increased or Decreased Amounts of the Antibiotic, Mycobacillin

S ummary : A simple, rapid and efficient technique is described for the isolation of mutants of Bacillus subtilis in which the synthesis of mycobacillin, an antifungal antibiotic, is blocked. Suspensions of a chosen culture were subjected to ultraviolet radiation and then plated; colonies failing to produce antibiotic were located by flooding the incubated plates with agar seeded with sensitive fungi such as Candida albicans and Aspergillus niger . The method is also applicable for the isolation of high potency strains.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

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.     

CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi,especially Metarhizium acridum,from soil

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic fungi from non-sterile substrates rely on N-dodecylguanidine monoacetate (dodine) as the selective fungicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum, from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum, from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons with selective media previously reported in the literature, the CTC medium afforded colonies that were larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-isolation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum, Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil samples inoculated (spiked) with fresh conidia in the laboratory.     

A method for both mass and individual rearing of fungivorous astigmatid mites (Acari)

Several species of common fungi were assessed as food for fungivorous astigmatid mites. Hypocrea nigricans, Botrytis cinerea and Flammulina velutipes were generally good food sources for most mites examined. Fungal mycelia growing on PDA (potato dextrose agar) medium were not only nutritionally adequate but the system also maintained high humidity through the water-based agar medium. Among acarid mites, most species of Rhizoglyphinae could be reared easily with the method. Although filter-feeding histiostomatid mites do not feed directly on hyphae, some species were successfully maintained with the same method through multiple generations. Presumably, these mites obtained sufficient nutrition from the agar medium and fungal metabolites leaching into it. Most species ultimately produced dispersing heteromorphic deutonymphs on these media. Individual mites were also maintained in isolation within glass rings on fungal colonies. Using this technique, we were able to compare developmental periods, fecundity and survival periods of mites reared under different conditions.     

A rapid plate culture method for screening of amylase producing micro-organisms

A simple and rapid method is described for the detection of amylase-producing micro-organisms on solid media using Remazol Brilliant Blue-starch as substrate. The blue starch is incorporated into nutrient agar and amylase production is detected by the disappearance of the colour around the colonies. The method which is non-destructive, allows for direct visualization and isolation of amylolytic micro-organisms from the environment without prior replication of colonies.     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.