A selective isolation method for Actinomadura viridis in soil

The rare actinomycete Actinomadura viridis was efficiently isolated from a variety of field soils by a new plate culture method in which a water suspension of dry-heated (110°C, 1h) soil was treated with 1.0% phenol and cultured on Humic acid-vitamin agar supplemented with kanamycin, josamycin, lysozyme and nalidixic acid. The method significantly eliminated unicellular bacteria and undesirable actinomycetes contaminants from the isolation plates, thereby achieving the highly selective isolation of A. viridis.     

A method for the selective isolation ofMyxococcus directly from soil

A new method is described for the selective isolation of species ofMyxococcus directly from soil by dilution plating. The method involves suppression of competing microorganisms with antibiotics combined with air drying and wet heat treatment of soils. Fungi were eliminated by supplementing the plating medium with cycloheximide and nystatin. Non-sporulating bacteria were controlled by air drying soils and then heating aqueous soil dilutions for 10 min at 56°C. The predominant sporulating bacteria in soil,Streptomyces andBacillus, were suppressed by adding either tiacumicin B, ristocetin or vancomycin to the medium. Swarming ofMyxococcus colonies was controlled with a casein digest-yeast extract plating medium (CY-C10 agar). Ultrasound treatment of soil suspensions gave the highest number ofMyxococcus colonies in the soils studied, but these cultures could be recovered without ultrasound. Strains ofMyxococccus fulvus, M. xanthus, M. coralloides, M. stipitatus andM. virescens were isolated from soil using this technique. Soils examined yielded one or twoMyxococcus species per sample.     

A new method for the selective isolation of actinomycetes from soil

Summary A coal-vitamin medium was developed to isolate actinomycetes from soil, which was superior to other currently used media. It increased the number of actinomycetes and inhibited the growth of other soil bacteria. The pretreatment of soil suspension with peptone (6%) and lauryl sulfate (0.05%) at 50°C for 10 min, also greatly increased the number of actinomycetes from soil prior to incubation with new medium.     

Use of membrane filters for selective isolation of actinomycetes from soil

Abstract A method using membrane filters of appropriate pore size, to selectively isolate actinomycetes from a mixed population of soil microorganisms, is described.
The method is based on the ability of actinomycetes to propagate and pass through the pores of filters while bacteria and fungi are retained on the membrane surface.     

A method for the selective isolation of Microtetraspora glauca and related four-spored actinomycetes from soil

An effective plate culture method for the isolation and enumeration of four-spored actinomycetes belonging to the Microtetraspora glauca group of Thiemann et al. is described, in which a water suspension of a soil sample given dry heat (110°C, 1 h) is subjected to a treatment with 0·05% benzethonium chloride (BC) and cultured on the new medium LSV-SE agar which is supplemented with kanamycin, norfloxacin and nalidixic acid. The LSV-SE agar, based on a commercial lignin (dealkaline; 1 g 1^-1 as the major carbon source, specifically supported adequate growth and good sporulation for the M. glauca -group species, thus facilitating the detection and enumeration of this rare actinomycete group in the sample. The addition of the antibacterial agents to the LSV-SE agar, as well as the dry heat and BC treatment, provided a selective effect of reducing the nonfilamentous bacteria and undesirable actinomycetes associates occurring on the isolation plates. A total of 26 different natural samples were examined. From 18 samples (16 of field and forest soils, and two of stream and lake sediments), the new method consistently achieved the selective isolation of the M. glauca group, which accounted for 2–27% of the total population recovered. Definite evidence of biological activity which degrades the grass lignocellulose was found in all of the 39 test M. glauca -group isolates. Of these, 14, or 36%, possessed antimicrobial activity.     

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.     

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.     

Modified method for selective enrichment and isolation of Bacillus thuringiensis from soil

Many colonies of Bacillus thuringiensis (Bt) were found to be lost using the isolation technique described by Martin and Travers modified by Morris (Morris method). Hence, a modified method for isolation of B. thuringiensis is described and compared with the Morris method. Screening methodology adopted by this method delivers an immensely rich and potentially more useful library of Bt colonies with ‘presumptive-positive’ morphology and 10-fold higher per cent frequency of Bt colonies as compared to the Morris method.     

A selective genome-guided method for environmental Burkholderia isolation

Journal of Industrial Microbiology & Biotechnology - The genus Burkholderia is an emerging source of novel natural products chemistry, yet to date few methods exist for the selective isolation...     

Baiting with Sclerotium rolfsii for selective isolation of Gliocladium virens from natural soil

A baiting technique for selective isolation of Gliocladium virens from natural soil was developed by mixing Sclerotium rolfsii —colonized sorghum grains with moist natural soil and incubating at 30 ± 5°C for 6–10 days. G. virens developed large, distinct colonies on the soil surface by colonizing S. rolfsii and was then easily isolated for use in biocontrol programmes. Trichoderma spp. were present in the soil but never developed conspicuous colonies on the soil surface, even when the soil was supplemented with large numbers of conidia.     

Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil

AIMS: To devise and evaluate a method for selective isolation of the less abundant actinomycetes, Nocardia spp. in soil. METHODS AND RESULTS: This newly developed method is based on differentiating Nocardia from other actinomycete taxa by centrifugation. A water suspension of air-dried soil is centrifuged through a gradient consisting of 10, 20, 30, 40 and 50% sucrose at 240 x g for 30 min. The 20% sucrose layer, which is enriched with Nocardia spp., is then diluted and plated on humic acid-vitamin agar supplemented with antibacterial agents. The proposed method consistently achieved selective isolation of Nocardia spp. in all 14 soil samples tested, which accounted for 5-89% of the total microbial population recovered. Tentative taxonomic characterization based on a restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal DNA suggested that many of the soil isolates could belong to N. asteroides, N. salmonicida or N. uniformis. CONCLUSIONS: Differential centrifugation can successfully and efficiently isolate soil Nocardia populations that are suppressed by conventional dilution plating approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to isolate less-explored actinomycete taxa is important for improving our knowledge about their taxonomy, ecology and industrial applications.     

Three new species of Chaetomium from soil

Chaetomium macrostiolatum sp. nov., Chaetomium olivicolor sp. nov. and Chaetomium tarraconensis sp. nov. isolated from Nigerian, Indian, and Spanish soils respectively, are described and illustrated. The first species is characterized by ascomata with a very broad ostiole and yellow hairs, and limoniform ascospores; the second by its thermotolerant growth, inconspicuous and short straight ascomatal hairs and fusiform ascospores; and the last by its unusual, irregularly-shaped ascospores and subglobose ascomata with flexuous to undulate, unbranched ascomatal hairs.     

A selective medium for the isolation of wood-rotting basidiomycetes

A selective medium for growing wood-rotting basidiomycetes is described. The medium is based on a mixture of benomyl and 2-phenylphenol which suppresses the growth of lower fungi and Sistotrema brinkmannii, a basidiomycete of frequent occurrence in timber but one which cannot decay wood. Use of the medium in isolation studies permits a higher recovery of wood-rotting basidiomycete fungi than can be achieved by the use of other selective media.     

A medium for the selective isolation of Edwardsiella ictaluri

A selective medium, called Edwardsiella ictaluri medium (EIM), has been formulated for the isolation of Edwardsiella ictaluri. The medium inhibits the growth of most gram-negative bacteria, except Proteus sp., Serratia marcescens and some isolates of Aeromonas hydrophila and Yersinia ruckeri. The bacteria that grow on the EIM are easily differentiated from E. ictaluri based on colony morphology. The EIM inhibits gram-positive bacteria with the exception of enterococci. The addition of fungizone to EIM suppressed the growth of most fungi. The EIM allows the evaluation of environmental reservoirs, levels of contamination and carrier states of E. ictaluri.     

A novel selective medium for isolation of Streptococcus mutans

The aim of this study was to improve the selective medium of Streptococcus mutans. A new selective medium, designated MS-MUTV, was prepared by adding 10 mg/l valinomycin to the MS-MUT medium previously described. The average recovery of S. mutans was 72.1%, and the growth of S. sobrinus and S. anginousus group was inhibited on MS-MUTV, but allowed on MS-MUT. One hundred and thirty-nine human saliva samples were examined and counted for S. mutans and non-S. mutans colonies. The recovery of S. mutans on MS-MUTV was similar to that on MS-MUT. Eighty-two and 7.9 percent of the saliva samples obtained S. mutans pure cultures, with no bacterial growth on MS-MUTV, respectively. The remaining 10.1% were contaminated with non-S. mutans, with low-level CFU. MS-MUTV is useful for the isolation of S. mutans alone from clinical samples in routine examinations.