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.     

Isolierung und Zählung von Bodenactinomyceten auf Erdplatten mit Membranfiltern

Summary A technique is described to separate actinomycetes from other micro-organisms with membrane filters on soil plates. A measured quantity of soil dilution was passed through a membrane filter and the filter with the adhering organisms was placed upside down on a medium provided with soil. After an incubation period of two weeks all actinomycetes had penetrated the filter developing colonies on the sterile surface, whereas the other organisms were retained by the filter. By this procedure the counting and isolation of actinomycetes has been facilitated. The selection of bacteriostatic active forms is practicable by stamping the crowded filter on a medium that contains test bacteria.

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THE FAILURE OF PHENOL TREATED ESCHERICHIA COLI TO GROW ON MEMBRANE FILTERS

SUMMARY: Counts of Escherichia coli were done on nutrient agar (control), on membrane filters on nutrient agar and on membrane filters on filter paper pads. With untreated bacteria counts were similar under all conditions, though membrane filters on nutrient agar tended to give slightly low counts. Phenol treated bacteria gave much lower counts when membrane filters were used: the mean counts for 3 strains of the test organism with filters on nutrient agar varied from 35–65% of the control, while counts with filters on filter paper pads were somewhat lower, varying from 30–47% of the control. The low counts on membrane filters on filter paper pads were not due to adsorption of phenol by the filters or to a low concentration of nutrients in the growth medium.     

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.     

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 new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

Selective isolation of members of the Streptomyces violaceoruber clade from soil

Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms "Streptomyces coelicolor" A3(2) and "Streptomyces lividans" 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.     

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.     

A new method for routine isolation of oral treponemes using U-tubes and pectin medium

Determination of the composition of the oral microflora has traditionally been based on cultivation. Treponemes are prevalent in many oral infections but, unfortunately, are not regularly cultured. In this study a new method was established for routine isolation of oral treponemes from clinical samples. Bacterial samples from 47 periodontal pockets and 4 endodontic infections were incubated anaerobically under nitrogen atmosphere at 37 degrees C in U-tubes containing pectin medium. In the U-tube a 'bacterial sample side' and a 'sterile medium side' were established on separate sides of a membrane filter and an agar plug. Using this method we were able to isolate viable treponemes from all bacterial samples. This was in contrast to previously established methods such as the agar dilution technique, the technique involving the membrane filter placed on the surface of solid agar media and the well in agar plate technique. We believe that the 'U-tube method' is a valuable supplement to previously described techniques in routine isolation of treponemes from clinical samples.     

Application of a single‐colony coculture technique to the isolation of hitherto unculturable gut bacteria

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.     

A nutrient medium for the isolation and cultivation of Campylobacter

Campylobacter agar, nutrient medium intended for the isolation of bacteria of the genus Campylobacter from clinical material, has been developed. The composition of the medium includes sprat hydrolysate, aerotolerant additive (ferric sulfate--oxide, sodium pyruvate, sodium pyrosulfite), sodium glutaminate, agar. The selective properties of the medium are ensured by introducing the mixture of antibiotics consisting of polymyxin B, rifampicin, amphotericin B, ristomycin. The balanced composition of Campylobacter agar ensures the aerotolerance of Campylobacter organisms and gives the optimal conditions for their growth when the inoculated material is cultivated in the atmosphere made up of the mixture of three gases (5% of oxygen, 10% of carbon dioxide, 85% of nitrogen), as well as under the conditions of a "candle vessel". The medium suppresses the development of the associative microflora diluted 10(-1). As shown in the trial of the quality of Campylobacter agar by the inoculation of material taken from patients with acute enteric infections, agricultural animals and monkeys, the medium has pronounced selective, properties with regard to extraneous microflora, while ensuring the isolation of Campylobacter on the level of the control medium.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of macrocolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0·89), pseudomonads (r = 0·93) and staphylococci (r = 0·92) after incubation at 30°C for 3 or 6 h (staphylococci) at contamination levels of above 10³ bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Improved medium for recovery and enumeration of the farmer's lung organism, Saccharomonospora viridis.

A new medium, which we propose to call R8, was developed for the isolation and enumeration of the thermophilic actinomycete, Saccharomonospora viridis. This organism has been implicated in a range of hypersensitivity pneumonitides, including farmer's lung, but is generally isolated in small numbers from contaminated environments. Recovery of S. viridis from moldy hay and mushroom compost on R8 medium was compared with recovery on conventional media. S. viridis was isolated from both substrates but in highest numbers and most consistently on the R8 medium. The selectivity of this medium was best observed when the sedimentation chamber method was used for hay samples. Here S. viridis accounted for up to 80% of the total number of actinomycetes recovered on R8 and could not be recovered on rifampin selective medium under the same conditions. R8 was also found to be an efficient recovery medium for a range of thermophilic actinomycetes from mushroom compost and for another allergenic species, Faenia rectivirgula, from moldy hay. Contamination of isolation plates by thermophilic bacilli was reduced on R8 compared with the activity on half-strength tryptone soy agar, supplemented with 0.2% casein hydrolysate, and this, together with specific improvements in S. viridis growth, accounts for the selective effect. It is possible that the occurrence of S. viridis and its role as a causative agent of hypersensitivity pnuemonitis have been underestimated by the use of suboptimal recovery protocols. It is hoped that use of R8 in conjunction with dilution plate techniques will generate information on the ecology of S. viridis and contribute to health risk assessment studies.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Improved medium for recovery and enumeration of the farmer's lung organism, Saccharomonospora viridis

A new medium, which we propose to call R8, was developed for the isolation and enumeration of the thermophilic actinomycete, Saccharomonospora viridis. This organism has been implicated in a range of hypersensitivity pneumonitides, including farmer's lung, but is generally isolated in small numbers from contaminated environments. Recovery of S. viridis from moldy hay and mushroom compost on R8 medium was compared with recovery on conventional media. S. viridis was isolated from both substrates but in highest numbers and most consistently on the R8 medium. The selectivity of this medium was best observed when the sedimentation chamber method was used for hay samples. Here S. viridis accounted for up to 80% of the total number of actinomycetes recovered on R8 and could not be recovered on rifampin selective medium under the same conditions. R8 was also found to be an efficient recovery medium for a range of thermophilic actinomycetes from mushroom compost and for another allergenic species, Faenia rectivirgula, from moldy hay. Contamination of isolation plates by thermophilic bacilli was reduced on R8 compared with the activity on half-strength tryptone soy agar, supplemented with 0.2% casein hydrolysate, and this, together with specific improvements in S. viridis growth, accounts for the selective effect. It is possible that the occurrence of S. viridis and its role as a causative agent of hypersensitivity pnuemonitis have been underestimated by the use of suboptimal recovery protocols. It is hoped that use of R8 in conjunction with dilution plate techniques will generate information on the ecology of S. viridis and contribute to health risk assessment studies.     

Direct identification of the common cheese contaminant Penicillium commune in factory air samples as an aid to factory hygiene

F. LUND. 1996. Creatine sucrose dichloran agar (CREAD) was used as a selective medium for Penicillium commune and related species found in air samples in a cheese factory. Using growth and simple colony characters on CREAD together with detection of indole metabolites with a filter paper method, it was possible to identify all 22 P. commune isolates from a total of 43 Penicillium isolates. Penicillium commune numbers on CREAD were compared with those found on a general isolation medium, dichloran 18% glycerol agar.     

A new selective medium for isolating Pseudomonas spp. from water.

A new medium, pseudomonas selective isolation agar, was developed to isolate Pseudomonas spp. from water. It consists of 350 micrograms of nitrofurantoin per ml and 2 micrograms of crystal violet per ml in a nutrient agar base. It is more selective for Pseudomonas spp. than are available commercial media. Its ingredients are inexpensive and readily available, and it is easy to prepare.     

A new selective medium for isolating Pseudomonas spp. from water

A new medium, pseudomonas selective isolation agar, was developed to isolate Pseudomonas spp. from water. It consists of 350 micrograms of nitrofurantoin per ml and 2 micrograms of crystal violet per ml in a nutrient agar base. It is more selective for Pseudomonas spp. than are available commercial media. Its ingredients are inexpensive and readily available, and it is easy to prepare.