Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Evaluation of Pseudosel Agar as an Aid in the Identification of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa and thirty-five other species of gramnegative bacilli was observed on 0.03% cetrimide in heart infusion agar medium and Pseudosel agar (BBL). The 0.03% cetrimide agar was more selective for growth of P. aeruginosa than was Pseudosel agar; however, certain bacteria other than P. aeruginosa also grew on the former medium. Although Pseudosel agar was not a highly selective medium for P. aeruginosa, it was preferable to technicolor agar for detection of the pyocyanin and pyorubin pigments produced by P. aeruginosa.     

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 micrograms/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 micrograms/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide - nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 10(2) to 10(5) colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 degrees C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 micrograms/mL. The medium appeared to be highly selective for P. aeruginosa with 95-100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.     

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.     

Selective media for isolating Pseudomonas aeruginosa

The comparative study of 4 selective media (medium containing N-cetylpyridinium chloride, acetamide agar, cetrimide agar, medium containing irgasane) showed that their use permitted one to enhance the isolation of P. aeruginosa, especially pigment-free forms, from pathological material and to reduce the time of their isolation by 24-48 hours. Of all the media subjected to testing medium containing N-cetylpyridinium chloride and acetamide medium proved to have the highest selectivity.     

Factors influencing detection and enumeration of Pseudomonas aeruginosa by most-probable-number and membrane filtration techniques.

Most-probable-number (MPN) and membrane filtration (mF) techniques were evaluated with respect to selectivity, sensitivity, and efficiency in recovering Pseudomonas aeruginosa strains in hospital fluids and extramural water environments. Known numbers of cells of a naturally occurring strain of P. aeruginosa maintained in distilled water or cells subcultured on Standard Methods agar were added to test samples containing various types and levels of background microbial contamiants. Environmental samples containing unknown numbers of P. aeruginosa strains also were tested. Asparagine and acetamide broths were employed as presumptive media in MPN tests, and mPA and Pseudosel agars were used in mF assays. Statistical analyses of data showed the superiority and comparability of the asparagine-MPN and mPA-mF systems. Greater precision and accuracy were consistently obtained in either assay technique by the use of naturally occurring cells as test organisms. The type of filter and nature of diluents employed, as well as pH of assay media, were found to greatly influence both recovery and developemnt of characteristic colonial morphology in the mPA-mF system.     

Glycine-containing selective medium for isolation of Legionellaceae from environmental specimens.

Glycine, at a final concentration of 0.3%, has been shown to be an excellent selective agent for the isolation of Legionellaceae. Stock cultures of Legionella pneumophila were not inhibited on buffered charcoal-yeast extract agar containing the amino acid. Among the other Legionellaceae tested, only one of two strains of L. dumoffii and two of six strains of L. micdadei were appreciably inhibited. This medium permitted the isolation of L. pneumophila from environmental specimens with marked inhibition of many non-Legionellaceae bacteria. The selectivity of the medium was subsequently improved by the incorporation of vancomycin (5 microgram/ml) and polymyxin B (100 U/ml). This selective medium, glycine-vancomycin-polymyxin B agar, should facilitate the recovery of Legionellaceae from environmental sources.     

A selective medium for Fusobacterium spp

A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 microgram/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Arginine medium for the isolation and primary identification of Pseudomonas aeruginosa in environmental objects

A culture medium for the isolation and primary identification of P. aeruginosa has been developed. The medium contains L-arginine and ions of K, Na, Mg, C and P; it has also an overlay of plain agar with 0.6% of 2,3,5-triphenyltetrazolium chloride added. The comparison of arginine medium with other media proposed for the isolation of P. aeruginosa from various objects in the environment has demonstrated the advantage of this highly selective medium permitting the primary identification of up to 95% of characteristic colonies.     

Tobramycin (Nebramycin Factor 6): In Vitro Activity Against Pseudomonas aeruginosa

Tobramycin (factor 6 of the nebramycin complex) is a new aminoglycoside antibiotic isolated from Streptomyces tenebrarius which is active against S. aureus, Enterobacteriaceae, and Pseudomonas aeruginosa. Susceptibility to tobramycin of 96 strains of P. aeruginosa, including 45 recent isolates from blood, was studied by using agar and broth dilution methods. The minimum inhibitory concentration (MIC) for 83 of 96 strains was 3.12 mug/ml or lower in Mueller Hinton agar; MIC values were two to eight times lower in Mueller Hinton broth tests. Agar dilution MIC values were generally lower than those obtained in parallel tests with gentamicin. Killing curves obtained from serial sampling of broth cultures showed a 100- to 10,000-fold decline in viability of log-phase organisms within 30 min of exposure to the drug. Two-dimensional agar dilution tests with carbenicillin and tobramycin with 79 strains showed additive or synergistic effects; no antagonism was documented. Seventy-eight of 79 strains were inhibited by a combination of 50 mug of carbenicillin per ml and 1.56 mug of tobramycin per ml, blood levels which seem attainable in man. Tobramycin appears to be a potent, rapidly bactericidal antibiotic against P. aeruginosa and merits clinical evaluation.     

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.     

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference ( P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus , (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.     

Effect of Recovery Medium on the Isolation of Campylobacter Jejuni Before and After Heat Treatment

The effect of various concentrations of polymyxin B and Colistin in Skirrow’s or Butzler’s type media, respectively, on the recovery rate of 13 strains of G. jejuni before and after heat treatment was studied. Prior to heating, a Butz-ler’s type medium containing 40 I.U. per ml Colistin was inhibitory for certain strains of C. jejuni. After heating at 48° G for 30 min, media containing 2.5 I.U. per ml polymyxin B or 10 I.U. per ml Colistin were not inhibitory for heat-injured cells. When the concentrations of polymyxin B were increased to 5.0 I.U. per ml or those of Colistin to 20 or 40 I.U. per ml in the selective media, the means of the differences of log cell counts between non-selective Brucella blood agar and the selective media was statistically significant (P < 0.05). The mean D-value of G. jejuni strains in Brucella broth at 48° G was 18.4±5.4 min.     

Evaluation of two selective media for the isolation of Bacillus anthracis

Aims:  To evaluate two selective media, polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate (PLET) agar and R&F Anthracis chromogenic agar (ChrA), for the isolation and selection of Bacillus anthracis .
Methods and Results:  Sixteen genotypically diverse B. anthracis strains were sub-cultured onto PLET and ChrA to test the sensitivity (ability of B. anthracis to grow and produce expected colony morphology) of both media. Fourteen of the 16 B. anthracis strains produced the expected morphology on PLET (88% sensitive) while 13/16 produced the expected morphology on the ChrA medium (81% sensitive). Seventeen other Bacillus strains and 18 non Bacillus spp. strains were used to evaluate the media's selectivity (ability to inhibit non- B. anthracis growth). PLET inhibited growth of 14/35 strains (40% selective), including six Bacillus strains, while ChrA inhibited 3/35 (9% selective). In addition, we did not observe any differences between the recovered CFU on PLET or ChrA when plating extractions of spiked soil.
Conclusions:  Polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate agar was more selective and sensitive than ChrA.
Significance and Impact of the Study:  Although both media are more expensive than sheep blood agar, for samples with high numbers of bacteria, they can be used to isolate B. anthracis with proper training and experience and with the knowledge that there are limitations to each media.     

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 novel strategy for the isolation and identification of environmental Burkholderia cepacia complex bacteria

The purpose of this study was to develop a novel strategy for the isolation and identification of Burkholderia cepacia complex bacteria from the home environment of cystic fibrosis (CF) patients. Water and soil samples were enriched in a broth containing 0.1% l-arabinose, 0.1% l-threonine, and a mixture of selective agents including 1 microgml(-1) C-390, 600U ml(-1) polymyxin B sulfate, 10 microgml(-1) gentamycin, 2 microgml(-1) vancomycin and 10 microgml(-1) cycloheximide. On selective media (consisting of the same components as above plus 1.8% agar), several dilutions of the enrichment broth were inoculated and incubated for 5 days at 28 degrees C. Isolates with different randomly amplified polymorphic DNA patterns were inoculated in Stewart's medium. Putative B. cepacia complex bacteria were confirmed by means of recA PCR and further identified by HaeIII-recA restriction fragment length polymorphism analysis. Our results suggest that these organisms may be more widespread in the home environment than previously assumed and that plant associated soil and pond water may be reservoirs of B. cepacia complex infection in CF patients.