Reversal of the silver inhibition of microorganisms by agar.

Increasing use of silver in the treatment of water has necessitated an examination of microbiological methods for the measurement of silver inactivation of microorganisms. Three common agar media were tested for their ability to neutralize the bacteriostatic effects of silver. Results suggested that growth media differed in their neutralizing capacity; that is, the non-inhibitory media tryptone glucose agar and Trypticase soy agar showed more neutralizing capacity than eosin methylene blue agar. Furthermore, the neutralizing effect appeared to be a function of the soluble component of the media and not of the agar itself.     

A rapid screening method for the isolation of metal-accumulating microorganisms

Summary An agar plate screening method was developed for the rapid isolation of heavy metal-accumulating microorganisms and preliminary estimation of their biosorption capacity. The test is based on the visulaization and interpretation of the metal distribution between agar and colonies by chemical preciptitation with hydrogen sulphide or ammonium sulphide. The heavy metals silver, thallium, lead, copper, nickel and cadmium have been tested successfully. The efficiency of the method is demonstrated for isolating silver-accumulating bacterian and estimating silver biosorption capacity.     

Comparative Studies on Enrichment And Selective Media for Isolation of Salmonellae from Faecal Specimens

Enrichment media (tetrathionate, selenite and Rapp ap ort broths) and selective media (desoxycholate citrate agar and brilliant green agar) were tested in different combinations to ascertain their capacity for isolation of salmonella bacteria. The material consisted of 299 samples of cattle faeces from two herds infected with salmonella (Table 1), and of 111 artificially contaminated samples of pig faeces (Table 3). The tetrathionate and selenite broths were equally useful for the material as a whole, whereas the results varied between different species of salmonella which is of great practical interest. The number of salmonella isolations was much lower when enrichment with Rappaport broth was used. The rate of salmonella isolations can often be increased by parallel enrichments with two different media. Of the selective agar media tested, brilliant green agar was superior to desoxycholate citrate agar.     

Evaluation of several selective media for recovery of Aeromonas hydrophila from polluted waters

Eleven media were studied for their suitability in the selective isolation of Aeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.     

Involvement of reactive oxygen species in the bactericidal activity of activated carbon fibre supporting silver; Bactericidal activity of ACF(Ag) mediated by ROS

An activated carbon fibre supporting silver (ACF(Ag)) was tested for its antibacterial capacity against Escherichia coli (E. coli). Water that has passed through ACF(Ag) demonstrated strong bactericidal ability. This activity decreased over the time suggesting that generated bactericidal species were short lifespan. Since formation of reactive oxygen species (ROS) might be catalysed by silver impregnated and/or ACF itself, implication of ROS and silver was evaluated by the use of ROS scavengers and a silver ions neutralizing agent. The role of ROS in the E. coli mortality was confirmed by the use of a molecular approach which revealed a strong expression of oxidative stress genes.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium.

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

Evaluation of several selective media for recovery of Aeromonas hydrophila from polluted waters.

Eleven media were studied for their suitability in the selective isolation of Aeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.     

Methodology for recovery of chemically treated Staphylococcus aureus with neutralizing medium

Recovery results of Staphylococcus aureus ATCC 6538 treated with phenolics and quaternary ammonium compounds on Dey and Engley (D/E) neutralizing medium at various time intervals were compared by the use of two commonly used media. Two recovery processes were utilized. In one, the chemically treated organisms were plated directly onto an agar medium. In the other, the aliquot was first put in broth and then was plated with agar. By either process, the numbers and the time period for recovery of organism were greater on D/E medium.     

Effect of silver nitrate and 2,4-D on anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

The response to anther culture, of six genotypes of Brussels sprouts was tested on six media which included two levels of 2,4-D and the presence or absence of silver nitrate. The presence of silver was usually beneficial, and with some genotypes had a very large effect. Increasing 2,4-D could be beneficial in the absence of silver nitrate, but was sometimes detrimental in the presence of silver. Replacing agar with highly purified agarose was not particularly beneficial. Genotype, medium and genotype × medium interactions were all significant factors, with genotype being the most important.     

Chemotaxis as a method for testing of the biological effects of silver nanoparticles

A method for assessing the abiotic efficiency of water-dispersed nano-sized silver particles is suggested. Nanoparticles were obtained by the method of biochemical synthesis in reversed micelles made from anionic surfactant bis-(2-ethylhexyl) sodium sulfosuccinate (AOT). A water dispersion of nanoparticles was prepared from the micellar solution in organic solvent by means of a special procedure. The abiotic efficiency assay is based on the capacity of cells to respond by the negative chemotaxis to chemical agents with harmful metabolic action. In plasmodium of myxomicete Physarum polycephalum, the biocide and repellent effects of silver nanoparticles, Ag+ ions, and AOT were tested in order to compare the abiotic efficiency of these substances in water solution and after introduction into agar substrate. The increase in the oscillation period, the reduction in the area of spreading, and the avoidance reaction in the spatial test, taken together, revealed a much higher repellent efficiency of silver nanoparticles as compared with that of Ag+ ions and AOT. The NSP concentrations lethal for Physarum were similar to those found earlier for bacteria and viruses. The chemotaxis-based tests applied in this study allow one to quantitatively assess cell reactions and monitor their time course. Besides, they have a much higher resolving capacity than the tests based on lethal effects of abiotic substances.     

Comparison of three rapid methods for identification of Salmonella spp

A study was carried out to compare three rapid methods for detection of Salmonella spp. The fluorogenic MUCAP test (Biolife, Italy), the SM-ID agar test (bioMérieux, France) and the Rambach agar test (Merck, Germany) were used in this study to examine 103 strains (69 Salmonella strains and 34 non- Salmonella strains). Two conventional culture media, Hektoen and Leifson agars, were also included. The sensitivities of the MUCAP, SM-ID, Rambach and Hektoen agar tests for pure strains were 100, 93, 88 and 99%, respectively, and their specificities were 74, 97, 76 and 59%, respectively. A total of 100 stool samples from patients with acute diarrhoea was also tested and showed great discrepancy between the different methods. In agreement with other investigators, it was found that the discriminating capacity of Rambach and SM-ID as primary plating media was very restricted. The MUCAP test was very sensitive, rapid and easy to perform but not very specific. In view of these results, it is essential to combine different methods for the accurate and reliable detection of Salmonella strains.     

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar.     

Quenching of the antibacterial activity of chlorhexidine and benzalkonium by Letheen broth and Letheen agar in relation to wild-type and envelope mutant strains of gram-negative bacteria

Letheen broth and Letheen agar have been investigated for their ability to act as neutralising and recovery media for wild-type and envelope mutants exposed to chlorhexidine diacetate and benzalkonium chloride. At high dilutions, untreated cells of the envelope mutant of Pseudomonas aeruginosa 799/61 were unable to produce colonies on Letheen agar. As a result of various procedures, it was concluded that dilution in Letheen broth and plating in Isosensitest agar was a suitable method for quenching cationic bactericides without harming the test strains, and that the increasing use of Gram-negative bacteria with outer membrane defects means that considerable care may be necessary in selecting media for evaluating bactericidal activity.     

Levels of fraction I and VW antigens in cultures of the plague microbe grown on yeast-casein, yeast-Hottinger broth and yeast-sunflower oil cake media

The content of fraction 1 and VW-antigens in Y. pestis cultures grown in different media (yeast-casein medium, yeast medium with Hottinger digest, and yeast medium with sunflower-seed protein) was studied over the course of their growth by means of the antibody neutralization and microprecipitation in agar tests. The media under study were not inferior to the casein sulfuric hydrolysate-based medium used for control in their capacity for ensuring the synthesis of VW-antigens. The maximum accumulation of fraction 1 was observed in yeast medium with sunflower-seed protein. In all media the maximum content of fraction 1 was registered on day 3 of cultivation, and the maximum accumulation of VW-antigens on days 8-9 of incubation at 37 degrees C. The data obtained in this study make it possible to regard fraction 1 and VW-antigens as the secondary metabolites of Y. pestis.     

Comparison of Media for the Enumeration of Clostridium perfringens

For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 10(4) organisms per g, but with 10(6) organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested.     

Analysis of human ascites effect on clonogenic growth of human tumor cell lines and NRK-49F cells in soft agar

We studied the factors that control the capacity of tumor cells to grow in soft agar. And, we analyzed the effect of cell-free ascites (CFA) obtained from ovarian cancer patients in combination with various media on the cloning efficiency of H-134 and OVCAR-3nu ovarian carcinoma cell lines and the WiDr colon carcinoma cell line. Seven batches of CFA consistently enhanced the soft agar growth of tumor cells more efficiently than tested sera. The addition of charcoal-treated bovine serum albumin (BSA) lowered the amount of CFA required for optimal tumor cell growth. As little as 1.25 ng of epidermal growth factor (EGF)/ml further improved OVCAR-3nu soft agar growth in combination with all of the amounts of CFA tested. Thus, neither BSA nor EGF seems to account for the colony-stimulating effect of ascites on tumor cells. Four batches of CFA were tested for stimulating soft agar growth of normal rat kidney (NRK-49F) fibroblasts; all induced colonies of different morphologies. This effect was potentiated by EGF, which suggests the presence of several transforming growth factor-like activities in CFA. The results show that differences in cloning efficiency of tumor cells of one or two orders of magnitude can be found between standard (anchorage-dependent growth-supporting) media and media optimalized for soft agar growth, such as CFA in the presence of EGF. This paper will discuss the similarity in effects of CFA on various tumor cells and NRK cells, and possible implications of the stimulatory effects of CFA.     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

Inhibitory Action of Various Plating Media on the Growth of Certain Salmonella Serotypes

The growth of 68 strains of Salmonella typhi , 697 other Salmonella strains and 213 strains of other Gram negative intestinal bacteria on 8 plating media was assessed semi-quantitatively. These media were found to be differentially inhibitory to different Salmonella serotypes. The combined use of two plating media, brilliant green MacConkey agar and deoxycholate citrate agar, allowed potentially the recovery of the maximum number of Salmonella strains. If only one medium was used, brilliant green MacConkey agar would appear to be the best plating medium for the isolation of non-typhoid salmonellas in general and S. choleraesuis in particular. Xylose lysine deoxycholate agar, on which a certain proportion of salmonellas failed to yield typical, recognizable colonies, was found not to be a good plating medium for their isolation.     

Evaluation of test media for routine monitoring of Escherichia coli in nonpotable waters.

Six test media, m-TEC, m-TEC with 4-methylumbelliferyl-beta-D-glucuronide (MUG), lauryl tryptose agar (LTA) with MUG, LTA with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Glue), EC medium with MUG, and lauryl tryptose broth with MUG, were evaluated for their usefulness in enumerating Escherichia coli in nonpotable waters on a routine basis. The media were chosen for their case of interpretation of target colonies, ability to allow enumeration at low and high concentrations, and ability to inhibit nontarget microorganisms. The recoveries on the test media were compared with those on three reference media, R2A, m-FC, and m-Endo, by analysis of spiked samples of filter-sterilized waters. The test media were then further tested for their ability to differentiate nontarget but closely related microorganisms. Statistical analysis indicated that the best recoveries were obtained with lauryl tryptose agar with added MUG and X-Gluc. The media were then tested with surface waters that could be expected to have high levels of total and fecal coliforms along with Escherichia coli.     

Neutralization of bacterial viruses by antibodies of young animals

The development of neutralizing antibodies against bacteriophage T 2 in newborn rabbits was investigated. On the seventh day after antigen injection only antibodies of 19 S type were detected following the fractionation of serum on a saccharose density gradient. The neutralizing activity of these antibodies was heavily complement-dependent: the neutralizing capacity was completely destroyed by inactivation at 56° C for 30 min; however, it was possible to renew it by the addition of fresh complement. Antibodies of the 7 S type detected on the fifteenth day after immunization by ultracentrifugation, were also dependent on complement in their neutralizing capacity. Antibodies of the 7 S type produced on the 28th day after immunization were still partially dependent on complement; however, antibodies produced in the secondary response no longer required complement for their neutralizing activity. The obtained results indicate that in the course of the primary and secondary response to bacteriophage T 2, antibodies of 19 S and 7 S are gradually formed in the newborn with higher ability to neutralize the phage particle alone without the potentiating action of complement.