Evidence for the involvement of thiocyanate in the inhibition of Candida albicans by Lactobacillus acidophilus

Lactobacillus acidophilus has been found to inhibit Candida albicans when grown on MRS agar plates. Attempts to isolate an active factor responsible for this inhibition from liquid culture and agar plates were not successful. The addition of sodium thiocyanate to the agar was found to increase the inhibition offered by the lactobacillus. The results indicate that hydrogen peroxide produced by the lactobacillus is being used to convert the thiocyanate to hypothiocyanate which is more toxic. The involvement of a lactobacillus peroxidase in this conversion is postulated.     

Fibronectin-binding capacity of staphylococci

The fibronectin-binding capacity of clinical isolates of the genus Staphylococcus has been studied in the flake-formation test on glass and by inoculation into agar with human fibronectin added. Most of 58 S. aureus strains have been found capable of binding fibronectin. None of coagulase-negative staphylococci has given flake formation with fibronectin. The possibilities of the quantitative evaluation of the fibronectin-binding by the above-mentioned methods have been shown. In the analysis of the monomers of bovine fibronectin its capacity for inducing the growth of compact colonies in semiliquid agar has been shown.     

Preparation of Agar-Agar from the Red Seaweed Pterocladia capillacea off the Coast of Alexandria, Egypt

The effect of different treatments on the quality of agar produced from Pterocladia has been studied, and the conditions for the production of material of good quality have been standardized. In the modified process, sun-bleached seaweed was washed well in water, soaked for 24 h, and then ground to a pulp and rinsed again in water. The pulp was then extracted with water (weed-to-water ratio, 1:30) under pressure for 2 h after adjusting the pH to 6 by the addition of acetic acid. The agar gel, after freeze thawing, was bleached with NaClO before drying in a current of hot air. Pretreatment of the seaweed with alkali at 80°C for 2 h prior to extraction was found to improve the quality of agar to a very great extent.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

Transparent solid nutrient medium for studying the lytic spectrum of bacteriophages of microbes of the genus Bordetella

A solid, transparent culture medium for the study of the lytic spectrum of the phages, active against B. pertussis and B. bronchiseptica, in respect to homologous and heterologous bacteria of the genus Bordetella has been developed. The Cohen-Wheeler liquid medium with nicotinic acid and nicotinamide added, solidified with agar, is nicotinamide added, solidified with agar, is used as the base of the new medium. This base ensures the growth of B. parapertussis and B. bronchiseptica. To stimulate the growth of B. pertussis, the tissue stimulant of B. pertussis growth (a transparent substrate obtained from the tissue of the large intestine of a rabbit) has been used. With 10% of this stimulant added, B. pertussis cells have been found to preserve their typical morphological and immunobiological properties.     

Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27^T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.     

A New Isolation Method for Labyrinthulids Using a Bacterium, Psychrobacter phenylpyruvicus

A new isolation method for labyrinthulids, marine microbes with spindle-shaped vegetative cells and gliding movement, is presented. The method for isolating labyrinthulids has been found to be more difficult and less reproducible than that for thraustochytrids, classified in the same order. So far serum seawater agar fortified with antibiotics has been proposed to be the best for isolation of labyrinthulids. The method presented here involves placing plant samples on an agar medium on which a marine bacterium, Psychrobacter phenylpyruvicus, has been grown. The new method, which utilizes fallen mangrove leaves as source material, was more than twice as effective as isolation agar medium without the bacterium. The increased effectiveness appears to derive partly from the bacterial colonies' delaying extension of fungal mycelium. The bacterium was more effective for the isolation of labyrinthulids than either the bacterium Shewanella sp. or the yeast Rhodotorula rubra.     

A Note on the Growth of Thiobacillus ferrooxidans on Solid Medium

An iron oxidizing strain of Thiobacillus ferrooxidans has been grown on solid medium using purified agar, carrageenan (Type 1) and agarose. This strain produces isolated and transferable colonies after 7 d incubation. Growth (increase in viable cells) by the direct plating method has been followed in relation to iron oxidation. Acidity, agar concentration and phosphate influenced colony development on solid medium.     

A new bioassay system for screening high berberine-producing cell colonies of Thalictrum minus

A new method of estimating the amount of berberine released from minute cell colonies of Thalictrum minus has been devised to facilitate the selection of high berberine-producing cell lines. In this system, cell aggregates obtained from a cell suspension culture are grown on small pieces of an agar culture medium and the concentration of berberine which has been released from the cells into the agar piece is assayed by the antibacterial activity against Bacillus cereus MT2026. Screening of 1000 cell colonies by the agar piece method has resulted in the isolation of four, high berberine-producing cell lines, although they have been found to be more or less unstable with respect to the biosynthetic capability during successive subcultures.     

Seasonal Variation in Sporulation of Phytophthora infestans

Sporulation ability of two isolates of Phytophthora infestans maintained on potato tuber slices of a susceptible cv. ‘Bintje’ and on-rye agar medium was studied for four years (1981–1984). This feature of the fungus was shown to vary in particular seasons during the year. Significantly higher sporulation density per cm² of aerial mycelium on potato tuber slices was observed in winter and late autumn while significantly lower sporulation was found in spring. Similar tendencies were observed when one of the isolates was cultivated on rye agar medium under controlled conditions. Positive correlation was found between sporulation patterns of isolates of the fungus maintained on rye agar and on tuber slices. Hypothesis has been proposed that these changes are due, to a biorhythm in the fungus.     

Effect of oxygenation level of virus-infected cell monolayer culture on the rate of viral multiplication

The dependence of the virus multiplication rate on the oxygenation level of a virus-infected cell culture was quantitatively evaluated by the influence of agar overlay thickness on the viral plaque diameter. Although the experimental curve was found to fit the exponential function it reflects an interaction of at least two processes: gas diffusion through the agar medium and active 'pumping' of respiring cells determining the oxygen influx. Therefore, it has been deduced that the dependence of the virus multiplication rate on the oxygenation level fits the linear function, which is not the case for the most metabolic (enzymatic) reactions. The concept of intracellular unbalanced molecular constellations is suggested as an explanation of the linear dependence.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.     

Screening of Marine Bacteria for the Production of Microbial Repellents Using a Spectrophotometric Chemotaxis Assay

A method for screening marine bacteria for the production of microbial repellents has been developed. The spectrophotometer provided quantitative information on bacterial chemotaxis in response to extracts from other strains of marine bacteria. Aqueous extracts were incorporated into an agar plug at the base of a cuvette, which was overlaid with a suspension of a motile strain. Negative chemotaxis of the motile strain in response to diffusion of repellent compounds from the agar could be measured by a fall in the optical density, allowing the direct screening of supernatants for repellent activity. Three strains producing metabolites with a repellent effect on a motile marine bacterium were identified. Antibiotic activity and the repellent effect of the supernatants were compared, with no significant correlation being found. The screening method will therefore allow the identification of bioactive metabolites that would be overlooked using traditional antibiotic screening strategies. Received March 4, 1998; accepted November 11, 1998.     

Studies upon the copper fungicides. IX. Investigations with the exudate from fungus spores

Determinations have been made of the copper dissolved from pure copper compounds by water, by a standardized solution of the exudate of Neurospora sitophila spores, by standard solutions of malic and succinic acids and their sodium salts, and by glycine and mannitol.
The results compared with the known fungicidal performances of the copper compounds show that the amounts of copper yielded to water and to the standardized spore extract or glycine solution provide an indication of fungicidal properties.
Although soluble copper in excess of that dissolved by water can only appear from alkaline Bordeaux deposit by complex ion formation, results show that this is not necessarily the only means by which copper can dissolve from other copper compounds.
In a chemical investigation of the exudate of N. sitophila spores, mannitol, succinate and fumarate have been isolated. None of these materials can dissolve an appreciable amount of copper from alkaline Bordeaux deposit. Malate, found by other workers to be a constituent of the exudate of N. sitophila spores cultured on potato dextrose agar, has not been detected in such a solution from spores grown on malt agar. The quantity of mannitol in spore exudate has been shown to depend on the nutrient medium on which the fungus is grown.     

On the importance of calcium and magnesium ions in yeast sporulation

Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.     

Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs

The oviducts of both oestrous and pseudopregnant rabbits can be used for the successful culture of mammalian embryos for short periods. This has alowed some selection to be made on the embryos as they are examined on at least two occasions before final transfer. Not only have pregnancy rates been normal, but in some instances they have been higher following a limited period (2–3 days) in the rabbit oviduct. It would appear that these higher pregnancy rates result from a more intensive selection of embryos at the time of transfer rather than from some substance acquired during storage in the oviduct. However, the system is not without disadvantages. There is some loss of embryos (15–30%) in the oviduct and all embryos recovered may not have developed at the normal rate.The rabbit oviduct has been used as a site of xenogenous fertilization. Initial reports indicate that success in that area is lower than when using large animals as the site of fertilization. With more widespread interest in the use of microsurgery in embryos, the rabbit oviduct has been used for the short term storage of agar cylinders and has been found to be unsuitable because of the high rate of degeneration of agar chips. However, the rabbit oviduct is still useful as an experimental tool in the manipulation of embryos from the domestic species.