14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 micrograms of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 μg of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

成都地区天然水域中异养微藻的分离及其特性研究

采用添加不同浓度和种类抑菌剂的平板进行多次平板划线分离和三角瓶培养,从成都地区四川大学望江及江安校区附近水域中分离得到了一株可在无光照条件下异养生长的微藻,经初步鉴定为小球藻并编号为Chlorella SCU-01.在此基础上初步研究了该藻株在不同葡萄糖起始浓度下的异养生长及色素合成特性.     

An improved method for the observation and enumeration of heterotrophic and photoautotrophic microplankton

Fluorescence microscopy is a preferred technique for discriminating between heterotrophic and photoautotrophic microorganisms in plankton studies. An improved technique is described which employs sample concentration by filtration onto Nuclepore filters followed by transfer to a glycerine jelly preparation for identification of phytoplankton by autofluorescence of photosynthetic pigments and examination of all microorganisms by phase-contrast microscopy. The method is rapid, and has been used routinely at sea to provide semipermanent preparations of pico-and nanoplankton including naked flagellates for subsequent enumeration onshore.     

Biotests for mineral waters with natural and recombinant luminescent microorganisms

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).     

An improved method for collecting heterotrophic microorganisms living on pebbles in streams

In microbiological studies in streams, pebble samples have until now been taken out of the water following the conventional method. However, this allows the loss of microorganisms as a result of the removal of overlying water. In the present study, to minimize the loss of microorganisms, we have developed a new sampling method, called the submerged method, for collecting microorganisms living on pebbles in streams. The abundance of microorganisms on natural pebbles and artificial clay tiles, both of which had biofilms developing on their surfaces, was measured using both the conventional and submerged methods and the results from the two methods were compared. The cell densities of bacteria (0.10–14.00 × 10⁸ cells cm^–2), heterotrophic nanoflagellates (0.36–50.30 × 10⁴ cells cm^–2), and ciliates (0.071–88.27 × 10² cells cm^–2) measured by the submerged method tended to be higher than those obtained by the conventional method, although there were only a few cases in which a significant difference existed between microbial abundances determined by the two methods. Also supported by microscopic observation, these results suggest the presence of planktonic and/or weakly attached microorganisms on substrate materials in streams. Significant correlations between the concentration of chlorophyll a and the cell densities of heterotrophic microorganisms and significant correlations among heterotrophic microorganisms suggest the presence of active microbial food webs in streams.     

Evaluation of Enterolert for enumeration of enterococci in recreational waters.

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.     

Corrosion of metals in natural waters influenced by manganese oxidizing microorganisms

Case histories and proposed mechanisms formicrobiologically influenced corrosion of metals andalloys by metal depositing microorganisms arereviewed. Mechanisms with indirect participation ofthese microorganisms, usually iron- and manganeseoxidizing species, are distinguished from anothermechanism which accounts specifically for theelectrochemical properties of deposits containingoxides and hydroxides of Mn in higher oxidationstates. The possible influence of such deposits whichwere formed microbiologically is evaluated. Theevaluation is based on the principles ofelectrochemical corrosion of metals and on theelectrochemical properties of Mn^3+/4+- compounds.After briefly reviewing the microbiologicalMn-oxidation, experimental evidence for the predictedcorrosion by such deposits is provided and a model formicrobiologically influenced corrosion by manganeseoxidizing microorganisms is proposed for stainlesssteel. Possible consequences of the model andpractical aspects of such a corrosion are discussed.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin, penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 micron pores were significantly more efficient than those with 0.45 micron pores in the isolation of BFG. A preliminary incubation period of 4 h at 30 degrees C prior to 44 h at 37 degrees C yielded significantly higher numbers of BFG than direct incubation at 37 degrees C for 48 h.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A llsop K. & S tickler D. J. 1984. The enumeration of Bacteroides fragilis group organisms from sewage and natural waters. Journal of Applied Bacteriology 56 , 15–24.
A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin. penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 μm pores were significantly more efficient than those with 0.45 μm pores in the isolation of BFG. A preliminary incubation period of 4 h at 30C prior to 44 h at 37C yielded significantly higher numbers of BFG than direct incubation at 37C for 48 h.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

A simplified method for coliphage detection in natural waters

The ARCAT (A Rapid Coliphage Analysis Technique) method for detecting coliphages in water has been modified. Modifications to the original method include media optimization, the use of frozen host cultures, the use of a single agar coliphage assay and optimized plaque resolution with 2, 3, 4-triphenyltetrazolium chloride. Detection of 5 coliphages per 100 ml of water sample is accomplished in 6 hours for rapid estimation of water quality.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Use of heterotrophic microorganisms in mineral biotechnology

A process for biological removal of iron from quartz sands, kaolins and clays was developed in which these industrial minerals were leached at 90°C with lixiviant produced as a result of the cultivation of acid-producing heterotrophic microorganisms, mainly strains of Aspergillus niger, at 30°C in a nutrient medium containing molasses as a source of carbon and energy. The lixiviant, i.e. the fermentation fluid, contained oxalic and citric acids as main components and after the cultivation was acidified to a pH of 0.5 by means of hydrochloric acid. The leaching was carried out in mechanically stirred acid-resistant vats for a period of from 1 to 5 hours. The iron content of some sands treated by this method was lowered from 0.035–0.088 to below 0.012% Fe₂O₃ making them suitable for the preparation of high quality glass. The iron content of different kaolins was lowered from 0.65–1.49 to 0.44–0.75% Fe₂O₃ and as a result of this their whiteness was increased from 55–87 to 86–92%. The iron content of a clay was lowered from 6.25 to 1.85% Fe₂O₃ and this increased the fireproofness of the clay from 1 670 to 1 750°C. Similar process was used for leaching of aluminium from aluminosilicates, mainly clays and kaolins. However, after the cultivation the fermentation fluid was acidified either by means of sulfuric or hydrochloric acid or by means of different mixtures of inorganic acids. For enhancing aluminium solubilization the aluminosilicates were heated before leaching at 600–650°C for 1–2 hours. Over 90% of the aluminium present in different clays and kaolins was leached within 3–6 hours in this way. “Silicate” bacteria related to the species Bacillus circulans and B. mucilaginosus were used to leach silicon from low-grade bauxite ores containing aluminosilicates as impurities. The bacterial action was connected with the formation of mucilaginous capsules consisting of expolysaccharides. The solid residues after leaching were characterized by higher values of alumina content and were suitable for processing by means of the BAYER process for recovering aluminium. Heterotrophic bacteria were used to leach manganese from oxide ores using different organic compounds as reducing agents.     

A new method for the detection and enumeration of manganese oxidizing and reducing microorganisms

Summary 1. Berbelin blue-I (N.N.-Dimethylamino-p, p-triphenylmethane-o-sulphonic acid) has been examined as a reagent for the detection of manganese oxidation and manganese reduction in microbial culture media.2. Berbelin blue in slightly acid solution may be added to media without influencing population growth of micro-organisms. The oxidation of Mn II solutions to Mn III and IV is detected by occurrence of deep blue colours.3. In case of long-term culture experiments, it is more advisable to add Berbelin blue-I solutions only after cultivation.4. Berbelin blue-I solutions react with manganese IV–VII in pH ranges between 3.5 and 10, thus allowing tests at desired pH values.5. Reduction of manganese VII or manganese IV by micro-organisms may be detected by addition of Berbelin blue-I solutions after cultivation periods. White halos within dark-blue coloured media indicate reduction of Mn-IV compounds better than occurrence of white spots in slightly violet or grey media, as produced by the incorporation of manganese oxides or permanganate solutions to test media.6. Ecological work and quantitative work on board research vessels is much easier and less dangerous with Berbelin-I solutions than withFeigl's Benzidinium reagent.7. Berbelin blue-I may be used for quantitative determination of manganese and oxygen when measuring optical density of the colour complex at 618 nm in wide concentration ranges and working in acetate buffer.

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Eine neue Methode zum Nachweis und zur Zählung von Mangan oxidierenden und reduzierenden Mikroorganismen

Kurzfassung Berbelinblau I (N.N-Dimethylamino-p,p-triphenylmethan-o-sulfonsäure) ist die Leukobase eines neuen Farbstoffes und bildet mit Mn(III)-Mn(VII)-kationen einen intensiv blau gefärbten Farbstoff. Die Reaktion verläuft quantitativ, indem die Leukobase (FH) zum Farbstoff (F+) oxidiert wird. Sie entwickelt sich am günstigsten in Acetatpuffer. Eine intensive und schnelle Reaktion mit anwesenden Mangan-III-VII-kationen läuft jedoch auch im pH-Bereich zwischen 4 und pH 7 ab. Bei Verwendung von konzentrierteren Lösungen von Berbelinblau I entstehen in Anwesenheit oxidierter Stufen des Mangans gut erkennbare Farbkomponenten noch bis pH 10. Die Verwendungsfähigkeit des neuen Farbstoffes als Beimengung zu Nährböden wurde getestet. Mangan oxidierende Bakterien und Pilze wachsen auf Nährböden, in denen der Farbstoff in Konzentrationen zwischen 10^–4 und 10^–5 g/100 ml imprägniert wurde, ohne erkennbare Behinderung gegenüber nichtbehandelten Nährböden. Bereits früher als die Bildung von schwarzbraunen Manganoxidhydratablagerungen im Nährboden tritt eine Blauverfärbung der Kolonien ein, die Mangan oxidieren. Berbelinblau I eignet sich deshalb ausgezeichnet für die Früherkennung von Mangan oxidierenden Mikroorganismen in selektiven Nährböden. Bei einem Vergleich der Benzidinmethode nachFeigl (1958) mit dem neuen Farbstoff ergab sich ein erkennbarer Vorteil von Berbelinblau I. Das Testreagens kann dem Nährboden oder den Kulturen in schwach saurem Puffer zugeführt werden und die Isolation von Mikroorganismen ist nach Anfärbung noch ohne weiteres möglich. Kulturen und Kolonien von Mangan reduzierenden Bakterien und Pilzen lassen sich deutlich von unspezifischen Kolonien unterscheiden, wenn nach der Anfärbung mit Berbelinblau ungefärbte Höfe gezählt werden. Auch in diesem Fall ist eine Isolation ohne weiteres möglich, während dies bei der Verwendung vonFeigls Reagens in konzentrierter Essigsäure auf Schwierigkeiten stößt. Quantitative Bestimmung von Mangan (und Sauerstoff über die Winklermethode) in Nährböden und bakteriell gefälten Manganablagerungen ist ohne weiteres möglich.