A tentative direct microscopic method for counting living marine bacteria.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.     

Adhesion of biodegradative anaerobic bacteria to solid surfaces.

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe(3+) on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Influence of substratum wettability on attachment of freshwater bacteria to solid surfaces

[This corrects the article on p. 811 in vol. 45.].     

Adhesion of bacteria from mixed cell suspension to solid surfaces

The attachment of four species of bacteria to solid surfaces was investigated to determine whether the attachment of one species of bacterium could be influenced by the presence of other attaching or attached species. Three types of experiment were done: (i) attachment of bacteria from suspensions containing two species (termed simultaneous attachment) was compared to attachment of each species in pure culture, (ii) the attachment of one species of bacterium to surfaces already colonized by a second species (termed sequential attachment) was compared to attachment of the bacteria to clean, uncolonized surfaces, and (iii) bacteria were allowed to attach to a surface already colonized by a second strain, and their effect on the stabilization of adhesion of the initial colonizing strain was determined. The bacteria were Acinetobacter calcoaceticus, a Staphylococcus sp., a coryneform (isolates from a canning factory), and Staphylococcus aureus. The surfaces were tin plate, glass, and nylon. The attachment of each species was either increased, decreased or not affected by the simultaneous or sequential attachment of another species. The results depended upon the species combination, the surface composition, and the sequence of attachment. The detachment of a primary colonizing species was either increased, decreased or not affected by the subsequent attachment of a second species, depending on the species combination and surface. The results demonstrate that bacterial attachment to a surface can be influenced by the composition of the attaching population and can differ considerably from the attachment of the component species in pure culture. This has implications for the control and removal of biofilms in food processing plants, as well as a wider significance for the composition and dynamics of biofilms in industrial and natural environments.Abbreviation PYE Peptone/yeast extract medium     

A disintegration method for direct counting of bacteria in clay-dominated sediments: dissolving silicates and subsequent fluorescent staining of bacteria

The masking of bacteria by abundant microparticles of the clay and silt fraction and cell losses due to sonication hampered direct enumeration of bacteria in sediments dominated by fine sediments. These problems can be circumvented by dissolving silicate fine particles using hydrofluoric acid and subsequent staining of bacteria by DTAF. The developed disintegration method partly replaces mechanical separation of bacteria from sediment particles by chemical disintegration of the silicates. Recovery efficiency ranged from 90% to 111% for different clays and clay-dominated sediments. Especially for the analysis of fine sediments and clays, this method circumvents both strong dilution of the sediment sample and harsh sonication. The method can also therefore be used in sediments where particle abundance is several orders of magnitude higher than bacterial abundance and simple dilution would not suffice in reliably counting bacteria.     

Simplified sample preparation using frame spotting method for direct counting of total bacteria by fluorescence microscopy

A new preparation method for direct counting of bacteria in liquid samples with fluorescence microscope was developed using a glass slide coated with 3-aminopropyltriethoxy silane and ring-shaped polyester seal as a retainer. The experimental steps of this method were spotting samples onto the coated slides with the seal, drying under vacuum, staining with SYBR Green II, drying and covering with immersion oil and coverslip to allow counting. This simplified method provided consistent results when compared with the conventional filtration method for fluorescence microscopy, and is rapid, inexpensive and reproducible.     

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims:  To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes , Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces.
Methods and Results:  Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10² CFU cm⁻². On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion.
Conclusions:  The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria.
Significance and Impact of the Study:  This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes , Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.     

Factors affecting survival of test bacteria in sea water: marine bacteria,test bacteria and solid surfaces

Summary 1. The antibacterial activity of raw sea water varied considerably during incubation of successive inocula ofEscherichia coli, Staphylococcus aureus, andSerratia marinorubra. In most cases inactivation of second inocula was stronger than that of first ones. However, withS. aureus, contradictory results were obtained also.2. The bactericidal effect of filter-sterilized sea water was strengthened by inactivated cells ofEscherichia coli andStaphylococcus aureus. Contradictory findings were obtained from autoclaved sea water.3. Inactivation of test bacteria was greatly influenced by solid surfaces. In most cases, the kill ofEscherichia coli andStaphylococcus aureus in raw and sterile-filtered sea water was stronger at increased surface/volume ratios than under standard conditions. More rapid inactivation of these test strains in sterile-filtered, than in raw, sea water occurred more often at enlarged ratios of solid surface per unit volume. The survival ofSerratia marinorubra was positively affected by solid surfaces.4. It is concluded that changes in nutritive conditions occurring during the experiments are more important in regard to antibacterial activity of sea water than production of harmful matter by marine bacteria.

---

Faktoren, welche das Überleben von Testbakterien in Meerwasser beeinflussen: Meeresbakterien, Testbakterien und feste Oberflächen

Kurzfassung Der Einfluß der vorstehend genannten Faktoren wurde auf die Überlebensfähigkeit vonEscherichia coli, Staphylococcus aureus undSerratia marinorubra in Meerwasser untersucht. Aktivitäten mariner Bakterien führten nicht generell zu verstärkter antibakterieller Wirkung rohen Meerwassers. Häufig waren sie für das Überleben vonE. coli undS. aureus förderlich. Inaktivierte Zellen vonE. coli undS. aureus erhöhten die bakterizide Wirkung rohen und filtersterilisierten Meerwassers gegenüber sekundär inokulierten, gleichartigen Testbakterien, während sie die inaktivierende Potenz autoklavierten Meerwassers verminderten. Durch erhöhtes Angebot an Glasoberfläche/Volumeneinheit, welches die adsorptive Anreicherung organischer Substanzen verstärkt, wurde die Inaktivation vonE. coli undS. aureus meistens beschleunigt, während sich diejenige vonS. marinorubra um so stärker verminderte, je größer das Verhältnis Oberfläche/Volumen war. Raschere Abtötung vonE. coli undS. aureus in Sterilfiltraten als in rohem Meerwasser trat bei erhöhtem Oberfläche/Volumen-Verhältnis häufiger auf als unter Standardbedingungen. Aus den Ergebnissen wird geschlossen, daß die während der Versuche eintretenden Veränderungen des Nährstoffangebotes, hervorgerufen durch Nährstoffverbrauch sowie durch Lysis inaktivierter Testbakterien, bezüglich der bakteriziden Wirkung von Meerwasser generell von größerer Bedeutung sind als bakterizide Stoffwechselprodukte mariner Bakterien.

     

Development of a direct viable count procedure for some Gram-positive bacteria

The direct viable count (DVC) is a procedure for enumerating viable-nonculturable cells. It should be noted, however, that bacteria demonstrating the viable but nonculturable phase have to date included only Gram-negative species, mainly because the DVC procedure does not lend itself to the analysis of Gram-positive bacteria since the DVC procedure is dependent on the bacterium being sensitive to nalidixic acid. The authors report here concerning studies on an analogous procedure for the direct enumeration of viable-nonculturable Gram-positive bacteria.
To facilitate a differential DVC for Gram-positive bacteria, ciprofloxacin, enoxacin, norfloxacin or isopropyl cinodine were substituted for nalidixic acid. These antibiotics were chosen because, like nalidixic acid, they are DNA gyrase inhibitors. The concentrations used for each antibiotic were 1000 μg ml^-1, 100 μg ml^-1 and 10 mg ml^-1. Pure cultures of Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis were obtained from the culture collection at the University of Wyoming and a faecal streptococcus was isolated from the Laramie wastewater treatment plant. An antibiotic and optimal concentration thereof was found which gave enlarged cells for all the organisms except the faecal streptococcus isolated from the wastewater plant for which no enlarged cells were ever seen. The antibiotic and concentration thereof which gave the optimal percent enlarged cells in the DVC procedure varied between organisms.     

Application of a microfluidic device for counting of bacteria

AIMS: To develop a miniaturized analytical system for counting of bacteria. METHODS AND RESULTS: Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 x 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer.     

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting bacteria in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally > 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1·5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.     

Electronic sizing and counting of bacteria

An electronic apparatus is described that permits rapid determination of the concentration and size distribution of bacteria in electrolyte suspensions by a resistance method. The resulting size-concentration distribution may be displayed on an oscilloscope and recorded with an X–Y plotter and an electric typewriter-tape punch unit. The paper tape is analyzed with a computer program. Comparisons are made between electronic measurements of bacterial cell concentration and size distribution and values obtained by other methods. Effects of heat-killing and disruption of the cell membrane on the electrical counting characteristics of the organisms are discussed.     

Enzyme catalysis on solid surfaces

Enzyme-catalysed reactions in which substrates are bound (immobilised) to solid surfaces are becoming increasingly important in biotechnology. There is a general drive for miniaturisation and automation in chemistry and biology, and immobilisation of the reaction intermediates and substrates, for example on microarrays or nanoparticles, helps to address technical challenges in this area. In bionanotechnology, enzyme catalysis can provide highly selective and biocompatible tools for the modification of surfaces on the nano-scale. Here, we review the range of enzyme-catalysed reactions that have been successfully performed on the solid phase and discuss their application in biotechnology.     

The counting of viable tubercle bacteria

Summary The publication ofChristensen, Robinson andWiddicombe (1953) was an occasion to draw renewed attention to the method of counting viable germs in rolling flasks since 1938 used in the Laboratory of Hygiene, State University at Utrecht and elsewhere (Julius, 1938). The way in which technique and medium are adapted to the viable counting of tubercle bacteria is described.