Marine diatom species harbour distinct bacterial communities

We examined bacterial dynamics in batch cultures of two axenic marine diatoms (Thalassiosira rotula and Skeletonema costatum). The axenic diatoms were inoculated with natural bacterial assemblages and monitored by 4,6-diamidino-2-phenolindole (DAPI) counts, denaturing gradient gel electrophoresis (DGGE) with subsequent analysis of excised, sequenced 16S rRNA gene fragments, and fluorescence in situ hybridization (FISH) with group-specific 16S rRNA oligonucleotide probes. Our results show that algal growth exhibited pronounced differences in axenic treatments and when bacteria were present. Bacterial abundance and community structure greatly depended on species, growth and physiological status of even closely related algae. Free-living and phytoplankton-associated bacteria were very different from each other and were dominated by distinct phylogenetic groups. The diatom-associated bacteria mainly belonged to the Flavobacteria-Sphingobacteria group of the Bacteroidetes phylum whereas free-living bacteria, which were rather similar in both cultures, comprised mainly of members of the Roseobacter group of alpha-Proteobacteria. Presence and disappearance of specific bacteria during algal growth indicated pronounced differences in environmental conditions over time and selection of bacteria highly adapted to the changing conditions. Tight interactions between marine bacteria and diatoms appear to be important for the decomposition of organic matter and nutrient cycling in the sea.     

Total Counts of Marine Bacteria Include a Large Fraction of Non-Nucleoid-Containing Bacteria (Ghosts)

Counts of heterotrophic bacteria in marine waters are usually in the order of 5 x 10(sup5) to 3 x 10(sup6) bacteria ml(sup-1). These numbers are derived from unspecific fluorescent staining techniques (J. E. Hobbie, R. J. Daley, and S. Jasper, Appl. Environ. Microbiol. 33:1225-1228, 1977; K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980) and are subsequently defined as total counts of bacteria. In samples from the Baltic Sea, the North Sea (Skagerrak), and the northeastern Mediterranean Sea, we found that only a minor fraction (2 to 32%) of total counts can be scored as bacteria with nucleoids. Lack of DNA no doubt means inactive cells; therefore, a much lower number of bacteria that grow at rates higher than those previously estimated must be responsible for the measured bacterial production in these seas. The remaining bacterium-sized and/or -shaped particles included in total counts may be cell residues of virus-lysed bacteria (ghosts) or remains of protozoan grazing.     

FURTHER OBSERVATIONS ON THE BACTERIAL CONTENT OF WATER IN WATERCRESS BEDS

SUMMARY: Observations on two watercress beds in Kent have confirmed previous findings (Jones & Baker, 1955) that even in comparatively well-protected beds the coli-aerogenes content of the water may be high in summer. They have also shown that in spite of circumstances which would lead one to expect pollution, as in the case of one bed, the coli-aerogenes content can be low in winter. The season appears to influence the content in the water of coli-aerogenes bacteria, pectate liquefying bacteria and those capable of growing at 37°, but in the case of those growing at 22° other factors can play a more important part, as higher counts were not uncommon in winter. Seasonal effects were less marked with inlet samples; they were less influenced by atmospheric temperature, and little extraneous contamination occurred. Numbers increased as the sampling point moved from inlet to outlet.     

Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and ecological significance

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 +/- 0.2) x 10(8) cells g(-1) and (53.1 +/- 16.0) x 10(8) cells g(-1) in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was "reactivated." Bacterial turnover rates estimated ranged from 0.01 to 0.1 day(-1) but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day(-1)). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.     

Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml⁻¹ at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Effect of freezing of water samples on viable counts of environmental mycobacteria

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at —75°C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0·9–10·4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.     

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

MARINE BACTERIA OF CARDIGAN BAY. I. BACTERIA OF AN OFF-SHORE AREA

SUMMARY: A study of marine bacteria from a fixed off-shore area in Cardigan Bay showed there was little significance in the variation between numbers from different levels within 8 fathoms deep. There was a suggestion of seasonal variation in surface water in colony counts at 15–18°. In general, the bacterial content was highest over the summer months, but showed little direct correlation with temperature, pH or light.
Strains were mainly obligate halophytes and small Gram-negative rods predominated (96%). Nearly 50% of strains isolated liquefied gelatin; otherwise, biochemical reactions were weak. The proportion of pigmented strains was low.
Alien bacteria were present in irregularly varying numbers in all samples and showed no seasonal variation. Coli-aerogenes strains were recorded in small numbers, at all depths and in all seasons, but Bact. coli type I only rarely occurred.     

Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.     

Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.     

The impact of temperature change on the activity and community composition of sulfate-reducing bacteria in arctic versus temperate marine sediments

Arctic regions may be particularly sensitive to climate warming and, consequently, rates of carbon mineralization in warming marine sediment may also be affected. Using long-term (24 months) incubation experiments at 0°C, 10°C and 20°C, the temperature response of metabolic activity and community composition of sulfate-reducing bacteria were studied in the permanently cold sediment of north-western Svalbard (Arctic Ocean) and compared with a temperate habitat with seasonally varying temperature (German Bight, North Sea). Short-term ³⁵S-sulfate tracer incubations in a temperature-gradient block (between −3.5°C and +40°C) were used to assess variations in sulfate reduction rates during the course of the experiment. Warming of arctic sediment resulted in a gradual increase of the temperature optima ( T _opt) for sulfate reduction suggesting a positive selection of psychrotolerant/mesophilic sulfate-reducing bacteria (SRB). However, high rates at in situ temperatures compared with maximum rates showed the predominance of psychrophilic SRB even at high incubation temperatures. Changing apparent activation energies ( E _a) showed that increasing temperatures had an initial negative impact on sulfate reduction that was weaker after prolonged incubations, which could imply an acclimatization response rather than a selection process of the SRB community. The microbial community composition was analysed by targeting the 16S ribosomal RNA using catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH). The results showed the decline of specific groups of SRB and confirmed a strong impact of increasing temperatures on the microbial community composition of arctic sediment. Conversely, in seasonally changing sediment sulfate reduction rates and sulfate-reducing bacterial abundance changed little in response to changing temperature.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections > or =70% of total cell counts, e.g., coastal surface waters during spring and summer.     

Large Fraction of Dead and Inactive Bacteria in Coastal Marine Sediments: Comparison of Protocols for Determination and Ecological Significance

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 ± 0.2) × 10⁸ cells g⁻¹ and (53.1 ± 16.0) × 10⁸ cells g⁻¹ in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was “reactivated.” Bacterial turnover rates estimated ranged from 0.01 to 0.1 day⁻¹ but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day⁻¹). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.     

The metabolism of hydrogen by extremely thermophilic, sulfur-dependent bacteria

Abstract In just the last few years, a group of bacteria have been discovered that have the remarkable property of growing near and above 100°C. These extremely thermophilic organisms, defined here as having the ability to grow at 90°C with optimum growth at 80°C and above, have been isolated mainly from sulfur-rich, marine geothermal environments, both shallow and deep sea. They comprise over a dozen different genera, and except for one novel eubacterium, all may be classified as archaebacteria. The majority of the extremely thermophilic genera metabolize elemental sulfur (S°) and a survey of the various organisms reveals that most of them also depend upon the oxidation of hydrogen gas (H₂) as an energy source. In addition, two extremely thermophilic genera are known that actively produce H₂ as end-products of novel fermentative metabolisms. The enzyme hydrogenase, which is responsible for catalysing H₂ activation and H₂ production, appears to play several roles in electron and energy transfer during the growth of these organisms. Hydrogenase has so far been purified from only one extremely thermophilic species, from Pyrococcus furiosus ( T _opt = 100°C), and hydrogenase activity has been exmained in cell-free extracts of only a few others. However, a comparison of their properties with those of hydrogenases from mesophilic bacteria suggests that (a) the hydrogenase responsible for catalysing H₂ oxidation in extremely thermophilic organisms may be an extremely thermostable version of the mesophilic enzyme, and (b) a new type of 'evolution' hydrogenase, lacking the Ni-S or Fe-S catalytic sites of the mesophilic enzymes, is required for catalysing H₂ evolution at temperatures near and above 100°C.     

Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4',6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of Proteobacteria as well as members of the Planctomycetales were detected in both freshwater and marine water in abundances <7%.     

Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.     

Environmental determinants correlated to Vibrio harveyi-mediated death of marine gastropods

Vibrio harveyi is an emerging pathogen that causes mass mortality in a wide variety of marine animal species; however, it is still unclear which environmental determinants correlate V. harveyi dynamics and the bacterium-mediated death of marine animal life. We conducted a correlation analysis over a 5-year period (2003–2007) analysing the following data: V. harveyi abundance, marine animal mortality and environmental variables (seawater temperature, salinity, pH, chlorophyll a , rainfall and total viable bacterial counts). The samples were collected from a coastal area in northern Japan, where deaths of a marine gastropod species ( Haliotis discus hannai ) have been reported. Our analysis revealed significant positive correlations between average seawater temperature and average V. harveyi abundance ( R  = 0.955; P  < 0.05), and between average seawater temperature and V. harveyi -mediated abalone death ( R  = 0.931; P  < 0.05). Based on the regression model, n °C rise in seawater temperature gave rise to a 21 ⁿ -fold increase in the risk of mortality caused by V. harveyi infection. This is the first report providing evidence of the strong positive correlation between seawater temperature and V. harveyi -mediated death of marine species.     

The Practical Significance in the Microbiological Examination of Cold Stored Foods of the Allegedly Low Heat Resistance among Psychrotrophic Organisms

Summary: The significance in food microbiology of the observation of Stapert, Sokolski & Northam (1962), that some bacteria occurring in water have such low heat resistances that they would be affected by the warm agar used for pouring plates, was tested. Forty-two samples of 6 different foods stored for 7 days at 3° to enrich their psychrotrophic microflora, were examined using the same medium, but in thin poured plates and on spread plates, respectively, incubated at 14°. The logarithmic average count of the foods was c. 10⁸/g and included about 60% Gram-negative rods and 30% cocci. In 90% of the samples no differences were observed between the results from the two methods of counting. The slightly higher spread plate counts in the remaining instances could be accounted for by the increased disruption of bacterial agglomerations always observed in this procedure. Hence there is no reason to query the results of poured plate counts in general. Where it is yet probably safer to use spread plates, rigorous asepsis in pouring and storing should be observed to prevent the development of colonies of contaminants too small to be detected when the plates are inoculated, but leading to erroneous counts after incubation.     

Evolution of bacterial flora from a subterranean saline well by graduated salinity changes in enrichment media

The bacterial flora of samples taken from a subterranean saline well was enriched (1) by periodic salinity increase and (2) by periodic salinity decrease, both at 25° and 35°C. During the enrichment process, bacterial flora, including halotolerant, marine, moderately and extremely halophilic bacteria, were enumerated. Results were similar at both temperatures. Marine bacteria and moderately halophilic microorganisms were the most favoured groups, predominating between 3 and 30% (w/v) salt content; extremely halophilic bacteria also appeared when salinity reached 30% (w/v) and halotolerant bacteria were poorly represented. The taxonomic distribution of 125 selected strains, chosen at random from counting media, was essentially similar to those from other hypersaline environments.