Direct Count Method Research Articles

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.

Survival and Recovery of Viable but Nonculturable Listeria monocytogenes Cells in a Nutritionally Depleted Medium

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4°C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37°C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.

Application of a rapid direct viable count method to deep-sea sediment bacteria

For the first time, a Live/Dead® (L/D) Bacterial Viability Kit (BacLight™) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250–5600 m) and sediment depth (0–5 cm). Percentage of viable (and thus potentially active) cells ranged between 20–60% within the first and 10–40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3±5.4 nmol cm −3 h −1) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations ( p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.

Rapid enumeration of virus-like particles in drinking water samples using SYBR green I-staining

We studied the suitability of SYBR green I-staining for determining total counts of virus-like particles and bacteria in drinking water. Low background fluorescence and lack of unspecific staining made drinking water samples an excellent matrix for SYBR green I-staining. Direct microscopic count method is a rapid and economical tool for assessing the total number of virus-like particles in aquatic samples, compared to culture-dependent or molecular biology methods. We applied this method to show the efficiency of a large-scale drinking water purification process in the removal of virus-like particles and bacteria from lake water.

Rapid enumeration of virus-like particles in drinking water samples using SYBR green I-staining

We studied the suitability of SYBR green I-staining for determining total counts of virus-like particles and bacteria in drinking water. Low background fluorescence and lack of unspecific staining made drinking water samples an excellent matrix for SYBR green I-staining. Direct microscopic count method is a rapid and economical tool for assessing the total number of virus-like particles in aquatic samples, compared to culture-dependent or molecular biology methods. We applied this method to show the efficiency of a large-scale drinking water purification process in the removal of virus-like particles and bacteria from lake water.

Factors Affecting Survival of Clavibacter michiganensis subsp. sepedonicus in Water

AbstractThe survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non‐sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non‐mucoid strain was also studied in sterile ditch water and simulated ‘drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non‐culturable state.

Population Dynamics of Free-Living Bacteria Related to the Microcystin-Degrading Strain Y2 in Lake Suwa and in Microcystin Amended Enrichments

We investigated the population dynamics of the free-living microcystin-degrading isolate Y2 (MCD-isolate) in an eutrophic lake (Lake Suwa, Japan) using fluorescence in situ hybridization (FISH) with a specific probe. Free-living MCD-isolate was successfully visualized by direct viable count method combined with FISH analysis. The highest concentration of MCD-isolate existed in 1999 when high concentrations of dissolved microcystin (Mcyst) and Microcystis populations were present. Mcyst degradation experiments with free-living bacteria collected at different times during a Microcystis bloom in 2000 indicated that three samples of free-living bacteria completely degraded Mcyst-LR and its isomer. The lag time before the degradation was however different for the free-living bacteria. Free-living bacteria present during the mid-bloom of Microcystis completely degraded Mcysts with the shortest lag time. The number of free-living cells of MCD-isolate detected by the FISH method significantly increased when Mcyst was degraded with the fastest degradation rate in the mid-bloom sample. Other bacterial populations collected at mid-and late-bloom increased during the experiments with the exception of the δ-Proteobacteria. However, the community structure was stable. Our findings suggest that MCD-isolate exists with various bacterial consortia in water and degrades Mcysts, the function of which is considered to be induced by exposure to Mcyst.

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.

The 1,5-H-shift in 1-butoxy: A case study in the rigorous implementation of transition state theory for a multirotamer system

The rigorous implementation of transition state theory (TST) for a reaction system with multiple reactant rotamers and multiple transition state conformers is discussed by way of a statistical rate analysis of the 1,5-H-shift in 1-butoxy radicals, a prototype reaction for the important class of H-shift reactions in atmospheric chemistry. Several approaches for deriving a multirotamer TST expression are treated: oscillator versus (hindered) internal rotor models; distinguishable versus indistinguishable atoms; and direct count methods versus degeneracy factors calculated by (simplified) direct count methods or from symmetry numbers and number of enantiomers, where applicable. It is shown that the various treatments are fully consistent, even if the TST expressions themselves appear different. The 1-butoxy H-shift reaction is characterized quantum chemically using B3LYP-DFT; the performance of this level of theory is compared to other methods. Rigorous application of the multirotamer TST methodology in an harmonic oscillator approximation based on this data yields a rate coefficient of k(298 K,1 atm)=1.4×105 s−1, and an Arrhenius expression k(T,1 atm)=1.43×1011 exp(−8.17 kcal mol−1/RT) s−1, which both closely match the experimental recommendations in the literature. The T-dependence is substantially influenced by the multirotamer treatment, as well as by the tunneling and fall-off corrections. The present results are compared to those of simplified TST calculations based solely on the properties of the lowest energy 1-butoxy rotamer.

Energy transfer rates and impact sensitivities of crystalline explosives

The energy transfer rates between phonons and vibrons in the “doorway region” could be the rate-determining step for impact-induced detonation of molecular crystalline explosives. To investigate the relationship between impact sensitivities and energy transfer, the overall rate of energy transfer in the doorway region is estimated on the basis of a simple theory in which the rate is proportional to a product of the number of states and the rate of population relaxation. We estimated frequencies of normal mode vibrations of PETN, β-HMX, RDX, Tetryl, TNT, FOX-7, m-DNB, ANTA, PN, NQ, NTO, and DMN by means of density functional theory calculations at the B3LYP/6-31G(d) level of theory. Normal mode vibrations of TATB were evaluated by using empirical intra-molecular potentials. The number of doorway modes in the regions of 200 to 700 cm −1 was evaluated by the direct counting method. It is found that the number of doorway modes shows a strong correlation with impact sensitivities obtained by drop hammer tests. This can be explained by the theory, if we assume that the rate of population relaxation is almost the same for all of the explosives investigated in the present work. This assumption is consistent with recent experimental measurements of energy transfer rates of explosives at low temperatures.

Akinete abundance of N2-fixing cyanobacteria in sediment of Lake Balaton (Hungary)

The progressive eutrophication of Lake Balaton was perceived in the early 1970s. From 1975 to 1994, the development of summer blooms of N&2inf;-fixing cyanobacteria, especially Cylindrospermopsis raciborskii, became a regular phenomenon in the western part and sometimes in the eastern part of the lake. Since 1995, the biomass of phytoplankton, particularly N&2inf;-fixing cyanobacteria, has decreased dramatically. This paper investigates the change in the akinete abundance of N&2inf;-fixing cyanobacteria in the lake sediment in the 1980s and 1990s. A direct counting method was developed for determination of the abundance of akinetes in the sediment. It was found that in parallel with the reduction in biomass of N&2inf;-fixing cyanobacteria, the abundance of akinetes decreased approximately two orders of magnitude after 1994. However, the results also suggest that the decreasing number of akinetes in the sediment could not cause a `disappearance' of cyanobacteria from the phytoplankton assemblage.

Elevated abundance of bacteriophage infecting bacteria in soil.

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 x 10(7) g(-1)), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 x 10(8) g(-1), which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.

Distribution and abundance of resting cysts of the toxic Alexandrium spp. (Dinophyceae) in sediments of the western Seto Inland Sea, Japan

: Sediment samples were collected from 135 stations in the western part of the Seto Inland Sea (Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada) to determine the horizontal distribution and abundance of resting cysts of Alexandrium spp. (A. tamarense + A. catenella). Enumeration of the cysts was performed using the primuline-staining direct count method. Cysts of Alexandrium spp. were rarely found in Iyo Nada, Suo Nada and Beppu Bay, but were widely distributed in Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada. Cyst concentrations ranged from not detected (ND) to 14, ND to 17, ND to 4, 93 to 8137, 8 to 4454, ND to 6, ND to 18 and 4–29 cysts/cm3 wet sediment in Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada, respectively. The majority of cysts occurred in Tokuyama Bay and Hiroshima Bay, where higher densities were observed in the inner bay and along the coastal margin. Relatively higher cyst concentrations were observed at stations with a higher mud content. The abundance of Alexandrium spp. cysts in western Seto Inland Sea is lower than in the eastern Seto Inland Sea, except for Tokuyama Bay and Hiroshima Bay. However, because sporadic blooms of Alexandrium have been observed, continuing monitoring is necessary to prevent paralytic shellfish poisoning outbreaks in this area.

Colorimetric quantitation of in vitro cell density using carmine, a chromosome-specific stain

Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 m NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 × 10 3 to 5 × 10 5 cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.

The structure of the micromycete complexes of oligotrophic peat deposits in the southern taiga subzone of West Siberia

The analysis of the micromycete complexes of oligotrophic peat deposits in the Vasyugan Marsh by direct count and culture methods showed that the micromycete carbon comprises no more than 3% of the total peat carbon and that the microscopic fungal biomass varies from 2 to 13 tons/hectare, depending on the season and the peat deposit thickness. Fungal spores were found in all layers of the peat deposits, whereas the mycelium was found only in the active peat layer. The high abundance of eukaryotic cells in the peats was due to the presence of yeastlike cells rather than fungal spores. Analyses by culture methods showed that micromycetes were present in all peat layers and that their abundance tended to decrease with depth, except for yeasts, which were uniformly distributed in a vertical direction. The micromycete complexes of the peat deposits were similar in the diversity and abundance of dominant species but differed in the composition of minor species. Peat yeasts were dominated by ascomycetes.

Microbial degradation of two carbamate insecticides and their main metabolites in soil

Degradation studies in soil of the insecticides aldicarb and carbofuran and their metabolites (aldicarb sulfoxide, aldicarb sulfone; 3-ketocarbofuran and 3-hydroxycarbofuran) were carried out using laboratory systems under controlled conditions (temperature, water content, light). The insecticides were added to soil samples and subsamples of the soil were analyzed at different times to assess both the bacterial abundance and the concentration of the different chemicals. The epifluorescence direct count method was applied to the subsamples to estimate microorganism numbers (N/g soil). Untreated samples of soil were used as controls for evaluating the effects of the application of the insecticides on microbial abundance. Subsamples treated with the pesticides were analyzed using HPLC and the DT 50 s of the different compounds studied were calculated. The DT 50 values show that neither the parent compounds nor the transformation products have a high persistence in soil and there is a general increase in the concentration of microorganisms as the pesticides diminish.

Measurement of microbial numbers and biomass contained in thermophilic anaerobic reactors.

This paper describes the determination of the microbial population in terms of the number, biomass, and composition of single- and two-phase, laboratory-scale thermophilic (55 degrees C) anaerobic reactors under steady-state conditions. Epifluorescence microscopy with 4',6-diamidine-2-phenylindole (DAPI) as fluorochrome was used to determine the total number of microorganisms in the reactors, and autofluorescence microscopy was used to determine the total methanogenic bacteria populations. The results obtained by the direct count methods were compared to the quantity of biomass contained in the system, which was determined by volatile suspended solids. The concentration of acidogenic bacteria was estimated by subtraction of the autofluorescence results from those of the DAPI epifluorescence microscopy. The viable bacterial population was determined by plating techniques using an anaerobic chamber. The total bacterial and methanogenic populations of single-stage digesters increase when the hydraulic retention time decreases; nevertheless, the percentages of the principal bacterial groups (acidogenic and methanogenic) remain constant at 87% and 13%, respectively. In the two-stage reactors, the percentages of the acidogenic and methanogenic groups are 99% and 26% of the total population in the acidogenic and methanogenic reactors, respectively. In the single-stage reactors, biomass determinations can be used to estimate microbial concentrations, and vice versa, as there is a high positive correlation between microorganism concentration and biomass. The syntrophic relationship between the bacteria involved in the anaerobic process is a possible explanation for the low values of viable population obtained in the reactors studied. Nevertheless, there is a high correlation between direct counts by epifluorescence microscopy and viable plate counts for the combined system studied.

23] Assessment of metabolic potential of biofilm-associated bacteria

Publisher Summary This chapter discusses the assessment of metabolic potential of biofilm-associated bacteria. In the study of the microbial ecology of bacteria involved in biofilm formation, two major questions concern the metabolic status and phylogenetic identity of community members. Identification of bacteria has been revolutionized by rRNA-based phylogeny, and the development of rRNA-targeted oligonucleotide probes for fluorescent in sire hybridization (FISH) in natural microbial communities. If combined with spectroscopy or flow cytometry, some of them might also be useful tools at the population level. Most measurements of metabolic activities can be roughly grouped into methods detecting activities of specific enzymes, synthesis of macromolecules, or membrane potentials. Some of these methods, however, are not presented here, either because they have not yet been applied to environmental samples of biofilm populations or because they are not widely used in studies of bacterial metabolic activities. They include detection of (1) esterase activity by fluorescent substrates such as fluorescein diacetate or sulfofluorescein diacetate; (2) cell division by microcolony formation; (3) membrane potential by rhodamine or by propidium iodide; (4) membrane integrity by plasmolytic response to osmotic stress; or (5) DNA and RNA degradation by lysozyme treatment and 4', 6-diamidino-2-phenylindole (DAPI) staining. Thus, this chapter starts with the presentation of two of the most widely applied procedures for the detection of bacterial metabolic potentials: the cyanoditolyl tetrazolium chloride (CTC) method, and the direct viable count (DVC) method..

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

Dépistage de mammites subcliniques chez des vaches Goudali en lactation au Nord Cameroun

L’objectif du présent travail a été d’étudier la prévalence des mammites subcliniquesdans les exploitations bovines laitières de la Vina. Le comptage des cellulessomatiques par la méthode microscopique directe et le test de Schalm(Cmt, California Mastitis Test) ont été utilisés pour analyser des échantillons delait prélevés. Les prélèvements de lait de mélange ont été effectués dans 37exploitations de la région. Un taux moyen de 1 240 ± 765 x 103 cellules parmillilitre de lait a été obtenu avec une variation significative liée à la taille dutroupeau (p &lt; 0,05). Le suivi a été régulier et s’est étalé sur six mois dans troisexploitations différentes. Il a consisté à analyser mensuellement le lait demélange et les échantillons individuels de lait de vaches en lactation. Les résultatsdu comptage cellulaire direct du lait de mélange ont montré un taux cellulairemoyen de 834 ± 204 x 103 cellules par millilitre de lait, soit l’équivalentd’un score Cmt2 révélateur d’une infection mammaire. Aucune différencemensuelle significative n’a été enregistrée. L’analyse de 504 échantillons individuelsa permis de mettre en évidence l’effet significatif du rang de lactation etdu stade de lactation sur les résultats du comptage cellulaire du lait. Les résultatsont montré que les femelles zébus Goudali des zones tropicales, bien quefaiblement productrices de lait, étaient prédisposées aux mammites subcliniques.Ainsi, le comptage des cellules somatiques a semblé être un outil utileà développer à petite ou à grande échelle pour un dépistage systématique etrégulier dans un programme intégré de lutte contre les mammites.