Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l^?1), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD® BacLight?). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l^?1 of free chlorine and in 10 min when the concentration is increased to 1.2 mg l^?1. However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.     

Determination of live and dead Komagataeibacter xylinus cells and first attempt at precise control of inoculation in nanocellulose production

The timely enumeration of cells of nanocellulose-producing bacteria is challenging due to their unique growth properties. To better understand the metabolism of the bacteria and better control the concentration of living cells during cultivation, a prompt cell counting technology is crucial and urgently required. In this work, two fluorescent dyes, the asymmetrical anthocyanidin dye SYBR Green I (SG) and propidium iodide (PI), were first combined for Komagataeibacter xylinus species to determine live/dead bacterial cells quantitatively and promptly. The number of live and dead K. xylinus cells determined using an epifluorescence microscope corresponded well to the results obtained using a fluorescence microplate reader. The R² values were 0.9986 and 0.9920, respectively, and were similar to those obtained with the LIVE/DEAD^® BacLight^TM commercial kit. SG/PI double-staining showed proper efficiency in distinguishing live/dead cells for the K. xylinus strain (R² = 0.9898). The technology was applied to standardize four different K. xylinus strains, and the initial cell concentration of the strains was precisely controlled (no significant difference among the strains, P> 0.05). The cellulose yield per live cell was calculated, and significant differences (P < 0.05) were found among the four strains in the following order: DHU-ATCC-1> DHU-ZCY-1> DHU-ZGD-1> ATCC 23770. The study shows (i) the application of the SG/PI staining to standardizing inocula for bacterial cellulose production so that a more accurate comparison can be made between different strains, and (ii) the lower cost of using SG rather than the SYTO 9 of the commercially available LIVE/DEAD^® BacLight^TM kit.     

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Viability of differentiated epilithic bacterial communities in the River Garonne (SW France)

Epilithic bacterial community viability was assessed on natural biofilm assemblages from environmentally contrasting locations over a 17-months period to determine if it reflects environmental conditions or conditions within the biofilm assemblage. Vital state was assessed by membrane integrity using LIVE/DEAD^® BacLight? staining kit. Samples were regularly collected in a large river, up and downstream of a large urban centre. Epilithic biomasses were similar between sites irrespective of the distinct water quality but varied temporarily, peaking up to 48 g AFDM m^?2. Bacterial community composition assessed by 16S rDNA based PCR-DGGE significantly differed between sites. Bacterial densities (median of 2.5 × 10¹¹ cell g AFDM^?1) were stable whatever the sample origin or biomass. Viable bacterial fractions ranged between 13 and 83% of the total bacterial densities and were correlated with hydrological stability indicators (average of 41.9% during stable water periods, 62.4% during intermediate flow regimes and 50.0% during flow instability) and seasonal parameters. At the river section and epilithic community scales, consistent bacterial densities per unit of biomass could reflect a biofilm assemblage carrying capacity while variable membrane integrity likely integrates changes in the vital state of the community under changing environmental conditions.     

Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.     

Analysis of cylindrospermopsin- and microcystin-producing genotypes and cyanotoxin concentrations in the Macau storage reservoir

The Macau storage reservoir (MSR) has experienced algal blooms in recent years, with high levels of Cylindrospermopsis and Microcystis and detectable concentrations of cyanotoxins. To analyze the cyanotoxin-producing genotypes and relate the corresponding cyanotoxins to the water quality parameters, a quantitative real-time polymerase chain reaction was developed and applied to the water samples in three locations of MSR. Cylindrospermopsin polyketide synthetase (pks) gene and a series of microcystin synthetase (mcy) genes were used for identifying and quantifying cylindrospermopsin- and microcystin-producing genes, and the corresponding water parameters were measured accordingly. Our results showed that high concentrations of cylindrospermopsin and low concentrations of microcystin were measured during the study period. There was a strong correlation between the pks gene numbers and cylindrospermopsin concentrations (R ² = 0.95), while weak correlations were obtained between the mcy genes numbers and microcystin concentrations. Furthermore, the pks gene numbers were strongly related to Cylindrospermopsis (R ² = 0.88), cyanobacterial cell numbers (R ² = 0.96), total algae numbers (R ² = 0.95), and chlorophyll-a concentrations (R ² = 0.83), consistent with the dominant species of Cylindrospermopsis among the cyanobacteria existing in MSR. NH₄–N (R ² = 0.68) and pH (R ² = 0.89) were the water quality parameters most highly correlated with the pks gene numbers. These results contribute to monitoring for potential cyanotoxins in raw water.     

Comparing BRIN-BD11 culture producing insulin using different type of microcarriers

This research was conducted to examine the growth profile, growth kinetics, and insulin-secretory responsiveness of BRIN-BD11 cells grown in optimized medium on different types of microcarriers (MCs). Comparisons were made on modified polystyrene (Hillex^® II) and crosslinked polystyrene Plastic Plus (PP) from Solohill Engineering. The cell line producing insulin was cultured in a 25 cm² T-flask as control while MCs based culture was implemented in a stirred tank bioreactor with 1 L working volume. For each culture type, the viable cell number, glucose, lactate, glutamate, and insulin concentrations were measured and compared. Maximum viable cell number was obtained at 1.47 × 10⁵ cell/mL for PP microcarrier (PPMCs) culture, 1.35 × 10⁵ cell/mL Hillex^® II (HIIMCs) culture and 0.95 × 10⁵ cell/mL for T-flask culture, respectively. The highest insulin concentration has been produced in PPMCs culture (5.31 mg/L) compared to HIIMCs culture (2.01 mg/L) and T-flask culture (1.99 mg/L). Therefore overall observation suggested that PPMCs was likely preferred to be used for BRIN-BD11 cell culture as compared with Hillex^® II MCs.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters

AIMS: The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). METHODS AND RESULTS: Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. CONCLUSIONS: This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. Significance and Impact of the Study: The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.     

The response of aggregated <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 cells to UV-C and UV-A/B disinfection

UV radiation is a spread method used worldwide for the disinfection of water. However, much of the research on the disinfection of bacterial cells by UV has focused on planktonic cells. Many bacterial cells in nature are present in clumps or aggregates, and these aggregates, which are more resistant to disinfection than their planktonic counterparts, can be problematic in engineered water systems. The current research used Pseudomonas putida (P. putida) CP1, an environmental and non-pathogenic microorganism which autoaggregates when grown under certain conditions, as a model organism to simulate aggregated cells. The study investigated the response of both the planktonic and the aggregated forms of the bacterium to UV-C (λ = 253.7 nm) and UV-A/B (λ > 300 nm) disinfection at laboratory scale in a minimal medium. The planktonic cells of P. putida CP1 were inactivated within 60 s by UV-C and in 60 min by UV-A/B; however, the aggregated cells required 120 min of UV-C treatment and 240 min of UV-A/B radiation to become inactive. The size of the aggregate was reduced following UV treatment. Although all the cells had lost culturability, viability as measured by the LIVE/DEAD^® stain and epifluorescence microscopy was not completely lost and the cells all demonstrated regrowth after overnight incubation in the dark.     

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Bac light bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation between the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcvs and Pseudomonas tested.     

Effect of a mouthrinse containing Malva sylvestris on the viability and activity of microcosm biofilm and on enamel demineralization compared to known antimicrobials mouthrinses

The purpose of this study was to evaluate the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of Malva sylvestris (Malvatricin^® Plus) compared with known antimicrobial mouthrinses. Microcosm biofilm was produced on enamel, using inoculum from pooled human saliva mixed with McBain saliva (0.2% sucrose) for 14 days. The biofilm was treated with mouthrinses for 1 min day^?1. Oral-B^® Complete, Listerine^® Zero and Malvatricin^® Plus had the greatest effect on the reduction of biofilm viability (p < 0.0001). On the other hand, lactic acid production was reduced significantly with PerioGard^®, Noplak^® Max and Listerine^® Zero compared with the control (p < 0.0001). No significant differences were found among the mouthrinses with respect to the colony-forming unit counting (total microorganisms, total streptococci, mutans streptococci and lactobacilli) and extracellular polysaccharide production. Enamel demineralization was reduced significantly with PerioGard^®, Noplak^® Max and Malvatricin^® Plus compared with the control (p < 0.0001). Malva sylvestris has a comparable anti-caries effect to chlorhexidine mouthrinses.     

Enumeration of viable CD34(+) cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit

Background aimsEnumeration of CD34⁺ cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences’ (BD) stem cell enumeration kit (SCE kit³), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit?.MethodsFresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur.ResultsResults from both assays were highly congruent, with an overall r² ≥ 0.99 (all specimen types included), a linear correlation across all CD34⁺ cell frequencies and concentrations, and an almost ideal steepness of the trend line.ConclusionsBoth assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34⁺ cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD? SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.     

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

Background aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 10⁴ to 28.3 × 10⁴ cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research.     

Reliability of cartilage digestion and FDA–EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA–EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA–EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA–EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA–EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

Bacterial Abundance, Activity, and Viability in the Eutrophic River Warnow, Northeast Germany

The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD BacLight bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4'6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different states of viable and active cells in natural bacterioplankton communities. However, it remains unclear why fractions of viable and active cells were rather low in this eutrophic river in comparison to similar waters. We recommend to carefully address cells as viable in contrast to nonviable, i.e., dead. As viable cells may be active or inactive with respect to many different activities, e.g., substrate uptake, respiration, hydrolysis, and cell deviation, it is necessary to choose the method to visualize active cells according to the question to be answered.     

Accuracy of the use of energy conversion factors for compound fish diets

Conversion factors of 23.6, 39.5 and 17.2 MJ/kg for protein, lipid and carbohydrate contents, respectively, are frequently used in fish studies to calculate the gross energy (GE) content of compound diets. Values predicted according to the above resulted in linear relationships of observed GE values with similar R ² and mean prediction error (MPE) values when using either nitrogen-free extract (NFE) (R ² = 0.5713, RMSE = 1.3134, MPE = 0.0741, n = 129, 32 studies) or starch (R ² = 0.5665, RMSE = 1.6768, MPE = 0.0839, n = 190, 45 studies) as measurements of carbohydrate content. Apparent digestible carbohydrate content (either NFE or starch) was found to be linearly-related (R ² values of 0.7531 and 0.7460, respectively) to its dietary content in compound fish diets. Predicted apparent digestible protein (ADP), lipid (DL) and carbohydrate contents, together with energy conversion factors, presented R ² and MPE values of 0.6205 (RMSE = 1.2606) and 0.2051, respectively, between observed and predicted apparent digestible energy (ADE) content with NFE as measurement of carbohydrate content (n = 97, 17 studies, eight fish species). However, with carbohydrates quantified by starch content, an R ² value of 0.7017 (RMSE = 1.7556) and MPE of 0.1055 were obtained (n = 37, 10 studies, five fish species).