A comparison of methods for assessing yeast viability

Summary Eight different methods were used to assess the cell viability of four strains of Saccharomyces. Staining with Mg-ANS, primuline yellow, FITC and methylene blue gave a good index of yeast cell viability. The standard plate count technique and microcolony formation also gave a good measure of cell viability. Fluorescent staining with acridine orange was the least useful of the methods tested. INT dye reduction gave a good index of respiring cells depending upon the yeast strain tested.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Systematic error and comparison of four methods for assessing the viability ofSaccharomyces cerevisiae suspensions

Four methods for the determination of cell viability were compared: the plate count technique, the flow cytometer, and two microscopic numerations- one after methylene blue staining and the other one with epifluorescence. The experimental error of these techniques was for the first time estimated: 8% for both numerations under microscope and 13% for the plate count technique. The staining mechanisms were explained by comparing the numerations under microscope and the flow cytometer analysis.     

A comparison of results obtained with two methods for assessing yield stability

Summary A major objective of the CIMMYT Maize Program is to develop open-pollinated varieties of maize (Zea mays L.) that are well adapted to a wide range of environments. To achieve this breeding goal, it is essential that the program use a stability technique that will identify high-yielding, stable genotypes accurately in international trials conducted under different environmental conditions. The objective of this study was to compare a spatial method with a modified conventional regression analysis method to determine the yield stability of 27 CIMMYT maize varieties evaluated at 37 locations. The methods also were compared on the basis of their consistency in assessing the stability of varieties when certain locations were omitted, and when subsets of varieties were analyzed. The varieties found to be stable by the spatial method with all sites included in the analysis were also stable (1) when the lowest and highest yielding sites were excluded from the analyses, and (2) when the varieties were considered, along with others, as a subset of the original group of materials. Stability parameters determined by regression analysis, however, varied for some varieties when (1) extreme sites were excluded, and (2) a subset of entries was considered in isolation. Because the spatial method was more consistent in identifying high-yielding stable varieties, it was considered the more useful of the two methods.     

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of methods for assessing toxicant effects on algal growth

Summary Ankistrodesmus braunii was used as the test organism to assess the effects of mercuric chloride on the growth of this species. Optical density measurements correlated highly with dry weight determinations, chlorophyll content, total cell counts, and respiring cell counts. Any one of the above methods may be used alone or in combination with other methods to assess the effects of toxicants on algal growth and survival.     

Effect of a bacteriocin-activated polythene film on Listeria monocytogenes as evaluated by viable staining and epifluorescence microscopy

AIMS: To evaluate the effect of a bacteriocin-activated polythene film on resting and growing populations of Listeria monocytogenes. METHODS AND RESULTS: The active polythene films were industrially obtained by coating a solution of bacteriocin 32Y from Lactobacillus curvatus upon the surface of the film to be in contact with the packaged material. The behaviour of live Listeria populations was examined in liquid suspensions directly in contact with the bacteriocin-activated film, packed in antimicrobial film, and in a challenge test of storage of frankfurters superficially contaminated by L. monocytogenes and packed in antimicrobial film. In all the experiments, live and dead cells of L. monocytogenes were counted in epifluorescence microscopy after viable staining, which proved to be a suitable method to evaluate the action of bacteriocins on populations of L. monocytogenes. The results showed that the direct contact between active film surface and L. monocytogenes cells is effective for a fast and irreversible inactivation of the population by determining a direct cell disruption. This was confirmed by the results of the challenge test indicating that the antimicrobial package was effective in inhibiting the growth and survival of the pathogen on the surface of frankfurters during storage. CONCLUSIONS: The use of the antimicrobial film is encouraged especially for solid food products where the superficial contaminants come immediately in contact with the antimicrobial film. SIGNIFICANCE AND IMPACT OF THE STUDY: A fast inactivation of the bacterial population, coupled with appropriate conditions of storage, can improve the quality and safety and prolong the shelf-life of the food products packed in antimicrobial films.     

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

New staining methods for sperm evaluation estimated by microscopy and flow cytometry

New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing accurate evaluation of sperm quality. However, before these test can be recommended for routine clinical and investigational use, in the stallion, they need to be confirmed on a larger number of stallions and their correlation with traditional semen parameters and with stallion fertility has to be demonstrated.     

A comparison of two methods for assessing critical flicker fusion frequency in hens

This paper compared two general methods for assessing critical fusion frequency in hens (gallus gallus domesticus). The first method was a conditional discrimination procedure with the stimuli presented successively. The second was a two-alternative forced-choice procedure with the stimuli presented simultaneously. It was found that both methods of stimulus presentation gave comparable results suggesting that either method is useful when investigating psychophysics in animals. The results also show that hens’ critical fusion frequency is considerably higher than that of humans which may account for hens’ inability to recognise images presented on standard computer or television monitors.     

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.     

A comparison of stereo-videos and visual census methods for assessing subtropical rocky reef fish assemblage

Environmental Biology of Fishes - Visual census conducted by divers has remained the most common method for obtaining quantitative data on reef fish assemblages since it was first applied in the...     

Ex-vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology

Ex-vivo fluorescence confocal microscopy (FCM) has been used on fresh tissue, but there is little experience on frozen sections. We evaluated the applicability of FCM on frozen sections of basal cell carcinomas (BCCs), stained with acridine orange and digitally colored to simulate hematoxylin and eosin (H&E) dyes. We compared our diagnostic accuracy in detecting and subtyping BCCs with FCM to our gold standard (H&E stained frozen sections used in 3D horizontal micrographic surgery). Fourty-six primary BCCs were analyzed for free margins as well as histological subtype with all FCM modes and conventional H&E staining. Adnexa, artifacts and diagnostic confidence were evaluated. Free margins were identified with a sensitivity and specificity of 92% and 91%. Concordance for tumor subtype was 88%. FCM may be used on both fresh tissue and frozen samples, although with reduced performance and different artifacts. The device is useful for the intraoperative diagnosis, subtyping and margin-mapping of BCCs.      

Fluorometric observation of viable and dead adhering diatoms using TO-PRO-1 iodide and its application to the estimation of electrochemical treatment

A rapid method for the direct measurement of viable and dead adhering diatoms was developed using a fluorescent dye, TO-PRO-1 iodide. By staining the marine diatom, Nitzchia closterium, with TO-PRO-1 iodide, viable and dead cells were identified as red and yellow cells respectively, under an epifluorescence microscope employing blue excitation. Only dead cells were stained with TO-PRO-1 iodide. Viable cells were observed as red because of autofluorescence arising from intracellular chlorophyll, whereas dead cells were observed as yellow because of the fluorescence of TO-PRO-1 iodide. The percentage of TO-PRO-1-iodide-stained was correlated with the percentage of dead cells in N. closterium cells exposed to heat (60 °C, 15 min). Other microalgae containing intracellular chlorophyll could be also distinguished as viable or dead cells by this fluorometric staining method. This method was applied for the assessment of N. closterium cells killed by the electrochemical treatment and used to monitor biofouling populations and their viability directly on the electrode surface. When 1.0 V was applied against a saturated calomel electrode, 99% of the cells attached to graphite electrode were killed in 1 h. Received: 7 August 1998 / Received revision: 16 October 1998 / Accepted: 7 November 1998     

Rapid quantification of planktonic ciliates: comparison of improved live counting with other methods

THE FOLLOWING EFFICIENT AND QUANTITATIVELY VALID METHOD TO FILTER CONCENTRATE AND COUNT LIVE PLANKTONIC CILIATES WAS DEVELOPED AND COMPARED WITH OTHER TREATMENTS: unconcentrated (raw) samples and centrifuged samples were counted live, and the effects of five different fixatives (HgCl(2), Lugol's iodine, formaldehyde, glutaraldehyde, and Champy-DaFano) on the counts were monitored. Samples originated from a eutrophic mountain lake (Lake Aydat, near Clermont-Ferrand, France). Overall, live filtered counts were similar to counts of raw samples, but they were significantly higher (2 to 2.3 fold, P < 0.05) by analysis of variance than counts from centrifuged samples. Nevertheless, some taxa, i.e., Halteria and Loxodes spp., were sensitive to filtration. The live filtered counts were also comparable to counts of raw HgCl(2)-fixed and settled samples. HgCl(2) and Lugol fixation consistently gave the highest total counts, while significantly lower counts were always obtained with Champy-DaFano-fixed samples. Losses due to fixation were insignificant for raw samples but were substantial and statistically significant in concentrated samples (15% after filtration and 71% after centrifugation, compared with counts from the corresponding live samples). Live counting of passively filter-concentrated ciliates has many advantages over other methods. It is two to four times quicker and more efficient. Ciliates are recognized with certainty, more species are identified, and enumeration of dead organisms (e.g., tintinnid loricas) is avoided. It should be recommended as a quantitatively valid alternative to classical methods for assessing planktonic ciliate populations.     

Quantitative intercomparison of transmission electron microscopy,flow cytometry,and epifluorescence microscopy for nanometric particle analysis

Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerated. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM); each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating < or =5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM.     

A comparison of two survey methods for assessing bird species richness and abundance in tropical farmlands

Capsule Although most birds do not need to refuel en route, many stop at Sandgerdi for a short period.